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
>fridge.
>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
