Bacillus is a genus of gram-positive, rod-shaped bacteria. It was isolated from soil samples taken 5 cm below the surface using a serial dilution technique on nutrient agar plates. The isolate was characterized as a gram-positive, rod-shaped bacterium that forms chains. It was identified as B. subtilis based on morphological and biochemical tests. B. subtilis was then mass multiplied in nutrient broth for use as a biopesticide formulated on talc powder to control fungi on various crops.

1. BACILLUS –
systemic position
,collection ,isolation
BY-ANAMIKA DEWANGAN
AGRIGRAD STUDENT

3. Systamic Position
• Kingdom = Bacteria
• Phylum = Firmicutes
• Class = Bacilli
• Order = Bacillales
• Family = Bacillaceae
• Genus = Bacillus

4. Characteristics
• It’s a Gram +ve bacteria.
• Rod shaped.
• Cell size :- (4-10 µm) × (0.25-1.0 µm).
• It can form an Endospore,to survive extreme
environmental conditions of temperature.

5. • B. subtilis is a Facultative anaerobe.
• It is heavily flagellated.
• Optimal temperature range is
25 – 35ºc .
• They produce colonics which are
dry , flat and irregular with lobate
margins.

6. Collection of soil sample
• The soil samples were
taken from 5 cm depth.
• A zigzag format of
sampling was used.

7. Isolation by-serial dilution technique
• Isolated in Nutrient Agar (NA) medium by serial dilution
technique.
• The procedure adopted was as follows :-
10 g soil sample diluted in 90 ml of
sterile distilled water in 250 ml conical flask and kept it a orbital
shaker at 150 rpm to get a homogenized soil suspension.

8. • Serial dilution was made.
• Dilution of 10−7 was inoculated into NA plates
• Incubated at 37°C for 24 h.
• Isolated colonies growing on each diluted plates
were transferred into freshly prepared nutrient agar
slants.
• The bacterial strains isolated on NA slants were kept
at in refrigerator for further study.

10. Identification and characterization
of the isolate
• The isolate was
characterized by Gram
staining technique.

11. •Based on Gram's staining,the strain was found
to be Gram-positive.
•Microscopic observation-Rod shaped
bacterium arranged in chain.
•( NOTE - Gram positive bacteria: Stain dark
purple due to retaining the primary dye called
Crystal Violet in the cell wall. )

12. Mass multiplication of B. subtilis
• Preparation of mother culture
The nutrient broth medium is prepared as detailed
below
Glucose : 5.0 g
Peptone : 5.0 g
Beef extract : 3.0 g
Sodium chloride : 3.0 g
Distilled water : 1000 ml

13. • The above medium is take in conical flasks.
• Autoclaved at 15 lb pressure for 15 min.
• A loop of B.subtilis is inoculated into the medium and
incubated for 2 days. This serve as the mother culture.
• Mass multiplication : The nutrient broth is prepared in
fermentor and sterilized at 15 lb pressure for 15 min.
• Then the mother culture is added @ 1 lit / 100 lit of the
medium and incubated at room temperature for 2 days.
• The medium containing the bacterial growth of B.subtilis is
used for mixing with talc powder.

14. Formulation of B. subtilis
Product Formulation type Primary target
Serenade WP, aqueous suspension Fungi, bacteria on
multiple vegetables, fruits
Kodiak WP (Conc.), flowable Fungi on cotton, large-
seeded legumes, soybeans
Subtilex WP (Conc.) Fungi on cotton, large-
seeded legumes, soybeans
Hi Stick L + Subtilex Flowable Fungi on soybeans, peanut

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