Biological Indicator Growth or False Positive?

Nov 15 / Chris Maughan
Liquid biological indicator ampoules are used as part of the validation of a sterilization cycle for fluids in sealed containers. The ampoules are placed in the load to be sterilised when the cycle is complete the are removed and placed in an incubator. 

Growth of the biological indicator is shown by a colour change in the ampoule from purple to yellow. No colour change in the indicator shows no growth.

So which BIs showed Growth in the image to the left?

How does a Self Contained Biological Indicator change colour?

Biological Indicators typically contain the test organism spores held in suspension, tryptic soya broth (TSB) and a pH Indicator. The distinctive purple colour of the liquid is from the pH indicator.

Any viable spores in the media will start to grow given suitable conditions. When placed in the incubator at 55-60c the spores will grown and start to metabolise the sugars in the media. the by product produced during the metabolization of the sugars in the media is acidic, changing the colour of the pH Indicator to Yellow.

Tryptic Soya Broth (TSB) Caramelization or darkening

When TSB is autoclaved, the colour of the media can darken. Normally manufacturers recommend a sterilization time of 15 minutes at 121c. 

This darkening effect is increased inline with the sterilization time, the media become darker the longer the cycle. We refer to this colour change as caramelization and exactly the same process happens in biological indicator ampoules.

BI Growth or Caramelization?

In answer to the question we posed above, there is only one biological indicator showing growth. This was the positive control we used in this study, all of the other indicators had a minimum of 15 minutes exposure.

You will notice that the longer the exposure phase the greater the effect of 'Caramelization'. This is easy to miss-interpret as Growth, which is why we recommend using 'Negative Controls' placed in they cycle in free space, to account for caramelization.
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