3.

Control of microorganisms

Sterilization

Sterilization is the process by which all living cells, spores, and acellular entities
(e.g., viruses, viroids, and prions)are either destroyed or removed from an object or
habitat.

Disinfection

Disinfection is the killing, inhibition, or removal of microorganisms that may

cause disease.

Microbes can be controlled by many mechanisms which can be physical or

chemical methods.

Physical agents

The six most frequently employed physical agents are:

1. Heat
2. Low temperatures
3. Desiccation
4. Osmotic pressure
5. Radiation
6. Filtration

1. Heat/High temperature

 Either moist or dry heat may be applied.

 Moist heat readily kills viruses, bacteria, and fungi .Moist heat is thought to
kill by degrading nucleic acids and by denaturing enzymes and other
essential proteins. It may also disrupt cell membranes.
  In order to destroy bacterial endospores, moist heat sterilization must be
carried out at temperatures above 100°C, and this requires the use of
saturated steam under pressure. Steam sterilization is carried out with an
autoclave (121°C and 15 pounds of pressure, 15 minutes), a device
somewhat like a fancy pressure cooker.
 Many substances, such as milk, are treated with controlled heating at
temperatures well below boiling, a process known as pasteurization.
 Many objects are best sterilized in the absence of water by dry heat
sterilization. Some items are sterilized by incineration.For instance,
inoculating loops, which are used routinely in the laboratory, can be
sterilized in a small, bench-top incinerator.
 Other items are sterilized in an oven at 160 to 170°C for 2 to 3 hours.
Microbial death apparently results from the oxidation of cell constituents
and denaturation of proteins.
 Dry air heat is less effective than moist heat.It 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 glassware and
pipettes with dry heat. Dry heat sterilization is slow and not suitable for heat
sensitive materials like many plastic and rubber items.
 Because heat is so useful in controlling microorganisms, it is essential to
have a precise measure of the heat-killing efficiency.
 Thermal Death Time (TDT) is the shortest time needed to kill all
organisms in a microbial suspension at a specific temperature and under
defined conditions.
 The decimal reduction time (D value) is the time required to kill 90% of
the microorganisms or spores in a sample at a specified temperature.
 The z value is the increase in temperature required to reduce D to 1/10 its
value
 The F value is the time in minutes at a specific temperature (usually 250°F
or 121.1°C) needed to kill a population of cells or spores.
 The food processing industry makes extensive use of D and z values. After a
food has been canned, it must be heated to eliminate the risk of botulism
arising from Clostridium botulinum spores.

2. Low temperatures

The most convenient control technique for microorganisms is to inhibit their

growth and reproduction by the use of either freezing or refrigeration.
 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.
 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.
 Refrigerated items may be ruined by growth of psychrophilic and
psychrotrophic microorganisms, particularly if water is present.Thus
refrigeration is a good technique only for shorter-term storage of food and other
items.

3. Desiccation/Drying

 Drying can be used to preserve foods because the absence of water inhibits the
action of enzymes. Many foods, including peas, beans, raisins, and other fruits,
are often preserved by drying.
 Drying also naturally minimizes the spread of infectious agents.
 Some bacteria, such as Treponema pallidum, which causes syphilis, are
extremely sensitive to drying and die almost immediately on a dry surface; thus
they can be prevented from spreading by keeping toilet seats and other
bathroom fixtures dry.
 Drying of laundry in dryers or in the sunshine also destroys pathogens.

4.Osmotic pressure

 High concentrations of salt, sugar, or other substances create a hyperosmotic

medium, which draws water from microorganisms by osmosis.
 Plasmolysis or loss of water, severely interferes with cell function and
eventually leads to cell death.
 The use of sugar in jellies, jams, and syrups or salt solutions in curing meat and
making pickles plasmolyzes most organisms present and prevents growth of
new organisms.

5.Radiation

 Microbiologists take advantage of the effects of non-ionizing and ionizing

radiation to sterilize or disinfect objects.
 Ultraviolet (UV) radiation around 260 nm is quite lethal but does not
penetrate glass,dirt films, water, and other substances very effectively.
 Ultraviolet light is especially effective in inactivating viruses. However, it kills
far fewer bacteria.
 It acts by destroying the nucleic acids.
 UV lamps are sometimes placed on the ceilings of rooms or in biological safety
cabinets to sterilize the air and any exposed surfaces.
 Ionizing radiation damages DNA and produces peroxides,which act as powerful
oxidizing agents in cells.
 Ionizing radiation(X ray and gamma rays) is an excellent sterilizing agent
and penetrates deep into objects. It will destroy bacterial endospores and
vegetative cells, both prokaryotic and eukaryotic; however, ionizing radiation is
not always effective against viruses.
 Gamma radiation from a cobalt 60 source is used in the cold sterilization of
antibiotics, hormones, and plastic disposable supplies such as syringes. Gamma
radiation has also been used to sterilize and “pasteurize” meat and other foods.
 Irradiation can eliminate the threat of such pathogens as Escherichia coli
O157:H7, Staphylococcus aureus, and Campylobacter jejuni.

6. 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.
 There are two types of filters.

1. Depth filters

Consist of fibrous or granular materials that have been bonded into a thick layer
filled with twisting channels of small diameter.Depth filters are made of
diatomaceous earth (Berkefield filters),unglazed porcelain (Chamberlain filters),
asbestos, or other similar materials.

