Sterilization and disinfection

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3 - STERILISATION AND DISINFECTION

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Microorganisms are present everywhere,more so in a
hospital where there are many persons suffering from
diseases . Through their secretions ,excretions,or any
other route microbes are contaminating their
surroundings .Various methods are used to destroy
these organisms.

3. STERILIZATION
Is defined as the process by which all microorganisms,vegitativeas well
as spores are removed from an article,surface or area.The material
subjected to this process is termed to be sterile.
DISINFECTION
Is the destruction of all organisms capable of causing infection.
ANTISEPSIS
Is the inhibition of growth of bacteria.
chemical agent used for this is called ANTISEPTICS OR DISINFECTANTS.
BACTERICIDAL AGENT
The agents which are able to kill the bacteria.
BACTERIOSTATIC AGENTS
The agents which only prevents the multiplication of bacteria and they
remain live .

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The method of sterilisation employed would
depend on the purpose for which sterilisation is
carried out,the material which has to be sterilised
and nature of organism that have to be get rid of.

5. METHODS OF STERILIZATION
PHYSICAL
Sulight
Drying
Dry heat : Flaming,
incineration ,
hot air
Moist heat:pasteurisation,
boiling,steam under pressure
Filtration
Radiation
ultra sonic and sonic
vibrations
CHEMICAL
Alcohols
Aldehydes
Ethylene oxide
Dyes
Halogens
Metallic salt
Phenols
Surface active agents

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PHYSICAL METHODS or AGENTS OF STERILISATION
SUN LIGHT
The sun's rays possess bactericidal activity and play an important
part in spontaneous sterilisation occuring under natural conditions.
The activity is due to the ultraviolet rays in them
Direct sun light has germicidal effect due to combined effect of UV
rays and heat rays
Bcateria suspended in water are readily destroyed by exposure to
sunlight- it is the natural method of sterilisation in river water,water
tanks etc...
Exposure of clothes to direct sunlight is an old method adopted by
people.

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DRYING
moisture is an important constituent of the bacterium.It is essential for its growth,so drying cause a
harmful effect on it.
spores are unaffected by drying.
susceptibility to drying depend on the type of organism,conditions of exposure such as cloth,furniture
etc..
HEAT
Heat is the most reliable method of sterilisation,it is always preferrd except when its use is
contraindicated.
materials liable to be damaged by heat can be sterilised at a lower temperature for a longer period
or by repeated cycles.
factors influencing sterilisation by heat are ;-
nature of heat -dry heat or moist heat
temperature and time
number of microorganisms present
characteristics of the organism-species,strain,sporing capacity
type of material from which the organisms have to be destroyed.

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The killing effect of dry heat is due to protien denaturation,oxidative damage and the toxic effect
of elevated levels of electrolytes.
The lethal effect of moist heat due to denaturation and coagulation of protien.
The advantage of steam lies in the latent heat generated when it condenses on a cooler surface,
raising temperature of that surface.
1600 ml of steam at 1000
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In completely moisture free atmosphere ,bacteria are more resistant to heat,so higher
temperature is required for their destruction.
THERMAL DEATH TIME ; It is the minimum time required to kill a suspension of
organisms at a predetermined temperature,in a specified environment.
The time required for sterilisation is inversely proportional to the temperature of exposure.this is
expressed as the thermal death time.

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The sterilisation time is directly related to the no.of organisms in the
suspension,the presence of spore,nature of spore,characteristics of the
strain and organism.
RECOMMENDED STERILISING TIME;- It is the time required for destroying the
organisms at the stated temperature (the organism have to be exposed at that
temperature throughout that period.)
A high content of organic substances ,dirt etc..generally tends to protect the
spores and vegetative organisms against the lethal action of heat,protien ,
starch,gelatin ,sugars,fats,oils...increase the time required.
The destruction of spores is helped by an acid or alkaline pH.

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DRY HEAT
FLAMING
INCINERATION
HOT AIR OVEN

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FLAMING ;- Inoculation loops and points of forceps may be heated
in the bunsen flame,till they are red hot ,for sterilising them.scalpels,
needles,glass slides,cover slips may be passed a few times through
the bunsen flame without allowing them to become red hot.The
bacteria get destroyed.
Inoculation loops carrying infective material may be dipped in a
disinfectant before flaming to prevent spattering.
INCINERATION ;- This is an excellent method for rapidly destroying
materials such as soiled dressings ,animal carcasses,non reusable
soiled bedding,pathological materials.
plastics such as PVC and polythene can be incinerated.polystyrene
emit clouds of dense black smoke and should incinerated in
appropriate containers.

