This chapter contains sterilization techniques : physical - heat , filtration and radiations and chemical methods of sterilization in simple concise way so that mode of action is understood .

1. STERILIZATION
(Heat-moist and dry heat ,filtration, radiation and by
chemicals)
SUMANGALA.C.H.
Lecturer
UCM

2. • Terms definition
• Physical methods of sterilization
a. Heat sterilization -Dry heat sterilization and Moist heat sterilization.
b. Filtration
c. Radiation (Ionizing and Non ionising)
• Chemical methods of sterilization
a. Alcohols
b. Aldehydes
c. Phenols
d. Halogens
e. Metallic salts
f. Quaternary ammonium compounds
g. Sterilizing gases

3. Sterilization
• is complete killing, or removal, of all living organisms from a
particular location or material.
• In this process all the living microorganisms, including bacterial
spores are killed.
• It can be achieved by Physical methods like - heat –moist and dry
heat ,filtration ,radiations and by use of chemicals.

4. Terms used in sterilization
• Antimicrobial agent - a natural or synthetic substance that kills or
inhibits the growth of microorganisms such as bacteria, fungi and algae
• Sanitizer is a substance or fluid used to kill germs on skin and objects.
E.g.: Alcohol is used as hand sanitizer .
• Germicide is an agent that kills pathogenic microorganisms .
The term germicide includes both antiseptics and disinfectants.
• Antiseptics are germicides applied to living tissue and skin
• Disinfectants are applied only to inanimate objects.

5. • Microbicidal is the process or an agent that kills the microorganism.
• Bactericidal agents that kill bacteria but not necessarily
bacterial endospores.
• Microbiostatic : Microbiostatic is an agent that inhibits the growth
and reproduction of the microorganism.
• Bacteriostatic: agents that inhibit the multiplication of the
bacteria without killing them.

6. Physical methods of Sterilization
a. Heat- Dry heat and Moist heat.
b. Filteration
c. Radiations

7. Factors affecting sterilization by heat:
• Nature of heat
• Temperature and time
• Number of microorganisms
• Nature of microorganism
• Type of material

8. a. Sterilization by Heat :
• Dry heat acts by protein denaturation and oxidative damage
• Moist heat acts by coagulation and denaturation of proteins.
• Moist heat is superior to dry heat in action.
• TDT(Thermal Death Time) is the minimum time required to kill a population
of microorganisms at a predetermined temperature in a specified
environment.
• TDP (Thermal Death Point ) is the lowest temperature required to kill a
population of microbes when applied for a specific time.

9. I .Dry Heat Sterilization
1. Red heat
2. Flaming
3. Incineration
4. Hot air oven

10. 1.Red heat
Here the article is sterilized by holding them in Bunsen flame till
they become red hot. E.g.: Inoculating Loops.
2. Flaming
Here the articles are sterilized by passing over a Bunsen flame.
E.g. : slides
3.Incineration:
Here contaminated material are destroyed by burning them in incinerator
E.g.: pathological material and bedding .
4. Hot air oven
Here sterilizing is by conduction. The heat is absorbed by the outside
surface of the item, then passes towards the centre of the item, layer by
layer. The entire item will eventually reach the temperature required for
sterilization to take place.

11. • Hot Air Oven consist of three walls and a door made of gun metal
that insulate and keep the heat in .
• An air circulating fan helps in uniform distribution of the heat.
• These are fitted with aluminum trays as well as they have
thermostat and controls for temperature and holding time.
• Articles to be sterilized are exposed to high temperature [160°c] for
duration of 1 hour.
• Dry heat causes oxidation of cell constituents and high
temperature causes denaturation of proteins and enzymes.

12. • Articles to be sterilized must be perfectly dry before placing them to
avoid breakage.
• Articles must be placed at sufficient distance so as to allow free
circulation of air in between.
• Mouth 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 cans and then placed.
• Individual glass articles must be wrapped in Kraft paper or aluminum
foils.

