Microbial growth

OUTLINE
• BACTERIAL CELL DIVISION

• GROWTH OF BACTERIAL POPULATIONS

• MEASURING MICROBIAL GROWTH

• TEMPERATURE AND MICROBIAL GROWTH

• OTHER ENVIRONMENTAL FACTORS AFFECTING GROWTH

 Cell Growth and Binary Fission
• In microbiology, growth is defined as an increase in
the number of cells.
• Bacterial cell growth depends upon a large number
of chemical reactions of a wide variety of types.
• The main reactions of cell synthesis are the
polymerization reactions that make macromolecules
from monomers. As macromolecules accumulate in
the cytoplasm of a cell, they are assembled into new
structures, such as the cell wall, cytoplasmic
membrane, flagella, ribosomes, inclusion bodies,
enzyme complexes, and so on, eventually leading to
cell division.
 Binary Fission
• Cell division following enlargement of a cell to twice its
minimum size
• There is partition called a septum separating the cell into
two daughter cells
• The time required for this process is called the generation
time. The time required for a generation in a given
bacterial species is highly variable and is dependent on
both nutritional and genetic factors. Under the best
nutritional conditions the generation time of a laboratory
culture of E. coli is about 20 min.
Binary fission in a rod-shaped prokaryote. Cell numbers double every
generation.
GROWTH OF BACTERIAL POPULATIONS
 The Microbial Growth Cycle
• For a culture growing in an enclosed vessel, such as a
tube or a flask, a condition called a batch culture
(closed-system microbial culture of fixed volume) ,
exponential growth cannot continue indefinitely.
Instead, a typical growth curve for a population of cells
is obtained

• The growth curve describes an entire growth cycle,

including the lag phase, exponential phase, stationary
phase, and death phase.
Typical growth curve for a bacterial population. A viable count measures
the cells in the culture that are capable of reproducing. Optical density
(turbidity), a quantitative measure of light scattering by a liquid culture,
increases with the increase in cell number.
 Lag Phase
• When a microbial population is inoculated into a
fresh medium, growth usually begins only after a
period of time called the lag phase.
• This interval may be brief or extended, depending
on the history of the inoculum and the growth
conditions.
• A lag is also observed when a microbial population is
transferred from a rich culture medium to a poorer
one; for example, from a complex medium to a
defined medium
 Exponential Phase
• During the exponential phase of growth each cell
divides to form two cells, each of which also divides
to form two more cells, and so on
• Cells in exponential growth are typically in their
healthiest state and hence are most desirable for
studies of their enzymes or other cell components.
• The rate of exponential growth is influenced by
environmental conditions (temperature,
composition of the culture medium), as well as by
genetic characteristics of the organism itself. In
general, prokaryotes grow faster than eukaryotic
microorganisms, and small eukaryotes grow faster
than large ones.
 Stationary Phase
• In a batch culture, such as in a tube or a flask,
exponential growth is limited.
• Typically, either or both of two things limit growth: (1)
an essential nutrient of the culture medium is used up,
or (2) a waste product of the organism accumulates in
the medium and inhibits growth. Either way,
exponential growth ceases and the population reaches
the stationary phase.
• In the stationary phase, there is no net increase or
decrease in cell number and thus the growth rate of the
population is zero. Some cells in the population grow,
whereas others die, the two processes balancing each
other out. This is a phenomenon called cryptic growth.
 Death Phase
• If incubation continues after a population reaches
the stationary phase, the cells may remain alive and
continue to metabolize, but they will eventually die.
When this occurs, the population enters the death
phase of the growth cycle.
• In some cases death is accompanied by actual cell
lysis.
• The rate of cell death is much slower than the rate of
exponential growth.
 Continuous Culture
• Unlike a batch culture, a continuous culture is an
open system.
• The continuous culture vessel maintains a constant
volume to which fresh medium is added at a
constant rate, and an equal volume of spent culture
medium is removed at the same rate.
• Once such a system is in equilibrium, the chemostat
volume, cell number, and nutrient status remain
constant, and the system is said to be in steady
state.
Schematic for a continuous culture device (chemostat). The population
density is controlled by the concentration of limiting nutrient in the reservoir, and
the growth rate is controlled by the flow rate. Both parameters can be set by the
experimenter.
MEASURING MICROBIAL GROWTH
 #1. Microscopic Count
• The most common total count method is the microscopic
cell count. Microscopic counts can be done on either
samples dried on slides or on samples in liquid. Dried
samples can be stained to increase contrast between cells
and their background. With liquid samples, specially
designed counting chambers are used. In such a counting
chamber, a grid with squares of known area is marked on
the surface of a glass slide
 Limitations: (1) without special staining techniques dead cells
cannot be distinguished from live cells; (2) small cells are difficult
to see under the microscope, and some cells are inevitably missed;
(3) precision is difficult to achieve; (4) a phase-contrast
microscope is required if the sample is not stained; (5) cell
suspensions of low density (less than about 106 cells/milliliter)
have few if any bacteria in the microscope field unless a sample is
first concentrated and resuspended in a small volume; (6) motile
cells must be immobilized before counting; (7) debris in the
sample may be mistaken for microbial cells.

