Introduction to Controlling Microbial Growth

Introduction

In the 19th century, surgery was risky and dangerous, and patients undergoing even the most
routine operations were at very high risk of infection. This was so because surgery was not
performed under aseptic conditions. The operating room, the surgeon's hands, and the surgical
instruments were laden with microbes, which caused high levels of infection and mortality.

Surgeons in the mid-1800s often operated wearing their street clothes, without washing their
hands. They frequently used ordinary sewing thread to suture wounds, and stuck the needles in
the lapels of their frock coats in between patients. Surgical dressings were often made up of
surplus cotton or jute from the floors of cotton mills. It was against this background that French
scientist Louis Pasteur demonstrated that invisible microbes caused disease

Louis Pasteur

Pasteur's work influenced the English surgeon Joseph Lister, who applied Pasteur's germ theory
of disease to surgery, thus founding modern antiseptic surgery. To disinfect, Lister used a
solution of carbolic acid (phenol), which was sprayed around the operating room by a handheld
sprayer.

Joseph Lister

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It was clear that Lister's techniques were effective in increasing the rates of surviving surgery,
but his theories were controversial because many 19th century surgeons were unwilling to accept
something they could not see. Also, perhaps another reason that surgeons were slow to pick up
on Lister's methods was the fact that during surgery they were required to breathe an irritating
aerosol of phenol.

Control of Microbial Growth

The control of microbial growth is necessary in many practical situations, and significant
advances in agriculture, medicine, and food science have been made through study of this area of
microbiology. "Control of microbial growth", as used here, means to inhibit or prevent growth of
microorganisms. This control is affected in two basic ways: (1) by killing microorganisms or (2)
by inhibiting the growth of microorganisms

The control of microbial growth may involve sterilization, disinfection, antisepsis, sanitization,
or degerming. Sterilization is the destruction of all forms of microbial life, with particular
attention to bacterial spores. Disinfection and antisepsis both refer to destruction of microbial
pathogens, although some organisms, such as bacterial spores, may remain alive. Disinfection
refers to the destruction of pathogenic organisms on an inanimate (lifeless) object, such as a
table-top, while antisepsis refers to that destruction on a living object, such as the skin surface

Sanitization refers to the reduction in the number of pathogens to a level deemed safe by public
health guidelines. Degerming is the physical removal of microorganisms by using such things as
soaps or detergents.

Any chemical agent that kills microorganisms is known as a germicide. An agent that destroys
bacteria is called a bactericide, one that kills fungi is a fungicide, and one that kills viruses is a
virucide. Agents which kill cells are called cidal agents; agents which inhibit the growth of cells
(without killing them) are referred to as static agents. Thus, the term bactericidal refers to
killing bacteria, and bacteriostatic refers to inhibiting the growth of bacterial cells.

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Among the conditions affecting the use of a germicide are temperature, the type of
microorganism, and the environment. Germicides are more effective at high temperatures
because the chemical breaks down at lower temperatures. Microorganisms vary in their
susceptibility depending on such things as the composition of their cell wall, the presence or
absence of a capsule, and the ability to form spores or cysts. The environment can affect the
activity of a germicide, as, for example, when organic matter is present. This material shields
microorganisms from germicides and often reacts with the germicide.

Physical methods:

• Heat (Dry and moist)

• Sunlight
• Vibration
• Radiation
• Filtration

Heat

Heat is considered to be most reliable method of sterilization of objects that can

withstand heat.Heat as Moist and Dry heat are the most common sterilizing methods
used in hospitals and are indicated for most materials.

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 Dry Heat:

Causes denaturation of proteins and oxidative damage.Techniques include:

• Red Heat (common uses: straight wires, bacterial loops and spatulas)
• Flaming (Common uses: bacterial loops, wires and spatulas)
• Incineration (common uses: soil dressing, pathological bedding)
• Hot Air oven (discovered by Louis Pasteur, common uses: in dairy industry)
• Infra red rays (common uses: heat glassware and metallic instruments)

Moist Heat: 

Moist heat is more efficient in contrast to dry heat; it causes coagulation and denaturation
of proteins. 

At temperature below 100°C:
• Pasteurization: Food (dairy) Industry
• Vaccine bath: (vaccine sterilization)
• Serum bath: (serum contaminants, does not kill spores survive)
• Inspissations: (egg and serum containing media, can kill spores)

At temperature 100°C:
• Boiling: Boiling water (100°C)
• Steam (100°C)
At temperature above 100°C:
• Autoclave

The temperature used in each method and effectiveness is clearly summarized in the table below.

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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.
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.

Radiation
There are 2 types of Radiation:

Non-ionizing: wavelength longer then visible light.

• UV Radiation has a wavelength of 200-280nm; it has a germicidal effect on
microorganisms.
• Common uses: Surface disinfection, in hospitals, operating theatre and laboratories.
Ionizing: 2 types:
• Particulate (Electron beam)
• Common uses: sterilization of instruments such as syringes, gloves, dressing packs,
foods and pharmaceuticals.
• Electromagnetic (Gamma rays)
• Common uses: sterilization of disposable Petri dishes, plastic syringes, antibiotics,
vitamins, hormones and fabrics.
The Other physical methods used in sterilization do not kill all the organisms hence
considered to be a form of disinfection.