2.Membrane filters

Have replaced depth filters for many purposes.These circular filters are porous
membranes, a little over 0.1 mm thick, made of cellulose acetate, cellulose nitrate,
polycarbonate, polyvinylidene fluoride, or other synthetic materials. 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.
 Other important examples are laminar air flow(LAF) biological safety
cabinets, which employ high-efficiency particulate air (HEPA) filters (a type
of depth filter) to remove 99.97% of 0.3 µm particles.
 Laminar flow biological safety cabinets or hoods force air through HEPAfilters,
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 and tumor
viruses. They are also employed in research labs and industries, such as the
pharmaceutical industry, when a sterile working surface is needed.

Chemical agents

 Chemicals are more often employed in disinfection and antisepsis.

 Chemicals also are employed to prevent microbial growth in food, and
certain chemicals are used to treat infectious diseases.

Characteristics of an ideal antimicrobial chemical agent

 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 low concentrations and in the
presence of organic matter.
 The chemical must be toxic for infectious agents, it should not be toxic to
people or corrosive for common materials.
 The ideal disinfectant should be stable upon storage, odorless or with a
pleasant odor, soluble in water and lipids for penetration into
microorganisms.
 It should have a low surface tension so that it can enter cracks in surfaces.
  It should be inexpensive.

The chemical agents commonly used are:

1. 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.
 Isopropyl alcohol is more often used because of legal regulation of ethyl
alcohol.
 It disinfects skin where injections will be made or blood drawn.
 Alcohol disinfects but does not sterilize skin because it evaporates
quickly and stays in contact with microbes for only a few seconds.It also
does not penetrate deeply enough into pores in the skin.
 Ethylene glycol and propylene glycol can be used in aerosols

2. 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 cross-linking and alkylating molecules.
 They are sporicidal
 Formaldehyde is usually dissolved in water or alcohol before use
 2% buffered solution of glutaraldehyde is an effective disinfectant.It is less
irritating than formaldehyde and is used to disinfect hospital and laboratory
equipment.
 Usually disinfects objects within about 10 minutes but may require as long
as 12 hours to destroy all spores.
3. Dyes

 The dye acridine, which interferes with cell replication by causing mutations
in DNA can be used to clean wounds.
  Methylene blue inhibits growth of some bacteria in cultures.
 Crystal violet (gentian violet) blocks cell wall synthesis, possibly by the same
reaction that causes this dye to bind to cell wall material in Gram staining.It
effectively inhibits growth of Gram-positive bacteria in cultures and in skin
infections. It can be used to treat protozoan (Trichomonas) and yeast
(Candida albicans) infections.

4. Halogens
 A halogen is any of the five elements (fluorine, chlorine, bromine,
iodine, and astatine) in group VIIA of the periodic table.

 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.
 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 an organic carrier to form an
iodophor.
 Iodophors are water soluble, stable, and nonstaining, and 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.
 Some popular brands are Wescodyne for skin and laboratory disinfection
and Betadine for wounds.
 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 (bleach), or calcium
hypochlorite, all of which yield hypochlorous acid (HClO) and then atomic
oxygen. The result is oxidation of cellular materials and destruction of
vegetative bacteria and fungi, although not spores.
 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 dichloramidobenzoic acid) slowly releases chloride
when added to water and disinfects it in about a half hour.

5. Phenolics

 Phenol was the first widely used antiseptic and disinfectant. In 1867 Joseph
Lister employed it to reduce the risk of infection during surgery.
 Today phenol and phenolics (phenol derivatives) such as cresols, xylenols, and
orthophenylphenol are used as disinfectants in laboratories and hospitals.
 Phenolics act by denaturing proteins and disrupting cell membranes.
 The commercial disinfectant Lysol is made of a mixture of phenolics.
 Phenolics are tuberculocidal, effective in the presence of organic material, and
remain active on surfaces long after application. However, they have a
disagreeable odor and can cause skin irritation.

6. Acids and Alkalis

 Soap is a mild alkali, and its alkaline properties help destroy microbes.
 A number of organic acids lower the Ph of materials sufficiently to inhibit
fermentation. Several are used as food preservatives.
 Lactic and propionic acids retard mold growth in breads and other products.
 Benzoic acid and several of its derivatives are used to prevent fungal
growth in soft drinks, ketchup, and margarine.
 Sorbic acid and sorbates are used to prevent fungal growth in cheeses and a
variety of other foods.
 Boric acid, formerly used as an eye wash, is no longer recommended
because of its toxicity.

7. Gases

 Many heat-sensitive items such as disposable plastic petri dishes and

syringes, heart-lung machine components and catheters are sterilized with
ethylene oxide gas .
 (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. 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.
 Vaporized hydrogen peroxide can be used to decontaminate biological safety
cabinets, operating rooms, and other large facilities.
 During the course of the decontamination process, it breaks down to water
and oxygen, both of which are harmless. Other advantages of these systems
are that they can be used at a wide range of temperature(4 to 80°C) and they
do not damage most materials.

8. Heavy Metals

 For many years the ions of heavy metals such as mercury, silver, arsenic,
zinc, and copper were used as germicides.
 In some hospitals, a 1% solution of silver nitrate is added to the eyes of
infants to prevent ophthalmic gonorrhea.
 Silver sulfadiazine is used on burns.
 Copper sulfate is an effective algicide in lakes and swimming pools.