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HOT AIR OVEN;-
Most widely used method by dry heat.
A holding period of 160 oc for one hour is used.
It is used to sterilise glassware,forceps,scissors,scalpels,all glass
syringes,swabs,some pharmaceutical products such as liquid paraffin,
sulphonamides,fats ,greases etc...
Hot air is a bad conductor and its penetrating power is poor.
The air oven must be fited with a fan to ensure even distribution of
hot air and elimination of air pockets.The material should be arranged
so that free circulation of air between materials is possible.
Glassware should be dry before placing in oven.
Test tubes,,flasks etc..should be plugged with cotton wool.petri dish,
pippets should be wrapped in craft paper.

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HOT AIR OVEN.....
for cutting instruments such as those used in ophthalmic surgery-2 hours
150 oc is recommended.
1 hour -150oc for oils,glyserol and dusting powder.
The oven must be allowed to cool slowly about 2 hours before the door is
opened ,since glasswares may get cracked by sudden or uneven cooling.
STERILISATION CONTROL ;-
The spores of a nontoxigenic strain of clostridium tetany are used as a
microbiological test of dry heat efficiency.paper strips impregnated with
the spores are placed in envelops and inserted in suitable packs.After
sterilisation is over the strips are sent for testing sterility.Easier method is
Browne's tube -green spot.After proper sterilisation a green colour is
produced (after 60 minute at 160oc or 115 minutes at 150oc

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HOT AIR OVEN

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MOIST HEAT

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MOIST HEAT
TEMPERATURE HIGHER THAN 100 OC
TEMPERATURE BELOW 100OC
TEMPERATURE AT 100 OC
STEAM UNDER PRESSURE

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21.  Temperature below 100 degree Celsius -
pasteurisation
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This technique was introduced by Louis Pasteur.
It involve the application of temperature at 1000c
Mainly used to kill pathogens in milk.
VARIOUS METHODS OF PASTEURISATION
Flash method : High temperature (720c)is used for short term (15 -20
sec.)
Low temperature holding method : Low temperature ( 630c) is used for
a long period of time (30 min. )
Ultra pasteurization : Very high temperature ( 137.80c) is used for short
period of time (2 sec )

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Pasteurisation of milk removes pathogenic bacteria ,with the advantage
that there is no unpleasant alteration in the taste which is likely to occur
on boiling.in both process the rapid cooling is important for keeping
properties of milk as it minimises multiplication of the surviving non
pathogenic bacteria.
A temperature of 80°c for 15 minutes destroys the vegetative forms of all
bacteria, yeasts and moulds.
Among the most heat resistant cells are the spores of clostridium
botulinum which require 120°C for 4 minutes or 100°C for 330 minutes.
Among viruses ,the polio virus requires 60°C for 30 minutes.
Clothing, bed sheets, eating utensils may be disinfected by washing in
water at 70-80° C For several minutes.
Cystoscope, specula etc.… can be disinfected at 75° C for 10 minutes.
Hard water must not be used.

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TEMPERATURE AT 100°C
BOILING;-
Vegetative bacteria are killed immediately at 90 -100°C, But
sporing bacteria require considerable period of boiling.
Boiling is not recommended for instruments used for surgical
procedures.
Sterilisation may be promoted by the addition of 2% sodium
bicarbonate to the water, hard water should be avoided.
If instruments are used, it should be immersed fully in boiling
water for 30 minutes to an hour at least.

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STEAM UNDER PRESSURE - AUTOCLAVE
The principle of the autoclave or steam steriliser is that water boils when its vapour
pressure equals that of the surrounding atmosphere.
Here when water is boiled in a closed vessel ,the pressure inside increases and the
temperature at which water boils also increases. Saturated steam has great
penetrating power.
When steam come into contact with a cooler surface it condenses in to water ,gives
up its heat to that surface and raises the temperature of that surface.
The large reduction in the volume occupied by the steam (as a result of
condensation)results in the suction of more steam to the area to fill up the void and
and the process continues till the temperature of that surface is raised to that of the
steam to which it is exposed. The condensed water ensures moist conditions for
killing the microbes present.

