BASIC FOOD MICROBIOLOGY

Micrograph of bacteria growing on alfalfa sprouts.

 BASIC FOOD
MICROBIOLOGY

Second Edition

George J. Banwart
Professor Emeritus
Department of Microbiology
The Ohio State University

CHAPMAN & HALL

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Basic tood microbiology, 2/e .
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Contents

Preface vii
1 General Aspects of Food 1
2 Estimating the Number of Microorganisms 11
3 Microorganisms Associated with Food 49
4 Factors That Affect Microbial Growth in Food 101
5 Sources of Microorganisms 165
6 Foodborne Agents Causing Illness 195
7 Indicator Organisms 371
8 Food Spoilage 393
9 Useful Microorganisms 433
10 Control of Microorganisms 505
11 Control of Microorganisms by Retarding Growth 545
12 Control of Microorganisms by Destruction 651
13 Regulations and Standards 725
Index 751
Preface

The second edition of Basic Food Microbiology follows the same general
outline as the highly successful first edition. The text has been revised
and updated to include as much as possible of the large body of infor-
mation published since the first edition appeared. Hence, foodborne ill-
ness now includes listeriosis as well as expanded information about
Campylobacter jejuni.
Among the suggestions for altering the text was to include flow sheets
for food processes. The production of dairy products and beer is now
depicted with flow diagrams.
In 1954, Herrington made the following statement regarding a review
article about lipase that he published in thejournal of Dairy Science: "Some
may feel that too much has been omitted; an equal number may feel that
too much has been included. So be it."
The author is grateful to his family for allowing him to spend the
time required for composing this text. He is especially indebted to his
partner, Sally, who gave assistance in typing, editing, and proofreading
the manuscript.
The author also thanks all of those people who allowed the use of
their information in the text, tables, and figures. Without this aid, the
book would not have been possible.
 1
General Aspects of Food

BASIC NEEDS

Our basic needs include air that contains an adequate amount of oxy·
gen, water that is potable, edible food, and shelter. Food provides us with
a source of energy needed for work and for various chemical reactions.
Food also supplies chemicals for growth, for repair of injured or worn·
out cells, and for reproduction. Food consumption can be a pleasurable
experience, and a time for meeting with family and friends. Food is so
necessary for our existence that the search for food has been the main
occupation of human beings throughout history.

FOOD NEEDS

In the United States, supermarket shelves are so well· stocked that an

uninformed observer might assume that our search for an adequate food
supply has been successful. However, it is believed that 12.5 percent of
the earth's people receive considerably less food than they need; as many
as 50 percent might be receiving a marginal level of food (Kahn 1981).
The reasons for this widespread hunger problem include unequal distri·
bution of food and money, as well as cultural, religious, and superstitious
beliefs.
The main problem facing the world today is its increasing population
(Fig. 1.1). In recent years the birth rate has declined, but so has the death
rate. The present world population is over 5 billion, and the annual in·
crease is estimated to be 60 to 80 million people. Predictions for the
future food supply range from very pessimistic, with famines expected
to start in the near future, to the very optimistic viewpoint that there will
be plenty of food for a population of almost limitless numbers. The most
widely held opinion seems to be that, unless the rate of population in·
crease can be substantially reduced, the demand for food will eventually
outrun the supply, regardless of the efficiency of food production.
Although world agricultural production has been increasing, the va·
garies of nature (floods, droughts, freezing, and other adverse climatic
conditions) could cause a severe setback. With the expected increase in
2 BASIC FOOD MICROBIOLOGY

z 4
2
~
-'-
~
a. c 3
.
~~
o§
-'
II:
0
~ 2

Figure 1.1. Estimated world population. Will we be able to feed everyone in the fu-
ture?

population, the search for food will continue to be the primary endeavor
of many food scientists, including food microbiologists.

SOURCES OF FOODS

Our supply of food depends upon the photosynthetic reaction be-

tween solar energy and plants that contain chlorophyll. Through photo·
synthesis, carbon dioxide and water are converted to glucose. Further
cellular reactions produce the various organic compounds-carbohy.
drates, fats, proteins, and vitamins.
Both the amount of food and its dietary quality can be increased. To
do this, we can improve the utilization of our land and fishery resources,
upgrade the quality of plant proteins, convert waste materials into edible
foods, and prevent losses or deterioration of our food supplies.

Land Resources
Increasing the productivity of land resources will require utilization
of more land and higher yields of foods. However, there is a finite
 GENERAL ASPECTS OF FOOD 3

amount of land, and not all of the land that is available can be used
for agriculture. Even less can be used for crop production. Experts have
estimated that the amount of land used for agriculture can be doubled.
This will not necessarily double production. These added lands are of
marginal quality and will require increased water, fertilizer, technology,
and energy to make them productive.
One problem in American food production is the loss of good agri·
cultural land for the construction of highways, airports, shopping cen·
tel's, housing, and factories. With an increasing population, there will be
an even greater demand for land for these purposes. The United States is
now losing about 3 million acres of arable land per year to development.

Fishery Resources
Oceans and seas cover more than 70 percent of the earth's surface,
yet in the United States less than 10 percent of our protein comes from
fishery resources. It is likely that this resource could be utilized more
effectively as a source of food for the future. As on land, the principal
food·producing organisms in the sea are plants (phytoplankton). These
plants use the photosynthetic process to produce food for herbivorous
animals in the sea. These, in turn, furnish food for small carnivorous
animals, larger fish, and ultimately human beings.

Microorganisms as Food
The use of microorganisms in food products is not a new idea. The
action of yeast in the fermentations producing wine and beer and the
leavening of doughs has been known for at least 4,000 or 5,000 years.
The nature of the action in these fermentations did not become estab·
lished until the latter part of the nineteenth century when the relation
of living yeast cells to fermentation was discovered. Microorganisms are
used in the fermentation of various foods and are consumed as part of
these foods. This is especially evident in cheese. Penicillium roquejorti, the
blue mold of Roquefort cheese, and Penicillium camemberti, the white mold
of Camembert cheese, are consumed with the cheese. Therefore, the con·
cept of using microorganisms as part of the food supply should not be
completely objectionable.
Although we can synthesize amino acids and polypeptides commer·
cially, microorganisms can produce not only these substances, but pro·
teins, antibiotics, vitamins, steroids, and many other products. The devel·
opment of microorganisms as a food source will involve many
disciplines, but food microbiologists will play an especially important
role.
Bacteria, yeasts, or molds cannot create foods, but they can grow on
4 BASIC FOOD MICROBIOLOGY

cellulosic compounds that would otherwise be wasted. Algae can utilize

solar energy to produce food. The production of microbial protein is
discussed in Chapter 9.

Wastes as Food
For every kilogram of food produced, between 5 and 10 kg of waste
materials are left in the field or at the processing plant. These substances
are considered wastes because their economic value is such that it is not
profitable to utilize them. Surprisingly, not long ago, fat was removed
from milk, fish, and oilseeds for human use, while the more valuable
protein was wasted or fed to animals. Hence, the "waste" products of
today may have food value in the future.
Some wastes are not readily usable because they are seasonal, diluted
with water, or require transportation to amass a large quantity for pro-
cessing. With waste disposal becoming an ever-increasing problem, and
food shortages becoming more critical, it is difficult to believe that the
problems involved in utilizing acceptable wastes cannot be resolved.
Processes such as reverse osmosis and ultrafiltration can be used to
concentrate dilute wastes (Moon 1980). Alternative systems for concen-
tration of wastes in the meat industry have been discussed (Hansen 1983).
Progress is being made in the recovery of wastes and in the utilization of
these materials in human foods and in animal feeds (Cherry, Young, and
Shewfelt 1975; Cooper 1976; Kamm et al. 1977; Knorr 1983; Toyama
1976).

Legal Aspects
U.S. food laws and regulations must be considered before any new
or novel food can be sold. The U.S. Department of Agriculture (USDA)
regulates red meat and poultry processing operations. In all other cases,
the U.S. Food and Drug Administration (FDA) decides what is an accept-
able food or food ingredient. Besides the dictates of these federal agen-
cies, the food laws of states and local jurisdictions must be followed.
If an ingredient is to be used in a food, it cannot create a health
hazard. The Food, Drug, and Cosmetic Act provides that a food shall be
deemed to be adulterated if it consists in whole or in part of any filthy
substances. What will be the FDA's reaction to single-cell protein ob-
tained from microorganisms grown on unconventional substrates? If a
food shortage does develop, we may need to reevaluate the aesthetic as-
pects of our foods. The health criteria for processed wastes have been
discussed (Taylor et al. 1974).
 GENERAL ASPECTS OF FOOD 5

Preventing Losses
The data on losses of plant and animal products are neither adequate
nor reliable enough to allow us to conduct investigations to fully deter·
mine the causes of the losses. We do know that deterioration, waste, and
loss occur in almost every step from production to consumption. Since
we already have a shortage of food and will need more food in the future,
we must make an increased effort to protect our food supply from fur·
ther losses. If losses could be reduced or prevented, our food supply
would increase with no additional utilization of land or sea resources.
The main losses of foods result from the action of microorganisms,
insects, rodents, birds, nematodes, and the enzymes inherent in the food.
One study (Ennis, Dowler, and Klassen 1975) estimated that 30 percent
of crops worldwide are lost to pests. Albrecht (1975) suggested that pests
destroy 25 percent of all crops in the United States. He believed that
government regulations prevent adequate pest controL Schweigert
(1975) estimated that food losses from production to consumption range
from 20 to 50 percent. The monetary loss from producer to consumer is
reported to be more than $30 billion in the United States.

Reasons for Food Preservation

Preservation can be defined as a process by which foods are treated
to retard decay or spoilage. There are many reasons for preserving foods.
Several plant foods are harvested only once each year. To have a supply
of these foods throughout the year, rather than only at harvest time, pres·
ervation is necessary. In case of a crop failure caused by a natural disaster
such as drought, wind, hail, flood, fire, freezing, or insect and disease
infestations, or by human disasters, such as war, the preservation of previ·
ously produced excess food becomes paramount.
With preservation, one can obtain a more varied diet because a crop
can then be used throughout the year and because crops native to only
a small area can be transported and used anywhere in the world. One of
the reasons developing countries have food shortages is that they do not
have facilities for preservation and transportation of foods. Thus, certain
areas have a temporary surplus of food while other areas have a shortage.
Flesh foods deteriorate rapidly if held at ambient temperatures. In
some countries, although fish is plentiful in the coastal areas, there is a
protein shortage inland because refrigeration and rapid transit are lack·
ing to transport the fish protein without spoilage.
Preservation allows the holding of foods so that they can be used as
ingredients for mixed foods. Many of our convenience foods are combi·
nations of various foods. Some systems used to preserve food also destroy
6 BASIC FOOD MICROBIOLOGY

many of the organisms and toxic factors that are hazards in food prod-
ucts.

METHODS OF FOOD PRESERVATION. The chief methods of food

preservation can be listed in four basic categories: asepsis (preventing
entry of microorganisms into foods); removing microorganisms; inhibit-
ing growth by controlling the environment; and destroying microorgan-
isms. Various systems have evolved from these basic procedures (Table
1.1).
Most methods of preserving food are merely modifications of systems
used in ancient times. The addition of salt as a chemical preservative,
fermentations, smoking, and cold storage have been practiced for over
2,000 years. Comparatively, canning might be considered a modern
method, although the process of preserving food by putting it into a
closed container and heat-treating it was patented by Appert in 1810. A
much newer system for preserving food involves the use of radiation; at
this time, however, irradiation has been approved by the FDA only for
specific purposes (see Chapter 12).

FOOD HAZARDS

From the beginning of life until death, a person is subjected to poten-

tially harmful environments. During one's lifetime, some hazards disap-
pear and others take their place, so that the problems of safety are not
static. The ingestion of food is no exception. Food may serve as a carrier
of chemical and biological substances, either added or acquired as con-
taminants from soil, water, air, food handlers, equipment, and other
sources. The possible subtle relationships between these substances in
foods and physical vigor, mental alertness, longevity, resistance to infec-
tion, and the onset of degenerative diseases are not fully understood.
Since we do not have all the answers regarding the safety of all possible
substances, there are many controversies concerning the overall safety of

TABLE 1.1. FOOD-PRESERVATION METHODS

Asepsis Gas or vacuum packing
Cen trifugation Acidification
Filtration Fermentation
Refrigeration Fumigation
Freezing Pasteurization
Drying Cooking
Freeze-drying Canning
Chemicals Radiation
Smoking
 GENERAL ASPECTS OF FOOD 7

food. Although absolute safety of food is an ideal goal, for all practical
purposes, it is unattainable. Since a human being is a biological system,
and since biological systems vary, a food that causes no ill effects in one
person may cause problems in another person.
Wodicka (1977) listed six principal categories of food hazards: micro·
biological hazards, malnutrition, environmental contaminants, naturally
occurring toxins, pesticides, and conscious food additives. Drug residues
or filth in foods might be hazardous when ingested. In addition, muta-
gens and carcinogens may be formed when certain foods are heated
(Grose et al. 1986; Jigerstad et al. 1983). Many chemicals that can be in-
gested relatively safely at low levels may be hazardous when ingested at
high levels. Even a high-fiber diet may reduce the absorption of essential
vitamins and minerals from the digestive tract.

Naturally Occurring Toxins

Several books and articles have been written concerning toxic agents
naturally present in foods (Coon 1975; Elton 1981; Gori 1979; Hatfield
and Brady 1975; Hironi 1981,Jadhav, Sharma, and Salunkhe 1981; Lewis
and Endean 1983; McMichael 1984; Munro 1976; Onoue et al. 1983; Op-
penheimer 1985; Panasiuk and Bills 1984; Shupe and James 1983; Swain,
Truswell, and Loblay 1984; Taylor 1982, 1985; van der Hoeven et al. 1983;
Wilson, McGann, and Bushway 1983). These naturally occurring toxins
include estrogens, tumorigens, carcinogens, cyanogens, as well as seafood
toxins, fungal toxins (mycotoxins), nutritional inhibitors, and antigens
that produce allergies. The quantities of these substances in foods are
usually low and, during processing, some of these substances are altered
to reduce their potency.
Problems resulting from the natural toxicants are often due to people's
eating raw foods or too much of only one type offood, or mistaking toxic
plants for similar, edible plants. Potatoes contain the alkaloid solanine,
a potent cholinesterase inhibitor that interferes with the transmission of
nerve impulses. The amount of potatoes an average person eats each
year contains enough solanine to be fatal if consumed in one dose.
At high levels, even polyunsaturated fats reportedly increase the inci-
dence of tumors and gallstones, increase the body's requirement of vita-
min E, and cause premature aging in laboratory animals. When exces-
sively heated, these fats are reported to contain toxic substances. One
product, malonaldehyde, is carcinogenic. Potentially carcinogenic lipid
peroxides are easily formed from polyunsaturated fats by autoxidation.
Many substances not generally considered toxic may present a poten-
tial hazard to some people. For example, some consumers of milk de-
velop severe distress of the digestive system because of an enzyme defi-
ciency that results in an intolerance to lactose.
8 BASIC FOOD MICROBIOLOGY

Microorganisms
Data compiled by the Centers for Disease Control (CDC 1981a, 1981b,
1983) show that microbiological hazards are by far the most common
type of food hazard (Table 1.2). Since not all foodborne illnesses are reo
ported, the CDC data are not exact, but they are the most complete data
presently available. Each year, more than 60 percent of foodborne out·
breaks are the result of bacterial etiologies, while less than 30 percent
are due to chemicals from various sources. The CDC has discontinued
listing foodborne outbreaks caused by unknown etiologies. In the past,
such outbreaks accounted for 25 to 30 percent of the total number.
Although not listed as causing any foodborne outbreaks, mycotoxins,
the toxins produced by molds, have received much attention in the past
ten to fifteen years. They are considered to be naturally occurring toxins
in foods.
Besides microorganisms, there are other biological entities that pre·
sent a potential health hazard. Trichinella spiralis (the agent of trichinosis),
Taenia solium (pork tapeworm), Taenia saginata (beef tapeworm), Ascarias
(a roundworm) and Entamoeba histolytica (which causes amoebic dysen·
tery) are a few of the agents that have been found in foods.

ROLE OF THE MICROBIOLOGIST

The food microbiologist is concerned with the biochemical reactions

of microorganisms in and on foods. These reactions result in spoilage,
public health hazards, and fermentation products. Determining the num·
bers and types of microorganisms associated with food, and knowing
the sources of microorganisms, factors affecting their multiplication, and
systems that can be used for their control are important to food micro·
biologists. However, we cannot look only at these aspects, but must also
examine other facets of foods, such as their chemical and physical charac·

TABLE 1.2. CONFIRMED FOODBORNE OUTBREAKS, 1978-1980

1978 1979 1980
Cause No. % No. % No. %
Bacterial 105 68.2 119 69.2 136 61.5
Viral 5 3.2 6 3.5 12 5.4
SL:IITOTAL 110 71.4 125 72.7 148 66.9
Parasitic 7 4.5 11 6.4 7 3.2
Chemical 37 24.0 6 20.9 66 29.9
TcrrAL 154 142 221
SOURCE: Data from CDC (l981a, 1981b, 1983)
 GENERAL ASPECTS OF FOOD 9

teristics and the various attributes that are referred to as quality. We can
sterilize a food to destroy all microorganisms, but if the process makes
the food inedible or depletes its nutritional value, then the sterilization
process is not satisfactory.
With an understanding of food science, a food microbiologist can
better relate his or her role to the very important endeavor of providing
all people with an adequate supply of safe, wholesome foods. The role
of the food microbiologist in the food industry was discussed by Bauman
(1982) and Winslow (1982).

REFERENCES

Albrecht,].]. 1975. The cost of government regulations to the food industry. Food Technol.
29(10): 61, 64-65.
Bauman, H. E. 1982. The food microbiologist's role in the decision·making process. Food
Technol. 36(12): 58-59.
CDC. 1981a. Foodborne Disease Surveillance, Annual Summary, 1978. Atlanta, Ga.: Cen·
ters for Disease Control.
- - . 1981 b. Foodborne Disease Surveillance, Annual Summary, 1979. Atlanta, Ga.: Cen·
ters for Disease Control.
- - . 1983. Foodborne Disease Surveillance, Annual Summary, 1980. Atlanta, Ga.: Cen·
ters for Disease Control.
Cherry,]. P.; Young, C. T.; and Shewfelt, A. L. 1975. Characterization of protein isolates
from keratinous material of poultry feathers.]. Food Sci. 40: 331-335.
Coon,]. M. 1975. Natural toxicants in foods.]. Amer. Diet. Assoc. 67: 213-218.
Cooper,]. L. 1976. The potential of food processing solid wastes as a source of cellulose
for enzymatic conversion. Proceedings of Biotechnol. Bioeng. Symp. 6: 251-271.
Elton, G. A. H. 1981. Additives and contaminants in the food supply. Food Technol. Aust.
33(4): 184-188.
Ennis, W. B.,Jr.; Dowler, W. M.; and Klassen, W. 1975. Crop protection to increase food
supplies. Science 188: 593-598.
Gori, G. B. 1979. Food as a factor in the etiology of certain human cancers. Food Technol.
33(12): 48-56.
Grose, K. R; Grant,]. L.; Bjeldanes, L. F.; Andresen, B. D.; Healy, S. K.; Lewis, P. R.; Felton,
]. S.; anrl Hatch, F. T. 1986. Isolation of the carcinogen IQ from fried egg patties.].
Agr. Food Ghern. 34: 201-202.
Hansen, C. 1983. Methods for animal waste recovery and energy conservation. Food Tech·
nolo 37(2): 77-80, 84.
Hatfield, G. M., and Brady, L. R. 1975. Toxins of higher fungi. Lloydia 38: 36-55.
Hironi, 1. 1981. Natural carcinogenic products of plant origin. Grit. Rev. Toxieol. 8(3):
235-277.
Jadhav, S.].; Sharma, R P.; and Salunkhe, D. K. 1981. Naturally occurring alkaloids in
foods. Grit. Rev. Toxieol. 8(3): 21-104.
Jiigerstad,.M.; Reutersward, A. L.; Oste, R; Dahlqvist, A.; Grivas, S.; Olsson, K.; and Nyham·
mar, T. 1983. Creatinine and Maillard reaction products as precursors of muta·
genic compounds formed in fried beef. In The Maillard Reaction in Foods and Nutrition
(G. R Waller and M. S. Feather, editors), pp. 507-519. ACS Symposium Series, No.
215.
10 BASIC FOOD MICROBIOLOGY

Kahn, S. G. 1981. World hunger: An overview. Food Technol. 35(9): 93-98.

Kamm, R.; Meacham, K.; Harrow, L. S; and Monroe, F. 1977. Evaluating new business
opportunities from food wastes. Food Technol. 31(6): 36,38-40.
Knorr, D. 1983. Recovery of functional proteins from food processing wastes. Food Tech·
nol. 37(2): 71-76.
Lewis, R.].; and Endean, R. 1983. Occurrence of a ciguatoxin·like substance in the Span·
ish mackerel (Scomberomoruscommersoni). Toxicon 21: 19-24.
McMichael, A.]. 1984. Dietary influences upon human carcinogenesis. Food Technol. Aust.
36(10): 460-463, 465.
Moon, N. J. 1980. Maximizing efficiences in the food system: A review of alternatives for
waste abatement.]. Food Prot. 43: 231-238.
Munro,1. C. 1976. Naturally occurring toxicants in foods and their significance. Clin.
Toxicol. 9: 647-663.
Onoue, Y.; Noguchi, T.; Maruyama,].; Hashimoto, K.; and Seto, H. 1983. Properties of
two toxins newly isolated from oysters.]. Agr. Food Chem. 31: 420-423.
Oppenheimer, S. B. 1985. Human·made carcinogens vs. natural food carcinogens:
Which pose the greatest cancer risk? Amer. Clin. Prod. Rev. 4(2): 16,18-19.
Panasiuk, 0., and Bills, D. D. 1984. Cyanide content of sorghum sprouts.]. Food Sci. 49:
791-793.
Schweigert, B. S. 1975. Food processing and nutrition-Priorities and needed outputs.
Food Technol. 29(9): 36, 38.
Shupe,]. L., and James, L. F. 1983. Teratogenic plants. Vet. Human Toxicol. 25: 415-421.
Swain, A.; Truswell, A. S.; and Loblay, R. H. 1984. Adverse reactions to food. Food Tech·
nolo A ust. 36: 467-468, 471.
Taylor,]. C.; Gable, D. A.; Graber, G.; and Lucas, E. W. 1974. Health criteria for processed
wastes. Fed. Proc. 33: 1945-1946.
Taylor, S. L. 1982. An overview of interactions between foodborne toxicants and nutri·
ents. Food Technol. 36(10): 91-95.
1985. Food allergies. Food Technol. 39(2): 98-105.
Toyama, N. 1976. Feasibility of sugar production from agricultural and urban cellulosic
wastes with Trichoderma viride cellulase. Proceedings of Biotechnol. Bioeng. Symp. 6: 207-
219.
van der Hoeven,]. C.; Laqerweij, W.].; Bruggeman, 1. M.; Voragen, F. G.; and Koeman,
]. H. 1983. Mutagenicity of extracts of some vegetables commonly consumed in the
Netherlands.]. Agr. Food Chem. 31: 1020-1026.
Wilson, A. M.; McGann, D. F.; and Bushway, R. ]. 1983. Effect of growth· location and
length of storage on glycoalkaloid content of roadside· stand potatoes as stored by
consumers.]. Food Prot. 46: 119-121, 125.
Winslow, R. L. 1982. The food microbiologist's role in the professional execution of in·
dustry's goals for a safe, wholesome food supply. Food Technol. 36(12): 60-62.
Wodicka, V. O. 1977. Food safety-rationalizing the ground rules for safety evaluation.
Food Technol. 31(9): 75-77, 79.
 2
Estimating the Number
of Microorganisms

An important aspect of food microbiology is the examination of food or

other materials for microorganisms.

NUMBERS OF MICROORGANISMS IN FOOD

The number of microorganisms in a food as determined by the aero·

bic plate count (APC) is variable because of the original contamination,
increase or decrease of microorganisms during processing, recontamina·
tion of processed product, and growth or death during storage, retailing,
and handling. The microbial flora are changing constantly. In foods such
as refrigerated fresh meat, the microbial numbers increase during stor·
age, whereas in dried or frozen foods, the viable organisms tend to de·
crease in number. The APes for a food may vary from less than 10 to
over 100,000,000 microorganisms per gram, depending upon the prod·
uct, how long it was stored, and the temperature of storage. The loga·
rithms of the range of APes reported for various foods are listed in Table
2.1. The microbial contents of various foods were reported by Pizzo,
Purvis, and Waters (1982).
The usual range of organisms in most animal products is 1,000 to
10,000 per gram. Ground meat is more contaminated than whole cuts of
meat because of the type of meat that is used in the product, the extra
handling during grinding, and the release of meat juices that allow bacte·
ria to multiply. Foods that receive a heat treatment generally have lower
microbial numbers than foods not heated. Even then, poor·quality ingre·
dients, poor sanitation, unsatisfactory heating, recontamination, or poor
handling and storage, cause some heated products to have high numbers
of microorganisms.
An estimate of the number of microorganisms in or on foods is
needed in order to determine if a product meets the microbial levels
expressed in specifications, guidelines, or standards. Spoilage of some
foods is imminent when the APe reaches very high numbers (10 7 -10 8 /g).
Hence, the microbial count can be used to help predict the shelf life of
11
TABLE 2.1. AEROBIC PLATE COUNTS OF VARIOUS FOODS
Food Overall Range" Usual Range"
Animal Products
Beef (steaks, roasts) 2-6 4
Beef (ground) 3-8 5-7
Pork sausage 4-6 5
Ham 1-8 4
Bacon 3-7 4
Dry sausage 3-7 4-5
Chicken carcasses (cm 2) 2-7 3-4
Fish (fresh) 2-8 4-5
Fish (smoked) 1-7 2-4
Fish sticks or crab cakes 2-6 3-4
Shrimp (raw) 2-7 4-5
Shrimp (raw, breaded) 2-8 4-6
Milk (raw, grade A) 2-5 3
Milk (pasteurized) 2-4 2
Milk (dry) 1-6 2-3
Butter 3-5 4
Plant Products
Raw
Almonds 0-4 3
Beans or peas 3-7 4-5
Broccoli or kale 6-7
Carrots, potatoes, or spinach 4-7
Corn or cucumbers 5-7
Tomatoes 3-7
Frozen
Asparagus, beans, or peas 2-5
Corn 2-7
Squash 2-4
Dried
Carrots 2-4
Garlic 4-6
Parsley 2-5
Spices
Cinnamon 1-5 2-3
Cloves 2-3 3
Ginger 2-7
Nutmeg 2-4
Oregano 2-6 3-4
Pepper 6-7 7
Sage 3
Mixed dried
Soup (meat· type) 3-5 4
Soup (vegetable·type) 2-5 3-4
Salads
Chicken or ham 1-7 3-5
Green 3-8 5-6
Macaroni 3-6 4-5
Shrimp 3-7 6
Tuna 2-6 3-4
a Reported in logarithms of bacteria per gram.

12
 ESTIMATING THE NUMBER OF MICROORGANISMS 13

certain foods. To a limited extent, the microbial numbers might be used

to evaluate the potential safety of foods. The count also might indicate
if the product was produced under sanitary conditions, or if the product
was mishandled during harvesting, processing, or storage. In general, as
the microbial count increases, the quality of the food is reduced. This
generalization does not apply to fermented foods, since microorganisms
are used in their production. There are cases in which the number of
microorganisms in a food has little or no relationship to potential shelf
life, spoilage, or a health hazard. Other factors to be considered include
the type of food, the type of microorganisms present, and the storage
conditions.
To ensure production of food with a low number of microorganisms,
the producer must assay not only the final food product but also such
things as ingredients, processing equipment, packaging, and environ·
mental samples. These determinations aid in the evaluation of general
sanitary practices prevailing during processing and handling of food,
and the potential sources of contamination. The determination of micro-
bial numbers is needed to evaluate the effectiveness of methods of pres·
ervation.
The presence of particular types of microorganisms, especially poten·
tial pathogens or toxin producers, is more important than the estimate
of the total number of microorganisms. In general, the main difference
in these analyses is that specific types of microorganisms are determined
with selective or differential media rather than with noninhibitory me·
dia. Thus, for purposes of simplicity, this discussion will be limited to
total number estimations. Some of the special procedures are discussed
with specific organisms in later chapters of this text.
Although the term total count has been used, no single method or me·
dium is capable of detecting all of the microorganisms in a food. Thus,
the counts that are obtained are merely estimates of the actual microbial
population. Errors of ± 90 percent in counts are not unusual when the
level is 10,000 to 100,000 per gram (Collins and Lyne 1976). Besides the
errors, many assumptions are involved in microbial estimations. Also,
there are factors that affect the growth of microorganisms and influence
the results when the viability of the cells is involved in the enumeration
technique. With all of these considerations, it is essential that the techni·
cian doing the testing does not further influence the results by using
poor technique.
For microbial analysis, a sample and a system for estimating the num·
ber of microorganisms in the sample are needed. After the data from the
evaluation are obtained, the information must be reported and, when
necessary, follow·up checks should be made. If the report is for manage·
14 BASIC FOOD MICROBIOLOGY

ment, an interpretation of the results might be included. What do they

mean? Are the levels of microorganisms acceptable, or are they too high?

THE SAMPLE

If the samples are not delivered to the laboratory, it might be neces·

sary to establish a sampling procedure. The samples of food might be
obtained from the processing line, from warehouse storage, or from reo
tail shelves. Food is processed as liquid, solid, mixed solid and liquid, or
semisolid, and in many shapes and sizes. Since there are many variables
in the food and many places of sampling, several sampling plans will be
needed. Sampling suggestions have been made for various factors in
food products (AOAC 1985; APHA 1984; Barrow 1983; FDA 1978; Jones
1979; Kilsby and Pugh 1981; Montagna 1982; Rao and Koehler 1979; Rob·
erts, MacFie, and Hudson 1980; Schutz 1984).
The sampling plan should reflect the ultimate use of the analysis, the
potential health hazard of the food, or potential for spoilage. If the reo
suits are needed to satisfy the requirements of a microbiological stan·
dard, the sampling plan as outlined in the standard should be followed.
If the results are for the producer's information, a less restrictive sam·
piing plan can be used. Sampling plans have been suggested for micro·
biological standards (Biltcliffe et al. 1983; ICMSF 1974; Martin 1979) and
for salmonellae (health hazard) testing (Olson 1975). Further discussion
of these sampling plans is presented in appropriate chapters of this text.
A sample will yield significant and meaningful information only if it
represents the mass of material being examined, is collected in a manner
that protects it against microbial contamination, and is protected from
changes in the population that might occur between collection and anal·
ysis.

Representative Samples
The need for a representative sample cannot be overemphasized. The
results of the analysis can be no more reliable than the sample on which
they were based. Usually microorganisms are not distributed homoge·
neously, so thorough mixing of the product prior to sampling is impor.
tanto Thorough mixing is not as easy for nonliquid foods as for liquid
foods.
The size of the particles being sampled may influence the sampling
procedure, since many particles of a product such as powdered milk can
be obtained; but if the product were sides of beef, a different procedure
would be necessary.
 ESTIMATING THE NUMBER OF MICROORGANISMS 15

Sampling material in motion, such as on a production line, tends to

minimize variables and gives a more representative sample than sam-
pling material at rest, such as in stacks in a warehouse or on retail shelves.
With on-line sampling, automatic sampling devices might be considered.
These devices usually give a more random and reliable sample and at
less cost than manual sampling of the product.
If cases or containers are stacked as a lot, the person collecting the
samples must randomly select containers throughout the entire pile. If
only containers around the edges or in front of the stack are selected, he
or she is introducing a bias into the results of the analysis.
The laboratory analysis is usually more expensive than obtaining the
sample, so cutting corners in sampling is not the way to save money.

Number of Samples
The number of samples needed, or the frequency of sampling, de-
pends upon many factors. The uniformity or homogeneity of the prod-
uct, the size of the many particles, previous knowledge of the material,
and experience will help dictate the amount of sampling needed. Either
too few samples or too many samples waste product, laboratory material,
and labor.
For microbiological standards, the number of samples to be obtained
and analyzed is included in the standard. One of the prime consider-
ations that influence the number of samples to be analyzed is the poten-
tial health hazard of the foods. Statistical sampling schemes will help en-
sure that the samples give an acceptable assessment of the microbial
conditions of the food, ingredient, or other substance being analyzed.

Aseptic Collection of Samples

Aseptic technique is needed when samples are collected. To prevent
possible contamination, if the samples are in individual containers, such
as cans, bottles, or boxes of food, they should be taken directly to the
laboratory for analysis. On the other hand, if the product is in bulk or
in containers of impractical size to submit directly to the laboratory, rep-
resentative portions must be transferred to sterile containers using asep-
tic technique.
Since there is little interest in bacteria associated with sampling de·
vices or sample containers, the instruments must be sterile. If possible,
the instruments should be sterilized in the laboratory rather than at the
place of sampling. After the sampling equipment is cleaned, the pre-
ferred methods of sterilization are (1) steam at 121.5°C in an autoclave
for 15 to 30 min (the time for exposure depends on how bulky the mate-
16 BASIC FOOD MICROBIOLOGY

rial is and how closely the material is packed in the chamber), or (2) a
hot air oven. The suggested conditions for hot air sterilization vary from
1 to 3 hr at 160° to 180°C. If protected from recontamination, the steri·
lized instruments may be stored. Alternative systems for sterilizing are
needed when neither an autoclave nor a hot air oven is available. These
include (1) expose to steam (l00°C) for 1 hr and use the same day, (2)
immerse in water at 100°C for 5 min and use immediately, (3) immerse
in 70 percent alcohol and flame to burn off alcohol immediately before
use, or (4) flame with hydrocarbon (propane or butane) torch so that all
working surfaces contact the flame before use. Using the alternative sys-
tems has been questioned. According to the FDA (1978), alcohol flaming
is unsatisfactory because the instrument does not get hot enough to be
effectively sterilized, and the flaming alcohol creates a fire hazard. The
FDA recommends using a propane torch. Tansey (1973) suggested using
a heavy-duty butane lighter rather than an unwieldy torch.
When obtained, the sample should be placed in a sterile container. A
wide-mouth screw-capped jar is recommended (APHA 1984; FDA 1978);
however, plastic bags or other acceptable containers can be used.
The methods of sampling and the types of instruments needed are
determined by the substance to be sampled.

LIQUIDS AND SMALL PARTICLES. These foods can be mixed and

sampled with sampling tubes, dippers, teaspoons, tablespoons, spatulas,
or similar instruments.

LARGE MATERIALS. If these substances can be cut, they may be sam-

pled with a knife or cheese trier. For many materials, such as animal
carcasses or processing equipment, the surface is sampled.

SURFACES. Since microorganisms are on the surfaces of equipment

as well as on foods such as animal carcasses, the sampling and analysis
of surfaces are important. The system to use for sampling depends upon
the type of surface, the amount of contamination, and the use of the data
that are obtained. Some of the surface sampling systems that have been
used are listed in Table 2.2. Each system has certain advantages and dis-
advantages. No single method is the best for all of the diverse surfaces of
foods and equipment. Hence, several have been proposed and compared
(Dewhurst, Rawson, and Steele 1986; Dickens, Cox, and Bailey 1986;
Goulet et al. 1983; Lee and Fung 1986; Scott, Bloomfield, and Barlow
1984; Speers, Lewis, and Gilmour 1984). Microorganisms become at-
tached to surfaces, which makes them difficult to recover for analysis.
Excision and maceration of tissue yield higher numbers of microorgan-
isms than do systems such as swabbing, rinsing, or contact agar or tape
 ESTIMATING THE NUMBER OF MICROORGANISMS 17

TABLE 2.2. SURFACE SAMPLING METHODS

Swab Contact systems
Cotton Agar·syringe
Alginate Agar-sausage
Glass sampler Agar plate (RODAC)
Cylinder template Tape
Scrape Membrane filter pad
Excise tissue Agar spray
Wash·rinse Drip or exuded juice
Vacuum probe Abrasive discs

(Anderson et al. 1987; Lillard and Thompson 1983; Morgan, Krautil, and
Craven 1985).

AIR. The two general methods for air sampling are solid and liquid
impingement. The systems for solid impingement include the settling
plate, slit samples, the sieve or Anderson sampler, the centrifugal sam-
pler, and the membrane sampler. Except for the settling plate, specific
volumes of air are sampled. Systems of air sampling have been evaluated
and compared (Lundholm 1982; Macher and First 1983; Placencia et al.
1982).

Holding of Sample
For best results, the sample should be analyzed immediately. When
this is not possible, the sample should be refrigerated to prevent growth
of any microorganisms. Alternatively, the sample can be packed in ice. If
shipment to another city is necessary, or if the sample is a frozen product,
dry ice should be placed in the package. Refrigeration is preferred to
freezing, because freezing may cause death or damage to some cells,
which may then give erroneous results when the sample is analyzed.

Preparation of Sample
Many methods of analysis require some preparation of the sample.
The main consideration is to get the bacteria into a homogeneous sus-
pension so they can be pipetted. If a food is a liquid such as milk, an
aliquot can be mixed and pipetted, but if the food is a solid, such as
hamburger, it is necessary to blend the food with a diluent to obtain a
suspension. The rinse or wash samples from surfaces are treated as liquid
samples, while swabs are placed in sterile diluent and shaken to suspend
the bacteria.
18 BASIC FOOD MICROBIOLOGY

SOLID FOOD. Solid food is generally mixed with a sterile diluent in a

sterile mechanical blender or other system to obtain a 1:10 dilution of
the food (Fig. 2.1). This 1:10 dilution also is referred to as Yio or 10- 1
dilution. A 1:10 dilution means that in 10 g of the mixture, there is 1 g
of food, or in 1 g of the mixture, there is 0.1 g of food, with associated
organisms. Thus, if 1 g of the 1:10 dilution is analyzed, the microbial
count is that of 0.1 g of food. To report the count as the number per
gram, it is multiplied by 10. The first dilution of the food may be 1:2 or
1:4, such as for shellfish (APHA 1970; Cook and Pabst 1984).
As an alternative to blending, a sterile plastic bag containing the sam-
ple and diluent is placed in a device called a stomacher (Fig. 2.2). In the
stomacher, the compression and shearing forces of the pounding result
in a homogeneous suspension of sample and microorganisms (Deibel
and Banwart 1982; Purvis et al. 1987; Sharpe and Jackson 1972; Thomas
and McMeekin 1980; Thrasher and Richardson 1980).
Various systems might be used to make further dilutions of the
blended food sample. One is the loop-tile method (Hudson, Roberts, and
Whelehan 1983)_

DILUENTS. Several diluents have been suggested and used_ Although

AOAC (1985) recommends the use of Butterfield's buffered phosphate,
0.1 percent peptone water is also accepted. Peptone water is easy to pre-
pare, and it protects the organisms during dilution and plating. One dis-
advantage of this preparation is that, if the prepared dilution is allowed
to remain at room temperature for extended periods, the organisms will
multiply. Not more than 20 min should elapse between the first dilution
in phosphate buffer until the last plate is poured in the series (APHA

Too,
'd450 ml DILUENT
10- 1

~ MIN, LOW SPEED

l" ~I,_,
IIml
,,_.
IIml
99m1_99ml~99ml ~99ml---+--
IIml IIml
99ml
DILUTION 10-2 10-3 10-4 10-5 10- 6

Figure 2.1_ Suspension and dilu- Iml Alml

tion of food sample for microbial
analysis_ 00
 ESTIMATING THE NUMBER OF MICROORGANISMS 19

Figure 2.2. Systems for blending food samples with diluents. From left to right:
Waring blender, stomacher, Osterizer.

1984). An increase in count up to 10 percent can be expected in this 20·

min period.
According to Harrewijn (1975), some pertinent aspects to be consid-
ered are the composition, temperature, and pH of the diluent; anaerobic
or aerobic condition; carryover of inhibitors with the food; and any treat·
ments needed to allow the recovery of cells injured during food process·
ing or preparation of the sample. The recovery of injured waterborne
coliforms is aided by diluents containing peptone or milk (McFeters,
Cameron, and Le Chevallier 1982).
The soaking of mustard seeds in a sterile diluent for 10 min prior to
analysis resulted in an increased aerobic plate count (Cowlen and Marshall
1982). Perhaps other such samples should be soaked before analysis.

DILUTIONS NEEDED. For the plate count, only plates with less than
250 or 300 colonies are considered to be countable (FDA 1978; Tomasie·
wicz et al. 1980). Hence, for moderately to highly contaminated foods,
dilutions are needed beyond the 1:10 ratio of the original suspension.
A 1:10 dilution of a 1:10 dilution is a 1:100 dilution. This 1:100 dilu·
tion is prepared by aseptically transferring 10 ml of the 1:10 dilution
20 BASIC FOOD MICROBIOLOGY

to a screw-capped bottle containing 90 ml of sterile diluent (or 11 ml

transferred to 99 ml)_ The bottle is shaken (twenty-five times through a
30-cm arc in 7 sec) to distribute the organisms homogeneously_ Further
dilutions can be 'made in this manner as far as needed.
The dilutions needed to estimate the number of microorganisms in
a food can be determined by experience or by the requirements of stan-
dards, guidelines, or specifications. If 50,000 organisms per gram are al-
lowed in a specification, a 1:1000 dilution can be used for the plate count.
If fewer than fifty organisms are observed on the incubated plate, the
food is within the limit, but if more than fifty colonies are observed, it
does not meet the requirement. Usually two or three dilutions are ana-
lyzed to increase the chances of obtaining an acceptable plate to count;
for the most probable number (MPN), at least three dilutions are needed.

ANALYSIS

Several procedures can be used to estimate a microbial population

(Table 2.3). Not all of these procedures are readily adaptable to all foods,
however. The ideal test should be accurate, rapid, inexpensive, and use-
ful for most types of samples.

TABLE 2.3. SYSTEMS TO ESTIMATE THE MICROBIAL LOAD OF FOOD

Direct microscopic count (DMC) Electrical
Breed clump count Conductance
Electronic particle count Impedance
Pour plate (APC, SPC) Capacitance
Spread plate Voltage drop
Spiral plate Spectrophotometric (optical density)
Drop plate Adenosine triphosphate (ATP)
Plate loop Reductase tests
Roll tube Easicult-TTC
Oval tube Respiration rates
Burri strip/slant Limulus amoebocyte lysate
Little plate Chemical indicators (decomposition prod-
Tube dilution ucts)
Most probable numbers (MPN) pH
Membrane filter Agar droplets
Hydrophobic grid (HGMF) Millipore sampler
Direct epifluorescent filter technique Bactoscan
(DEFT) Microcalorimetry
Microtiter-Spot plate Flow cytometry
Dry rehydratable film
Petrifilm
NOTF.: For the systems not discussed in text, see the following references: Ackland, Manvell,
and Bean 1984; Bailey and May 1979; Ginn, Packard, and Fox 1984; King and Mabbitt
1984; Kramer 1977; O'Toole 1974; Schoon et al. 1970; Sharpe and Kilsby 1971; Swientek
1981. 1983.
 ESTIMATING THE NUMBER OF MICROORGANISMS 21

Total Cell Counts

Some systems make no differentiation of living or dead cells. All mi·
crobial cells are counted. Two of these are the direct microscopic count
and the electronic particle count.

