Physical and Chemical methods to control microorganisms

Dr. Chhaya Sawant

Chapter 8
Physical and Chemical methods to control microorganisms
Sterilization is defined as the process where all the living microorganisms, including
bacterial spores are killed.
Disinfection is the process of elimination of most pathogenic microorganisms (excluding
bacterial spores) on inanimate objects.
Some methods of disinfection such as filtration do not kill bacteria, they separate them out.
Decontamination is the process of removal of contaminating pathogenic microorganisms
from the articles by a process of sterilization or disinfection.
Sanitization is the process of chemical or mechanical cleansing, applicable in public health
systems. Usually used by the food industry. It reduces microbes on eating utensils to safe,
acceptable levels for public health.
Asepsis is the employment of techniques (such as usage of gloves, air filters, UV rays etc.) to
achieve microbe-free environment.
Antisepsis is the use of chemicals (antiseptics) to make skin or mucus membranes devoid of
pathogenic microorganisms.
Bacteriostasis is a condition where the multiplication of the bacteria is inhibited without
killing them.
Bactericidal is that chemical that can kill or inactivate bacteria. Such chemicals may be
called variously depending on the spectrum of activity, such as bactericidal, virucidal,
fungicidal, microbicidal, sporicidal, tuberculocidal or germicidal.
Antibiotics are substances produced by one microbe that inhibits or kills another microbe.
Often the term is used more generally to include synthetic and semi-synthetic antimicrobial
agents.

Physical methods of sterilization

Sunlight: The microbicidal activity of sunlight is mainly due to the presence of ultra violet
rays in it and heat. Helps in spontaneous sterilization in natural conditions.
Sunlight is not sporicidal; hence it does not sterilize.

Use of Heat:
• Heat is the most practical, reliable, and inexpensive method of sterilization. It is used
for sterilization of objects and materials that can withstand high temperatures.
• It can be either:
– Dry heat
– Moist Heat

Factors affecting sterilization by heat are:

• Nature of heat: Moist heat is more effective than dry heat
• Temperature and time: Temperature and time are inversely proportional. As
temperature increases the time taken decreases.
• Number of microorganisms: More the number of microorganisms, higher the
temperature or longer the duration required.
• Nature of microorganism: Depends on species and strain of microorganism,
sensitivity to heat may vary. Spores are highly resistant to heat.
 Physical and Chemical methods to control microorganisms
Dr. Chhaya Sawant

• Type of material: Articles that are heavily contaminated require higher temperature
or prolonged exposure. Certain heat sensitive articles must be sterilized at lower
temperature.
• Presence of organic material: Organic materials such as protein, sugars, oils and fats
increase the time required.

Heat is a method of sterilization of material that can withstand heat, by oxidative effects as
well as denaturation and coagulation of proteins.

Dry heat:
• Red Heat. Wire loops used in microbiology laboratory are sterilized by heating to
'red' in Bunsen burner or spirit lamp flame. Temperature is above 100°C. It leads to
sterilization.
• Flaming. The article is passed through flame without allowing it to become red hot,
e.g., scalpel. Temperature is not high to cause sterilization.
• Incineration. This is a method of destroying contaminated material by burning them
in incinerator. Articles such as soiled dressings; animal carcasses, pathological
material and bedding etc. should be subjected to incineration.

HOT AIR OVEN:

Introduced by Louis Pasteur. Articles to be sterilized are exposed to high temperature (160o
C) for duration of one hour in an electrically heated oven.
Working Principle: Sterilization by dry heat is performed by conduction. The temperature is
consumed by the surface of the objects, then moves towards the core of the object, coating
by coating. The object will attain the temperature needed for sterilization to take place.

Articles sterilized: Metallic instruments (like forceps, scalpels, scissors), glassware (such as
petri-dishes, pipettes, flasks, all-glass syringes), swabs, oils, grease, petroleum jelly and
some pharmaceutical products.
Sterilization cycle: Different temperature-time relations for holding time
– 60 minutes at 160oC,
– 40 minutes at 170oC
– 20 minutes at 180oC.
Advantages: It is an effective method of sterilization of heat stable articles. The articles
remain dry after sterilization. This is the only method of sterilizing oils and powders.
Disadvantages:
– Since air is poor conductor of heat, hot air has poor penetration.
– Cotton wool and paper may get slightly charred.
– Glasses may become smoky.

