enumeration of anaerobes - (Jan/14/2019 )

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.

All,

I've solved the enumeration of glycerol stocks problem. Reduced 0.9% NaCl was the culprit; with no buffering capacity as the pH was extremely low due to using cysteine-HCl as the reducing agent. Switched over to reduced PBS and no longer had a problem.