Lab Preparations

Day 1 in the Lab
Made up two additions of MYPG agar in 500 ml volumes. One was with the addition of CaCO3 and the other without CaCO3. MYPG w/CaCO3 is similar to Dekkera media agar recommended for use in culturing Brettanomyces spp. by the European Brewery Convention (EBC). The calcium carbonate is supposed to help with long term storage and neutralizing the effects of acids produced by the Brettanomyces yeasts. Another ingredient which could be included is Thiamine, a water-soluble vitamin of the B-complex. The phosphate derivatives are involved in many cellular processes and the best characterized form is thiamine diphosphate a coenzyme used in the catabolism of sugars and amino acids. In yeast, thiamine diphosphate is required in the first step of alcoholic fermentation. It has been reported that Brettanomyces spp. lacks the ability to produce Thiamine and therefore exogenous additions are required for adequate growth.

Agar media ingredients list:
MYPG agar – 500 ml
-Malt extract 1.5 g
-Yeast extract 1.5 g
-Peptone 1 g
-Glucose 5 g
-Agar 7.5 g
adjusted to pH=5.0

MYPG w/CaCO3 agar – 500 ml
-Malt extract 1.5 g
-Yeast extract 1.5 g
-Peptone 1 g
-Glucose 5.0 g
-CaCO3 (2%) 10 g
-Agar 7.5 g
adjusted to pH=5.0

Autoclaved at 121°C for 15 minutes at 15 psi. After letting the media cool in a water bath I prepared 100 mm petri dishes in a sanitary room. From these two solutions I poured 21 petri dishes of MYPG agar and 23 petri dishes of MYPG w/CaCO3 agar. I will need to work on my pouring skills… The solution with CaCO3 dropped a precipitate very quickly upon cooling and required shaking to keep in solution. Once the plates had solidified they were turned up side down and angled on the lid to allow the medium to dry for 24 hours before inoculation with the dregs of Avery 15, a 100% Brettanomyces fermented beer.

UPDATE 2/9/09
I found the plates containing CaCO3 to be an unnecessary culture medium. There were fewer colony forming units (CFU’s), combined with slower overall growth on every MYPG w/CaCO3 plates compared with the standard MYPG plates. This could be a coincidence but I no longer feel it advantageous to use this medium. For further plating of cultures I will only be using MYPG agar plates.

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~ by Chad on February 3, 2009.

 
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