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An autoclave is a modified pressure cooker
in which sterilization by saturated steam
under high pressure is achieved.
an autoclave may be horizontal or
vertical it is a double walled or jacketed
chamber made up of stainless steel or
gunmetal with supporting frame

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sterilisation by steam under pressure is carried out at temperatures of between
1080C and 1470C .
By using appropriate temperature and time ,a variety of materials such as dressings,
instruments,laboratory ware,media and pharmaceutical products can be sterilised.
Aqueous solutions are sterilised between 108 0C and 1260C
Heat is conducted through the walls of the sealed containers until the temperature of
the fluid inside is the same as that of the steam outside.
several types of steam sterilisers are in use;-
laboratory autoclaves
hospital dressing sterilisers
bowl and instrument sterilisers
rapid cooling sterilisers
even the domestic pressure cooker can be used as a steriliser.

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In its simplest form ,the laboratory autoclave consist of a vertical or horizontal cylinder of gunmetal
or stainless steel ,in a supporting sheeet iron case .The lid or door is fastened by screw clamps and
made airtight using a suitable washer.The autoclave has on its lid or upperside a discharge tap for air
and steam,a pressure gauge and a safety valve that can be set to blow off at any desired pressure.
Heating is by gas or electricity .
sufficient water is added to the cylinder ,the material to be sterilised is placed on the tray,and the
autoclave is heated.The lid is screwed tight with the discharge tap open.The safety valve is adjusted to
the required pressure.
The steam -air mixture is allowed to escape freely till all the air has been displaced.This can be
tested by leading the escaping steam into a pail of water through rubber tubing,when no more air
bubbles come out in the pail the discharge tap is closed.
The steam presuure rises inside and when it reaches the desired set level ,the safety valve opens
and excess steam escapes.from this point the holding period is calculated.when the holding period is
over ,the heater is turned off and the autoclave allowed to cool till the pressure gauge indicates that
pressure inside is equal to the atmospheric pressure.

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The discharge tap is opened slowly and air is allowed to enter into autoclave.If the tap is
opened when the pressure inside is high ,liquid media will tend to boil violently and spill from
their containers and some times explosion may occur.
If not opened till the pressure inside has fallen below atmospheric pressure ,an excessive
amount of water will be evaporated and lost from the media.
The defects in this type of autoclave are ; -
The method of air discharge is inneficient and it is difficult to decide when the discharge is
complete.If the air is not completely removed ,the desired temperature would not be attained.
There is no facility for drying the load after sterilisation and before taking it out
The time taken for cooling after holding time is long

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Sterilisation control
for determining the efficacy of moist heat sterilisation ,
spores of Bacillus stearothermophilus are used as the test
organism.Its spores are killed in 12 minutes at 1210 C.paper
srips impregnated with 106 spores are dried at room
temperature and placed in paper envelops.These envelops
are inserted in different parts of a load and after sterilisation
the strips are tested for viability of the spoers
Browne's tube,autoclave tapes and thermocouples can also
be used.
sterilisation is no substitute for cleaning,dirt must be
removed before sterilisation.

35. METHOD TEMPERATURE IN 0C
HOLDING TIME IN
MINUTES
AUTOCLAVE
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121
126
134
15
10
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HOT AIR OVEN
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160
170
180
190
45
18
7.5
1.5

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FILTRATION
Filtration helps to remove bacteria from heat labile liquids such as sera and solutions of
sugars or antibiotics used for preparation of culture media.
But this does not sterilise the fluids as viruses are able to pass through ordinary filters.By
using membrane filters of appropriate pore diameter we can keep back viruses.But this is not a
method of sterilisation for routine use.
types of filters used in microbiology;-
earthenware candles - Berkefeld,chamberland,Mandler
asbetose filters eg;seitz
sintered glass filters
collodion or membrane filters

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RADIATION
Two types of radiation are used for sterilising purpose;-
Ionising and nonionising
infrared radiation ;- nonionising type,a form of hot air sterilisation ,organisms are
killed by oxidation as a result of heat generated.It is a convenient method for rapid
mass sterilisation of syringes and other materials,and this method is in commercial
use.
Ultraviolet radiation ;- non ionising type,used for disinfecting enclosed areas like
operation rooms,hospital ward,virus laboratories etc...it is not a reliable method.Direct
exposure causes conjuctival damage.
gamma radiation and high energy electrones;- ionising type ,used in commercial
plants and not used as a steirlisation method in hospitals and laboratories.
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There are a very large number of chemical agents used as antiseptics and
disinfectants
There is no ‘ ideal disinfectant ‘ or “ miracle fluid”
Chemical disinfectant should be considered only when disinfection by heat
is impossible, and thorough cleaning is not sufficient.
An ideal antiseptic or disinfectant should have the following characteristics;
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A wide spectrum of activity and be effective against all
microorganisms ; bacteria including spores,fungi,viruses,
etc..
Be active in the presence of organic matter.
Be effective in acid as well as alkaline media.
Have a speedy action.