13. Hot Air Oven

14. II. Moist Heat Sterilization
1.Sterilization at a temperature below 100°C (Pasteurization)
2.Sterilization at a temperature of 100°C ( boiling)
3.Sterilization at a temperature above100°C ( Autoclave, pressure
cooker)
4.Intermittent sterilization(Tyndallisation, Fractional Sterilization )

15. 1.Sterilization at a temperature below 100°C (Pasteurization)
• Pasteurization: is a process that kills the pathogenic bacteria by
heating liquids to a certain temperature for a set period of time.
• Here liquids flavor and food value are retained. It kills only
vegetative forms of bacteria.
1.Low Temperature Long Time (LTLT)- 62.3degree for 30 mins.
2. High Temperature Short Time(HTST)- 72degree for 15 secs.
it is more effective against certain resistant pathogens, such
as Coxiella and Mycobacterium.
3.Ultra High Temperature (UHT)-130 degree for 1 second .

16. 2.Sterilization at a temperature of 100°C ( boiling)
• Boiling: Simple boiling of water for 10–30 minutes kills most of the
vegetative forms of bacteria but not bacterial spores.
• Sterilization by boiling is facilitated by addition of 2% sodium
bicarbonate to water.
• The greatest disadvantage of this method is that the items sterilized
by boiling can be easily recontaminated when removed from water
after boiling

17. 3.Sterilization at temperature above 100°C(Autoclave, Pressure cooker)
A) Autoclave works using steam under pressure.
It kills bacteria, viruses, and even spores present in the material put
inside of the vessel.
• Temperature employed is 121 degree for 20 mins at 15 lbs pressure.
• The high pressure increases the boiling point of water and thus
helps achieve a higher temperature for sterilization
• and moisture present in the steam causes the coagulation of
proteins.

18. An autoclave has:
a. Pressure Chamber :Inner chamber and an outer jacket. Inner chamber
is made up of stainless steel or gunmetal .Outer chamber -iron case
b. Lid/ Door : The purpose of the lid is to seal off the outside the
atmosphere and create a sterilized condition on inside of the autoclave.
Pressure gauge indicate the pressure . Pressure releasing unit/
Whistle:-controls the pressure inside the chamber.
Safety valve :The valve has a thin layer of rubber that bursts itself to
release the pressure and to avoid the danger of explosion when
pressure inside increases.

19. B) Pressure cooker is used in the process of boiling water or any
other liquid to create pressure within the cooker. This pressure
enables temperature to rise quickly and therefore save energy.
• Because of the high temperature, these pressure cookers can be
used for sterilization purposes. Steam can be released from within
the pan to keep a steady temperature.
• A pressure cooker works just as well as autoclaving, especially for
small batches. Just like an autoclave, the pressure cooker
chamber reaches temperatures high enough to kill bacteria and
mold spores.

20. 4.Intermittent sterilization( Tyndallisation)
Tyndallization : It is called intermittent sterilization as it has start and
stop operation. It is also called fractional sterilization Liquids are
sterilized by this method at 100 degree for 30 minutes per day for
three successive days. Here vegetative cells and a few spores are
killed during the first warming and that the more safe spores
subsequently develop and are killed amid either the second or the
third warming. Heat-sensitive culture media containing such materials
as sugars, egg or serum are sterilized by this method.

21. Autoclave Membrane filters

22. b. Sterilization by Filteration
Filtration is the preferred method of sterilizing heat sensitive liquid and
gases without exposure to denaturing heat.
• Rather than destroying contaminating microorganisms, it simply
removes them.
• A. Filteration of Air
• B. Filteration of Liquids

23. A.Filteration of Air (Laminar Air Flow)
• Laminar Air flow is used for reducing the danger of infection while
working with pathogenic microbes.
• The laminar flow apparatus sucks the air in the room continuously
and blows out the air through a pack of filters called HEPA
Filters.(High Efficiency Particulate Air Filters ).
• HEPA filters the air and does not allow particle above 0.3 mm
dimension to go out.
• Used for transferring of cultures ,inoculation of culture.