DIRECT MICROSCOPIC COUNT (DMC). With this method, the reo

suIts are obtained sooner than with most other procedures because no
incubation period is needed for the cells to metabolize and multiply.
Liquid foods may be determined directly, but solid foods must be put
into a suspension (1:10 dilution) before analysis. A counting chamber
can be used, but for food, usually a portion (0.01 ml) of the material
(measured with a standard loop or micropipet) is spread uniformly over
a prescribed area on a glass slide (usually 1 cm 2 ).
For products such as eggs or cream, xylene or another suitable solvent
is added prior to staining to remove the fat from the material. After dry·
ing, the slide is then fixed by dipping in ethyl alcohol for 1 to 2 min
before staining.
Several stains have been suggested for and used in the DMC. The
stained films are examined with a microscope, using the oil· immersion
objective. The number of fields to be examined and counted is inverse
to the number of cells and clumps observed in each field.
To calculate the organisms per gram of food, the diameter of the field
that is examined must be known. The diameter (d) is measured with a
stage micrometer to the nearest 0.001 mm. Since the field is a circle, the
area can be calculated (A = 7r r2).
The average number of cells or clumps per field is calculated and
divided by the area of the field to obtain the number per mm 2 • To deter·
mine the number of cells or clumps per cm2 , the number per mm 2 must
be multiplied by 100 (since there are 100 mm 2 per cm 2 ). The resultant
number is then multiplied by the dilution factor, which, in the case of
liquid food (milk) is 100 (0.01 ml was used), or 1,000 for solid food (0.01
ml of a 1:10 dilution).
Values of the DMG. The DMC is a rapid method because an estimate of the
bacterial load can be obtained in a short time. This is of value when on-
the-spot alterations or adjustments are needed in the processing opera-
tion to remedy any problems.
Other values of the DMC that have been suggested include the follow-
ing: (1) little work is required; (2) the test is not too difficult; (3) very
little apparatus or equipment is needed except for a microscope; (4) the
prepared slide can be stored and maintained as a permanent record; (5)
some idea as to the type of organism (cocci or rods) is obtained; (6) counts
represent organisms in the original product (if it has been treated, such
22 BASIC FOOD MICROBIOLOGY

as by heat); (7) preservatives can be added to the sample for holding prior
to analysis, for shipment, or to hold for futher study, so that organisms
do not multiply; (8) only a small amount of sample is needed, which is
of value if the product is expensive.
Since both living and dead cells are counted, there is some question
as to the value of the microscopic count. However, high numbers of cells,
whether living or dead, in pasteurized products indicate poor quality of
product before processing, survival or multiplication of bacteria during
processing, or recontamination or growth, or both, after processing.
The value of the DMC is limited to samples with high cell loads. How-
ever, with increased technology and efforts in sanitation, the bacterial
load in many foods has been reduced. The DMC has little or no value
for foods with low microbial loads.
Besides being used to evaluate the microbial content of a food, the
DMC can be used to evaluate the number of body cells (leucocytes or
lymphocytes) in milk. This is especially valuable in indicating mastitis in
cows.
The value of the DMC depends upon the type of food and the type
of organisms associated with the food. For products that have received a
treatment such as heat to control the microorganisms, it is doubtful that
the DMC could predict shelf life of the product. It is also doubtful tha.
the DMC would have any value in determining the public health hazard
of the product.

Assumptions and Errors. In any microbiological method of analysis, many

assumptions and errors are made. There are errors inherent in the ana-
lytical procedure, as well as those introduced by the technician doing the
test.
Assumptions are also made regarding the sample. It is assumed that
(1) the sample is representative of the entire lot of product; (2) the sub-
sample used for analysis is representative of the sample; (3) cells are dis-
tributed homogeneously in the sample as well as the subsample and, if
not originally homogeneous, that operations such as mixing, blending,
or shaking have produced a homogeneous mixture; (4) the weighing or
measuring of the subsample, diluents, and aliquots is accurate; and (5)
the sample or subsample has been handled so that there is no contamina-
tion or multiplication of cells during sampling or analysis.
Errors in the DMC can occur during the preparation and staining of
the cells, counting the cells or clumps, or in the calculations involved in
converting the raw data into the count per gram of product.
It is easier to spread the sample uniformly in circles than in squares.
If the material is not spread uniformly, the cells will not be distributed
homogeneously. It is assumed that the smear on the slide will dry into
 ESTIMATING THE NUMBER OF MICROORGANISMS 23

flat layers of uniform density. However, the smears have been found to
vary in thickness from one area to another. For some foods, such as liquid
egg, the film on the slide may be of such thickness that it obscures many
bacterial cells, which cannot then be counted. Organisms that are lightly
stained are difficult to discern, and with an unevenly stained back·
ground, it is difficult to distinguish dirt or other particles from bacterial
cells. Improper illumination with resulting eye strain and fatigue is
another cause of errors in cell counting. The staining process itself can
also wash some cells from the slide or can result in the counting of pre·
cipitated stain as cells.
For other errors, see APHA (1978).

ELECTRONIC PARTICLE COUNT. The electronic counter is based

on the principle that cells are poor electrical conductors as compared to
an electrolyte solution. A dilute suspension of cells in saline or other
suitable electrolyte is drawn through a minute aperture conducting an
electric current between two electrodes-usually platinum. Each cell
passing through the aperture displaces an equal volume of the electrolyte
solution and causes a momentary increased impedance to the flow of
electric current. The resulting voltage pulse is proportional to the size
or volume of the particle passing through the aperture. These pulses are
amplified and counted. They simultaneously appear on the screen of an
oscilloscope.
Since background particles, such as those that occur in foods, also
would produce pulses as they pass the aperture, or could clog the aper·
ture, the particles must be removed. Although electronic particle
counters are used successfully to count somatic cells in milk (Dijkman et
al. 1979), blood cells, and mammalian cells, much work needs to be done
before they are useful in determining microorganisms in foods. However,
Kogure and Koike (1987) reported satisfactory results when a particle
counter was used to determine the bacterial biomass of seawater.

Viable Counts
Several methods exist to estimate the number of viable microorgan·
isms in foods. Most of the systems are based on the plate count or tube
dilution methods.

PLATE COUNT (POUR). The standard plate count (SPC) has been the
usual technique for estimating the living microorganisms in foods. The
procedure is relatively simple. Appropriate dilutions are plated immedi·
ately by transferring a measured aliquot to a sterile petri plate and add·
24 BASIC FOOD MICROBIOLOGY

ing sterile melted and cooled (42° to 45°C) agar. The type of agar used
for the SPC is non inhibitory and nutritious, unless specific microbial
types are to be determined. For the aerobic plate count the media usually
used are plate count agar or tryptone glucose extract agar, but various
other agars have been employed. The medium and inoculum should be
mixed thoroughly to distribute the cells uniformly. After solidification of
the agar, the prepared plates are inverted (turned upside down) to pre·
vent condensation of moisture on the agar surfaces, and then incubated.
The temperature and time of incubation will vary, depending upon the
type of cells that are being determined (psychrotrophs, mesophiles, or
thermophiles). A temperature of 32°C for three days is used for eggs and
egg products, while 35°C for 48 ± 2 hr is listed for frozen, chilled, or
prepared foods (AOAC 1985). Huhtanen (1968) found the highest bacte-
rial counts in raw milk when the plates were incubated at 27°C. However,
these counts were not significantly different from those obtained at a
range from 10° to 30°C.
During the incubation period, growth and multiplication of cells will
occur until a visible colony is formed. These colonies are then counted
on the plates that contain from 30 to 300 colonies (AOAC 1985; FDA
1978). Cowell and Morisetti (1969) furnished evidence that greater preci·
sion is obtained from plates containing from 80 to 320 colonies. A range
of 25 to 250 colonies was suggested in a later study (Tomasiewicz et ai.
1980). The number of colonies is multiplied by the dilution factor and
reported as the number of colony-forming units (CFU) per gram of food.

Desirable Characteristics. The pour plate procedure is simple, can cover a

large concentration range, and, at present, is probably the most precise
method for determining those bacteria that will grow in an agar medium
(Gilchrist et al. 1973). Besides these virtues, the organisms can be recov·
ered for further study. The results should reflect the level of viable micro-
organisms in the food at the time of sampling.
The data obtained from the pour plate should reveal information
such as the source of microorganisms, potential shelf life, or possible
public health hazards of the product. With the present aerobic plate sys-
tem, the source of microorganisms generally is not determined (Blanken-
agel 1976).
In most foods, microbial growth causes undesirable changes. Hence,
the plate count might be used as an indicator of potential shelf life or of
incipient spoilage. No relationship was found to exist between the bacte-
rial count and potential shelf life of iced shrimp (Cobb et al. 1973), or
pasteurized milk (Watrous, Barnard, and Coleman 1971). The usual plate
count system does not differentiate types of organisms that cause
spoilage.
 ESTIMATING THE NUMBER OF MICROORGANISMS 25

It is generally agreed that any potential health hazard is not deter-

mined by the aerobic plate count. Some people believe that a high micro-
bial count indicates improper handling with possible pathogens being
present. Quite often the reverse is true, and low-count products contain
potential pathogens. Microbial toxins can be present after the bacteria
are destroyed by processing.
Undesirable Characteristics. There are many facets of the pour plate system
that are undesirable. Of most concern are time, expense, technical re-
quirements, information obtained, and accuracy.
The prepared plates must be incubated so that the organisms can
produce a visible colony prior to counting. This incubation period may
range from two to ten days. For highly perishable products, or for deter-
mining production or processing conditions, it is desirable to obtain the
results as soon as possible. If a ten-day incubation period is needed, the
potential shelf.life of a food can be determined more easily by incubating
the food directly.
Since the pour plate system is so common in the United States, we
might not realize that it is rather expensive compared to other methods.
In some countries, other, less expensive methods are used in preference
to the plate count.
The pour plate method seems simple to do, but a trained technician
is needed to perform the test. The accuracy of the pour plate depends
upon the ability of the technician as well as on assumptions and errors
inherent in the technique.
Assumptions and Errors. The same assumptions and errors in sampling as
discussed for the DMC apply equally to the pour plate. The technical
ability and concern of the technician during cleaning of glassware, prep-
aration of dilutions and media, sampling, plating, counting, and calculat·
ing can influence the reported CFU.
Two major assumptions of the pour plate system are that (1) microor-
ganisms are in suspension as dissociated single-cell units so that each
colony on the plate arises from an individual cell; and (2) all cells planted
in the medium will multiply and produce a visible colony. Neither as-
sumption is accurate.
Quite often bacteria grow in chains or clusters. Mixing, shaking, and
other procedures do not always separate these chains or clumps into in·
dividual cells. Hence, when plated, a colony may arise from not only one,
but several bacterial cells.
The environment in which the organisms are placed (medium, tem-
perature, oxygen) as well as previous treatments of the cells (sublethal
heating, freezing, radiation) and even the presence of other types of mi-
croorganisms influence the ability of the cell to multiply and produce a
26 BASIC FOOD MICROBIOLOGY

visible colony. No one environmental condition will support the growth

of all of the types of microbial cells that might be present in a food prod-
uct. Hence the bacterial count should be referred to as CFU per gram
of food.
The main value of a plate count is to be able to compare the results
of various samples taken at different times from the different laborato-
ries. This is possible only when the results are reproducible. It is impor-
tant that standardized procedures be followed so that results can be com-
pared.

PLATE COUNT (SURFACE). In this system, the sterile melted and

cooled agar is poured in sterile petri plates. After solidification, the
plates are preincubated overnight. The incubation dries the surface of
the agar so that, when planted, the organisms do not coalesce. Before
using, the dried agar surface should be observed for any possible contam-
ination.
Aliquots of dilutions are added to the dry surface and uniformly
spread over the agar by means of a sterile glass rod, bent into the shape
of a hockey stick. Various amounts of aliquots have been suggested. Inas-
much as we usually work with dilutions in the order of 10, it is much
easier to calculate the results per gram of product if O.l-ml aliquots are
used.
For simplification, calibrated loops can be used in place of pipets for
preparing dilutions as well as for inoculating the pour plate or the sur-
face of the spread plate. In the plate loop system, a calibrated loop is
fitted into the barrel of a repeating syringe. The loopful of sample is
flushed onto the agar surface in a petri plate with sterile diluent in the
syringe. According to O'Connor (1984), the precision and accuracy of the
plate loop system are within acceptable limits.
With the drop plate method, 0.02 ml of inoculum is allowed to drop
onto the surface so that it spreads over an area 1.5 to 2.0 cm in diameter.
Six to eight drops are placed on an agar surface in a petri plate, with no
further manual spreading. After inoculation, the plates are inverted and
incubated, and the resultant colonies counted as with the pour plate
method.
Automated devices for distributing the samples over the agar surface
have been described and evaluated (Gilchrist et aL 1973; Gilchrist et aL
1977; Jarvis, Lach, and Wood 1977; Kramer, Kendall, and Gilbert 1979;
Tilton and Ryan 1978; Trotman and Byrne 1975). One type of automatic
plating system is the spiral plater (Fig. 2.3) (Gilchrist et aL 1973; Gilchrist
et aL 1977)_ This system was adopted as an official method by the AOAC
(1981). With this mechanical device, a stylus dispenses the sample, or di-
lution, in a spiral, in varying amounts, on an agar surface from the center
 ESTIMATING THE NUMBER OF MICROORGANISMS 27

Figure 2.3. The spiral plater.

of the dish to the outer edge. By varying the amount of inoculum, the
equivalent of three dilutions can be plated on one agar surface. After
incubation of the inoculated plates, a laser colony counter, developed for
the spiral plater, follows the spiral from the outer edge toward the center,
counts the colonies, and determines the CFU for the inoculum. Also, the
colonies can be counted manually with the use of a spiral grid system.
Surface VS. Pour Plates. The desirable aspects listed for the pour plate are
equally applicable to the surface plate.
It is well recognized that higher counts are obtained by surface spread
plates than by pour plates. The possibility of heat-sensitive organisms
being damaged by hot agar during the preparation of pour plates is over-
come by using the spread plate technique. Obligate aerobic organisms
will grow faster on the surface than in the depth of agar in pour plates.
Surface colonies are always detectable sooner and are much larger and
easier to count than are colonies in a pour plate.
The main advantage of surface plates as compared to pour plates is
that surface plating can be automated. With automated analyses such as
the spiral plate system, both work and materials are saved. One problem
with the spiral plate system is that the stylus is easily clogged if food
28 BASIC FOOD MICROBIOLOGY

particles are present in the sample. Hence, filtration of the sample may
be needed to remove these particles prior to plating. Hoben and Soma-
segaran (1982) found the drop plate method to be more economical than
either the spread or pour plate systems.
The undesirable characteristics of the spread plate are similar to
those discussed for the pour plate. With the spread plate system, some
of the organisms might cling to the glass rod used for spreading. Treating
of the glass rod with silicone helps to overcome the problem. Reportedly,
precision with the pour plate is better than with the spread plate.
Dry, Rehydratable Film. As an alternative to the petri plates used in the
aerobic plate count systems, plastic films with a dry, rehydratable me-
dium coated upon them (Petrifilm SM) have been developed. The dry
medium contains nutrients, a cold water-soluble gel, and 2,3,5-triphenyl-
tetrazolium chloride that is reduced by microbial growth from white to
red. The prepared samples or dilutions are added at the rate of 1.0 ml
per plate. The sample is spread over an area of about 20 cm 2 by applying
pressure with a plastic spreader on the overlay film. The liquid in the
sample rehydrates the medium, then the gel is allowed to solidify before
the prepared films are incubated for bacterial growth. Reportedly, the
Petrifilm SM system was a satisfactory alternative to the aerobic plate
count for poultry (Bailey and Cox 1987), pasteurized fluid milk (Senyk
et aL 1987), and ground beef (Smith, Fox, and Busta 1985). Petrifilm
methods were adopted as official first action by the AOAC (Brickey et aL
1986).

ROLL TUBE. The basic idea of the roll tube is the same as for the pour
plate method, except that screw-capped test tubes or bottles are used in
place of petri plates. Test tubes are sterilized with 2 to 4 ml of plate count
agar (with 2 percent agar). When the melted agar is cooled to 42° to
45°C, 0.1 ml of the appropriate dilution of the sample is added and the
tube rolled in cool water in a horizontal position until the agar is solidi-
fied in a thin layer on the inner wall of the tube.
The roll tubes are incubated upside down so that any water that con-
denses collects below the inoculated agar and does not smear the colo-
nies. After incubation, the colonies that develop are counted with the aid
of a low-power magnifier. Multiplying the colony count by the dilution
factor yields the number of organisms per gram of food.
Although the basic idea of the roll tube is similar to the plate count,
there are obvious differences. Since test tubes are used rather than petri
plates, the cost of the procedure may be lower or higher, depending upon
the relative cost of these items. Less plate count agar is used in the roll
tube method.
 ESTIMATING THE NUMBER OF MICROORGANISMS 29

Hartman (1968) stated that the roll tube requires less space, materials,
and time, with less risk of contamination and less desiccation of the me-
dia in the tubes than in plates during long incubation periods_ He also
reported that there is no waiting for agar to solidify to invert and incu-
bate such as in the pour plate system_ There are machines for rolling the
tubes_
It would seem that the colonies would be more difficult to discern
and count in the roll tube than in the pour or spread plate techniques_
In his review, Hartman (1968) did not find counting of the colonies to be
a problem in the roll tube_ Devices are available to assist in the counting
of colonies in roll tubes_ The roll tube technique can be used to deter-
mine anaerobic types of microorganisms in foods (Gray and Johnson
1976)_

BURRI STRIP OR SLANT. This method involves the spreading of a

sample over an agar slant with a calibrated loop. Test tubes can be used,
but the oval tube gives a larger surface for the growth of colonies. The
agar surface must be dry to prevent colonies from coalescing_ After incu-
bation (32° or 37°C for 24 hr) in a horizontal position, the surface is
examined for microbial growth. Colonies may be counted or compari-
sons can be made as to the extent of growth that occurs so that high- and
low-count products can be distinguished.
The Burri slant method is a simple test for the evaluation of plant
sanitation.

LITTLE PLATES. Since Frost introduced the little plate system in

1916, many modifications to the system have been proposed. The origi-
nal procedure was to mix 0.1 ml of milk with about 2 ml of nutrient agar,
and this was spread uniformly over a 4 cm 2 area on a glass slide. After
incubation for 3 to 8 hr in a moist chamber, the slides were air-dried,
flame-fixed, and stained for counting. The colonies were observed and
counted with a microscope.
Modifications have been suggested in the procedure, such as the types
of slide used, the method of inoculation and incubation, as well as type
of stains. A similar procedure was described by Postgate (1969) to distin-
guish viable cells from dead cells, since to observe colonies on the slide,
the cells must be viable.
This system is a more rapid method than the plate count, since only
3 to 8 hr of incubation are used. Besides being rapid, an estimate of the
viable number of cells is obtained, which is not the case with DMC. The
little plate, slide plate, and microplate methods give results comparable
those for the plate count.
30 BASIC FOOD MICROBIOLOGY

MEMBRANE FILTERS. When fluids are filtered through a membrane

filter (MF), all particles, bacteria, or cells larger than the pores are re-
tained on the filter surface.
The procedure has been useful for analyzing processed water, various
beverages, or air when the microbial count is relatively low. Such low
contamination is difficult to evaluate with the APC. More recently, MF
systems have been used to analyze foods with relatively high numbers of
bacteria. Prefilters are used to remove food particles that might clog the
MF. In some cases, surfactants and enzymes, such as proteases, are used
to degrade the food so that it can be filtered (Bourgeois et al. 1984; Entis,
Brodsky, and Sharpe 1982; QALL 1981).
The retained microorganisms can be cultured by aseptically transfer-
ring the filter onto a nutrient agar or one that is selective, differential,
or both. After incubation for 6 to 8 hr, the microcolonies can be counted
with a microscope similarly to that used in the little plate or microplate
method. After incubation for 24 to 48 hr, the colonies can be counted
similarly to the APC.
The bacterial cells can be stained with 0.1 percent toluidine blue
(O'Toole 1983a, 1984). After destaining the filter and making it transpar-
ent, the dye retained by the cells is determined with a spectrophotometer.
Reportedly, this reading is related to the number of cells on the filter.
A membrane filter with hydrophobic material in a grid pattern is
called a hydrophobic grid membrane filter (HGMF). The grids are com-
partments of equal and known size, and the hydrophobic material deters
the spreading of colonies. After the organisms are grown on the filter,
the number of squares containing colonies is enumerated and converted
to a most probable number- The results can be determined manually or
with an automated counting system (sample analyzer). A disposable filter
unit has been developed for the HGMF (Tsuji and Bussey 1986). The
HGMF system was given official status by the AOAC (AOAC 1983; Entis
1986).
In one system, the microorganisms on the filter are subjected to a
fluorescent dye, acridine orange, which stains viable cells, and then ob-
served with an epifluorescent microscope. This direct epifluorescent fil-
ter technique (DEFT) was reviewed by Pettipher (1986). The DEFT is a
rapid method and is especially useful for samples of food containing
high numbers of organisms (Pettipher 1987; Qvist and Jakobsen 1985;
Shaw et al. 1987). The method was not suitable for heated samples
(Hunter and McCorquodale 1983; Rodrigues and Kroll 1986). However,
a double staining system using acridine orange and janus green B al-
lowed the differentiation of viable and heat-killed cells (Rodrigues and
Kroll 1986). A modified Gram-staining procedure using acridine orange
as the counterstain allows the differentiation of Gram-positive and Gram-
 ESTIMATING THE NUMBER OF MICROORGANISMS 31

negative cells (Rodrigues and Kroll 1985). Microorganisms on DEFT

slides can be counted automatically (Pettipher 1986).

TUBE DILUTION. The tube dilution method is essentially the aseptic

inoculation of a series of tubes of sterile nutrient broth with a series of
dilutions of the food. After incubating the inoculated tubes, the broth is
observed for turbidity, which indicates growth of organisms. If no turbid·
ity is evident, it is assumed that no microorganisms were present or were
able to multiply. With broth that appears turbid due to inoculated food,
growth can be detected by streaking on an agar surface and observing
growth after a few hours of incubation, or by spreading some turbid
broth on a slide and looking for microorganisms with the aid of a micro·
scope.
If the tube with the 1:100 dilution showed growth and the tube with
1:1000 had no growth, there were between 100 and 1,000 organisms in
the food. Sometimes this rough estimate is all that is needed. It gives only
an estimate of the range of bacteria that are present.

MOST PROBABLE NUMBERS (MPN). By using several tubes at each

dilution and recording the positive (showing growth) tubes and negative
(no growth) tubes, you get a more accurate estimate of the number of
organisms present. In the tube dilution example, if you inoculated 10
tubes with 1 ml of the 1:1,000 dilution, there would be as much total
inoculum as in the 1:100 tube which showed growth. Theoretically, one
or more of the 10 tubes with the 1:1,000 dilution also should be turbid.
The relationship of positive and negative tubes has been determined
mathematically and MPN tables have been derived (Tables 2.4 and 2.5).
To use the MPN system, at least three dilutions are needed. Ideally, the
least dilute tubes should all be positive and the most dilute tubes (of the
three dilutions) should all be negative. This is not always the case, so the
rule that has been established is to select the highest dilution in which
all portions tested are positive (no lower dilution giving negative results),
and the two succeeding dilutions are then chosen. The more tubes that
are used in each dilution, the more accurate is the estimate, but for rea·
sons of convenience, three·tube or five· tube series are adopted. After se·
lecting the three series of dilutions, consult the appropriate MPN table,
obtain a most probable number that satisfies the number of positive
tubes, and multiply this by the dilution factor to obtain the MPN per
gram of product.

Assumptions and Errors (MPN). The assumptions and errors due to sam·
pIing and diluting apply to the MPN technique. It is assumed that a single
viable cell inoculated into a tube of broth will multiply so that a change
32 BASIC FOOD MICROBIOLOGY

TABLE 2.4. MOST PROBABLE NUMBER (MPN) PER GRAM OF SAMPLE

Two·sided 95% One·
Number of Positives Program Values Conf. Limits sided
Upper 95%
1.0 0.1 O.oI MPN St. Error Lower Upper Limit
0 0 0 < 0.03
1 0 0 0.36 0.36 0.05 2.54 1.85
1 1 0 0.74 0.52 0.18 2.94 2.36
1 1 1 1.12 0.64 0.36 3.47 2.89
2 0 0 0.92 0.65 0.23 3.67 2.94
2 1 0 1.47 0.85 0.47 4.55 3.80
2 1 1 2.05 1.02 0.77 5.46 4.66
2 2 0 2.11 1.05 0.79 5.61 4.79
2 2 1 2.76 1.24 1.15 6.64 5.76
2 2 2 3.48 1.42 1.56 7.74 6.80
3 0 0 2.31 1.33 0.74 7.17 5.98
3 1 0 4.27 2.14 1.60 11.38 9.72
3 1 I 7.49 3.35 3.12 17.99 15.63
3 2 0 9.33 4.17 3.88 22.41 19.47
3 2 1 14.94 6.10 6.71 33.25 29.23
3 2 2 21.46 8.11 10.23 45.02 39.97
3 3 0 23.98 17.41 5.78 99.49 79.15
3 3 1 46.22 17.47 22.03 96.96 86.07
3 3 2 109.89 38.87 54.94 219.82 196.65
3 3 3 > 110.00
SOURCE: Data Courtesy of Robert J. Parnow (personal communication).
NOTE: Standard error, upper and lower 95% confidence limits, and one·sided upper 95%
confidence limits when three dilutions are used with three tubes in each dilution at levels
of 1.0, 0.1. and 0.01 g per tube.

such as turbidity or production of acid or gas can be observed. Because

dilution to extinction is necessary, good aseptic technique is needed,
since any contamination during inoculation of the tubes of broth could
result in growth. The MPN is less precise than the agar plating methods
(Pike et al. 1972).
Some people become confused when 1 g of sample is added to a tube
with 9 ml of broth for the MPN series. They feel that since this is a 1:10
dilution, somehow it has to be considered when the dilution factor for
the MPN is determined. It does not make any difference if there are 8,
9, 10, or 11 ml of nutrient media per tube. The only consideration is the
amount of original sample that is added to the tube (0.01, 0.001, 0.0001
g, or whatever).

Advantages of the MPN. The MPN is, in some ways, easier or simpler to do
than the plate count. Broth can be dispensed into tubes with an auto·
matic pipetter. Selective or differential media can be used so that certain
types of organisms can be determined. The MPN is particularly useful
TABLE 2.5. FREQUENTLY OCCURRING MOST PROBABLE NUMBER (MPN) PER
GRAM OF SAMPLE
Two·sided 95%
One·sided
Number of Positives Program Values Conf. Limits
Upper 95%
1.0 0.1 0.01 MPN St. Error Lower Upper Limit
0 0 0 <0.18
1 0 0 0.19 0.19 0.03 1.34 0.98
1 1 0 0.40 0.28 0.10 1.60 1.28
1 1 1 0.60 0.35 0.19 1.86 1.55
2 0 0 0.44 0.31 0.11 1.76 1.41
2 1 0 0.68 0.39 0.22 2.11 1.76
2 1 1 0.92 0.46 0.34 2.45 2.09
2 2 0 0.93 0.46 0.35 2.48 2.12
2 2 1 1.17 0.52 0.49 2.81 2.44
2 2 2 1.42 0.58 0.64 3.16 2.78
3 0 0 0.77 0.44 0.25 2.39 1.99
3 1 0 1.07 0.54 0.40 2.85 2.44
3 1 1 1.36 0.61 0.57 3.27 2.84
3 2 0 1.38 0.62 0.57 3.32 2.88
3 2 1 1.69 0.69 0.76 3.76 3.31
3 2 2 2.02 0.76 0.96 4.24 3.76
3 3 0 1.72 0.70 0.77 3.83 3.37
3 3 1 2.05 0.77 0.98 4.30 3.82
4 0 0 1.27 0.64 0.48 3.38 2.89
4 1 0 1.68 0.75 0.70 4.04 3.50
4 1 1 2.11 0.86 0.95 4.70 4.13
4 2 0 2.16 0.88 0.97 4.81 4.23
4 2 1 2.64 1.00 1.26 5.54 4.92
4 2 2 3.17 U2 1.58 6.34 5.67
4 3 0 2.70 1.02 1.29 5.66 5.03
4 3 1 3.25 U5 1.62 6.50 5.81
4 3 2 3.86 1.29 2.01 7.42 6.68
4 4 0 3.35 U8 1.68 6.70 5.99
4 4 1 3.98 1.33 2.07 7.65 6.89
5 0 0 2.31 1.03 0.96 5.55 4.82
5 1 0 3.29 1.34 1.48 7.32 6.44
5 1 1 4.56 1.72 2.17 9.56 8.49
5 2 0 4.93 1.86 2.35 10.34 9.18
5 2 1 6.99 2.47 3.50 13.98 12.50
5 2 2 9.43 3.14 4.91 18.12 16.32
5 3 0 7.92 2.80 3.96 15.84 14.17
5 3 1 10.86 3.62 5.65 20.87 18.79
5 3 2 14.05 4.44 7.56 26.11 23.64
5 3 3 17.49 5.27 9.68 31.58 28.72
5 4 0 12.99 4.33 6.76 24.97 22.48
5 4 1 17.23 5.45 9.27 32.02 28.99
5 4 2 22.11 6.67 12.24 39.92 36.31
5 4 3 27.80 8.02 15.79 48.95 44.70
5 4 4 34.54 9.58 20.06 59.49 54.51
5 5 0 23.97 7.58 12.90 44.55 40.33
5 5 1 34.76 10.48 19.25 62.77 57.08
5 5 2 54.22 15.65 30.79 95.48 87.18
5 5 3 91.78 25.46 53.28 158.09 144.86
5 5 4 160.94 43.06 95.26 271.90 249.92
5 5 5 > 1,600
SOURCE: Data Courtesy of Robert]. Parnow (personal communication).
NOTE: Standard error, upper and lower 95% confidence limits, and one·sided upper 95%
confidence limits when three dilutions are used with five tubes in each dilution at levels
of 1.0, 0.1 and 0.01 g.
33
34 BASIC FOOD MICROBIOLOGY

for samples with only a few organisms and can be used to detect orga-
nisms in samples larger than 1 g_

Estimations Based on Metabolism

Microbial metabolism is used in general microbiology to determine
fermentation of sugars, starch hydrolysis, production of hydrogen sul-
fide, indole, or reduction of nitrate_ The metabolic products that are pro-
duced can be determined and used to estimate microbial populations or
the quality of the food_

REDUCTASE TESTS. Organisms obtain energy from chemical reac-

tions involving either organic or inorganic compounds. This involves an
oxidation-reduction reaction; the energy source becomes oxidized, while
another compound is reduced. Oxygen mayor may not be involved be-
cause oxidation-reduction reactions concern electron transfers. When a
compound loses an electron it becomes oxidized, and another com-
pound which accepts this electron is reduced.
Compounds vary in their oxidation-reduction potential, which is the
tendency for a compound to give up electrons. Since these reactions con-
sist of electron transfers, they can be measured electrically with a potenti-
ometer and are expressed by the electrical unit, the volt. The oxidation-
reduction potential also is called the redox potential.
Besides being determined potentiometrically, the redox potential can
be determined with indicators or dyes. Many compounds undergo color
changes when oxidized or reduced. If such a compound is added to a
substrate containing metabolizing bacteria, electrons may be transferred
to the indicator, and its color will be altered.
Since the color change of the indicator depends on the metabolic rate
of a microbial culture, the larger the number of cells, the sooner the
indicator will show a color change. The reduction time is inversely pro-
portional to the number of cells present (Fig. 2.4). Although several oxi-
dation-reduction indicators could be used, methylene blue, resazurin,
and the tetrazoliums are the ones used most often in food analysis. The
reductase tests usually are called dye reduction tests, apparently because
the dye methylene blue is used. However, resazurin and the tetrazoliums
are not dyes, but indicators (Conn 1961).
During reduction, methylene blue becomes colorless. This dye has
been used to determine the bacterial quality of milk and dairy products
such as ice cream (Anderson and Whitehead, 1974). Also, it has been
suggested as a means to predict the sterility of heated food (Hall 1971)
and to estimate the number of bacteria in ground beef (Emswiler et al.
1976).
 ESTIMATING THE NUMBER OF MICROORGANISMS 35

""0;
z=
5~ 7
(.)'0
..J~
<t"
_D
a:E
1oJ'"
.... c 6
(.).,.
<t.3
ID~

!5

2 6
DYE REDUCTION TIME (HOURS)

Figure 2.4. Relationship between reduction time and microbial

load. The slope of the line depends upon the types of microorgan-
isms that are present.

Two color changes occur during the reduction of resazurin. The blue
color goes through various shades of purple and mauve to pink. This
color change is not due to an electron transfer, but to the loss of a loosely
bound oxygen atom. This color change to pink is not reversible by atmo·
spheric oxygen. The second color change results in the indicator becom·
ing colorless and is reversible by atmospheric oxygen. The resazurin test
is a simple, relatively rapid, inexpensive system to determine the quality
of fresh scallop meat (Webb et al. 1972) and frozen shrimps (Kiimmerlin
1982).
The tetrazolium salt most often used for testing food is 2,3,5-triphen·
yltetrazolium chloride (TTC), since it is less toxic to bacteria than are
other tetrazolium salts. The TTC is colorless in the oxidized state but
forms intensely colored pink to red pigments (formazans) when reduced.
The red color that develops when TTC is sprayed onto a surface indicates
36 BASIC FOOD MICROBIOLOGY

sites of bacterial activity. This indicator can be used to distinguish bacte·

rial colonies from food particles in an SPC.
Comparison of Reductase Tests to Viable Count Tests. The reductase test gener·
ally gives an estimate of the bacterial contamination in a shorter time
than the SPC. The information obtained from reductase tests can, at best,
be used to obtain a rough estimate of the number of microorganisms
present in or on a food.
Not all organisms cause a lowering of the redox potential at the same
rate. If a clump or chain of bacteria is plated in agar, a single colony will
develop, but the metabolic activity in the reductase test will be the sum
of the total number of cells in the clump or chain. This will result in a
more rapid color change in the indicator than the plate count would
suggest.
For a cell to be counted in the SPC, it must multiply and form a visible
colony. Cells may be metabolizing, but not reproducing. These cells
could cause a color change in the redox indicator and not be included
in the SPC.
Methylene blue and tetrazolium are inhibitory to certain microorgan·
isms. Sometimes tetrazolium is added after the organisms have grown,
such as by flooding an agar surface, due to its potential for inhibiting the
cells. It has been suggested that reducing enzymes naturally present in
foods can cause color changes of these indicators. In this case, the reduc·
tase test would indicate more contamination than is present.

CHEMICAL INDICATORS OF DECOMPOSITION. Everyone makes

organoleptic evaluations of food by sight, smell, taste, or touch. The food
industry relies on these organoleptic tests to determine certain quality
attributes of foods. This type of analysis is very subjective, and many ar·
guments can develop between seller and buyer. Chemical indicators can
be used to evaluate the quality of food in a more objective manner. Food
is composed of various chemical compounds that are subject to biochem·
ical changes. These changes may be desirable or undesirable, depending
upon the food, the microorganisms that are present, and the end prod-
ucts of the reaction. Decomposition of a food with resulting quality dete-
rioration is an undesirable change.
The main reactions occurring in foods are catalyzed by enzymes.
These enzymes may be tissue enzymes naturally present in the food, they
may be produced by microorganisms associated with the food, or they
may be added to catalyze a desirable reaction (see Chapter 9). Some
chemical reactions, such as oxidation, occur in foods without specific en-
zymes to catalyze them. The extent of change occurring in the food may
or may not be related to the number of microorganisms present.
The type and amount of metabolic products formed depends upon
 ESTIMATING THE NUMBER OF MICROORGANISMS 37

the kind of food (protein, carbohydrate or fat), the type of microorgan-

ism (proteolytic, saccharolytic, or lipolytic), the availability of oxygen
(aerobic-oxidation, decay, or oxidative rancidity; anaerobic-fermentation,
putrefaction, or hydrolytic rancidity), the temperature (psychrotrophic,
mesophilic, or thermophilic organisms) and the types of inhibitors that
might be present.
Criteria for Chemical Indicators. For a chemical to be a useful indicator, it
must meet the following criteria: (1) it must be absent or at very low levels
in sound food; (2) it should be produced by the predominant spoilage
flora and not used as a nutrient; (3) it should not be detected quantita-
tively with simple and rapid tests and the tests should never yield false
positive results; (4) it should not have a useful function in the food; and
(5) it should preferably be able to distinguish poor quality from poor
processing operations.
Possible Chemical Indicators_ Fields, Richmond, and Baldwin (1968) pre-
sented a comprehensive review of chemical indicators. Some potential
chemicals for estimating the microbial or other quality of food are listed
in Table 2.6.
Because of variations in a food and its microbial flora, none of these
chemical indicators is entirely satisfactory_ However, the presence of cer-
tain indicators in some foods does correlate with the microbial count or
organoleptic evaluation. In general, a group of compounds such as vola·
tile reducing substances, total volatile acids, or bases gives a better indica-
tion of quality than a single indicator such as ammonia, indole, or alco-
hol. One problem with many of these indicators is that by the time there
is a significant change in the amount that is present, deterioration of the
food is very evident. Perhaps incubation of the food for a few hours be-
fore analysis could be used to develop the presence of indicators more
rapidly. However, this higher temperature could alter the dominant
spoilage flora so that the metabolic products would differ from those
expected to develop under normal storage conditions.

TABLE 2.6. POTENTIAL CHEMICAL INDICATORS OF FOOD QUALITY

Ammonia Indole
Trimethylamine Ethanol
Dimethylamine Furfural
Total volatile bases Hydrogen sulfide
Total volatile acids Total reducing substances
Free fatty acids Volatile reducing substances
Water insoluble acids (oleic, palmitic) Histamine
Organic acids (acetic, lactic, pyruvic, suc- Hypoxanthine
cinic) Diacetyl
Acetylmethylcarbinol
38 BASIC FOOD MICROBIOLOGY

PHYSICAL TESTS. Microbial growth causes alterations in foods, such

as acid content or pH. As the pH is varied, the water-binding capacity of
protein changes. This difference can be determined by the extract re-
lease volume test.
The fluorescence of liquid egg is related to mustiness and growth of
certain bacteria, while pyoverdine, a fluorescent pigment produced by
pseudomonads, has been determined in frozen whole egg and on poultry
carcasses_
pH. Since basic compounds such as ammonia and amines are formed
during deterioration of protein foods, the pH will tend to rise. If carbo-
hydrates are present and fermented, the pH will tend to decline. Shelef
and Jay (1970) recommended blending 10 g of meat with water and titrat-
ing to pH 5.0 with 0.02N HCl. If more than 2.0 ml of acid is required,
the meat is in some form of incipient spoilage.
Extract Release Volume (ERV). As meat deteriorates, there is an increase in
the amount of water retained and a decrease in ERY. Shelef (1974) re-
ported the relationship between pH and ERY. Regardless of the micro-
bial quality of the meat, the maximum ERV occurred at pH 5.5.

ADENOSINE TRIPHOSPHATE (ATP). During metabolism, cells form

high-energy phosphate bonds stored in ATP. Not only microbial cells, but
also other living cells contain ATP.
When an animal dies and the muscle glycogen is utilized by anaerobic
glycolysis, the amount of ATP decreases. It has been established that
when bacterial cells are killed, the ATP disappears. In starved cells, the
ATP falls to low levels before the loss of viability is evident. Since all
microbial cells contain ATP, it should be possible to estimate the number
of cells by quantitating the ATP in a system.
The method for determining ATP is based on the firefly reaction as
shown in Figure 2.5. A purified extract from the firefly, containing luci-
ferin and luciferase, when reacted with ATP in the presence of magne-
sium ions, causes a light emission. Crude extracts from the firefly, when
reacted with adenosine diphosphate (ADP) have caused light emission.
When the purified firefly extract is added to bacterial ATP, the light emis-
sion can be measured with a photometer. For the assay, it is necessary to
eliminate the nonbacterial ATP and then to release the bacterial ATP to
react with the luciferin-luciferase system.
One method used to eliminate nonbacterial ATP is to rupture the
somatic cells and hydrolyze the released ATP (Anon. 1974; Thore et al.
1975). Another method is the centrifuging and/or filtration of a food to
separate the bacterial cells from food cells (Jarvis 1982; Picciolo et al.
1981; Stannard and Wood 1983).
After elimination of food ATP, the microbial ATP can be released
 ESTIMATING THE NUMBER OF MICROORGANISMS 39

Mg++
+ E + ATP
:::;::::!=
Luciferin Luciferase Adenosine
triphosphate

E· LH 2 • AMP + PP

Luciferyl adenylate Pyrophosphate

complex

E· LH2 • AMP + O2 - - E + LO + CO2 + AMP + Light

Oxyluciferin Adenosine
monophosphate
Figure 2.5. Reactions involved in adenosine triphosphate determinations.

from the cells and assayed with the luciferin·luciferase system, and an
estimate of the number of cells is calculated.
The ATP system is relatively simple and rapid. The system is report·
edly reliable and acceptable for food and water analysis (J arvis 1982; Pic·
ciolo et al. 1981). Firefly bioluminescence was reviewed by McElroy and
DeLuca (1983).

MEASUREMENT OF GAS PRODUCTION. When organisms metabo·

lize compounds, carbon dioxide is produced as a metabolic product and
oxygen is consumed. One method for determining CO 2 production uses
14C·labeled glucose and measures the reactivity of the released 14C0 2 • The
detection time of 14C0 2 is proportional to the logarithm of the original
inoculum (Waters 1972). This procedure has been used for determining
various bacteria in water and food (Hatcher et al. 1977; Lampi et al. 1974;
Mafart et al. 1978; Rowley, Previte, and Srinivasa 1978).
Headspace gas of bottled fruit juice was analyzed for CO 2 by an infra·
red system (Threlkeld 1982). Infrared analysis detected 89 percent of the
samples that were positive by conventional methods. Most of the false
negative samples lacked the presence of fermentative organisms.

IMPEDANCE. The metabolic activity of cells changes the chemical

composition of a medium, a process that results in altering the imped·
ance (Firstenberg·Eden and Zindulis 1984). The impedance is the opposi·
40 BASIC FOOD MICROBIOLOGY

tion to the flow of alternating electrical current and can be measured by

a sensitive meter. This system has been used to estimate microorganisms
in cooked food (Rowley et al. 1979), cereal grain (Sorrells 1981), milk
(Firstenberg·Eden 1984; Firstenberg-Eden and Tricarico 1983; Gnan and
Luedecke 1982; Martins et al. 1982), meat (Firstenberg-Eden 1983; Mar-
tins and Selby 1980), frozen vegetables (Hardy et al. 1977), and orange
juice (Weihe, Seibt, and Hatcher 1984).

OTHER INSTRUMENTATION OR METHODS. Besides those systems

described, other systems have been used by microbiologists to develop
rapid methods for microbial detection. A pyrolysis·gas chromatography-
mass spectrometry experiment was used to analyze Martian soil for or-
ganic compounds that would indicate life on Mars (Klein 1976; Sim·
monds 1970). Sanders and Parkes (1970) attempted to correlate infrared
analysis of swabbings from broiler skin with bacterial numbers.
Gas chromatography or gas·liquid chromatography has been used to
detect metabolic products of microorganisms which can be used to differ·
entiate bacterial species or estimate the count or quality (Carlsson 1973;
Staruszkiewicz and Bond 1978).
Limulus amoebocyte lysate forms a gel in the presence of small
amounts of endotoxin from Gram-negative bacteria. This assay has been
suggested as a system to estimate Gram·negative bacteria in various sam-
ples such as meat Gay 1981; Seiter and Jay 1980), milk (Hansen, Mik-
kelsen, and M<t>ller-Madsen 1982), fish (Sullivan et al. 1983), cooked
turkey (Dodds, Holley, and Kempton 1983), and drinking water (Sykora
et al. 1980).
It is not possible to discuss all of the procedures that might be used
to determine microorganisms in foods. Some other procedures were de-
scribed by Bordner (1981), Feldman (1982), Goldschmidt and Fung (1978,
1979), Jarvis (1982), Newsom (1978), and Southern (1979).
Although agar is the usual solidifying agent in solid media, various
substitutes have been suggested (Cranston 1983; Kang et al. 1982; Lin
and Cas ida 1984; Shungu et al. 1983).