•In microbial ecology, total cell counts are often performed on

natural samples using stains to visualize the cells. The stain DAPI
stains all cells in a sample because it reacts with DNA.
Direct microscopic counting procedure using the Petroff–Hausser
counting chamber. A phase-contrast microscope is typically used to count the
cells to avoid the necessity for staining.
 #2. Flow Cytometry

A second method of enumerating cells in liquid

samples is with a flow cytometer. This is a machine
that employs a laser beam and complex electronics to
count individual cells.
Source: Internet
 #3. Viable Cell Counting (CFU
Counting)
• A viable cell is one that is able to divide and form
offspring. Usually, we determine the number of cells
in the sample capable of forming colonies on a agar
medium  also called a plate count.

• There are at least two ways of performing a plate

count: the spread-plate method and the pour-plate
method
Two methods for the viable count. In the pour-plate method, colonies form
within the agar as well as on the agar surface. In the fourth column is shown a
photo of colonies of Escherichia coli formed from cells plated by the spread-
plate method (top) or the pour-plate method (bottom).
 Diluting Cell Suspensions before Plating
• The number of colonies developing on the plate
must not be too many or too few. It is statistically
valid to count colonies only on plates that have
between 30 and 300 colonies.

• To obtain the appropriate colony number, the

sample to be counted must almost always be
diluted. Several 10-fold dilutions of the sample are
commonly used.
Procedure for viable
counting using serial
dilutions of the
sample and the pour-
plate method. The
sterile liquid used for
making dilutions can
simply be water, but a
balanced salt solution
or growth medium may
yield a higher recovery.
The dilution factor is the
reciprocal of the
dilution.
0. Calculate the number of bacteria (CFU) per mil iliter or gram of sample by
number of colonies by the dilution factor multiplied by the amount of spec
to liquefied agar.
number of colonies (CFUs) = # of bacteria/ml
dilution X amount plated
1. Record your results.

BIDIMETRY DETERMINATION OF BACTERIAL NUMBERS

 #4. Measurements of Microbial Mass:
Turbidimetric Method
• During exponential growth, all cellular components
increase in proportion to the increase in cell
numbers. Thus, instead of measuring changes in cell
number over time, we could instead measure the
increase in protein, DNA, or dry weight of a culture
as a barometer of growth.

• Because cell mass is proportional to cell number, we

can use turbidity as a measure of cell numbers in a
growing culture.
Turbidity measurements of microbial growth. (a) Measurements of turbidity are
made in a spectrophotometer. (b) Typical growth curve data for two organisms growing
at different growth rates. (c) Relationship between cell number or dry weight and
turbidity readings.
 #5. Dried Cell Weight (DCW)

Microbial cells will be collected from a

submerged culture by centrifugation. The
pellets will be dried until a constant weight is
obtained.
 A new method for microbial growth
measurement

Geographic information system (GIS)-based image analysis for assessing

growth of Physarum polycephalum on a solid medium (Tran et al. 2015)
TEMPERATURE AND MICROBIAL GROWTH
 Effect of Temperature on Microbial
Growth
• Temperature is probably the most important
environmental factor affecting the growth and
survival of microorganisms.

• The minimum and maximum temperatures for

growth vary greatly among different microorganisms
and usually reflect the temperature range and
average temperature of their habitats.
 Cardinal Temperatures
• Be the minimum, maximum, and optimum growth
temperatures for a given organism  characteristic
for any given microorganism.

