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enumeration of anaerobes

enumeration glycerol stocks anaerobes

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#1 sarcidia

sarcidia

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Posted 14 January 2019 - 04:20 PM

Hello,

 

I am trying to use plate count to enumerate glycerol stocks of anaerobic bacteria without success.   Its unclear why and I'm hoping someone can help.

 

Cells were prepped as follows:

1.  Organisms grown to OD600 0.5-1 (approx. 1E8 CFU/ml, previously validated by plate count) in preferred culture medium.

2.  An aliquot is removed and 10-fold serial dilutions in reduced 0.9% NaCl are plated.

2.  Cells are pelleted and the broth removed.

3.  Cells are resuspended in reduced phosphate buffered 15% glycerol and the OD rechecked.

4.  10-fold serial dilutions are plated on culture medium/agar plates.

 

Results:

Plate count from culture: approx. 1E8

OD600 from glycerol: 0.5-1

Plate count from glycerol: No growth

The glycerol stocks were successfully used for bioreactor inoculation.

 

I'm surprised at this result, and it's happened with several different organisms. 

 

Any comments/thoughts are appreciated.

 

Thank you.

 

Steve A.

 

 







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