This is beneficial for all nursing students and paramedical students.

1. Sterilisation and disinfection
• Definitions
Sterilisation:- it is a process by which an article,
surface or medium is made free of all
microorganisms either in the vegetative or spore
form.
Disinfection:- it means the destruction of all
pathogen or organisms capable of producing
infections but not necessarily spores. All
organisms may not be killed but the number is
reduced to a level that is no longer harmful to
health.

2. • Uses of sterilisation/disinfection:-
 sterilization of materials, instruments used in
surgical and diagnostic procedures.
 for media and reagents used in the
microbiology laboratory.

3. Methods of sterilization
A) Physical methods
1) Sunlight a) Dry heat
2) heat
b) moist heat
3) ozone
4) filtration
5) radiation

4. B) chemical methods:-
1. alcohols
2. aldehydes
3. phenols
4. halogens
5. oxidising agents
6. surface active agents
7. Dyes
8. gases

5. A) Physical methods
1) Sunlight:- sunlight has an active germicidal
effect due to its content of ultraviolet rays. It
is a natural method of sterilisation in cases of
water in tanks, rivers and lakes.
2) heat:- heat is the most reliable and
commonly employed method of sterilisation.
Two types of heat are used, dry heat and
moist heat.

6. a) Dry heat sterilisation:- the following
procedures are used for sterilisation by dry
heat:-
i. Red heat
ii. Flaming
iii. incineration
iv. hot air oven

7. • 1) Red heat:- inoculating wires or loops, tips of forceps
and needles are held in the flame of a Bunsen burner
till they become ret hot.
• 2) flaming:- glass slides, scalpels and mouth of culture
tubes are passed through Bunsen flame without
allowing them to become red hot.
• 3) incineration:- By this method, infective material is
reduced to ashes by burning, instrument named
incinerator may be used for this purpose, soiled
dressings, animal carcasses, bedding and pathological
materials are dealt with this method.

8. 4) Hot air oven
• It is the most widely used methods 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 a
thermostat that maintains the chamber air at
a chosen temperature.
• Temperature and time:- 160’C for two hours is
required for sterilization.

10. • Uses:- it is used for sterilization of :-
 Glass-wares like glass syringes, pertidishes,
flasks, pipettes and test tubes.
 surgical instruments like scalpels, scissors,
forceps etc.

11. Precautions
i. it should not be overloaded
ii. the material should be arranged in a manner which
allows free circulation of air.
iii. material to be sterilised should be perfectly dry.
iv. Test tubes flasks etc. should be fitted with cotton
plugs.
v. petridishes and pipettes should be wrapped in paper.
vi. rubber material or any inflammable material should
not be kept inside the oven.
vii. the oven must be allow to cool for two hours before
opening the doors, since the glassware may crack by
sudden cooling.

12. b) Moist heat sterilisation
• This method of sterilisation may be used at
different temperatures as follows:-
A. At a temperature below 100’C
B. At a temperature of 100’c
C. At a temperature above 100’C

13. At a temperature below 100’c
a) Pasteurisation of milk:- Two types of method,
holder method (63’C for 30 minutes) and flash
method (72’C for 20 seconds) are used.
b) Inspissation- some serum or egg media, such as
lowenstein-jensen’s and loeffler’s serum, are
rendered sterile by heating at 80-85’C
temperature for half an hour daily on three
consecutive days. This process of sterilisation is
called inspissation. The instrument used is called
inspissator.

14. At a temperature of 100’C
• A) Boiling:- Boiling for 10 to 30 minutes may
kill most of the vegetative forms but many
spore withstand boiling for a considerable
time. When better method are not available,
boiling may be used for glass syringes and
rubber stoppers. It is not recommended for
the sterilistation of instruments used for
surgical procedures.

15. • B) Tyndallisation:- steam at 100’C for 20 minutes
on three successive days is used. This is known as
tyndallisation or intermittent sterilization. The
principle is that the first exposure kills all the
vegetative forms, in the intervals between the
heating the remaining spores germinate into
vegetative forms which are killed on subsequent
heating. It is used for sterilisation of egg, serum
or sugar containing media which are damaged at
higher temperature of autoclave.

