STERILIZATION

AND
DISINFECTION

Why do we disinfect?
To protect:

Lab workers
General public
The environment
To stop the spread of disease
To stop the release of recombinant or exotic
organisms into the environment.
Our research

STERILIZATION

Sterilization is the total destruction of all

microbes, including the more resilient forms such
as:
bacterial spores,
mycobacteria,
nonenveloped viruses,
and fungi.

DISINFECTION

Disinfection is the process of elimination of

most pathogenic microorganisms (excluding
bacterial spores) on inanimate objects.

DECONTAMINATION

Decontamination is the process of removal of

contaminating pathogenic microorganisms from
the articles by a process of sterilization or
disinfection.

DEFINITIONS

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.

DEFINITIONS

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 semisynthetic antimicrobial agents.

Microbes in decreasing order of resistance

to germicidal chemicals

DISINFECTION

Disinfectants are those chemicals that destroy

pathogenic bacteria excluding bacterial spores,
from inanimate surfaces.
Those chemicals that can sterilize are called
chemisterilants.
Those chemicals that can be safely applied over
skin and mucus membranes are called
antiseptics.

DISINFECTION

Disinfection can be accomplished with:

1. Heat disinfection
2. Chemical disinfection

Objects that can be disinfected are: bedpans,

patient skin before operation and surgeon
hands before putting gloves.

HEAT DISINFECTION

It is accomplished by boiling water at atmospheric

pressure for at least 5 mins.

CHEMICAL DISINFECTION
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)

Chlorine-Containing Compounds

Bleach

Active ingredient = Sodium Hypochlorite (NaOCl) (5%)

1 g/l available chlorine = general lab decon (1:50)

5 g/l AC = stronger lab cleanup (1:10)

Laboratory decontamination recommendation is a 10% dilution

10% = 0.5% NaOCl = 5 g/l available chlorine

Unstable: degrades over time after opening and/or dilution

Make dilutions fresh daily

Reactive: Corrosive to metals (stainless)

Alcohol (ethyl or isopropyl)

Broad spectrum antimicrobial

Vegetative bacteria (incl. mycobacteria)
Viruses
Fungi

Not effective against spores

Non- toxic (when used in moderation)

Most effective at a dilution of 70% in water

Causes membrane damage and rapid protein denaturation

Phenolic Compounds

Some of the earliest used germicides

Active against vegetative bacteria and viruses

Can be used against mycobacteria

Not sporicidal

Membrane active induce leakage of intracellular components

Quaternary Ammonia Compounds

Effective against vegetative bacteria and some lipid-containing

viruses

Mycobacteriostatic and sporostatic

Commonly used as low-level disinfectants or sanitizing agents

Active against microbial membranes

Formaldehyde and Gluteraldehyde

Formaldehyde (Paraformaldehyde/Formalin)
Effective against vegetative bacteria, mycobacteria, and spores
Used to decontaminate large equipment/spaces
Suspected carcinogen

Gluteraldehyde
Broad spectrum of activity: vegetative bacteria and spores; fungi,
viruses
Considered safer than formaldehyde

Active against microbial membranes and cell walls

Chemical disinfection
Number of different agents are used according to
tolerance of objects and infectious agents.
Using phenol with cleaning component destroy the
membrane of microorganisms.
Using 70% alcohol for skin which denatures proteins
of microorganisms.
Use soap containing hexachlorophene for hands.
In case of hepatitis use 5% solution of chloramines
or heat disinfection

TESTING OF DISINFECTANTS
Kochs 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.
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).

STERILIZATION
CLASSIFICATION OF METHODS

Physical Agents
1. Heat
2. Radiation
3. Filtration

Chemical Agents
In practice, certain methods are placed under sterilization which in fact do
not fulfill the definition of sterilization such as boiling for 1/2 hr and
pasteurization which will not kill spores.

HEAT
Factors affecting sterilization by heat

Nature of heat Moist heat is more effective than dry heat.

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.

DRY HEAT

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.

DRY HEAT

Flaming This is a method of passing the article over a Bunsen flame.

DRY HEAT

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

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 min. at 160C
40 min. at 170C
20 min. at 180C.
Increasing temperature by 10 degrees shortens
the sterilizing time by 50 %.
The hot air oven must not be opened until the
temperature inside has fallen below 60C to
prevent breakage of glasswares.