• The cardinal temperatures of different

microorganisms differ widely; from below freezing to
well above the boiling point of water. The range for
any given organism is typically 25–40 degrees.
The cardinal temperatures: Minimum, optimum, and maximum. The actual
values may vary greatly for different organisms
 Temperature Classes of Organisms
• It is possible to distinguish at least four groups of
microorganisms in relation to their growth temperature
optima:
– Psychrophile: An organism with a growth temperature optimum of
15°C or lower and a maximum growth temperature below 20°C
– Mesophile: An organism that grows best at temperatures between 20
and 45°C
– Thermophile: An organism of which growth temperature optimum lies
between 45 and 80°C
– Hyperthermophile: A prokaryote that has a growth temperature
optimum of 80°C or greater
• Mesophiles are widespread in nature.
 Microbial Growth at Cold Temperatures
• Extremophile: An organism that grows optimally
under one or more chemical or physical extremes,
such as high or low temperature or pH

• Even in frozen materials there are often small

pockets of liquid water where solutes have
concentrated and microorganisms can metabolize
and grow.

• Psychrophiles are found in environments that are

constantly cold and may be rapidly killed by
warming, even to as little as 20°C.
Temperature and growth relations in different temperature classes of
microorganisms. The temperature optimum of each example organism is
shown on the graph.
Antarctic microbial habitats and microorganisms. (a) A core of frozen seawater from
McMurdo Sound, Antarctica. The core is about 8 cm wide. Note the dense coloration
due to pigmented microorganisms. (b) Phase-contrast micrograph of phototrophic
microorganisms from the core shown in (a). Most organisms are either diatoms or green
algae (both eukaryotic phototrophs). (c) Transmission electron micrograph of
Polaromonas, a gas vesiculate bacterium that lives in sea ice and grows optimally at
4°C. (d) Photo of the surface of Lake Bonney, McMurdo Dry Valleys, Antarctica.
Snow algae. (a) Snow bank in the Sierra
Nevada, California, with red coloration
caused by the presence of snow algae. Pink
snow such as this is common on summer
snow banks at high altitudes throughout the
world. (b) Photomicrograph of red-pigmented
spores of the snow alga Chlamydomonas
nivalis. The spores germinate to yield motile
green algal cells. Some strains of snow algae
are true psychrophiles but many are
psychrotolerant, growing best at
temperatures above 20°C.
 Psychrotolerant Microorganisms
• Capable of growing at low temperatures but having an
optimum above 20°C

• Psychrotolerant microorganisms are more widely distributed

in nature than are psychrophiles and can be isolated from
soils and water in temperate climates as well as from meat,
milk and other dairy products, cider, vegetables, and fruit
stored at refrigeration temperatures (∼4°C).

• Various Bacteria, Archaea, and microbial eukaryotes are

psychrotolerant.
 Microbial Growth at High Temperatures
• Thermophiles and hyperthermophiles can survive at high
temperature:
– First, their enzymes and other proteins are much more heat-stable
than are those of mesophiles and actually function optimally at high
temperatures
– Second, the cytoplasmic membranes of thermophiles and
hyperthermophiles must be heat-stable.

• A classic example of a heat-stable enzyme of great

importance to biology is the DNA polymerase isolated from
Thermus aquaticus. Taq polymerase, as this enzyme is known,
has been used to automate the repetitive steps in the
polymerase chain reaction (PCR) technique
Growth of hyperthermophiles in
boiling water.
(a) Boulder Spring, a small boiling
spring in Yellowstone National
Park. This spring is superheated,
having a temperature 1–2°C
above the boiling point. The
mineral deposits around the spring
consist mainly of silica and sulfur.
(b) Photomicrograph of a
microcolony of prokaryotes that
developed on a microscope slide
immersed in a boiling spring such
as that shown in (a).
Growth of thermophilic cyanobacteria in a hot spring in Yellowstone National
Park. Characteristic V-shaped pattern (shown by the dashed red lines) formed by
cyanobacteria at the upper temperature for phototrophic life, 70–74°C, in the thermal
gradient formed from a boiling hot spring. The pattern develops because the water cools
more rapidly at the edges than in the center of the channel. The spring flows from the
back of the picture toward the foreground. The light-green color is from a high-
temperature strain of the cyanobacterium Synechococcus. As water flows down the
gradient, the density of cells increases, less thermophilic strains enter, and the color
becomes more intensely green.
 Psychrotolerant Microorganisms
• Capable of growing at low temperatures but having an
optimum above 20°C

• Psychrotolerant microorganisms are more widely distributed

in nature than are psychrophiles and can be isolated from
soils and water in temperate climates as well as from meat,
milk and other dairy products, cider, vegetables, and fruit
stored at refrigeration temperatures (∼4°C).