24. • Filteration of Liquids
• The liquid is passed through a filter, a device with pores too small
for the passage of microorganisms, but large enough to allow the
passage of the liquid .It is an effective method of sterilization for heat
sensitive liquids.
• Seitz Filters (Asbestos filter)
• Porcelain Filters (Chamberland Filters)
• Sintered Glass Filters (Fritted Glass Filters)
• Diatomaceous earth Filters (Berkefeld )
• Membrane filters.

25. • Seitz Filters (Asbestos filter)
• A bacterial filter made of asbestos and used to sterilize solutions with
out the use of heat.
• Porcelain Filters (Chamberland Filters)
• The filter consists of a permeable unglazed porcelain tube (called
bisque) that contains a ring of enameled porcelain through which the
inflow pipe fits.
• The core of the porcelain is made up of a metal pipe with holes
through which water flows out and is collected.

26. • Sintered Glass Filters (Fritted Glass Filters)
• It is a filter that has undergone the process of sintering. It is made
by sintering together glass particles into a solid but porous body. This
porous glass body is called a frit.
• Sintered glass are prepared from the fine particles of ground glass
which are heated to a sintering point so that they form a disc. They are
made of borosilicate glass.

27. • Diatomaceous earth Filters (Berkefeld )
• These filter are water filters made of diatomaceous earth
(Kieselguhr- fossilized hard walled algae called diatoms )
• These filters are simple to operate and are effective in removing
cysts, algae, and asbestos from water.

28. • Membrane filters.
Membrane filters - used for liquid sterilization in the microbiology
laboratory. Membrane filters are composed of cellulose acetate or
cellulose nitrate.
• commonly used membrane filter has the pore size of 0.22μm and
0.45μm.
•The membranes are held in special holders The solution to be sterilized
is pulled or forced through the filter and is collected in previously
sterilized containers. Sterilization of fluid materials and identification and
enumeration of microorganisms.

29. c. Sterilization by Radiation
Based on wavelength and penetration power radiations are of two
types:
1. Ionizing radiation: have high energy and they ionize the target
molecules.
2. Non-Ionizing radiation: less energy and do not ionize the target
molecules.

30. 1.Ionizing radiation:
These radiations penetrate deep into objects and destroy bacterial
endospores and vegetative cells, both prokaryotic and eukaryotic.
These are, however, not that effective against viruses.
Ionizing radiations include
(a) X-rays,
(b) gamma rays, and
(c) cathode rays

31. a) X-rays
• The X-rays are high-energy, high-frequency, short-wavelength
radiations.
• X-ray sterilization uses irradiation to disrupt the DNA of
microorganisms on the product.The lethal dose of X-ray (350-500 nm)
breaks the phosphodiester bonds between the DNA and thus results in
the strand breakages.
• X-rays are able be used on products made of all types of materials
including metals, liquids and packaging materials.

32. b. Gamma rays
• Gamma radiation is a natural radiation that is emitted from an atom or
a molecule when its energy level drops.
• Gamma irradiation kills bacteria by breaking down bacterial DNA,
inhibiting bacterial division. Energy of gamma rays passes through
the equipment, disrupting the pathogens that cause contamination.
These causes the death of organisms or make organisms incapable of
reproduction.
• Eg: cobalt-60 source is used for sterilization of antibiotics, hormones,
metal foils, and plastic disposables, such as syringes.

33. C )Cathode Rays (Electron-Beam Radiation)
• Produced when a high voltage electrical charge is released in a
cathode ray tube
• It is called cathode rays as electrons are emitted from cathode
• Cathode rays or electron-beams can sterilize materials at room
temperature with brief exposure.
They have limited penetrating power and are used for the sterilization of
surgical supplies, drugs, and other materials.

34. 2.Non-ionizing Radiation
• Non-ionizing radiations are quite lethal but do not penetrate glass,
dirt, films, water; hence their use is restricted for disinfection of
clean surfaces in operation theaters, laminar flow hoods as well as
water treatment.
• The recommended dose is 250-300 nm wavelength, given for 30
minutes.
Non Ionizing radiation include
(a) Infra-Red Radiation
(b) Ultraviolet Light radiation

35. a) Infra-Red Radiation
• Infra-red radiations are low energy type electromagnetic rays, having
wavelengths longer than those of visible light.
• They kill microorganisms by oxidation of molecules as a result of
heat generated.
• Infra-red rays are used for the rapid mass sterilization of syringes
and catheters.