Sterilization control

Physical Temperature chart recorder and

thermocouple.

Chemical
Brownes tube No.3 green spot color
changes from red to green.
Dry Heat Indicator Labels (DHI Labels)
green color changes from green to darkbrown.

Biological: 10 6 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 and incubated at 37C
for 3-5 days. Proper sterilization should
kill the spores and there should not be
any growth.

MOIST HEAT
At temperature below 100C
Pasteurization

The holder method: heated at 63C for 30 min.

The flash method: heated at 72C for 15 sec.followed by quickly cooling to 13C .
(Ultra-High Temperature (UHT): heated at 140C for 15 sec and 149C for
0.5sec. This method is suitable to destroy most milk borne pathogens like
Salmonella, Mycobacteria, 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 60C for one hour.

Serum bath
The contaminating bacteria in a serum preparation can be inactivated by heating
in a water bath at 56C for one hour on several successive days.

Only vegetative bacteria are killed and spores survive.

MOIST HEAT
At temperature 100C

Tyndallization
Steaming for aprox. 20 min. on 3 or 4 successive occasions, separated
by 24 hour intervals at room temperature.
The incubation intervals permit any dormant, resistant endospores to
germinate and become active.

MOIST HEAT
At temperature above 100C

AutoclaveSterilization can be effectively achieved at a temperature

above 100C using an autoclave.
In an autoclave the water is boiled in a closed chamber. As the
pressure rises, the boiling point of water also raises.

134C

3.0 atm

5 min

MOIST HEAT

2
3

atm
atm

121C
134C

A simple autoclave has vertical or horizontal cylindric.

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

Sterilization control

Physical method includes automatic process

control, thermocouple and temperature chart
recorder.

Chemical method 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 Ampoule biological tests

includes a paper strip containing 10 6 spores of
Geobacillus stearothermophilus.

RADIATION

Non-ionizing wavelength
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.

Sunlight The microbicidal activity of sunlight is mainly due to the presence of ultra
violet rays in it. It is responsible for spontaneous sterilization in natural conditions.

RADIATION

Ionizing wavelength
Particulate (Electron beam)
Common uses: sterilisation of instruments such as syringes, gloves,
dressing packs, foods and pharmaceuticals.
Electromagnetic (Gamma rays)
Common uses: sterilisation of disposable petri dishes, plastic
syringes, antibiotics, vitamins, hormones and fabrics.

FILTRATION

Filtration does not kill microbes, it separates them out. Membrane

filters with pore sizes between 0.2-0.45 m 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.
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.

Types of filters

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. The newer ones are composed of cellulose diacetate. These
membranes have a pore diameter ranging from 0.015 m to 12 m. These
filters are sterilized by autoclaving.
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 m in diameter.

Asepsis

Sterilization

Heat

Disinfection

Chemical

Steam

Dry heat

Radiation

Gas

Heat

Gamma

Chemical

Boiling water

Phenol

Ethylene oxide

70% Alcohol

Formalin

Hexachlorophene

Heat or
chloramines solution

Liquid

Glutaric
Aldehyde

Microbiological Waste Management

Biohazardous and infectious (or potentially infectious) agents are

routinely handled and manipulated in biomedical and animal
laboratories.

Biohazards include human samples and tissue cultures, bacteria,

viruses, prions, parasites, toxins, recombinant DNA (rDNA) - which
is used in expression vectors such as plasmids, infected or
transfected/transduced cells, infected human or animal tissues,
transgenic animals, and any material contaminated with a
biohazardous agent.

To prevent the escape of biohazards into the environment,

waste from biohazardous operations must be treated and/or
disposed of in a special way.

Microbiological Waste Management

Microbiological waste includes:
Discarded cultures and stocks of infectious agents and
associated biologicals
Discarded cultures of specimens from medical, clinical,
commercial, and industrial laboratories
Discarded, used disposable culture dishes
Discarded, used disposable devices used to transfer,
inoculate, and mix cultures

Acceptable Methods of Treatment

of Microbiological Waste

Steam Sterilization
1. temperature of at least 121C
2. pressure of at least 2 atm.
3. time of at least 30 minutes

Identifying Biohazardous Waste

All biohazardous waste containers

must be labeled with a biohazard label
(red or red/orange label
with the universal biohazard symbol and
the word BIOHAZARD underneath).

Biohazard containers