The document discusses sterilization and microbial control methods, defining key terms such as sterilization, disinfection, sepsis, and aseptic techniques. It elaborates on physical and chemical methods of sterilization, including the use of heat, radiation, and specific chemicals, alongside historical references to the development of the autoclave. Additionally, it highlights various agents used in the disinfection process and the operation principles of sterilization equipment.

1. Lecture 5

2. Sterilization
 ‘Sterilization is a process by which an article,
surface, or medium is freed of all living
microorganisms either in vegetative or spore
state.

3. Disinfection
 It is a process of destruction of pathogenic
organisms capable of giving rise to infection, but
not necessarily spores.

4. Sepsis: Comes from Greek for decay or putrid.
Indicates bacterial contamination.
Asepsis: Absence of significant contamination.
Aseptic techniques are used to prevent
contamination of surgical instruments, medical
personnel, and the patient during surgery.

5. Bacteriostatic Agent:
An agent that inhibits the growth of bacteria,
but does not necessarily kill them.

6. Bactericide:
An agent that kills bacteria. Most do not kill
Endospores.
Sporicide:
An agent that kills spores.

7.  Sterilization of of materials, instruments
used in surgical and diagnostic procedures.
 For media and reagents used in the
microbiology laboratory.

8. Microbial
Control
Methods
Physical
METHODS
Chemical
METHODS

9.  Physical methods.
1) Sunlight
2) Heat - dry heat
- moist heat
3) Radiation
4) Filtration

10.  Chemical methods
1. Alcohol
2. Aldehyde
3. Phenols
4. Halogens
5. Oxidising agens
6. Dyes
7. Gases.

11. PHYSICAL METHODS

12.  1. SUNLIGHT
 Sunlight has an active germicidal effect due
to its content of ultraviolet rays.
 It is a natural method of sterilization in cases
of water in tanks, rivers and lakes.

13. 2. HEAT
 Heat is the most reliable and commonly employed
method of sterilization.
 Two types of heat are used, dry heat and moist
heat.
Principles:
1) Dry heat kills the organisms by denaturation
of bacterial protein, toxic effect or elevate
levels of electrolyte.
2) Moist heat kills the microorganisms by
denaturation and coagulation of bacterial
proteins.

14. Heat method
Dry heat Moist heat

15.  The following procedures are used for
sterilization by dry heat.
1. Red heat
2. Flaming
3. Incineration
4. Hot air oven
Dry heat

16.  Red heat:
 Inoculating wires or loops, tips of forceps and
needles are held in the flame of a bunsen
burner till they become red hot.
 Flaming
 Glass slides, scalpels, and mouths of culture
tubes are passed through bunsen flame without
allowing them to become red hot.

17.  Incineration:
 By this method, infective material is reduced to
ashes by burning. Instrument named incinerator
may be used for this purpose. Soiled dressing,
animal carcasses, bedding and pathological
materials are delt with this method.

18.  Hot air oven:
 It is the most widely used method of
sterilization by dry heat.
 The oven is electrically heated and is fitted
with a fan to ensure adequate and even
distribution of hot air in the chamber
 It is also fitted with the thermostat that
maintains the chamber air at a chosen
temperature.

20.  This method of sterilization may be used at
different temperatures as follows.
1. At temperature below 100 C
2. At temperature of 100 C
3. At temperature above 100 C
Moist heat

21. 1) Pasteurization of milk:
 Two types of methods are used are
 1-Holder method= (63 C for 30 minutes )
 2-Flash method= (72 C for 20 seconds
followed by cooling)
2) inspissation:
 Some serum or egg media are sterilize by this
method
 The instruments used for sterilization is
called inspissator.

22. 1) Boiling:
 Boiling for 10-30 minutes may kill most of the
vegetative forms but not spore forms
 When other method is not available then boiling
is effective in sterilization
 It is used for glass syringes and rubber stoppers.

23. 2) vaccine bath):
 It is used for killing non sporing bacteria
which may be present in vaccine.
 In vaccine bath the vaccine is treated with
moist heat for 1 hour at 60 C.

25.  Autoclave is a equipment
used to remove
microorganisms
( viruse,bacteria,fungus)
 And spores using high
pressure and high
temperature steam
sterilization

26. Denis Papin
 French-born British physicist
 invented a precursor known as the steam
digester in 1679.
 was essentially a cooking instrument.

27.  Role of Chamberland
 Autoclave was invented by Charles
Chamberland in 1879.
 Colleague of Pasteur
 Building up on Papin’s marmite.
 began work on reforming the steam digester.
 credited for starting the research project.

28.  Autoclave is a pressurized device designed to
heat aqueous solutions above their boiling point
at normal atmospheric pressure to achieve
sterilization.
 Auto= self
 Clavis= self locking device
Autoclave Machine

29.  Principle of working Autoclave:
 Autoclave and pressure cooker are working
on a same principles.
 Water boils when its vapour pressure equals to
the surrounding atmosphere.
 When the atmospheric pressure is raised then
the boiling temperature is also raised.
 At normal pressure water boils at 100 C but
when pressure inside a closed vessel
increases, the temperature at which water
boils also increases.
 This principle is applied in autoclave…..

30.  Physical structure:
 Autoclave is a modified pressure cooker or
boiler.
 It consist of a vertical or horizontal cylinder of
gunmetal or stainless steel in a supporting iron
case.
 The lid is fastened by screw clamps and
rendered air tight by an asbestos washer.
 The lid having discharge tap for air and
steam.
 A pressure gauze and safety valve.