Comparison of Methods
There are many procedures that can be used to estimate the total
microbial population of a food product. The test that is selected depends
upon the use of the information that is obtained. If the results are to be
used to satisfy a microbiological standard, then the specified method,
usually the standard plate count, must be used. If the results are for inter-
nal quality control, some other method that is simpler, faster, or less ex·
pensive might be used. The procedure selected will depend on the accu·
 ESTIMATING THE NUMBER OF MICROORGANISMS 41

racy, reliability, or precision that is needed. The laboratory equipment

and personnel, both now and in the future, will probably dictate the type
of analysis that can be conducted. The cost of supplies, materials, instru-
ments, and labor must be compared with other factors to determine
which method or methods give the desired information with ease, sim-
plicity, speed, and low cost.
In selecting the test, not only precision but also accuracy must be
considered. Precision is an index of the random error in a group of de-
terminations and is not related to accuracy. The term accuracy considers
the relationship of the determined value and the actual value. Since there
is no way to know the actual or true number of microorganisms in a
sample, it is difficult to assess the accuracy of a method. Quite often, the
results of a method are compared to those obtained with the APC. The
results obtained with the APC may not be accurate, but the method yields
acceptable reproducibility or precision.
Rapid tests such as those based on metabolic products and instrumen·
tation are valuable for control purposes. Time is important in analyzing
highly perishable foods. Also, it is desirable to keep the inventory of
processed food at a reasonable level. If unacceptable product is shipped
from storage, it might need to be recalled. A recall of a product and the
bad publicity that often results is not desirable for a processor.
If microbial inhibitors are present in the food, these will be carried
over into the growth medium. In these cases, as the sample is diluted,
higher counts might be obtained due to dilution of the inhibitors. If a
food containing glucose is added to a medium designed to determine
lactose fermentation, erroneous results may be obtained due to fermen-
tation of the added glucose.
In processed foods, there may be cells that are sublethally injured.
These are of special importance when the types of organisms are deter·
mined with selective media.
Comparisons of various methods of estimating microbial counts have
been published (Greenwood et al. 1984; O'Toole 1983b). Perhaps food
microbiologists have emphasized the so·called total count too much,
since spoilage organisms and potential pathogens are more important in
a food than are other types of organisms. However, at the present time,
the total count is usually the first microbial characteristic determined for
almost any food.

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 ESTIMATING THE NUMBER OF MICROORGANISMS 47

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48 BASIC FOOD MICROBIOLOGY

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 3
Microorganisms Associated
with Food

The significance of microorganisms in foods depends upon several con-

ditions: (1) the numbers found; (2) the types of microorganisms; (3) the
type of food; (4) the treatments to which the food has been exposed; (5)
the processing or storage treatments the food will receive; (6) whether
the food is to be eaten as is or heated; and (7) the individuals who might
consume the food. In this text, it is necessary to limit the discussion of
microorganisms in foods to bacteria, fungi, and viruses.
Microorganisms may have at least one of four functions in a food.
They may have a useful function, cause spoilage, be a health hazard, or
be inert. The inert microorganisms do not find an environment favor-
able for growth. In most cases of foodborne illness, spoilage, or useful
activity, the microorganisms grow and multiply. Organisms that cause
foodborne illness are of more concern than are other types of microorga-
nisms.
Spoilage is the result of undesirable changes in the odor, color, flavor,
texture, or appearance of food. Some organisms that do not directly
cause changes in a food may alter the flora so that spoilage organisms
can grow. An example is the bacteriophages that attack useful organisms,
allowing undesirable organisms to grow and cause spoilage.
Useful organisms are those which produce desirable changes in food,
such as converting milk to cheese, sugar to alcohol, and cabbage to sauer-
kraut. These changes are referred to as fermentations. The microorganism
does not always have to be present to have a useful function, since en-
zymes can be separated from the organism, and the enzymes used to pro-
duce the desired reaction. Another function of useful organisms is the
production of single-cell protein which can be used as food. Spoilage
organisms and useful organisms are similar in that they both produce
changes in the food. The difference between them is that one is desirable
and the other is undesirable.
Although it is easy to establish categories for microorganisms, it is
difficult to place an organism in only one category, since it may have
different functions in different foods. An organism may spoil one food
but be inert in another, because of the different characteristics of foods.
49
50 BASIC FOOD MICROBIOLOGY

However, we do tend to associate certain organisms with particular func-

tions in food, and these will be considered as much as possible_

DETERMINING TYPES OF MICROORGANISMS

The basic systems of microbial analysis discussed in Chapter 2 can be

used to determine the specific types of microorganisms that are present
in or on a food_ However, differential or selective media are substituted
for the noninhibitory, nonselective media_ Quite often, prior to deter-
mining whether certain types of organisms are present, an enrichment
procedure is used to increase the probability of detection_ After these
enrichment processes, the organisms are detected on differential or
selective agar.
Various tests are used to differentiate organisms isolated from selec-
tive or differential agars_ The usual staining reactions and morphological
characteristics are determined_ The required biochemical tests depend
upon the microorganisms being considered_ Various commercial kits and
systems for determining biochemical reactions have been evaluated (Costi-
gan and Hollick 1984; Cox and Mercuri 1979; Cox, Bailey, and Thomson
1983; Cox et aL 1984; Ferraro, Edelblut, and Kunz 1981; Fung, Gold-
schmidt, and Cox 1984; Griffiths and Phillips 1982; Izard et aL 1984; Kelly
and Latimer 1980; Odlaug et aL 1982)_ Serological reactions can utilize
cellular, flagellar, or other antigens_ The development of monoclonal an-
tibodies (L0vborg 1984) and immunoassays such as radioimmunoassay
(RIA), enzyme immunoassay (EIA) (Milby and Zare 1984), and enzyme-
linked immunosorbent assay (ELISA) are used to detect specific microor-
ganisms as well as toxins and other antigenic agents_ The production of
pigment, antimicrobial resistance pattern, phage typing, and bacteriocin
typing can be useful for the differentiation of strains of some organisms
(Anderson and Engley 1978; Kaneko and Hashimoto 1982)_
In the future, it is likely that genetic relatedness will become the sys-
tem to identify microorganisms (Krieg and Holt 1984)_ This includes de-
oxyribonucleic acid (DNA) and ribonucleic acid (RNA) homology (Paller-
oni 1983), as well as DNA colony hybridization and gene-specific DNA
probes (Claus, Moseley, and Falkow 1983)_
Besides the genetic information in the chromosome, many bacteria
contain extrachromosomal elements called plasmids_ The genetic mate-
rial in the plasmids is not absolutely essential for the cells to survive, but
it may give the cell a selective advantage in certain environments_
When a bacterium that contains plasmids divides, the plasmids are
replicated so there is at least one copy of the plasm ids in each of the
 MICROORGANISMS ASSOCIATED WITH FOOD 51

daughter cells. Under certain conditions, the plasmids from one cell can
be transferred to another cell by conjugation.
The plasmids have been separated into several classes according to
the characteristics they confer upon the cells. Class F plasmids, the fertil·
ity plasmids, have the ability to transfer genetic material by conjugation.
Other plasmids are involved with antibiotic, chemical, or radiation resist·
ance; enzyme coding, so that metabolic reactions different from the usual
ones occur; virulence and production of toxins, such as enterotoxins; and
various other traits (Helinski 1976; Mach and Grimes 1982; Murray et al.
1983; Taylor, Levine, and Kouvelos 1982; Veltkamp 1979).

MICROBIAL TYPES IN FOOD

The microbial analysis of food products yields many diverse types of

microorganisms. However, we are concerned with the predominant types
and those which may cause spoilage or be a health hazard.
Various genera of bacteria have been isolated from red meat shortly
after slaughter of the animal. Lee, Fung, and Kastner (1982) reported the
prevalent genera on hot· boned beef as Staphylococcus, Micrococcus, Brevi·
bacterium, and Bacillus. Others that have been isolated from red meat in·
clude Lactobacillus, Corynebacterium, Flavobacterium, Pseudomonas, Alcaligenes,
Streptococcus, Pedicoccus, Acinetobacter, Microbacterium, and Aerococcus. Quite
often coliforms, salmonellae, and Clostridium perfringens are found on red
meats. The temperature of holding and the packaging materials influ·
ence the types of organisms that become dominant on fresh meat. Spe·
cies of Micrococcus and Lactobacillus are important on cured meat. Also,
Bacillus, Vibrio, coliforms, fecal streptococci, and various yeasts can be
isolated from cured meats.
The same genera found on red meat tend to be present on poultry
and fishery products. Although the potential pathogens, such as Salmo·
nella and Campylobacter, have been isolated from poultry, generally less
than 25 percent of the carcasses are contaminated (Cunningham 1982;
Kraft et al. 1982; McKinley and Avens 1981; Norberg 1981). Fishery prod·
ucts, especially those from the oceans, contain Vibrio species (Sobsey et
al. 1980; Sochard et al. 1979).
Animal products such as milk and eggs tend to have bacteria similar
to those of meat and poultry (Coghill 1982; Cousin 1982; Johnston and
Bruce 1982; Sashihara et al. 1979; Schwab et al. 1982).
Many types of microorganisms are associated with plant products. The
most common bacteria in vegetables are Lactobacillus and Leuconostoc. The
genus Erwinia is important in vegetable spoilage. Other genera include Aer-
52 BASIC FOOD MICROBIOLOGY

omonas, Alcaligenes, Bacillus, Corynebacterium, Enterobacter, Flavobacterium,

Klebsiella, Pseudomonas, Serratia, and Xanthomonas. Molds on vegetables in·
clude Alternaria, Aspergillus, Aureobasidium, Botrytis, Chaetomium, Cladospor·
ium, Epicoccum, Fusarium, Mucor, Penicillium, Phoma, and Rhizopus (Andrews
et al. 1982; Geeson 1979; Webb and Mundt 1978). During processing, the
initial microflora are distributed throughout the product and are supple·
mented with other microorganisms.
The important organisms on fruits are molds (Penicillium, Alternaria,
Aspergillus). Lactobacillus species cause spoilage in fruit juices.
Molds such as Aspergillus, Fusarium, and Penicillium are found on dry
products such as grain and peanuts.
Several genera of yeasts are important in the spoilage of foods, espe·
cially fruits and foods containing sugar. Yeasts are also useful in fermen·
tation and the production of single·cell protein.

BACTERIA

Compared to the total number of species of bacteria, relatively few

have any importance in food, and most of these are the common types
that are discussed in general microbiology. Individual species of bacteria
are discussed with their functions in later chapters, so we will be con·
cerned mainly with the genera that are important.
In Bergey's Manual of Determinative Bacteriology (Buchanan and Gibbons
1974), the bacteria are grouped according to the Gram reaction, mor·
phology, and relation to growth with or without oxygen. In essence, that
system is used by Krieg and Holt (1984) as well as in this text for bacteria
found in food. Various morphological types of bacteria are shown in Fig·
ures 3.1 and 3.2.

Aerobic/Microaerophilic, Motile, Helical/Vibroid

Gram-Negative Bacteria
In this group only one genus, Campylobacter, is important in food.

CAMPYLOBACTER. These Gram·negative, oxidase-positive organisms

have a singular polar flagellum at one or both ends and are motile with
a typical corkscrew motion. Unlike most bacteria, campylobacters use
amino acids or tricarboxylic acid intermediates rather than carbohy-
drates as a source of energy. The species of concern is C. jejuni. This or-
ganism is found on and in animal products and is a cause of gastroenter-
itis in human beings. (See Chapter 6.)
 MICROORGANISMS ASSOCIATED WITH FOOD 53

o 8fj m
tP flJ b

Cocci , I'-i", llllllIods 01

multiplication. G, SftptOCOCC.. ;
b, Mic r _ ; e ...,;, SaIaro

0 0 0 CD Q? 00

a:m(¢J ~
c::l c:::> CO a Cl

Bac1eroal Cell dl.ision

Hic;lher bclc:,.,;a

Figure 3.1. Morphological forms of bacteria.

Courtesy of Weiser, Mountney, and Gould (1971).

"--Qa~p
QI()
~~OsJ
gOODt?~f)

Gtanul" in _'eria
BacterIa wi'" flGQe lla
Figure 3.2. Structures of Bacteria,
Courtesy of Weiser, Mountney, and Gould (1971).

Gram-Negative, Aerobic Rods, and Cocci

Included in this group are the family Pseudomonadaceae with the
genera Pseudomonas and Xanthomonas; the family Halobacteriaceae with
the genera Halobacterium and Halococcus; the family Acetobacteriaceae
with the genera Acetobacter and Gluconobacter; the family Neisseriaceae
with the genus Acinetobacter and four other genera (Alcaligenes, Alteromonas,
Brucella, and Flavobacterium) of uncertain status (Krieg and Holt 1984).
54 BASIC FOOD MICROBIOLOGY

PSEUDOMONAS. This genus is divided into four sections on the basis

of RNA homology and a fifth section which contains species whose rela-
tionships to the others are generally unknown (Krieg and Holt 1984).
Oyaizu and Komagata (1983) divided the pseudomonads into nine groups,
primarily on the basis of cellular fatty acid composition.
The pseudomonads are straight to curved, motile (polar flagella) rods.
They are widely distributed in nature and are found on both animal and
plant products. Some pseudomonads are pathogenic for plants (Wong
and Preece 1980). Others, such as P aeruginosa, produce toxic substances
and are opportunistic pathogens for human beings (DeBell 1979;
Homma 1978). Some Pseudomonas species have been suggested as caus-
ative agents of foodborne illness, but there is incomplete proof that this
is so (Bryan 1973).
The pseudomonads are noted for their biochemical activity, being
able to attack a wide variety of organic compounds, including aromatic
types, and some synthetically produced chemicals. Some species (P aerugi-
nasa and P cepacia) have been found to grow in distilled water. The
pseudomonads produce catalase and most produce oxidase. They can
produce acids oxidatively from glucose, maltose, or both.
Some species of pseudomonads produce pyoverdine or fluorescein,
which are water-soluble fluorescent pigments (Leisinger and Margraff
1979). These pigments can be observed in spoiled foods by using an ultra-
violet light. They are usually yellow-green but may appear blue or orange,
depending on the species and environmental factors.
The pseudo monads produce enzymes that catalyze proteolytic (Mc-
Kellar 1982; Patel, Bartlett, and Hamid 1983) and lipolytic (Roy 1980)
reactions that contribute to spoilage of refrigerated fresh animal prod·
ucts (Shaw and Latty 1982). Some pseudomonads produce pectolytic en-
zymes that can cause soft rot of fleshy vegetables (Cuppels and Kelman
1980; Palleroni and Holmes 1981).
The psychrotrophic pseudomonads are found in almost all types of
refrigerated and frozen foods. Since they are not very heat resistant, they
are not found in heat-processed foods unless the food is recontaminated
after heating. They are not very resistant to drying or gamma irradiation.
P aeruginosa is notoriously resistant to quaternary ammonium com-
pounds.
Several recent reports of selective media for Pseudomonas have been
published (Gould et al. 1985; Katoh and Itoh 1983; Wu and Thompson
1984).

XANTHOMONAS. The organisms in this genus are plant pathogens.

They are straight, motile (polar flagellum) rods and are catalase positive.
 MICROORGANISMS ASSOCIATED WITH FOOD 55

A medium to isolate a strain of X. campestris from walnuts was described

by Mulrean and Schroth (1981).
Being plant pathogens, these organisms have been involved with var·
ious types of rots of these products. X. campestris produces xanthan gum,
which is used in the food industry.

HALOBACTERIACEAE. The two genera Halobacterium and Halococcus

are extreme halophiles requiring about 15 percent salt for growth. They
are found in solar salt (produced by evaporation of seawater).
Their halophilic character prevents them from growing in most
foods. However, in foods preserved by salting, these organisms can pro·
duce a red pigment, bacteriorubein.
The high salt concentration is necessary for activity of the enzymes,
stability of membranes and ribosomes, and synthesis of protein. A low
salt concentration results in Halobacterium changing from rods to spheri·
cal forms.

ACETOBACTER. These cells are ellipsoidal to straight or slightly

curved rods. Young cultures are Gram negative, while older cultures are
Gram variable. They are motile (peritrichous flagella) or nonmotile.
The Acetobacter are noted for the oxidation of ethanol to acetic acid.
They oxidize acetate and lactate to CO 2 and H 2 0. They are found on
fruits and vegetables and are involved in the souring of fruit juices and
alcoholic beverages (beer and wine).
A selective medium for differentiating Acetobacter and Gluconobacter
was described by Cirigliano (1982).

GLUCONOBACTER. G. oxydans represents this genus. This organism is

ellipsoidal to rod·shaped, Gram negative to weakly Gram positive in
older cultures. The cells occur singly, in pairs, or in chains. They are
strictly aerobic and oxidize ethanol to acetic acid.
G. oxydans is found in various food products such as vegetables, fruits,
baker's yeast, beer, wine, cider, and vinegar. It is involved in spoilage,
causing the souring of fruits.

ACINETOBACTER. These organisms are very short, plump rods or

coccobacilli, predominantly in pairs or short chains. They are strictly aero
obic, mesophilic saprophytes.
They are found in soil and water and also in animals and human
beings. Organisms identified as Acinetobacter have been isolated from var·
ious types of raw and prepared foods, including beef and poultry car·
casses (Lahellec et aL 1975). They are potential spoilage organisms. Meth·
56 BASIC FOOD MICROBIOLOGY

ods for enumeration of Acinetobacter were described by LaCroix and

Cabelli (1982) and Spino and Geldreich (1981).

ALCALIGENES. The four species in this genus are motile (four to

eight peritrichous flagella) rods, coccal rods, or cocci. They are strict aero
obes and oxidase positive.
They are widespread in nature, being found in soil, water, decaying
matter, and the intestinal tract of animals. These organism are involved
with the spoilage of protein foods (eggs and dairy products).

ALTEROMONAS. This genus comprises Gram·negative, motile (polar

flagellate), nonfermentative bacteria with a guanine + cytosine (GC) ra·
tio between 43.2 and 48 mol percent (Baumann et al. 1972). This ratio is
listed as 38:50 mol percent by Krieg and Holt (1984).
These organisms are common in marine environments and on fish,
causing fish spoilage (Chan et al. 1978; Gillespie 1981; Gray and Stewart
1980). Alteromonas was isolated from ground beef (Parker and Levin
1983).

BRUCELLA. These coccobacilli or short rods are nonmotile. Brucella

melitensis (pathogenic for goats and sheep), B. abortus (pathogenic for cat·
tle), and B. suis (pathogenic for pigs) can affect people, causing bru·
cellosis (undulant fever). The sources of infection are raw milk or dairy
products, uncooked meat or sausage products, discharges from infected
animals, and human' carriers.
Since these organisms are susceptible to the heat treatment used for
pasteurization of milk and cooking of meat, they should be of limited
importance in food.
There are between 100 and 200 cases of brucellosis in the United
States annually. The majority of cases involve meat· packing· plant work·
ers, and most of these are involved with hog·slaughter operations. Most
of the other cases involve livestock producers, veterinarians, and govern·
ment inspectors.

FLAVOBACTERIUM. This genus contains diverse bacteria. The spe·

cies includes nonmotile rods that produce yellow, orange, or greenish·
yellow pigment (Hayes 1977). Environmental factors (substrate and
temperature) affect the synthesis of pigment and resultant hue. These
organisms prefer temperatures below 30°C, although some strains can
grow at 37°C.
Flavobacterium species have been isolated from water, soil, animals, hu·
mans, and various food products. They can produce discoloration on
some foods. The organisms have been found on thawing frozen vegeta·
 MICROORGANISMS ASSOCIATED WITH FOOD 57

bles, fresh vegetables, refrigerated fish and shellfish, meat, meat prod-
ucts, poultry, and in cannery environments.

Gram-Negative, Facultative Anaerobic Rods

In this group, the family Enterobacteriaceae contains several genera,
including Escherichia, Shigella, Salmonella, Citrobacter, Klebsiella, Enterobacter,
Erwinia, Serratia, Edwardsiella, Proteus, and Yersinia. The family Vibrionaceae
includes Vibrio and Aeromonas. There are other genera in these families
and in this section, but they are not very important in food microbiology.

ESCHERICHIA. The principal species is E. coli. The organisms are

members of the coliform group, which are indicators of fecal contamina-
tion (see Chapter 7). E. coli may cause spoilage of food, and certain strains
are enteropathogenic or enterotoxigenic.
E. coli is found in soil and water, on plants, in the intestinal tract
of animals, and in various foods, especially animal products and foods
handled by people. Since the cells are heat sensitive, their presence in
heat-pasteurized or cooked products indicates recontamination after the
treatment.

SHIGELLA. The four species in this genus are nonmotile, nonspore-

forming rods. The normal habitat is the intestinal tract of human beings
and other primates. Rarely are these organisms isolated from other ani-
mals.
The shigellae are the causative agent of shigellosis, a foodborne gas-
troenteritis. The number of foodborne illnesses due to these organisms
is not great, but the illness can be severe. Additional information about
Shigella and shigellosis is in Chapter 6 and in Dodd and Jones (1982).

SALMONELLA. The genus Salmonella is divided into five subgenera

(I-V). These subgenera are differentiated on the basis of minor biochemi-
cal differences. Subgenus I includes the majority of serotypes of this ge-
nus, especially the more important serotypes, and subgenus III includes
the organisms previously called the Arizona group. Most of the different
types were differentiated on the basis of serological reactions, so they are
called serotypes or serovars. The number of known serovars is approach-
ing 1900.
The salmonella organisms are catalase positive and oxidase negative,
with both respiratory and fermentative metabolism. Most of the sero-
types are motile (peritrichous flagella), do not ferment lactose, and grow
well in simple media containing glucose, inorganic nitrogen, and mineral
salts. Although salmonellae do not ferment sucrose, strains of S. arizonae
58 BASIC FOOD MICROBIOLOGY

contain a plasmid that gives them the ability to ferment this sugar (Bart·
lett and Trust 1980).
The salmonellae are said to be ubiquitous, being worldwide and
found in or on soil, water, sewage, animals, humans, processing equip·
ment, feed, and various food products. The natural habitat is the intesti·
nal tract of humans and animals. It is logical that humans, animals, and
their environments are the primary sources of salmonellae. Some sero·
types seem to be localized in a region or a country, but with national and
international travel and trade, the organisms are easily disseminated.
The serotypes of Salmonella are an important cause of foodborne ill·
ness. Further information about these organisms is found in Chapter 6.

CITROBACTER. The species in this genus are motile (peritrichous

flagella) rods that utilize citrate as the sole carbon source. Most ferment
lactose. They are members of the coliform group of indicator organisms.
They are normal intestinal inhabitants and are found on various foods,
especially animal products. These organisms are listed as causing enteri·
tis in humans, but proof of transmission by foods is inconclusive (Bryan
1973). They have been associated with various human infections. Citro·
bacter can also cause spoilage of foods.
Cross·reactions with antisera of other Enterobacteriaceae indicate
that there are close relationships between Citrobacter, Salmonella, and Esch·
erichia.

KLEBSIELLA. The klebsiellae are nonmotile, encapsulated rods. They

are found on grain and fresh produce and in frozen foods. They are
members of the coliform group, can cause food spoilage, and are poten·
tial health hazards, causing gastroenteritis. According to Bryan (1973),
proof that transmission of these organisms is by means of food is incon·
clusive.

ENTEROBACTER. Organisms in the Enterobacter genus are similar to

the klebsiellae, except they are motile (peritrichous flagella).
They can be found in soil, water, sewage, the intestinal tract of people
and animals, and in various food products. The organisms are important
in food as a potential health hazard and as indicators of spoilage.

ERWINIA. Organisms in this genus are small, mostly motile (peritrich·

ous flagella), predominantly single, straight rods. They are oxidase nega·
tive and catalase positive.
The organisms cause various plant diseases and soft rots. E. carotovara
is the main spoilage organism of stored vegetables (Chapter 8). At least
one species is associated with humans and animals.
 MICROORGANISMS ASSOCIATED WITH FOOD 59

SERRATIA. The cells of Serratia are motile (peritrichous flagella) rods.

They are important as potential food·spoilage organisms. S. marcescens is
noted for the production of red pigment and is used as a biological
marker (Yu 1979).

EDWARDSIELLA. These organisms have peritrichous flagella. They

produce indole from tryptophan and produce H 2 S on triple sugar iron
agar.
Although these organisms cause enteritis in humans, proof that trans-
mission is by means of food is not conclusive (Bryan 1973). E. tarda has
a wide geographical distribution and infects numerous animal species.

PROTEUS. Organisms in this genus are highly motile (peritrichous

flagella) rods that occur singly, in pairs, or in short chains. On moist agar
surfaces, some Proteus species tend to swarm, which makes it difficult to
pick isolated colonies for further study. Swarming is more evident at
20°C than at 37°C.
The organisms are widely distributed. They are common inhabitants
of the gastrointestinal tract, and are found in sewage, soil, in decompos-
ing animal protein, and in various foods.
The importance of Proteus in food includes potential health hazards
and spoilage. Although strains of Proteus can cause enteric infection in
humans, according to Bryan (1973), proof of transmission by food is in-
conclusive.

YERSINIA. The cells in this genus are ovoid to rod shaped. Included
in this genus is Y. enterocolitica, which is associated with gastroenteritis,
meseteric lymphadenitis, terminal ileitis, and pseudoappendicitis (Berco-
vier et al. 1980)_ This organism has been isolated from red meat, chicken,
shellfish, milk, ice cream, and vegetables. These organisms may be more
prevalent in food than reported.

VIBRIO. This genus is in the family Vibrionaceae. The cells are short,
motile, oxidase positive, curved, or straight rods. Some strains fail to grow
without the presence ofNaCI, the optimum concentration being 3.0 per-
cent.
Strains of V. costicola can tolerate NaCI concentrations of 23 percent.
This species has been found in cured meats and curing brines (Gardner
1980-1981). A medium for isolating these salt-tolerant organisms was de-
scribed by Gardner (1973). Methods for enumeration, phage typing, and
characterizing V. cholerae were discussed by various researchers (Bocke-
muhl and Meinicke 1976; Morris et al. 1976; Salles and Momen 1981).
V. cholerae and V. parahaemolyticus are important pathogens, causing
60 BASIC FOOD MICROBIOLOGY

gastroenteritis in humans. V. cholerae is found in the intestinal tract of

people and animals, in water, and occasionally in food. V. parahaemolyticus
is found in the ocean, in seafoods, and in the intestinal contents of in·
fected humans. Various other vibrios have been incriminated in out-
breaks of foodborne illness (Roberts and Gilbert 1979).

AEROMONAS. The cells are rods with rounded ends to coccoid. They
are motile (polar flagella), and are oxidase and catalase positive.
These organisms are frequently mistaken for members of the family
Enterobacteriaceae because of their similarity in growth and biochemical
characteristics. A positive oxidase test and nitrate reduction help differ-
entiate the Aeromonas from Enterobacteriaceae.
The main habitat of Aeromonas is water. Some strains cause disease
(hemorrhagic septicemia) in fish, eels, and frogs. Some strains cause en-
teritis in humans. One source of this infection may be fish or other sea-
food. Aeromonas may playa role in spoilage of fish and other animal prod-
ucts.

Gram-Negative, Anaerobic Straight, Curved, and

Helical Rods
In this category, the family Bacteroidaceae contains twelve genera,
including Bacteroides. These organisms are nonmotile, straight rods. Be-
ing nonsporeforming obligate anaerobes, they probably cause no
changes in foods. However, they are found in fecal material in very high
numbers. Hence, they might be useful as indicators of fecal contamina·
tion of food as well as water (Fiksdal et al 1985).

Gram-Positive Cocci
The Gram-positive cocci include aerobic or facultative anaerobic bac·
teria in the family Micrococcaceae with the genera Micrococcus and Staphy-
lococcus. Other Gram-positive cocci include the genera Streptococcus, Leuco-
nostoc, Pediococcus, and Aerococcus.

MICROCOCCUS. These spherical cells are strict aerobes, catalase posi-

tive, occur singly or in pairs, and characteristically divide in more than
one plane to form irregular clusters, tetrads, or cubical packets. They can
grow in the presence of 5 percent salt.
The micrococci are found in soil, water, and 'dust, and on the skin
of people and other animals. They are found in several types of foods,
especially milk and dairy products, on animal carcasses, and in meat
products. They are important as potential spoilage organisms.
 MICROORGANISMS ASSOCIATED WITH FOOD 61

STAPHYLOCOCCUS. These nonmotile cells occur singly, in pairs, or

in irregular clusters. They are facultative anaerobes with respiratory and
fermentative metabolism. Most strains can grow in 7.5 percent to 15 per-
cent salt. The organisms usually are sensitive to chlorine, chloramine,
iodine, and iodophors. Although usually sensitive to heat, they are mod-
erately resistant to radiation.
Both S. aureus and S. epidermidis are commonly found on the skin and
mucous membranes of humans and warm-blooded animals. They are po·
tential pathogens, being either the primary pathogen or secondary in-
vader.
The staphylococci are found in many types of food products. In gen-
eral, they are not able to compete very well with other organisms. S.
aureus produces enterotoxins, which are one of the main causes of food·
borne illness. S. aureus is found in pimples, boils, acne, wound infections,
and in the nose. It is easily transferred to foods by careless food handlers.
This species is differentiated by its ability to produce coagulase (which
clots blood plasma) and a heat·stable nuclease. S. aureus is described fur-
ther in Chapter 6.
Other species have been found in food (Devriese et al. 1983; Schleifer
and Fischer 1982). S. hyicus reportedly produces an enterotoxin (Hoover,
Tatini, and Maltais 1983).

STREPTOCOCCUS. The bacterial species in this genus have various

characteristics and functions. These organisms have been divided into
several groups (Bridge and Sneath 1983). The groups of primary impor-
tance in food are the lactic streptococci and enterococci. Researchers
have suggested that organisms in the latter group be put into the genus
Enterococcus (Collins et al. 1984; Schleifer and Kilpper·Balz 1984).
These spherical cells occur in pairs or chains. With the exception of
some strains, they are not motile. They are facultative anaerobes with
a fermentative metabolism, fermenting glucose primarily by the hexose
diphosphate pathway and producing mainly lactic acid. Thus, they are
called homofermentative.
The fermentation of carbohydrates to lactic acid is desirable in prod-
ucts such as cultured milk, cheese, and sauerkraut, but it is undesirable
in products such as fresh milk. They can utilize sugar in an alcohol fer-
mentation, resulting in lower alcohol and higher acid than is wanted.
Some strains utilize citric acid and form acetoin and diacetyl. The ability
to produce diacetyl in milk is desirable in the manufacture of cultured
sour cream, buttermilk, and butter.
Various functions such as lactose metabolism, proteolytic activity, and
hemolysin and bacteriocin production are mediated by plasmids (Ander-
son and McKay 1977; Gasson 1983; McKay 1983; Oliver, Brown, and
Clewell 1977).
62 BASIC FOOD MICROBIOLOGY

The streptococci are widely distributed, being found in air, water, sew-
age, soil, on plants, in the intestinal tract of people and animals, and in
various food products_
Some species and strains are somewhat heat resistant, surviving 60°C
for 30 min_ The enterococci can survive freezing and frozen storage in
food products_ This makes these organisms acceptable as potential indi-
cator organisms in frozen foods_
Although some pathogenic types may be transmitted to foods, there
is more interest in the streptococci as possible indicators of fecal contam-
ination, as useful fermentative organisms, and as potential spoilage bac-
teria_

LEUCONOSTOC. These spherical to lenticular cells occur in pairs or

chains. They often have complex nutrient requirements such as vitamins,
amino acids, and a fermentable carbohydrate. They ferment glucose to
lactic acid, ethanol, and CO 2 _
The organisms are important in fermentation and spoilage of foods.
They are not pathogenic. Although they have physiological differences,
Garvie (1983) has suggested that L. cremoris, L. dextranicum, and L mesentero-
ides belong to a single deoxyribonucleic acid (DNA) homology group and
should be considered subspecies of L. mesenteroides.

PEDIOCOCCUS. These homofermentative cocci occur as single cells,

pairs, or tetrads. They have rather complex nutritional requirements,
such as vitamins and amino acids, which makes them useful to assay for
these nutrients. These organisms are found in pickles, sauerkraut, beer,
wine, and other fermenting materials_

AEROCOCCUS. This genus has only one recognized species, A. viri-

dans, which used to be a Pediococcus (P. homari) (Buchanan and Gibbons
1974). The genus is very similar to Pediococcus. On blood agar, the colonies
are surrounded by a green zone, hence the name viridans (green).
The aerococci have been found in air and dust, human infections,
meat-curing brines, and on raw and processed vegetables.

Endospore-forming Rods and Cocci

This group includes the genera Bacillus, Clostridium, Sporolactobacillus,
and Desulfotomaculum. Although Desulfotomaculum is listed with dissimila-
tory sulfate or sulfur-reducing bacteria (Krieg and Holt 1984), the discus-
sion of this genus is included with endospore-forming bacteria. The
spores that these organisms produce are different from the vegetative
cells (Fig. 3.3) so there is another entity to consider. The spores are an
inactive or dormant state of the organisms.
 MICROORGANISMS ASSOCIATED WITH FOOD 63

Figure 3.3. Spores formed in the

center of the rod: Bacillus subtilis
at left, Clostridium sporogenes at
right.
Courtesy of Weiser, Mountney, and
Gould (1971).

The organisms go through certain stages to change from a vegetative

cell to a spore and back to a vegetative cell. The stages we can consider
are cell sporulation, germination, and outgrowth.

SPORULATION. The mechanisms that trigger spore formation are not

fully known. Most strains of clostridia produce spores when incubated
in a good medium under anaerobic conditions, 3° to 8°C below their
optimum growth temperature.
Muhammed, Morrison, and Boyd (1975) stated that demonstrating
the complete sporulation requirements of an organism is impossible be-
cause some nutrients essential for spore formation also may be required
for growth.
For all species, not all cells sporulate, regardless of the conditions.
There is no information as to why some cells in a culture sporulate and
others do not. However, there is agreement that spore formation begins
after the exponential growth phase during the stationary phase. Spore
formation can be arbitrarily divided into seven stages: (1) development
of axial chromatin filament; (2) spore septation; (3) engulfment of the
spore protoplast; (4) cortex formation; (5) coat formation; (6) maturation;
and (7) the free spore stage. The spore consists of a core surrounded by
several layers of mucopeptide and proteinaceous outer coats (Aronson
and Fitz:James 1976).
An organism can initiate sporulation without completing the process
and reverting to a vegetative cell. However, there is a stage at which the
cell becomes irreversibly engaged in sporulation and is committed to
completing the process. The composition of the substrate and the tem-
perature of sporulation can affect the resistance characteristics of the
resultant spores (Bayliss, Waites, and King 1981).
The main characteristic that is important to food microbiologists is
the resistance of spores to heat, radiation, chemicals, desiccation, and
freezing (Gould 1977). Quite commonly, the heat resistance of spores is
about 10 5 times, and radiation resistance is about 10 times more than
that of the corresponding vegetative cells. Spores can be stored for long
periods and retain their ability to germinate and produce vegetative cells.

GERMINATION. Since spores are dormant, they must be converted to

vegetative cells to be important in food spoilage or toxin production.
64 BASIC FOOD MICROBIOLOGY

The conversion of the heat-resistant spore to vegetative cells would allow

less severe thermal processes for food preservation.
Spores may need a conditioning treatment prior to germination. This
process, called activation, may be induced by aging, heating, radiation,
altering the pH, or with chemicals. Low numbers of viable spores do not
always germinate immediately. Thus, canned foods that apparently pass
short storage tests to determine potential spoilage, can show spoilage
after extensive storage.
Activation is a reversible process and, if conditions do not allow
germination, the spore reverts to its dormant state. Although some
spores may germinate without heat shock, low levels of heat treatment,
such as 65° to 85°C for 10 to 30 min, will activate most spores and induce
germination. The heat treatment used depends upon the species and
strain. Spores of species such as C. botulinum type E strains are heat sensi-
tive and should not be heated above 70°C. Higher temperatures (10° to
115°C) for 3 to 10 min can be used to activate spores of the thermophilic
B. stearothermophilus.
During germination, the bright, refractile spores become dark; dipi-
colinic acid is released and the cortex disintegrates. Although respiration
in the spore is undetectable, there is an abrupt onset of respiration dur-
ing germination. There is activity of a variety of enzymes, typical of the
vegetative form. The spores lose their resistance to heat, desiccation,
chemical agents, electric shock, and hydrostatic pressure. The germinat-
ing spores show a temporary rise in resistance to ultraviolet light and
ionizing radiation followed by a rapid fall in resistance.
Although pasteurized milk supported germination of B. cereus, raw
milk supported little or none (Wilkinson and Davies 1973). This helps to
explain the spoilage defect due to B. cereus in pasteurized milk but not
in raw milk. This inability to germinate in raw milk might be due to
natural inhibitory agents found in raw milk that are inactivated by pas-
teurization.
A review of bacterial spore germination by Smoot and Pierson (1982)
is suggested for further information on this subject.

OUTGROWTH. The development of a vegetative cell through the first

cell division is called outgrowth. Outgrowth proceeds by the swelling of
the spore, emergence from the spore coat, elongation, and cell division.
Germination and outgrowth can be followed by determining the synthe-
sis of RNA, proteins, and DNA, in that order.
Outgrowth occurs when germination takes place in a substrate capa-
ble of supporting vegetative growth. If the germinating medium is not
sufficient to support growth, either development is stopped or the out-
growing cell may form a second spore with no intervening cell division.
 MICROORGANISMS ASSOCIATED WITH FOOD 65

This cycle of spore-cell-spore, is called a microcycle. Microcycle sporu·

lation can be induced by dilution of an acceptable medium or by sus·
pending germinated spores in a glucose·free medium.
The effects of various factors on the germination and outgrowth is
important in food microbiology. It is the ability to determine viable from
nonviable spores that allows us to establish thermal processes that will
destroy spores in food. Also, if we could cause all of the spores to germi·
nate prior to heat processing, the thermal treatment could be reduced.

BACILLUS. These organisms are usually Gram·positive rods, but older

cultures may appear as Gram negative. The majority are motile, produce
catalase, and produce acid but not gas from glucose.
The cells in this genus vary from strict aerobes to facultative anaer·
obes. The nutrient requirements vary from simple to complex. There are
psychrotrophs, mesophiles, and thermophiles. Thus, for growth, the min·
imum temperature varies from - 5°C to about 45°C. The maximum for
some species is 25°C and for others up to 75°C. The minimum pH for
growth varies from pH 2.0 for B. acidocaldarius to pH 7.5 to 8.0 for B.
alcalophilus. The salt tolerance is 2 percent or less for some species. Oth·
ers can grow in 25 percent salt. Due to the diversity of the species, there
have been suggestions that the genus should be divided. Five genera have
been proposed, but this differentiation has not been adopted.
Bacillus can be found in soil, water, fecal material, decaying materials,
and in various foods. Ingredients can serve as a source of Bacillus. Spices,
flour, starch, and sugar have been incriminated as sources of spores for
contamination of fermented sausages, bread, and canned food. The reo
sistance of the spores of Bacillus to various agents makes these organisms
important in food preservation. Some species, such as B. subtilis, decom·
pose pectin and polysaccharides of plant tissue, causing spoilage of fresh
plant products. Low·acid canned foods are spoiled by B. stearothermophilus,
and B. coagulans causes spoilage of tomato products. B. cereus is involved
in foodborne gastroenteritis and B. anthracis causes anthrax of both ani·
mals and man.
Besides potential spoilage and health hazards, some species of Bacillus
can be useful in foods. The bacilli are a source of proteolytic enzymes
that might be used to clot milk for cheese production. Some bacilli have
been suggested for use in the production of single·cell protein. Some
species are insect pathogens, which makes them useful in food produc·
tion.

CLOSTRIDIUM. The species in this genus are divided into four groups
on the basis of spore position and gelatin liquefaction (Buchanan and
Gibbons 1974). The cells are usually Gram positive, at least in the early
66 BASIC FOOD MICROBIOLOGY

stages of growth. They are catalase negative and, except for a few aerotol·
erant species, are strictly anaerobic.
The critical tolerance of N aCI is 2.5 to 6.5 percent. Sodium nitrate at
0.5 to 1.0 percent inhibits these organisms. The lethal chlorine concentra·
tion is 2.5 p,g/ml.
The principal metabolic products of a species can be determined by
chromatography (column, thin·layer, or gas). In conjunction with other
tests, gas chromatography is a valuable aid in differentiating the clos-
tridia. The analysis of soluble cellular proteins with polyacrylamide gel
electrophoresis was used to differentiate species of clostridia (Cato et al.
1982). Anaerobic methods of analysis were reviewed by Anderson and
Fung (1983).
The primary source of clostridia is soil. They are found in the intesti-
nal tract of people and animals, as well as in various foods.
The species in this genus include two involved with foodborne illness,
several that cause food spoilage, and many of no concern to food micro-
biologists. Some are free-living nitrogen-fixing organisms, some cause se-
rious illness (tetanus, gas gangrene), and others are used to produc~ com-
mercial chemicals such as butyric acid, butanol, acetone, and enzymes.
The spores of some species are very heat resistant and may survive
the heat treatment of canned foods. If the surviving spores can germinate
and the vegetative cells grow, spoilage will occur. If C. botulinum spores
survive the heat treatment, germination, and outgrowth of the spores
may result in the production of potent toxins.

DESULFOTOMACULUM. The species in this genus are similar to the

clostridia. However, they are Gram negative and have a higher DNA base
composition (G+C is 41-46 mol percent as compared to 23-43 mol per-
cent). The clostridia do not reduce sulfate, but these organisms reduce
sulfur compounds (sulfates, sulfites, and other reducible sulfur com-
pounds) to H 2 S.
D. nigrificans is a thermophilic spore former that causes sulfide spoil-
age of canned foods.

Regular, Nonsporing, Gram-Positive Rods

In this group, the genera Lactobacillus is the most important to food
microbiologists. Other genera in food are Brochothrix, Kurthia, and Lis-
teria.

LACTOBACILLUS. These organisms are straight to curved rods occur-

ring singly or in chains. The rods vary from long and slender to short
coccobacilli. (Figs. 3.4 and 3.5.) Generally they are nonmotile. Although
considered to be Gram positive, as the culture ages, the cells may become
 MICROORGANISMS ASSOCIATED WITH FOOD 67

Figure 3.4. Lactobacillus bulgari-

cus, the high-acid-producing long
rod of sour milk preparations such
as yogurt (magnification = x
2,250).
Courtesy of Pederson (1979).

Gram-negative. Growth is enhanced by 5 to 10 percent CO 2 . These orga-

nisms generally have complex nutrient requirements. Both homofermen-
tative and heterofermentative types are in this genus.
Lactobacilli are found in plant and animal material, in various places
in the body of human beings and other warm-blooded animals (including
the intestinal tract) and in various foods.
In food microbiology, the lactobacilli are useful but also cause spoil-
age. They are useful in fermentations in which lactic acid production is
desirable, whether in plant or animal products (sauerkraut, pickles, ol-
ives, fermented sausages). Some lactobacilli form slime and spoil sugar
cane. Others cause green discolorations of sausage products. Vacuum-
packaged meat becomes sour due to lactobacilli. Even in fermented vege-
tables, they may be a problem, causing pink sauerkraut or bloater for-
mation in fermented cucumbers. The lactobacilli can cause spoilage of
vinegar-preserved products, such as catsup and mayonnaise.

Figure 3.5. Lactobacillus brevis,

the common heterofermentative
species of the genus (magnifica-
tion = x 2,250).
Courtesy of Pederson (1979).
68 BASIC FOOD MICROBIOLOGY

Due to their complex nutrient requirements, lactobacilli are used in

assays of food for vitamins, amino acids, and other nutrients. The lysine·
excreting mutants of lactobacilli have been suggested for use in food and
feed enrichment (Sands and Hankin 1974). Vandercook and Smolensky
(1976) suggested using L. plantarum to detect adulteration of orange juice
with imitation orange beverages. The imitation juice does not support
the growth of this organism as well as does real orange juice.

BROCHOTHRIX. The species B. thermosphacta was previuusly named Mi·

crobacterium thermosphacta. It is a Gram·positive, facultatively anaerobic
psychrotroph. It is associated with red meat and meat products, and may,
in certain cases, cause spoilage (a sweet off.odor) of these foods.

