This document discusses the history and methods of sterilization. It begins by describing the contributions of Ignatz Semmelweis and Joseph Lister in introducing antisepsis through hand washing and wound treatment. It then defines various sterilization, disinfection, and antisepsis terms. The remainder of the document details various physical and chemical sterilization methods such as heat, radiation, filtration and their mechanisms and appropriate uses.

2. HISTORICAL BACKGROUND
Ignatz Semmelweis (1816 –1865), a
Hungarian physician, was the pioneer in
introducing hand washing with chlorinated
lime water to avoid infection.
Joseph Lister (1827–1912), a British
physician, treated his patients’ wounds with
phenol to avoid wound infection.

3. STERILIZATION
It is defined as the process that refers to removal, killing,
or deactivating all forms of life (in particular referring to
microorganisms such as fungi, bacteria, viruses, spores,
unicellular eukaryotic organisms such as Plasmodium,
etc.)
It involves a biocidal agent or microbial removal process
from any product or surface
Disinfection: Destruction of microorganisms, especially
potential pathogens, on the surfaces of inanimate
objects or in the environment

4. STERILIZATION
 Antisepsis: Destruction or inhibition of microorganisms on
living tissues by chemicals (non-toxic and non-irritating)
 Chemical agents- Antiseptic
 Germicide/microbicide: chemical agent that kills pathogenic
microorganisms
 Sepsis: growth of microorganisms in the body or the presence
of microbial toxins in blood and other tissues
 Asepsis: Practice to prevent entry of infectious agents into
sterile tissues and thus prevents infection

5. STERILIZATION
 Sanitization: Cleansing technique that mechanically removes
microorganisms to reduce the level of contaminants.
 Sanitizer- compound (e.g., soap or detergent).
 Bacteriostasis- condition where the multiplication of the
bacteria is inhibited without killing them.
 Bactericidal- 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.

6. Uses of Sterilization
1. Sterilization for Surgical Procedures and
medicines: Gloves, aprons, surgical instruments,
syringes, drugs and other supplies etc.
2. Sterilization in Microbiological works:
Preparation of culture media, reagents and
equipments.

8. PHYSICAL METHODS OF STERILIZATION:
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.
 In tropical countries, the sunlight is more effective in killing
germs due to combination of ultraviolet rays and heat.
 By killing bacteria suspended in water, sunlight provides natural
method of disinfection of water bodies such as tanks and lakes.
Sunlight is not sporicidal, hence it does not sterilize.

9. TEMPERATURE (HEAT)
 It is the most common, effective, reliable and productive mean of sterilization
 It is used to sterilize laboratory glassware, laboratory media, surgical
instruments etc.
 ADVANTAGE
 It is economical and easily controllable means of microbial growth
 MECHANISM
 It usually kill microbes by causing denaturation of respective enzymes or
coagulation of
 LIMITATION
 Heat resistance capacity of organisms must be studied carefully and taken
into consideration

10. Factors affecting sterilization by heat
 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.

11. Characterization of Heat Sterilization
 Thermal Death Time (TDT)
 Minimum time required to kill all microbes at a specified temperature.
 Thermal Death Point (TDP)
 Lowest temperature at which all microbes are killed in 10 minutes.
 Heat resistance of each organism is variable hence TDP determines the sensitivity of each organism
to temperature.
 Both TDT and TDP are employed to determine the heat resistance of microbes before sterilization.
 Decimal Reduction Time (DRT)
 It is also termed as D-Value.
 It represents the extent of heat resistance of each microbe.
 It is the time at which almost 90% microbes are killed
 It is usually employed in canning industry for sterilizing fruit concentrates, pulps, vegetables, fish
etc.

12. 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. 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.

13. DRY HEAT
 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.

14. DRY HEAT
 Incineration:
 This is a method of destroying contaminated material by burning them
in incinerator.
 Articles such as soild 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.

