Bacillus subtilis is a Gram-positive bacteria found in soil.B. subtilis is used as a soil inoculate in horticulture and agriculture.Simpler methods for isolation of a pure culture include: (i) spread plating on solid agar medium with a glass spreader and (ii) streak plating with a loop to isolate individual bacterial cells (colony-forming units) on a nutrient medium

Bacillus is a spore-forming species. You can obtain a soil sample from an agricultural field and let it dry (to induce sporulation), then do a suspension and heat treatment (80°C for 15 min) to eliminate any remaining vegetative cells of other species.

The resulting sample theoretically contains bacterial spores from different soil bacteria. Plating and further biochemical tests are needed to verify that a particular isolate is what you're looking for. Keep in mind, differentiating B. subtilis from other Bacillus species requires cell size measurements (micrometry) after growth on recommended media.

Better to do a quick literature search for the most convenient protocol. If you want a detailed discussion, kindly refer to any of the following texts:

1. Bergey's manual for systematic bacteriology (Wiley).

2. The Prokaryotes (Springer).

Hi Sheraz Ali,

There is no such defined protocol for the isolation of Bacillus subtilis from soil samples. But in this case, as Jameel M. Abduljalil mentioned, you may heat the soil specimens at 60-80 degrees in an oven for 2-3 hours followed by preparing dilutions in sterile saline (0.85% NaCl) and plating them on Nutrient agar plates. This high-temperature treatment would possibly kill other non-sporulating bacterial populations, while Bacillus species and other spore formers will remain. It will be easy for you to isolate this species from soils with rotting vegetables/fruits or manure-rich soil. Nevertheless, you will have to confirm your isolate by isolating its DNA, amplifying its 16S rDNA by PCR, and subjecting this 1500bp amplicon to DNA sequencing. This will confirm the identity of your strain. Good luck!

Try this https://microbialcellfactories.biomedcentral.com/track/pdf/10.1186/1475-2859-12-3.pdf