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28 December 2001

Techniques in Microbiology: Sterilization

Source: World Health Organization.

Editor's Note: What follows is the first in a series of basic informational articles on techniques of microbiology. -SG

Sterilization is defined as the destruction or removal (by filtration) of all microorganisms and their spores, whereas disinfection is the destruction of many microorganisms but not usually the bacterial spores. Sterilization is usually achieved with the help of heat whereas chemical agents are employed to effect disinfection.

Sterilization and disinfection are part of the daily routine of microbiological laboratories and constitute a vital activity which ensures that cultures, containers, media and equipment are treated in such a way that only the inoculated organisms will grow while all others will be eliminated.

Sterilization by heat

This can be achieved by autoclaving, by exposing articles to dry heat in hot air ovens or boiling.

Autoclaving

Autoclaves can sterilize anything that can withstand a temperature of 121°C for 30 minutes. A pressure cooker used in homes for cooking purposes can also be used as a makeshift autoclave.

The containers having clinical material are subjected to heat treatment in the autoclave after which these are emptied and washed and put back into service.

Only autoclaves designed for laboratory work and capable of dealing with a mixed load should be used. Porous load and bottle fluid sterilizers are rarely satisfactory for laboratory work. There are two varieties of laboratory autoclaves:

• Pressure cooker type.
• Gravity displacement models with automatic air and condensate

discharge.

Pressure-cooker type laboratory autoclaves

The most common type is a device for boiling water under pressure. It has a vertical metal chamber with a strong metal lid which can be fastened down and sealed with a rubber gasket. An air and steam discharge tap, pressure gauge and a safety valve are fitted in the lid. Water in the bottom of the autoclave is heated by external gas burners, an electric immersion heater or a steam coil.

Operating instructions

• Ensure that there is sufficient water inside the chamber.
• Load the autoclave and fasten the lid keeping the discharge tap open.
• Adjust the safety valve to the required temperature and turn the heat on.
• Allow the mixture of air and steam to pass out freely till all air has been discharged.
• Close the air discharge tap and let the steam pressure rise within the chamber till it attains a temperature of
• 121°C (1.5 kg/cm2).
• Hold on the pressure for 15 minutes.
• Turn off the heat and let the autoclave cool.
• Slowly open the air and steam discharge taps after the pressure gauge has reached zero.
• Allow the material to cool before these are handled (usually agar bottles take hours before these become safe to handle).

Autoclave with air discharge by gravity displacement

These are usually rectangular in shape and arranged horizontally. These autoclaves have a jacket around the chamber.

Operating instructions

• Bring the jacket of the autoclave to operating temperature.
• Load the chamber, close the door and open the steam valve so that steam can freely enter the top of the chamber. Air and condensate shall automatically flow out through the drain at the bottom.
• When the drain thermometer reaches the required temperature, allow further period for the load to reach that temperature (this has to be determined initially and periodically for each autoclave).
• Continue the autoclave cycle for the holding period.
• Close the steam valve and let the autoclave cool till a temperature of 80°C is reached.
• Gradually and softly open the autoclave enabling the steam to escape and allow the load to cool further.

Hot air oven

A hot air oven is electrically operated and should be equipped with a fan to ensure uniform temperature inside. The required temperature for sterilization is generally 160°C for one hour.

Operating instructions

• Arrange the material to be sterilized loosely and evenly on the racks of the oven allowing free circulation of air and thereby even heating
• Do not pack the load tightly since air is a poor conductor of heat.
• Switch on the power supply and control the temperature of the oven by adjusting the thermostat.
• Note the time when the desired temperature is reached (heating-up time).
• Hold the load in the oven at this temperature for a definite period of time (holding period). This is usually 60 minutes at 160°C.

Do not overheat since it would char the cotton plugs and paper wrappings.

Autoclaves and hot air ovens can be used for disinfection of infectious waste before it is discarded. In addition, waste can be disposed of by boiling in detergent or by burial.

Boiling in detergent

In the absence of an autoclave, most specimen containers can be boiled in water having detergents to decontaminate. This process kills the vegetative bacteria but fails to destroy the spores and certain viruses. The easiest way to get best results is to add washing powder or sodium carbonate crystals, 60 grams to one litre of water in a big container and boil specimen containers in it for a minimum of 30 minutes.

Disinfection

Disinfection can be undertaken either chemically or by boiling. Boiling is an effective method to disinfect equipment e.g. needles and syringes, if autoclaving facilities are not available. Equipment which has already been cleaned should be boiled for 20 minutes. Chemical disinfection is used for heat-sensitive equipment that is damaged at high temperatures. Commonly-used chemical disinfectants include chlorine releasing compounds; ethyl and isopropyl alcohol, quaternary ammonium compounds and gluteraldehyde.

The synopsis of a few commonly-used disinfectants is given in Table 1.

Preferred methods of sterilization for common articles are given in Table 2.

Decontamination of some of the commonly reusable equipment has been briefly presented in Table 3.