15. HOT AIR OVEN
 Introduced by Louis Pasteur.
 Articles are exposed to high temperature (160º C) 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.
 Conduction is the process by which heat energy is transmitted through collisions between
neighboring atoms or molecules.
 Convection is a process of displacement of cooler air by hot air.

17. HOT AIR OVEN (cont..)
 Metallic instruments (like forceps, scalpels, scissors)
 Glasswares (such as petridishes, pipettes, flasks, all-glass
syringes)
 Swabs, oils, grease, petroleum jelly and some pharmaceutical
products
 Unsuitable for rubber and plastic

18. HOT AIR OVEN (cont..)
 The oven should be fitted with a thermostat control, temperature
indicator, meshed shelves and must have adequate insulation.
 Oven not overloaded
 Materials perfectly dry and arranged to allows free circulation of
air inside the chamber
 Mouths of flasks, test tubes and both ends of pipettes must be
plugged with cotton
 Petri dishes and pipettes wrapped in a paper
 60 minutes at 160º C, 40 minutes at 170ºC and 20 minutes at
180º C.

19. HOT AIR OVEN (cont..)
 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 glassware
 To determine the efficacy of sterilization - Thermocouples,
chemical indicators, and bacteriological spores of Bacillus
subtilis as sterilization controls

20. HOT AIR OVEN (cont..)
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:
Hot air has poor penetration
Cotton wool and paper may get slightly charred.
Glasses may become smoky.
Takes longer time compared to autoclave.

21. INFRARED STERILIZATION
 Sterilization by generation of heat
 Articles placed in a moving conveyer belt
and passed through a tunnel that is heated
by infrared radiators to a temperature of
180o C •
 The articles are exposed to that
temperature for a period of 7.5 minutes •
Articles sterilized: metallic instruments and
glassware
 Requires special equipment
 Efficiency can be checked using Browne’s
tube No.4 (blue spot)

23. Moist Heat
 Moist heat invariably kills microbes by coagulation of
proteins which is caused by Hydrogen bond cleavage
 It has high penetration power
 Moist heat sterilization is achieved by following methods:
1. At temperature below 100º C
2. At temperature 100º C
3. At temperature above 100º C

24. At temperature below 100º C
Pasteurization:
• originally employed by Louis Pasteur
• employed in food and dairy industry
• Two methods of pasteurization
1. • holder method (heated at 63o C for 30 minutes)
2. • flash method (heated at 72o C for 15 seconds) followed by quickly cooling to 13º C
• Suitable to destroy most milk borne pathogens (Mycobacteria, Streptococci, Staphylococci,
Brucella etc.)
• Inactivates most viruses and destroys the vegetative stages of 97–99% of bacteria, fungi, does
not kill endospores or thermoduric species
• Efficacy tested by phosphatase test and methylene blue test
• Newer techniques: Ultra-High Temperature (UHT), 134°C for 1–2 second

25. At temperature below 100º C
Vaccine bath:
 The contaminating bacteria in a vaccine preparation can be inactivated by
heating in a water bath at 60o C for one hr
 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 56o C for one hour on several successive days
 Proteins in the serum will coagulate at higher temperature
 Only vegetative bacteria are killed and spores survive

26. At temperature 100º C
Boiling
 Boiling water (100o C) for 10–30 minutes kills most vegetative
bacteria and viruses
 Certain bacterial toxins such as Staphylococcal enterotoxin heat
resistant
 Some bacterial spores are resistant to boiling and survive
 Not a substitute for sterilization
 The killing activity can be enhanced by addition of 2% sodium
bicarbonate
 Certain metal articles and glasswares disinfected by placing them in
boiling water for 10-20 minutes
 The lid of the boiler must not be opened during the period

27. At temperature 100º C
Steam at 100º C
 Subjected to free steam at 100o C
 Traditionally Arnold’s and Koch’s steamers were used
 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 generated sterilizes the articles when exposed for a period
of 90 minutes
 Media such as TCBS, DCA and Selenite broth are sterilized by
steaming

