As in most bacteria the most common isolation method is by titration. It is not certain that you will isolate the specific species, although you have to try. If you know that your sample is contaminated with B. subtilis then titration is your answer. Should you need further information about titration do not hesitate to ask.

You could use MPN method. Serially dilute your sample and spread on to nutriant agar or LB agar. If you expect spores of B. subtilis will be there, just pasteurize your sample (80oC for 15-30 min) before spreading. Most of the times B. subtilis swarm on low % agar plates, so i would recommend using a 2% agar. You then might want to check your cultures under microscope to find rods and possibly gram staining, followed specific biochemical tests. One straight forward method could be to sequence the 16S gene form each of the isolated colonies. Ofcourse what you want to do depends on your budget!

Thanks for the information, but I wonder whether nutrient agar or LB is good? as a rule, the food and feed are high contaminated with various bacteria and just because I was thinking more about selective agar or more specific method.

Thanks for the information, but I mean about plate counting method. I want to determine the number of Bacillus subtilis in food and feed. To be honest, I've never heard about your method. If you giv me more intormation i will be grateful.

Titration is synonym to plate counting method. As far as i know there is no special, selective agar for Bacillus species. Viduthalai R. Regina' s method with the serial dilutions of your initial sample is the most efficient, cheap and easy method for your problem. If you are familiar with B. subtilis macroscopic morphology then titration will solve the problem. I work with B. thuringiensis and realised that best growth medium is nutrient agar.

Am also afraid that there may be no selective media for B. subtilis. NA should host BS verywell, ofcourse you need to deal with the other bacteria.

You can use Mossel agar plates. (mannitol + yolk+ polymyxin) this media is selective and differential to B. cereus (mann- lec+) but B.subtilis also developes in this agar as yellow colonies without halo (mann+ lec-)

You can treat the processed sample/ or Sample suspended in physiological saline to 100 degree Celsius for 10 minutes to kill all the vegetative forms of bacteria. Bacillus spp. being spore formers withstand this temperature and the you can isolate the culture on Nutrient agar first. You also have to perform Gram staining as it helps in identifying genus and also to some extent B. subtilis, it is Gram positive and slender rods in chains whereas B.Cereus are thick swollen rods.

Then you can isolate the culture or individual colony on Mosel agar.

For further identification you have to employ few biochemical tests such as: Glucose nitrate agar: Usually abundant, cream-colored growth.

Citrates utilized.

Nitrites produced from nitrates. No gas produced from nitrate broth under anaerobic conditions.

You can also refer Bergey's manual of determinative bacteriology.

I hope this will help you.

You can use MYP medium and recognize B. subtillis according to color of colonies:

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/2/m1928dat.pdf

http://www.neogen.com/Acumedia/pdf/ProdInfo/7741_PI.pdf

Use HiChrome Agar media (HiMedia) which contains a chromogenic substrate and specific color colonies appear for different genera...... identify them using the givene data sheet...... then use HiBacillus Identification Kit (Himedia) to identify B.sutilis

Very informative discussion.  Thank you!

you can use Bacillus Differentiation Agar (differentiate between B. cereus and B. subtilis) from HIMEDIA. I am attaching its technical specification sheet.

Kunal