Physical Methods of Control

Physical methods for controlling the growth of microorganisms can be divided into heat methods
and nonheat methods. The lowest temperature at which all microorganisms are killed in 10
minutes is the thermal death point, while the minimum amount of time required to kill
microorganisms at a given temperature is known as the thermal death time. The time for
destruction of 90 percent of the microbial population is the decimal reduction time

Moist heat:
Moist heat is used to kill microorganisms in such things as boiling water. Most vegetating
microorganisms are killed within two or three minutes, but over two or three hours may be
required for destruction of bacterial spores. In moist heat, the microbial proteins undergo

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denaturation, a process in which the three-dimensional form of the protein reverts to a two-
dimensional form, and the protein breaks down.

At temperature below 100oC:

Pasteurization: This process was originally employed by Louis Pasteur. Currently this
procedure is employed in food and dairy industry. There are two methods of
pasteurization, the holder method (heated at 63oC for 30 minutes) and flash method
(heated at 72oC for 15 seconds) followed by quickly cooling to 13oC. 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, Mycobacterium, Streptococci, Staphylococci and Brucella, however Coxiella
may survive pasteurization. Efficacy is tested by phosphatase test and methylene blue
test.

Vaccine bath: The contaminating bacteria in a vaccine preparation can be inactivated by

heating in a water bath at 60oC for one hour. Only vegetative bacteria are killed and
spores survive.

Serum bath: The contaminating bacteria in a serum preparation can be inactivated by

heating in a water bath at 56oC for one hour on several successive days. Proteins in the
serum will coagulate at higher temperature. Only vegetative bacteria are killed and spores
survive.

Inspissations: This is a technique to solidify as well as disinfect egg and serum containing
media. The medium containing serum or egg are placed in the slopes of an inspissator
and heated at 80-85oC for 30 minutes on three successive days. On the first day, the
vegetative bacteria would die and those spores that germinate by next day are then killed
the following day. The process depends on germination of spores in between inspissation.
If the spores fail to germinate then this technique cannot be considered sterilization.

At temperature 100oC:

Boiling: Boiling water (100oC) kills most vegetative bacteria and viruses immediately.
Certain bacterial toxins such as Staphylococcal enterotoxin are also heat resistant. Some
bacterial spores are resistant to boiling and survive; hence this is not a substitute for
sterilization. The killing activity can be enhanced by addition of 2% sodium bicarbonate.
When absolute sterility is not required, certain metal articles and glasswares can be
disinfected by placing them in boiling water for 10-20 minutes. The lid of the boiler must
not be opened during the period.

Steam at 100oC: Instead of keeping the articles in boiling water, they are subjected to free
steam at 100oC. Traditionally Arnold‟s and Koch‟s steamers were used. An autoclave
(with discharge tap open) can also serve the same purpose. A steamer is a metal cabinet
with perforated trays to hold the articles and a conical lid. The bottom of steamer is filled
with water and heated. The steam that is generated sterilizes the articles when exposed

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 for a period of 90 minutes. Media such as TCBS, DCA and selenite broth are sterilized by
steaming. Sugar and gelatin in medium may get decomposed on autoclaving; hence they
are exposed to free steaming for 20 minutes for three successive days. This process is
known as Tyndallisation (after John Tyndall) or fractional sterilization or intermittent
sterilization. The vegetative bacteria are killed in the first exposure and the spores that
germinate by next day are killed in subsequent days. The success of process depends on
the germination of spores.

At temperature above 100oC:

Autoclave is the modern equipments with many controls and safeguards to manage day to day
sterilization in the Clinics, Hospitals, O.T.s, and Labs. Autoclave is economically designed,
automatic and convenient to operate. After placing instruments in the sterilizer one needs to just
switch on and the equipments controls the rest. It is very simple to install, use and maintain.

Different types of autoclave:

Simple “pressure-cooker type” laboratory autoclave, Steam jacketed downward displacement
laboratory autoclave and high pressure pre-vacuum autoclave

Construction
Triple walled with steam jacket and separate boiler. Inner chamber and steam jacket are made
our of Heavy gauge S.S. sheet with leak proof argon welding. The Autoclave has single piece
door made of stainless steel. Back plate and rising is also made of thick stainless steel sheet. All
the Autoclaves are hydraulically tested to withstanding 2.5 times the working pressure. It is
mounted on tubular steel frame with ground leveling screws. The Outer jacket is wrapped with
asbestos sheet or glass wool to minimize the heat losses due to radiation and is covered by
polished stainless steel for the elegant appearance.

Steam Generator
Made of heavy stainless steel sheet, Heavy ring mounted in front of the boiler with folding thick
stainless steel plate fitted with heating elements and low water level cutoff device to protect the
former from burning out dry. Front folding plate system provides for easy cleaning of the
deposited scale on the elements and the walls for long life and efficiency. Fitted with water
Gauge glass for water level indication, water inlet and outlet valves

Pressure Control

The pressure control device is incorporated in all electrically operated sterilizers. It economics on
power consumption and reduces the frequent opening of the steam release valve and prevents the
release of steam in the room, it cuts off the power supply to heaters when the set pressure is
achieved an re-energizes the heating when the set pressure is achieved and re-energizes the
heating elements when pressure falls below the set point.