31.  Heating is generally done by electricity
 The steam circulate within the jacket and is
supplied under high pressure to the inner
chamber where materials are kept for
sterilization.

34.  Sterilization condition:
 temperature 121 C
 Chamber pressure-15 pounds (lbs) per square
inch.
 Holding time -15 minutes.

35. 1) To sterilize culture media, rubber
materials, gowns, dressing, gloves etc.
2) It is particularly useful for materials which
can not be withstand the higher temperature
of hot air oven.
3) For all glass syringes, hot air oven is a
better sterilizing method.

36.  The air must be allowed to escape from the
chamber as temperature of air steam mixture
is lower than that of pure steam.
 Materials should be arranged in such a
manner to ensure free circulation of steam
inside the chamber.

37. Sterilization indicator:
 Thermocouple- it is to record the
temperature directly by a potentiometer.
 Bacterial spores- spores of bacillus
stearothermophilus are used as a test
organism. The spore of this organism are killed
at a temperature 121 C for 12 minutes
 an envelop containing a filter paper strip
impregnated with 10 spores is placed inside
the autoclave during sterilization.

38.  Chemical indicator:Browne’s tube contains red
solution which turns green when exposed to
temperature of 121 C for 15 minutes.

39.  This method of sterilization is useful for
substances which get damaged by heat
process
 E.g sera, sugars, antibiotics solutions.etc.
 Principle: as viruses pass through the
ordinary filters, filtration can be
used to obtain bacteria-free
filtrates of clinical samples for
virus isolation.

40. Types of filters:
 Earthenware candles
 Asbestos disk filters
 Sintered glass filters
 Membrane filters

41.  Candle filter
 Types-
 Unglazed ceramic filters
 Diatomaceous earth filters
 Asbestos filters
 Disposable, single-used disc
 Usage is discouraged because of its carcinogenic
property.
 Eg: Seitz and Sterimat filters
 Sintered glass filters
 has low absorptive properties
 Brittle and expensive
 Membrane filters
 Made of cellulose esters or other polymers
 Usually used for water purification and analysis,
sterilization and sterility testing and preparation of
solutions for parenteral use.

42.  Two types of radiation method are used
 Ionizing
 Non-ionizing
 Ionizing radiations: ionizing rays including
gamma rays , X-rays and cosmic rays
 Non-ionizing radiations: these include
infrared and ultraviolet (UV) radiations.
 Both methods work by damage to DNA of a
bacteria.

43. CHEMICAL METHODS

44.  Chemical methods
1. Alcohol
2. Aldehyde
3. Phenols
4. Halogens
5. Oxidising agens
6. Dyes
7. Gases.

45.  Factors determine the potency of disinfectants:
 Concentration of the substance
 Time of action
 pH of the medium
 Temperature
 Nature of the organisms
 Presence of extraneous material

46. Alcohol
 Frequently used:
 Ethyl alcohol
 Isopropyl alcohol
 Must be used at concentration 60-90%
 Isopropyl alcohol used in disinfection of
clinical thermometer.
 Methyl acohol is effective against fungal
spores, treating cabinets and incubators.
 Methyl alcohol is also toxic and inflammable.

47. Aldehyde
 Formaldehyde:
 Bactericidal, sporicidal and has lethal effect
on viruses.
 Used to preserve anatomical specimens,
destroying anthrax spores on hair and wool.
 Glutaldehyde:
 Effective against tubercle bacilli, fungi,
viruses.
 Less toxic and irritant to eyes, skin
 Used to treat corrugated rubber anaesthetic
rubber, face masks, plastic endotracheal
tubes, metal instruments and polythene
tubing.

48. Dyes
 2 groups of dyes:
 Aniline dye
 Acridine dye
 Both are bacteriostatic in high dilution but
are of low bactericidal activity.
 Aniline dye is more active against gram +ve
than gram-ve organisms.
 Used in microbiology labs as selective agents
in culture media.

49. Halogens
 Iodine
 Skin disinfectant
 Active bactericidal, moderate action on spores.
 Chlorine
 Water supplies, swimming pools and food and
dairy industries.
 Along with hypochlorides are bactericidal. Also
act on viruses.

50. Phenols
 Obtained from distillation of coal tar
between 170-270°C.
 Lethal effect:
 Capacity to cause cell membrane damage,
releasing cell contents and causing lysis.
 Low concentration will precipitate proteins.

51. Gases
 Types of gases
 Ethylene oxide
 Formaldehyde gas
 Beta propiolactone (BPL)
 Ethylene oxide
 Action is due to its alkylating the amino,
carboxyl, hydroxyl and sulphydryl groups in
protein molecules. Also on DNA and RNA.
 Items: heart-lung machines, respirators, sutures,
dental equipment, books, clothing.

52.  Formaldehyde gas
 Employed for fumigation of OT and other rooms.
 After fumigation, the doors should be sealed and
left unopened for 48 hours.
 BPL
 Product of ketane and formaldehyde with a
boiling point of 163°C.
 Rapid biocidal activity but carcinogenic.
 Capable of killing all microorganisms and is very
active against viruses.

53. surface-active agents
 Def
 substance that alter the energy relationship at
interfaces, producing a reduction of surface or
interfacial tension.
 Widely used as wetting agents, detergents and
emulsifiers.
 4 main groups:
 anionic
 Cationic
 Nonionic
 amphoteric

54. metallic salts
 Salts of heavy metals have a greater action.
 Eg: salts of silver, copper and mercury
 Protein coagulant and have capacity to
combine with free sulfhydryl group of cell
enzymes.