KURTHIA. These Gram·positive cells are regular, unbranched rods in

young cultures but become coccoid in older cultures by fragmentation
of the rods. The cells are strict aerobes.
These organisms are found in intestinal contents, stagnant water,
fresh and spoiling meat and meat products, meat· processing plants, and
milk. According to Gardner (1969), K. zopfii is not known to cause spoil·
age of refrigerated meat, but its presence indicates that the meat was
exposed to temperatures higher than refrigerator temperatures during
processing, distribution, or retailing. In meat held at 2°C, Kurthia is over·
grown by Pseudomonas species and other organisms. Although present in
food, their importance might be only as an indicator of mishandling.

Irregular, Nonsporing, Gram-Positive Rods

This group of bacteria includes the genera Corynebacterium, Arthro·
bacter, Brevibacterium, and Propionibacterium.

CORYNEBACTERIUM. This genus is divided into three sections: (1)

human and animal parasites and pathogens; (2) plant pathogens; and (3)
nonpathogenic.
The coryneform bacteria are characterized by their pleomorphism.
The Corynebacterium cells are straight to slightly curved rods but have a
tendency to form club and pointed shapes. Generally they are not motile
and are Gram positive. The best growth is aerobic, but they can grow in
anaerobic conditions.
These organisms are widely distributed in nature. They are found in
water, soil, plants, and animals. These organisms can be found in food
products derived from both animals and plants. They have been associ·
ated with spoiling food, but it is doubtful they are primary spoilage orga·
nisms.
 MICROORGANISMS ASSOCIATED WITH FOOD 69

ARTHROBACTER. Organisms in this genus show considerable pleo-

morphism_ The coccal form may appear as spheres, ovoid, or slightly
elongated_ When large cocci are transferred to a fresh medium, from
one to three (seldom four) germination tubes arise from the celIs- These
develop into rods which vary in size and shape_ Upon aging, the rods
change almost completely into cocci- The cocci are Gram positive and the
rods have Gram-positive granules surrounded by Gram-negative cellular
materials.
The organisms are found in soil, in and on meat and poultry prod-
ucts, in milk products, dairy waste, activated sludge, and brewery and fish
slime.
According to Buchanan and Gibbons (1974), the organism referred
to as Brevibacterium linens, which is important in cheese, is related to and
should perhaps be placed in the genus Arthrobacter.

BREVIBACTERIUM. Although this genus is listed in Bergey's Manual

(Buchanan and Gibbons 1974), no species are recognized. However, Su-
zuki and Komagata (1983) listed several species of Brevibacterium, includ-
ing B. linens. Brevibacterium linens is important in flavor production in
cheese, especially limburger cheese.

PROPIONIBACTERIUM. Generally these organisms are Gram-positive

rods, but they may be diphtheroid, club-shaped, coccoid, elongate, bifid,
or branched. They are anaerobic to aero tolerant.
Propionibacters are found on people and in the intestinal tract of
people and animals. During fermentation, propionbacters produce pro-
pionic acid and acetic acid, with lesser amounts of other organic acids.
The species of most interest in food microbiology is P. freudenreichii subsp.
shermani. This is used in the manufacture of Swiss cheese. Due to the
production of propionic acid and CO 2, the organism is responsible for
the characteristic flavor and the eyes in this cheese. These bacteria syn-
thesize large quantities of vitamin B12 and produce propionic acid and
can be used in the commercial production of these compounds.

Mycobacteria
Mycobacterium tuberculosis causes tuberculosis. So that this disease
could be controlled, pasteurization of milk was inaugurated. Today, tu-
berculosis is primarily an airborne disease, rather than foodborne. Myco-
bacterium strains are found in raw milk, oysters, pork, and vegetables
sprayed with sewage effluent (Hosty and McDurmont 1975; Thoen, Jar-
nagin, and Richards 1975; Van Donsel and Larkin 1977).
70 BASIC FOOD MICROBIOLOGY

Streptomycetes and Their Allies

Some Streptomyces may be useful. Streptomyces produce extracellular
ex·galactosidase (Lyons, Pridham, and Hesseltine 1969). According to
these workers, this enzyme may be useful during beet·sugar processing
(aids in crystallization of sucrose) and be used for determining raffinose
and fot eliminating the agent in beans that induces flatulence. Streptomyces
was suggested as a possible source of single·cell protein, protease en·
zymes, and glucose isomerase.

The Rickettsias and Chlamydias

In this grouping, the family Rickettsiaceae includes the genus Coxiella.

COXIELLA. These cells are short rods, occasionally appearing as diplo·

bacilli or spheres. There are no flagella or capsules. The cells are Gram
negative, but under certain conditions they may appear to be Gram posi·
tive. They are resistant to chemicals and elevated temperatures that nor·
mally inactivate Rickettsia species. These organisms will not grow on agar
media but require host cells.
Since these organisms do not grow outside a host cell, they are not
important in food spoilage. However, food can serve as a carrier of the
organisms so that they may infect humans. Coxiella burnetii is the causative
agent of Q fever. Infected cows, sheep, and goats shed the organism in
their milk, which is the food source for human infection if raw milk is
consumed. This organism is more heat resistant than the vegetative cells
of most pathogens. Therefore, the time and temperature for pasteuriza·
tion of milk are determined primarily to destroy these organisms. Heat·
ing at 62.SoC for 30 min or 71.7°C for 15 sec is adequate to eliminate C.
burnetii from milk.

MOLDS

Molds are part of a larger group of microorganisms called fungi. The

exact number of fungi is not known. Some fungi have not been isolated
and identified, while others have been given more than one name. The
fungi are ubiquitous, but soil, air, water, and decaying organic matter are
prime sources.
Fungi are heterotrophic organisms that lack the definite root, stem,
or leaves of higher plants. They possess a thallus and are called thallo·
phytes. They are differentiated from the algae and higher plants by their
lack of chlorophyll. Hence, they are saprophytic or parasitic. They differ
 MICROORGANISMS ASSOCIATED WITH FOOD 71

from bacteria by their more complex structure and greater size. The
fungi may be multicellular or unicellular.
The fundamental structural units of molds are filaments or tubes
called hyphae. By formation of crosswalls or septa, some hyphae form
chains of cells that are septate. Others may not form septa and the hy.
phae are nonseptate or coenocytic. The septa have pores that allow the
movement of cytoplasm from one cell to another. As the hyphae elon·
gate, they intertwine. A mass of these intertwined branched hyphae is
called a mycelium. Part of the mycelium grows into the substrate and
absorbs food. This is known as the vegetative mycelium. The mycelium
that remains in the air above the substrate and bears spores is called the
aerial or reproductive mycelium. When the spores find a proper sub·
strate, the cycle is repeated.
Molds can reproduce sexually, asexually, or by both systems. A fungus
that has a sexual phase is known as a perfect fungus, while one that has
no sexual phase is an imperfect fungus.
There are several types of spores formed by the fungi. The asexual
fungi produce spores directly from or by the mycelium. These are called
thallospores, conidiospores, or sporangiospores. There are three types
of thallospores: blastospores, chlamydospores, and arthrospores. Sexual
spores include oospores, zygospores, ascospores, and basidiospores.

Enumeration
With bacteria, one cell is usually responsible for the growth of a col·
ony. However, a mold colony may result from a single cell, a spore, a
piece of mycelium, or a number of cells. Thus there is a poor one·to·one
relationship for molds. The problems involved with estimating growth
by evaluation of the mycelium were discussed by Calam (1969) and Sut·
ton and Starzyk (1972).
In the past, media for enumerating molds and yeasts were acidified to
pH 4 or 5. The low pH inhibited bacteria and allowed the fungi to grow.
However, fungi that have experienced a sublethal treatment might not
grow on an acidified medium. Hence, media incorporating antibiotics
rather than acidification to inhibit bacteria have been developed and
tested for fungal enumeration (Baggerman 1981; Beuchat 1979; Henson
et al. 1982; Mossel, Vega, and Put 1975; Nelson 1972). Dichloran has been
added to media to inhibit the spreading of mold colonies (Henson 1981).
The addition of sodium thiosulfate and sodium tetrathionate to media
reduced the effect of heavy metal toxicity on the growth of fungi (Wain·
wright and Grayston 1983). Higher fungal counts were obtained using
the spiral plater than the pour plate method (Zipkes, Gilchrist, and
Peeler 1981). Microscopic methods are used to enumerate mold filaments
72 BASIC FOOD MICROBIOLOGY

in tomato products as well as in other canned fruits and vegetables

(AOAC 1985; Bandler and Cichowicz 1981). Determining the fungal
chitin by conversion to glucosamine is useful in estimating fungal my·
celia in tomato products (Bishop et al. 1982). These and other methods
used for fungal analysis were reviewed by Jarvis et al. (1983).

Fungi in Food
Several types of fungi have been isolated from food. The extent of
contamination is influenced by the prevalence in the environment. Peni·
cillium and Aspergillus enjoy favorable conditions throughout the year,
while some other fungi are limited to warm temperatures. Cladosporium
and Alternaria are prevalent during the summer and early autumn.
Although important in all types of foods, molds are more apt to cause
spoilage or be a health hazard in foods such as grain, flour, nuts, or fruit,
which do not support the growth of bacteria due to low water activity or
low pH.
Throughout the world, fungi rank second only to insects in causing
the loss of stored products (Christensen and Kaufman 1974). In countries
that have implemented rodent and insect controls, fungi destroy more
stored products than does any other agent.

Importance of Molds
We usually consider that the appearance of mold on a food is an
indication of spoilage. However, even before growth is evident to the
naked eye, these organisms can be working to cause degradation of
foods.
Although molds associated with food are not considered to be patho·
genic, some produce mycotoxins. These toxic substances pose a potential
health hazard to humans.
Molds can be useful in the processing of many foods such as cheese.
Their enzyme systems can be isolated and used in many food processes.
Some fungi are used to convert wastes into usable food (protein) for ani·
mal food or human use. Also, they are a source of vitamins.
The production of antibiotics by molds is well known. Antibiotics
have been of great value in medicine, and they have been investigated
for use as food preservatives.
Obviously many inert molds are associated with foods. They simply
cannot grow due to an unsatisfactory environment or the overgrowth by
bacteria that cause spoilage before the molds get a chance to multiply.
 MICROORGANISMS ASSOCIATED WITH FOOD 73

Zygomycetes
These fungi produce asexual spores (sporangiospores, arthrospores,
and conidiospores) or sexual spores (zygospores). The hyphae contain
no septa, but older hyphae may have septa.
The organisms of interest in food microbiology are in the order Mu·
corales. The family Mucoraceae include the genera Mucor and Rhizopus.
The family Thamnidiaceae includes the genus Thamnidium.

MUCOR. Like other dimorphic fungi, species of Mucor can develop as

either individual spherical cells that multiply by budding (as yeasts), or
they form typical mycelia of coenocytic hyphae (Fig. 3.6). In normal aero·
bic conditions, growth is usually filamentous. Stoloniferous growth does
not occur in mucor, which differentiates this genus from Rhizopus.
Organisms in this genus occur in soil and manure, and in fruits, vege·
tables, stored grain, and other foods. Mucor species are used in the Orient
in food fermentations. M. pusillus produces an extracellular protease that
has milk clotting activity. Mucor is a spoilage organism of berries.

Figure 3.6. Hyphae and sporangia of Mucor.

Courtesy of Continental Can Co.
74 BASIC FOOD MICROBIOLOGY

RHIZOPUS. These organisms have coenocytic mycelia and spread by

stolons. Usually they reproduce asexually with sporangiospores or ar-
throspores_
These are common spoilage organisms of various stored foods. Rhizo-
pus stolonifer is the common bread mold. Pectinolytic enzymes are se-
creted by Rhizopus species. The degradation of pectin results in the soft
rot of various plant products. Due to the heat stability of the pectinolytic
enzyme, the infection of fruit before heat processing can result in post-
process softening.
Rhizopus produces high yields of fumaric acid from fermentable
sugars. Species are used in the production of fermented foods such as
tempeh.

THAMNIDIUM. Organisms in this genus have coenocytic mycelia.

They are found on meat as well as on soil and animal excrement. They
grow on refrigerated meat, causing a defect referred to as whiskers. A
process using T. elegans to tenderize meat was patented by Williams
(1957). However, Kotula, Campano, and Kinsman (1982) found that the
lipolytic and proteolytic enzymes of this organism had little effect on
meat at 4°C, the typical temperature at which beef is held in coolers.

Ascomycetes
Both molds and yeasts are present in this class of fungi. Ascomycetes
develop sexual ascospores in a saclike structure called an ascus. Besides
this sexual phase, they may grow an extension of the hyphal tip or have
an imperfect state in which asexual spores are produced. Those fungi for
which no perfect state has been observed are listed in Deuteromycetes
(Fungi Imperfecti). When a sexual phase is found for a fungus in Fungi
Imperfecti, it is given a name according to the structure and form of the
perfect state. This name takes precedence over the imperfect, asexual
state.
There are between 1,900 and 2,000 genera in Ascomycetes. Only a
few are of importance in foods.

BYSSOCHLAMYS. Besides asexual reproduction, the two species of

importance (B. fulva and B. nivea) sexually produce heat-resistant as co-
spores. These organisms can grow at relatively low pH levels and in re-
duced oxygen tension. They produce strong pectolytic enzymes. With
this combination of properties, they have been implicated in the spoilage
of canned fruit and fruit juice. Spoilage mayor may not be accompanied
by gas. If gas is formed, the can may bulge slightly.
Besides spoilage, B. fulva produces a mycotoxin that is toxic to brine
 MICROORGANISMS ASSOCIATED WITH FOOD 75

shrimp, chicken embryos, and rats (Kramer, Davis, and Diener 1976).
Chu, Nei, and Leung (1973) studied a renninlike enzyme (byssochlamyo·
peptidase A) that might be useful in milk clotting for cheese manufac·
ture.

CLAVICEPS. The species Claviceps purpurea is of interest to food micro·

biologists because it produces toxic alkaloids on cereals. These contain a
tetracyclic ring called lysergic acid. When ingested, these alkaloids cause
numerous symptoms, especially hallucinations. With improved grain
handling, the illness is rare in humans. The last major outbreak was in
1951.

NEUROSPORA. The name Neurospora is derived from the characteristi·

cally ribbed ascospores. The ascospores of N. crassa remain viable for
many years. Heat shock for 20 min at 60°C aids in the germination of
the spores.
In asexual reproduction, branched chains of pink conidia develop
from upright stalks. Budding of the terminal conidia of the chain pro·
duces further conidia. When the terminal conidia form two buds, the
chain branches.
Neurospora crassa and N. sitophila are two well·known species of this
genus. Wild type strains of Neurospora have simple nutrient requirements.
They have been used as tools in genetics and biochemical research. Neuro·
spora can be found in warm, humid environments. N. sitophila causes
problems in bakeries and is the red or pink bread mold. N. sitophila is
used in the fermentation of red or orange ontjom, a fermented peanut
press cake used in the Orient.

Deuteromycetes
This heterogeneous group of fungi has branching, septate hyphae
and reproduces asexually by conidia or sclerotia. There are various sys·
terns used to classify the Fungi Imperfecti. Rather than attempt to segre·
gate them, those of importance in food microbiology are discussed in
alphabetical order.

ALTERNARIA. These organisms are characterized by muriform, dark·

colored spores. The aerial mycelia are described as wooly, gray, brown,
and olive·green (Fig. 3.7).
Alternaria is one of the most prevalent of the molds that cause spoilage
of tomatoes in the field, attacking injured or weakened tissue. Due to
darkening of the tissues, the defect is called black rot. Late-harvested to·
matoes are particularly susceptible. These organisms cause defects ofvar-
76 BASIC FOOD MICROBIOLOGY

Figure 3.7. Hyphae and spores of Alternaria, a cause of rot of tomatoes.

Courtesy of Continental Can Co.

ious plant products, including internal mold of peppers (Halfon·Meiri

and Rylski 1983). Also they are involved in the development of rancid
flavors in dairy products.
A. alternata produces the enzyme (3'D-galactosidase which, according
to Macris (1982), is useful in reducing the lactose level in dairy products.
The Alternaria may be a health hazard because it produces mutagenic
substances (Scott and Stoltz 1980).

ASPERGILLUS. There are more than 100 species in this genus. These
organisms, along with penicillia, are known as storage fungi of grain.
They discolor infected grain and reduce or destroy germination of the
seed. These organisms can cause spoilage of a wide range of food prod-
ucts.
Certain aspergilli are very useful in food microbiology. Species such
as A. oryzae are used to break down rice starch to glucose in the manufac-
ture of sake and similar alcoholic beverages. Some strains are used in the
production of shoyu (soy sauce) and miso (bean paste).
 MICROORGANISMS ASSOCIATED WITH FOOD 77

Strains of Aspergillus are used in the commercial production of citric,

gluconic, and gallic acids. Almost all of the commercial citric acid is pro·
duced by A. niger growing in sucrose solutions. The organisms are a
source of amylase and pectinolytic enzymes. A proteolytic enzyme of
Aspergillus is able to clot milk and might be a substitute for rennet in
cheesemaking.
These fungi may be useful as a source of protein. The mycelium of
A. niger grown on brewery wastes contained 29 percent crude protein
and might be used as a feed supplement (Hang, Splittstoesser, and Wood-
ams 1975). Reade and Gregory (1975) described the use of A. fumigatus to
convert the starchy root cassava to microbial protein for food or feed.
Several species of Aspergillus produce substances that are toxic to
other biological systems Strains of A. jlavus and A. parasiticus produce
aflatoxins. There is considerable information that implicates aflatoxins
with human illness.
The aspergilli are common contaminants of organic materials and
soils. They are found on fruits, vegetables, stored grain, peanuts, and
other food products. Aspergillus is shown in Figure 3.8.

BOTRYTIS. The asexual form of Botrytis reproduces by conidia. The

conidiophores develop from a sclerotium and are irregularly branched.
The conidia occur on short sterigmata. The positions and numbers of
sterigmata cause the conidia to appear in grapelike clusters (Fig. 3.9).
B. cinerea is the common species of Botrytis. It is the gray mold of var-
ious plants and plant products, especially lettuce, tomato, strawberry,
raspberry, and grape. It can be classed as a field mold, since it is a com-
mon soil contaminant and attacks fruits and vegetables in the field. It
enters fruits through cracks and injured areas.

CLADOSPORIUM. These organisms are common in the soil. They can

grow on connective tissue or the fat covering of meat when it is refriger·
ated for several days. This results in black spots on the meat. C. carpophi-
tum causes peach scab, numerous dark circular lesions on the fruit. The
organisms are associated with stored grains and dairy products.

COLLETOTRICHUM. Molds in this genus are involved with spoilage

of foods. C. circinans causes onion smudge. Colored onions are resistant.
C. phomoides causes anthracnose rot of tomatoes. Invasion by this or-
ganism is not dependent upon an injury to the fruit.

FUSARIUM. The conidia produced by these organisms have various

shapes. The colonies may be fluffy and spreading, with colors varying
78 BASIC FOOD MICROBIOLOGY

Figure 3.8. Aspergillus, showing mycelia and conidial heads.

Courtesy of Continental Can Co.

from white, through shades of pink, red, brown, yellow, orange, and blue,
to purple.
The fusaria are widespread in nature, being found in soil, decaying
material, and foods. Some fusaria are associated with plant diseases. A
disease in rice due to F. moniliforme led to the discovery of gibberellic acid,
a plant growth stimulant.
Fusaria cause tomato rot, entering the fruit through insect or other
damage of the skin. F. solani causes a decay of potatoes called powdery
rot, dry rot, or white rot.
The fusaria produce mycotoxins that affect various animals and possi-
bly human beings. Animals refuse to eat feed that is highly infected with
fusaria.

GEOTRICHUM. This yeastlike fungus grows rapidly at room temper-

ature, forming white to cream-colored colonies. The septate, branching
 MICROORGANISMS ASSOCIATED WITH FOOD 79

Figure 3.9. Mycelia and spores of Botrytis.

Courtesy of Continental Can Co.

hyphae fragment into chains of rectangular, barrel·shaped, or spherical

arthrospores that readily break apart. These arthrospores are a means of
reproduction. Geotrichum is depicted in Figure 3.10.
Geotrichum is a spoilage organism. It has been called dairy mold, be-
cause it is found growing on dairy products. It also causes watery rot, a
common spoilage of tomatoes.
G. candidum is called machinery mold because it will grow on
equipment on which are attached food particles or juices . .As the food
product is processed, it becomes contaminated with the mold. Hence, the
mold is found in many types of processed foods. The mold is killed by
heat used in thermal processing of food, but the hyphae can be deter-
80 BASIC FOOD MICROBIOLOGY

Figure 3.10. Geotrichum-machinery mold.

courtesy of Continental Can Co.

mined by microscopic examination. The presence of these mold fila·

ments in processed foods has been considered to be an adulterant and
an indication of inadequate sanitation in the processing plant. However,
Splittstoesser et al. (1980) found little correlation between the aerobic
plate count and the incidence of the mold in frozen blanched vegetables.

PENICILLIUM. There are many species of Penicillium. They are closely

related to the aspergilli. The penicillia are characterized by branching
of the conidiophore to form a brushlike conidial head (Fig 3.11). The
organisms in this genus can be divided into three groups on the basis of
the type of conidiophore branching. The conidia may be white, green,
gray·green, blue·green, or yellow·green.
These organisms are widely distributed in nature and are found on
many foods. They are important spoilage organisms of various fruits.
This is one of the storage fungi of grain. The species most often en·
countered are P. cyclopium and P. viridicatum. This is probably because they
are able to grow at relatively low levels of moisture and temperature.
 MICROORGANISMS ASSOCIATED WITH FOOD 81

Figure 3.11. Mycelia and conidial heads of Penicillium.

Courtesy of Continental Can Co.

P. martensii and P. viridicatum are associated with blue-eyes, a blue-green

discoloration of corn germs. Species of Penicillium are found growing on
the fatty layer or connective tissue of meat that is stored in a refrigerator
for several days, and on moldy bread.
Species of Penicillium are useful in various ways. Antibiotics produced
by penicillia include penicillin_ Some species are used in cheese manu-
facture. P. camemberti and P. caseicolum are important in Camembert, Brie,
and similar cheeses, while P. roqueforti is used in Roquefort, Gorgonzola,
and blue-veined cheeses. Schwimmer and Kurtzman (1972) suggested us-
ing P. crustosum to remove caffein from coffee. The penicillia produce
enzymes such as glucose oxidase as well as proteins that can be used by
the food industry.
Certain species of penicillia can be a health hazard_ Some species
have been associated with pulmonary and urinary tract infections. Peni-
cillia produce mycotoxins_ P. islandicum, P. citrinum, and P. citreoviride were
involved in yellow rice disease which caused the deaths of several people.
Although P. roqueforti is used in cheese processing, toxin-producing
strains of these species have been isolated (Scott et al. 1977), and roque-
82 BASIC FOOD MICROBIOLOGY

fortine, a neurotoxin, has been isolated from blue cheese (Scott and
Kennedy 1976).

SCOPULARIOPSIS. This genus produces conidia in brushlike clusters

similar to Penicillium. The colonies appear cottony and may be cream·
colored, yellow, brownish, chocolate brown, or almost black. They are
never green like the Penicillium. One characteristic of S. brevicaulis is that
it produces a poisonous gas (diethylarsine) with a garliclike odor when
grown in the presence of arsenical compounds.
Scopulariopsis is found on all types of decaying matter and grows well
on high·protein substrates.
Being proteolytic, Scopulariopsis is involved with the development of
off·flavors in dairy products (especially Camembert cheese), and spoilage
of meat. It has been detected on ham (both on the surface and in deeper
tissues), stored eggs, and peanuts.

SPOROTRICHUM. The colonies are usually white but may be yellow,

gray, pink, red, or green. It is a common soil inhabitant. S. carnis grows
at low temperatures (- 5° to - SoC) and can cause a defect, called white
spot, of refrigerated meat.
S. thermophile has an optimum growth temperature near 40°C. It pro·
duces cellulase and has been suggested for use in converting cellulose to
simple carbohydrates (Coutts and Smith 1976). The use of S. pulverulen-
tum as a potential food source was studied by Eriksson and Larsson
(1975).

TRICHODERMA. This genus has irregularly branched conidiophores.

The conidia are produced in slime balls.
Species are common in soil and on organic matter. The nitrogen con-
tent of the cells is about 5 percent, of which 60 to 70 percent is protein.
Since the species T. viride is cellulolytic and has a high protein content,
it has been considered for converting cellulosic wastes into foods.

YEASTS
The yeasts have been defined as fungi in which the usual dominant,
or conspicuous, form is unicellular. The unicellular form gives yeasts an
advantage over the mycelial form of molds. There is a greater surface-to-
volume ratio that allows a higher metabolic activity. Also, the unicellular
form is more readily distributed than is the mycelial form. Some yeasts
produce a true mycelium by fission of cells that remain attached. A
 MICROORGANISMS ASSOCIATED WITH FOOD 83

pseudomycelium, formed by budding, has constrictions between the

cells.
There are some 350 species recognized and classified into thirty·nine
genera (Kreger·van Rij 1969; Lodder 1970). The classification of yeasts is
based on morphological, cultural, sexual, and physiological characteris·
tics.
On the basis of method of reproduction, the yeasts can be separated
into four groups. Only two of these groups contain yeasts involved with
foods. One group produces sexual ascospores in asci and is in the class
Ascomycetes. These are the so· called true yeasts. The yeasts in the other
group form no sexual spores and have no sexual life cycle. These are the
false yeasts, Fungi Imperfecti, or Deuteromyces. All yeasts can reproduce
asexually, and this is the only method for about 50 percent of them. The
usual asexual (vegetative) reproduction is by budding (Fig. 3.12). Some
yeasts reproduce by fission or by an intermediate system called bud fis·
sion. If the new cell or bud appears at the short end of the mother cell,
it is called polar budding. If a bud forms at both ends, it is bipolar bud·
ding. The appearance of buds any place on the mother cell is multilateral
budding.
Nearly all of the yeasts that produce sexual ascospores and are associ·
ated with food are in the class Ascomycetes, order Endomycetales, and
family Saccharomycetaceae. For most yeasts, the maximum number of
spores per ascus is four, but a few yeasts can produce eight. Strains of
Kluyveromyces may produce a large number of spores (up to 100) per as·
cus. These ascospores have many shapes, including spheroidal, ovoid,

Figure 3.12. Cells of Saccharomyces cerevisiae, showing budding.

Courtesy of Amerine, Berg, and Cruess (1979).
84 BASIC FOOD MICROBIOLOGY

reniform, elliptical, bean-shaped, cylindrical, and needle-shaped. Some

spherical or oval spores have a ledge in the middle or to one side, which
makes them appear saturn-shaped or helmet-shaped.
The production of ascospores may playa role in survival or in adapta-
tion to altered environmental conditions. However, the main purpose is
the rearrangement of heritable qualities.
The vegetative cells may have shapes similar to those of ascospores.
Young yeast cells may be small and spherical and, as they grow older and
larger, they may assume other shapes. The older cells become misshapen
and scarred due to budding. Aged cells tend to become shriveled.
Some molds are dimorphic and have a yeast-like phase. Some yeasts
produce a pseudomycelium or true mycelium. Hence, these organisms
may be confused when a culture is examined, especially for borderline
genera. The mold Geotrichum resembles the yeast Trichosporon. They both
form a true mycelium and arthrospores. However, Trichosporon can repro-
duce by budding.
Most yeast cultures are cream, tan, or gray. However, some are yellow,
pink, red, green, or brown.
The physiology of yeasts was discussed by Kreger-van Rij (1969) and
MacMillan and Phaff (1973). Information about fermentation, assimila-
tion, and nutritive requirements aids in identification of yeast species.
This information was compiled by Lodder (1970) and is too extensive to
repeat. The heat resistance of various yeasts has also been documented
(Put et al. 1976; Put and De Jong 1982)_
Yeasts are associated with nearly all types of food products. Foods
such as fresh vegetables, meat, poultry, and cheese often contain yeasts,
but in these foods, bacteria usually outgrow the yeasts. When bacterial
inhibitors are added, yeasts can dominate. Osmophilic yeasts such as Sac-
charomyces rouxii can tolerate high sugar concentrations. These yeasts are
found in foods such as honey, molasses, sugar, and fruit. Salt-tolerant
yeasts grow as films on brined food and on salted food and ham.
Certain strains of yeasts are very important in the baking industry, in
chemicals, in single-cell protein, in sewage disposal, and in the fermenta-
tion of alcoholic beverages.

Methodology
Sublethally stressed yeast is recovered at maximum levels with a me-
dium at pH 8.0 or over (Nelson 1972). Therefore, the use of potato dex-
trose agar (PDA) acidified to pH 3.5 is not satisfactory for enumeration
of yeasts in many food products. To inhibit bacteria and to allow yeasts
to grow, a combination of antibiotics is used in place of acidification.
 MICROORGANISMS ASSOCIATED WITH FOOD 85

Fluorescent microscopy can be used to detect yeasts in certain products

(Andrews 1982; Aries 1976; Cranston and Carver 1974).

Classification
In this text, the genera and species of yeasts are those listed in Lodder
(1970). As more information accumulates regarding the relationships of
yeast such as DNA base composition and serological reactions, there may
be some revisions in the classification (Price, Fuson, and Phaff 1978).
The yeasts are divided into Ascomycetes (the ascosporogenous yeasts)
and Deuteromycetes (the asporogenous yeasts).

ASCOMYCETES. These yeasts produce sexual ascospores as well as reo

produce asexually. Unless otherwise noted, asexual reproduction is by
budding. Certain genera such as Citeromyces, Dekkera, and Endomycopsis
have been isolated from food and may be involved with spoilage or fer·
mentation. Due to space limitations, these are not discussed in this text.
Debaryomyces. The vegetative cells are often spherical to globose and reo
produce by multilateral budding. The sexual ascospores, produced by
conjugation of the mother cell and bud, are spherical or oval. There are
usually one or two spores per ascus. Fermentation is weak, slow or absent.
Nitrate is not assimilated.
D. hansenii has a high salt tolerance, being able to grow in media with
18 to 20 percent salt. It has been isolated from brined food and salted
meat products and can use nitrate as the sole source of nitrogen. Debaryo-
myces species are perhaps the most widely distributed film-forming yeasts
associated with food brines.
Species of Debaryomyces have been associated with spoiled mushrooms,
cheeses, cider, white wine, tomato puree, sausage, and salted beans.
Hanseniaspora. These yeast cells are diploid, lemon-shaped, ovoid, or long
ovoid. They are the ascospore stage of the genus Kloeckera. They have a
vigorous fermentation, but have a low tolerance to alcohol (about 4 to 6
percent). All species require inositol and pantothenate for growth. Due
to this need, they can be used to assay for these compounds.
These yeasts are found in the soil of orchards and vineyards and in
all insects. They have been isolated from grape berries and grapes and
have been associated with fermenting spoiled fruit.

Hansenula. This genus reproduces by multilateral budding and asco-

spores. A pseudomycelium or a true mycelium may be formed. These
yeasts assimilate nitrate.
86 BASIC FOOD MICROBIOLOGY

Species of Hansenula have been isolated from foods such as grain,

fruit, shrimp, and brines of fermenting cucumbers and olives.
Some species and strains are useful. According to Batra and Millner
(1974), certain species are used in India to produce kanji (a beerlike bev·
erage) and murcha (rice wine). A strain of Hansenula grows on methanol
to produce single·cell protein (Levine and Cooney 1973).

Kluyveromyces. These organisms reproduce by multilateral budding. The

cells may be spherical, ellipsoidal, cylindrical, or elongate. Some species
produce a red pigment, but the colonies are grayish·white, brownish·
cream, yellowish· gray, sometimes tinged with pink. The organisms show
a vigorous fermentation. They grow at temperatures of from 5° to 46°C.
This genus has a widespread distribution. They are found in various
foods such as fruits, preserves, corn meal, grape must, milk, and other
dairy products. The species that ferment lactose, such as K. lac tis, are
found in dairy products. Some species are osmophilic. Organisms of this
genus cause spoilage of foods, especially figs and dairy products. Certain
strains have been suggested as a source of the enzyme polygalacturonase.

Pichia. The cells have many shapes. Asexual reproduction is by multilat·

eral budding, with most species forming a pseudomycelium. There is lim·
ited formation of a true mycelium.
The colonies are slimy or pasty and colored yellowish·white, yellowish·
brown, tan, white, cream, grayish·white, or red.
These are common yeasts found in various sources. Their association
with foods includes both fermentations and spoilage. Foods in which
these yeasts are found and can cause spoilage include fruit, beer, dairy
products, sorghum, and wine. They can occur as films on brined foods.

Saccharomyces. This genus is considered to be a rather heterogeneous

group of organisms. The cells are spheroidal, ellipsoidal, cylindrical, or
elongate. Vegetative reproduction is by multilateral budding. Pseudo·
mycelia may be formed, but true mycelia are absent. On agar, the colon·
ies are generally white or cream colored, with a typical yeasty odor. Co·
wan and Bryant (1981a, 1981b) developed a serological classification of
brewing yeasts using a microimmunoelectrophoresis technique.
The name Saccharomyces means sugar fungus. All species have a vigor·
ous fermentation. Some species are among the most useful yeasts, due to
the production of alcohol (brewing) and carbon dioxide (baking). Strains
of S. uvarum and S. cerevisiae are most often used in these fermentations.
Besides these important uses, species and strains have been used to reo
move glucose from egg white prior to drying, to remove the mucilage
layer from coffee beans during processing, to assay for vitamins (biotin,
pyridoxine, pantothenic acid, thiamin), as a source of enzyme (invertase),
 MICROORGANISMS ASSOCIATED WITH FOOD 87

as autolyzed yeast extract for flavoring in place of meat extracts, as a

source of ergosterol, and as a source of single·cell protein.
Sinai et aL (1974) fed brewer's yeast (S. cerevisiae) to Rhesus monkeys
and found that the yeast significantly enhanced the monkeys' resistance
to respiratory and enteric infections. They believed the resistance was
due to the stimulation of phagocytes.
The organisms in this genus have a widespread distribution. They are
associated with a variety of foods and can cause spoilage in fruit and fruit
products, sugar, syrup, honey, vinegar, mayonnaise, salad dressing, dairy
products (buttermilk, cheese), and fermenting foods such as cucumbers
for pickles.
S. aceti, found in wine, can convert alcohol to acetic acid. S. bailii var
osmophilus is found in substances with a high concentration of sugar, alco·
hoI, sulfur dioxide or acetic acid. S. bailii var bailii has been associated
with spoiled wine, vinegar, salad dressing, and mayonnaise. Two sugar·
tolerant (osmophilic) species, S. rouxii and S. bisporus, are the main spoil·
age organisms of dates and can cause spoilage of honey, syrup, and other
foods with up to 60 percent sugar (Restaino et aL 1983).
Occasionally, species of Saccharomyces exhibit pathogenic behavior
(Eschete and West 1980).

Schizosaccharomyces. The cells are spheroidal to cylindrical. Vegetative reo

production is by fission. The absence of budding distinguishes this genus
from other yeasts. The cells may form a rudimentary true mycelium that
can break up into arthrospores. There are four to eight spores per ascus.
The name Schizosaccharomyces indicates the close relationship to Sac·
charomyces (fermentation of glucose to alcohol), but also a difference (fis·
sion as compared to budding).
As with other yeasts, these species are widespread. They have been
associated with spoilage of prunes, figs, raisins, and wine.
Yang (1973) suggested using S. pombe in the fermentation to produce
wine from grapes with a low pH. The yeast converts malic acid to lactic
acid, resulting in a less acid wine than when Saccharomyces cerevisiae is
used. S. malidevorans is noted for its ability to convert malic acid to lactic
acid.

DEUTEROMYCETES. These yeasts do not produce sexual spores. Un·

less otherwise stated, these asporogenous yeasts reproduce by budding.
Certain genera, such as Brettanomyces and Kloeckera, are found in foods
and may be involved in spoilage or fermentation.
Candida. This is a rather large genus of yeasts. All species form pseudomy·
celia; by fission, some form true mycelia.
Organisms in this genus are widespread. Besides being found in soil,
88 BASIC FOOD MICROBIOLOGY

water, and air, they are found in plants, insects, higher animals, humans,
sewage, on processing equipment, and in food products.
These organisms have been involved with spoilage of various foods
such as frankfurters, fresh fruits, vegetables, dairy products, brines, and
alcoholic beverages. C. vini (formerly Mycoderma vini) forms "flowers" on
wine and causes spoilage. C. mycoderma is a film· forming yeast on ferment·
ing olive and cucumber brines. The psychrophilic strains tend to become
dominant in refrigerated fruit juices.
This genus contains strains of yeast that are very useful in food indus·
tries and in associated industries. They are used as food yeasts, a source
of lipid, vitamins, invertase, lactose, and lysine. C. utilis is used as a fodder
yeast for animals and is a potential food yeast for humans.
There are potentially pathogenic species of Candida for both people
and animals. However, there is no information that food is the source of
these infective agents. Candida species are found in the intestinal tract,
and it has been suggested that they may cause diarrhea in malnutrition
(Gracey et al. 1974).
Rhodotorula. The cells (spheroidal, ovoidal, elongate) reproduce by multi·
lateral budding. There may be a rudimentary pseudomycelium. The orga·
nisms form red or yellow carotenoid pigments. These organisms are
widespread and are involved in the spoilage of a wide range of foods.
Species of this genus may be useful as a source of lipids, cystine, and
methionine and for degrading wastes in the production of single·cell pro-
teins.
Torulopsis_ Torulopsis has been defined as a heterogeneous group of imper-
fect yeasts that cannot be placed in the homogeneous genera. The cells
are spherical or oval to elongate and reproduce by multipolar budding.
Torulopsis can be differentiated from Candida by not being able to form
a pseudomycelium, from Cryptococcus by not producing starch, and from
Rhodotorula by not producing carotenoid pigments.
The colonies are usually white to cream and are usually glossy or
shiny but may be dull. The tolerance to sodium chloride varies from 2
percent to 21 percent (W/v) for different species_ At 30°C, T. halonitrato-
philia is an obligate halophile. There are osmophilic species in this genus.
Species of Torulopsis are found in various types of foods. They are
credited with causing surface slime on cottage cheese, spoilage of refrig-
erated beef, cream, butter, sweetened condensed milk, and various food
brines_
Torulopsis magnoliae has been suggested for use in the production of
glycerol. T. ernobii is a source of lipase. T. utilis is a source of single-cell
protein. Torulopsis strains have been found in sourdough, apparently aid-
ing in the leavening of this product.
 MICROORGANISMS ASSOCIATED WITH FOOD 89

Trichosporon. The cells of Trichosporon species are of various shapes.

Pseudomycelia and true mycelia may be formed. The presence of ar·
throspores differentiates this genus from Candida.
These organisms are found on various foods such as fresh shrimp,
crab, butter, cheese, fruit, fruit juice, and rice. They are part of the flora
used in the production of idli (Batra and Millner 1974), and may be a
source of protein.

VIRUSES

Viruses are obligate intracellular parasites. They generally have a lim·

ited host range. However, nearly all forms of life are susceptible to some
type of virus. To survive and replicate, a virus must contact and invade an
acceptable host cell. If a host cell is not available, the virus may become
inactivated. This inactivation is influenced by temperature, humidity,
pH, and substrate composition as well as other factors.
There is some doubt that viruses are alive. Cliver (1971) stated that
they are not alive, but borrow life from the host cell and direct it to
produce more viruses.
Viruses have a genetic system with either RNA or DNA. This is sur·
rounded by a protein coat, called the capsid. The capsid protects the
genetic material from nucleases, serves as the vehicle of transmission
from one host cell to another, and forms a bond with the host cell during
the attachment stage of infection.
For a virus to infect a host cell, there must be an attachment site on
the host cell. The attached virus injects its nuclear material into the host.
This can result in the viral genetic material causing the cell to manufac·
ture new viruses, or the genetic material may attach to the cellular chro·
mosome and the cell becomes lysogenic. If new viral particles are pro·
duced, usually the cell is lysed upon release of the new viruses. Then
these viruses must contact another acceptable host.
If the virus material becomes attached to the chromosome of the cell,
the genetic material of the virus is reproduced along with that of the
cell (Campbell 1976). This lysogenic state may continue for an indefinite
period. Eventually some disturbance triggers the viral genetic material to
become activated, and the cell produces new viruses, which are subse·
quently released.
Although the viruses are inactive as far as the reaction on food is
concerned, they are important. Since they are obligate parasites, and
some cause disease in human beings, the presence of viruses can be con·
sidered potentially harmful. They can be put into the category of an envi·
ronmental condition that allows spoilage, since they, as bacteriophages,
90 BASIC FOOD MICROBIOLOGY

can attack useful fermentative organisms, resulting in an unsatisfactory

product being developed. Since they are involved with genetic transfer
(transduction), the viruses might be considered helpful in the develop·
ment of better strains of useful microorganisms. However, transduction
may be undesirable when it results in unacceptable mutant strains that
are less sensitive to inhibitors, are more virulent, or lose the characteris·
tics that made them useful. Transduction is known to occur in only a few
groups of bacteria.

Intestinal Viruses
The primary viruses that can infect people and are foodborne are
the intestinal or enteric viruses. Cliver (1971) included the enteroviruses,
adenoviruses, reoviruses, and the agents that cause infectious hepatitis
and epidemic viral diarrheas as intestinal viruses.
The enteroviruses are spherical and about 20 to 30 nm. They have
single·stranded RNA. These viruses are stable at pH 3.0. This tolerance
to low pH allows them to survive passage through the stomach. They
replicate in the intestinal tract, are shed in the feces and, if they contami·
nate food that is subsequently ingested, the process is repeated. Wilner
(1973) included poliovirus, coxsackievirus A, coxsackievirus B, and echo·
virus as enteroviruses.
The adenoviruses are hexagonal, about 70 to 90 nm in diameter. They
contain double·stranded DNA. They are acid stable. There are thirty·
three serotypes of adenoviruses that infect human beings (Wilner 1973).
The reoviruses are hexagonal, and about 70 to 80 nm in diameter.
They contain double· stranded RNA. The reoviruses, like the other intesti·
nal viruses, are stable at pH 3 to 5.
The agents that cause infectious hepatitis have been described (Prov·
ost et al. 1975). They are spherical, about 27 nm in diameter. Viral diar·
rheal agents have been isolated or characterized. However, these agents
pass through bacterial filters and can be transmitted through several
people and still cause diarrhea. If the agent were merely a chemical toxin,
this transmission would dilute it beyond the ability to cause illness.
Since these viruses are intestinal, they are found in feces of infected
persons and resultant sewage. Present sewage treatment does not inacti·
vate all of the viruses from the effluent or the sludge (Goddard, Bates,
and Butler 1982; Irving and Smith 1981; Larkin, Tierney, and Sullivan
1976). When sewage wastes are used to irrigate or fertilize crops, the vege·
tation is contaminated. Larkin, Tierney, and Sullivan (1976) found that
poliovirus persisted on radishes and lettuce for up to thirty·six days. Such
foods are eaten raw, so any contaminating viruses that are not removed
by washing are ingested. When sewage contaminated with viruses is
 MICROORGANISMS ASSOCIATED WITH FOOD 91

dumped into coastal water, the shellfish growing in these waters tend to
bioaccumulate these viruses (Metcalf et al. 1980). Shellfish have been the
source of enteric viruses in several outbreaks of gastroenteritis (Eyles,
Davey, and Huntley 1981).
The procedure for the detection of viruses in or on foods consists of
several parts. First, the viruses are separated from a food suspension by
differential filtration or centrifugation. Then, the viruses are concen·
trated by centrifugation or by filtration. Bacterial cells are destroyed in
the viral concentrate by adding antibiotics. Aliquots of the treated viral
suspension are planted on a monolayer of susceptible host cells. After
incubation, the monolayer of cells is observed for plaques (holes) pro·
duced by lysis of cells due to the viruses. The number of plaque· forming
units (PFU) is determined by counting the plaques and multiplying by
the calculated dilution factor. Several modified and improved methods
for concentrating viruses from water have been suggested (Guttman·Bass
and Armon 1983; Keswick et al. 1984; Lipson and Stotzky 1983; Shields,
Berenfeld, and Farrah 1985; Wait and Sobsey 1983). Absorption, concen·
tration, and detection of viruses in fecal samples were described by Pon·
tefract and Bergeron (1985). The methods used to concentrate and detect
viruses were reviewed by Cliver, Ellender, and Sobsey (1983a, 1983b) and
by Richman et al. (1984). Enzyme·linked immunosorbent assays have
been described for the detection of viruses (DeBokx and Maat 1979; Lo·
camini et al. 1979; Marco and Cohen 1979). Fortunately, the viral contam·
ination of food is very low to nonexistent (Kostenbader and Cliver 1977;
Larkin 1981).