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Heating Elements

Flanged type/ immersion heating element are made of high grade material Operating Temp. &
Pressure Sterilizing temp: 121" Sterilizing pressure: 1.2o 1.5 kg/cm2 (15 psi to 22 psi) Power
Requirement: Suitable to operate on 440V volts, 3ph, 50Hz/Ac supply

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Effectiveness of Autoclave or Optimum Conditions:

Sterilization in an autoclave is most effective when the organisms are either contacted by the
steam directly or are contained in a small volume of aqueous (primarily water) liquid. Under
these conditions, steam at a pressure about 15 psi; attaining temperature (121oC) will kill all
organisms and their endospores in about 15 minutes.

Principle of Autoclaving:

A basic principle of chemistry is that when the pressure of a gas increases, the temperature of the
gas increase proportionally. For example, when free flowing steam at a temperature of 100oC is
placed under a pressure of 1 atmosphere above sea level pressure – that is, about 15 pounds of
pressure per square inch (Psi) --- the temperature rises to 121oC. Increasing the pressure to 20 psi
raises the temperature to 126oC. The relationship between temperature and pressure is shown in
table 2. In this way steam is a gas, increasing its pressure in a closed system increases its
temperature. As the water molecules in steam become more energized, their penetration
increases substantially. This principle is used to reduce cooking time in the home pressure
cooker and to reduce sterilizing time in the autoclave. It is important to note that the sterilizing
agent is the moist heat, not the pressure.

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 The Relationship Between the Pressure and
Table
Temperature of Steam at Sea Level*
Pressure (psi in excess of atmospheric
Temperature (oC)
pressure)
0 psi 100
5 psi 110
10 psi 116
15 psi 121
20 psi 126
30 psi 135

Rules implied for Autoclaving:

Sterilization by autoclaving is invariably successful if properly done and if two common-sense

rules are followed:

First, articles should be placed in the autoclave so that steam can easily penetrate them.
Second, air should be evacuated so that the chamber fills with steam.

Working of Autoclave:

Most autoclaves contain a sterilizing chamber into which articles are place and a steam jacket
where steam is maintained. As steam flows from the steam jacket into the sterilizing chamber,
cool air is forced out and a special valve increases the pressure to 15 pounds/square inch above
normal atmospheric pressure. The temperature rises to 121.5oC, and the superheated water
molecules rapidly conduct heat into microorganisms. The time for destruction of the most
resistant bacteria spore is now reduced to about 15 minutes. For denser objects, up to 30 minutes
of exposure may be required. The conditions must be carefully controlled or serious problems
may occur.

Uses of Autoclave:

Autoclaving is used to sterilize culture media, instruments, dressings, intravenous equipment,

applicators, solutions, syringes, transfusion equipment, and numerous other items that can
withstand high temperatures and pressures. The laboratory technician uses it to sterilize
bacteriological media and destroy pathogenic cultures. The autoclave is equally valuable for
glassware and metal ware, and is among the first instruments ordered when a microbiology
laboratory is established. Autoclaves are also used on large industrial scale. Large industrial
autoclaves are called retorts, but the same principle applies for common household pressure
cooker used in the home canning of foods

Limitations and Disadvantages of Autoclave:

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The autoclave also has certain limitations. For example, some plastic ware melts in the high heat,
and sharp instruments often become dull. Moreover, many chemicals breakdown during the
sterilization process and oily substances cannot be treated because they do not mix with water.

Heat requires extra time to reach the center of solid materials, such as canned meats, because
such materials do not develop the efficient heat-distributing convection currents that occur in
liquids. Heating large containers also requires extra time. Drenching and wetting or articles may
occur; trapped air may reduce the efficacy, takes long time to cool

The effect of Container Size on Autoclve

Table
Sterilization Times for Liquid Solutions*
Sterilization Time
Container Size Liquid Volume
(min)
Test Tube:
10 ml 15
18×150 mm
Erlenmeyer Flask:
95 ml 15
125 ml
Erlenmeyer Flask:
1500 ml 30
2000 ml
Fermentation Bottle:
6750 ml 70
9000 ml

Indicator of Sterilization Achievement:

Sterilization control:
Physical method includes automatic process control, thermocouple and temperature chart
recorder.
Chemical method includes Browne‟s tube No.1 (black spot) and succinic acid (whose
melting point is 121oC) and Bowie Dick tape. Bowie Dick tape is applied to articles being
autoclaved. If the process has been satisfactory, dark brown stripes will appear across the
tape.
Biological method includes a paper strip containing 106 spores of Bacillus
stearothermophilus.

DRY HEAT

Dry heat kills microorganisms by reacting with and oxidizing their proteins. Dry heat can be
used in incineration devices, such as the Bunsen burner or the hot-air oven. In the hot-air oven,
a temperature of about 170°C for two hours will bring about sterilization.

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Red heat: Articles such as bacteriological loops, straight wires, tips of forceps and searing
spatulas are sterilized by holding them in Bunsen flame till they become red hot. This is a simple
method for effective sterilization of such articles, but is limited to those articles that can be
heated to redness in flame.

Flaming: This is a method of passing the article over a Bunsen flame, but not heating it to
redness. Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips are
passed through the flame a few times. Even though most vegetative cells are killed, there is no
guarantee that spores too would die on such short exposure. This method too is limited to those
articles that can be exposed to flame. Cracking of the glassware may occur.