Infra red rays:

Infrared rays bring about sterilization by generation of heat.
Articles to be sterilized are exposed to infrared radiators to a temperature of 180oC The
articles are exposed to that temperature for a period of 7.5 minutes.

Articles sterilized included metallic instruments and glassware. It is mainly used in central
sterile supply department. It requires special equipment.
 Physical and Chemical methods to control microorganisms
Dr. Chhaya Sawant

MOIST HEAT:
Moist heat acts by coagulation and denaturation of proteins.

AT TEMPERATURE BELOW 100OC:

Pasteurization: This process was originally employed by Louis Pasteur. Currently this
procedure is employed in food and dairy industry.

Two methods of pasteurization,

(i) Flash Method. It is "high temperature- short time method". Heating is done at 72°C for
15 seconds.
(ii) Holding Method. Heating is done between 63°C and 66°C for 30 minutes.
Other pasteurization methods include Ultra-High Temperature (UHT), 140oC for 15 sec and
149oC for 0.5 sec.
This method is suitable to destroy most milk borne pathogens like Salmonella,
Mycobacteria, Streptococci, Staphylococci and Brucella, however Coxiella may survive
pasteurization.

How a pasteurizer works

A typical pasteurizer is completely automatic. You pour milk in one end and it flows
between a set of heating pipes or plates for a set period of time (long enough to kill off most
of the harmful bacteria), then between a set of cooling pipes, before emerging from an
outlet pipe into the bottles

STERILIZATION AT 100°C
Boiling. Boiling at 100°C for 30 minutes is done in a water bath. Syringes, rubber goods and
surgical instruments are sterilized by this method. All bacteria and certain spores are killed.
It leads to disinfection.

Steaming: Steam (100°C) is more effective than dry heat at the same temperature as:
(a) Bacteria are more susceptible to moist heat,
(b) Steam has more penetrating power,
(c) Steam has more sterilizing power as more heat is given up during condensation.
Steam Sterilizer. It works at 100°C under normal atmospheric pressure i.e. without extra
pressure. It is ideally suitable for sterilizing media which may be damaged at a temperature
higher than 100°C.

Sterilization at 100oC is done by two methods:

(a) Single Exposure for 1 and1/2 hours. It leads to disinfection.
(b) Tyndallisation (Fractional Sterilization). Heat labile media like those containing sugar,
milk, gelatine can be sterilized by this method.

Steaming at 100°C is done in steam sterilizer for 30 minutes followed by incubation at 37°C
overnight. This procedure is repeated for another 2 successive days. That is 'steaming' is
done for 3 successive days. Spores, if any, germinate to vegetative bacteria during
incubation and are destroyed during steaming on second and third day. It leads to
sterilization.
 Physical and Chemical methods to control microorganisms
Dr. Chhaya Sawant

AT TEMPERATURE ABOVE 100OC:

Autoclave: Sterilization can be effectively achieved at a temperature above 100oC using an
autoclave.
Water boils at 100oC at atmospheric pressure, but if pressure is raised, the temperature at
which the water boils also increases.

Autoclaving (steam under pressure) : The most effective and most efficient means of
sterilization.
• All autoclaves operate on a time/temperature relationship.
• Standard temperature/pressure employed is 121ºC/15 psi for 15 minutes. Longer
times are needed for larger loads, large volumes of liquid, and more dense materials.
• Used for sterilizing bio-hazardous waste, surgical dressings, glassware, many types of
microbiologic media, liquids, and many other things.
• However, certain items, such as plastics and certain medical instruments (e.g. fibre-
optic endoscopes), cannot withstand autoclaving and should be sterilized with
chemical or gas sterilants.