Bacterial Viruses (Bacteriophages)

The taxonomy of bacteriophages was discussed by Ackermann and
Eisenstark (1974). The phages can have at least four relationships with
their host cells. These are as follows: (1) Virulent phages cannot combine
with the host's chromosomes to establish a lysogenic relationship; (2)
pseudolysogenic phages can establish a carrier state in the host and may
act as transducing agents; (3) defective temperature phages form parti·
cles with morphological attributes of a normal phage, but cannot repli·
cate; and (4) true temperate phages are able to replicate and lysogenize
their host cells.
The genetic material of temperate phages can become attached to the
chromosome of the host cell. The properties of the phage are modified
and it becomes a prophage. The prophage acts as a bacterial gene, divid·
ing the bacterial chromosome and being transmitted to daughter cells. A
bacterium that carries prophage is called lysogenic. Agents such as UV
light, X·rays, organic peroxides, nitrogen mustard, and mitomycin C can
92 BASIC FOOD MICROBIOLOGY

cause the release of phage material from the chromosome, with resultant
production of phage and lysis of the cell when the phage is released.
Bacteriophages can be stored by freezing with liquid nitrogen, using or
not using glycerol (19 percent) as a protective additive, and holding
at -196°C. They can be held for about two years in a broth lysate stored
at 4°C, with only slight loss of titer. Freeze-drying or aerosolization re-
sults in a loss of titer of phage (Cox, Harris, and Lee 1974). The loss
occurs during rehydration of the phage_
Bacteriophages can be enumerated by determining the clearance of
susceptible bacterial cultures in broth or by the formation of plaques on
lawns of the bacteria_ In some cases, the phages must be concentrated
prior to enumeration (Seeley and Primrose 1982)_
Perhaps the most important function of phages in foods is their role
in destroying cultures of lactic acid bacteria during the fermentation of
dairy products_
There is a possibility that phages could be used to control unwanted
bacteria in various food products. The main problems appear to be the
host specificity of the phages and the development of lysogenic bacte-
ria. The lysogenic bacteria can continue metabolizing and cause spoil-
age of foods.
Phages can be useful in epidemiological work. Since phages are
rather host specific, a set of phages can be devised and used for typing
bacterial species or strains. Phage typing can aid in tracing the origin
of contamination or infection of foodborne outbreaks of gastroenteritis.
One reason that phages can lyse an entire bacterial culture is that
they have a high rate of replication. If a bacterium has a generation time
of 20 min, in 1 hour there will be eight cells. A phage may require from
40 to 80 min for replication, but the burst size (number of phages re-
leased on lysis of cell) may be from 2 to over 100. It is obvious that
with a large burst size, in only a short time the phages will outnumber
susceptible bacterial cells_

Fungal Viruses
Lemke and Nash (1974) reviewed the fungal viruses. According to
them, viruses have been reported for more than sixty species of fungi_
They believe the metabolism and genetics of fungi may be influenced by
these viruses. All of the fungal viruses have double-stranded RNA, and
most have a polyhedral shape. In a review of viruslike particles of yeast,
Bruenn (1980) questioned referring to these agents as viruses. According
to him, no extracellular particles are present and they lack the criterion
of infectivity normally associated with viruses.
 MICROORGANISMS ASSOCIATED WITH FOOD 93

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96 BASIC FOOD MICROBIOLOGY

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 4
Factors That Affect Microbial
Growth in Food

Food microbiologists need a knowledge of the factors that influence mi·

crobial growth. Desirable growth conditions are needed for enumera·
tion, fermentations, or the production of single·cell protein. Undesirable
conditions are used for food preservation.
Discussions concerning environmental conditions for growth attempt
to relate the macroenvironment of the system to the microenvironment
in which a microorganism exists. This has been necessary because little or
no information exists about the microenvironments in foods and other
products. There are many microenvironments with different conditions
in a food product. The microenvironments in a liquid food are less het·
erogeneous than are those in a solid food. Some microorganisms that
could not grow in the measured macroenvironment might be able to
grow in a microenvironment of the food.
The microenvironments in food products are changing constantly.
The reactions catalyzed by enzyme systems naturally inherent in some
foods result in heat being generated, atmospheric oxygen used, and car·
bon dioxide and other gases given off. These changes in the food affect
the processes of microbial systems. The environment determines the con·
ditions under which the microorganisms exist, and microorganisms influ·
ence the conditions prevailing in the environment. In any food envi·
ronment, certain microbial species survive and become dominant.
Organisms that lack the ability to withstand stresses induced by an unfa·
vorable environment will succumb. Microorganisms can respond rapidly
to changes in the growth conditions.
With optimum conditions for growth, some bacteria will reproduce
every 15 to 20 min. Although they may grow in conditions above or below
the optimum, the lag phase or the generation time may be longer (Fig.
4.1). If the stresses become too severe or increase in number, the organ·
isms might not grow or might fail to survive.
The optimum conditions for microbial growth are influenced by the
enzyme systems. The enzymes are organic catalysts that increase the rate
of reactions. The rates of metabolic reactions are influenced by the envi·
ronmental conditions that affect the activity of the enzymes.
101
102 BASIC FOOD MICROBIOLOGY

stationary or
resting phase

./'
/"
/'
~ less than ideal ",
• growth ",
logarithmic growth •/ conditions~
phase / /'
./. ",/
./ ",/

./ ",
//
./ ./
../ .,......"."
._-.,.....
~

Time

Figure 4.1. Bacterial growth curves. Under less·than-optimum conditions, the lag
phase, generation time, or both may be extended.

The conditions that affect the metabolism and multiplication of mi-

croorganisms include nutrients, water, pH, inhibitors, oxygen, light, tem-
perature, and time. Since organisms are part of the environment and can
alter it, microbial interactions (the effect of one organism on another)
are important. The previous stresses (sublethal heating, freezing, or radi-
ation) that a microorganism has endured will affect its ability to cope
with the environment.

NUTRIENTS

All biosystems require certain chemicals and chemical reactions in

order to survive and reproduce. The microbial cell must be able to obtain
a source of energy and synthesize cellular protoplasm from its environ-
ment. Organisms require a source of carbon and nitrogen, growth fac-
tors, such as vitamins, minerals, and water. The ability of organisms to
utilize compounds and synthesize cellular components depends upon
the enzyme systems that the organism can make, according to its genetic
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 103

code. Since the genetic code can change (mutate) there may be slight or
important differences between strains of a species of microorganism.
The substrate and other environmental factors affect the amount of en-
zymes produced by microorganisms. Some enzymes of microorganisms
are listed in Table 4.1 and are discussed more fully in Chapter 9.

Energy and Carbon Sources

In general microbiology a distinction is made between the types of
energy sources and carbon sources of microorganisms. The organisms of
primary concern in food microbiology are the chemoorganotrophs that
use organic compounds as their energy and carbon sources. By defini-
tion, these microorganisms are heterotrophic.

Nitrogen Source
Amino acids are needed to produce cellular proteins, including en-
zymes. A number of organisms, such as Escherichia coli, can utilize nitro-
gen in the form of nitrates or ammonia to produce amino acids. Other
organisms need one or several amino acids supplied to them in the sub·
strate.

Other Growth Factors

Some organisms need growth factors, such as vitamins, purines, or
pyrimidines. For some fastidious organisms, metabolites must be sup-
plied as preformed molecules. In general, the Gram-positive bacteria reo
quire more preformed growth factors than do other microorganisms. Vi-
tamins function in coenzyme systems as listed in Table 4.2.
The obligate parasites, such as viruses, require a living organism to
supply the machinery to synthesize their components.

TABLE 4.1. SOME ENZYMES ASSOCIATED WITH MICROORGANISMS

Enzyme Substrate Major Product
Invertase Sucrose Glucose, fructose
Maltase Maltose Glucose
Ribonuclease Ribonucleic acid Nucleotides
Lactic dehydrogenase Lactic acid Pyruvic acid
Proteinases Proteins Peptides, amino acids
Amylase Starch Dextrins, maltose, glucose
Lipases Fats Glycerol, fatty acids
Catalase Hydrogen peroxide Water, oxygen
Urease Urea Carbon dioxide, ammonia
104 BASIC FOOD MICROBIOLOGY

TABLE 4.2. FUNCTIONS OF VITAMINS

Vitamin Coenzyme Function·Reaction
Biotin Biotin coenzyme Carboxyl transfer, deamination, CO 2 fixa-
tion
Pantothenic acid Coenzyme A Acyl transfer or exchange
Folic acid Tetrahydrofolate Transfer of one-carbon units, methylation,
purine, and pyrimidine synthesis
Thiamin pyrophos· Accepts carboxyl groups from decarboxyl-
phate ation of keto acids; TCA cycle, lipid me-
tabolism
Riboflavin (B 2 ) Flavin mononu· Dehydrogenation, electron transfer-oxida-
cleotide (FMN) tions
Flavin adenine di- Dehydrogenation
nucleotide
Nicotinamide (B,,) Nicotinamide aden- Dehydrogenation, energy generation
(Niacin) ine dinucleotide
Nicotinamide aden- Dehydrogenation, hydrogen acceptor, syn-
ine dinucleotide thesis of fatty acids
phosphate
Pyridoxine (B6) Pyridoxyl phos- Deamination, racemization, transamina-
phate tion, decarboxylation of amino acids
Cob amide (Bd Cob amide coen- Transfer of methyl groups
zyme, deoxyaden-
osyl (Bd

It would be difficult to list the essential metabolites of specific micro-

organisms. There are shifts in the nutritional requirements of the strains
comprising a culture. Not only do species of organisms have different
essential metabolites, but strains within the species differ in their require-
ments.

Elements and Minerals

Certain elements or minerals found in cellular components are
needed in trace amounts by microorganisms. Somewhat larger amounts
of sodium, potassium, calcium, and magnesium are needed, as compared
to iron, copper, manganese, zinc, cobalt, and molybdenum.
Phosphorus and sulfur are also needed by microorganisms. Trace ele·
ments enhance the activity of some enzyme systems and are needed for
the production of toxins and other secondary metabolites.

Foods as Nutrients
Foods contain carbohydrates, fats, proteins, vitamins, minerals, water,
and other factors which microorganisms need for growth. Since they are
derived from biological systems, foods vary in their composition. The
components of the soil can affect the chemical composition of plants and
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 105

the food derived therefrom. Variations in composition are also due to

geography, climate, season, maturity, processing methods, variety, and
market conditions. The type of diet eaten by an animal also can affect
the composition of the food derived from this source.
From their general composition, foods are classed as protein, carbo·
hydrate, or fat. The protein foods are primarily of animal origin, the
carbohydrate foods of plant origin, and fats or oils are derived from ani·
mals and plants.
Fresh meats contain essentially no carbohydrate (less than 1 percent),
since most is converted to lactic acid by glycolytic reactions during rigor.
The vitamin and mineral content of most foods is sufficient for the
growth of most organisms. The composition of foods influences the types
of microorganisms that will grow, as well as the products that are formed
during microbial growth and spoilage.

MOISTURE

Some microorganisms can remain alive in a dried condition but can·

not carry out their normal metabolic activities or multiply without water.
Microorganisms can grow only in aqueous solutions. They cannot grow
in pure water or in the absence of water. Water dissolves more substances
than any other solvent. In this capacity, water is used to bring nutrients
into the cells and to dispel waste products. Water is involved in the chem·
ical reactions that break down substrates to usable molecules. These reac·
tions include the hydrolysis of the peptide bonds in protein, the ester
bonds in fats, and the conversion of polysaccharides to monosaccharides.
Water is a donor of hydrogen ions, contributes to the regulation of cellu-
lar pH and temperature, and is the lubricant of tissue (Beall 1983).
The water in a food is both bound and free. Bound water is held by
physical forces to macromolecules and is not available to act as a solvent
or to participate in chemical reactions. Hence, it is not available to micro-
organisms for metabolic activity.
When solutes are dissolved in water, the freezing point is lowered, the
boiling point is increased, the vapor pressure of the water is reduced,
and there is potential development of an osmotic pressure.

Water Activity
This is an index ofthe availability of water for chemical reactions and
microbial growth. Water activity (a w ) is the ratio of the vapor pressure
(VP) above a material or solution (P) and that of pure water (Po) at the
same temperature. The values of water activity range from 0 to 1.
106 BASIC FOOD MICROBIOLOGY

The VP of a pure liquid depends upon the rate of escape of molecules

from the surface. The escape of water to the air is measured by the equi·
librium relative humidity (ERH). Thus, VP and ERR are related. When
pure water is altered by the addition of a solute, the concentration of
water is decreased and the rate of escape from the surface is diminished.
The water activity of a solution is defined in terms of VP and ERR by
the formula

Many systems have been discussed for calculating, predicting, or de·

termining the water activity of various substances, including foods (Fa-
vetto et al. 1983; Ferro Fontan and Chirife 1981; Labuza et al. 1976; Nor-
tholt and Heuvelman 1982; Nunes, Urbicain, and Rotstein 1985; Stamp
et al. 1984; Teng and Seow 1981; Troller 1983a, 1983b; Wiebe et al. 1981).
Volatile compounds in a food may result in erroneous measurements of
aw when an electric hygrometer is used (Favetto et al. 1984). The a w of
food generally decreased as the temperature was raised from 23° to 27°C
(Scott and Bernard 1983).

Water Activity and Microbial Growth

Microorganisms have a maximum, optimum, and minimum aw for
growth. Since the aw of pure water is 1.00, and microorganisms cannot
grow in pure water, the maximum or upper limit for microbial growth is
an aw somewhat less than 1.00. Some foods have aw values greater than
0.995, which are rounded to 1.00. However, microorganisms can grow in
these foods.
The prevention of microbial growth is of importance to food micro-
biologists. Hence, the minimum aw at which growth can occur has been
of most interest. In general, for growth, bacteria require a higher aw than
yeasts, and yeasts require a higher aw than molds. The minimum aw values
for the growth of various microorganisms are listed in Table 4.3. Even
different strains of the same species appear to have different minimum
aw for growth. The limiting aw for growth is influenced by other environ-
mental factors which should be at optimum values for the organism be-
ing tested if the absolute minimum water activity is to be determined. If
these factors are not optimum during testing, the resultant minimum aw
for growth of an organism will appear to be higher than the true value.
The solute used to lower the aw affects the minimum aw for growth of
microorganisms. Osmotolerant organisms grow at a lower aw when the
solute is sugar rather than salt. The reverse is true for halophilic or halo-
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 107

TABLE 4.3. APPROXIMATE MINIMUM WATER ACTIVITIES FOR GROWTH

OF MICROORGANISMS
Organism Minimum aw Organism Minimum aw

Most spoilage bacteria 0.90-0.91 Staphylococcus albus 0.88-0.92

Acinetobacter 0.95-0.98 S. aureus 0.83-0.92
Aeromonas 0.95-0.98 Streptococcus 0.92-0.98
Alcaligenes 0.95-0.98 Vibrio parahaemolyticus 0.94-0.98
Arthrobacter 0.95-0.98 Halophilic bacteria 0.75
Bacillus 0.90-0.99 Most yeasts 0.87-0.94
B. cereus 0.92-0.95 Osmophilic yeasts 0.60-0.78
Citrobacter 0.95-0.98 Most molds 0.70-0.80
Clostridium botulinum 0.90-0.98 Xerophilic molds 0.60-0.70
Type A 0.93-0.95 Aspergillus 0.68-0.88
Type B 0.93-0.96 A. glaucus 0.70-0.75
Type E 0.94-0.97 A. flavus 0.78-0.90
C. perfringens 0.93-0.97 A. halophilicus 0.68
Corynebacterium 0.95-0.98 A. niger 0.80-0.84
Enterobacter 0.95-0.98 Botrytis cinerea 0.93
Escherichia coli 0.94-0.97 Debaryomyces 0.87-0.91
Flavobacterium 0.95-0.98 Fusarium 0.80-0.92
Klebsiella 0.95-0.98 Hansenula 0.89-0.90
Lactobacillus 0.90-0.96 Mucor 0.80-0.93
Leuconostoc 0.96-0.98 Penicillium 0.78-0.90
Micrococcus 0.90-0.95 Rhodatoruia 0.89-0.92
M. roseus 0.90-0.93 Saccharomyces cerevisiae 0.90-0.94
Pseudomonas aeruginosa 0.96-0.98 S. rouxii 0.62-0.81
P. fluorescens 0.94-0.97 Xeromyces bisporus 0.60-0.61
Salmonella 0.93-0.96

tolerant microorganisms. Organisms tend to grow better in a substrate

in which the aw is altered by desorption rather than adsorption (Labuza,
Cassil, and Sinskey 1972).
The minimum aw for most bacteria is 0.90 to 0.91, except for S. aureus
and the halotolerant bacteria. The minimum aw for these latter bacteria
is listed as 0.75, which also is the approximate aw of a saturated NaCI
solution (Table 4.4). The growth of S. aureus at various a.,u levels is shown
in Figure 4.2. As the aw is reduced below 0.99, a reduced growth rate, as
well as less total growth is evident.
A very high aw may limit the growth rate. A typical curve relating the
growth rate to aw is shown in Figure 4.3. The optimum growth for most
organisms is slightly below 1.00, and for halophiles, or xerotolerant orga-
nisms, the optimum is lower. The halophile Vibrio costicola did not grow
at aw values above about 0.98 (Forsyth and Kushner 1970). The optimum
aw for the growth of Penicillium species varied from 0.93 to 0.98 (Hocking
and Pitt 1979).
108 BASIC FOOD MICROBIOLOGY

TABLE 4.4. RANGES OF EQUILIBRIUM RELATIVE HUMIDITIES OF SATURATED

SOLUTIONS AT 20°C
Chemical Range ofERH Chemical Range ofERH
LiBr 6.6 NaN0 2 65.6-66.0
NaOHoH 20 7.0-8.9 KI 68.2-70.0
LiCloH 20 10.2-15.0 NaN0 3 74.0
K(C2 H s0 2) 20.0-23.0 NaCI 75.0-77.5
CaCI2°6H20 32.3 Na2S203 78.0
MgCbo6H 20 32.5-33.6 (NH')2S0• 80.0-81.5
Cr03 35.0-38.7 KCI 80.3-88.2
Zn(N 03)2°6H 20 42.0 ZnSO.o7H 20 89.0-90.5
K2C0 3°2H20 43.0-44.0 BaCI2 90.0-91.0
KN02 45.0-49.1 K2HPO. 92.0
LiNOso3H 20 47.0 KN03 92.0-95.0
Mg(N03ho6H20 54.9 NH.H 2P04 93.0-93.9
Na2Cr207°2H20 55.2 N a2HP04012H20 95.0-96.2
NaBro2H 20 55.3-59.3 K 2 S0 4 96.4-98.2
NH.N03 64.9 CuSO.o5H 20 98.0
Mg(C2H302)2°4H20 65.0 K 2Cr20 7 98.3

OTHER ASPECTS. Other aspects of water activity besides the mini-

mum activity for growth are important. These aspects include the germi-
nation of spores, toxin production, resistance to heat, and survival of
microorganisms.
Pitt and Christian (1968) listed the 0". requirements of thirty-six molds
for germination and for the formation of asexual and sexual sporulation.
Often a higher water activity is needed for sporulation than for germina-
tion and growth. Sexual sporulation usually requires a higher 0". than
does asexual sporulation. According to a review by Sperber (1983), the
aw requirement for bacterial sporulation is the same as for growth, but
germination usually can occur at lower 0". values.
The production of enterotoxin by S. aureus requires a higher 0". than
for growth (Smith et al. 1983). Higher 0". levels are needed for the produc-
tion of mycotoxins than for growth by several species of Aspergillus and
Penicillium (Beuchat 1981, 1983).
In general, lowering the 0". of the substrate increases the heat resist-
ance of microorganisms. However, at extremely low levels of aw, the resist-
ance may be decreased. Bacterial spores are most heat resistant at 0". val-
ues of 0.2 to 0.4.
Generally, the lower the 0"., the longer the organisms survive during
storage. The tolerance to low 0". values results from the ability of the
organism to accumulate compatible solutes within the cell. These solutes
include proline and 'Y-aminobutyric acid for bacteria, and polya1cohols
for fungi.
 II

10

9
t-
Z
;:)
oU
l&J 8
t-
c:t
..J
Q.

~ 7
o...J

4
o 20 40 60 80 100
HOURS
Figure 4.2. Growth of Staphylococcus aureus at various levels of water
activity.
Courtesy of Troller (1971).

109
110 BASIC FOOD MICROBIOLOGY

EQUILIBRIUM RELATIVE HUMIDITY (%)

100 99 9B 97 96 95 94

1.8

a:
:;)
0
x
a:
w
Cl.
(fJ
z
52
(fJ
:>
i5

....w4
a:
X
....
~
a:
C)

1.00 0.99 0.98 0.97 0.96 0.95 0.94

WATER ACTIVITY (a w)

Figure 4.3. Effect of water activity on bacterial growth rate.

Water Activity of Food

The aw of food can be lowered by removing water (dehydration), by
adding solutes (sugar, salt), or by freezing. The usual aw levels of various
foods are listed in Table 4.5. Fett (1973) reported variations of aw of
±0.003 to ±0.01l, depending upon the system of measurement; other
researchers reported that different methods of measurement may vary
±0.02 aw units (Labuza et al. 1976). Dewpoint hydrometer measurements
are reproducible to 0.003 aw and accuracy of 0.006 aw (Northolt 1979).
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD III

TABLE 4.5. APPROXIMATE WATER ACTIVITIES OF SELECTED FOODS

Food aU' Food aw
Fresh fruit or vegetables 0.97-1.00 Jelly 0.82-0.94
Fresh poultry or fish 0.98-1.00 Rice 0.80-0.87
Fresh meats 0.95-1.00 Fruitcake 0.80-0.87
Pudding 0.97-0.99 Flour 0.67-0.87
Eggs 0.97 Cake icing 0.76-0.84
Juices-fruit and vegetable 0.97 Molasses 0.76
Bread 0.95-0.96 Honey 0.54-0.75
White 0.94-0.97 Dried fruits 0.51-0.89
Crust 0.30 Chocolate candy 0.69
Cheese, most types 0.91-1.00 Rolled oats 0.65-0.75
Parmesan 0.68-0.76 Toffee 0.60-0.65
Cured meat 0.87-0.95 Caramels 0.60-0.65
Baked cake 0.90-0.94 Noodles 0.50
Refrigerated biscuit dough 0.94 Dried whole egg 0.40
Maple syrup 0.90 Biscuits 0.30
Salted egg yolk 0.90 Dried vegetables 0.20
Soft, moist pet food 0.83-0.91 Cereals 0.10-0.20
Nuts 0.66-0.84 Crackers 0.10
Jam 0.75-0.80 Sugar 0.10

Fresh foods, such as fruit, vegetables, meat, poultry, and fish have aw
values of 0.98 to over 0.99, which will allow the growth of most microorgan·
Isms.
The rate of growth of bacteria is greater than that of yeasts or molds.
Hence, with foods of high aw, bacteria will outgrow the fungi (yeasts and
molds) and cause spoilage, while at aw values that restrict or prevent bac·
terial growth, the fungi grow and become dominant. Exceptions to these
generalizations include fruits spoiled by fungi because of the acidity of
the product restricting bacterial growth. Products that have low Ow due
to sugar content Uams, jellies, or honey) will be subject to attack by os·
mophilic yeasts, while products that contain high salt concentrations
(lowa,v) will be spoiled by halotolerant or halophilic bacteria. Dried foods
generally have aw values below 0.75. A safe aw level for storage is usually
considered to be 0.70 or less. In foods protected by low aw, enzymatic
changes can occur, but at a slow rate.

SORPTION ISOTHERMS. The moisture content of foods is often de·

termined and reported. The relationship between moisture and aw values
is desirable. This relationship is described by a sorption isotherm that
has the shape ofa sigmoid curve (Fig. 4.4). Numerous mathematical equa·
tions have been used to describe sorption isotherms (Aguerre, Suarez,
and Viollaz 1983; Ferro Fontan et al. 1982; King et al. 1983).
The curve may be an isotherm of adsorption (a moistening process),
or an isotherm of desorption (a drying process). The isotherms vary with
112 BASIC FOOD MICROBIOLOGY

EQUILIBRIUM RELATIVE HUMIDITY (%)

o 20 40 60 80 100

o 0.20 0.40 0.60 0.80 1.00

WATER ACTIVITY (a w)
Figure 4.4. Typical sorption isotherm. 1·Monomolecular film of water, no
growth of organisms; 2·some added water to monomolecular film, slight
growth in upper range; 3·water activity is sufficient for microbial growth.

temperature for different products. The adsorption and desorption iso·

therms shown in Figure 4.4 have a hysteresis "loop." According to Troller
(1983a), equilibrium will occur, given sufficient time, so that only one
line is evident.
Three areas are apparent in most isotherms. In the lowest part of the
curve, the water is in a monomolecular layer on the food product, or so·
called bound water, and is not available for biological activity. In the mid·
dIe of the curve, additional layers of water are added to the monomolecu·
lar film, and there is a rapid increase in aw with a moderate increase in
moisture content. In the upper regions of this section of the curve, some
water becomes available to support the growth of some molds and yeasts.
In the upper part of the isotherm, the moisture content increases rapidly
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 113

with comparatively small increases in water activity, and the food con·
tains free water that is available for bacterial as well as fungal growth.
If food is not stored in moisture· proof containers, it will equilibrate
with the relative humidity of the surrounding air according to its sorp·
tion or desorption isotherm. The direction will be determined by the
moisture in the food and the relative humidity of the air. Dry foods may
pick up moisture to the extent that molds will be able to grow, while
foods with high a., stored in low· humidity air may lose moisture, causing
shrinkage and other organoleptic changes (Labuza and Contreras·
Medellin 1981). The rate of equilibration depends upon the relative hu·
midity of the air, the initial a., of the food, and temperature.
In mixtures of foods such as cereals and raisins, or marshmallow top·
ping on a cookie base, the moisture from one food will transfer to the
drier product. For raisins in cereals, this results in the cereal becoming
less crisp and the raisins becoming hard. Coating the raisins with bees·
wax, oils, or sugar has been used to retard the transfer of moisture.
Since many of our foods are combinations of various components,
the a., values and isotherms of the ingredients should be considered in
order to avoid potential problems either with organoleptic quality or
with possible microbial growth. Although a product may have an a., not
conducive to growth of microorganisms, syneresis may occur, increasing
the a., in microenvironments, thus allowing microbial growth.
Case hardening, the phenomenon in which the moisture content of
the exposed surface is different from that in the unexposed portion, may
act as a barrier for moisture sorption or desorption. This may influence
the measurement of the a., of a food so that, although the determined a.,
of the food would appear in a safe range, the interior of the food will
support microbial growth.
Foods that can be eaten without rehydration but are microbiolog·
ically stable without refrigeration are called intermediate moisture foods.
These foods are discussed in Chapter II.

pH
In many texts, pH is defined as the negative logarithm of the hydro·
gen ion concentration. The pH is actually a function of apparent concen·
trations or activity of the electrolytes in solution. Thus, pH is the negative
logarithm of the activity of the hydrogen ions. Since pH is usually deter·
mined by means of a pH meter, it might be defined as a value calculated
from the electromotive force (emf) of an electromotive cell in which the
substance being tested is one of the electrolytes. In the food industry, the
determination and maintenance of pH are important in quality control
and processing requirements.
114 BASIC FOOD MICROBIOLOGY

Effect on Microorganisms
Microorganisms have a minimum, optimum, and maximum pH for
growth. Most bacteria show optimum growth at a pH near 7.0, while oth·
ers are favored by an acid environment, probably due to the inhibition
of other organisms, thus eliminating microbial competition. The acid for·
mers (Lactobacillus and Streptococcus) can tolerate moderate acidity, while
proteolytic types (Pseudomonas) can grow in moderately alkaline sub·
strates. In general, molds can grow at lower pH values than yeasts, and
yeasts are more tolerant of low pH values than are bacteria. Bacteria
usually grow faster than yeasts in a neutral or slightly acid substrate, but
at pH 5.0 or less, yeasts can compete with or outgrow bacteria.
Microorganisms can grow in a wide pH range (Table 4.6). The vari·
ations in pH values for growth may be due to different strains of a species
or different species in a genus, the type of substrate, the acid or base
used to adjust the pH, or other factors. There is an interrelationship be·
tween pH and other environmental factors.
Alteration of the pH of a substrate may relate indirectly to growth.
For example, the availability of metallic ions is altered. Although coexist·
ing in the free form at low pH, magnesium and phosphorus form an
insoluble complex at higher pH values. In an alkaline medium, ferric,
zinc, and calcium ions become insoluble.
The pH of the substrate may influence cell permeability. At low pH,
the membrane becomes saturated with hydrogen ions, thus limiting pas·
sage of essential cations. Conversely, at a high pH, saturation of the mem-
brane with hydroxyl ions will limit the passage of essential anions.
The toxicity of adverse pH values is due partly to the penetration of
undissociated molecules of acid or basic substances into the cells. At low
pH values, undissociated weak acids can enter the cell, then ionize and
alter the internal pH. In alkaline solutions, undissociated weak bases can
enter the cell. If the internal pH is changed to basic pH values, amino
acid transferase RNA is inhibited and protein synthesis is stopped. The
pH of the substrate influences the activity of enzyme systems and the
products of metabolism of microorganisms.

ALTERATION OF pH BY MICROORGANISMS. The environment in-

fluences microorganisms and the microorganisms influence the environ-
ment. During growth, metabolic products are formed. These can be
either acidic or alkaline, depending upon the substrate, the organisms
involved, and the time allowed for growth. The initial reaction of most
organisms is acidic because of the breakdown of carbohydrates and the
formation of organic acids. The alteration of pH by production of acids
is used in the food-fermentation industries. The lactic acid bacteria tend
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 115

TABLE 4.6 APPROXIMATE pH RANGES FOR MICROBIAL GROWTH

pH
Organism Minimum Optimum Maximum
Bacteria (most) 4.5 6.5-7.5 9.0
Acetobacter 4.0 5.4-6.3
Bacillus subtilis 4.2-4.5 6.8-7.2 9.4-10.
Clostridium botulinum 4.8-5.0 6.0-8.0 8.5-8.8
C. perfringens 5.0-5.5 6.0-7.6 8.5
C. sporogenes 5.0-5.8 6.0-7.6 8.5-9.0
Erwinia carotovora 4.6 7.1 9.3
Escherichia coli 4.3-4.4 6.0-8.0 9.0-10.
Gluconobacter oxydans 4.0-4.5 5.5-6.0
Lactobacillus (most) 3.0-4.4 5.5-6.0 7.2-8.0
L. acidophilus 4.0-4.6 5.5-6.0 7.0
L. plantarum 3.5 5.5-6.5 8.0
Leuconostoc cremoris 5.0 5.5-6.0 6.5
L. oenos 4.2-4.8
Pediococcus cerevisiae 2.9 4.5-6.5 7.8
Propionibacterium 4.7 6.2-7.0 7.5
Proteus vulgaris 4.4 6.0-7.0 8.4-9.2
Pseudomonas (most) 5.6 6.6-7.0 8.0
P. aeruginosa 5.6 6.6-7.0 8.0-9.0
Salmonella (most) 4.5-5.0 6.0-7.5 8.0-9.6
S. typhi 4.0-4.5 6.5-7.2 8.0-9.0
S. choleraesuis 5.0 7.0-7.6 8.2
Serratia marcescens 4.6 "6:0":'1.0 8.0
Staphylococcus aureus 4.0-4.7 6.0-7.0 9.5-9.8
Streptococcus lactis 4.1-4.8 6.4 9.2
Vibrio 5.5-6.0 9.0
V. cholerae 8.6
V. parahaemolyticus 4.8-5.0 7.5-8.5 11.0
Yeasts 1.5-3.5 4.0-6.5 8.0-8.5
Hansenula 4.5-5.5
Kluyveromyces 1.5-2.0
Pichia 1.5
Saccharomyces cerevisiae 2.0-2.4 4.0-5.0
S. rouxii 1.5 3.5-5.5 8.5-10.5
Molds 1.5-3.5 4.5-6.8 8.0-11.
Aspergillus niger 1.2 3.0-6.0
A. oryzae 1.6-1.8 9.0-9.3
Bot1ytis cinerea 2.5 7.4
Mucor 3.0-6.1 9.2
Penicillium 1.9 4.5-6.7 9.3
Rhizopus nigricans 4.5-6.0

to lower the pH by production of lactic acid, while proteolytic types such

as pseudomonads tend to raise the pH by production of ammonia or
other basic chemicals.
Bacillus coagulans grew in tomato juice, raising the pH from 4.5 to 5.0
(Anderson 1984). Molds growing on tomatoes raised the pH to levels that
allowed bacterial growth in the food (Mundt and Norman 1982).
116 BASIC FOOD MICROBIOLOGY

SURVIVAL. The pH for survival of cells is somewhat different from

that for growth. Some organisms show greater survival at slightly acid pH
levels 5.6 to 6.5 and optimum growth at 6.8 to 7.2. Microorganisms can
survive at pH levels too acid or too basic for metabolism and growth. The
effect of pH on survival during heating is very evident.

pH of Food
The pH of a food, along with other environmental factors, will deter·
mine the types of microorganisms that are able to grow and dominate,
and eventually cause spoilage, a desired fermentation, or a potential
health hazard. Besides the microbial aspects of the pH of foods, the acids
in foods play other important roles.
The food may be naturally acid, acids may be added, or acids may
be produced in the food by enzymatic action with or without microbial
growth.
The pH of a food is determined by the balance between the buffering
capacity and the acids or alkaline substances it contains. Since protein
has a high buffering capacity, protein foods have greater buffering than
do fruits or vegetables. This is important in the fermentations producing
lactic acid because the production of small amounts of acid in sauerkraut
or pickle processes will lower the pH significantly.
The approximate pH values for selected foods are listed in Table 4.7.
The pH values are only approximations because there is considerable
variation in the pH of some foods and the pH of food products can
change during ripening, processing, or storage. The pH values of various
foods were reported by Sapers, Phillips, and Divito (1984).
Foods have been categorized according to their pH as follows:

High·acid foods pH below 3.7

Acid foods pH 3.7-4.6
Medium·acid foods pH 4.6-5.3
Low or nonacid foods pH over 5.3

EGG WHITE. This is one of the most alkaline biological solutions. The
albumen of a freshly laid chicken egg is approximately pH 7.6. When the
egg is stored in air, the carbon dioxide from carbonic acid in the albumen
is released through the egg shell. When this occurs, the pH increases and
has been reported to reach levels of 9.4 to 9.7. Although the high pH
is desirable from a microbiologist's viewpoint, these high pH levels are
associated with thinning of albumen and a decrease in the strength of
the vitelline (yolk) membrane, or a general decrease in egg quality. Egg
quality is maintained at an albumen pH near 8.2, which is above the
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 117

TABLE 4.7. APPROXIMATE pH RANGES OF SELECTED FOODS

Food pH Range Food pH Range
Egg white 7.6-9.5 Cabbage 5.2-6.3
Shrimp 6.S-S.2 Turnip 5.2-5.6
Crab 6.S-S.0 Spinach 5.1-6.S
Scallops 6.S-7.1 Asparagus 5.0-6.1
Cod, small 6.7-7.1 Cheeses, most 5.0-6.1
Cod, large 6.5-6.9 Camembert 6.1-7.0
Catfish 6.6-7.0 Cottage 4.1-5.4
Soda crackers 6.5-S.5 Gouda 4.7
Maple syrup 6.5-7.0 Bread 5.0-6.0
Milk 6.3-6.S Carrots 4.9-6.3
Brussels sprouts 6.3-6.6 Beets 4.9-5.S
Whiting 6.2-7.1 Bananas 4.5-5.2
Haddock 6.2-6.7 Dry sausages 4.4-5.6
Cantaloupe 6.2-6.5 Pimientos 4.3-5.2
Dates 6.2-6.4 Tomato juice 3.9-4.7
Herring 6.1-6.6 Mayonnaise 3.S-4.0
Butter 6.1-6.4 Tomatoes 3.7-4.9
Honey 6.0-6.S Jams 3.5-4.0
Mushrooms 6.0-6.5 Apricots 3.5-4.0
Cauliflower 6.0-6.7 Apple sauce 3.4-3.5
Lettuce 6.0-6.4 Pears 3.4-4.7
Egg yolk 6.0-6.3 Grapes 3.3-4.5
Corn, sweet 5.9-6.5 Cherries 3.2-4.7
Oysters 5.9-6.6 Pineapple 3.2-4.1
Celery 5.7-6.0 Peaches 3.1-4.2
Peas 5.6-6.S Rhubarb 3.1-3.2
Turkey 5.6-6.0 Strawberries 3.0-4.2
Chicken 5.5-6.4 Grapefruit 2.9-4.0
Halibut 5.5-5.S Raspberries 2.9-3.7
Beans, lima 5.4-6.5 Apples 2.9-3.5
Potatoes, Irish 5.4-6.3 Plums 2.S-4.6
Walnuts 5.4-5.5 Oranges 2.S-4.0
Pork 5.3-6.4 Cranberries 2.5-2.S
Beef 5.3-6.2 Lemons 2.2-2.4
Onions 5.3-5.S Limes 1.S-2.0
Sweet potatoes 5.3-5.6

maximum for the growth of many microorganisms. Storage in an atmo·

sphere of CO 2 or oiling the egg shell maintains the pH at an intermediate
level.

RED MEAT. The pH of living animal tissue is near neutral (pH 7.0-
7.2). The circulating blood brings nutrients and oxygen to the cells and
removes the waste products of metabolism. When an animal is slaugh·
tered, blood no longer circulates, anaerobic conditions develop, and any
metabolic products accumulate. The inherent tissue enzymes ferment the
muscle glycogen to lactic acid, which lowers the pH. When glycogen sup'
118 BASIC FOOD MICROBIOLOGY

plies are abnormally low in the muscle prior to slaughter, less acid is
produced, with a higher·than·normal ultimate pH. The temperature at
which a beef carcass is held determines the rate of glycolysis and pH
changes.
Immediately after slaughter, the pH of most beef muscles is 6.9 to 7.2
(Hamm 1982). After 24 hr postmortem, most muscles from well·rested
animals have a pH of 5.4 to 5.6 (Currie and Wolfe 1980; Tarrant and
Sherington 1980). It is generally believed that pH 5.3 is the limit below
which glycolysis is inhibited, even though some glycogen may be present.
The pH of most pork muscles with normal glycolysis ranges from 5.3 to
5.8 (Warriss 1982).
It might be expected that a higher glycogen content is present in the
muscles of well· fed, rested, unexcited animals than in starved, tired, or
stressed animals.
Preslaughter stress conditions, such as fighting among hogs, have
been associated with a rapid pH drop and low ultimate pH. This rapid
lowering of the pH while the muscles are still warm results in pale, soft,
exudative (PSE) pork. Preslaughter exercise or stress causes muscles to
be deficient in glycogen and results in ultimate higher pH values (above
pH 6.0). This meat is called dark, firm, dry (DFD) pork, or dark cutting
beef. With a higher·than·normal ultimate pH, these muscles have a tend·
ency to spoil more readily than do muscles with a lower pH.
Subjecting beef carcasses to an electrical current of various intensities
promotes tenderness of the muscles as well as a rapid drop in pH (Martin
et al. 1983). Also, a rapid pH reduction was noted when beef muscle was
pressurized (Elkhalifa et al. 1984).
The pH of commercial dry sausages was reported as ranging from 4.4
to 5.6 (Acton and Dick 1976). Fermentation is responsible for the lower
pH values of sausages as compared to fresh meat. The addition of acid
(acetic, lactic, citric) results in a low pH of pickled meats.
Microbiologically, a low pH in fresh meat is desired. The minimum
pH for the growth of most pseudomonads that spoil meat is 5.6. If meat
has an ultimate pH less than 5.6, it would be expected to have a longer
shelf life. On the other hand, higher pH values have been associated with
enhanced water·holding capacity, nutrition,juiciness, tenderness, and in·
creased emulsifying properties and binding strength of meat, which are
desirable attributes.

CHICKEN. The pH of chicken muscle varies similarly to that of red

meats. Postslaughter chicken muscle is approximately pH 7.0, which is
reduced during glycolysis to 5.5 to 5.9. A rapid glycolysis at high temper·
ature can result in tough meat. The pH of meat from heat·stressed birds
was 5.46, from cold·stressed, 5.51, and from control birds, 5.53 (Lee et al.
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 119

1976). This indicates that this type of stress has little influence on the
final pH of chicken meat.

SEAFOODS. The pH offish (7.0-7.3) is lowered during rigor to pH 5.5

to 6.5, depending on the species of fish and the initial amount of glyco·
gen in the muscle. Fish muscle generally has less glycogen than poultry
or red meat.
The pH of cod was found to be lower at the end of the spawning
season, and large cod had a lower pH than small cod (Love et al. 1972).
They found that a low pH was related to gaping in cod. This is a condi·
tion in which the tissue that holds the muscle segments together becomes
weakened and causes slits to appear across the fillets.
According to Love and Haq (1970), haddock has a lower pH than
whiting. These researchers postulated that the difference may be in their
eating habits. The whiting chases food, while the haddock tends to graze.
The haddock, being a less active fish, probably does not struggle as much
as a whiting when caught, so there would be greater carbohydrate reo
serve, which results in more lactic acid production postmortem and a
lower pH. Of all the fish for which data are available, the halibut has the
lowest ultimate pH and the best keeping quality.
The pH of canned crab is usually pH 6.8 to 7.4 but may be higher.
Motohiro and Inoue (1970) observed that the pH of fresh king crab was
6.8 for the hard shell and 7.4 for the paper shell intermolt stages.
A pH range of 7.1 to 8.2 was determined on brown shrimp obtained
from commercial fishing boats at landing (Cobb et al. 1973). The upper
limit, pH 8.2, may seem rather high, since pH 7.95 has been indicative
of shrimp spoilage. At the time of spoilage, the pH of brown shrimp was
found to be 7.3 to 8.5 (Cobb et al. 1973). This indicates that pH 7.95 has
little, if any, significance. At a pH of about 8.5, the bacterial numbers
were estimated at 10 5 per gram (Flores and Crawford 1973). This level of
microorganisms usually does not indicate spoilage.

FRUITS AND VEGETABLES. Fruits have a lower pH than do most

foods. Unripe fruit generally has a lower pH than ripe fruit. As the fruit
matures, the moisture and sugars increase, and the acids decrease or are
diluted. The ripening temperature influences the ultimate pH, generally
in a direct relationship. The pH of fruit influences not only the growth
of microorganisms, but also quality factors, such as softening and discol-
oration of the canned product. Since the pH is low, fruits are usually
spoiled by mold growth. The pH of the tomato is influenced by variety,
maturity, geographic area of growth, and storage conditions. Vegetables
generally have a higher pH than fruits and are therefore subject to bacte-
rial spoilage.
120 BASIC FOOD MICROBIOLOGY

OXIDATION-REDUCTION POTENTIAL

When a substance is oxidized, it loses electrons, and these electrons

must be accepted by another substance which then becomes reduced.
The oxidation-reduction (OR) potential is a measure of the tendency of
a reversible system to give or receive electrons.
The OR potential is referred to in Chapter 2 in regard to the use of
reductase tests to estimate microbial numbers in foods. By use of indica-
tors, the OR potential of a system can be estimated. The indicator should
give a distinct color to the solution at the pH of the system being studied.
Unfortunately, some indicators behave as both OR and pH indicators.
They may give an intense color in an alkaline solution, but in neutral or
acid solutions yield only a dull tint. The usual method of measuring OR
is with a platinum redox electrode attached to a pH/mV meter.
Eh is a measure of intensity, not of capacity of a system. The capacity
is the poising ability, which acts in a manner similar to buffers in main-
taining pH at a constant value. A system is said to be poised if it resists
change in potential.