44. 5. Have high penetrating power.
6. Be stable.
7. Be compatible with other antiseptics and disinfectants
8. Not corrode metals
9. Not cause local irritation or sensitisation.
10.Not interfere with healing.
11.Not be toxic if absorbed in to circulation.
12.Be cheap and easily available.
13.Be safe and easy to use.

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The factors which determine the potency of
disinfectants are;-
CONCENTRATION
TIME OF ACTION
pH OF THE MEDIUM.
TEMPERATURE
NATURE OF THE ORGANISMS
PRESENCE OF EXTRANEOUS MATERIAL

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ALCOHOLS
Ethyl alcohol (Ethanol) isopropyl alcohol are most frequently used. They are mainly
used as skin antiseptics and act by denaturing bacterial proteins.
They have no action on spores .
They must be used in a concentration of 70% alcohol in water.
Protein slows their action ,1% mineral acid or alkali enhances their activity.
Isopropyl alcohol is preferred because it is more bactericidal and less volatile.it is
used for disinfection of clinical thermometer and skin.
Methyl alcohol (methanol) is effective against fungal spores ,and is used for treating
cabinets and incubators affected by them.
The insides of the chambers are liberally wiped with methanol , a pad moistened with
it along with a dish of water (to ensure humidity) is placed inside and the apparatus
left in a working condition for several hours. Methyl alcohol vapour is toxic and
inflammable

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ALDEHYDES
Formaldehyde ;- In aqueous solution it is bactericidal ,sporicidal and
has a lethal effect on viruses.it is used to preserve pathological
specimens.
A solution of 10% formaldehyde containing 0.5% sodium tetraborate is
used to sterilise clean metal instruments .formaldehyde gas is used to
sterilise instruments and heat sensitive catheters.
It is used for fumigating wards, sick rooms and laboratories
Under properly controlled conditions ,clothing ,beddings ,furniture and
books can be disinfected.
Disadvantages;- pungent smell and it is irritating to skin, eye and
mucous membrane. And it is toxic when inhaled.

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ALDEHYDES……
GLUTARALDEHYDE
Similar action as that of formaldehyde.
Specially effective against tubercle bacilli,fungi,viruses.
It is less toxic and less of an irritant to eyes ,skin than
formaldehyde.
It has no deleterious effect on cement or the lenses of
instruments such as bronchoscope, cystoscopes
It can be used to treat rubber anaesthetic tubes ,face
masks, plastic endotracheal tubes, metal instruments, and
polythene tubing.

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ETHYLENE OXIDE
It is a colourless liquid with boiling point of 10.70C
Very penetrating gas with a sweet ether like smell.
highly infammable
It is effective against all all types of miocroorganisms, including
viruses and spores.
It is used for sterilising heart-lung machines ,respirators ,sutures,
dental equipments,books,clothings etc...
It is an irritant and personnel working with it have to take strict
precautions.
It is unsuitable for fumigating rooms because of its explosive
property.

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DYES
The aniline and acridine dyes are extensively used as skin and wound antiseptics.
Both are bacterio static in high dilution,but low bactericidal activity.
ANILINE DYES ;- The aniline dyes in use are brilliant green,malachite green,crystal
violet.
They are most effective against Gram positive .They have no activity aginst tubercle
bacilli.
Non irritant and non toxic to tissues.
They are inhibited by organic material such as pus.
ACRIDINE DYES ;- Acridine dyes in use are proflavine,acriflavin,euflavin, and
aminacrine.more active against gram positive .They are affected very little by the
presence of organic matter.If impregnated in gauze the dyes release slowly in a moist
environment and used in clinical medicine.

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HALOGENS
Iodine ;-
2.5% iodine in 70 % alcohol is an effective skin disinfectant.
It is active bactericidal agent with moderate activity against spores.
It is active against tubercle bacilli and many viruses.
It is irritant with stinging action on raw surfaces,it stains skin
Many people are sensitive to iodine and application on skin causes allergic
reactions(urticaria,conjunctivitis)
combination of iodine with nonionic wetting or surafce active agents known as
iodophores eg;povidon -iodine (Betadine)- non staining,iess toxic,less irritant

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chlorine;-
chlorine and its compounds such as chlorinated lime (bleaching powder)
,chloramines and hypochlorites have ben extensively used as
disinfectants.
water supplies,swimming baths ,food and dairy industries are some of
the areas of their use.
chlorine and hypochlorites are bactericidal.
Eusol is a chlorinated lime and boric acid compound used for dressing
wounds.
Dakin's solution (surgical chlorinated soda solution )used for wound
irrigation and dressing.
sodium hypochlorite commonly used to disinfect infant feeding bottles
and foe kitchen hygiene.
organic chloramines are used as antiseptics for dressing wounds.

