Control of Microorganisms

Microbiology
• Although many microorganisms are beneficial and necessary
for human well-being, microbial activities may have
undesirable consequences such as food spoilage and disease.
• Therefore it is essential to be able to kill a wide variety of
microorganisms or inhibit their growth to minimize their
destructive effects.

The goal is two fold:

(1) to destroy pathogens and prevent their transmission,
(2) to reduce or eliminate microorganisms responsible for the
contamination of water, food, and other substances.
Sterilization [Latin sterilis, unable to produce offspring or barren] is the
process by which all living cells, viable spores, viruses, and Viroids are
either destroyed or removed from an object or habitat.
• A sterile object is totally free of viable microorganisms, spores, and other
infectious agents.
• When sterilization is achieved by a chemical agent, the chemical is called
a sterilant.
Disinfection:
• It is the process of killing, inhibition, or removal of microorganisms that
may cause disease.
• The primary goal is to destroy potential pathogens, but disinfection also
substantially reduces the total microbial population.
• Disinfectants are agents, usually chemical, used to carry out disinfection
and are normally used only on inanimate objects.
• A disinfectant does not necessarily sterilize an object because viable
spores and a few microorganisms may remain.
Sanitization
• Sanitization is closely related to disinfection.
• In sanitization, the microbial population is reduced to levels that are
considered safe by public health standards.
• The inanimate object is usually cleaned as well as partially disinfected. For
example, sanitizers are used to clean eating utensils in restaurants.

Antisepsis
• Antisepsis [Greek anti, against, and sepsis, putrefaction] is the prevention
of infection and is accomplished with antiseptics.
• Antiseptics are chemical agents applied to tissue to prevent infection by
killing or inhibiting pathogen growth; they also reduce the total microbial
population.
• Because they must not destroy too much host tissue, antiseptics are
generally not as toxic as disinfectants.
Germicide:

• Substances that kill organisms often have the suffix –cide [Latin cida, to
kill]: a germicide kills pathogens (and many non pathogens) but not
necessarily endospores.

• A disinfectant or antiseptic can be particularly effective against a specific

group, in which case it may be called a bactericide, fungicide, algicide, or
viricide.

• Other chemicals do not kill, but they do prevent growth. If these agents
are removed, growth will resume. Their names end in -static [Greek
statikos, causing to stand or stopping]—for example, bacteriostatic and
fungistatic.
The Use of Physical Methods in
Control
Heat
• Fire and boiling water have been used for sterilization and disinfection
since the time of the Greeks, and heating is still one of the most popular
ways to destroy microorganisms.
• Either moist or dry heat may be applied.
• Moist heat readily kills viruses, bacteria, and fungi. Exposure to boiling
water for 10 minutes is sufficient to destroy vegetative cells and
eukaryotic spores.
• Unfortunately the temperature of boiling water (100°C or 212°F) is not
high enough to destroy bacterial endospores that may survive hours of
boiling.
• Therefore boiling can be used for disinfection of drinking water and
objects not harmed by water, but boiling does not sterilize.
 Moist heat sterilization

• Moist heat sterilization must be carried out at temperatures

above 100°C in order to destroy bacterial endospores, and
this requires the use of saturated steam under pressure.

• Steam sterilization is carried out with an autoclave.