• Various Bacteria, Archaea, and microbial eukaryotes are

psychrotolerant.
OTHER ENVIRONMENTAL FACTORS
AFFECTING MIROBIAL GROWTH
 Microbial Growth at Low or High pH
• Most organisms show a growth pH range of 2–3
units. Most natural environments have pH values
between 4 and 9, and organisms with optima in this
range are most commonly encountered.

• Only a few species can grow at pH values of lower

than 3 or greater than 9.
 •How pH affects microorganisms

•Ionization- solubility of many sunstances-

nutrient availability
•Stability of membrane
•Enzyme activity (protein denaturation)

The pH scale. Although some microorganisms can live at very low or very high
pH, the cell’s internal pH remains near neutrality.
 Acidophiles
• Organisms that grow optimally at low pH, typically below pH
6, are called acidophiles.

• Fungi as a group tend to be more acid tolerant than bacteria.

Many fungi grow best at pH 5 or below, and a few grow well
at pH values as low as 2.

• A critical factor governing acidophily is the stability of the

cytoplasmic membrane. When the pH is raised to neutrality,
the cytoplasmic membranes of strongly acidophilic bacteria
are destroyed and the cells lyse.
 Alkaliphiles
• A few extremophiles have very high pH optima for
growth, sometimes as high as pH 11.
Microorganisms showing growth pH optima of 9 or
higher are called alkaliphiles.

• Alkaliphilic microorganisms are typically found in

highly alkaline habitats, such as soda lakes and high-
carbonate soils

• The most well-studied alkaliphilic prokaryotes have

been Bacillus species, such as Bacillus firmus.
 Internal Cell pH
• The optimal pH for growth of any organism is a measure of
the pH of the extracellular environment only. The intracellular
pH must remain relatively close to neutrality to prevent
destruction of macromolecules in the cell.

• In acidophiles and alkaliphiles the internal pH can vary from

neutrality. For example, the internal pH has been measured
at pH 4.6 and 9.5, they are extremely close to the limits . This
is because DNA is acid-labile and RNA is alkaline-labile; if a cell
cannot maintain these key macromolecules in a stable state,
it obviously cannot survive.
 Buffers
• In a batch culture, the pH can change during growth as the
result of metabolic reactions of microorganisms that consume
or produce acidic or basic substances. Thus, buffers are
frequently added to microbial culture media to keep the pH
relatively constant.

• For near-neutral pH ranges, potassium phosphate (KH2PO4)

and calcium carbonate (CaCO3) are good buffers.

• The best buffering system for one organism or enzyme may

be considerably different from that for another. Thus, the
optimal buffer for use in a particular situation must usually be
determined empirically.
 Osmotic Effects on Microbial Growth
• Water is the solvent of life, and water availability is
an important factor affecting the growth of
microorganisms.

• Water availability not only depends on the absolute

water content of an environment, that is, how moist
or dry it is, but it is also a function of the
concentration of solutes such as salts, sugars, or
other substances that are dissolved in the water.
 Water Activity and Osmosis

• Water activity, abbreviated aw, is defined as the ratio

of the vapor pressure of the air in equilibrium with a
substance or solution to the vapor pressure of pure
water. Thus, values of aw vary between 0 and 1

• Water activities in agricultural soils generally range

between 0.90 and 1.
 Halophiles and Related Organisms
• Halophile is a microorganism that requires NaCl for growth

• Halotolerant: Not requiring NaCl for growth but able to grow

in the presence of salt, in some cases, substantial levels of salt

• Extreme halophile: A microorganism that requires very large

amounts of salt (NaCl), usually greater than 10% and in some
cases near to saturation, for growth

• The terms mild halophile and moderate halophile are used to

describe halophiles with low (1–6%) and moderate (7–15%)
NaCl requirements, respectively
Effect of sodium chloride concentration on growth of microorganisms of
different salt tolerances or requirements. The optimum NaCl concentration
for marine microorganisms such as V.fischeri is about 3%; for extreme
halophiles, it is between 15 and 30%, depending on the organism.
 Compatible Solutes
• When an organism grows in a medium with a low water
activity, it can obtain water from its environment only by
increasing its internal solute concentration.