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METALIC SALTS
The salts of silver ,copper,and mercury are used as disinfectant.
perchloride of mercury as 1/ 1000 solution is used for skin and body surfaces.
The organic compounds ,thiomersal,phenyl mercuric nitrate and mercuro chrome
are less toxic,used as mild antiseptics- and have a marked bacteriostatic,limited
fungicidal, and weak bactericidal activity.
copper salts are used as fungicides
silver nitrate 1 % used in burns treatment. - it stains clothing.
the instillation of a drop of 2 % solution of silver nitrate into each of the eyes of a new
born baby is used as prophylaxis against gonorrhoea (crede's method)

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PHENOLS
It is obtained by the distillation of coal tar.
their use in surgery first introduced by LISTER -father of antiseptic surgey
phenol;- (carbolic acid ) powerful microbicidal substance,and also very corrosive
lysol;- is a saponified solution of cresols.
the phenolics should be used with care and are toxic.
they are not readily inactivated by the pesence of organic matter.
chlorophenols,chloroxyphenols -less toxic and less irritant, inactive against
pseudomonas
hexachlorophene;- effective against gram positive, less against gram negative
particularly pseudomonas.it is toxic.
Chlorhexidine (hibitane);- non toxic skin antiseptic ,most active against gram
positive ,aqueous solution used in the treatment of wounds.
chloroxylenol ;- dettol-type ,inactivated by organic matter and hard water.

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SURFACE ACTIVE AGENTS
Substance which produce reduction in surface tension.
They are widely used as wetting agents,detergents, emulsifiers.
Types;- ANIONIC,CATIONIC,NON-IONIC,AMPHOTERIC
The most important antibacterial agents are cationic surface acting agents.
The cationic aompounds in the form of quaternary ammonium compounds
are bactericidal,active against grampositive.No action against spores,tubercle
bacilli and most viruses.
The common compounds are cetyl trimethyl ammonium bromide(cetavlon or
cetrimide) and benzalkonium chloride - these are most active in alkaline pH
and inactivated by acid,organic matter,amonic surafce agents like ordinary
soaps.

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ANIONIC COMPOUNDS ;- Eg -COMMON SOAP, have a moderate activity,they are
prepared from saturated fatti acids(coconut oils) are more effective against gram
negative bacilli.while those prepared from unsaturated fatty acid eg; oleic acid -
have greater activity against gram positive and Neisseria group of organisms.
AMPHOTERIC OR AMPHOLYTIC COMPOUNDS ;- known as 'TERGO COMPOUNDS'
active against wide range of gram positive and gram negative organisms and some
viruses, they are not generally used

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some important precautions while using disinfectants
Measure your disinfectant correctly.
Add the right amount of water to make the solution.
use clean vessels for making solutions
Remove dirt before applying the disinfectant
Throw away the disinfectant solution at the end of the day's work
Remember that improperly used disinfectants breed microbes and
spread infection
Don't use a disinfectant where sterilisation is required

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Dont store instruments or cleaning tools in a disinfectant
Dont top up a disinfectant
Dont use yesterday's disinfectant,use a freshly made solution
Don't mix two disinfectants together unless one is alcohol
Don't mix detergents and disinfectants.both may be inactivated
Always remember that a disinfectant does not make dirt safe.

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A POLICY FOR STERILISATION AND DISINFECTION
Hospital infection control committee
central sterile supplies Department (CSSD) ;- Every large hospital should have a central sterile
supplies department in which dressings,theatre clothings and instruments are serviced ,packed, and
sterilised and distributed or made available for all sections in the hospital.
functions of CSSD ;-
Supply of sterile materials for dressings and procedures,carried out in wards and
departments such as cisterna puncture sets ,biopsy sets,catheterisation packs,
anesthetic sets etc...
supply of sterile linen and instruments for operation theatres
Disinfection or sterilisation of equipment such as ventilators,oxygen tents and baby
incubators
selection and distribution of disposable sterile supplies ,if available.
provision of sterile fluids (in some places the pharmacist is responsible for this)

62. The success of a CSSD depends on collaboration,mutual trust and
cooperation between nursing staff and CSSD staff.