Sterilization by Ananthnarayana &

Apurba S Sastry.
1. Introduction
 Objective: To remove & destroy microorganisms present in the environment.
 Sterilisation is defined as the process by which an article, surface or medium is freed of all
living microorganisms either in, the vegetative or spore state.
 Results in reduction of ≥106 log colony forming units (CFU) of microorganisms and their
spores.
 The agents which achieve sterilization are called as sterilants.
 Disinfection is the process that destroys or removes most if not all pathogenic organisms but
may or may not destroy bacterial spores.


 Results in reduction of ≥103 log CFU of most microorganism but not spores.
 Achieved by a physical agent or a chemical agent and are normally used only on inanimate
objects, not on body surfaces
 Asepsis is the state of complete absence of viable pathogenic microorganisms in any
environment.
 Antiseptics are agents that can be safely applied the skin or mucous membrane to prevent
infection by inhibiting the growth of bacteria.
 Bactericidal agents (or germicides) are substances that can kill bacteria.
 Bacteriostatic agents prevent the multiplication of bacteria which may, however, remain
alive. A chemical which is bactericidal at a particular concentration may become
bacteriostatic at a higher dilution.
 Decontamination is the process of rendering an article or area free of contaminants,
including microbial chemical, radioactive and other hazardous materials from an area object
or body surface.

2. STERILISING AGENTS
1. Physical agents
 Dry heat: By flaming, incineration or using hot air
 Moist heat: By boiling, steam at atmospheric pressure, steam above atmospheric pressure
 Filtration: Using candles, asbestos pads, membranes
 Radiation
2. Chemical agents
 Alcohols: ethyl, isopropyl, trichlorobutanol
 Aldehydes: formaldehyde, glutaraldehyde
 Orthophthalaldehyde
 Peracetic acid,
 Hydrogen peroxide
 Hypochlorous acid
 Dyes
 Halogens
 Phenols
 Surface-active agents
 Metallic salts

Heat
 Applying heat to an object is the most reliable method of sterilisation.
 Materials that may be damaged by heat can be sterilised by exposing them to low heat for
longer periods or by repeated cycles.
The factors influencing sterilisation heat are:
 Nature of heat: Dry or moist
 Temperature and time
 Number of microorganisms present
 Characteristics of the organisms, such as species, strain, presence of spores.
 Type of material from which the organisms must be eradicated.
Mechanism of action:
 The killing effect of dry heat is due to protein denaturation, damage by oxidising molecules,
destroying cell constituents and the toxic effect of elevated levels of electrolytes.
 The lethal effect of moist heat is due to denaturation and coagulation of proteins.
 The advantage of steam lies in the latent heat liberated when it condenses on a cooler surface,
raising the temperature of that surface.
 In the case of spores, the steam condenses on them, increasing their water content resulting in
the ultimate hydrolysis and breakdown of the bacterial protein.
 The time required for sterilization is inversely proportional to the temperature of exposure and
can be expresses as thermal death time, which is the minimum time required to kill a
suspension of organisms at a predetermined temperature in a specific environment. It doesn’t
include time taken to reach specific temperature.
 The nature of material in which the organisms are present affects the rate of killing.
 The presence of organic substances, proteins, nucleic acids, starch, gelatin, sugar, fats and oils
increases the thermal death time.
 The presence of disinfectants and high acid or alkaline pH hastens bacterial killing.

Dry heat
 Flaming: An inoculating loop or wire, the tip of forceps and searing spatulas can be sterilised
by holding them over a Bunsen flame till they become red hot.
 Inoculation loop carrying infective material may be dipped in a disinfectant before flaming to
prevent spattering.
 Incineration: This is an excellent method for terminal sterilisation for destroying biomedical
waste.
  Plastics such as PVC and polythene can dealt with similarly, but polystyrene materials emit
clouds of dense toxic smoke which pollute the environment.
 Hot air oven: This is the most widely used method of sterilisation by dry heat.
 Sterilisation is achieved by conduction.
 The heat is observed by the surface of the item to be sterilised, which then penetrates to the
centre, until the entire item reaches the desired temperature.
 A holding period of 160°C for two hours is necessary to sterilise glassware, forceps, scissors,
scalpels, all glass syringes, swabs and some pharmaceuticals products such as liquid paraffin,
dusting powder, fats and grease.
 The oven is usually heated by electricity.
 A fan is fitted inside usually heated by electricity.
 A fan is fitted inside to ensure even distribution of air and elimination of air pockets.
 The chamber should not be overloaded.
 Free circulation od air in between the objects should be ensured.
 Glassware should be perfectly dry before being places in the oven.
 Test tubes and flasks should be wrapped in craft paper.
 Rubber materials, except silicon rubber, will not withstand the temperature.
 At 180°C, cotton plugs may not get charred.
 Sharp instruments, such as those used in ophthalmic surgery, should ideally be sterlised for 2
hours at 150°C.
 The British pharmacopoeia recommends a holding time of 1 hr at 150°C for oils, glycerol and
dusting powder.
 The oven must be cooled down slowly for about 2 hours before opening the door.

Sterilization control:
 Physical: temperature monitoring by thermocouples.
 Chemical: A Browne’s tube (Green spot) is used.
 A green colour is produces after 60 min at 160°C or 115 min at 150°C, suggesting complete
sterilisation.
 Biological: Heat resistant spores of a non-toxigenic stain of clostridium tetani or bacillus
subtilis subsp. Niger are used to indicate efficiency of dry hear sterilisation.