Autoclave:
• Advantages of steam: It has more penetrative power than dry air, it moistens the
spores (moisture is essential for coagulation of proteins), condensation of steam on
cooler surface releases latent heat, condensation of steam draws in fresh steam.
Advantage: Very effective way of sterilization, quicker than hot air oven.
Disadvantages: Drenching and wetting or articles may occur, trapped air may reduce the
efficacy, takes long time to cool

RADIATION
Two types of radiation are used, ionizing and non-ionizing.
– Non-ionizing rays are low energy rays with poor penetrative power
– Ionizing rays are high-energy rays with good penetrative power.

Non-ionizing rays:
Rays of wavelength longer than the visible light are non-ionizing.
Most effective Microbicidal wavelength of UV rays is 260 nm, generated using a high
pressure mercury vapour lamp.
UV rays induce formation of thymine-thymine dimers, which ultimately inhibits DNA
replication. UV readily induces mutations in cells irradiated with a non-lethal dose.
UV rays are employed to disinfect hospital wards, operation theatres, virus laboratories,
corridors, etc.
Disadvantages of using UV rays include low penetrative power, limited life of the UV bulb,
some bacteria have DNA repair enzymes that can overcome damage caused by UV rays,
organic matter and dust prevents its reach, rays are harmful to skin and eyes. It doesn't
penetrate glass, paper or plastic.

Ionizing rays: Electromagnetic rays such as gamma rays emanate from nuclear
disintegration of certain radioactive isotopes (CO60, Cs137).
• More penetrative power than electron beam but require longer time of exposure.
 Physical and Chemical methods to control microorganisms
Dr. Chhaya Sawant

• These high-energy radiations damage the nucleic acid of the microorganism.

• A dosage of 2.5 megarads kills all bacteria, fungi, viruses and spores. It is used
commercially to sterilize disposable petri dishes, plastic syringes, antibiotics,
vitamins, hormones, glasswares and fabrics.

Disadvantages include; unlike electron beams, they can’t be switched off, glassware tend to
become brownish, loss of tensile strength in fabric. Gamma irradiation impairs the flavour of
certain foods. Bacillus pumilus E601 is used to evaluate sterilization process.

FILTRATION STERILIZATION
Filtration process does not destroy but removes the microorganisms.
• Use : Both the clarification and sterilization of liquids and gases as it is capable of
preventing the passage of both viable and non viable particles.

The major mechanisms of filtration

• Sieving, adsorption and trapping within the matrix of the filter material.
• Sterilizing grade filters are used
– In the treatment of heat sensitive injections and ophthalmic solutions,
biological products and air and other gases for supply to aseptic areas.
– They are also used in industry as part of the venting systems on fermentors,
centrifuges, autoclaves and freeze driers.
– Membrane filters are used for sterility testing.

Different types of filters are:

Earthenware filters: These filters are made up of diatomaceous earth or porcelain. They are
usually baked into the shape of candle.
Asbestos filters: These filters are made from chrysotile type of asbestos, chemically
composed of magnesium silicate. They are pressed to form disc, which are to be used only
once. The disc is held inside a metal mount, which is sterilized by autoclaving.
Sintered glass filters: These are made from finely ground glass that are fused sufficiently to
make small particles adhere to each other. They are usually available in the form of disc
fused into a glass funnel.
Membrane filters: These filters are made from a variety of polymeric materials such as
cellulose nitrate, cellulose diacetate, polycarbonate and polyester.
o Millipore (membrane) filters:
▪ Synthetic membranes made from cellulose diacetate.
o Syringe filter:
▪ Membrane filters 13 – 25 mm in diameter in a small holder connected
to a syringe containing the fluid to be filtered
Air Filters: Air can be filtered using HEPA (High Efficiency Particle Air) filters. They are
usually used in biological safety cabinets. HEPA filters are at least 99.97% efficient for
removing particles >0.3 μm in diameter. HEPA filter efficiency is monitored with the
dioctylphthalate (DOP) particle test using particles that are 0.3 μm in diameter.
 Physical and Chemical methods to control microorganisms
Dr. Chhaya Sawant

Chemical Sterilization: Classification of disinfectants is based on its consistency, spectrum of

activity and mechanism of action

1. Based on consistency
a. Liquid (E.g., Alcohols, Phenols) b. Gaseous (Formaldehyde vapour, Ethylene oxide)
2. Based on spectrum of activity
a. High level b. Intermediate level c. Low level