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. This technique results in the loss of the article,
hence is suitable only for those articles that have to be disposed. Burning of polystyrene
materials emits dense smoke, and hence they should not be incinerated.
Hot air oven: This method was introduced by Louis Pasteur. Articles to be sterilized are
exposed to high temperature (160oC) for duration of one hour in an electrically heated oven.
Since air is poor conductor of heat, even distribution of heat throughout the chamber is achieved
by a fan. The heat is transferred to the article by radiation, conduction and convection. The oven
should be fitted with a thermostat control, temperature indicator, meshed shelves and must have
adequate insulation.
It has a triple walled inner chamber made of special grade stainless steel. Two perforated
Stainless Steel shelves are provided for keeping the samples. The inner walls are made of
Stainless Steel and outer walls of mild steel sheet, duly powder coated for striking appearance.
The forced air circulation system, with a horizontal blower, keeps the temperature in the inner
chamber uniform and consistent. The Temperature Range is from ambient to 250 º C with an
accuracy of ± 2 º C. It has 2 special Heaters of 750 watts each. The Heaters are made of high-
quality chrome wire and finest ceramic Beads. The Heaters are duly placed in, specially
fabricated deep drawn ribs, in the Inner Stainless Steel Chamber, for uniform Temperature in the
Oven.
The insulated Door is fixed with toughened Glass view panel, so that the sample inside can be
viewed without disturbing the Temperature. The complete circuitry is on the top, with a special
cooling Fan, to let the Oven run continuously for unusually longer hours. A specially fabricated
door with specially designed handle keeps the inside temperature completely stable. The
specially fabricated footrest keeps the whole oven in correct equilibrium.

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1. Handle to open the oven.
2. Mains lead with plug.
3. Ventilation port for moisture out let.
4. Control panel.
5. Top cover of the unit

Articles sterilized: Metallic instruments (like forceps, scalpels, and scissors), glassware‟s (such
as Petri-dishes, pipettes, flasks, and all-glass syringes), swabs, oils, grease, petroleum jelly and
some pharmaceutical products.

Sterilization process: Articles to be sterilized must be perfectly dry before placing them inside to
avoid breakage. Articles must be placed at sufficient distance so as to allow free circulation of air
in between. Mouths of flasks, test tubes and both ends of pipettes must be plugged with cotton
wool. Articles such as petri dishes and pipettes may be arranged inside metal canisters and then
placed. Individual glass articles must be wrapped in Kraft paper or aluminum foils.

Sterilization cycle: This takes into consideration the time taken for the articles to reach the
sterilizing temperature, maintenance of the sterilizing temperature for a defined period (holding
time) and the time taken for the articles to cool down. Different temperature-time relations for
holding time are 60 minutes at 160oC, 40 minutes at 170oC and 20 minutes at 180oC. Increasing
temperature by 10 degrees shortens the sterilizing time by 50 percent. The hot air oven must not
be opened until the temperature inside has fallen below 60oC to prevent breakage of glass wares.
Sterilization control: Three methods exist to check the efficacy of sterilization process, namely
physical, chemical and biological.

Physical: Temperature chart recorder and thermocouple.

Chemical: Browne‟s tube No.3 (green spot, color changes from red to green)
Biological: 106 spores of Bacillus subtilis var niger or Clostridium tetani on paper strips
are placed inside envelopes and then placed inside the hot air oven. Upon completion of
sterilization cycle, the strips are removed and inoculated into thioglycollate broth or

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 cooked meat medium and incubated at 37oC for 3-5 days. Proper sterilization should kill
the spores and there should not be any growth.

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.
Takes longer time compared to autoclave.

RADIATION:

Two types of radiation are used, ionizing and non-ionizing. Non-ionizing rays are low energy
rays with poor penetrative power while ionizing rays are high-energy rays with good penetrative
power. Since radiation does not generate heat, it is termed "cold sterilization". In some parts of
Europe, fruits and vegetables are irradiated to increase their shelf life up to 500 percent.

Non-ionizing rays:

Rays of wavelength longer than the visible light are non-ionizing. Microbicidal
wavelength of UV rays lie in the range of 200-280 nm, with 260 nm being most effective.
UV rays are generated using a high-pressure mercury vapor lamp. It is at this wavelength
that the absorption by the microorganisms is at its maximum, which results in the
germicidal effect. 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. Microorganisms such as bacteria, viruses, yeast, etc. that are exposed
to the effective UV radiation are inactivated within seconds. Since UV rays don‟t kill
spores, they are considered to be of use in surface disinfection. 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:

Ionizing rays are of two types, particulate and electromagnetic rays.

a) Electron beams are particulate in nature while gamma rays are electromagnetic in
nature. Highspeed electrons are produced by a linear accelerator from a heated
cathode. Electron beams are employed to sterilize articles like syringes, gloves,
dressing packs, foods and pharmaceuticals. Sterilization is accomplished in few
seconds. Unlike electromagnetic rays, the instruments can be switched off.
Disadvantage includes poor penetrative power and requirement of sophisticated
equipment.

