The one conundrum I'm facing is how to maintain and propagate the culture. Anecdotal reports suggest propagation using a simple sugar solution such as apple juice, and in lab cultures, Rogosa medium or MRS medium is used. I'll be doing a small series of experimental plating to explore if there is any suitable medium that might be easily-used in a hobbyist set-up. The following are the media compositions that I'll be trying out:
1) Apple juice agar
Apple juice
0.5% Fermaid (yeast nutrient)
1% agar
2) TAU: Treatment-As-Usual (Dried malt extract agar)
1% DME
0.5% Fermaid
1% agar
3) Modified MRS agar
2.0% glucose
0.1% polysorbate 80
0.5% sodium acetate
1.0% agar
0.5% Fermaid
4) Lactose-MRS agar
2.0% lactose
0.1% polysorbate 80
0.5% sodium acetate
1.0% agar
0.5% Fermaid
My microscope is most likely not powerful enough to do a proper cell count, so I will be doing the enumeration via a dilution method on spread plates. The culture contains about 80 million cells/ml, which means a serial dilution of 1:100 repeated 3 times will yield a solution of 80 cells/ml. A 250uL sample of this final dilution will be spread on each of the plates and incubated at 30C. The total number of colony forming units (CFUs) will then be counted. Of course I don't have a real anaerobic chamber for incubation, but the plan is to flood an airtight box with CO2 and incubate the cultures inside. An alternative method would be to use a pour plate instead of using a spread plate technique, but I'm not confident of being able to control the agar temperature with any precision in a home setting, which will be essential in doing pour plates. Stab cultures will of course be the simplest, but this does not allow for enumeration of CFUs.
In the meantime, I'll be pitching most of the culture into a Berliner Weiss wort today, reserving some for the above study and also some in a ghetto-style bug culture farm - essentially a clean growler filled with apple juice, and 'fed' every 2 months to keep it going.
1 comments:
he's back
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