Effect on Microorganisms
In microbial cultures, the simultaneous oxidations and reductions are
the sources of energy for cell processes. Since energy is needed by the
cell to function normally, OR reactions and OR potentials are important.
The broad classifications of organisms into aerobes and anaerobes,
as discussed in general microbiology, is based on their apparent toler-
ance to oxygen during growth_ Strictly aerobic organisms grow only in
the presence of free atmospheric oxygen. Strictly anaerobic organisms
survive and increase only in the absence of free oxygen. Facultative an-
aerobes can grow with or without free oxygen. Some facultative orga-
nisms prefer growth in atmospheric oxygen; others do not. Microaero-
philic organisms cannot multiply in either entirely aerobic or anaerobic
conditions. They grow best in a limited amount of free oxygen.
In discussing growth-limiting effects on anaerobic bacteria, Hentges
and Maier (1972) cited references to show that some obligate anaerobic
types of organisms failed to grow if exposed to oxygen in the air during
the manipulation of diluting and plating. These obligate anaerobes
rarely are reported in foods, probably because not enough care is taken
to find them. If they are such strict anaerobes, they probably cause few,
if any, serious problems in foods_
In one study, anaerobes were classed as intolerant, moderately toler-
ant, or tolerant, on the basis of their tolerance to atmospheric oxygen
(Rolfe et al. 1978). When exposed to oxygen, intolerant types survived
less than 2 hr, moderately tolerant for 4 to 8 hr, and tolerant anaerobes
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 121

at least 48 hr. Of the eleven anaerobic species the researchers tested,

Clostridium perfringens was the most oxygen tolerant.
From preliminary studies, Hentges and Maier (1972) stated that the
OR potential is unimportant regarding the multiplication of Bacteroides
species. When oxygen is excluded from the environment, Bacteroides are
unaffected by a high OR potential of the medium. Walden and Hentges
(1975) studied the growth and survival of Bacteroides fragilis, Clostridium
perjringens, and Peptococcus magnus at low and high Eh with and without
oxygen. Even at an Eh of - 50 mv these organisms did not grow in the
presence of oxygen. No inhibition was observed in the absence of oxygen
even at an Eh of + 325 mv. This indicates that oxygen, rather than a
positive potential, was the limiting factor for growth of these anaerobes.
Of these three organisms, P. magnus was the most sensitive to oxygen and
C. perfringens the least sensitive.
Other researchers reported that B. fragilis was not affected by an Eh
of + 300 mv in the absence of oxygen, but cell death occurred with oxy·
gen at + 250 mv (Onderdonk et al. 1976). They suggested that dissolved
oxygen has an inhibitory effect on B. fragilis, which may be independent
of changes in Eh alone. Similar effects of oxygen at low Eh were noted
for sporulation and growth of Clostridium butyricium (Douglas, Ham·
bleton, and Rigby 1973), and C. botulinum (Lund and Wyatt 1984).
Many anaerobic clostridia (c. botulinum, C. histolyticum, C. sporogenes)
do not need a negative Eh for growth. These organisms will grow at Eh
levels from + 85 to + 160 mv, especially in the absence of oxygen.
Due to complexities in maintaining and measuring OR potentials,
there'is not as much information on this parameter as on pH. Also, the
results vary considerably from one experiment to another, between
strains of species, and between different researchers.

Effect of Organisms on Eh
It has been well established that, as organisms metabolize, a lower Eh
value is produced in the medium or substrate. The lowering of the poten·
tial has been ascribed to the consumption of oxygen or the production
of reducing substances.
With aerobic bacteria, there is a slight lowering of the OR potential
during the lag phase of growth due to the consumption of oxygen. As
the bacteria enter the log phase of growth, this oxygen usage increases
and causes a rapid drop in Eh. As the Eh becomes negative, the growth
rate of the bacteria decreases. An overall lowering of the potential is
between 400 and 500 mv.
At the beginning of growth, facultative anaerobes alter the Eh of the
substrate in a manner similar to aerobes, but the rate of reduction may
be somewhat slower. After the bacteria enter the log phase of growth, the
122 BASIC FOOD MICROBIOLOGY.

Eh decreases very rapidly. This overall potential drop may be from 700
to 800 mv or more. As the culture reaches the stationary phase, and as the
age of the stationary phase increases, the observed OR potential shifts to
more positive values.
Oxygen is removed from media prior to inoculation and growth of
anaerobes. As a result, the initial Eh is lower than that for aerobes or
facultative anaerobes. During the germination and emergence of spores
or the lag phase of growth, the Eh is lowered by 500 to 700 mv (Douglas
and Rigby 1974). There is little further reduction during the lag phase
of growth. Although the total reduction of Eh is generally less for anaer-
obes than for facultative anaerobes, a lower Eh is attained due to the
lower initial Eh of the substrate.

Redox Potential of Food

The OR potential of a food depends upon the composition (oxydiz-
ing and reducing agents present), the pH, the oxygen tension in the at-
mosphere, and the access that the atmosphere has to the food. Due to
the complications of determining the Eh of foods and the variations ob-
tained due to atmospheric influences, the OR potentials of only a few
foods are available.
Harrison (1972) stated that in a conglomerate mixture with different
systems, not in equilibrium, an overall redox potential cannot be con·
ceived. It is evident that most foods are conglomerate mixtures. Regard-
less, there have been attempts to measure and compare the redox poten-
tial of foods. Living cells tend to have a low OR potential due to -SH
groups in animal products and ascorbic acid and reducing sugars in
plants. In dairy products, the redox potential is related to fat oxidation.
Thus, there is an inverse relation of OR potential and keeping quality.
The contamination of milk with cupric or ferric ions tends to increase
the OR potential, resulting in less shelf life of the product.
The OR potential of cheese depends on the type, with Emmenthal
(Swiss) cheese varying from - 200 to - 50 mv and Cheddar cheese from
+220 to +340 mv (Mossel and Ingram 1955).
The Eh of meat has been reported to range from -150 to + 250 mv.
There is an oxygen requirement for postmortem tissue. The myoglobin
of muscle can bind oxygen to form oxymyoglobin, or it can be oxidized
by oxygen to form metmyoglobin. To oxygenate the pigment requires
about 5 ttl of oxygen per gram of meat, while oxidation to metmyoglobin
requires about 13 ttl of oxygen per gram of meat. Other oxygen require-
ments are tissue respiration, lipid oxidation, tissue fluids with low oxy-
gen tension dissolving O 2 and bacterial demands. DeVore and Solberg
(1974) found that tissue respiration accounted for 80 percent of the oxy-
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 123

gen uptake of postmortem meat, while bacterial demands were insignifi-

cant.
The Eh of plant products varies from + 383 to + 436 mv for fruit
juices, + 74 mv for spinach, + 225 mv for barley, and - 470 mv for wheat
germ (Mossel and Ingram 1955)_ The Eh of cherries and peaches was
determined to be + 179 mv and + 175 mv, respectively (Ross, Bartlett,
and Hard 1953)_ The addition of ascorbic acid lowered the Eh of the
fruit_ The Eh of canned foods ranged from -18 mv for sliced carrots
packed in glass to - 446 mv for beef stew packed in cans (Montville and
Conway 1982).
The oxygen content of the atmosphere and the access of the atmo-
sphere to the food influences the OR potentiaL Large pieces of foods,
such as carcasses, have less total surface area than do smaller-sized foods.
Food in a deep vat has less surface exposed proportionately than does
food in shallow vats. Food at rest in a vat has a lower Eh than does liquid
being stirred or mixed. Food that is packaged in a material impermeable
to oxygen should have a lower Eh than unpackaged food_ Vacuum pack-
aging will reduce or prevent the access of free oxygen to the food.

Relationships of Food OR and Microorganisms

The relatively high Eh of fruit juices favors the growth of aerobic mi-
croorganisms. Since these products have a low pH, the aerobic yeasts and
molds are the predominant organisms that cause spoilage.
In meat, the surfaces exposed to the atmosphere allow the growth of
aerobic bacteria, but in deeper tissue, the Eh is lower so that anaerobes
can grow. In prerigor meat, the Eh is sufficiently high to prevent the
growth of anaerobic types, but during rigor, the Eh is reduced to the
extent that allows clostridia to grow_ The Eh of fresh raw milk inhibits
the growth of C. botulinum. The heating of milk lowers the Eh so that C.
botulinum can grow (Kaufmann and Marshall 1965).
Although molds are considered to be strictly aerobic, some types can
grow in low levels of oxygen_ One of the reasons that Penicillium roqueforti
can grow in the inside of Roquefort-type cheese is its tolerance for low
p02' Byssochlamys fulva grows in grape products at extremely low levels of
oxygen.

INHIBITORY SUBSTANCES

When you hear inhibitory substances in foods mentioned, you might

envision some chemical preservative added by the processor. However,
microbial inhibitors were present long before human beings appeared
on earth.
124 BASIC FOOD MICROBIOLOGY

Action of Inhibitors
Generally, inhibitors affect microorganisms by acting on the whole
cell, cell wall, or cell membranes, by interfering with the genetic mecha-
nism of the cell, by interfering with the enzyme systems of the cell, or by
binding essential nutrients_
Many microbial inhibitors have been reported to be naturally present
in foods_ The factors involved with the growth of microorganisms, such
as pH, that have been discussed in this chapter, are partially due to chem-
icals naturally present in foods. Besides these, there are other inhibitors
in both animal and plant products.

Inhibitors in Animals Products

Microbial inhibitors have been found in egg white, milk, and various
tissues from animals.

EGG WHITE. The growth of bacteria in egg white is limited by the

presence of lysozyme, enzyme inhibitors (such as ovomucoid), avidin,
conalbumin, and a high pH (Table 4.8).
Lysozyme. In 1922, Fleming reported a lytic agent in egg white and other
biological systems, which was named lysozyme. This enzyme has been
referred to as muramidase, N-acetylmuramide glycanohydrolase, gluco-
hydrolase, mucopeptide glycohydrolase and {j-glucosaminidase. To help
alleviate confusion about the name of this and other enzymes, the Com-
mission on Enzymes has assigned numbers to the various enzymes. Ly-
sozyme is 3.2.1.17.
Lysozyme is found in egg white, milk, many tissues and secretions of
animals, some molds, and in the latex of various plants. This widespread
distribution has made it a very popular substance for study. Lysozymes
from different animal species and from different organs of the same ani-

TABLE 4.8. MICROBIAL INHIBITORS IN EGG WHITE

Egg White
Inhibitor Effect on Microorganisms Solids (%)
Lysozyme Lysis of cell walls (Gram·positive bacteria) 3.5
Ovomucoid Enzyme inhibitor 11.0
Conalbumin Chelates metal, ions, especially ferric ions 13.0
(ovotransferrin)
Ovoflavoprotein Sequesters riboflavin
Avidin Binds biotin 0.05
pH Alkaline conditions restrict growth, accen-
tuate iron chelation
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 125

mal differ chemically and immunologically, but they possess the same
type of biological activity. The lysozyme content varies not only between
species, but also between strains of the same species. On a dry· weight
basis, chicken egg white contains about 3.5 percent lysozyme. This is the
most readily available source, and hence, the most studied of the lyso·
zymes.
Bacteria display various sensitivities to the lysis of the cell wall by
lysozyme. Certain micrococci are rapidly lysed by 1 J.tg/ml of lysozyme.
Bacillus megaterium requires 50 J.tg/ml and B. cereus barely is attacked by 50
Itg/ml of lysozyme.
Gram·negative bacteria are rather insensitive to lysozyme because of
their lipoprotein·lipopolysaccharide layer, which acts as a barrier pro·
tecting the sensitive mucopolysaccharides of the cell. If the barrier is
damaged by certain treatments, the enzyme can penetrate to the muco·
peptide layer and cause lysis, or partial lysis, of the cell wall. Starving
Gram·negative cells at an abnormal pH, treatment with polymyxin B, or
ethylenediamine tetraacetic acid, as well as reacting with complement, os-
motic injury or aerosolization can render these cells susceptible to lyso-
zyme action.
Enzyme Inhibitors. Enzyme inhibitors found in egg white are the ovomu-
coids, ovoinhibitors, and a ficin-papain inhibitor.
Rhodes, Bennett, and Feeney (1960) divided ovomucoids into four
classes according to their inhibitory activities. Some ovomucoids, such as
the chicken and goose, inhibit trypsin. Another group inhibits chymo-
trypsin (golden pheasant). A third group (turkey) inhibits approximately
equal amounts of trypsin and chymotrypsin, and the fourth group in·
cluded those from duck and emu, which inhibit twice as much trypsin as
chymotrypsin. A duck ovomucoid inhibited a commercial bacterial pro-
teolytic enzyme.
Matsushima (1958) discovered an inhibitor in egg white which he
termed ovoinhibitor. This substance inhibited fungal proteinase of an
Aspergillus and a bacterial proteinase prepared from a strain of Bacillus
subtilis.
There is no evidence that these enzyme inhibitors have any signifi·
cant effect on microbial growth in egg white.
Avidin. Each mole of avidin combines with two moles of biotin. Thus,
organisms having a strict requirement for biotin as a nutrient are in-
hibited by this compound. The binding of avidin to bacterial cells has
been discussed in detail (Korpela et al. 1984).
Conalbumin. This protein comprises about 12 percent of the total solids
of egg white. It forms a stable complex with two ferric or ferrous ions
per molecule, and by this action it inhibits organisms that require iron
126 BASIC FOOD MICROBIOLOGY

for growth. This includes Gram-negative and Gram-positive bacteria as

well as yeasts. It is evident that conalbumin does not destroy the microor-
ganisms but merely inhibits growth by extending the lag phase. Feeney
and Nagy (1952) postulated that dissociation of the complex would al-
ways result in traces of free iron which, when used by the organism,
would allow the release of further iron from the complex. This results in
slower-than-normal growth of the organism.
pH. The high pH established when carbon dioxide leaves egg albumen
inhibits the growth of many microorganisms.
It might be postulated that because of lysozyme, and inhibition of
Gram-positive bacteria, spoilage of eggs is primarily due to Gram-
negative types. In the egg, conalbumin is the main inhibitor of Gram·
negative bacteria. Although it does not prevent growth, it does inhibit or
delay it. The albumen was more inhibitory at 39.5°C than at 30°C
(Tranter and Board 1984). It is important that microbial growth be in-
hibited, because it is necessary to have shell eggs in commercial channels
for the time needed for grading, packing, distribution, and retailing.

DAIRY PRODUCTS. Several substances may be present in milk that

inhibit microorganisms. These include antibiotics, pesticides, bacterial
viruses, sanitizing and cleaning compounds, and various natural inhibi-
tors.
The naturally occurring microbial inhibitors in raw milk include lyso-
zyme, cationic proteins, agglutinins or antibodies, leucocytes, lactenins,
lactoperoxidase, lactoferrin, and perhaps, fatty acids. Many of the natural
inhibitors in milk are heat labile and not found in pasteurized milk.
Of thirty-seven samples of cow milk tested, only one contained lyso-
zyme at a level of 5 mg/kg (Carroll 1979). Only 10 percent of 200 samples
of camel milk inhibited one or more of six test organisms (Barbour et al.
1984). The lysozyme of these samples averaged 6.48 mg/kg.
The antibiotic nisin is produced by certain strains of Streptococcus
lactis, a common dairy organism, and may be present in small amounts
in raw milk and certain milk products.
Cationic Proteins. These proteins carry a positive charge at physiological
pH values and react with the anionic binding sites of cell walls and mem-
branes of bacteria (MacMillan and Hibbitt 1973). This causes an in-
creased permeability of the bacterial membranes, resulting in leakage
from the cells. Hibitt, Brownlie, and Cole (1971) isolated cationic proteins
from bulk milk samples. The antimicrobial activity was not destroyed by
heating to 70°C for 30 min but was almost completely destroyed at
100°C. These proteins have activity to both Gram-positive and Gram·
negative bacteria.
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 127

Lactoperoxidase. Lactoperoxidase is the peroxidase enzyme in milk. Peroxi·

dases catalyze the oxidation of molecules, with peroxide (usually hydro·
gen peroxide) as the oxygen source. For antibacterial effects, besides per·
oxidase and H 20 2, the presence of thiocyanate (SCN-) is required. Reiter
and Harnulv (1984) suggested adding minute amounts of H 2 0 2 and
SCN- to milk to enhance lactoperoxidase effect and the shelflife of milk.
The action on cells is greatest at pH 5.0 or less (Reiter et al. 1976).
According to the review of Reiter and Harnulv (1984), this system may
be bacteriostatic or bactericidal. Bacterial enzymes are inhibited, the cy·
toplasmic membrane is damaged, and synthesis of protein, DNA, and
RNA are inhibited.

Lactoferrin. This is an iron·binding protein in milk that acts on bacteria

similar to conalbumin in egg white. Normal milk contains 0.1 to 0.3 mgl
ml. This is increased from four to ten times in mastitic milk and in colos·
trum (Harmon et al. 1975). Antibodies in milk enhance the activity of
lactoferrin (Dolby and Honour 1979; Spik et al. 1978). The addition of
citrate reduces the inhibitory effect of lactoferrin (Bishop et al. 1976).

Fatty Acids. Fatty acids may be inhibitory or stimulatory, or they may have
no effect on microorganisms. The action depends upon the type of or·
ganism, the type and concentration of the fatty acid, the pH of the growth
medium, or the presence of certain other compounds. Short·chain fatty
acids are about equally effective toward either Gram·negative or Gram·
positive cells. Fatty acids with six or more carbon atoms are more inhibi·
tory to Gram·positive cells than to Gram·negative cells. Long·chain fatty
acids cannot penetrate the lipopolysaccharide layer of Gram·negative
cells.
The mode of action and the type of inhibition depend upon the fatty
acid and the concentration. At low concentration, some fatty acids are
stimulatory, while at higher concentrations they are inhibitory to the
same organism. The toxic effect is, in general, bacteriostatic, but for cer·
tain organisms the fatty acids may be bactericidal at high concentrations.
The bacteriostatic effect may be due to the blockage of adsorption of
essential nutrients. With extended incubation, the cells seem to over·
come this difficulty, and have, at times, shown total cell growth greater
than when no fatty acids were present. Besides affecting the permeability
of the cell membranes and inhibiting transfer mechanisms, fatty acids
inhibit the action of enzyme systems.
Although fatty acids are found in various foods containing fats, they
have been of special concern to the dairy industry because of their preva·
lence in butter and cheese. The amount of free fatty acids increases dur-
ing the aging of cheese. Some common fatty acids are listed in Table 4.9.
128 BASIC FOOD MICROBIOLOGY

TABLE 4.9. SOME COMMON FATTY ACIDS

Common Name Systematic Name Chemical Formula
Saturated Acids
Formic acid' Methanoic acid HCOOH
Acetic acid Ethanoic acid CH,COOH
Propionic acid Propanoic acid CH,CH.COOH
Butyric acid Butanoic acid CH,(CH.).COOH
Caproic acid Hexanoic acid CH.(CH 2).COOH
Caprylic acid Octanoic acid CH.(CH2)6COOH
Capric acid Decanoic acid CH.(CH2)"COOH
Lauric acid Dodecanoic acid CH.(CH2)IOCOOH
Myristic acid Tetradecanoic acid CH.(CH2)'2COOH
Palmitic acid Hexadecanoic acid CH.(CH 2h.COOH
Stearic acid Octadecanoic acid CH.(CH2)'6COOH
Arachidic acid Eicosanoic acid CH 3 (CH.h"COOH

Unsaturated Acids
Crotonic acid trans·2·Butenoic acid CH,CH=CHCOOH
Palmitoleic acid g·Hexadecenoic acid CH 3 (CH 2),CH = CH(CH 2),COOH
Oleic acid cis-9-Octadecenoic acid CH 3 (CH 2),CH = CH(CH 2),COOH
Linoleic acid cis-9, cis-12-0ctadecadi- CH.(CH 2MCH 2 CH = CH).
enoic acid (CH 2),COOH
Linolenic acid 9,12,15-0ctadecatrie- CH 3 (CH.CH = CH),(CH.),COOH
noic acid
Arachidonic acid 5,8,11,14-Eicosatetraenoic CH.(CH 2 MCH.CH = CH).(CH.laCOOH
acid
'Formic, acetic, and propionic acids are included because they are listed as fatty acids in
some publications, although others consider butyric acid to be the simplest fatty acid_

TISSUE FOODS. Various natural microbial inhibitors have been found

in tissue foods (meat, poultry, fish). Lysozyme, discussed in relation to
inhibitors in egg white, is found in various tissues. Tolerances for antibi-
otic residues in meats have been established by the World Health Organi-
zation. Even though these inhibitors are at low levels, some microorga-
nisms may be inhibited.
A living animal has defense mechanisms to inhibit the invasion of
microorganisms. These include various antibacterial substances as well
as natural and immune antibodies. Although, after slaughter, the mecha-
nism for producing these materials is lost, those substances already pro-
duced in the tissue will remain. The extent to which they have an antibac-
terial function depends primarily upon their stability.
Tissues from cattle (brain, spleen, heart, kidney, and liver) possess
anti staphylococcal substances. Basic polypeptides with antibacterial
properties have been extracted from tissues (thymus, spleen, and thy-
roid). Synthetic basic polyamino acids are active against both bacteria
and viruses. Spermine and spermidine are polyamines found widely dis-
tributed in animal tissues and are inhibitors of many types of microorga-
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 129

nisms (Bachrach and Weinstein 1970; Fair and Wehner 1971). The anti·
bacterial action of basic polyamino acids is thought to be disruption of
normal cell functions due to their combining with components of the
cell wall.
Some hormones are bacteriostatic. Progesterone (progesterol) is anti·
bacterial to Gram·positive organisms, and diethylstilbestrol (DES) has a
bactericidal effect on Staphylococcus aureus (Yotis and Baman 1969) and
Candida albicans (Yotis and Haan 1978). Deoxycorticosterone inhibits
Gram·positive bacteria, yeasts, and molds. but not Gram·negative bac·
teria.

Plant Products
Since the discovery of antibiotics, there have been several surveys in
which crude plant extracts or juices were tested for antibacterial activity.
The extracts of several thousand plant species have been tested and, de·
pending upon the plants surveyed and the test organisms used, from 30
to 50 percent of the extracts showed some antimicrobial activity. Juices
and extracts from cabbage, carrot, green beans, celery, chicory, cucumber,
chard, okra, rhubarb, corn, and turnip have shown antimicrobial activity
to one or more microorganisms.
To be fair, it should be mentioned that some extracts have shown
stimulatory effects. Pea medium is used to detect heat-stressed anaerobes
in canned foods_
Several microbial inhibitors (flavanols, tannins, enzyme inhibitors,
lectins, cyclopropene fatty acids, phytic acid) found to be naturally pres-
ent in certain seeds, are discussed in a review by Halloin (1983). Although
they do have inhibitory properties, these compounds either impart unsat-
isfactory properties to foods, are toxic, or may enhance carcinogenic
properties of other compounds. These and some other selected micro-
bial inhibitors are listed in Table 4.10.
Besides those inhibitors, lysozyme is found in some plant products,
but at low levels. Enzyme inhibitors are found in various plant products.
Gossypol, found in cotton seeds, inhibits Gram-positive bacteria, yeasts,
and molds but has little or no effect on Gram·negative bacteria. The to·
mato contains tomatin or a-tomatine, which inhibits various fungi and
bacteria. Anthocyanin pigments in cocoa possess antibacterial proper-
ties. Tannins comprise a group of natural phenolic compounds. English
walnut meats, carob pods, sorghum grains, malt, mint, and peanuts in-
hibit microorganisms due to the presence of tannins. There is evidence
that tannins interfere with enzyme activity, as well as absorbing on the
cell surface and altering the permeability of the cell wall.
Benzoic acid and its salts are used as food preservatives and are found
130 BASIC FOOD MICROBIOLOGY

TABLE 4.10. MICROBIAL INHIBITORS NATURALLY PRESENT IN PLANTS OR FOOD

FROM PLANTS
Inhibitory Agent Plant or Food Organism Inhibited
Anthocyanin pigment Fruits Bacteria (enzymes)
Acetaldehyde Fruit tissue Yeast
Dimethoxyisoflavone Peanut Aspergillus flavus
Purothionins Wheat endosperm Pseudomonas, Xanthomonas,
Erwinia amylovora, Corynebac-
terium
Juglone Pecan Fusicladium effusum
Protease inhibitor Barley Aspergillus
Trypsin inhibitor Corn Fungi
Enzyme inhibitor Bean Fungal cellulase
Lectins "Seeds" Virus
Wheat germ Trichoderma viride
Lipids Peas Fungi
Free fatty acids Cocoa Aspergillus parasiticus
Tannin(s) Peanuts Aspergillus parasiticus
Mint Antiviral
Phenolic White potato A. parasiticus
Lupulone Hops Gram-positive bacteria
Humulone
Isohumulone
Methylxanthines Cocoa beans Aspergillus parasiticus
Cinnamaldehyde Oils (essential) Fungi
Citral
Perillaldehyde
Citronellal
Extract Carrot root Aspergillus parasiticus
Phytic acid (binds zinc) Soybeans Aspergillus parasiticus
H ydroxycinnamates Fruits, vegetables
p-coumaric acid Grapes Saccharomyces cerevisiae, Escher-
ichia coli, Staphylococcus au-
reus, Bacillus cereus
ferulic acid S_ cerevisiae
p-methoxy cinnamate Curcurma zedoaria Trichophyton rubrum
Aspergillus niger, S. cerevisiae
Caffeic acid Fusarium nivale
Chlorogenic acid Chicory Bacteria
Antiviral Grape juice Human enterovirus "inactiva-
tion"
Enteric virus
Grape juice, apple juice, Poliovirus
tea

naturally in many berries. Cranberries are especially prominent for con-

taining benzoic acid. Other compounds in cranberries with antimicrobial
properties were described by Chu, Clydesdale, and Francis (1973).
Dallyn and Everton (1970) found that some vegetable oils contain a
sporistatic or sporicidal agent. They believe that a peroxide precursor of
a carbonyl is involved in the antimicrobial activity.
Pectin is a component of plant products. Although pectin shows no
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 131

inhibitory properties to microorganisms, a product of hydrolysis of pec-

tin, ex methyl D-galacturonate, inhibits the growth of many Gram-negative
dysentery bacteria_
Olives contain oleuropein, a phenolic compound_ Juven, Henis, and
Jacoby (1972) reported that oleuropein was inhibitory to species of Lacto-
bacillus, Leuconostoc, yeasts, and molds_ An ethyl acetate extract of oleuro-
pein reveals two components (glycoside oleuropein and its phenolic agly-
cone), which have antibacterial activity_ Fleming, Walter, and Etchells
(1973) found that oleuropein was not inhibitory to lactic acid bacteria,
but two hydrolysis products, the aglycone and elenolic acid, were inhibi-
tory for four species of lactic acid bacteria_ The acid hydrolysate of oleuro-
pein showed inhibitory properties to species of Lactobacillus, Pediococcus,
Leuconostoc, Staphylococcus, Bacillus, Salmonella, Pseudomonas, Erwinia, and
Xanthomonas, but no inhibition of the yeasts that were used_ The inhibi-
tion of growth of Lactobacillus plantarum by oleuropein aglycone and elen-
olic acid also was detected by Federici and Bongi (1983)_ The inhibition
is due to membrane damage and leakage of cellular constituents_
Homogenates and extracts of onions and garlic have antimicrobial
activity_ It is thought that, when the bulbs are crushed, enzymes react on
compounds containing cysteine sulfoxide and form inhibitory thiosulfi-
nates_ One of the active compounds is allicin (thio-2-propene-1-sulfinic
acid-5-allyl ester)_ It is not known whether its action is due to enzyme
inhibition or to membrane disruption (Bogin and Abrams 1976)_ Of the
factors in onion, the lachrymatory factor (thiopropanal-S-oxide) had the
maximum inhibitory activity to spores of Aspergillus parasiticus (Sharma
et aL 1981)_ The inhibitory action was lost by heating, drying, aeration,
agitation, or prolonged storage_ Neither garlic nor onion oil inhibited
toxin production of Clostridium botulinum types Band E (DeWitt et aL
1979)_ Garlic extracts reportedly inhibit the growth of Cryptococcus neofor-
mans and Candida albicans_ Aqueous extracts inhibited the growth of Bacil-
lus cereus (Saleem and AI-Delaimy 1982)_ Higher microbial counts of on-
ion and garlic products are obtained when 0_05 percent K 2 SO:J is added
to the dilution water_
Fruits contain various organic acids that inhibit the growth of micro-
organisms_ A natural isoflavone found in some fruits has antifungal activ-
ity (Fukui et aL 1973)_ The essential oils of citrus fruits are found in the
peel and can be recovered from this waste product_ The disinfectant
power of lemon and orange oil against spore-bearing organisms is re-
portedly greater than that of phenoL Orange oil at 0_1 percent and 0_2
percent was found to inhibit both Gram-positive and Gram-negative bac-
teria (Subba, Soumithri, and Rao 1967)_ At 0_2 to 0_3 percent orange and
lemon oil suppressed growth of Aspergillus parasiticus and aflatoxin pro-
duction (Alderman and Marth 1976)_
132 BASIC FOOD MICROBIOLOGY

Several spices or herbs and especially their essential oils inhibit var-
ious microorganisms (Shelef 1983)_ M ycotoxigenic molds were inhibited
by cloves, allspice, anise seed, eugenol extracted from cloves, thymol
from thyme, and anethol from anise seed (Hitokoto et aL 1980), by cloves,
cinnamon, mustard, allspice, and oregano (Azzouz and Bullerman 1982),
by o-methoxycinnamaldehyde from cinnamon (Morozumi 1978), and by
various essential oils (Benjilali et aL 1984).
Low levels of oregano or cloves stimulated acid production of Lactoba·
cillus plantarum and Pediococcus cerevisiae, while higher concentrations in·
hibited acid production (Zaika and Kissinger 1979,1981). Lactic acid bac·
teria developed a resistance to the herbs oregano, rosemary, sage, and
thyme (Zaika, Kissinger, and Wasserman 1983). Gram·positive bacteria
were more sensitive than Gram-negative bacteria to the spices and herbs
tested (Shelef, Jyothi, and Bulgarelli 1984).
Eugenol from oil of cloves was more effective than vanillin, an alco-
holic extract of the vanilla bean, in inhibiting medically important yeasts
(Boonchird and Flegel 1982). According to Davis and Cooks (1982), eu·
genol is also a constituent of nutmeg.
Apparently there are inducible resistance responses in plants to
pathogenic bacteria (Caruso and Kut: 1979; Goodman 1978), which might
carryover to the fruit.

Summary
There are many natural microbial inhibitors in foods. These inhibi-
tors give the living animal or plant some protection from invading para-
sites. The food products, being an extension of the animal or plant, have
a residual amount of these agents. Most of these natural compounds in-
hibit Gram-positive more than Gram-negative organisms. Perhaps this is
one reason that spoilage is more often associated with Gram-negative
organisms.
The microbial inhibitors naturally present in foods may maintain the
food in a satisfactory state for transportation, processing, and distribu-
tion.

PROTECTIVE BARRIERS

Some foods are protected from direct contact with microorganisms

by a natural cover or barrier. Examples of barriers are the shell and shell
membrane of eggs, the testa of seeds, and the cuticle of intact plant
organs.
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 133

Egg Shell and Membranes

An invisible, natural, proteinlike film on the egg shell, called the cuti·
cle, or bloom, is considered by some to be the first line of defense against
microbial penetration of the egg. The second physical barrier is the egg
shelL Inside the shell are two membranes, the outer and inner shell memo
branes, which are the third and fourth barriers to microbial penetration.
The structure of the egg and egg shell is shown in Figures 4.5 and 4.6.

CUTICLE. Washing of eggs and the use of abrasives to remove dirt

disrupt the cuticle layer and allow easier penetration by microorganisms.
There is a diversity of opinion on the effect of washing eggs on the result·
ant contamination of the interior. The cuticle tends to crack and deterio·
rate with time. Hence, it is not much of a barrier for stored eggs.

EGG SHELL. The egg shell is not a homogeneous structure. It consists

of an organic framework of fibers and an interstitial substance of inor·
ganic materiaL The two main layers of the shell are the outer or spongy
layer and the inner or mammillary layer. The outer layer is thicker and
contains most of the minerals.
The shell contains from 6,000 to 8,000 microscopic pores. These
pores allow the exchange of water vapor and gases between the contents
of the shell and the outer atmosphere. The pores vary in size, with ex·
tremes being reported as 1.6 to 7.47 p.m. The average pore size is prob·

ALBUMEN YOLK
GI,. iul disc (Bludod,,.)
Latebll
Lltht yolk Ilyer
Oark yolk Ilyer
Yolk (Vit,lIine) .,.brut

MEMBRANE
SHELL Air etll
Out" shell .e.brln,
Inn" .h,1I ••• bllne

Figure 4.5. The parts of an egg.

Courtesy of USDA.
134 BASIC FOOD MICROBIOLOGY

POR;~NALS

"", / \',
I CUTICLE

2. SPONGY LAYER

:: } 3 . MAMMILLARY LAYE"

_ ___
-- ---=- -7'.- - - -•., ___.. ~ ) 4 . SHELL MEMBRANE
'" ,'''
, !
5. MAMMILLA (MAMMILLARY KNOB) 6 . PROTE IN MATRIX MATERIAL FORMING
CORE OF THE MAMMILLA

Figure 4.6. Magnified radial section through an egg shell.

Courtesy of USDA.

ably between 20 and 45 p.m. The size of most pores will allow the passage
of many microorganisms. Even yeast cells can be forced through pores,
and mold mycelia grow through the pores. There is a linear relationship
between shell porosity and infection of the eggs.
Shell weight and shell thickness do not correlate with penetration of
microorganisms. However, thin shells have a tendency to crack more
readily than thicker shells. Eggs with damaged shells are more subject to
penetration of bacteria and egg spoilage.
If the temperature ofthe egg is higher than the surroundings, a condi-
tion that exists when the egg is laid, there is a greater possibility of pene-
tration and, as the temperature differential increases, the potential for
infectivity increases. As the egg contents cool, they contract, which results
in a higher pressure outside the shell than inside. This pressure differen-
tial will force microorganisms through the pores of the shell. For further
information about the egg shell, refer to Board (1980) and Tung, Gar-
land, and Gill (1979).
Moisture on the egg shell, such as occurs during washing of eggs or
when a cold egg is brought into a warm and humid room, increases the
potential for microbial infection of the egg.

SHELL MEMBRANES. The outer shell membrane is in contact with

the egg shell, and the inner shell membrane, sometimes called the egg
membrane, is in contact with the albumen. They are composed mainly
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 135

of protein fibers (fibrin and mucin) strengthened with an albuminous

cementing material. The membranes have been considered effective bac·
terial filters. The inner membrane is more effective than the outer memo
brane because of its closely knit fibers. Organisms inoculated onto the
shell surface are found on the membranes almost instantly, but micoor·
ganisms inoculated into the air cell and onto the inner membranes are
not recoverable from the albumen for several days. This apparent reten·
tion of bacteria on the inner membrane may be partly due to conalbumin
in the albumen, since adding iron to the inoculum or to the interior
albumen of the egg hastens the penetration of the bacteria through the
inner membrane. Wedral, Vadehra, and Baker (1971) found no differ·
ence in the permeability of the inner shell membrane before and after
passage of either Pseudomonas aeruginosa or Salmonella typhimurium, indicat·
ing that no enzymatic activity is needed for penetration. With the condi·
tions of their experiment, both bacterial species were able to penetrate
from the shell surface through the inner membrane within two hours. By
36 hr after shell inoculation, all except one egg showed contaminated
albumen. Although there are no pores, per se, in the membranes, there
are apparently openings between the intermeshed fibers similar to a
maze, which allow passage of bacteria through the membranes.
Even if bacteria can penetrate these barriers, they are confronted with
the antimicrobial mechanisms of the albumen before they can attack the
nutrients in the yolk.

Barriers in Plant Products

Foods such as fruits, vegetables, grains, and nuts contain a peel, skin,
or hard surface that covers the interior food and protects it from micro·
bial attack (Halloin 1983). The effectiveness of these barriers is evidenced
by the growth of mold primarily on the stem end of fruit, where injury
to the cover may occur during picking. Also, when injury occurs to the
barrier by insects, birds, or bruising, the microbial population in that
area is high and rotting of the fruit occurs there, before spreading to the
remainder of the fruit.
In the section on inhibition, the presence of essential oils in the peel
of citrus fruit is discussed. There are inhibitors in the waxlike coating on
many fruits. Cabbage leaves contain wax esters on their surface. The wax·
like coating of apples increases during storage, the extent of the increase
depending upon the cultivar.
The protective coverings are not only microbial barriers, but also
help control diffusion of materials into and out of the fruits or nuts. The
sale of nuts in the shell has remained fairly constant, while purchases of
shelled nuts have increased. Removing the nuts from the shell subjects
136 BASIC FOOD MICROBIOLOGY

them not only to microbial contamination but also to oxidative rancidity

of the fatty material and other deteriorative changes.
The seed coat and peri carp of grains, which allow the long· term stor·
age of these foods, were important in establishing human civilization.
According to Halloin (1983), the seed coat is the most important compo·
nent in the resistance of seeds to deterioration.

TEMPERATURE

Since water, as the solvent, is necessary for microbial growth, the tem·
peratures that will support microorganisms are limited to those at which
water is a liquid. Pure water freezes at oDe, but the addition of solutes
lowers the freezing point. The exact minimum temperature for microbial
growth is difficult to determine, because the solutes that are added to
prevent freezing of water below -lODe may inhibit the microorganisms.
Water boils at lOOoe and is no longer acceptable for growth at that
temperature. The boiling point of water is increased by adding solutes
or by increasing the pressure above atmospheric. By increasing the pres·
sure in a chemostat, a culture of Bacillus caldolyticus grew at 105°e
(Heinen and Lauwers 1981). With further increased pressure and higher
boiling points, bacteria might grow at much higher temperatures.
The absolute highest and lowest temperatures for growth remain un·
known, primarily because of problems of incubation at extremely high
or low temperatures.
Temperature is one of the most important environmental factors that
regulates the growth of microorganisms. Temperature is related to the
ability of an organism to grow and to survive. The environmental temper·
ature also has an effect on cell size, metabolic products such as pigments
and toxins, nutritional requirements, enzymatic reactions, and the chem·
ical composition of cells.

Ranges for Growth

Each organism has a mInimUm, optimum, and maximum temper·
ature for growth. The minimum and maximum temperatures are those
beyond which the organism ceases to grow. The optimum temperature is
more difficult to describe, since it may be the optimum for total cell yield,
rate of growth, rate of metabolism, respiration, or the production of
some metabolic product. Usually the optimum temperature is based on
the rate of growth.
In general microbiology, the microorganisms are usually divided into
three arbitrary classes: psychrophilic (low temperature), mesophilic (me·
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 137

dium or middle temperature), and thermophilic (high temperature). Psy·

chrophiles and thermophiles are divided into facultative and obligate
types.
The temperature ranges for the various types are listed in Table 4.11.
Microbiologists tend to disagree on the exact ranges of growth for the
different types. The fact that some organisms grow at low, some at inter·
mediate, and others at high temperatures is more important than the
exact temperature for these ranges. The approximate temperature
ranges for the growth of selected microorganisms are listed in Table 4.12.
One should remember that the growth temperature depends upon the
strains of a species and on the chemical and physical properties of the
substrate.
In general, organisms can survive and show some growth at temper·
atures considerably lower than the optimum temperature. When the tem·
perature is increased above the optimum, there is usually a very rapid
decline in the growth rate. The maximum temperature is usually only a
few degrees (5 0 -12°C) above the optimum. Above the maximum temper-
ature, growth ceases and the life of the cell is in jeopardy.

PSYCHROPHILES AND PSYCHROTROPHS. There are many defini

tions of psychrophilic organisms. One of the simplest definitions is that
psychrophiles grow well at OOC. They should produce a visible colony at
that temperature in seven, ten, or fourteen days. This definition does not
consider optimum, minimum, or maximum temperatures. Perhaps some
distinction should be made between those organisms with a maximum
temperature of 20°C or less and those that can grow at a higher maxi-
mum. Morita (1975) suggested that psychrophiles have an optimal tem-
perature for growth at about 15°C or lower, a maximum at about 20 o e,
and a minimum at ooe or lower. Organisms that grow at low temper-
atures but do not meet the definition are called psychrotrophs. The term

TABLE 4.11. APPROXIMATE TEMPERATURE RANGES OF GROWTH

FOR ARBITRARY CLASSES OF MICROORGANISMS
Temperatures roC)
Minimum Optimum Maximum
Psychrophilic -15-5 10-30 20-40
Obligate -15-0 10-20 20-22
Facultative -5-5 20-30 30-40
Psychrotrophic -5-5 25-30 30-40
Mesophilic 5-25 25-40 40-50
Thermophilic 35-45 45-65 60-90
Obligate 40-45 55-65 70-90
Facultative 35-40 45-55 60-80
138 BASIC FOOD MICROBIOLOGY

TABLE 4.12. TEMPERATURE RANGES OF SELECTED MICROORGANISMS

Temperature (O°C)
Microorganism Minimum Optimum Maximum
Bacteria
Acetobacter 5 42
Aeromonas 0-5 25-30 38-41
Bacillus cereus 10 47-50
Brevibacterium 5 42
Clostridium 0-45 60
C. botulinum 3-10 30-40 42-45
C. perfringens 15-20 30-40 45-51
C. putrefaciens 0 20-25 30
C. thermosacchamlyticum 43-45 55-62 70-71
Escherichia coli 3-10 37-41 48-50
Lactobacillus 5 30-40 53
Leuconostoc 10 20-30 40
Micmcoccus 10 25-30 45
Moraxella -1-2 30 41-42
Pmpionibacterium 2-3 30-37 45
Pmteus 10 37 43-45
Pseudomonas -7-4 20-30 31-43
P. aeruginosa 8 42
P. fluorescens 0-6 20-25 40
Salmonella 5-10 35-37 46-49
Staphylococcus 5-10 35-40 46-48
S. aureus 5-10 35-39 44-48
Streptococcus c?'emoris 25-30
S·faecalis 5-10 37 49-51
S.lactis 10-15 25-30 40
S. thermophilus 20 40-45 52
Vibrio 10-37
V. parahaemolyticus 3-13 35-37 42-44
Xanthomonas 0-5 25-31 40
Yersinia enterocolitica 0-4 37
Molds
Aspergillus fumigatus 30-40
Botrytis cinerea -1 20 30
Cladosporium -5--8
Penicillium rubrum 25-28
Rhizopus stolonifer 5 25
Yeasts
Candida 0 29-48
C. lipolytica 5 25 35-40
Hansenula 37-42 50
Saccharomyces 0-7 20-35 40
Torulopsis 0 17-25 30-35

psychrotroph was suggested by Eddy (1960) to apply to microorganisms

capable of multiplying at SoC and below, regardless of the optimum tem·
perature. This definition does not include the minimum or maximum
temperature. Eddy's definition of psychrotroph is essentially that of a
psychrophile, except that the temperature has been increased from 0° to
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 139

5°C. There are few, if any, obligate psychrophiles in foods. Most microor·
ganisms that grow in food at low temperature are psychrotrophic.
The spoilage of refrigerated food (meat, milk, eggs) is generally due
to psychrotrophic growth. Psychrotrophs are found in many genera and
include aerobes and anaerobes.
The pseudomonads are an important group of psychrotrophs. Some
members of the genus can grow at 4°C, while others grow at 43°C. Over·
all, the optimum temperature is somewhere between 20° and 30°C. P.
fluorescens grows at 4°C or less, but not at 41°C. The usual range for P.
aeruginosa is 8° to 42°C. The optimum temperature for pigment produc-
tion of P. jluorescens is 20°C. The diameter of colonies of P. jluorescens in-
creases at a more rapid rate at 30°C than at 25°C or less (johnson et al.
1970).