Table 1: Disinfectants and their mode of application*

Target	Disinfectant	Strength to use (disinfectant/ material V/V)	Application	Time of exposure
Skin	Ethanol Iodine Povidone iodine Quaternary ammonium comp	70% 1% 1%	Direct Direct Direct Direct	2 minutes 2 minutes 2 minutes 2 minutes
Blood	Cresol (pH 9) Ca hypochlorite	5% 1%	2:1 2:1	6 hours 6 hours
Urine	Cresol (pH 9)	5%	1:1	4 hours
Sputum	Cresol (pH 9)	5%	1:1	4 hours
Faeces	Cresol (pH 9) Hypochlorite (Na/Ca) Ca hydroxide	5% 1% 20%	2:1 3:1 2:1	6 hours 6 hours 6 hours
Work benches	Lysol Cresol Hypochlorite Chloramine-T	5% 1% 5%	Direct Direct Direct Direct	4 hours 4 hours 4 hours 4 hours
Glassware	Hypochlorite	1%	Direct	4 hours
Lab instruments	Hypochlorite Isopropanol	0.1% 70%	Direct Direct	4 hours 4 hours

* Based upon: Basics of quality assurance: WHO/EMRO, 1992, page 162

Table 2: Preferred methods of sterilization for common-use articles

Autoclaving Animal cages Sugar tubes Lab. coats Cotton Filters Instruments Culture media	Hot air oven Glass ware Beakers Flasks Petridish Pipette Slides Glass syringes Test tubes Powders
Rubber Gloves, stopper, tubing	Wood Tongue depressor, applicator
Glass Slides, syringes, test tubes Enamel metal trays Wire baskets

Table 3: Disinfection of specific equipment

Container/material	Method of choice for decontamination	Alternative method of decontamination
Reusable stool container	Autoclaving 121°C for 30 minutes	Fill the jar having stool with 5% solution of phenol and keep for 24hours
	Empty into lavatory*	Empty into lavatory*
Reusable containers of CSF, pus, sputum	Autoclaving	Boiling in detergent
Urine bottles (after emptying in lavatory*)	Autoclaving	Fill with 2% phenol or 1% bleach, leave for 4 hours, clean with detergent
Blood containers	Autoclaving	Soak overnight in strong disinfectant(5% cresol; 1% Ca hypochlorite, 1:2 V/V)
Glass microscope slides**	Autoclaving	Soak overnight in 5% phenol

* If the lavatory is connected to a septic tank, phenol or other antiseptics should not be put into the lavatory.

** Glass microscope slides which have been used for the diagnosis of tuberculosis should be discarded after keeping them soaked in detergent overnight.

Biohazard waste management

Waste is defined as any solid, liquid or gaseous material that is no longer used and will either be recycled, disposed of or stored in anticipation of treatment and/or disposal.

Storage

Prior to disposal, all biohazardous waste should be maintained and stored separately from the general waste stream and from other hazardous wastes. The containers used to store biohazardous waste should be leak-proof, clearly labelled with a red or orange universal biohazard symbol and sealed tightly when transported. In certain cases, it may be necessary to double-bag the waste to prevent leakage. Any biohazardous sharps, such as infectious needles and scalpels, must be placed in containers that are puncture-resistant, leak-proof on all sides and the bottom, and close-able. These containers can then be placed in a standard biohazard bag.

Disposal options

There are three main disposal options:

• render the waste noninfectious by autoclaving and dispose it in the
• general waste stream. If autoclaving is not possible, decontaminate with
• chemical disinfectants or by boiling for 20 minutes before disposal.
• on-site incineration, if possible.
• transportation of locally-generated waste to a distant appropriate
• facility.

Incineration is the preferred disposal option. Not only does this method render the waste noninfectious but it also changes the form and shape of the waste. Sterilization is an effective method for decontaminating waste, but it does not alter the appearance of the waste. Steam sterilization in an autoclave at a temperature of 121°C for at least 15 minutes destroys all forms of microbial life, including high numbers of bacterial spores. This type of complete sterilization can also be accomplished using dry heat which requires a temperature of 160-170°C for 2-4 hours. However, it must be ensured that heat comes in contact with the material to be rendered sterile. Therefore, bottles containing liquid material should have loosened caps or cotton plug caps to allow for steam and heat exchange within the bottle. Biohazard bags containing waste should be tied loosely. Once sterilized, biohazardous waste should be sealed in appropriate containers, labelled as disinfected waste and disposed of in an approved facility.

Biological waste should be clearly labelled prior to disposal and complete records should be maintained.

Burial

It is not a decontaminating process per se. However, it does prevent the infectious material from becoming a reservoir of infection if properly buried. It requires digging a pit of almost 5 meters depth and 2 meters width and having a tightly fitted heavy lid on top. Disposable containers with clinical material are thrown daily into it and the lid is replaced immediately after throwing the specimens. Once a week, the refuse is covered with a layer of quicklime. If quicklime is not available, the refuse is covered with almost 10 cm thick layer of dried leaves once a week.

Further reading

1. El-Nageh MM et al. Basics of Quality Assurance for Intermediate and Peripheral Laboratories. WHO Regional Publication, Eastern Mediterranean Series No 2, 156-166, 1992.