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 b) Electromagnetic rays such as gamma rays emanate from nuclear disintegration of
certain radioactive isotopes (Co60, Cs137). They have more penetrative power
than electron beam but require longer time of exposure. 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, glasswares 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:

Filtration does not kill microbes, it separates them out. Membrane filters with pore sizes between
0.2-0.45 μm are commonly used to remove particles from solutions that can't be autoclaved. It is
used to remove microbes from heat labile liquids such as serum, antibiotic solutions, sugar
solutions, urea solution. Various applications of filtration include removing bacteria from
ingredients of culture media, preparing suspensions of viruses and phages free of bacteria,
measuring sizes of viruses, separating toxins from culture filtrates, counting bacteria, clarifying
fluids and purifying hydatid fluid. Filtration is aided by using either positive or negative pressure
using vacuum pumps. The older filters made of earthenware or asbestos are called depth filters.

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. Different types of earthenware filters are:
a) Pasteur-Chamberland filter: These candle filters are from France and are made up
of porcelain
b) (sand and kaolin). Similar filter from Britain is Doulton. Chamberland filters are
made with various porosities, which are graded as L1, L1a, L2, L3, L5, L7, L9
and L11. Doulton filters are P2, P5 and P11.
c) Berkefeld filter: These are made of Kieselguhr, a fossilized diatomaceous earth
found in Germany. They are available in three grades depending on their porosity
(pore size); they are V (veil), N (normal) and W (wenig). Quality of V grade filter
is checked using culture suspension of Serrtia marcescens (0.75 μm).
d) Mandler filter: This filter from America is made of kieselguhr, asbestos and
plaster of Paris.

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. They
are available in following grades; HP/PYR (for removal of pyrogens), HP/EKS (for
absolute sterility) and HP/EK (for claryfying).

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 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. Filters of Grade 5 have average pore diameter of 1-
1.5 μm. They are washed in running water in reverse direction and cleaned with warm
concentrated H2SO4 and sterilized by autoclaving.

Membrane filters: These filters are made from a variety of polymeric materials such as
cellulose nitrate, cellulose diacetate, polycarbonate and polyester. The older type of
membrane, called gradocol (graded colloidion) membrane was composed of cellulose
nitrate. Gradocol membranes have average pore diameter of 3-10 μm. The newer ones are
composed of cellulose diacetate. These membranes have a pore diameter ranging from
0.015 μm to 12 μm. These filters are sterilized by autoclaving. Membrane filters are made
in two ways, the capillary pore membranes have pores produced by radiation while the
labyrinthine pore membranes are produced by forced evaporation of solvents from
cellulose esters. The disadvantages of depth filters are migration of filter material into the
filtrate, absorption or retention of certain volume of liquid by the filters, pore sizes are
not definite and viruses and mycoplasma could pass through. The advantages of
membrane filters are known porosity, no retention of fluids, reusable after autoclaving
and compatible with many chemicals. However, membrane filters have little loading
capacity and are fragile.

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. Examples of areas where HEPA filters are
used include rooms housing severely neutropenic patients and those operating rooms
designated for orthopedic implant procedures. HEPA filter efficiency is monitored with
the dioctylphthalate (DOP) particle test using particles that are 0.3 μm in diameter.

SONIC AND ULTRASONIC VIBRATIONS: Sound waves of frequency >20,000

cycle/second kills bacteria and some viruses on exposing for one hour. Microwaves are not
particularly antimicrobial in themselves, rather the killing effect of microwaves are largely due to
the heat that they generate. High frequency sound waves disrupt cells. They are used to clean and
disinfect instruments as well as to reduce microbial load. This method is not reliable since many
viruses and phages are not affected by these waves.

CHEMICAL METHODS OF DISINFECTION:

Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces.
Some chemical have very narrow spectrum of activity and some have very wide. Those
chemicals that can sterilize are called chemisterilants. Those chemicals that can be safely applied
over skin and mucus membranes are called antiseptics.

An ideal antiseptic or disinfectant should have following properties:

Should have wide spectrum of activity
Should be able to destroy microbes within practical period of time
Should be active in the presence of organic matter
Should make effective contact and be wettable

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 Should be active in any pH
Should be stable
Should have long shelf life
Should be speedy
Should have high penetrating power
Should be non-toxic, non-allergenic, non-irritative or non-corrosive
Should not have bad odour
Should not leave non-volatile residue or stain
Efficacy should not be lost on reasonable dilution
Should not be expensive and must be available easily

Such an ideal disinfectant is not yet available. The level of disinfection achieved depends on
contact time, temperature, type and concentration of the active ingredient, the presence of
organic matter, the type and quantum of microbial load. The chemical disinfectants at working
concentrations rapidly lose their strength on standing.

Classification of disinfectants:
1. Based on consistency
a. Liquid (E.g., Alcohols, Phenols)
b. Gaseous (Formaldehyde vapor, 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 sulphydryl 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)

ALCOHOLS:
Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein.
Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol
Application: A 70% aqueous solution is more effective at killing microbes than absolute
alcohols. 70% ethyl alcohol (spirit) is used as antiseptic on skin. Isopropyl alcohol is preferred to
ethanol. It can also be used to disinfect surfaces. It is used to disinfect clinical thermometers.
Methyl alcohol kills fungal spores, hence is useful in disinfecting inoculation hoods.
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