3. Based on mechanism of action

a. Action on membrane (E.g., Alcohol, detergent)
b. Denaturation of cellular proteins (E.g., Alcohol, Phenol)
c. Oxidation of essential sulfhydryl groups of enzymes (E.g., H2O2, Halogens)
d. Alkylation of amino-, carboxyl- and hydroxyl group (E.g., Ethylene Oxide,
Formaldehyde)
e. Damage to nucleic acids (Ethylene Oxide, Formaldehyde)

Different chemicals used are

ALCOHOLS: Mode of action-Alcohols dehydrates cells, disrupt membranes and cause
coagulation of protein. Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol
Disadvantages: Skin irritant, volatile (evaporates rapidly), inflammable

ALDEHYDES: Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl

group, and probably damages nucleic acids. It kills all microorganisms, including spores.
Examples: Formaldehyde, Gluteraldehyde,
Disadvantages: Vapours are irritating (must be neutralized by ammonia), has poor
penetration, leaves non-volatile residue, activity is reduced in the presence of protein.
Gluteraldehyde requires alkaline pH and only those articles that are wettable can be
sterilized.

PHENOL: Mode of action: Act by disruption of membranes, precipitation of proteins and

inactivation of enzymes. Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol),
hexachlorophene, chlorhexidine, chloroxylenol (Dettol)
Disadvantages: It is toxic, corrosive and skin irritant. Chlorhexidine is inactivated by anionic
soaps. Chloroxylenol is inactivated by hard water.

HALOGENS: Mode of action: They are oxidizing agents and cause damage by oxidation of
essential sulfydryl groups of enzymes. Chlorine reacts with water to form hypochlorous acid,
 Physical and Chemical methods to control microorganisms
Dr. Chhaya Sawant

which is microbicidal. Examples: Chlorine compounds (chlorine, bleach, hypochlorite) and

iodine compounds (tincture iodine, iodophores)
Disadvantages: They are rapidly inactivated in the presence of organic matter. Iodine is
corrosive and staining. Bleach solution is corrosive and will corrode stainless steel surfaces.

SURFACE ACTIVE AGENTS: Mode of actions: They concentrate at interfaces between lipid
containing membrane of bacterial cell and surrounding aqueous medium. Having long chain
hydrocarbons that are fat soluble and charged ions that are water-soluble, they concentrate
on the surface of membranes. They disrupt membrane resulting in leakage of cell
constituents.
Examples : Anionic detergents contains negatively charged long chain hydrocarbon, Soaps
and bile salts, Cationic detergents Quaternary ammonium compounds - Cetrimide and
benzalkonium chloride
Disadvantages: Their activity is reduced by hard water, anionic detergents and organic
matter. Pseudomonas can metabolise cetrimide, using them as a carbon, nitrogen and
energy source.

DYES: Mode of action: Acridine dyes are bactericidal because of their interaction with
bacterial nucleic acids. Examples: Aniline dyes such as crystal violet, malachite green and
brilliant green. They may be used topically as antiseptics to treat mild burns and bacterial
infections and as selective agents in certain selective media.

HYDROGEN PEROXIDE: Mode of action: It acts on the microorganisms through its release of
nascent oxygen. Hydrogen peroxide produces hydroxyl-free radical that damages proteins
and DNA.
Disadvantages: Decomposes in light, broken down by catalase, proteinaceous organic
matter drastically reduces its activity

ETHYLENE OXIDE (EO): Mode of action: It is an alkylating agent. It acts by alkylating

sulfydryl-, amino-, carboxyl- and hydroxyl- groups.
Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and
carcinogenic.

HEAVY METALS: Mode of action: Act by precipitation of proteins and oxidation of sulfydryl
groups. They are bacteriostatic. Examples: Mercuric chloride, silver nitrate, copper sulfate,
organic mercury salts (e.g., mercurochrome, merthiolate)
Disadvantages: Mercuric chloride is highly toxic, are readily inactivated by organic matter.

BETA-PROPIOLACTONE (BPL): Mode of action: It is an alkylating agent and acts through

alkylation of carboxyl- and hydroxyl- groups.
Disadvantages: It has poor penetrating power and is a carcinogen