MESOPHILES. The mesophiles are organisms that grow in the middle

temperature range. We can define mesophiles as those organisms with
an optimum temperature of 25° to 45°C. There are two groups of organ-
isms in the mesophilic range. The saprophitic organisms have an opti·
mum temperature of 25° to 30°C, and potential pathogens have an opti-
mum between 35° and 45°C.

THERMOPHILES. These organisms have been defined as growing at

high temperatures, with an optimum of 45°C or higher. The information
in Table 4.11 shows the minimum temperature for thermophiles some-
where between 35° and 45°C, the optimum between 45° and 65°C, and
the maximum between 60° and 90°C. Since the minimum temperatures
for thermophiles overlap the higher mesophilic range, the thermophiles
have been divided into facultative and obligate types. The facultative
thermophiles can grow in the mesophilic range of 37°C or less, but the
obligate thermophiles cannot grow at 37°C.

ENUMERATION. If the arbitrary temperature classes have any mean-

ing, methods for determining the organisms should follow the defini·
tions. The incubation condition for psychrotrophs is listed as 7°C for ten
days (APHA 1984).
Juffs (1972) compared the extremes of the British Standards Institute
method of incubating at 5° to 7°C for seven to ten days to determine
psychrotrophs. The high extreme (7°C for ten days) is the same as the
APHA (1984) method.Juffs found that 7°C for seven days or 5°C for ten
days gave counts that were not significantly different. However, counts
from plates incubated at 7°C for ten days were significantly higher, and
those using 5°C for seven days were significantly lower.
The incubation period of seven or ten days becomes impractical as
140 BASIC FOOD MICROBIOLOGY

a control measure for perishable refrigerated foods such as milk. By

the time the count is obtained, the product is consumed or may have
developed defects. As a continuing control procedure, it is of value by
revealing potential problems. To obtain a psychrotrophic count sooner,
incubation at 21°C for 25 hr yields counts similar to the standard psy·
chrotrophic method (Oliveria and Parmellee 1976).
Comparing the incubation temperature of 7°C to the definitions of
classes, it fits neither the OOC given for psychrophiles nor the 5°C given
for psychrotrophs. Since the term psychrotroph has been substituted for
psychrophile, and using the APHA plating system, we might define psychro·
trophs as those organisms that grow at 7°C and produce a visible colony
in ten days.
The mesophilic count can be related to the standard plate count, or
aerobic plate count. Incubation temperatures of 25° to 37°C have been
used. The 37°C temperature indicates the potential pathogen popula·
tion better than the lower temperature; however, since spoilage organ-
isms are a problem and higher counts are obtained, a temperature of
32° or 35°C is used (APHA 1984).
Thermophiles are enumerated by incubating the culture at 55°C
± 2°C for 48 to 72 hr (APHA 1984).

RELATIVE IMPORTANCE. The relative importance of each of the

temperature classes (psychrophiles, psychrotrophs, mesophiles, and ther-
mophiles) depends upon the field of microbiology. Much of our food
supply is held in cold storage, which makes psychrotrophs important as
potential spoilage organisms.
Psychrotrophic microorganisms are ubiquitous in nature. Although
they are favored by cold, they are not confined to temperatures of 5°C
or less. Psychrotrophs are in fresh and salt water, in soil, and in or on
nearly all raw food. Stokes (1968) reported that psychrophiles constituted
35 to 95 percent of the bacterial population on meats and 67 percent of
the population on fresh chicken. At low temperatures (0°-5°C), carbo-
hydrate fermenters generally are inhibited and acids are not formed. Pro-
teolytic and lipolytic organisms grow at low temperatures, resulting in
food defects.
Mesophilic species can be found in nearly every genus of organisms
important in foods. Although spoilage of foods by me sop hiles is signifi-
cant, there is more concern with the mesophilic organisms that cause
foodborne illness. These include species of Salmonella, Staphylococcus, Clos-
tridium, Shigella, and Bacillus.
Since the rate of growth of microorganisms and enzyme activity is
higher in the mesophilic range than in the psychrophilic range, spoilage
of food occurs sooner.
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 141

Thermophiles can cause spoilage whenever foods are held at 50° to

70°C. This may occur during heating of the food while cooking, process·
ing, or pasteurizing. Their growth in hot syrup may cause undesirable
effects in sugar·processing plants. In some tropical countries, thermo·
philes-not psychrotrophs-are the important spoilage organisms.
The generation time is shorter for thermophiles than for either psy·
chrophiles or mesophiles when each is grown at its optimum temperature.
This results in a more rapid spoilage of food by thermophiles.
The thermophiles may be more important than is presently realized.
Since food is not normally held at temperatures conducive for growth of
thermophiles, they have not been given the attention that has been allo·
cated to the other classes. Due to the rapid growth and metabolism at
high temperatures, the new biotechnology is examining thermophiles for
direct use in fermentations or as a source of enzymes (see Chapter 9).

Effect of Organisms on Temperature

Just as the environmental temperature alters the growth of microor·
ganisms, the growth of microorganisms alters the temperature. When the
organisms metabolize organic compounds, they not only grow and multi·
ply, they also produce heat as a by·product. In a compost pile, the temper·
ature may rise to a level at which only thermophilic bacteria can grow.
In the production of compounds by microbial fermentation, it is often
necessary to use a cooling system in the fermenter to maintain the tem-
perature so that the maximum amount of desired product is developed.

GASEOUS ATMOSPHERE

The type of gas in the atmosphere surrounding the food may deter-
mine the types of organisms that become dominant. Oxygen in the atmo-
sphere will favor the growth of aerobic types. The lack of oxygen, or a
vacuum, will allow facultative anaerobes to become dominant.
Microorganisms vary widely in their tolerance to carbon dioxide. In
a CO 2 atmosphere, the growth of some microorganisms is completely
suppressed, while others are less affected. The lag period for growth of
spoilage organisms on beef is increased in a 10 percent CO 2 atmosphere.
Also, these organisms need a higher minimum water activity for growth
in 10 percent CO 2 as compared to the normal atmosphere (0.033 percent
CO 2). On the other hand, it is well documented that the presence of low
concentrations (1 percent or less) of CO 2 stimulates oxygen uptake of
bacteria. The amount of stimulation varies with bacterial species.
142 BASIC FOOD MICROBIOLOGY

According to King and Nagel (1975), CO 2 inhibits the synthetic proc-

ess of Pseudomonas aeruginosa, since resting cell metabolism is not affected_

MICROBIAL INTERACTIONS

Microorganisms can inhibit or stimulate the growth of one another.

Although pure cultures are usually used in laboratory studies, mixed cul-
tures are found in nature and on food products_ Since the main goals of
all living forms are self-preservation and perpetuation of the species, the
microorganisms have to compete for food and the other necessities. As
a result, there are various actions and reactions (or coactions) that may
be harmful or beneficial. Burkholder (1952) listed nine possible coac-
tions when organisms are in a mixed population:

Predation. In this relationship, the strong predators damage the weak

prey.
Parasitism. The weak parasite benefits at the expense of the strong
host.
Commensalism. The coaction results in the weak benefiting, and the
strong is unaffected.
Amensalism. The opposite of commensalism. The strong benefits and
the weak is unaffected.
Allotrophy. In this relationship, the strong feeds the weak.
Allolimy. The strong starves the weak.
Symbiosis. In symbiosis, there is mutual aid, with both organisms
benefiting from the relationship.
Synnecrosis. There is mutual conflict resulting in the death of both
organisms.
Neutrality. Neither organism benefits or loses from the relationship.
There is no coaction between them.

Systems for determining interactions of microorganisms have been

reported (MacFarlane and Makrides 1982; Nordbring-Hertz 1983).

Metabolism
The growth of microorganisms depends on the chromosomally deter-
mined primary metabolism. The loss of primary metabolism results in
the death of the cell. Secondary metabolism is not essential for the
growth and multiplication of microorganisms. Both primary and second-
ary metabolism produce metabolic products. Quite often the production
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 143

of secondary metabolites is induced by the presence of plasmids. The

plasmids are composed of extrachromosomal nucleic acids that can be
eliminated from the cell by various treatments and, under acceptable
conditions, can be transferred from one cell to another. Simple and rapid
methods for isolation and preparation of bacterial plasmids have been
described by Anderson and McKay (1983) and Summerton, Atkins, and
Bestwick (1983). By using plasmids to transfer genetic information, we
may be able to develop more useful organisms than we have now.
The products of metabolism may stimulate the growth of other organ·
isms. A strain of Staphylococcus aureus that required thymine and trypto·
phan was able to grow in mixed cultures or with culture filtrates of
Pseudomonas aeruginosa (Gadbois, De Repentigny, and Mathieu 1973). The
Pseudomonas supplied the thymine and tryptophan essential for the
growth of the S. aureus.
U sing a chemically defined medium that supported the growth of the
yeast Saccharomyces cerevisiae, but not Proteus vulgaris, researchers found
that both organisms grew in mixed culture (Shindala et al. 1965). A niacin·
like factor, produced by the yeast, made it possible for the bacterium to
grow. When niacin was added, the Proteus was no longer dependent upon
the yeast and promptly outgrew it.
Several bacteria isolated from bananas stimulated the germination of
the conidia of Colletotrichum musae on this fruit (McCracken and Swin·
burne 1980). On an agar medium deficient in L-cysteine, Legionella pneu-
mophila did not grow, but when Flavobacterium breve was present, the Legio-
nella grew as satellites around the Flavobacterium colonies (Wadowsky and
Yee 1983).
Although these citations show one organism aiding another or-
ganism, the dominance of one species can result in the utilization of the
nutrients so there is little or none left for other species.

pH. Microorganisms can alter the pH upward or downward, and

thereby influence the growth of other microorganisms. The lactic acid
bacteria (lactobacilli, streptococci, pediococci, leuconostoc) inhibit sev-
eral bacteria (both spoilage types and foodborne pathogens) and viruses
partially by acid formation with low pH and also by some undetermined
factors (Abdel-Bar and Harris 1984; Collins-Thompson, Wood, and Be-
veridge 1983; Gilbert et al. 1983; Moon et al. 1982; Rao and Pulusani
1981; Reddy and Ranganathan 1983; Ross 1981; Talon, Labadie, and Lar-
pent 1980). Montville (1982) reported that, in a model system at pH 4.4,
growth of Bacillus licheniformis raised the pH sufficiently to allow growth
and toxin production by C. botulinum. However, he found no toxin in
canned tomatoes with elevated pH values.
144 BASIC FOOD MICROBIOLOGY

INHIBITORS. The production of microbial inhibitors by microorgan·

isms is well known. Besides the chemicals called antibiotics, there are
other metabolic compounds that have antibioticlike or inhibitory charac·
teristics. Several enzyme inhibitors are produced by microorganisms, but,
according to Umezawa (1982), most of them have no significant antimi·
crobial activity. Some microbially produced inhibitors are listed in Table
4.13.
The pseudomonads produce several antibiotic substances, including
pyocyanine and pyrrolnitrin. Species of Pseudomonas influence the
growth of strains of Vibrio parahaemolyticus, S. aureus, Bacillus, as well as
some molds and yeasts (Collins·Thompson, Aris, and Hurst 1973; Goat·
cher and Westhoff 1975; Hemming et al. 1982; Scannell et al. 1972; Van·
denbergh et al. 1983).
Strains of species vary in their interactions with other organisms. In
comparing the interactions of four strains of Streptococcus cremoris and
four strains of S. lac tis, it was found that two strains of S. cremoris domi·
nated the four S. lac tis, one strain of S. cremoris was compatible with the
S. lac tis, and one strain of S. cremoris was dominated by the four S. lactis
strains (Reddy et al. 1971).
Bacillus species interact with other microorganisms. B. subtilis pro·
duces an antibiotic called subtilin. This polypeptide has a marked action
against a wide range of Gram'positive, acid·fast, and certain Gram·
negative bacteria. Depending on the concentration, subtilin is bacterio·
static or bactericidal. Barr (1975) found ten antimicrobial metabolites
produced by a strain of B. subtilis. Some of these were antibacterial, while
others were antifungal. Bacillus species isolated from mud inhibited the
growth of C. botulinum type C (Graham 1978). A thermophilic species of

TABLE 4.13. SOME TYPICAL MICROBIALLY PRODUCED MICROBIAL INHIBITORS

Inhibitory Substance Organism
Organism Produced Inhibited Reference
Serratia marcescens Prodigiosin E. coli, B. subtilis, Kalesperis, Prahlad,
Enterobacter aero· and Lynch
genes, S. aureus, P. (1975)
aeruginosa
P. aeruginosa Amino acid anti· Bacillus sp. Scannell et al.
metabolite (1972)
Saccharomyces Glycoprotein Torulopsis glabrata Bussey and Skipper
cerevisiae (1976)
Lactobacilli Hydrogen peroxide Pseudomonas sp. Price and Lee
Bacillus sp. (1970)
Proteus sp.
C. perfringens Unknown C. botulinum Smith (1975)
S.lactis Nisin C. botulinum Scott and Taylor
(1981)
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 145

Bacillus not only provided some protection of mushroom beds from the
mold Chaetomium olivaceum but also increased the yield of Agaricus bisporus
(Tautorus and Townsley 1983). A B. subtilis controlled the onion white
rot organism, Sclerotium cepivorum (Utkhede and Rahe 1983).
Volatile metabolites of certain strains of bacteria were able to inhibit
the growth, sporulation, and mycotoxin production of Penicillium and
Aspergillus species (Barr 1976).
Aflatoxin, a substance produced by the mold Aspergillusflavus, inhibits
various Bacillus species (Lillehoj and Ciegler 1970). Also, fusariotoxin
T·2 produced by Fusarium inhibits the growth of Saccharomyces cerevisiae
and S. carlbergensis (Schappert and Khachatourians 1983).
Certain strains of Saccharomyces cerevisiae produce a toxin that kills sen·
sitive strains of this yeast (Wickner 1983; Young 1981).
Antagonism among microorganisms may be due simply to competi·
tion for space or nutrients. One organism may gain an advantage over
another because of its motility (Lauffenburger and Calcagno 1983) or to
environmental effects (temperature, moisture) favoring one organism
over the others.
Fatty Acids. These substances are discussed as inhibitors naturally present
in foods. They also are formed by microorganisms during metabolism.
The growth of Clostridium botulinum is inhibited on surface·ripened
cheese due to the formation of fatty acids by Brevibacterium linens. Salmo·
nella gallinarum is inhibited by Leuconostoc citrovorum due to the acidic pH
and production of acetic acid (Sorrells and Speck 1970).
Hydrogen Peroxide. The mechanism of hydrogen peroxide (H 20 2) forma·
tion by streptococci was described by Anders, Hogg, andJago (1970). The
amount of H 2 0 2 that accumulates varies among strains. The addition of
catalase or ferrous sulfate to milk prevents peroxide accumulation and
results in an increased rate of acid production by the lactic streptococci
(Gilliland and Speck 1969).
Organisms such as the micrococci produce catalase. It was reported
by Nath and Wagner (1973) that in the presence of a Micrococcus species,
the growth of, and acid production by, six cultures of lactic acid bacteria
is stimulated. The stimulation is greater than that observed with the addi·
tion of catalase, indicating that factors besides hydrogen peroxide reo
moval are involved.
Hydrogen peroxide produced by Streptococcus mitis and Lactobacillus
acidophilus, in combination with a peroxidase and a halide, has viricidal
activity to polio virus (Klebanoff and Belding 1974).

BACTERIOCINS. The bacteriocins are bactericidal substances pro·

duced by various species of bacteria. Although considered antibiotics,
146 BASIC FOOD MICROBIOLOGY

they are not like the classical antibiotics since they are macromolecular,
including or consisting of polypeptide or protein, and they generally act
on strains of the same, or closely related, species (Vidaver 1983).
The colicins have been studied the most extensively. They are found
in E. coli and other Enterobacteriaceae. There are over twenty types of
colicins. In some respects, they act similarly to phage. They can be in·
duced by UV light, similarly to prophage. In some cases, they are held
intracellularly, and when released, the cell is lysed.
When bacteriocins attach to the receptor sites of a sensitive cell, one
or more things may occur, depending upon the bacteriocin and the cell.
One of the effects is the inhibition of macromolecular synthesis, such as
proteins, RNA, and DNA (Konisky 1982). Some colicins inhibit only pro·
tein synthesis, while others inhibit all three. Inhibition of respiration may
occur with some colicins, while others may block the function of per·
meases. Interference with the formation of ATP, or enhancement of its
breakdown, has been reported. Leakage of cellular constituents has been
reported but this may be due to secondary reactions and not caused di·
rectly by the bacteriocin. Colicins disrupt transport mechanisms of
amino acids, which results in a lack of protein synthesis.
Bacteriocins of Gram·positive organisms generally have a wider spec·
trum of activity than those produced by Gram-negative cells.

Parasites
Two of the parasites of bacteria are the bacterial viruses or bacterio-
phages and the bacterial genus, Bdellovibrio.
Some phages have a limited number of microbial species that are sus-
ceptible hosts. In a species of bacteria there may be susceptible and resist-
ant strains of bacteria. For other phages, there may be several species
and even different genera of bacteria that contain susceptible cells. For
instance, some pasteurella phages also attack strains of Salmonella and
Shigella. The limited hosts for phages makes it possible to use phage typ-
ing to differentiate bacteria.
The Bdellovibrio resemble the virulent bacteriophages in their ability
to lyse bacterial cells. However, unlike the phages, these microorganisms
are actively motile (a single flagellum), small (0.25-0.4 p.m wide and 0.8-
1.2 p.m long), vibroid, Gram-negative bacteria. The formation of plaques
(holes in a host lawn due to lysis of cells) is usually developed in 12 to
24 hr by phage, but Bdellovibrio plaques are visible only after two to four
days, and they enlarge up to six days of incubation. Host-independent
Bdellovibrio populations have been grown, which has not been possible
with phage. About one in a million cells is host independent, and these
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 147

host· independent cultures can revert to host·dependent varieties at

about the same rate.

SUBLETHAL STRESS

During the processing of foods, microorganisms can be subjected to

various physical or chemical stresses. These include treatments such as
heat, freezing, drying, osmotic effects, irradiation, and various chemicals.
To maintain the quality attributes of the food, the treatments may be
minimal and the effect on the microorganisms is of secondary consider·
ation. In other cases, the treatment may be an attempt to control certain
organisms, but others may be affected. These treatments that do not kill,
but damage or injure the cell, are called sublethal.
The damages or injuries to the organism due to sublethal treatments
are called lesions. As a result of the injuries, the growth capabilities of
the organisms may be altered, both in the food and on media during
enumeration. The stressed cells may have an extension of the lag phase,
more exacting growth requirements, or increased sensitivity to inhibitors
or to selective agents in media. The longer·than·normallag phase can be
called the recovery period, during which the damage to the cell is reo
paired. Although the cells usually can recover in a noninhibitory growth
medium, they can repair malfunctions in media in which growth is not
evident. In other words, growth is not needed for repair of the lesion.
It has been stated that injured organisms that normally cause food·
borne illness or food spoilage, when in a substrate conducive for repair,
can, after repair, be involved in these unacceptable activities. However,
in most populations with injured cells there are also some cells with no
injury. Hence, given sufficient time for multiplication, the uninjured cells
can cause foodborne illness or spoilage, without any assistance from the
repaired cells.

Heating
In several processes (scalding, blanching, pasteurization, cooking, and
spray drying), the food and associated microbial flora are subjected to
heat. Since none of these processes are designed to obtain a sterile prod·
uct, some organisms may be killed, many damaged, and others unaffected
by the treatment.
Heat·induced lesions include damage of the cytoplasmic membrane,
with leakage of cellular components, alteration of metabolic capabilities
148 BASIC FOOD MICROBIOLOGY

Qf the cell, impairment Qf enzyme activity, and degradatiQn Qf ribQsQmes

and ribQnucleic acid.
Heat-induced damage results in the IQSS Qf ability to. grow in cQndi-
tiQns in which nQrmal cells can grQw. N Qrmal S. aureus cells can grQw in
media with 7.S percent NaCI, but heat-damaged cells cannQt tQlerate 4
percent salt (SmQlka, NelsQn, and Kelley 1974). Repair, with the return
Qf salt tQlerance, Qccurs withQut grQwth in either a dilute synthetic Qr a
rich cQmplex medium (Hurst and Hughes 1981). The pH range fQr Qpti-
mum grQwth is mQre narrQW fQr heat-stressed cells than fQr nQrmal cells.
The additiQn Qf catalase Qr superoxide dismutase to. tryptic SQy agar pro-
tected heat-stressed cells frQm hydrQgen perQxide (Bucker and Martin
1982). This results in higher CQunts Qf sublethally injured cells (Egan
1979). For increased recQvery Qf injured E. coli cells in fQQd samples,
McDQnald, Hackney, and Ray (1983) suggested adding 3, 3'-thiQ-diprQpiQ-
nic acid to. supplement media fQr the repair process.
The temperature Qf incubatiQn befQre and after heating influences
the viability Qf E. coli (Katsui et al. 1982). They suggested that the fluidity
Qf the cell membrane was invQlved in this reactiQn. The repair Qf the
nucleQid structure Qf E. coli cells after heat shQck (SO°C fQr S min) is
different than that after mQre severe heat treatments (PellQn 1983).
All SQlid media were more inhibitQry to. heat-stressed cells Qf Salmo-
nella typhimurium than were the corresPQnding liquid media (Mackey and
Derrick 1982). HQwever, the additiQn Qf either catalase Qr pyruvate to.
nutrient agar resulted in increased recQvery and cQmparable grQwth to.
that in nutrient brQth.
Yeasts heated in water near the maximum temperature fQr grQwth are
essentially undamaged, but the presence Qf glucQse induces leakage Qf
cell CQntents (Hagler and Lewis 1974). Calcium iQns tempQrarily protect
the yeasts against glucQse-induced leakage. This indicates damage to. the
cytQplasmic membrane. If heated in the presence Qf glucQse, injury af-
fecting endQgenQus respiratiQn is irreversible.
Yeasts have been enumerated Qn acidified (to. pH 3.5) media; hQwever,
when twelve species Qf yeast were heat stressed, the IQwest Qptimum pH
fQr any Qf these was pH 6.8 and the maximum Qptimum was as high as
pH 10 fQr two. yeasts (N elsQn 1972). KQburger (1972) fQund that mQst
retail fQQd samples yielded maximum counts Qf yeasts and mQlds when
the medium was adjusted to. pH 8. FQr enumeratiQn Qffungi, the additiQn
Qf antibiQtics to. media, rather than acidification, to. cQntrQI bacteria, is
nQW the usual prQcedure. The effects Qf variQus chemicals in media Qn
recQvery or inhibitiQn Qf heat-stressed S. cerevisiae were discussed by Beu-
chat (1982). CQnner and Beuchat (1984) fQund that variQus species Qf
heat-stressed yeasts CQuid nQt repair their irUuries when eXPQsed to. rela-
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 149

tively low levels of essential oils of plants (allspice, cinnamon, clove, gar·
lie, onion, oregano, savory, thyme).
Heat injury can occur in the spores of Bacillus and Clostridium. The
germination system of B. subtilis is impaired by heating. Primary activa·
tion of spores at 90°C for 60 min, reduction of the incubation temper·
ature by 10° to 15°C or addition of CaCh and sodium dipicolinate, in·
creases germination and colony formation of heat·damaged spores. The
addition of lysozyme to enumeration media improves the recovery of
severely heated Clostridium spores. Bacterial spore injury and recovery
were reviewed by Foegeding and Busta (1981). Michels and Kagei (1983)
recommended using trypticase soy agar supplemented with cysteine,
methylene blue, and egg yolk emulsion for the enumeration of heat·
damaged spores of Clostridium sporogenes.

Cold Effects
Freezing of cells can cause an apparent increase not only in nutri·
tional requirements but also to sensitivity to selective agents used in me·
dia. Cells in the logarithmic growth phase are highly susceptible to cold
shock and show an apparent loss in viability, which can be recovered by
incubation with magnesium ions at 30°C. They gradually lose their ca·
pacity to recover if kept in cold magnesium·free buffer. Loss of viability
due to cold shock is apparently due to damage of DNA (Sato and Taka·
hashi 1970). The lethal effect of cold shock depends on the exposure
time to low temperature, the growth phase, and physiological condition
of the cells, the substrate, and the type of cell.

Drying
It is well established that organisms in dried foods may have meta·
bolic injuries that impair the proliferation of the cells in selective media
containing inhibitors in a concentration well tolerated by normal cells
of the same species. These stressed cells can recover their tolerance to
inhibitors if allowed to recuperate or repair in a nonselective medium.
Dried organisms are stressed by freezing (freeze·drying or lyophiliza·
tion), aerosolization (spray·drying), or heat (roller, drum, tunnel or spray·
drying), as well as existing in an environment of very low water activity.
The initial physiological state, the composition of the food, the loca·
tion of the organisms in the food, the processing history, storage condi·
tions, and the method of rehydration, will influence the survival and reo
covery of organisms from dried food.
Freeze·dried cells have altered permeability. Freeze·dried E. coli are
150 BASIC FOOD MICROBIOLOGY

susceptible to antibiotics that do not affect normal cells. Also, there is

leakage of RNA from cells stressed by freeze· drying. The alterations in
the stressed cells are reversible, since during or immediately after recov·
ery of cellular permeability, the damaged RNA, as well as metabolic dam·
age, is repaired (Sinskey and Silverman 1970). After freeze·drying, resyn·
thesis of cell wall and membranes and the reestablishment of transport
mechanisms are needed before cellular growth occurs. Many freeze·dried
cells require pyruvate, hematin, and menadione for recovery. The effec·
tiveness of the three compounds during repair of the cells is additive. It
is believed these compounds may aid in reestablishing the permeability
barrier of the cell.
Besides the need for nonselective nutrients in the recovery medium,
due to the need for rehydration, the temperature at which this is per·
formed is important. Ray, Jezeski, and Busta (1971a) recovered a higher
number of cells at 15° to 25°C, but there was an earlier initiation of
growth and more rapid growth if rehydration was done at 35°C. The rate
of repair is reduced by lowering the temperature of the recovery medium
from 35°C to lOoC and is extremely low at 1°C (Ray, Jezeski, and Busta
1971b).
Usually dried food is prepared for the analysis of microorganisms by
rapidly blending the food with a sterile diluent. However, a more gradual
system of rehydration such as soaking the food in diluent at a 1:1 ratio
for 30 to 60 min allows the microorganisms to adapt to a normal mois·
ture level. Rapid rehydration can cause lethal or sublethal injury to the
dried cells. This results in a lower count of cells when the sample is plated
onto a selection medium (Ting and Banwart 1985).

Chemical Inhibitors
Since preservation processes cause injuries to microorganisms, per·
haps chemical inhibitors may do likewise. Besides chemical preservatives,
organisms on equipment surfaces are exposed to cleaning and sanitizing
agents. If these organisms remain on the equipment, they may contami·
nate the food; if injured, they may be difficult to enumerate.
The action of physical factors can be readily altered and the microor·
ganisms tested for injury, but it is more difficult to eliminate chemicals
if they are sorbed to the cell or are inside the cell. In these cases, even
dilution may not eliminate the chemical effect on the cell, and death can
result.
Injury by acid and recovery of Salmonella bareilly was reported by Blan·
kenship (1981). For recovery, it was indicated that synthesis of protein
and RNA and electron transport are needed. The exposure of E. coli to
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 151

hydrogen peroxide or to low levels of cadmium causes single-strand

breakage in DNA (Ananthaswamy and Eisenstark 1977; Mitra and
Bernstein 1978)_ The transfer of injured E. coli to a cadmium-free liquid
medium resulted in recovery without significant synthesis of DNA (Mitra
1984)_
Chlorine injury to coliforms (E. coli) was manifested as inhibition of
uptake of metabolites, reduced adhesion and ability to colonize the small
intestine, and inhibition in selective media for enumeration (Camper
and McFeters 1979; Graham and Brenniman 1983; Walsh and Bisson-
nette 1983).

Ultraviolet Light
The ultraviolet portion of the spectrum includes radiations from 150
to 390 nm. When living cells are irradiated with this light, some may be
"killed" and others may be mutated. The UV wavelengths (around
260 nm) most active in producing either of these effects are those most
readily absorbed by the nucleic acids. Both lethal and mutagenic effects
of ultraviolet light can be partially reversed by repair or reactivation_
Although the ultraviolet light may be absorbed by many cellular com-
ponents, absorption by the nucleic acids results in damage to cellular
mechanisms for division. Repair of DNA in the light is called photoreacti-
vation. Reactivation of UV-irradiated cells also can occur in the dark
(dark repair, dark reactivation, or excision repair).
Various compounds have been given credit for activation or protec-
tion of irradiated cells. One such, the enzyme catalase, breaks down the
microbial inhibitor, peroxide, that is formed during irradiation. Another
action of ultraviolet light is the inactivation of disulfide enzymes due
to disruption of cystine_ Inactivation of enzymes can affect microbial
growth but not necessarily cause death.
Nalidixic acid (Eberle and Masker 1971), coumarin, pyronin Y, 6, 9-
dimethyl 2-methylthiopurine and caffeine (Grigg 1972; Koukalova and
Reich 1981) inhibit or block repair of UV-treated cells_

Other Repair Systems

Microoganisms can repair damage by gamma rays (Foegeding and
Busta 1981; Yatvin, Wood, and Brown 1972), fluorescent and photo light
(Eisenstark and Ruff 1970), as well as water stress (McFeters, Cameron,
and LeChevallier 1982; Rhodes, Anderson, and Kator 1983). The injury
and repair of these sublethal treatments are similar to those discussed
for other treatments.
152 BASIC FOOD MICROBIOLOGY

Summary
Various treatments as discussed in this section can cause lllJury to
microorganisms. Often these injuries can be repaired, but the treatment
rendered to the organism can be lethal depending upon the extent or
amount of treatment. A truly lethal injury cannot be repaired, but micro·
organisms given a sublethal treatment, under certain conditions, can reo
cover, grow, and multiply. In some instances, the treatment appears to
kill the cells, since they are not able to multiply. However, they may still
be alive, since they are metabolizing and even growing, such as the fila·
mentous forms of E. coli.
The sublethal injuries are important since, without repair, the organ·
isms are not able to grow on selective media used in enumeration of a
particular type. Microorganisms in a stressed condition are more suscep·
tible to inhibition and death by other stress factors.

OTHER FACTORS

Environmental conditions, such as pressure, surface tension, surfaces,

light and photosensitizers, and pesticides, as well as colloidal characteris·
tics, emulsion structure, and concentration of nutrients, might help deter·
mine the microorganisms that are able to metabolize, grow, reproduce,
and spoil food products or result in a public health hazard. Compared
to the other factors listed, these are of minor consequence in foods.

INTERACTIONS OF FACTORS

In a few instances, only one factor causes stresses on microorganisms.

The interations of these factors are important in foods.

Water Activity and Nutrition

The minimum aw for growth of a microorganism depends upon the
nutrients present in the growth medium. Christian (1955) showed that
the range of aw for growth of Salmonella oranienburg was appreciably
smaller in a chemically defined glucose·salts medium than in a nutrient
broth. The addition of small amounts of proline and four other amino
acids to the glucose·salts medium stimulated the growth of S. oranienburg
at 0.97 a w and permitted growth down to 0.96 au,. Addition of vitamins
showed further stimulation, and growth occurred at 0.95 a,u- Thus, the
minimum aw levels for microorganisms should be determined in a me·
dium with ample nutrients for the growth of the organism tested.
 FACTORS THAT AFFECT MICROBIAL GROWTH IN FOOD 153

Since foods differ in their nutrient content, microorganisms may be

able to grow in some foods at lower au, than required in other foods.

Water Activity and Temperature

The greatest tolerance to low aw occurs at the optimum temperature.
As the temperature is changed from optimum, the range of au, is reduced
in which spore germination and growth occur. Studying the growth of
fungi on jam, Horner and Anagnostopoulos (1973) found the interaction
of aw and temperature had a significant effect.
For Salmonella survival and multiplication, there was a close correla·
tion between au, and storage temperatures of meat and bone meal (Liu,
Snoeyenbos, and Carlson 1969). The relationships of aw and temperature
as well as pH and antifungal agents on growth of Aspergillus flavus and A.
parasiticus were determined by Holmquist, Walker, and Stahr (1983).

Water Activity and pH

As the pH is increased or decreased from the optimum, the minimum
aw needed for growth is increased. Decreasing the pH of the growth me·
dium increases the minimum au, at which Clostridium botulinum spores will
germinate and initiate growth (Baird-Parker and Freame 1967).

pH and Temperature
Outgrowth of spores of Clostridium botulinum type E occurred at
15.6°C and a pH of 5.4 to 5.6. When the temperature was lowered to
5°C, a pH of 6.2 to 6.4 was needed for outgrowth to occur (Emodi and
Lechowich 1969).
The optimum pH for B. subtilis spore germination is markedly
changed by altering the incubation temperature (Ishida, Ishido, and Ka-
dota 1976). The optimum pH was 7.4 at 37°C and 5.4 at lO°C.

Eh and pH
There is a definite relationship between the pH and the functional
OR systems in bacterial cultures. Staphylococci are more acid tolerant
aerobically than anaerobically (Barber and Deibel 1972). Most staphylo-
coccal strains initiate growth and produce detectable enterotoxin at pH
5.1 when grown aerobically. In anaerobic conditions, most strains fail to
produce enterotoxin below pH 5.7. When held at 5°C, Brochothrix grew
aerobically at pH as low as 5.4, but anaerobically no growth was evident
154 BASIC FOOD MICROBIOLOGY

below pH 5.8. It grew readily at pH 6.0 or higher (Campbell et al.

1979).

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162 BASIC FOOD MICROBIOLOGY

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 5
Sources of Microorganisms

The microbial flora of a food consists of the microorganisms associated

with the raw material, those acquired during handling and processing,
and those surviving any preservation treatment and storage.
Since these microorganisms do not arise by spontaneous generation,
they must contaminate the food at some stage of production, harvesting,
handling, processing, storage, distribution, or preparation for consump'
tion. Most foods are subjected to many potential sources of microorgan·
isms.
Why should we be concerned with sources of contamination? Primar-
ily so that we can control contamination and keep the microbial load on
or in the food as low as possible. By doing this, we obtain a longer shelf
life for the food; we hope that this reduces the chance of microbial food-
borne illness when the food is ingested. By keeping the contamination
low, we can more easily control or eliminate the microorganisms with
food-preservation techniques.
The potential sources of contamination are soil, water, air, plants,
feed or fertilizer, animals, human beings. sewage, processing equipment,
ingredients, product to product, and packaging materials.
Microorganisms can be exchanged between these sources. For exam-
ple, animals contaminate the soil with fecal material. Then rain washes
the microorganisms into the creeks and rivers. This water may be used
for irrigation and contaminate plants used for food. Thus, although wa-
ter is the carrier, the microorganisms originally come from animals.
For some foods, it is difficult to determine how many of the organisms
in the flora are contaminants and how many are the result of multiplica-
tion on or in the food.

SOIL

Soil is the natural habitat of many microorganisms which, at times,

may be found there in high numbers. The microbial density is greater
nearer the soil surface than at deeper levels. The types and numbers of
microorganisms vary with the type of soil (sandy, clay), as well as with
165
166 BASIC FOOD MICROBIOLOGY

environmental conditions. The environment is changing constantly, es·

pecially moisture and temperature.

Growth of Microorganisms
The microbial growth in soil is limited to areas of organic material.
These areas include the roots of plants, plant debris falling onto the soil,
animal carcasses, fecal deposition by animals, and dead microorganisms.
Besides the animal carcasses on the soil surface, dead earthworms, in·
sects, and other small animals are in the soil.
The growth of microorganisms is influenced by the chemical compo·
sition of the materials undergoing decomposition, the rate of decomposi·
tion of the chemical constituents of these substances, and the environ·
mental conditions. Fats, waxes, and lignins may gradually accumulate in
the soil because of their relative resistance to decomposition. This resid·
ual, called humus, is subject to slow and gradual attack by a variety of
microorganisms. The main factor affecting multiplication is nutrient
depletion. In general, the soil environment is not a good medium for the
growth of microorganisms. However, they tend to survive.

Number and Types of Microorganisms

The microbial numbers can vary from a few organisms in sandy and
desert soil to as many as 10 10 /g in fertile soil. Due to the usually poor
environmental conditions, microorganisms in soil are in some form of
resting stage or in a reduced state of metabolic activity. Spores of Bacillus
and Clostridium are quite prevalent in some soils. Generally, bacteria out·
number other types of microorganisms. Those that are common in food
and found in soil include Acinetobacter, Alcaligenes, Arthrobacter, Bacillus,
Clostridium, Corynebacterium, Flavobacterium, Micrococcus, Pseudomonas, and
Streptomyces. Yeasts are prevalent in the soil of orchards and vineyards.
Molds, especially spores, are found in some soils (Christensen, Raper,
and States 1978; McKenzie and Taylor 1983).
The natural habitat of Clostridium botulinum types A and B is soil. C.
botulinum type E was not found in cultivated soil or woodlands (Huss
1980). Thermophilic sporeformers that can spoil canned foods are found
in soil, usually in low numbers.
Enteroviruses survive sewage treatment and can be found in soil on
which treated wastewater is discharged. The viruses are adsorped to soil
particles, which makes them relatively resistant to inactivation. Another
important factor that influences inactivation is temperature. At 4°C,
poliovirus persisted at least 180 days in saturated soil (Yeager and
 SOURCES OF MICROORGANISMS 167

O'Brien 1979a). No infectivity was found in dried soil, regardless oftem·

perature. The loss of infectivity was due to irreversible damage including
dissociation of viral genomes and capsids as well as degradation of viral
RNA (Yeager and O'Brien 1979b). Poliovirus survives in soil longer in
winter than in summer (Tierney, Sullivan, and Larkin 1977).
Other microorganisms become inactivated in soil by predators (Cas·
ida 1980, 1983), bacteriolytic enzymes (Hemelt, Mares, and Upadhyay
1979), or toxins (Lynch 1982). In dry organic soil Ustilago spores remain
viable for one year, but in wet organic soil they should remain viable for
less than one week (Andreis 1980). St. John and Matches (1982) reported
survival of C. perfringens for at least twenty·four weeks in tilled soil.

Contamination of Food
Microorganisms in the soil can contaminate tubers or root crops by
direct contact. Also, dirt is blown by the wind or is splashed by rain fall·
ing onto the soil, so that the dirt can contaminate crops such as strawber·
ries, beans, cabbage, or peas that grow near the ground level. The micro·
bial numbers and types on crops are influenced by the degree of
contamination of the soil in which they are grown.
Mechanical harvesting has increased the amount of soil contamina·
tion as well as breakage of fruits and vegetables. Cereal crops are contam·
inated mainly during harvesting.
Marine sediments have microbial counts in the range of 104 to 109/g.
The numbers are usually higher near the shore than at deeper levels. The
bacteria found in these sediments include Aeromonas, Bacillus, Chromobac·
terium, Citrobacter, Escherichia, Pseudomonas, and Vibrio. These sediments
serve as a source of microorganisms for water as well as for fish and
shellfish. During trawling along the bottom with nets, the sediment is
disturbed so that it contaminates the fish or shellfish that are caught in
the nets.

WATER

Water is a potential source of microbial contamination of food. Rain

contains microorganisms that are washed from the air. As the water lands
on the ground, it is further contaminated by soil microorganisms. In the
ocean, organisms are in greater abundance near the shore than in reo
gions distant from land. The dumping of wastes such as sewage and the
runoff from animal feedlots result in considerable microbial contamina·
tion of waterways with enteric types of bacteria.
168 BASIC FOOD MICROBIOLOGY

Types of Microorganisms
The genera of bacteria that tend to be part of the "normal" flora of
water include Pseudomonas, Flavobacterium, Cytophaga, Acinetobacter, Mor-
axella, Aeromonas, Corynebacterium, Streptococcus, Klebsiella, Alcaligenes, Bacil-
lus, and Micrococcus. Also, enteroviruses are quite resistant to the treat-
ment given to sewage and therefore can be found in water supplies.
Escherichia coli is used as an indicator of fecal pollution of water in
temperate climates. This organism supposedly dies rapidly in the rela-
tively hostile environment of rivers due to low temperature, sunlight,
toxic chemicals, and a lack of nutrients. However, some reports indicate
that the coliforms and E. coli seem to maintain their population
(Coleman et al. 1974; Goodrich et al. 1970). Water is still the main carrier
of organisms that cause gastroenteritis in humans. From 1971 to 1982,
there were 387 outbreaks of gastroenteritis caused by consumption of
contaminated water in the United States_ Of these, 150 outbreaks were
caused by municipal water supplies (CDC 1983).

Food Contamination
Water contacts food during production, harvesting, and processing.
If the water used for irrigation of various crops is contaminated or is
sewage effluent, the fruits and vegetables can be potential health hazards
(Rosas, Baez, and Coutiiio 1984; Tamminga, Beumer, and Kampelmacher
1978; Tierney, Sullivan, and Larkin 1977; Van Donsel and Larkin 1977).
Seafoods are harvested from water. The microorganisms in the water
contaminate the surface, gills, and intestinal tract of fish and shellfish.
The occurrence of fecal coliforms in fish is a reflection of the pollution
level of their water environment. The skin of Atlantic salmon was ana-
lyzed by Horsley (1973). The predominant genera were Cytophaga, Flavo-
bacterium, Moraxella, and Pseudomonas. Other organisms found were Acineto-
bacter, Bacillus, Aeromonas, Vibrio, coryneforms, members of the families
Enterobacteriaceae and Micrococcaceae.
When bivalve mollusks feed, they filter large quantities of water and
concentrate bacteria and viruses that are present in the water environ-
ment. Shellfish are normally found in water near the shore. This water
is subject to contamination of runoff water carrying soil microorganisms
and sewage outfalls. The accumulation and concentration of these micro-
organisms from the water is of particular concern when they are poten-
tial pathogens such as salmonellae and human enteric viruses.
Potential pathogens in the drinking water of animals can be a health
hazard to the animals and to people who work with the animals. These
 SOURCES OF MICROORGANISMS 169

organisms from infected animals also can contaminate the carcass during
the slaughter operations.
During harvesting, water may be used for hydrocooling of vegetables.
Irrigation or surface water that is polluted with sewage or animal wastes
can serve as a source of inoculation with various organisms, including
potential pathogens. The water can redistribute organisms already on
the vegetables from high levels of contamination or spoilage to other
areas so that all of the vegetables are contaminated. Since many vegeta·
bles and fruits are eaten raw, the use of untreated water to wash these
foods can serve as a vehicle of transmission of pathogenic organisms.
Ice is used to cool and maintain coolness in a variety of fresh foods.
As the ice melts, the organisms associated with it are passed on to the
foods and vice versa. The reuse of ice is neither a recommended nor an
accepted procedure, since this ice is contaminated. Chen and Chai (1982)
reported that ice·melt drainage in fishholds of fishing vessels contained
bacteria at levels of 10 7 to 109 /ml.
The use of water in a food· processing plant can be a source of micro·
organisms for contaminating food. Water is the food industry's number·
one raw ingredient. Water enters into the processing of most foods for
cleaning equipment and processing areas, for washing food, for convey·
ing (fluming) food, or as an ingredient.
The safety of water that is used in foods is of major importance. How-
ever, although considered to be safe for drinking (potable), municipal
water supplies are not always acceptable for food processing. This is be-
cause of the presence of food-spoilage microorganisms as well as chemi-
cals that may produce objectionable odors and flavors in food. The
organisms that tend to be present in municipal water supplies are
psychrotrophs such as pseudo monads which can grow in water with a
limited nutrient content. Hence, when a food processor stores water in
small tanks, the growth of the pseudo monads may result in microbial
levels of 10 5 to 10 6 per milliliter.
In the dairy industry, these psychrotrophs are especially troublesome.
The wash water may contain up to 10 3 psychrotrophs per milliliter and
be one of the most important sources of microbial contamination. Psy-
chrotrophs canse spoilage of refrigerated milk and milk products.
In poultry-processing plants, water is used in the scald tank for wash-
ing of the carcass and for cleanup of the building and equipment. There
has been much speculation that water from the scald tank is a source of
contamination of the carcass. This is due to the fact that the dirt from
the head, feet, and feathers, as well as feces (due to defecation at death)
contaminate the scald water. The temperature of the water (53° to 61°C)
can kill many pathogenic and spoilage types but is not sufficient to kill
170 BASIC FOOD MICROBIOLOGY

all of the microorganisms that are present, and thermophilic types can
grow. Bacterial counts of the scald water range from 10 2 to 10 6 per milli-
liter. When the birds enter the scald tank, they may be taking their last
breath and the heart may be pumping. The contaminated water then
can contaminate the internal portions of the bird through the lungs and
vascular system. The addition ofNaOH to raise the scald water pH to 9.0
effectively reduced the level of bacterial population (Humphrey, Lan-
ning, and Beresford 1981).
Ice water used to cool the carcasses after processing can be a source
of microorganisms that can penetrate deep into the skin surface (Thomas
and McMeekin 1982).
In food-canning operations, water is used to cool the cans of food
after heat processing. Due to the heat and expansion of metal, the seams
and seals on the cans are under stress, and leakage can occur. Hence, it
is important that the cooling water be maintained with as few microor-
ganisms as possible. Methods of monitoring this water for microorgan-
isms have been described (Rey et aL 1982). Thompson and Griffith (1983)
found clostridial spores at low levels in about 10 percent of their samples
of cooling water. Since no further processing occurs after cooling, any
contamination of the food by microorganisms that can grow in the
canned product may cause spoilage or, if pathogenic, foodborne illness.
Water is used as an ingredient in many foods. In these cases, the water
is a direct source of microbial contamination.
With the present shortages and high cost of water, there is an effort
to reduce water consumption and to recycle water in the food-processing
plant. Caution must be used so that contamination or high microbial
loads on foods do not result from these pratices.