• The development of the autoclave by Chamberland in 1884

tremendously stimulated the growth of microbiology.
• Water is boiled to produce steam, which is released through the jacket
and into the autoclave’s chamber.
• The air initially present in the chamber is forced out until the chamber is
filled with saturated steam and the outlets are closed.
• Hot, saturated steam continues to enter until the chamber reaches the
desired temperature and pressure, usually 121°C and 15 pounds of
pressure.
• At this temperature saturated steam destroys all vegetative cells and
endospores in a small volume of liquid within 10 to 12 minutes.
• Treatment is continued for about 15 minutes to provide a margin of
safety.
• Of course, larger containers of liquid such as flasks and carboys will
require much longer treatment times.
• Moist heat is thought to kill so effectively by degrading nucleic acids and
by denaturing enzymes and other essential proteins.
• It also may disrupt cell membranes.
• Autoclaving must be carried out properly or the processed materials will not be
sterile.
• The chamber should not be packed too tightly because the steam needs to
circulate freely and contact everything in the autoclave.
• Bacterial endospores will be killed only if they are kept at 121°C for 10 to 12
minutes.
• When a large volume of liquid must be sterilized, an extended sterilization time
will be needed because it will take longer for the center of the liquid to reach
121°C; 5 liters of liquid may require about 70 minutes.
Pasteurization
• Many substances, such as milk, are treated with controlled heating at temperatures well
below boiling, a process known as pasteurization, in honor of its developer Louis Pasteur.
• Pasteur examined spoiled wine under the microscope and detected microorganisms that
looked like the bacteria responsible for lactic acid and acetic acid fermentations.
• He then discovered that a brief heating at 55 to 60°C would destroy these microorganisms
and preserve wine for long periods.
• Milk pasteurization was introduced into the United States in 1889. Milk, beer, and many
other beverages are now pasteurized.
• Pasteurization does not sterilize a beverage, but it does kill any pathogens present and
drastically slows spoilage by reducing the level of nonpathogenic spoilage microorganisms.
• Milk can be pasteurized in two ways.
• In the older method the milk is held at 63°C for 30 minutes. Large quantities of milk are now
usually subjected to flash pasteurization or high-temperature short-term (HTST)
pasteurization, which consists of quick heating to about 72°C for 15 seconds, then rapid
cooling.
• The dairy industry also sometimes uses ultrahigh-temperature (UHT) sterilization. Milk and
milk products are heated at 140 to 150°C for 1 to 3 seconds. UHT-processed milk does not
require refrigeration and can be stored at room temperature for about 2 months without
flavor changes. The small coffee creamer portions provided by restaurants often are
prepared using UHT sterilization.
• Many objects are best sterilized in the absence of water by dry heat sterilization.
• The items to be sterilized are placed in an oven at 160 to 170°C for 2 to 3 hours.
• Microbial death apparently results from denaturation of proteins.
• Although dry air heat is less effective than moist heat— Clostridium botulinum
spores are killed in 5 minutes at 121°C by moist heat but only after 2 hours at
160°C with dry heat—it has some definite advantages.
• Dry heat does not corrode glassware and metal instruments as moist heat does,
and it can be used to sterilize powders, oils, and similar items.
• Most laboratories sterilize glass petri dishes and pipettes with dry heat.
• Despite these advantages, dry heat sterilization is slow and not suitable for heat
sensitive materials like many plastic and rubber items.
 Low Temperatures
• Often the most convenient control technique is to inhibit their growth and
reproduction by the use of either freezing or refrigeration.
• This approach is particularly important in food microbiology. Freezing items at
20°C or lower stops microbial growth because of the low temperature and the
absence of liquid water.
• Freezing is a very good method for long-term storage of microbial samples when
carried out properly, and many laboratories have a low-temperature freezer for
culture storage at 30 or 70°C. Because frozen food can contain many
microorganisms, it should be prepared and consumed promptly after thawing in
order to avoid spoilage and pathogen growth.
• Refrigeration greatly slows microbial growth and reproduction, but does not halt it
completely. Fortunately most pathogens are mesophilic and do not grow well at
temperatures around 4°C.
• Thus refrigeration is a good technique only for shorter-term storage of food and
other items.
 Filtration
• Filtration is an excellent way to reduce the microbial population in solutions of
heat-sensitive material, and sometimes it can be used to sterilize solutions. Rather
than directly destroying contaminating microorganisms, the filter simply removes
them.
• Membrane filters are now used for many purposes.
• These circular filters are porous membranes, a little over 0.1 mm thick, made of
cellulose acetate, cellulose nitrate, polycarbonate or polyvinylidene fluoride.
• Although a wide variety of pore sizes are available, membranes with pores about
0.2 m in diameter are used to remove most vegetative cells, but not viruses, from
solutions ranging in volume from 1 ml to many liters.
• Membrane filters remove microorganisms by screening them out much as a sieve
separates large sand particles from small ones.
• These filters are used to sterilize pharmaceuticals, ophthalmic solutions, culture
media, oils, antibiotics, and other heat-sensitive solutions.
• Air also can be sterilized by filtration. Two common examples are surgical masks
and cotton plugs on culture vessels that let air in but keep microorganisms out.
• Laminar flow biological safety cabinets employing high-efficiency particulate air
(HEPA) filters, which remove 99.97% of 0.3 m particles, are one of the most
important air filtration systems.
• Laminar flow biological safety cabinets force air through HEPA filters, then project
a vertical curtain of sterile air across the cabinet opening. This protects a worker
from microorganisms being handled within the cabinet and prevents
contamination of the room.
• A person uses these cabinets when working with dangerous agents such as
Mycobacterium tuberculosis, tumor viruses, and recombinant DNA.
• They are also employed in research labs and industries, such as the
pharmaceutical industry, when a sterile working surface is needed for conducting
assays, preparing media, examining tissue cultures.
 Radiation
• Ultraviolet (UV) radiation around 260 nm is quite lethal but does not
penetrate glass, dirt films, water, and other substances very effectively.
Because of this disadvantage, UV radiation is used as a sterilizing agent
only in a few specific situations.