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Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of
rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms etc.
Fumigation is achieved by boiling formalin, heating paraformaldehyde or treating formalin with
potassium permanganate. It also sterilizes bedding, furniture and books. 10% formalin with 0.5%
tetraborate sterilizes clean metal instruments. 2% gluteraldehyde is used to sterilize
thermometers, cystoscopes, bronchoscopes, centrifuges, anasethetic equipments etc. An exposure
of at least 3 hours at alkaline pH is required for action by gluteraldehyde. 2% formaldehyde at
40oC for 20 minutes is used to disinfect wool and 0.25% at 60oC for six hours to disinfect animal
hair and bristles.
Disadvantages: Vapors 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)
Applications: Joseph Lister used it to prevent infection of surgical wounds. Phenols are coal-tar
derivatives. They act as disinfectants at high concentration and as antiseptics at low
concentrations. They are bactericidal, fungicidal, mycobactericidal but are inactive against
spores and most viruses. They are not readily inactivated by organic matter. The corrosive
phenolics are used for disinfection of ward floors, in discarding jars in laboratories and
disinfection of bedpans. Chlorhexidine can be used in an isopropanol solution for skin
disinfection, or as an aqueous solution for wound irrigation. It is often used as an antiseptic hand
wash. 20% Chlorhexidine gluconate solution is used for pre-operative hand and skin preparation
and for general skin disinfection. Chlorhexidine gluconate is also mixed with quaternary
ammonium compounds such as cetrimide to get stronger and broader antimicrobial effects (eg.
Savlon). Chloroxylenols are less irritants and can be used for topical purposes and are more
effective against gram positive bacteria than gram negative bacteria. Hexachlorophene is
chlorinated diphenyl and is much less irritants. It has marked effect over gram positive bacteria
but poor effect over gram negative bacteria, Mycobacterium, fungi and viruses. Triclosan is
organic phenyl ether with good activity against grampositive bacteria and effective to some
extent against many gram negative bacteria including Pseudomonas. It also has fair activity on
fungi and viruses.
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, which is microbicidal.
Examples: Chlorine compounds (chlorine, bleach, and hypochlorite) and iodine compounds
(tincture iodine, iodophores)
Applications: Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. Iodine can be
combined with neutral carrier polymers such as polyvinylpyrrolidone to prepare iodophores such
as povidone-iodine. Iodophores permit slow release and reduce the irritation of the antiseptic.

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For hand washing iodophores are diluted in 50% alcohol.10% Povidone Iodine is used undiluted
in pre and postoperative skin disinfection. Chlorine gas is used to bleach water. Household
bleach can be used to disinfect floors. Household bleach used in a stock dilution of 1:10. In
higher concentrations chlorine is used to disinfect swimming pools. 0.5% sodium hypochlorite is
used in serology and virology. Used at a dilution of 1:10 in decontamination of spillage of
infectious material. Mercuric chloride is used as a disinfectant.
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.

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)
Applications: 1% silver nitrate solution can be applied on eyes as treatment for opthalmia
neonatorum (Crede‟smethod). This procedure is no longer followed. Silver sulphadiazine is used
topically to help to prevent colonization and infection of burn tissues. Mercurials are active
against viruses at dilution of 1:500 to 1:1000. Merthiolate at a concentration of 1:10000 is used
in preservation of serum. Copper salts are used as a fungicide.
Disadvantages: Mercuric chloride is highly toxic, are readily inactivated by organic matter.

SURFACE ACTIVE AGENTS:

Mode of actions: They have the property of concentrating at interfaces between lipid containing
membrane of bacterial cell and surrounding aqueous medium. These compounds have long chain
hydrocarbons that are fat soluble and charged ions that are water-soluble. Since they contain both
of these, they concentrate on the surface of membranes. They disrupt membrane resulting in
leakage of cell constituents.
Examples: These are soaps or detergents. Detergents can be anionic or cationic. Detergents
containing negatively charged long chain hydrocarbon are called anionic detergents. These
include soaps and bile salts. If the fat-soluble part is made to have a positive charge by
combining with a quaternary nitrogen atom, it is called cationic detergents. Cationic detergents
are known as quaternary ammonium compounds (or quat). Cetrimide and benzalkonium chloride
act as cationic detergents.
Application: They are active against vegetative cells, Mycobacteria and enveloped viruses. They
are widely used as disinfectants at dilution of 1-2% for domestic use and in hospitals.
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. Acridine dyes
such as acriflavin and aminacrine. Acriflavine is a mixture of proflavine and euflavine. Only
euflavine has effective antimicrobialproperties. A related dye, ethidium bromide, is also
germicidal. It intercalates between base pairs in DNA. They are more effective against gram
positive bacteria than gram negative bacteria and are more bacteriostatic in action.

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Applications: They may be used topically as antiseptics to treat mild burns. They are used as
paint on the skin to treat bacterial skin infections. The dyes are used 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.
Application: It is used at 6% concentration to decontaminate the instruments, equipments such
as ventilators. 3% Hydrogen Peroxide Solution is used for skin disinfection and deodorising
wounds and ulcers. Strong solutions are sporicidal.
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.
Properties: It is a cyclic molecule, which is a colorless liquid at room temperature. It has a sweet
ethereal odor,readily polymerizes and is flammable.
Application: It is a highly effective chemisterilant, capable of killing spores rapidly. Since it is
highly flammable, it is usually combined with CO2 (10% CO2+ 90% EO) or
dichlorodifluoromethane. It requires presence of humidity. It has good penetration and is well
absorbed by porous material. It is used to sterilize heat labile articles such as bedding, textiles,
rubber, plastics, syringes, disposable petri dishes, and complex apparatus like heart - lung
machine, respiratory and dental equipments. Efficiency testing is done using Bacillus subtilis var
niger.
Disadvantages: It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and
carcinogenic.