36. • Short-wavelength UV is the most damaging type of UV radiation.
 Ultraviolet (UV) radiation with wavelength of 240–280 nm is quite
lethal and has a marked bactericidal activity.
 It acts by denaturation of bacterial protein and also interferes with
replication of bacterial DNA by formation of thymine dimers .
b).Ultraviolet Light radiation

37. Chemical methods of Sterilization
An ideal Antimicrobial agent should have the following characteristics:
1. Be fast acting even in the presence of organic substances.
2. Be effective against all types of infectious agents.
3. Easily penetrate material to be disinfected without damaging or
discoloring material.
4. Be easy to prepare and stable even when exposed to light, heat or
other environmental factors.
5. Not have unpleasant odor.

38. • Chemical methods used for sterilization:
a. Alcohol
b. Aldehydes
c. Phenols
d. Halogens
e. Metallic salts
f. Quaternary ammonium compounds
g. Sterilizing gases

39. a.Alcohol
• Alcohols are chemicals commonly used as disinfectants and antiseptics.
• Most commonly used microbicidal alcohols are Ethyl alcohol and Isopropyl
alcohol. Ethanol effective at 50-90% but ideal concentration is 70%.
• Mode of action : Denatures proteins and dissolves lipids thus interferes with
membrane functions of the microbes and kills them. Alcohols kills the
vegetative cells but not spores.
• Used to reduce the surface microflora of skin and for disinfection of clinical
oral thermometers. One common clinical use of alcohols is swabbing the skin
for degerming before needle injection.

40. b. Aldehydes
Formaldehyde and Glutaraldehyde are used. These are microbicidal .
• Formaldehyde :- Aqueous form of formaldehyde is called Formalin
which contains 37 to 40 % formaldehyde.
• Formaldehyde gas obtained by heating a concentrated solution of
formaldehyde.
• A 2% solution of Glutaraldehyde has a wide spectrum of antimicrobial
activity. It kills even viruses.
.

41. Mode of action :- They denature proteins and inactivate nucleic acids.
• Formaldehyde is used to preserve anatomical specimens, and for
destroying anthrax spores in hair and wool.
• 10% formalin containing 0.5% sodium tetraborate is used to sterilize
clean metal instruments.
• Formaldehyde gas is used for fumigation of wards, sick rooms and
laboratories.
• Glutaraldehyde used for sterilizing urological instruments, lensed
instruments, respiratory therapy equipment and other special
equipment.

42. c. Phenols
Phenolic compounds used as antiseptics or disinfectants include pure
phenol and substitution products with halogens and alkyl groups.
• Phenol (carbolic acid) is one of the oldest antiseptic agents. Phenol
is bacteriostatic at concentrations of 0.1%–1% and is
bactericidal/fungicidal at 1%–2%.
• Derivatives of Phenol used as disinfectant are Cresol,
Hexachlorophene. Lysol is a cresol solution used for disinfecting
inanimate objects like floor furniture etc at 1-5% concentration.

43. • Hexachlorophene is a combination of phenol with chlorine
.Antiseptic soaps and handwashes contain hexachlorophenes.
Mode of Action: Phenols act on cell membrane and disrupt them.
Phenols cause disruption of cells, precipitation of cell protein, and
inactivation of enzymes and leakage of amino acids from the cells.
• Derivatives of phenol diluted in detergents are used in many
antiseptic and disinfectant preparations.

44. d. Halogens
Halogens like Chlorine and Iodine are germicidal.
Chlorine is widely used disinfectant in form of gas or in chemical
combination. The convenient forms are hypochlorite- calcium
hypochlorite, and Sodium hypochlorite. Solutions of Sodium
hypochlorite of a 1% concentration are used for personal hygiene and
as a household bleaches and disinfectants.

