I have grown my strain on nutrient broth agar(12.5 g/l) and incubated it for 5 days at 30C and I was able to obtain spores using spore staining method, however I am not sure how would you obtain pure spores i.e. do I need to incubate at 80c for 15 mins in order to kill vegetative cells??
also, how would you determine how much have sporulated i.e. 20, 50%, 60% and so on????
finally, how would you perform spore viability ??? is it by inoculating a specific amount , such as 10^5, into a fresh nutrient broth then perform a cell counting after 24H ???
I would be grateful for your enlightenment.
thank you so much for you time.
Dear Wasef,
in order to test the sporulation rate in Bacillus you streak out cell suspensions (at different dilutions) on agar plates before and after a heat treatment at 80°C for 30 min. After 12-24 hours you can count colonies and determine the spore fraction in %. There are nutrient sporulation media available which are pretty effective producing spores.
To trigger sporulation numerous stress factors are known: nutrient limitation (C, N or P), amino acid depriviation or slow growth rate in continuous culture (see https://www.ncbi.nlm.nih.gov/pmc/articles/PMC248122/).
Good luck
Michael
Dear , Wasef Yousef
You mean Bacillus sphaericus ?
You can see the following attachments
With my best regards
Dear , Wasef Yousef
Also you can find the following article , it may be useful .
Best greetings
Hi ,Wasef Yousef
Please return to the links and article below . I hope they help you.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2648361/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1169001/
Good luck
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