BETA-PROPIOLACTONE (BPL):
Mode of action: It is an alkylating agent and acts through alkylation of carboxyl- and hydroxyl-
groups.
Properties: It is a colorless liquid with pungent to slightly sweetish smell. It is a condensation
product of ketane with formaldehyde.
Application: It is an effective sporicidal agent, and has broad-spectrum activity. 0.2% is used to
sterilize biological products. It is more efficient in fumigation that formaldehyde. It is used to
sterilize vaccines, tissue grafts, surgical instruments and enzymes
Disadvantages: It has poor penetrating power and is a carcinogen.

PHYSIO-CHEMICAL METHOD:
Mode of action: A physio-chemical method adopts both physical and chemical method. Use of
steam formaldehyde is a physio-chemical method of sterilization, which takes into account
action of steam as well as that of formaldehyde. Saturated steam at a pressure of 263 mm has a
temperature of 70oC. The air is removed from the autoclave chamber and saturated steam at sub-
atmospheric pressure is flushed in. Formaldehyde is then injected with steam in a series of
pulses, each of 5-10 minutes. The articles are held at this holding temperature for one hour.
Formaldehyde is then flushed by inflow of steam.

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Disadvantages: Condensation of formaldehyde occurs and induction of large volume of
formaldehyde wets the steam resulting in loss of latent heat.
Sterilization control: using paper strips containing 106 spores of G.stearothermophilus.

Table: Common antiseptics and disinfectants

Chemical Action Uses

Denatures proteins and
Ethanol (50-70%) Antiseptic used on skin
solubilizes lipids
Denatures proteins and
Isopropanol (50-70%) Antiseptic used on skin
solubilizes lipids
Reacts with NH2, SH and COOH Disinfectant, kills
Formaldehyde (8%)
groups endospores
Antiseptic used on skin
Tincture of Iodine (2% I2
Inactivates proteins Disinfection of drinking
in 70% alcohol)
water
Forms hypochlorous acid Disinfect drinking water;
Chlorine (Cl2) gas
(HClO), a strong oxidizing agent general disinfectant
General antiseptic and used
Silver nitrate (AgNO3) Precipitates proteins
in the eyes of newborns
Disinfectant, although
Inactivates proteins by reacting
Mercuric chloride occasionally used as an
with sulfide groups
antiseptic on skin
Detergents (e.g. quaternary Skin antiseptics and
Disrupts cell membranes
ammonium compounds) disinfectants
Phenolic compounds (e.g. Antiseptics at low
carbolic acid, lysol, Denature proteins and disrupt concentrations;
hexylresorcinol, cell membranes disinfectants at high
hexachlorophene) concentrations
Disinfectant used to
sterilize heat-sensitive
Ethylene oxide gas Alkylating agent
objects such as rubber and
plastics
Purification of water,
Ozone Generates lethal oxygen radicals
sewage

TESTING OF DISINFECTANTS:
A disinfectant must be tested to know the required effective dilution, the time taken to effect
disinfection and to periodically monitor its activity. As disinfectants are known to lose their
activity on standing as well as in the presence of organic matter, their activity must be
periodically tested.

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Different methods are:
1. Koch‟s method
2. Rideal Walker Method
3. Chick Martin test
4. Capacity use dilution test (Kelsey-Sykes test)
5. In-use test

Koch’s method: Spores of Bacillus anthracis were dried on silk thread and were subjected to
action of disinfectants. Later, it was washed and transferred to solid medium.

Rideal Walker method: This method relies on the estimation of phenol coefficient. Phenol
coefficient of a disinfectant is calculated by dividing the dilution of test disinfectant by the
dilution of phenol that disinfects under predetermined conditions. Both the phenol and the test
disinfectant are diluted from 1/95 to 1/115 and their bactericidal activity is determined against
Salmonella typhi suspension. Subcultures are performed from both the test and phenol at
intervals of 2.5, 5, 7.5 and 10 minutes. The plates are incubated for 48-72 hours at 37°C. That
dilution of disinfectant which disinfects the suspension in a given time is divided by that dilution
of phenol which disinfects the suspension in same time gives its phenol coefficient.
Disadvantages of the Rideal-Walker test are: No organic matter is included; the microorganism
Salmonella typhi may not be appropriate; the time allowed for disinfection is short; it should be
used to evaluate phenolic type disinfectants only.

Chick Martin test: This test also determines the phenol coefficient of the test disinfectant.
Unlike in Rideal Walker method where the test is carried out in water, the disinfectants are made
to act in the presence of yeast suspension (or 3% dried human feces). Time for subculture is
fixed at 30 minutes and the organism used to test efficacy is S.typhi as well as S.aureus. The
phenol coefficient is lower than that given by Rideal Walker method.

