Fundamentals of Microbiology

Autoclave

Autoclave:  It is a double walled vessel made up of thick stainless steel or copper.  It is fitted with gauges which measure temperature and pressure, an exhaust valve (steam cock) for release of steam from the chamber, a safety valve to avoid accidents due to development of excess pressure, controls for adjusting temperature and pressure.  A rubber seal inside the lid maintains airtight condition.  Screw fasteners or fly nuts are used to secure the lid.  Autoclave is used to sterilize all those substances that cannot be sterilized by dry heat such as, nutrient media, water, reagents, metabolites, instruments etc.  It uses the principle of steam sterilization.  Steam sterilization is a process of killing microorganisms through the application of moist heat (saturated steam) under pressure.  Saturated steam is the water vapour at the temperature at which it is produces. As compared to hot air at the same temperature it is 2500 times more efficient.  Heat damages the cell’s essential structures including the cytoplasm and membrane rendering the cell nonviable.

          The rate at which bacterial cells are thermally inactivated depends on the temperature and the time of heat exposure to which they are exposed.  In practical terms, this means that it would take a longer amount of time at lower temperatures to sterilize a population than at a high temperature.  Additionally, the higher the concentration of organisms that need to be killed, the longer it will take to kill all of the cells in that population at the same temperature. At 15 psi steam pressure the temperature reaches 121 ºC. Around the working range of 15 psi, each psi raises the autoclave temperature by approximately 1ºC.

  Autoclave operating procedures

The operating procedures of an autoclave are as follows:

·        Fill the autoclave and heat it till steam starts building up

·        Place items to be sterilized in the chamber

·        Close and lock the autoclave door.

·        When temperature reaches 100ºC, close the operating valve.

·        Check the jacket pressure

·        Measure the sterilization time from the moment temperature reaches 121ºC rather than 15 lbs pressure.

·        Sterilize for 15-20 minutes

·        After 15-20 minutes, close the steam supply valve and wait till the pressure falls to zero

·        Unlock and open the autoclave door

·        Remove autoclaved items from the chamber

·        Clean up

Substances which should not be autoclaved

·        Heat susceptible products such as plastics cannot be sterilized this way as they will melt.

·        Paper, chemicals such as sugars, vitamins, enzymes and other products may be damaged by steam.

·        Inoculum should never be autoclaved as it will be killed and will be of no use.

Items should be autoclaved in autoclave bags and a rigid secondary container (typically polypropylene or stainless steel).

Precautions

The following points should be noted while using an autoclave:

·        Do not open the door unless the chamber pressure comes down.

·        Do not open the lid immediately as pressurized steam is hotter than boiling water and causes severe burns.

·        Sudden release of pressure causes the liquids to boil up, which will wet the plugs or blow them out.

·        Let the pressure fall gradually when liquids are being sterilized.

·        If air has not been completely displaced from the autoclave, even though the pressure gauge may indicate 15 lbs pressure the temperature will not reach 121ºC.

·        Heat of vaporization released upon the condensation of steam causes much more severe damage than does the same quantity of boiling water.

·        Wear insulated gloves when handling hot autoclaved items.

·        Clean and disinfect the autoclave after every use.

·        Large loads require increased sterilization time.  Therefore it is advised to sterilize in small convenient loads.

·        Divide 5-litre medium in five one - litre flasks rather than autoclaving it in one 5 -litre flask.

·        Cotton plugs should be tightly fitted.  Cotton fibers absorb moisture and after drying stick to each other, therefore the plug becomes loose later on.

·        Plastic caps, rubber screws and stoppers should be set slightly loose so as to allow air to escape, otherwise the flasks or tubes will burst due to pressure of steam generated within.

·        In case of burn immerse burn area in cold water and get medical aid.