• Compatible solute: A molecule that is accumulated in the

cytoplasm of a cell for adjustment of water activity but that
does not inhibit biochemical processes

• Osmophile: An organism that grows best in the presence of

high levels of solute, typically a sugar

• Xerophile: An organism that is able to live, or that lives best,

in very dry environments
 Oxygen Classes of Microorganisms
• Aerobe: An organism that can use oxygen (O2) in respiration;
some require oxygen

• Microaerophile: An aerobic organism that can grow only

when oxygen tensions are reduced from that present in air

• Facultative with respect to oxygen, an organism that can grow

in either its presence or absence

• Anaerobe: An organism that cannot use O2 in respiration and

whose growth is typically inhibited by O2

• Obligate anaerobe: An organism that cannot grow in the

presence of O2
 Culture Techniques for Aerobes and
Anaerobes
• For the growth of many aerobes, it is necessary to provide
extensive aeration. This is because the oxygen that is
consumed by the organisms during growth is not replaced
fast enough by simple diffusion from the air. Aerobes typically
grow better with forced aeration than with oxygen supplied
from simple diffusion.

• For the culture of anaerobes, the problem is not to provide

air, but to exclude it. Obligate anaerobes vary in their
sensitivity to oxygen, and procedures are available for
reducing the oxygen content of cultures.
Growth versus oxygen concentration. (a–e) Aerobic, anaerobic, facultative,
microaerophilic, and aerotolerant anaerobe growth, as revealed by the position
of microbial colonies (depicted here as black dots) within tubes of thioglycolate
broth culture medium. A small amount of agar has been added to keep the
liquid from becoming disturbed, and the redox dye, resazurin, which is pink
when oxidized and colorless when reduced, is added as a redox indicator.
Incubation under anoxic conditions. (a) Anoxic jar. A chemical reaction in
the envelope in the jar generates H2 + CO2. The H2 reacts with O2 in the jar on
the surface of a palladium catalyst to yield H2O; the final atmosphere contains
N2, H2, and CO2. (b) Anoxic glove bag for manipulating and incubating cultures
under anoxic conditions. The airlock on the right, which can be evacuated and
filled with O2-free gas, serves as a port for adding and removing materials to
and from the glove bag.
 Toxic Forms of Oxygen
• Oxygen is a powerful oxidant and the best electron
acceptor for respiration.

• But oxygen can also be a poison to obligate

anaerobes because the toxic derivatives of oxygen
can damage cells
 Oxygen Chemistry
• Oxygen in its ground state is called triplet oxygen (3O2).
However, other electronic configurations of oxygen are
possible, and most are toxic to cells.

• Singlet oxygen is produced both photochemically and

biochemically, the latter through the activity of various
peroxidase enzymes

• Organisms that frequently encounter singlet oxygen, such as

airborne bacteria and phototrophic microorganisms, often
contain pigments called carotenoids, which function to
convert singlet oxygen to nontoxic forms.
 Superoxide and Other Toxic Oxygen
Species
• Besides singlet oxygen, many other toxic forms of
oxygen exist, including superoxide anion (O2−),
hydrogen peroxide (H2O2), and hydroxyl radical
(OH•). All of these are produced as by-products of
the reduction of O2 to H2O in respiration