• UV lamps are sometimes placed on the ceilings of rooms or in biological

safety cabinets to sterilize the air and any exposed surfaces.

• Because UV radiation burns the skin and damages eyes, people working in
such areas must be certain the UV lamps are off when the areas are in
use.

• Commercial UV units are available for water treatment. Pathogens and

other microorganisms are destroyed when a thin layer of water is passed
under the lamps.
The Use of Chemical Agents in Control
• Although objects are sometimes disinfected with physical agents, chemicals are more often
employed in disinfection and antisepsis.

• Many factors influence the effectiveness of chemical disinfectants such as the kinds of
microorganisms potentially present, the concentration and nature of the disinfectant to be
used, and the length of treatment should be considered. Dirty surfaces must be cleaned
before a disinfectant or antiseptic is applied.

• Many different chemicals are available for use as disinfectants, and each has its own
advantages and disadvantages.

• In selecting an agent, it is important to keep in mind the characteristics of a desirable

disinfectant.

• Ideally the disinfectant must be effective against a wide variety of infectious agents (gram-
positive and gram-negative bacteria, acid-fast bacteria, bacterial endospores, fungi, and
viruses) at high dilutions.

• Although the chemical must be toxic for infectious agents, it should not be toxic to people or
corrosive for common materials.

• In practice, this balance between effectiveness and low toxicity for animals is hard to
achieve.
• Some chemicals are used despite their low effectiveness because they are
relatively nontoxic.
• The disinfectant should be stable upon storage, odorless or with a
pleasant odor, soluble in water and lipids for penetration into
microorganisms. If possible the disinfectant should be relatively
inexpensive.
• One potentially serious problem is the overuse of triclosan and other
germicides.
• This antibacterial agent is now found in products such as deodorants,
mouthwashes, soaps, cutting boards, and baby toys. Triclosan seems to be
everywhere. Thus leading to the emergence of triclosan-resistant
bacteria, E.g. Pseudomonas aeruginosa actively pumps the antiseptic out
the cell.
• Bacteria seem to be responding to antiseptic overuse in the same way as
they reacted to antibiotic overuse.
• Thus overuse of antiseptics can have unintended harmful consequences.
 Phenolics
• Phenol was the first widely used antiseptic and disinfectant. In 1867
Joseph Lister employed it to reduce the risk of infection during
operations.
• Today phenol and phenolics (phenol derivatives) such as cresols, xylenols,
and orthophenylphenol are used as disinfectants in laboratories and
hospitals.
• The commercial disinfectant Lysol is made of a mixture of phenolics.
• Phenolics act by denaturing proteins and disrupting cell membranes. They
have some real advantages as disinfectants: phenolics are tuberculocidal,
and remain active on surfaces long after application. However, they do
have a disagreeable odor and can cause skin irritation.
• Hexachlorophene has been one of the most popular antiseptics because
it persists on the skin once applied and reduces skin bacteria for long
periods.
• However, it can cause brain damage and is now used in hospital nurseries
only in response to a staphylococcal outbreak.
 Alcohols

• Alcohols are among the most widely used disinfectants and

antiseptics.
• They are bactericidal and fungicidal but not sporicidal; some
lipid-containing viruses are also destroyed.
• The two most popular alcohol germicides are ethanol and
isopropanol, usually used in about 70 to 80% concentration.
• They act by denaturing proteins and possibly by dissolving
membrane lipids.
• A 10 to 15 minute soaking is sufficient to disinfect
thermometers and small instruments.
 Halogens
• A halogen is any of the five elements (fluorine, chlorine, bromine, iodine, and
astatine) in group VII of the periodic table.

• They exist as diatomic molecules in the free state and form salt like compounds
with sodium and most other metals.