28. At temperature 100º C
 Tyndallisation:
 Heat labile media like those containing sugar, milk, gelatin
 Tyndallisation (after John Tyndall)/ fractional sterilization/ intermittent
sterilization
 Steaming at 100°C is done in steam sterilizer for 20 minutes followed
by incubation at 37°C overnight
 Repeated for another 2 successive days
 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

29. At temperature above 100º C
Autoclave:
 Sterilization can be effectively achieved at a temperature above
100º C using an autoclave.
 In an autoclave the water is boiled in a closed chamber.
 At a pressure of 15 lbs inside the autoclave, the temperature is 121º
C.
 Exposure of articles to this temperature for 15 minutes sterilizes
them.
 To destroy the infective agents associated with spongiform
encephalopathies (prions), higher temperatures or longer times are
used; 135o C or 121o C for at least one hour are recommended.

30. At temperature above 100º C
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.
Different types of autoclave:
Simple “pressure-cooker type” laboratory autoclave, Steam
jacketed downward displacement laboratory autoclave and
high pressure pre-vacuum autoclave

31. At temperature above 100º C
Autoclave
Vertical or horizontal cylindrical body
Articles sterilized: Culture media, dressings, certain equipment, linen, rubber (gloves), heat-resistant
plastics, liquids etc. Ineffective for sterilizing substances that repel moisture (oils, waxes, or powders)
Kills all the vegetative as well as spore forms of bacteria
Sterilization controls: (a) Thermocouples (b) Chemical indicators- Brown’s tube No.1 (black spot) (c)
Bacteriological spores- Bacillus stearothermophilu

32. Autoclave
 Vertical or horizontal cylindrical body
 Articles sterilized: Culture media, dressings,
certain equipment, linen, rubber (gloves), heat-
resistant plastics, liquids etc.
 Ineffective for sterilizing substances that repel
moisture (oils, waxes, or powders)
 Kills all the vegetative as well as spore forms of
bacteria
 Sterilization controls: (a) Thermocouples (b)
Chemical indicators- Brown’s tube No.1 (black
spot) (c) Bacteriological spores- Bacillus
stearothermophilu

33. Autoclave
Advantage:
 Very effective way of sterilization, quicker than hot air oven.
Disadvantages:
Drenching and wetting or articles may occur, trapped air may reduce the efficacy,
takes long time to cool

34. RADIATION:
 Ionizing and non-ionizing
Non-ionizing rays
 Low energy rays, poor penetrative power
 Rays of wavelength longer than visible light
 Microbicidal
 Wavelength of UV rays: 200-280 nm, 260 nm most effective
 UV rays induce formation of thymine-thymine dimers, ultimately inhibits DNA replication
 UV readily induces mutations in cells
 Don’t kill spores

35. Uses of UV radiation:
 Disinfection of closed areas in microbiology laboratory, inoculation
hoods, laminar flow, and operating theaters
 Harmful to skin and eyes
 Doesn't penetrate glass, paper or plastic
 Of use in surface disinfection
 Generated using a high-pressure mercury vapor lamp

36. RADIATION (cont..):
Ionizing rays
 High-energy rays, good penetrative power
 Radiation does not generate heat- "cold sterilization“
 e.g. (a) X-rays, (b) gamma rays, and (c) cosmic rays
 Gamma radiation from cobalt-60 sourcesterilization of antibiotics, hormones,
vitamins, sutures, catheters, animal feeds, metal foils, and plastic disposables, such
as syringes, petri dishes
 A dosage of 2.5 megarads kills all bacteria, fungi, viruses and spores
 Also used for meat and other food items
 Damage the nucleic acid of the microorganism

