agar plates

Agar plates: I've been asked to make up agar plates with different pH values from 3–11—any information I can find on the net suggests the agar won't set at a low pH. Any ideas?

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Publication Date: 14 July 2015
Asked By: amanda.murchie
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agar plates

The ability of agar to set is affected by the concentration of the agar, the sugar content and the pH. Varying the pH of the agar too far (more than 1 unit) away from the neutral (pH 6–8) could result in the agar not setting. In an acid pH range, this is due to the hydrolysis, or chemical breakdown, of the agarose polysaccharides, the units that bind together to form the agar gel. In addition, the effect of pH on the gelling process is more evident after heating. For a detailed explanation of the chemistry see: http://www.agargel.com.br/agar-tec-en.html.

Through further correspondence we understand that the proposed activity is to investigate the effect of pH on probiotics. We have the following suggestions.

  1. Narrow the pH range in the investigation from 6.0–8.0 rather than 3–11.
  2. Trial increasing the agar concentration.
  3. Trial lowering the pH by adding acid under sterile conditions following the heat sterilisation of the agar and before the gel reaches its setting temperature. This may require filter sterilising of the added solution, which may not be possible in a school setting.
  4. Add the probiotic to the buffer solution for a period of time and then add a sample of this to a standard agar plate.
  5. Using aseptic techniques, inoculate agar plates with a lawn culture of a probiotic such as yoghurt culture and place discs of filter paper soaked in various ranges of pH buffer solutions onto this lawn culture. Incubate and look at the growth patterns demonstrating any signs of inhibition.

Note: After inoculation and incubation, the agar plates should not be opened or subcultured but should be sterilised in a pressure cooker or autoclave prior to being disposed of in the regular waste. See AIS: Sterilising agar and SOP: Operating a pressure cooker and autoclave and related questions such as Agar plate experiments and Inoculating agar plates and sealing them.

It is important that microbiology activities are supervised by staff who are aware of the safety procedures required in dealing with biohazards.

References:

‘Agar-Agar: Properties and Specifications’ Agargel website.  http://www.agargel.com.br/agar-tec-en.html (Accessed July 2015)

Starr, M.P., H. Stolp, H.G. Trüper, A. Balows and H.G. Schlegel (Eds.). 1981  The prokaryotes: A handbook on habitats, isolation and identification of bacteria Pg 143, Springer-Verlag: Berlin. https://books.google.com.au/books?id=RpPuCAAAQBAJ&pg=PA143&lpg=PA143&dq=...

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