• With so many toxic oxygen derivatives to deal with,

organisms have evolved enzymes that destroy these
compounds
Four-electron reduction of O2 to H2O by stepwise addition of
electrons. All the intermediates formed are reactive and toxic to
cells
Enzymes that destroy toxic oxygen species.
Method for testing a microbial culture for the presence of
catalase. A heavy loopful of cells from an agar culture was mixed
on a slide (right) with a drop of 30% hydrogen peroxide. The
immediate appearance of bubbles is indicative of the presence of
catalase. The bubbles are O2 produced by the reaction H2O +
H2O2 → 2 H2O + O2.
Inhibiting Microbial Growth
 Definition of Terms
• Sterilization is the complete destruction of all
microorganisms, including cells, spores and viruses.
– Accomplished by dry heat, autoclaving (steam under
pressure), gas, various chemicals and certain types of
radiation.
• Disinfection is the destruction or removal of pathogens
from nonliving objects by physical or chemical
methods; example = pasteurization.
– Disinfectants are chemical substances that eliminate
pathogens on inanimate objects.
– Antiseptics are solutions used to disinfect skin and other living
tissues.
 Definition of Terms (cont.)
• The suffix –cide or –cidal refers to “killing.”
• Germicidal agents, biocidal agents and microbicidal
agents are chemicals that kill microbes.
• Bactericidal agents are chemicals that specifically kill
bacteria, but not necessarily endospores.
– Sporicidal agents kill bacterial endospores.
– Fungicidal agents kill fungi, including fungal spores.
– Algicidal agents kill algae.
– Viricidal agents destroy viruses.
 Definition of Terms (cont.)
• A microbistatic agent is a drug or chemical that inhibits
growth and reproduction of microorganisms.
• A bacteriostatic agent is one that specifically inhibits
the metabolism and reproduction of bacteria.
• Lyophilization is a process that combines dehydration
(drying) and freezing. This process is widely used in
industry to preserve foods, antibiotics, microorganisms
and other biological materials.
• Sepsis refers to the presence of pathogens in blood or
tissues, whereas asepsis means the absence of
pathogens.
• Antisepsis is the prevention of infection.
 Using Physical Methods to Inhibit
Microbial Growth
• Heat
– 2 factors – temperature and time - determine the
effectiveness of heat for sterilization.
– The thermal death point (TDP) of any species is the lowest
temperature that will kill all of the organisms in a
standardized pure culture within a specified time.
• Types of Heat
– Dry heat – electrical incinerator or flame
– Moist heat – boiling or use of an autoclave
 Dry heat sterilization.

Using an electrical heating

Using a Bunsen burner device.
flame.
 Using Physical Methods to Inhibit
Microbial Growth (cont.)
• The autoclave
– A large metal pressure cooker that uses steam under pressure
to completely destroy all microbial life.
– Increased pressure raises the temperature above the
temperature of boiling water (above 100oC) and forces steam
into material being sterilized.
– Autoclaving at a pressure of 15 psi at 121.5oC for 20 minutes
kills vegetative microorganisms, bacterial endospores and
viruses.
– Can use pressure-sensitive tape or spore strips as a quality
control measure to ensure proper autoclaving.
A large, built-in autoclave.
Pressure-sensitive autoclave tape showing dark stripes after
sterilization.
 Using Physical Methods to Inhibit
Microbial Growth (cont.)
• Cold; most microorganisms are not killed, but their
metabolic activities are slowed.
• Desiccation; many dried microorganisms remain viable,
but they cannot reproduce.
• Radiation; an ultra-violet (UV) lamp is useful for
reducing the number of microorganisms in the air.
• Ultrasonic waves; used in hospitals and medical and
dental clinics to clean and sterilize equipment.
• Filters; used to separate cells/microorganisms from
liquids or gases.
• Gaseous atmosphere; can be altered to inhibit growth.
 Using Chemical Agents to Inhibit
Microbial Growth
• Chemical disinfection refers to the use of chemical
agents to inhibit the growth of pathogens, either
temporarily or permanently.
• Disinfectants are affected by:
– Prior cleaning of the object or surface
– The organic load (e.g., feces, blood, pus)
– The bioburden; type and number of microbes
– Concentration of disinfectant
– Contact time
– Physical nature of the object being disinfected
– Temperature and pH
 Using Chemical Agents to Inhibit
Microbial Growth (cont.)
Characteristics of an ideal chemical antimicrobial agent:

• Should have a broad antimicrobial • Soluble in water and easy to apply

spectrum • Inexpensive and easy to prepare
• Fast acting • Stable as both a concentrate and a
• Not affected by the presence of working solution
organic matter • Odorless
• Nontoxic to human tissues and
noncorrosive
• Should leave a residual
antimicrobial film on surface
 Using Chemical Agents to Inhibit
Microbial Growth (cont.)
• Antiseptics
– May safely be used on human tissues.
– Reduce the number of organisms on the surface of
the skin; do not penetrate pores and hair follicles.
• Antiseptic soaps and scrubbing are used by
healthcare personnel to remove organisms
lodged in pores or folds of the skin.