16. At a temperature above 100’c (under
pressure)
• Water boils when its vapour pressure equals
that of the surrounding atmosphere. When
the atmospheric pressure is raised then the
boiling temperature is also raised. At normal
pressure inside a closed vessel increases, the
temperature at which water boils also
increases. This principle has been applied in
autoclave and pressure cooker.

17. Autoclave

19. Principle
• Steam above 100’c or saturated steam has a better killing
power than dry heat. Bacteria are more susceptible to
moist heat as bacterial protein coagulates rapidly.
Saturated steam can penetrate porous material easily.
• when steam comes into contact with a cooler surface it
condenses to water and liberates its latent heat to that
surface, for examples, 1600 ml of steam at 100’c and at
atmospheric pressure condenses into one ml of water at
100’c and releases 518 calories of heat. The large reduction
in volume sucks in more steam to the same site and the
process continues till the temperature of the article is
raised to that of steam.
• the condensed water produces moist condition for killing
the microbes present.

20. Components of autoclave
• 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
bears a discharge tap for air and steam, a
pressure gauge and a safety valve. Heating is
generally done by electricity. The steam circulates
within the jacket and is supplied under high
pressure to the inner chamber where materials
are kept for sterilisation.

21. Sterilisation condition
• Temperature -121’C
• Chamber pressure- 15 pound (lbs) per square
inch
• Holding time -15 minutes
• these condition are generally used, however,
sterilisation can also be done at higher
temperatures, at 126’C (20 lbs/square inch)
for 10 minutes or at 133’C (30 lbs/square inch)
for 3minutes.

22. • Uses:-
 to sterilise culture media, rubber material,
gown, dressing, gloves etc.
 it is particularly useful for materials which
cannot withstand the higher temperature of
hot air oven.
 for all glass syringes, hot air oven is a better
sterilising method.

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

24. Ozone
• Low temperature sterilistion by ozone:-
• ozone steriliser uses oxygen, water and
electricity to produce ozone with in the
steriliser and provide sterilistion without
producing toxic chemicals. It runs at lower
temperature, i.e. 25’C-35’C.

25. Filtration
• This method of sterilsation is useful for
substances which get damaged by heat process
e.g. sera, sugars, antibiotics solution etc.
• Uses of filtration:-
 to sterilise sera, sugars and antibiotic solution.
 separation of toxins and bacteriophages from
bacteria.
 sterilisation of hydatid fluid.

26. Type of filters
i. Earthenware candle
ii. asbestos disc filter ( Seitz filter)
iii. sintered glass filters
iv. membrane filters

27. 1) Earthenware candles:- these filters are
usually made in the form of hollow candles.
The liquid to be filtered is passed through
the candle.
2)Asbestos disc filters (Seitz filter):-
• these are made of asbestos (magnesium
silicate) the filter disc is supported in a metal
mount. After use, the filter disc is discarded.

28. 3) Sintered glass filters:- these are prepared by
fusing finely powered glass particles. These
available in different pore sizes.
4) Membrane filters:- membrane filters are
made up of cellulose esters. Nitrocellulose
membrane filters are widely used. Membrane
filters are available in pore sizes of 0.015 to
12mm

29. Radiations
• Two type of radiations are used in sterilisation:-
A) ionising radiation B) non-ionising radiation.
• A) ionising radiations:- ionising radiations include
gamma rays, X-rays and cosmic rays. They are
hi8ghly lethal to all cells including bacteria. They
damage DNA by various mechanisms. Gamma
radiations are commercially used for sterilisation
of disposable items such as plastic syringes,
swabs, culture plates, cannulas, catheters etc.

30. • B) Non-ionising radiation:- these include
infrareds and ultraviolet radiation. Infrared is
used for rapid mass sterilisation of syringes
and catheters. Ultraviolet radiation has
marked bactericidal activity. It acts by
denaturation of bacterial protein and
interference with DNA replication. UV
radiation is used for disinfecting area such as
bacteriological laboratory, operation theaters.

31. Chemical method
• A variety of chemical agents are used as
antiseptics and disinfectants.
• A) Alcohols:- Ethyl alcohol and isopropyl
alcohol are the most frequently used. They act
by denaturing bacterial proteins. They are
used mainly as skin antiseptics. To be
effective, they should be used at a
concentration of 60-70 percent in water.

