Caramelisation Or Darkening In The Colourof Autoclaved Liquid Bi Ampoules

Russ Nyberg, Director of Manufacturing and Production, Mesa Labs, explains glucose and other sugars that may be present in the media slightly darken and the overall appearance of the media will look more of a dark honey or light brown colour.The shear presence of the additional purple pH indicator used in these ampoules make the colour look even darker than media would without the pH indicator.

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WHEN AUTOCLAVING liquid agar or tryptic soy broth, many times the sterilised media takes on a darker than expected colour due to the sterilisation and resulting exposure to heat. This presents a concern when working with sealed glass ampoule type biological indicators (BI) that contain tryptic soy broth (TSB). With most ampoule type self-contained biological indicators, the bacterial spores are contained in the ampoule as a suspension of spores and TSB. Once the ampoule BI has been autoclaved in a typical cycle being monitored, the ampoule is then incubated to see if all the spores are dead or if some survived the cycle. This growth/no growth testing is evaluated by looking at the colour and clarity of the TSB within the ampoule. The ampoules also contain a pH indicator that gives the TSB a purple colour.

Upon incubation, if the spores were not killed in the cycle they will germinate to vegetative cells, metabolise the TSB and begin to grow. As part of the metabolism of the sugars present in the TSB, acidic waste products are released and the colour of the ampoule changes from purple towards a yellow colour as a result of a lowering of pH. When one is using the self-contained ampoule the question arises as to whether the normal darkening of colour that occurs when ampoules are exposed to extended or high temperature cycles will still promote growth and detect any badly damaged spores that may have survived the cycle. This is mainly being questioned since some individuals have stated that the darker colour ampoules are caramelised and will no longer function due to an inability to promote growth.