This document discusses various methods for decontamination, sterilization, and disinfection important for laboratory biosafety. It defines key terms like sterilization, which aims to eliminate all microbial life, versus disinfection, which aims to eliminate pathogens but not bacterial spores. Various physical methods for sterilization are covered, including heat/thermal methods like autoclaving, radiation methods, and filtration. Specific sterilization techniques using heat, chemicals, and gases are then described in more detail. The document emphasizes the importance of choosing the appropriate sterilization or disinfection method based on the level and type of contamination present.

1. BIOSAFETY
AND IPR
DECONTAMINATION
STERILIZATION
AND
DISINFECTION

2. • Decontamination is essential for laboratory biosafety, as the accumulation of
microbial contamination in the laboratory can lead to the transmission of disease.
• The degree of decontamination can be classified as either disinfection or
sterilization.
• Disinfection aims to eliminate all pathogenic microorganisms, with the exception of
bacterial spores on lab surfaces or equipment.
• Sterilization, on the other hand, aims to eliminate all microbial life.
• Different methods are available which include chemicals, heat, and radiation, and
once again depend on the degree of decontamination, as well as the concentration
of the contaminating microorganisms, presence of organic matter, and type of
equipment or surface to be cleaned.
• Each method has its advantages and cautionary measures that need to be taken to
avoid hazards.
DECONTAMINATION

4. STERILIZATION
• A sterile surface/object is completely free of living microorganisms and viruses.
• Sterilization procedures kill all microorganisms. Methods used in sterilization
procedures include heat, ethylene oxide gas, hydrogen peroxide gas, plasma, ozone,
and radiation.
• Sterility Assurance Level - the probability of a microorganism surviving on an item
subjected to treatment is less than one in one million.
The various methods of sterilization are:
1. Physical Method
(a) Thermal (Heat) methods
(b) Radiation method
(c) Filtration method
2. Chemical Method
3. Gaseous method

5. PHYSICAL STERILISATION
Physicalsterilisation
heat
filtration
radiation

6. i) HEAT
• DRY HEAT-
Dry heat method of sterilization acts by protein denaturation, oxidative damage
and toxic effects of elevated levels of electrolytes.
• MOIST HEAT-
Moist heat method of sterilization acts by coagulation and denaturation of
proteins.
***Thermal death time is the minimum time required to kill a suspension of
organisms at a predetermined temperature in a specified environment***
DRY HEAT METHOD:
• Red heat
• Flaming
• Incineration
• Hot air oven

7. 1.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.
2. 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. Cannot kill
spores.

8. 3. Incinerator: This is an excellent method for destroying materials such as
contaminated clothes, cotton wool stoppers, animal carcasses and pathological
materials. It involves burning of materials in incinerators.

9. 4. Hot air oven: This is the most widely used method of sterilization using dry
heat. The oven is usually heated by electricity and it has a thermostat that
maintains the chamber air constantly at the chosen temperature.
It has a fan or turbo-blower to assist the circulation of air and to ensure rapid,
uniform heating of the load. In Hot Air Oven, the air is heated at a temperature
of 160oC for one hour.
This is the best method of sterilizing dry glass ware such as test tubes, petri
dishes, flasks, pipettes and instruments such as forceps, scalpels and scissors. It
is also used to sterilize some pharmaceutical products such as liquid paraffin,
dusting powder, fats and grease.
Quality control of dry heat sterilization:
The spores of a non toxigenic strain of Clostridium tetani are used to test
the efficiency of dry heat sterilization.

10. MOIST HEAT METHOD:
• Temperature below 100°C.
• Temperature at 100°C.
• Temperature above 100°C.
1. Temperature below 100°C:
Pasteurization-The process of heating a liquid food or beverage either at 62.8°C for 30
minutes or 72°C for 15 seconds to enhance their shelf life and destroy harmful
microorganisms. It should be noted that pasteurization process kills only vegetative cells but
not the spores.
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.

11. 2. Temperature at 100°C:
Water at 100°C (Boiling):
Boiling is one of the moist heat sterilization methods. It kills vegetative forms of
bacterial pathogens, almost all viruses and fungi (including their spores) within 10
minutes, usually much faster.
Most vegetative bacteria will die in 5-10 minutes when immersed in boiling water,
but some spores will survive at this temperature for several hours.
Steaming at 100°C (Tyndallisation):
It is a process discovered by John Tyndall in 19th century for sterilizing substances
to kill the spores of bacteria. The process of exposure of materials to steam at
100°C for 20 min for three consecutive days is known as tyndallisation.
First exposure kills all the vegetative forms and in the intervals between heating,
the remaining spores germinate into vegetative forms which are killed on
subsequent heating. Tyndallisation is also called fractional sterilization or
intermittent boiling.

12. 3. Temperature above 100°C:
Autoclave
Sterilization using an autoclave is most effective when the organisms are either
contacted by the steam directly or contained in a small volume of aqueous liquid
(primarily water). The temperature used in autoclave is 121°C at 15 lbs (pounds)
pressure for 15 minutes.
Autoclaving is used in sterilizing
culture media, instruments,
dressings, applicators, solutions,
syringes, transfusion equipment,
pharmaceutical products,
aqueous solutions and numerous
other items that can withstand
high temperatures and pressures.
The same principle of autoclaving
applies for the common household
pressure cooker used for cooking food.

