Dear Elballat, very nice and commercially important question. We have been working on same line with Azotobacter chroococcum since last two years. Following few things can be noted. IMP Note: We consider the grey color is sign of cyst formation, but we have never done microscopy, but it improves stability.

Hope that works fine....

I've cultured  Bacillus species including subtilis from sample. They grow well on dilute Tryptic soy broth/Agar, you can dilute the medium to 0.1-0.2%(1-2g TSB per litre) since they will tolerate low saline conditions, keep pH between 7-8 and add agar(1.5%) for solid medium.

Some Bacillus (including subtilis) are thermophilic and will grow happily at 50 degrees Celsius  that could make them easier to isolate from the general mesophilic bacteria.

A general procedure I would use to isolate bacillus from soil sample:

1. add 1g of soil sample to 9ml sterile water and shake mix for 2 minutes.

2. let the solution settle and serially dilute the supernatant.

3. inoculate dilute TSB agar with dilutions using spread plate technique, incubate inoculated plates 24-48 hours.

Many bacillus will form irregular (sometimes spreading) colonies that will start out partially translucent and become opaque-white in colour as they produce spores.