45. Mode of Action:
• The antimicrobial action is due to hypochlorous acid formed when
free chlorine is added to water.
• Hypochlorite and Chloramines undergo hydrolysis with the
formation of hypochlorous acid.
• The hypochlorous acid formed in each case is further decomposed:
HClO  HCl + O to release nascent oxygen.
• The nascent oxygen released in this reaction is a strong oxidizing
agent and through its action on cellular constituents,
microorganisms are destroyed.

46. Iodine in the form of tincture of Iodine is used as antiseptic to treat
wounds. 2%solution is used .
• Iodophores are organic compounds containing Iodine.
eg :Betadine
• Mode of Action: Oxidize essential metabolic compounds such as
proteins with sulfhydryl groups and causes protein inactivation.
• Iodine mainly used as antiseptic and sanitization of food utensils

47. e. Metallic salts(Compounds of Heavy metals)
Compounds of Mercury , Iron , Zinc ,silver and copper are antimicrobial
heavy metals used.
• Mode of Action: These compounds combine with cell proteins and
inactivate them. Mercuric chloride act upon sulfhydryl group of
enzymes.
• Inorganic Mercuric chloride is Bactericidal in dilutions
1:1,000;limited use due high toxicity. It is used in ointments as
antiseptics. Organic mercury compounds like Metaphen used as
antiseptics on skin, wounds etc these are microbiostatic.

48. Silver compounds are germicidal.
• Silver nitrate at concentration of 1:1000 dilution is bactericidal and
used in few drops for the eyes of new born babies to prevent
ophthalmia neonatorum .
• Organic preparations like Protargol are used in 20%concentrations
• Copper compounds are microbicidal.
• Copper sulphate (Bordeaux mixture)is used as a fungicide .
• It is also used to control algal growth in lakes and swimming pools.

49. f. Quaternary Ammonium compounds
• Quaternary Ammonium compounds (QACs) are cationic
surfactants (positively charged surface-active agents) that disrupt cell
walls and membranes .
• Their permanent positive charge makes them bind readily to
the negatively charged surface of most microbes. e.g. Cetrimide,
Zephirol .They have microbicidal action .
• Most effective against Gram Positive bacteria.

50. Mode of Action: These cause denaturation of proteins, interfere in
glycolysis and damage to cytoplasmic membrane, alter permeability of
cell structures.
• Used as skin disinfectants, as a preservative in ophthalmic
solutions and in cosmetic preparations.
• Used widely in hospitals to disinfect surfaces, food processing
plants and as sanitizers of utensils in restaurants.

51. g. Sterilizing gases
Sterilizing gases are typically used when exposure to other methods
(heat or radiation) could damage the materials or equipment.
• The most common gases used for sterilization include Formaldehyde,
ethylene oxide , β-Propiolactone .
• Formaldehyde fumes are a gaseous agent. It is good disinfectant .
Ethylene oxide: It is a liquid at temperatures below 10.8°C and
vaporizes quickly above this temperature. It is highly inflammable.
• For use, it is prepared as a non-flammable mixture of 11% ethylene
oxide and 89% halogenated petroleum ( Freon). This is microbicidal.

52. • Mode of Action: The killing effect of ethylene oxide is due to
alkylation with organic compounds and enzymes.
• Alkylation is replacement of hydrogen atom in organic compound
with an alkyl group.
• It is used for sterilizing agent for heat and moisture sensitive
materials.
• It is used on spices, biological preparations, soil, plastics certain
medical preparations and contaminated laboratory equipment.

53. • β-Propiolactone
• Beta-propiolactone (BPL) is a liquid which exerts a strong bactericidal
action on the surfaces of objects immersed in aqueous solutions of it
or exposed to its vapor phase.
• Only 2 to 5mg / litre of β-Propiolactone is enough for sterilization
purposes.
• Mode of action of this is also due to alkylation. Since it has a low
penetration power and carcinogenic, it is of restricted use as a
sterilizing agent.

54. Thus sterilization is important in order to reduce the risk of
contamination and minimize the growth of microorganisms on
culture medium ,to prevent the spread of disease causing
organisms .