13. Quality control of moist heat sterilization:
To check the efficiency of moist heat sterlization, the indicator commonly used is the
paper strips containing spores of Bacillus sterothermophilus.
ii) 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".
Non-ionizing rays: (infra red and UV) Rays of wavelength longer than the visible light
are non-ionizing. Microbicide 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
vapour lamp. It is at this wavelength that the absorption by the microorganisms is at its
maximum, which results in the germicidal effect.
Ionizing rays: (gamma rays and cosmic rays) Ionizing rays are of two types, particulate
and electromagnetic rays. Electron beams are particulate in nature while gamma rays
are electromagnetic in nature. High speed 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.

14. iii) FILTRATION:
Filtration is an effective and reasonably economical method of sterilization. It is
used to sterilize heat-sensitive fluids, and air. It is particularly useful for solutions
containing toxins, enzymes, drug, serum and sugars. 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.
Different types of filters are:
1. Earthenware filters: These filters are made up of diatomaceous earth or
porcelain. Eg: Berkefeld filter, Mandler filter.
2. Asbestos filters: These filters are made from chrysotile type of asbestos,
chemically composed of magnesium silicate. Can be used only once.
3. Sintered glass filters: These are made from finely ground glass that are fused
sufficiently to make small particles adhere to each other.
4. Membrane filters: These filters are made from a variety of polymeric materials
such as cellulose nitrate,cellulose diacetate, polycarbonate and polyester.

16. CHEMICAL STERILIZATION
Chemical sterilization is the elimination of all viable microorganisms and their spores
using liquid or gaseous compounds.
The method you use for chemical sterilization varies with the form of chemical you use.
Liquid sterilization involves submerging equipment in a chemical fluid for enough time
to kill all viable microorganisms and their spores. Gas sterilization involves exposing
equipment to chemical gases in an enclosed heated or pressurized chamber.
Liquid sterilants include glutaraldehyde, ortho-phthaldehyde, peracetic acid, hydrogen
peroxide, and hypochlorite. Evaporating chemical sterilants may be toxic, so it is a good
idea to place them in covered containers.
At times, liquid sterilizing agents are impractical or ineffective for sterilizing items.
Gaseous chemicals are more effective sterilants because they are able to permeate
small openings and crevices easily. Gas chemicals also sterilize faster than liquids
because they usually are combined with high heat. Gas residue is also easier to remove
from sterilized articles, but requires much more expensive equipment.
Gaseous sterilants include ethylene oxide, ozone, chlorine dioxide, nitrogen dioxide,
etc.

17. DISINFECTION
Disinfection reduces the level of microbial contamination. Chemical disinfection does
not kill spores, unlike chemical sterilization. Some common laboratory disinfectants
include 10% bleach, 70% ethanol. Disinfectants are those chemicals that destroy
pathogenic bacteria from inanimate surfaces.
An ideal antiseptic or disinfectant should have following properties:
1. Should have wide spectrum of activity
2. Should be able to destroy microbes within practical period of time
3. Should be active in the presence of organic matter
4. Efficacy should not be lost on reasonable dilution
5. Should be active in any pH
6. Should be stable
7. Should have long shelf life
8. Should be speedy
9. Should have high penetrating power
10. Should be non-toxic, non-allergenic, non-irritative or non-corrosive

18. Classification of disinfectants:
1. Based on spectrum of activity
(a) High level-
Comprises high concentrations of chemical germicides (ex: concentrated sodium hypochlorite).
o Kills vegetative microorganisms and inactivates viruses
o Does not kill high numbers of bacterial spores
o High level disinfectants are typically used for short time periods (10-30 min) for disinfection
purposes, but may achieve sterilization if left in contact with the surface for long time periods
(6-10 hours).
o Not for use on environmental surfaces like floors or lab benches
(b) Intermediate level-
Kills vegetative microorganisms, including Mycobacterium tuberculosis, all fungi, and
inactivates most viruses
o EPA Approved Hospital Disinfectants which are also tuberculocidal fall into this category
o May be used for housekeeping and disinfection of laboratory benches

19. (c) Low level-
oKills most vegetative bacteria, some fungi, and inactivates some viruses.
o Does not kill M. tuberculosis
o Also known as “hospital disinfectants” or “sanitizers”
2. Based on consistency
(a) Liquid (E.g., Alcohols, Phenols)
(b) Gaseous (Formaldehyde vapour)
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)
(d) Alkylation of amino-, carboxyl- and hydroxyl group (E.g.,
Formaldehyde)
(e) Damage to nucleic acids (Formaldehyde)

20. Chemical Disinfectants
• Alcohol
• Chlorine and chlorine compounds
• Formaldehyde
• Glutaraldehyde
• Hydrogen peroxide
• Iodophors
• Ortho-phthalaldehyde (OPA)
• Peracetic acid
• Peracetic acid and hydrogen peroxide
• Phenolics
• Quaternary ammonium compounds

21. IMPORTANT TERMS
• Decontamination: Decontamination removes pathogenic
microorganisms from objects so they are safe to handle, use, or
discard.
• Disinfection: Disinfection describes a process that eliminates
many or all pathogenic microorganisms, except bacterial spores,
on inanimate objects.
• Sterilization: Sterilization describes a process that destroys or
eliminates all forms of microbial life and is carried out in health-
care facilities by physical or chemical methods.
• Antisepsis : Antisepsis is the application of a liquid antimicrobial
chemical to skin or living tissue to inhibit or destroy
microorganisms. It includes swabbing an injection site on a
person or animal and hand washing with germicidal solutions .

22. THANK YOU !
By:
BHAVINI K
II BSc Biotechnology
Nehru Arts and Science College, Coimbatore