Autoclaving and Sterile Preparation

Death in micro-organisms can be defined as the irreversible loss of reproductive ability. As will be seen later, the death of bacteria is exponential, and in practical terms sterility is defined as the probability that the material treated should not contain a survivor in an infinitely large sample. This is usually defined in practice as the probability of less than one bacteria per million units (1 in 10 ). 

Thermally stable material such as glassware or metal instruments may be sterilised by heating them in an oven at 185°C for two hours. The material is wrapped in autoclave paper prior to heating, and after removal remains sterile until the wrapping paper is removed. Steam treatment in an autoclave is normally used for the sterilisation of aqueous material. The autoclave uses steam at a pressure greater than atmospheric and laboratory systems normally operate at 15 lbs in which corresponds to a temperature of 121 °C. This makes the assumption that the atmosphere inside the autoclave is composed only of steam and therefore it is necessary to expel all the air before the sterilisation process commences. 

The time taken to sterilise materials depends on the load, but typically loads made up of relatively small volumes would be treated adequately within 20 minutes. Loads containing large volumes take longer and suitable times can be determined by the use of thermal probes or spore strips (see page 86). 

The actual operational sequence of autoclaves varies considerably from manufacturer to manufacturer and the appropriate instructions should be followed. 

Material which is thermally unstable cannot be sterilised by autoclaving, and solutions should be sterilised by membrane filtration through a membrane with a pore size of less than 0.45 pm. Membrane filtration is dealt with in more detail in the chapter on water testing. It should be noted that membrane filters will only remove bacteria and do not necessarily remove viruses. 

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