Bacillus Subtillus culture

Lesley Robertson l.a.robertson at stm.tudelft.nl
Thu Nov 23 03:52:33 EST 1995

dwimmer at smartnet.net wrote:
>Derek & Nikki ALBERT wrote:
>> Could anyone tell me the best way to culture and then store this
>> bacteria in any numbers
>> Regards Derek
>1. Get your pure culture and inoculate a 100ml flask of an enriched 
>broth media. Incubate 24hr at 37dC on a shaker so you get a good, 
>turbid solution.
>2. To eight flasks with 250mL each of a "sporulation broth" which I 
>think has low carbohydrates, inoculate with 10mL of the culture.
>3. Shake n bake at R.T. for a couple of weeks. There will be a film 
>on the top. Gram stain should show 90%+ sporulation.
>4. Spin down the stuff to get a nice thick tan paste.
>5. Bring up to about 50mL with sterile saline.
>6. Heat shock at about 60dC for a half hour or so. Store in the 
>This will give a culture containing about 8 logs per 0.1mL and 
>should last at least a couple of years.

I've used much the same procedure, except that we spread the suspension 
after point 1 on peptone or tryptone agar plates - incubated for a week, 
and then washed the growth off and carried on as at point 4. One point to 
watch - don't use very pure agar. For some reason, top quality (Difco) 
agar didn't give sporulation, where the cheap stuff did.
Lesley Robertson

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