INTRODUCTION

Microorganisms are ubiquitous. They are found in the surroundings, on inanimate objects and on the
surface of the human body. Since they cause con-tamination, infection and decay, it becomes neces-sary
to remove or destroy them. This is the object of sterilisation. Methods to remove or kill microor-ganisms
are known as sterilisation. The methods of sterilisation used depend on the purpose for which it is
carried out, the material to be sterilised and the nature of the microorganisms to be removed a
destroyed. Sterilisation is defined as the process by which article, surface or medium is freed of all living
micr organisms either in the vegetative or spore state.

Disinfection is the destruction of all pathogenic organ. isms. Asepsis is the state of complete absence of
viable pathogenic microorganisms in any environment. Antiseptics are agents that can be safely applied
on thc 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.

STERILISING AGENTS

Methods used for sterilising an object are:

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, trichlorobutand

Aldehydes: formaldehyde, glutarakichyde

• Orthophillalaidehyde

• Pcracetic acid,

• Hydrogen peroxide
• Hypochlorous acid

• Dyes

• Halogens

• Phenols

• Surface-active agents

• Metallic salts

Gases: ethylene oxide, formaldehyde, beta propiolactone Some of the agents mentioned above are also
used as disinfectants.

PHYSICAL AGENTS

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 by 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 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 the denaturation and coagu-lation 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, steam condenses on them, increasing their water content resulting in the
ultimate hydrolysis and breakdown of the bacterial protein. In a completely moisture-free atmosphere
(as in dry heat), bacteria, like many proteins, are more resistant to heat. They are killed when oxidation
of the cell constituents occurs, and this requires much higher temperatures than that needed for
coagulation of proteins (by moist heat). The time required for sterilisation is inversely pro-portional to
the temperature of exposure and can be expressed as thermal death time. This is the minimum time
required to kill a suspension of organisms at a predetermined temperature in a specified environment.
The sterilisation time is related to the number of organ-isms in the suspension, presence or absence of
spores, and the strain and characteristics of the organism. It does not include the time taken to reach
the specified temperature. The nature of the 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 ther-mal death time. The presence of disinfectants and high acid or alkaline pi I
hastens bacterial killing.

Dry heat

• Flaming: An inoculating loop or wire, the tip of forceps and scaring spatulas can be sterilised by holding
them over a Bunsen flame till they become red hot. Inoculation loops carrying infective mate-rial may be
dipped in a disinfectant before flaming to prevent spattering.

• Incineration: This is an excellent method for ter-minal sterilisation for destroying biomedical waste.
Plastics such as PVC and polythene can be dealt with similarly, but polystyrene materials emit clouds of
dense toxic smoke which pollute the environment.

In order to check the proliferation of poorly designed bio-medical waste incinerators, guidelines on
'Design and Construction of Biomedical Waste Incinerator' I recommend various design features of an
incinerator as I well as the air pollution control device. +no.n.mxwm.

Hot-air oven: This is the most widely used method of sterilisation by dry heat. Sterilisation is achieved by
conduction. The heat is absorbed 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 lassware, forceps, it scissors, scalpels, all gases syringes, swabs and some
pharmaceutical products such as liquid paraf-fin, dusting powder, this and grease. The oven is usually
healed by electricity. A fan is fitted inside to ensure even distribution of air and elimination of air
pockets (I lg. 3.1). The chamber should not be overloaded. Free circulation of air in between the objects
should be ensured. Glassware should be per-fectly dr.) before being placed in the oven. Test tubes and
flasks should be wrapped in craft paper. Rubber materials, except silicon rubber, will not withstand the
temperature. At I 80°C, cotton plugs may get charred. Sharp instruments, such as those used in
ophthalmic surgery, should ideally be sterilised for two hours at 150°C. The British pharmacopoeia
recommends a holding time of one hour at I 50°C for oils, glycerol and dusting powder. The oven must
be allowed to cool slowly for about two hours before the door is opened, since the glassware may crack
due to sudden or uneven cooling. Table 3 I lists the recommended temperature and duration for heat
sterilisation.

Sterilisation control: * Physical: Temperature monitoring by thermocouples

• Chemical: A Browne's tube (green spot) is used. A green colour is produced after 60 minutes at 160°C
or 115 minutes at 150°C, suggesting complete sterilisation.

Biological: Heat-resistant spores of a non-toxi-genic strain of Clostridium tetani or Bacillus subtilis

subsp. niger are used to indicate efficiency of dry heat sterilisation.

Moist heat

Temperatures below 100°C

• Pasteurisation of milk: The milk is heated at either 63°C for 30 minutes (the holder method) or 72°C for
15-20 seconds (the flash process), followed by cooling quickly to 13c or lower. By the processall non-
sporing pathogens such as mycobacteria, brucellae and salmonellae are destroyed. Coxiella burnetii is
relatively heat resistant and may survive the holder method.

• Inspissation: Media such as Lowenstein—Jensen and Loefflefs serum are rendered sterile by heating
at 80--85°C for half an hour on three successive days in an inspissator

Bacterial vaccines are heat-inactivated in special 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 bath on
several successive days.

All non-sporing bacteria are killed at 60°C in 30 minutes.

* Streptococcus faecalis and Staphylococcus aureus are kilted at 60°C for 60 minutes.

Yeasts, moulds and vegetative bacteria are killed at 80°C in 5-10 minutes.

Spores of Clostridium botulinum require 120°C for minutes or 100°C for 330 minutes.

Most viruses are killed rapidly at 60°C except for polio and Hepatitis B viruses which may survive at this
temperature for 30 minutes and 10 hours respectively.

Temperature at 100°C

* Boiling:

Vegetative bacteria are killed almost immediately at 90-1 00°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 I 0-30 min-utes. The lid of the steriliser should not be opened during this
period. Boiling is not recommended for sterilisation of instruments used for surgical proceo dures. Hard
water should not be used for soiling. Addition of 2% sodium bicarbonate to the water mayrender it soft
and may it itable for sterilisation.

• Steam at atmospheric pressure (100°C) : An atmos-phere of free steam is used to sterilise culture
media which may decompose if subjected to higher tempera-tures. 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 1 00°C for 20 minutes on three suc-cessive 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 boils, its vapour
pressure equals that of the surrounding atmosphere. Hence, when pressure inside a closed vessel
increases, the tem-perature 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 atmos-pheric pressure condenses into I ml of water at 100°C and releases 518
calories of heat). The large reduction in volume sucks in more steam and the process contin-ues 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 auto-clave. Aqueous solutions are sterilised
between 108°C and I 26°C. Heat is conducted through the walls of the sealed container untill the
tempeof the fluid inside is the same as that of the steam outside.

Autoclaves (steam sterilisers) used in a healthcare setup:

a Laboratory autoclaves

• Hospital dressing sterilisers

• instrument sterilisers

a Rapid cooling steriliers

Two types of autoclaves arc av8ilable: gravity disei placcmcnt I) pe 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 clamps 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 auto-clave. Heating is by electricity .