Moist heat
Temperatures below 100°C
 Pasteurisation of milk: The milk is heated at either 63°C for 30 minut,es (the holder method)
or 72°C for 15-20 seconds (the flash process), followed by cooling quickly to 13°C or lower.
 By the processes, all non sporing pathogens such as mycobacteria, brucellae and almonellae
are destroyed. Coxiella burnetii is relatively heat-resistant and may survive the holder
method.
 Inspissation: media such as Lowenstein hensen and loeddlers serum are rendered sterile by
heating at 80- 85°C for half an hour/on three successive days.
 
 Bacterial vaccines are heat-inactivat in vaccine baths at 60°C for one hour.
 Serum or body fluids containing coagulable proteins can be sterilised by heating for one hour
at 56°C in a water hath on several successive days.

Temperature at 100°C
 Boiling: Vegetative bacteria are killed immediately a 90-100°C but bacterial spores can
withstand long periods of boiling, In cases where boiling is considered adequate, the material
should be immersed in the water and boiled for 10-30 minutes.
 The lid of the steriliser should ,not be opened during this period is not recommended for
sterilisation of instruments used for surgical procedures.
 Hard water should not be used for boiling.
 Addition of 2% sodium bicarbonate to the water may render it soft and make it suitable for
sterilisation.
 Steam at atmospheric pressure (100°C): An atmosphere of free steam is used to sterilise
culture media which may decompose if subjected to higher temperatures. A Koch or Arnold
steamer is usually used.
 Tyndallisation or intermittent sterilisation: This method is used for media containing
sugars or gelatin.
 An exposure to 100°C for 20 min on three successive days is used.
 The principle is that the first exposure kills all vegetative bacteria, and the spores,
 since they are in a favourable medium, will germinate and be killed on subsequent exposure
to 100°C for 20 minutes.
 However, this method may fail to kill spores - of certain anaerobes and thermophiles.
 Steam under pressure: The equipment used is an autoclave. The principle of the autoclave
or steam steriliser is that when water hails, its vapour pressure equals that of the surrounding
atmosphere.
 Hence, when pressure inside a closed vessel increases, the temperature at which water boils
also increases, generating steam.
 When steam comes into contact with a cooler surface, it condenses and transmits its latent
heat to that surface (1600 ml steam at 100°C and at atmospheric pressure condenses into 1 ml
of water at 100°C and releases 518 calories of heat).
 The large reduction in volume sucks in more steam and the process continues till the
temperature of that surface equalises with that of steam. Condensed water ensures moist heat
for killing the microbes present on the material.
 Sterilisation by steam under pressure is carried out at temperatures between 108°C and
147°C.
 Materials such as linen, instruments, laboratory ware, media and pharmaceutical products can
be sterilised in an autoclave.
 Aqueous solutions are sterilised between 108°C and 126°C. 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.
 Autoclaves (steam sterilisers) used in a healthcare setup:
 Laboratory autoclaves
 Hospital dressing sterilisers
 Instrument sterilisers
 Rapid cooling sterilisers
 Two types of autoclaves are available: gravity displacement type and high vacuum sterilisers.
 The laboratory autoclave consists of a vertical or horizontal cylinder of gunmetal or stainless
steel, in a supporting sheet iron case.
 The lid is fastened by screw damps and made airtight.
 An exit tap for air and steam, and a pressure gauge are attached to the autoclave.
 A safety valve that can be set to blow off, if pressure rises beyond the desired level, is also
attached to the autoclave.
 Heating is by electricity.

 Mechanism: Sufficient water is added to the cylinder, and the material to be sterilised is
placed on the tray.
 The lid is screwed tight with the discharge tap open, and the autoclave is heated. 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 pressure rises inside and, then it reaches the desired set level, the safety valve
opens and the 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 is allowed to cool
till the pressure reaches atmospheric pressure.
 The discharge tap is opened slowly, and air is let into the autoclave.
 If the tap is opened when the internal pressure is high, liquid media boils violently, spills from
the container and may explode.
 If opened after the pressure inside has fallen below atmospheric pressure, an excessive
 amount of water will evaporate and be lost from the media.
 The defects in this type of autoclave are:
 The method of air discharge is inefficient, and it is difficult to decide when the discharge is
complete.
 Materials remain moist after removal from the autoclave.
 Vacuum sterilisers do not have this disadvantage.
 They usually have automatic cycle control. The air is drawn out, whereby steam
penetrates faster and the time required for sterilisation is shorter. Once the sterilisation is
over, post-cycle vacuum can be programmed for quick drying.
 The pressure cooker serves as a miniature autoclave and may be used for sterilising small
articles in clinics and small establishments.
 Sterilisation control: Sterilisation control is similar to that of dry heat sterilisation as
described above except for the use of Geobacillus stearothermophilus, which is used as the
test organism for checking sterilisation by steam under pressure.
 This is a thermophilic organism with an optimum growth temperature of 55- 60°C and its
spores require an exposure of 12 minutes at 121 °C to be killed.
 Biological indicators are the only process indicators that directly monitor the lethality of a
given sterilisation process.
 Rapid readout biological indicators, which provide results within one to three hours, are
available for shorter turnaround time for reading the results.
Filtration
 Filtration helps remove bacteria from heat labile liquids such as sera and solutions of sugars
or antibiotics.
 As viruses can pass through ordinary filters, filtration only renders the material bacteria-free.
 Bacterial toxins can be obtained by passing cultures through filters.
 Asbestos filters are no longer used due to their carcinogenic property