37. Filtration
 It is the process of removing particles from a solution by allowing the liquid or
gas through a membrane or particle barrier.
 Serum, antibiotic solutions, sugar solutions, urea solution are sterilized
by filtration.
 Two types of filtration are employed
 High Efficiency Particulate Air
It is used to purify environmental air such as ICU, Burns unit, Industrial sterile
zones etc.
 Membrane Filtration
Cellulose esters
Plastic Polymers
 It is employed to sterilize heat sensitive substances, culture media, vaccines,
enzymes and several antibiotic solutions

38. Types of filters
 1. Earthenware filters:
 Made up of diatomaceous earth or porcelain
 usually baked into the shape of candle
 a. Pasteur-Chamberland filter:
Candle filters from France, of porcelain
Various porosities, graded as L1, L1a, L2, L3, L5, L7, L9 and L11
Similar filter from Britain is Doulton; P2, P5 and P11

39. Types of filters
 b. Berkefeld filter:
 made of Kieselguhr, a fossilized diatomaceous earth found in
Germany
 Three grades depending on their porosity (pore size)
 V (veil), N (normal) and W (wenig)
 c. Mandler filter:
 from America
 made of kieselguhr, asbestos and plaster of Paris

40. Types of filters
 Asbestos filters:
 Made up of asbestos such as magnesium silicate
 Examples- Seitz and Sterimat filters
 Disposable, single-use discs in different grades
 Tend to alkalinize the filtered fluid
 Use limited, carcinogenic potential of asbestos Sintered glass filters:
 Made up of finely powdered glass particles, which are fused together
 Available in different pore sizes

41. Types of filters
 Membrane filters:
 made up of
 (a) Cellulose acetate
 (b) Cellulose nitrate,
 (c) Polycarbonate
 (d)Polyvinylidene fluoride,
 (e) Other synthetic materials
 Widely used, circular porous membranes, usually 0.1 mm thick
 Variety of pore sizes (0.015–12 μm)
 Membranes with pores about 0.2 μm are used, smaller than the size of bacteria

42. Uses of membrane filters
 Sterilize pharmaceutical substances, ophthalmic
solutions, liquid culture media, oils, antibiotics,
and other heat-sensitive solutions
 To obtain bacterial free filtrates of clinical
specimens for virus isolation
 To separate toxins and bacteriophages from
bacteria

43. DESICCATION
 Microbes require water for their optimum growth
 Desiccation is a process to expel or extract out the water content.
 When applied to in sterilization, desiccation can temporarily
retard or restrict the growth and reproduction by disrupting
metabolism but it does not completely kill them.
 It is usually applied with Lyophilization

44. OSMOTIC PRESSURE
 Osmosis is another important parameter required for optimum
growth and reproduction.
 The sterility is provided by plasmolysis
 Osmotic pressure is the pressure that develops when two
solutions are separated by semi-permeable membrane.
 This process is applied in food industry for preservation using
salt or sugar solutions to retard growth and reproduction of
microbes

45. CHEMICAL STERILIZATION
 Different chemicals are used to manage and control usual
growth of microorganisms
 Chemical sterilants/ disinfactants should have following
properties:
 wide spectrum of activity
 able to destroy microbes within practical period of time
 active in the presence of organic matter
 effective contact and be wettable
 active in any pH
 Stable, speedy, and long shelf-life
 high penetrating power
 non-toxic, non-allergenic, non-irritative or non-corrosive

46. Effective Disinfection
 Disinfectant
 Substance that prevents infection by killing an organism
 Selection of disinfectant to be an effective sterilizing agent, following factors must be taken into
consideration:
 The concentration of disinfectant determines the effectiveness
 Disinfectant should be diluted as instructed by manufacturer on the label
 Diluted solutions may act as bacteriostatic rather than a bactericidal
 Nature of the material must be taken into account whether it is organic substance or inorganic
substance and the pH of that disinfectant
 Accessibility of microbes must be taken into consideration
 Higher the temperature used for actual application of disinfectant higher would be its effectiveness or
versatility

47. 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)

48. DISINFECTANT VARIANTS