Quantifying Sourdough Microbial Diversity

Five grams of sourdough were supplemented with sterile 0.85% NaCl solution up to a volume of 50 ml. The mixture was then homogenized by vortexing. Decimal dilutions were plated on to both sourdough bacteria (SDB) agar (maltose, 2.0%; yeast extract, 1%; Tween 80, 0.03%, trypticase 0.6%; pH 5.6) and de Man, Rogosa and Sharpe (MRS) agar (Lab M Ltd, UK) with 100 µg/ml cycloheximide (Sigma-Aldrich, USA).
The plates were incubated at the same temperature the sourdough was fermented at (20 or 30°C). Incubation was carried out for 48 h under anaerobic conditions (AnaeroGen, Oxoid). Colony forming units (CFU) were counted from the agar media using suitable dilutions.
For each of the six sourdough samples collected on day 56, 20 colonies were picked from the MRS and SDB agar plates (ten from each medium) for further analysis by rep-PCR. Colony picking was performed in succession from one sector of the plate. On day 56 samples were also plated on Yeast Extract Peptone Dextrose (YPD) agar (dextrose, 2.0%; peptone 2%; yeast extract, 1%) with 100 µg/ml chloramphenicol (Sigma-Aldrich, USA) and incubated at the same temperature the sample was fermented at (20 or 30°C). Ten colonies per sample were picked in succession from YPD agar plates and analyzed using RAPD-PCR.