Rideal -Walker Chick-Martin

Volume medium  5.0 ml 10.0 ml
Diluent for test disinfectant Water Yeast suspension
Reaction temperature  17.5±0.5ºC 30ºC
Organism Salmonella typhi, Staphylococcus aureus
Sampling times  2.5, 5.0, 7.5, 10 and 30 minutes.
Calculation of coefficient Dilution test killing in 7.5 min divided by same for phenol Mean
concentration of phenol showing no growth after 30 min. divided by same for test. The classical
tests such as Rideal - Walker or Chick - Martin are not practicable.

Capacity use dilution test (Kelsey-Sykes test):

Inoculums of four different test organisms, namely Staphylococcus aureus, Escherichia coli,
Pseudomonas aeruginosa and Proteus vulgaris are added to the disinfectant in three successive.
Dried yeast is included to simulate presence of organic matter. The method can be carried out
under 'clean' or 'dirty' conditions. The dilutions of the disinfectant are made in hard water for
clean conditions and in yeast suspension for dirty conditions. Test organism alone or with yeast
is added at 0, 10 and 20 minutes interval. The contact time of disinfectant and test organism is 8
min. The disinfectant is evaluated on its ability to kill microorganisms or lack of it and the result

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is reported as a pass or a fail and not as a coefficient. The capacity test of Kelsey and Sykes gives
a good guideline for the dilution of the preparation to be used. Disadvantage of this test is the
fact that it is rather complicated.

In-use test:
The routine monitoring of disinfectant in use can be done by the „in use‟ test of Maurer. This test
is intended to estimate the number of living organism in a vessel of disinfectant in actual use.
The disinfectant that is already in use is diluted 1 in 10 by mixing 1 ml of the disinfectant with 9
ml of sterile nutrient broth. Ten drops of the diluted disinfectant (each 0.02 ml) is placed on two
nutrient agar plates. One plate is incubated at 37oC for 3 days while the other is held at room
temperature for 7 days. The number of drops that yielded growth is counted after incubation. If
there growth in more than five drops on either plate, it represents failure of disinfectant.

Just for Knowledge not exam portions

Preservatives: static agents used to inhibit the growth of microorganisms, most often in foods. If
eaten they should be nontoxic. Examples are calcium propionate, sodium benzoate,
formaldehyde, nitrate and sulfur dioxide. Below Tables are lists of common preservative and
their uses.

Some common preservatives added to processed foods

Salt - retards bacterial growth. Not good for blood pressure.

Nitrates - can be found in some cheeses, adds flavor, maintains pink color in cured meats
and prevents botulism in canned foods. Can cause adverse reactions in children, and
potentially carcinogenic.
Sulfur Dioxide and Sulfites - are used as preservatives and to prevent browning in
alcoholic beverages, fruit juices, soft drinks, dried fruits and vegetables. Sulfites prevent
yeast growth and also retard bacterial growth in wine. Sulfites may cause asthma and
hyperactivity. They also destroy vitamins.
Benzoic Acid and Sodium Benzoate - are used to preserve oyster sauce, fish sauce,
ketchup, non-alcoholic beverages, fruit juices, margarine, salads, confections, baked
goods, cheeses, jams and pickled products. They have also been found to cause
hyperactivity.
Propionic Acid and Propionates - used in bread, chocolate products, and cheese for
lasting freshness.
Sorbic Acid and Sorbates - prevent mold formation in cheese and flour confectioneries

Table: Common food preservatives and their uses

Preservative Effective Concentration Uses

Propionic acid and Antifungal agent in breads,
0.32%
propionates cake, Swiss cheeses

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 Antifungal agent in
Sorbic acid and
0.2% cheeses, jellies, syrups,
sorbates
cakes
Antifungal agent in
Benzoic acid and
0.1% margarine, cider, relishes,
benzoates
soft drinks
Sodium diacetate 0.32% Antifungal agent in breads
Antimicrobial agent in
Lactic acid unknown cheeses, buttermilk, yogurt
and pickled foods
Antimicrobial agent in
Sulfur dioxide,
200-300 ppm dried fruits, grapes,
sulfites
molasses
Antibacterial agent in cured
Sodium nitrite 200 ppm
meats, fish
Prevents microbial spoilage
Sodium chloride unknown
of meats, fish, etc.
Prevents microbial spoilage
Sugar unknown of preserves, jams, syrups,
jellies, etc.
Prevents microbial spoilage
Wood smoke unknown
of meats, fish, etc.

Antibiotics

Chemotherapeutic agents (synthetic antibiotics): antimicrobial agents of synthetic origin

useful in the treatment of microbial or viral disease. Examples are sulfonilamides, isoniazid,
ethambutol, AZT, nalidixic acid and chloramphenicol. Note that the microbiologist's definition
of a chemotherapeutic agent requires that the agent be used for antimicrobial purpose and
excludes synthetic agents used for therapy against diseases that are not of microbial origin.
Hence, pharmacology distinguishes the microbiologist's chemotherapeutic agent as a "synthetic
antibiotic".

Antibiotics: antimicrobial agents produced by microorganisms that kill or inhibit other

microorganisms. This is the microbiologist's definition. A more broadened definition of an
antibiotic includes any chemical of natural origin (from any type of cell) which has the effect to
kill or inhibit the growth of other type‟s cells. Since most clinically-useful antibiotics are
produced by microorganisms and are used to kill or inhibit infectious Bacteria, we will follow
the classic definition. Note also (above), pharmacologists refer to any antimicrobial chemical
used in the treatment of infectious disease as antibiotic.

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