• The halogens iodine and chlorine are important antimicrobial agents. Iodine is
used as a skin antiseptic and kills by oxidizing cell constituents and iodinating cell
proteins. At higher concentrations, it may even kill some spores.

• Iodine often has been applied as tincture of iodine, 2% or more iodine in a water-
ethanol solution of potassium iodide. Although it is an effective antiseptic, the skin
may be damaged, a stain is left, and iodine allergies can result.
• More recently iodine has been complexed with solubilizing agent or surfactants to
form an iodophor.
• Iodophors are water soluble, stable, and non staining agents, release iodine slowly
to minimize skin burns and irritation. They are used in hospitals for preoperative
skin degerming and in hospitals and laboratories for disinfecting.
• Chlorine is the usual disinfectant for municipal water supplies and swimming pools
and is also employed in the dairy and food industries.
• It may be applied as chlorine gas, sodium hypochlorite, or calcium hypochlorite, all
of which yield hypochlorous acid (HClO) and then atomic oxygen.
• Since organic material interferes with chlorine action by reacting with chlorine and
its products, an excess of chlorine is added to ensure microbial destruction.
• One potential problem is that chlorine reacts with organic compounds to form
carcinogenic trihalomethanes, which must be monitored in drinking water.
• Chlorine is also an excellent disinfectant for individual use because it is effective,
inexpensive, and easy to employ.
• Small quantities of drinking water can be disinfected with halazone tablets.
Halazone (parasulfone dichloraminobenzoic acid) slowly releases chloride when
added to water and disinfects it in about half an hour.
• Chlorine solutions make very effective laboratory and household disinfectants.
 Heavy Metals
• For many years the ions of heavy metals such as mercury, silver, arsenic,
zinc, and copper were used as germicides.
• More recently these have been superseded by other less toxic and more
effective germicides (many heavy metals are more bacteriostatic than
bactericidal).
• There are a few exceptions. A 1% solution of silver nitrate is often added
to the eyes of infants to prevent ophthalmic gonorrhea (in many
hospitals, erythromycin is used instead of silver nitrate because it is
effective against Chlamydia as well as Neisseria).
• Silver sulfadiazine is used on burns.
• Copper sulfate is an effective algicide in lakes and swimming pools.
• Heavy metals combine with proteins, often with their sulfhydryl groups,
and inactivate them.
• They may also precipitate cell proteins.
 Aldehydes
• Both of the commonly used aldehydes, formaldehyde and glutaraldehyde,
are highly reactive molecules that combine with nucleic acids and proteins
and inactivate them, probably by crosslinking and alkylating molecules.

• They are sporicidal and can be used as chemical sterilants.

• Formaldehyde is usually dissolved in water or alcohol before use.

• A 2% buffered solution of glutaraldehyde is an effective disinfectant. It is

less irritating than formaldehyde and is used to disinfect hospital and
laboratory equipment.

• Glutaraldehyde usually disinfects objects within about 10 minutes but

may require as long as 12 hours to destroy all spores.
 Sterilizing Gases
• Many heat-sensitive items such as disposable plastic petri dishes and syringes, heart-lung
machine components are now sterilized with ethylene oxide gas.

• Ethylene oxide (EtO) is both microbicidal and sporicidal and kills by combining with cell
proteins.

• It is a particularly effective sterilizing agent because it rapidly penetrates packing materials,

even plastic wraps.

• Sterilization is carried out in a special ethylene oxide sterilizer, very much resembling an
autoclave in appearance, that controls the EtO concentration, temperature, and humidity.

• Because pure EtO is explosive, it is usually supplied in a 10 to 20% concentration mixed with
either CO2 or dichlorodifluoromethane.

• A clean object can be sterilized if treated for 5 to 8 hours at 38°C or 3 to 4 hours at 54°C
when the relative humidity is maintained at 40 to 50% and the EtO concentration at 700
mg/liter.
• Extensive aeration of the sterilized materials is necessary to remove residual EtO
because it is so toxic.

• Betapropiolactone (BPL) is occasionally employed as a sterilizing gas.

• In the liquid form it has been used to sterilize vaccines and sera.

• BPL decomposes to an inactive form after several hours and is therefore not as
difficult to eliminate as EtO.

• It also destroys microorganisms more readily than ethylene oxide but does not
penetrate materials well and may be carcinogenic.

• For these reasons, BPL has not been used as extensively as EtO.