AIR

Today, the main concern with air pollution is with chemicals (carbon
monoxide, hydrocarbons, soot, fly ash) rather than with biological agents.
Nature is the major contributor of not only the chemical pollutants but
of biological agents such as plant cells, animal hair, pollen, algae, proto-
zoa, bacteria, yeasts, mold spores, and viruses. Food is subjected to air-
borne contamination until it is sealed in a package.

Types and Numbers of Microorganisms

There is no natural or normal microflora of air, which is contami-
nated from various sources. Generally, mold spores are more prevalent
than are other microorganisms. The main source of microorganisms, es-
 SOURCES OF MICROORGANISMS 171

pecially mold spores, is decaying plant materials near the ground surface.
Contamination of the air is caused by gusts of wind picking up the micro-
organisms or spores_ Compared to mold spores, there are relatively few
yeast cells in the atmosphere, and these are mainly near ground level.
One would expect yeasts to be prominent in the air of orchards and
vineyards. However, even here, they may comprise only 25 percent of the
fungal population of air.
There are many sources of microorganisms for contamination of the
air. Liquids are aerosolized by spraying, splashing, the bursting of bub-
bles, by forcing through a small orifice, or by vibrations. Trickling filters
used in sewage-treatment plants produce aerosols by the spraying action
and by the splashing of liquid sewage. The application of wastewater to
land by spraying under pressure causes aerosolization of organisms.
Teltsch et al. (1980) devised equations to predict aerosolized pathogenic
microorganisms downwind from a wastewater spray or aeration site.
The types of organisms in air are often associated with the type of
activity in the area. Downwind from a sewage-treatment plant, Pereira
and Benjaminson (1975) found species of Klebsiella, Bacillus, Flavobacter-
ium, Streptococcus, and Micrococcus. Streptocci are found near dairies, and
yeasts are found near bakeries and breweries. The microbial flora of the
air in a food· processing plant reflects the sanitary condition of the plant
unless the air is purified. Incinerators, if not operated properly, can be
a source of aerosols that may contain infectious microorganisms.
Human beings shed organisms and also produce aerosols during talk-
ing, coughing, and sneezing. The microbial load of the air is proportional
to the numbers of persons in an enclosed space, their activity, and the
rate of air circulation. Although humans might not contribute signifi-
cantly to the microorganisms in outdoor air, they can be a significant
source for air in a processing room.
Gerba, Wallis, and Melnick (1975) reported that bacteria and viruses
become airborne when a toilet is flushed. These organisms settle out on
surfaces throughout the bathroom.
Thus, air is subjected to a number of sources of microorganisms (de-
caying matter, water, soil, human beings, animals, sewage). The numbers
and types of organisms present in the air or atmosphere depend upon
many factors, such as tendency to settle out and maintenance of viability.
There is a considerable variation of the microbial load of air. It has
been suggested that country air in the summer contains an average of
10,000 fungal spores/m3. Lacey (1973) suggested the level may reach 108 /m 3
during haymaking or harvesting. The handling of moldy grain or hay
indoors can result in a level of 109 spores/m3. One study found more
bacteria over an alfalfa field than over bare soil (Lindeman et al. 1982).
At 46 m downwind from a wastewater spray irrigation site there were
172 BASIC FOOD MICROBIOLOGY

10 4 bacteria/m3 (Bausum etal. 1982). Clark, Rylander, and Larsson (1983)

reported a level of 10 5 bacteria/m3 in both poultry· and swine·confine·
ment buildings. Lenhart, Olenchock, and Cole (1982) stated that the at·
mosphere in a poultry. processing plant contained bacteria that might be
a potential health hazard to the workers.
Lacey (1973) listed several genera of fungi as being present in rural
air, with Cladosporium predominating. This dominance was also reported
by Ogunlana (1975). Other researchers have reported that species of
yeasts, Aspergillus, Penicillium, Cladosporium, Alternaria, Helminthosporium,
and Fusarium are the most dominant fungi in corn dust (Hill et al. 1984).
The air near the earth is more contaminated than at higher altitudes,
although the microbial population at 2,000 to 3,000 meters is more stable
than that at lower altitudes. Air over land is more contaminated than
air over the ocean, although wind currents can carry micoorganisms for
several hundred miles out to sea. The upper atmosphere over the ocean
is more contaminated than air near the ocean surface.
Clouds often contain high levels of bacteria and fungal spores. Turbu·
lent air tends to carry high numbers of cells aloft. Temperature inver·
sions can affect the microbial load at various altitudes. Rain and snow
tend to wash the microflora from the air. The atmosphere is more con·
taminated in the summer than in the winter.

Survival
Although Dimmick, Wolochow, and Chatigny (1979) reported that,
with special conditions of growth and aerosolization, bacterial cells in air
can divide, this has not yet been shown for naturally occurring microor·
ganisms in air.
The stability of microorganisms in air is affected by the relative hu·
midity, temperature, oxygen, solar factors including ultraviolet radiation,
and chemical components. Spores of molds and bacteria retain their via·
bility better than do vegetative cells. Also, capsules help protect cells in
the atmosphere.
Aerosols of microorganisms have been produced in the laboratory
and the viability determined. Aerosolization damages the cytoplasmic
membrane and at least some of the associated transport mechanism (Ben·
bough et al. 1972).
The stability of bacteria in aerosols is similar to that of viruses. There
is rapid death during aerosolization, followed by a less rapid death rate.
The method of recovery and enumeration influence the apparent effect
of relative humidity and oxygen on survival of bacteria in aerosols.
When air is sampled downwind from a source of contamination,
there is a significant decrease in the microbial load as the distance from
 SOURCES OF MICROORGANISMS 173

the source increases. This is because the organisms settle out onto var·
ious surfaces, the organisms lose viability, and the air is contaminated by
less contaminated air. However, bacterial spores were found to travel in
air from near the Black Sea to Sweden (Bovallius et al. 1978).
Sunlight reduces the number of viable organisms in air. By analysis
of air near a spray irrigation system, Teltsch and Katzenelson (1978)
found higher counts at night than during the day.

Food-Processing Operations
The contamination of air in food·processing plants was reviewed by
Heldman (1974). The effect that air has on the microflora of food de·
pends upon the level of contamination of air and the time of contact of
air with food.
Aerosols are produced in food· processing plants by spray washing or
spray cooling of food, by high·pressure sprays used in cleaning, by flood·
ing of floor drains, by mixers and motors, and by the operation of various
other equipment. Workers in the area produce aerosols. The movement
of equipment, supplies, and people in a food plant causes turbulent wind
currents that increase the microbial load of the air.
There is considerable variation in the microbial load of air in various
areas of a processing plant. In clean areas there are very few organisms
in the air. In areas in which live animals are handled or raw products are
brought into the processing operation, the microbial load can be quite
high.
One method that is used to control the microbial load in the air of a
processing plant is to move air from clean areas to dirty areas or by using
positive air pressure in clean areas. With positive air pressure, if a door
is opened, air flows out of a room and outside air does not come in.
Fresh air entering the clean areas is filtered to remove dirt as well as
some microorganisms.

ANIMAL AND PLANT FOOD

Animal and plant food can serve as a source of potential chemical

and biological contamination of food.
Microorganisms in animal feed can contaminate the feet, hide, hair,
and feathers of animals. Consumption of the feed adds organisms to the
digestive tract. Those organisms that survive the rigors of digestion, upon
elimination, can contaminate exterior portions of the animal. When the
feed contains potential pathogens such as salmonellae, these may cause
illness in the animal, invade the body and locate in lymph nodes, contam·
174 BASIC FOOD MICROBIOLOGY

inate the carcass during slaughter, as well as contaminate external por-

tions of the animal (MacKenzie and Bains 1976)_
Plant food may contain organisms that can contaminate the surfaces
of plants and associated human foods. When animal or human wastes
are used as fertilizer, pathogens from these sources can contaminate the
plant and associated foods_ Since fruits and vegetables are eaten raw, they
can be a source of foodborne illness. In countries that use night soil as
a fertilizer for food crops, washing of the food is suggested. The decom-
position of plant tissue can cause an increase of potential toxin-forming
molds in fields (Griffin and Garren 1976).
Although pathogens may be present in manure or sewage used to
fertilize crops, sunlight tends to eliminate microorganisms from the
plant parts (Bell, Wilson, and Dew 1976; Jones 1976).

PLANTS

Plants are contaminated by microorganisms from several sources

(dirt, water, air, fertilizer, animals, and humans). Once contaminated, cer-
tain microorganisms can grow on the plant surfaces and plant pathogens
can attack their host plants. The microbial flora on plant surfaces varies
with the kind of plant.
One study found a total count of 10 9 /g including 10 7 coliforms, 10 8
psychrotrophs, 10 7 streptococci, 10 5 yeasts, and 10 6 molds per gram on
wild rice (Goel et al. 1970).
Pseudomonas species are quite prevalent on vegetables. Kominos and
colleagues (1972) isolated P aeruginosa from tomatoes, radishes, celery,
carrots, endive, cabbage, cucumbers, onions, and lettuce. They believe
that raw vegetables are a source of this organism contaminating patients
in hospitals. Various bacteria, including pseudo monads, can adhere to
and colonize the surface of leaves (Ercolani 1978; Hildebrand, Alosi, and
Schroth 1980; Leben and Whitmoyer 1979).
The flowers of fruits are inhabited by many genera of yeasts, includ-
ing the ascospore formers Saccharomyces and Hansenula, as well as the im-
perfect Torulopsis, Candida, Rhodotorula, and Kloeckera. In some fruits such
as grapes, the yeasts might not be present on the flowers but are found
on the ripe fruit.
A Pseudomonas and an Arthrobacter introduced into open flowers of
soybean plants were isolated from 24 of 177 resultant beans pods (Leben
1976).
There is evidence that not only the surface but also the interior tissue
of plants can be contaminated. Vegetables can harbor fecal streptococci
within unopened pods, heads, and other structures. Meneley and Stan-
ghellini (1974) found that 44 percent of apparently healthy cucumbers
 SOURCES OF MICROORGANISMS 175

contained bacteria internally. Enterobacteria (Proteus, Citrobacter, and

Enterobacter) were present in 10 percent of the cucumbers.
Thus, live plants can serve as a source of microorganisms. When the
plant dies, the decaying vegetation becomes an important source of air-
borne, waterborne, and soil microorganisms, which then contaminate
plants the following year.

ANIMALS

Animals have a normal or natural microflora that is established very

early in life. Besides this micro flora, animals tend to harbor the types of
organisms found in their environment, since they are contaminated by
soil, water, air, feed, and excreta.
Animals harbor organisms that can cause food spoilage or foodborne
disease. Organisms are found in animals' gastrointestinal tract, nasal pas·
sages, cutaneous lesions, and on the skin, feet, hair, or feathers on the
outer surface. These organisms are readily transferred to the edible por·
tions of the animal during processing.
Wild animals contaminate growing crops and stored products. One
of the means by which viruses are transmitted to plants is by insects (Cou-
driet, Kishaba, and Carroll 1979). Insects, birds, and rodents destroy the
protective covering on foods, so that not only do they contaminate the
food, they also make the food more susceptible to microorganisms that
cause spoilage. These animals also carry potential human pathogens that
may be transferred to the foods they contaminate (Kapperud 1975; Stek
1982).
Flies, much more so than many other types of common food plant
insects, are a serious potential carrier of disease-producing organisms.
Flies pick up organisms on their hairy legs and feet. They then contami·
nate human food by walking or leaving their excreta on it. Flies have a
part in the spreading of Salmonella, Shigella, Vibrio, Escherichia, and other
disease·causing organisms as well as food·spoilage types.
The muscle tissue of most live, healthy animals is essentially sterile.
In a diseased state or after slaughter (or after harvesting in the case of
fish), bacterial invasion of the muscle tissue can occur. Most of the micro·
organisms in tissue are thought to result from surface or intestinal con·
tamination or from processing operations. Vanderzant and Nickelson
(1969) removed muscles from slaughtered animals aseptically. The maxi·
mum number found in any sample was 1,400 per gram in beef. Of sixty·
five samples analyzed, forty· four did not yield isolates when incubated at
37°C. Hence, it is evident that even after slaughter, a majority of muscle
tissues are sterile.
One of the sources of microorganisms in the interior portion of ani·
176 BASIC FOOD MICROBIOLOGY

mals is the lymph nodes. These nodes tend to filter out bacteria from the
lymphatic system. Microbial counts of 105/g have been obtained from the
lymph nodes, with many types of organisms being present. Both cattle
(61 percent) and sheep (14 percent) at slaughter contained Salmonella in
lymph nodes (Samuel et al. 1981). Of forty·two hog carcasses passed by
federal meat inspectors, Brown and Neuman (1979) found mycobacterial
infection of the lymph nodes.
The microbial flora that grows on refrigerated beef is composed
mainly of soil organisms derived from the hide, hair, and hooves of the
animals. The hides of animals may contain from 10 3 to over 10 9 microor·
ganisms per square centimeter. Although they do not penetrate the intact
skin, these microorganisms are a source of contamination of the edible
portions of the carcass during processing.
The predominant organisms in the intestinal flora of both animals
and humans are obligate anaerobes such as species of Bacteroides and Pep·
tostreptococcus. They may reach levels of 10 10 to lOll per gram. The faculta·
tive anaerobes may reach levels of 10 7 to 10 9 per gram of intestinal con·
tents. These include coliforms, enterococci, and lactobacilli. The natural
habitat of the human pathogen Salmonella is the intestinal tract of animals
as well as of humans. Other organisms, such as fungi and viruses, can be
found in the intestinal tract. There is a possibility that microorganisms
can pass through the intestinal wall into other organs and tissues. Viruses
were found in chicken tissues that appeared normal (Robertson, Wilcox,
and Kibenge 1984).
During life, the intestinal wastes can contaminate the outer surface
of the animal. During slaughter and processing, the sphincter muscle be·
comes nonfunctional, and also the intestines can be punctured acciden·
tally. In either case, the contents and associated organisms can contami·
nate the carcass.
The circulatory system is sterile except for the occasional invader or
during an infection. Although a majority of samples of hemolymph from
the horseshoe crab contained no detectable microorganisms, 27 percent
did reveal low numbers of bacteria (Brandin and Pistole 1985).
The respiratory system acquires microorganisms from the air during
breathing. The nose filters out many microorganisms, and it tends to be
the most contaminated part of the system. The respiratory system may
serve as a passageway for microorganisms to contaminate internal tis·
sues.
The surface of fish may contain 10 2 to 105 bacteria per square centi·
meter and the intestinal contents vary from 10 4 to 10 7 organisms per
gram. These microorganisms reflect the condition of the environment
from which the fish are obtained (Austin 1982). This is especially evident
for shellfish (Ellender et al. 1980; Larkin and Hunt 1982; Metcalf et al.
1980; Perkins et al. 1980).
 SOURCES OF MICROORGANISMS 177

Milk in a healthy udder has few microorganisms, if any. However,

aseptically drawn milk usually ranges from less than 500 to 5,000 and
occasionally, up to 10,000 organisms per milliliter (Thomas, Druce, and
Jones 1971). The flora is predominantly coagulase·negative staphylo-
cocci, micrococci, and corynebacteria. Infection of the bovine mammary
glands is called mastitis. With this condition, counts of 10 5 or more per
milliliter of milk have been found. A variety of organisms can cause mas-
titis, and organisms such as coagulase-positive staphylococci, coliforms,
and streptococci, will dominate in the milk. Aseptically drawn milk con-
tains mesophiles, but few psychrotrophs or thermophiles. When milk is
obtained normally, it is contaminated by bacteria in the teat canal and
on the surface of the teat. The surfaces of the four teats, even after wash-
ing with disinfectant and drying, had a mean colony count of 10 6 bacteria
(Thomas, Druce, and Jones 1971). Perhaps this is due to the strong attach-
ment of bacteria to the teat surfaces (Notermans, Firstenberg-Eden, and
Van Schothorst 1979). Hibbitt, Benians, and Rowlands (1972) found an
average of 22,500 viable microorganisms per teat canal. McKinnon, Cous-
ins, and Fulford (1973) analyzed milk obtained normally from twenty-five
cows and found bacterial counts from 44 to 11,400 per milliliter, which
is similar to the so-called aseptically drawn milk referred to by Thomas,
Druce, and Jones (1971). Due to microorganisms normally present in the
teat canal, the first drawn milk contains more organisms than the last
portion from any particular quarter of the udder.
When an egg is laid, it is essentially sterile. Some bacteria (salmonel-
lae) and avian viruses may invade the ovaries of hens and contaminate
the yolk before the egg is formed. Even for those few eggs that might be
contaminated, the bacterial number is relatively low. Most of the contami-
nants on the egg shells are of intestinal origin. Other sources of orga-
nisms are nesting material, feather dust, the feet and body of the bird, as
well as subsequent handling and storage. Eggs obtained from poultry
flocks on wire have less bacterial contamination than do eggs from flocks
in houses with floor litter. This is because nesting material, including
feces, has less chance of coming into contact with the shell.

HUMANS

A human embryo develops in a relatively sterile environment. At

birth the baby is subjected to a massive invasion of microorganisms from
the mother, other people, bedding, air, food, water, and other materials.
The skin is the most available part of the body for colonization after
birth. Staphylococci are predominant on normal infant skin (Carr and
Kloos 1977). The colonization is followed by microbial invasion of the
nose, oral cavity, throat, and the respiratory, digestive, and urogenital
178 BASIC FOOD MICROBIOLOGY

tracts. Many microorganisms are merely transients, while others become

established as a normal, perhaps permanent, flora. Besides the microbial
population on or in a human, clothing can be contaminated by external
sources or by the person, and then serve to pass the microorganisms on
to the person or to food products.
The skin is never free of bacteria, and the dirtier the skin, the greater
the contamination. Normal human skin contains a relatively stable mi·
croflora. However, the numbers and types of microorganisms vary from
person to person, from site to site, and from day to day. There are differ-
ences in the environment at various places on the body. Some areas are
too dry and, in other areas, the pH is not satisfactory for bacterial growth.
The palm of the hand has primarily surface organisms, whereas the fore-
head has both surface and subsurface flora, but mainly subsurface. Sur-
face organisms consist of transient and normal resident types, while sub-
surface organisms are primarily normal resident types.
Washing the skin removes most of the transient microorganisms, but
it is practically impossible to remove all of the normal flora. Some micro-
organisms associate with the sweat glands, sebaceous glands, and hair
follicles, where they not only can grow but also are difficult to remove.
The predominant bacteria on the skin are staphylococci, corynebacteria,
and propionibacteria. Also, species of Micrococcus, Bacillus, Alcaligenes,
Pseudomonas, Enterobacter, Klebsiella, Proteus, Escherichia, and Citrobacter are
quite prevalent (Adams and Marrie 1982; Sunga, Heldman, and Hedrick
1970). S. aureus is found more often on the hands and face than on other
parts of the body. The organism is associated with the nose and is spread
when people handle their faces and noses. S. aureus is associated with
infections such as acne, pimples, and boils.
Humans are a source of airborne microorganisms as well as an impor·
tant source of food contamination through handling of food. The hands
are subject to contamination by considerable numbers of bacteria, most
of which are unable to multiply on the hands and usually die. However,
these transient bacteria are passed on to food products when they are
handled. Many of the transient bacteria result from the handling of foods
(DeWit and Kampelmacher 1982; Seligmann and Rosenbluth 1975).
Workers in meat- and chicken-processing plants had rather high levels of
E. coli, and several had salmonellae on their hands.
The hair covering the skin is a potential source of microorganisms.
There is no normal flora of the hair, but hair acts as a carrier to retain
and shed organisms. Beards, sideburns, and mustaches may be especially
bad if worn in a food-handling area. Barbeito, Mathews, and Taylor
(1967) found that, although washing with soap and water reduced the
level of contamination of beards, it did not eliminate microorganisms
and toxins from them. If a person grows a beard to hide acne or pimples,
 SOURCES OF MICROORGANISMS 179

or a nasal carrier has a mustache, these hairy growths will merely serve
for continued dissemination of S. aureus. Besides the microorganisms that
may be shed into foods, the hairs and hair clippings may contaminate
the food.
Clothing is directly associated with humans and can serve as a carrier
to contaminate foods with human microflora or can contaminate people
with environmental microorganisms. Laundering does not necessarily re-
move all of the microorganisms from clothing. The effectiveness of laun·
dering depends upon the type of organism, fabric, temperature, deter·
gent, antibacterial agents, bleaches, flushes and rinses, drying, ironing,
and final packaging. Viruses tend to adsorb to the fabric and are difficult
to remove. Microorganisms tend to remain viable in wool longer than in
cotton. Synthetic fabrics require less drastic laundering, allowing greater
microbial survival. Microorganisms tend to survive storage for a shorter
time on these fabrics than on either cotton or wool.
During laundering, microorganisms are transferred among the cloth·
ing in the same wash load, and it is conceivable that microorganisms can
carryover from one load to the next. Even dry cleaning does not assure
the sterility of clothes.
The gastrointestinal tract may be considered a hollow tube within the
body, but the internal portion is outside the body. The gastrointestinal
tract is important as a potential source of organisms, as well as the pri-
mary site of action of foodborne organisms causing illness (gastroenter·
itis). Factors that can influence the flora of the gastrointestinal tract in-
clude the diet, antibiotics or other chemotherapeutic agents, and certain
diseases.
Although enormous numbers of microorganisms are ingested with
food and with mucus from the respiratory tract, most do not survive in
the digestive system.
In the normal stomach, the microbial flora increases and decreases.
The ingestion of food or mucus causes a rise in numbers. The flora is
reduced by acid and digestive enzymes and by emptying of the stomach
into the duodenum. There are usually low numbers of organisms in the
normal stomach, duodenum, jejunum, and upper ileum. The pH of the
stomach varies among individuals. Higher pH values will allow the sur·
vival of more organisms. Foods tend to react with stomach acids and raise
the pH. Also, some microbial cells are imbedded in food particles, which
protects them from the acid.
Normal peristalsis, bile, and immunoglobulins tend to remove organ-
isms from the upper small intestine. However, drugs such as morphine
slow peristalsis; this and several other conditions can result in coloniza·
tion (Gracey 1979). Some organisms are able to adhere to the epithelial
cells of the small intestine by at least two systems (Kleeman and Klaen·
180 BASIC FOOD MICROBIOLOGY

hammer 1982). Most of the organisms that cause foodborne illness must
attach to the epithelium and become established in the intestine, where
they produce toxins (see Chapter 6).
As the cells move down the intestines, the conditions improve for
microbial growth. Active multiplication begins in the lower small intes·
tine and continues through the large intestine. Bacterial levels of more
than 1010/g of contents are attained in the colon and excreta. The redox
potential varies from - 150 mv in the duodenum to - 250 mv in the
colon (Holdeman and Moore 1972). This low level in the colon favors
anaerobic types of organisms.
The fecal flora of infants is composed primarily of species of Bifido-
bacterium. Other organisms include the anaerobic Bacteroides as well as
coliforms, enterococci, lactobacilli, and staphylococci (Mitsuoka and
Keneuchi 1977). In the adult, Bacteroides surpasses the anaerobic bifido-
bacteria. These two groups range from 10 9 to 10 10 cells per gram of feces.
The eubacteria and anaerobic lactobacilli are also at levels of 10 9 per
gram of feces. The remaining organisms consist of enterobacteria, enter-
ococci, lactobacilli, clostridia, bacilli, veillonellae, pseudomonads, yeasts,
molds, and viruses (Haenel 1970). The relationship of viruses to the fecal
microflora has not been examined thoroughly. Enteric viruses are pres-
ent in the intestines and feces of apparently healthy people.
Speck, Calloway, and Hadley (1970) tested the effect of diets on the
fecal flora. Their results indicated that a change in diet influences the
fecal microflora. The type of diet affected the aerobic population more
than the anaerobic microflora. A protein diet supported a higher aerobic
population than did either a carbohydrate or fat diet. Haenel (1970)
found no dramatic differences in the intestinal microbial flora with meat-
egg, milk-vegetable, vegetarian, or raw vegetarian diets. Even the con-
sumption of 1 kg of Bulgarian yogurt per day failed to dramatically alter
the microbial flora in most individuals. Conn and Floch (1970) fed eight
to twenty capsules, each containing five (10 10 ) viable Lactobacillus acidophi-
Ius for seven to ten days. They found no significant increase in fecallacto-
bacilli, but they did notice a decrease in total anaerobic bacteria. Similar
results were reported by Paul and Hoskins (1972). A comparison of Sev-
enth Day Adventists revealed few statistically significant differences in
the fecal flora of vegetarians and nonvegetarians (Finegold et al. 1977).
Although some reports indicate that diet does influence the fecal flora,
in a review, Hentges (1980) stated that there are minor and inconsistent
changes in fecal flora composition in response to dietary alterations.
People with acute diarrhea may show the following features of intesti-
nal dysfunction: (1) bacterial colonization of the small intestine with de-
fective microbial clearing systems; (2) malabsorption of fat, carbohydrate,
and vitamin B 12 ; (3) fluid accumulation due to alterations in electrolytes
and water transport; and (4) alteration of the mucosal morphology. Those
 SOURCES OF MICROORGANISMS 181

abnormalities are not necessarily manifested in all persons with acute

diarrhea or in all cases of diarrhea.
Mendes and Lynch (1976) found fecal organisms on various surfaces
in restrooms (door handles, flush handles, tap handles, toilet seat, floor).
The tap handles were more contaminated than the door handles. The
wash basin overflow was more contaminated than the floor and the area
under the rim of the toilet. They believed that their findings showed that
a person with Salmonella or Shigella infection could contaminate various
surfaces in a washroom or toilet. These areas could conceivably serve as
a source of contamination for other people.
Since people are carriers of pathogenic types of microorganisms, they
are especially hazardous when handling processed (cooked or pasteur-
ized) products that may be held for short periods and eaten with no fur-
ther treatment.
The hands of workers have been cited as increasing the microbial
load of many products_ Poultry products show increased counts when the
carcass is hand transferred from the picking to the eviscerating line, dur-
ing eviscerating, cutting up the carcass, and deboning by hand. In the
shellfish industry, the hand shucking of oysters and clams, shelling of
shrimp, and picking of crabmeat from the shell are associated with in-
creased bacterial counts. Slicing, weighing, and hand packaging of meat
products increase the contamination through human handling of the
product. The unpacking, trimming, sorting, and repackaging of fresh
produce (fruit and vegetables) provide an opportunity for contamination
of the food with human pathogens. Fresh produce may be eaten raw,
which makes contamination a potential hazard.
The carelessness of humans is an important cause of microbial con-
tamination of foods. Failure to properly clean and sanitize equipment,
failure to wash one's hands, working and handling food with an infec-
tion, poor personal hygiene, lack of care in handling of food, making
unsatisfactory alterations of equipment, and failure to keep foods at the
proper temperature are some of the careless things that people do that
can increase the microfloral load of foods.
Some shoppers have been seen opening containers of foods. This is
a deplorable practice that can cause contamination of the food with re-
sultant spoilage or a health hazard.
During the analysis of foods, microbiologists can be a source of con-
tamination (Denny 1972).

SEWAGE

Animal manure or, in some cases, human wastes are used as a fertil-
izeron crops. These biologically produced substances contain microorgan-
182 BASIC FOOD MICROBIOLOGY

isms, including human pathogens. When added to soil, these pathogens

may survive for periods sufficient to contaminate the harvested crop.
In rural areas, septic tanks are used. Quite often these are not prop·
erly installed and do not operate effectively, and raw sewage leaks into
the soil. Salmonellae are quite prevalent in raw sewage. Processing in
sewage· treatment plants lowers the incidence, but even at 99 percent reo
duction, if the original level of salmonellae is high, they remain in the
effluent and sewage sludge (Farrah and Bitton 1984; Langeland 1982;
Nabbut, Barbour, and AI·Nakhli 1982; Yaziz and Lloyd 1982).
Enteric viruses survive the treatment process, may remain infective
when bound to solids, and can contaminate soil and groundwater when
sewage sludge is applied to agricultural land (Goyal, Keswick, and Gerba
1984; Larkin et al. 1978; Ward and Ashley 1980).

EQUIPMENT

During the Industrial Revolution, machines were developed to do

most of the work of humans. Hence, there is less contact with food by
people and more contact by machines and equipment.
Thousands of small pieces of equipment, such as knives, cutting
boards, and bins, are used in food· processing or ·handling establish-
ments. Although most equipment is metal, some parts may be made of
rubber or plastic. In some cases, cardboard is used in boxes used to hold
harvested fruits or vegetables. Cardboard or paper is used as packaging
material for bulk shipment and storage of various ingredients.
Metal processing equipment does not support the growth of microor-
ganisms. It has no natural or normal microbial flora. Yet, food-processing
equipment is one of the major sources of contamination of foods.
Equipment may be cleaned and sanitized, but this does not mean that
it is sterile. Even on washed, visibly clean surfaces, the survival and
growth of bacteria are possible. Even visibly clean surfaces may have food
deposits or films that provide microenvironments acceptable for survival
and growth of microorganisms. The potential for microbial buildup on
equipment is enhanced when equipment is improperly cleaned and sani-
tized. If food residues are visible on cleaned equipment, one can be as-
sured of a potentially high microbial level.
Pitted surfaces or poorly soldered joints are places in which foods
can lodge on the equipment. During the course of the daily operation,
bacterial growth will occur in these food films and deposits. This then
serves as a source of contamination when food contacts these surfaces.
It is well established that microorganisms adhere to surfaces. Strepto-
coccus thermophilus adheres to stainless steel during the pasteurization
 SOURCES OF MICROORGANISMS 183

process (Driessen, DeVries, and Kingma 1984). However, it is easier to

remove soil and bacteria from stainless steel than from rubber or plastics.
Although stainless steel is more expensive than some other materials, it
pays for itself through savings in cleaning, as well as durability and prod·
uct quality. When flexibility is needed, such as in milking machines or in
chicken-picking machines, rubber or plastic becomes an essential part of
the equipment.
Gilbert and Maurer (1968) surveyed equipment in shops and cafes.
Swabbings from a meat-slicing machine revealed bacterial loads of 10 6
to 107/cm2 • The cleaning methods used were ineffective in reducing the
bacterial loads to an acceptable level. When we consider that most out-
breaks of foodborne illness are due to contamination of the food in
foodservice units or in the home, it should be obvious that the equip-
ment in the home and foodservice institutions needs more attention.

Meat
The contamination of meat by equipment begins with slaughtering
the animal. Contaminants from the knife used to cut the artery may circu-
late through the body during the bleeding process. Humane slaughter by
immobilizing the animal with CO 2 or electric stunning before bleeding
helps reduce the heart action and hence, contamination.
During the slaughtering operation, the carcass contacts various equip-
ment surfaces. Various pieces of equipment and humans can cause the
spread of Salmonella throughout the slaughter operation (Smeltzer,
Thomas, and Collins 1980a, b; Stolle 1981). When the carcass is cut up
and packaged for retail, further transfer of organisms is made from the
carcass surface by means of saws, knives, slicers, and cutting blocks. These
increased surfaces with their associated juices support large populations
of bacteria.
The grinding of meat further increases the surface area, releases
juices, and distributes the original surface bacteria throughout the meat.
Equipment may be involved in the distribution and increased counts of
the product. Meat grinders are highly contaminated with millions of bac-
teria (Dempster 1973).

Poultry
During processing there may be a tenfold increase in the microbial
count on the skin of poultry carcasses. Some of the increase is due to
contaminated scald water and to human factors. The equipment used in
processing the birds plays a role in contaminating the carcasses. At every
stage of the processing operation (Fig. 5.1) there is ample opportunity
184 BASIC FOOD MICROBIOLOGY

Slaughter (shackle, stun, sever throat)

~------------------------------------~
-I Bleed
+
I
I I+-'E---I
Spray Wash Defeather 1+-(---1 Scald I
t
1 I-I
Singe Cut hocks and vent 1 ~ I Eviscerate I

+
~-I Spray Wash \-1 Remove head and neck \
.
I Package and weigh I-I Refrigerate or freeze

Figure 5.1. Poultry-processing operation.

for microbial contamination (McMeekin and Thomas 1979; Thomas and

McMeekin 1980).
Schuler and Badenhop (1972) sampled seventeen equipment sites on
two sampling days in thirteen poultry-processing plants. On only five of
the pieces of equipment was the average bacterial count less than
1,000/cm2 on both sampling days prior to start of the operation. Sites of
contamination were the flexible fingers on the picking machine (11,000
to 99,000/cm 2 ), the hock cutter (7.000 to 97,000/cm 2 ), the transfer belt be-
tween dressing and eviscerating lines (5,000 to 56,000/cm2 ), lung gun
(3,500 to 73,000/cm 2 ), and cut-up belt (2,000 to 35,000/cm 2 ).
The picker fingers are difficult to clean properly, and the contamina-
tion of these fingers increases significantly during plant operation. There
is increased contamination of poultry carcasses during feather removal,
which can be due to leakage from the vent and the bacteria from the
feathers contaminating the skin via the flexible rubber fingers.
Further processing (cutting up the carcass and deboning) can increase
the microbial level of poultry meat (Denton and Gardner 1981). Sources
of organisms include both equipment and humans.

Milk
Since milk is a liquid, it is in contact with some type of equipment or
surface from the time it is removed from the cow until it is packaged.
Unsatisfactorily washed and sanitized milking and milk-handling equip-
ment constitute the main source of bacteria in milk at the farm level.
 SOURCES OF MICROORGANISMS 185

The bacteria in farm milk tanks include many psychrotrophs and few
thermodurics (MacKenzie 1973; Thomas, Druce, and Jones 1971). Ther·
modurics are more prevalent than psychrotrophs in pipeline milking
plants (MacKenzie 1973).
Psychrotrophs can grow and cause spoilage of refrigerated milk, but
few thermodurics grow below 10°C. Pasteurization of milk destroys most
of the psychrotrophs. Contamination of pasteurized milk by psychro·
trophs from poorly cleaned pipes and filling machines can result in spoil·
age of the refrigerated milk.

Seafood
When fish are caught they are subjected to contamination from fish
boxes (plastic, wood, or metal) and other fish·holding systems on the
ship. However, there are few, if any, bacteria in the flesh of fish before
filleting and subsequent handling.
Filleting may be done manually or by filleting and skinning machines.
When fish are filleted by hand, the knives and cutting boards provide a
source of microorganisms, and filleting machines become contaminated
by the surface microflora on the fish, which is transferred to the flesh of
the fillet. The bacterial count on the filleting equipment varies from 10 3
to 108 bacterialcm2 • When the bacterial load on the surface of the fish is
high, there is a higher bacterial count on the filleting boards. Gloves
worn by the filleters often have a higher bacterial load than the incoming
fish.

Fruits and Vegetables

The microbial load of fruits and vegetables increases during harvest·
ing and processing. Peas aseptically removed from the pods are essen·
tially sterile. When the peas are separated by a viner, the bacterial count
may reach 10 6 /g. The microbial load of most vegetables is reduced by
washing and by blanching. After blanching, the bacterial load increases
due to contamination by equipment and may attain levels of 10 4 to
105/g. The equipment causing contamination includes conveyor belts,
hoppers, and filling machines.
Splittstoesser (1973) stated that the source of most organisms on fro·
zen vegetables is contaminated equipment surfaces. His data show that
corn cutters, French bean slicers, spinach choppers, and inspection belts
for peas significantly increased the microbial level of these vegetables. In
one instance, he found that moving peas to the freezer by an airlift (to
186 BASIC FOOD MICROBIOLOGY

conserve water) resulted in significantly more bacterial contamination of

the peas than when they were flumed.

INGREDIENTS

The quality of a processed food is influenced by the quality of the

ingredients. Although ingredients may constitute a small part of the total
food, they may add a substantial number of microorganisms. Hence,
specifications, including acceptable microbiological levels, are needed
for the purchase or production of ingredients.
Spices or seasonings are often the source of high microbial numbers.
Spices are parts of plants such as dried seeds, buds, fruit or flower parts,
bark, or roots, usually of tropical origin. These seasonings and flavors
may contain over 108 aerobic bacteria per gram. Also, they contain aero·
bic and anaerobic spores. A sample of ginger contained 48 million total
bacteria, 12 million yeasts and molds, 26 million aerobic sporeformers,
and 0.72 million anaerobic sporeformers (Tjaberg, Underdal, and Lunde
1972). Pepper is usually highly contaminated with bacteria and fungi.
Flannigan and Hui (1976) reported that fourteen of twenty spices con·
tained Aspergillus jlavus and four of these spices supported growth of this
mold and the production of aflatoxin. Mean standard plate counts of
over 10 6 per gram were obtained for black pepper, ginger, and paprika
(Julseth and Deibel 1974). These spices also contained over 10 6 bacterial
spores per gram. Psychrotrophic spores were absent in herbs and spices
analyzed by Michels and Visser (1976). A nationwide survey of ten spices
and herbs revealed less contamination at retail than had been reported
for these ingredients at import (Schwab et al. 1982). Some samples of all
ten spices and herbs had an aerobic plate count over lOG/g. However,
many samples had less than 100/g. Thus, the geometric means of individ·
ual spices and herbs ranged from 1,400 for cloves to 820,000 for pepper.
Thermophilic bacteria, usually as spores, are added to foods with in·
gredients such as spices, starch, flour, and sugar. Thermophilic spores
are important when added to canned foods. The higher the level of heat·
resistant spores, the greater the chance that some may survive the heat
treatment and be a potential spoilage problem.
The total aerobic count of flour sampled at the mills is generally in
the range of 10 2 to 10 3 /g (Graves et al. 1967). They reported levels of total
aerobic thermophilic spores from 0 to 1,200 per 10 g of flour, and al·
though thermophilic flat· sour spores were not present in 10 g of most
flour samples, one 10·g sample contained 730 flat·sour spores.
Since sugar is a potential source of spores, the National Canner's As·
 SOURCES OF MICROORGANISMS 187

sociation has set limits of aerobic thermophilic flat-sour and anaerobic

thermophilic spores when this ingredient is to be added to canned foods.
Eskin, Henderson, and Townsend (1971) suggested that infections of
confectionery items by osmophilic yeasts are due to contaminated nuts,
fruits, chocolate, flour, or flavoring syrup. Bakery products can be con-
taminated by the ingredients in the doughs or by adding materials to the
baked food. Icing and nuts are added to already baked sweet rolls. The
toasting of almonds and pecans is not sufficient to assure sterility. These
products may contain 10 4 to 10 5 bacteria and 104 yeasts and molds per
gram.
Batters used in the breading of seafoods, onion rings, or other prod-
ucts are a potential source of high levels of microorganisms. Batters used
in the manufacture of breaded onion rings had a total bacterial count of
2,500 to 8,500,000/g (Maxcy and Arnold 1972). There were 110 to 32,000
coliforms per gram of the batters they examined.
Since ingredients are an important source of microorganisms, food
processors must establish acceptable microbiological limits for these sub·
stances. Then sufficient testing is needed to assure the processor that the
ingredients are acceptable and, if they are used in the manufacture of a
food, adjustments of the process are needed to take care of any excessive
bacterial load.

PRODUCT TO PRODUCT

There is an old saying that one' bad apple can spoil the barrel of
apples. This is very true, because the spoilage organisms from the bad
apple can contaminate and cause spoilage of surrounding apples until
all of the apples are spoiled.
In this example, the transfer of the organisms can be disastrous be·
cause they are known to cause spoilage of apples. Although the transfer
of microorganisms from one food to another can occur directly if the
foods contact each other, more often it occurs through contamination of
wash water, equipment, or handling.
Researchers have reported that the shelf life of vacuum-packed sliced
cooked meats would be improved if the meats were handled and sliced in
an area strictly separated from raw cured meats (Mol et al. 1971).
People handling both cooked and raw foods can transfer microor·
ganisms from the raw to the cooked product. With no further treatment,
this is a potential health hazard. The cook who cuts up a raw chicken on
a cutting board that is then used for cutting up raw vegetables for a salad
may transfer Salmonella from the raw chicken to the raw vegetables. The
188 BASIC FOOD MICROBIOLOGY

meat department in a retail store may use the same knife and block to
cut fish, cold meat, chicken, beef, and other types of food. Some of these
foods may be eaten with no further treatment or only a minimal heating.
A potential health hazard can result from this. Cross·contamination of
food is one of the ten main factors that contribute to foodborne illnesses.

PACKAGING

Packaging is a potential source of contamination. There are many

studies concerning the effect of different types of packaging materials on
the shelf life of food. The possibility that the packaging material might
contribute to the flora is not mentioned. This is probably because the
number of flora on most foods is much greater than that on packaging
material. In their survey of microorganisms on poultry· processing equip·
ment, Schuler and Badenhop (1972) reported the total bacterial count of
bulk containers for ice·packed poultry. The counts ranged from 290/4 in. 2
(U/cm2) to 9,770/4 in. 2 (376/cm 2 ). They stated that the holding of boxes
overnight led to contamination by dust, rodents, and birds.
Korab (1963) tested one-trip glass bottles used for carbonated bever-
ages. He reported that a majority of bottles had no bacteria, yeasts, or
molds when they were received at the beverage company, but about 6.5
percent had more than fifty bacteria per bottle. Storage of uncovered
bottles for one week increased the microbial contamination. Storage out-
side resulted in more contamination than storage inside, and the contam-
ination increased with storage time. Compared to returnable and reused
bottles, the one·trip bottles have very low microbial counts.
Plastic materials are used for packaging many food products. During
manufacture and handling, electrostatic charges can occur on the plastic.
These charges attract airborne material such as dust and microorganisms
(Baribo, Avens, and O'Neill 1966). Although plastics are essentially ster-
ile when made into packaging material, they may become contaminated
if not handled in an acceptable manner. The static charges add to the
problem of maintaining plastic free of microbial contamination.
There was a time when containers used for shipping poultry were
reused to pack fresh vegetables for shipment. Due to the potential for
contamination of the containers by salmonellae from the poultry and
then the transfer of these pathogens from the container to the vegetables,
this practice was halted.
Packages serve as a protective covering to limit or prevent microbial
contamination; however, they do not prevent microbial growth. It is im-
portant that preparation of the food for packaging is designed to limit
 SOURCES OF MICROORGANISMS 189

or prevent microbial contamination prior to packaging. Also, the pack-

age must be durable enough to maintain its integrity during storage and
distribution.

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