32. • B) Aldehydes:-
• 1) Formaldehyde:- it is used both as aqueous
solution and in gaseous form. A 10% aqueous
solution of formalin is routinely used.
• uses :-
To sterilise bacterial vaccines.
 to prepare toxoide from toxin.
 for killing of bacterial cultures and suspensions.

33. • 2) Glutaraldehyde:- it is used as 2% buffered
solution. It is available commercially as ‘cidex’. It
can be used for delicate instruments having
lenses.
• Uses:-
 for sterilisation of cystoscopes, endoscopes and
bronchoscope.
 to sterilise plastic endotracheal tubes, face
masks, corrugated rubber an anesthetic tubes
and metal instruments.

34. Phenols
• Phenol (1%) has bactericidal action. Certain phenol
derivatives like cresol, chlorhexidine, chloroxylenol and
hexachlorophane are commonly used as antiseptics.
1) Cresols:- Lysol is a solution of cresols in soap. It is
most commonly used for sterilization of infected glass
wares, cleaning floors, disinfection of excreta.
2) chlorhexidine :- savlon (chlorhexidene and cetrimide)
is widely used in wounds, pre-operative disinfection
of skin, as bladder irrigant etc.
3) chloroxylenol :- it is an active ingredient of dettol.

35. Halogens
• Chlorine and iodine are two commonly used
disinfectants.
• Chlorine is used in water supplies and swimming pools.
Chlorine compounds in the form of bleaching powder,
sodium hypochlorite and chloramines are also used.
The disinfection action of all the chlorine compounds is
due to release of free chlorine. Bleaching powder or
hypochlorite solution are the most widely used for
human immunodeficiency virus (HIV) infected
material.
• Iodine in alcoholic and aqueous solution is used as a
skin disinfectant . Betadine is one example of
commonly used iodine compound.

36. Oxidising agents
I. Hydrogen peroxide
II. Peracetic acid
III. Plasma sterilisation

37. Oxidising agents
I. Hydrogen peroxide:- hydrogen peroxide
(H2O2)is effective against most organism at
concentration of 3-6%, while it kills all
organism including spores at higher
concentration (10-25%). Hydrogen peroxide
is used to disinfect contact lenses, surgical
prostheses and plastic implants. It is used for
high level disinfectant and sterilization.

38. II. Peracetic acid:- peracetic acid is an oxidising
agent. it is one of the high level disinfectants.
The end product (acetic acid & oxygen) of
the agent are non toxic. It also used in
plasma sterilisation procedure. It is a more
potent germicidal agent than hydrogen
peroxide.

39. III. Plasma sterilisation:- plasma refers to any gas
which consists of electrons, ions or neutral
particles. The formation of a low temperature
plasma requires several devices.
chemical disinfectant such as H2O2 alone or a
mixture of h2o2 and peracetic acid is used in
these devices to induce the plasma. Two
commercial plasma sterilisers available are
sterrad 100s steriliser and plazlyte steriliser. It is
used for sterilisation of surgical instruments.

41. Surface active agents
• Surface active agents or surfactants are
classified into anionic, cationic, nonionic and
amphoteric compounds. Of these, the cationic
surfactant have been the most important
antibacterial agents. Quaternary ammonium
compounds are the most important
surfactants. The common compounds used
are acetyl trimethyl ammonium bromide and
benzalkonium chloride.

42. Dyes
• Two group of dyes, aniline dyes and acridine
dyes have been used extensively as skin and
wound antiseptics. Aniline dyes include crystal
violet, brilliant green and malachite green.
Acridine dyes include acriflavine, euflavine,
proflavine and aminacrine.

43. Gases
Formaldehyde gas
Ethylene oxide (ETO)
Betapropiolactone (BPL)

44. Formaldehyde
• This is employed for fumigation of operation
theatres, wards etc. formaldehyde gas is
generated by adding KMnO4 to formalin. The
doors should be sealed and left unopened for
48 hours. Sterilisation is achieved by
condensation of gas on exposed surface.

45. Ethylene oxide (ETO)
• It is specially used for sterilising plastic and
rubber articles, plastic syringes, respirators,
heart-lung machines, sutures, dental equipments
and clothing. It is suitable for fumigation of
rooms because of its explosive nature.
• Betapropiolactone (BPL)
• it is more efficient for fumigation than
formaldehyde. BPL is used for inactivation of
vaccines.