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STANDARD OPERATING

PROCEDURE

FOR ANALYSIS OF

Bacillus subtilis

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SPECIFICATION & METHOD OF ANALYSIS OF Bacillus subtilis

S.NO. CHARACTERISTICS SPECIFICATIONS

1. Form & Appearance Physical state and colour of samples

2. Moisture content 12.0 % maximum

3. pH 6.0-8.0

4. Viable spore count per g. or per ml 1 x108

5. Pathogenic contaminants Absent

6. Other microbial contaminants <1x104 /gm

ESTIMATION OF COLONY FORMING UNITS (CFUs) COUNT

Take 1 g. of product and mix it in 9 ml of sterilized distilled water in a

clean and sterilized test tube to make 10-1 dilution (1:10). Shake well and take 1

ml. of the suspension to 9 ml. of sterile water in a tube to make 10-2 dilution

(1:100). Make six more serial dilutions in the same way to get 10-8 dilution.

Transfer 1 ml. of this suspension to sterile Petri Plates and add 15 ml. of sterilized,

melted and cooled Bacillus subtilis selective media. Rotate the plates gently and

allow it to solidify. Incubate the Perti plates in BOD incubator under the

fluorescent illumination at 35 oC ± 2 oC and R.H. at 65% ± 5 % for two days.

Observe the development of typical Bacillus subtilis colony and calculate the

number of colony unit per gram of the sample as the following formula.

CFU/g.= Average number of colonies/dilution factor

(A) PRECAUTIONS:-

The following precaution may be taken during the analysis of Bacillus subtilis

samples:-

1. Chemicals & Glasswares:- Analytical Grade (AR) quality chemicals and

Schott Duran or Borosil glassware should be used.

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2. Cleaning and Sterilization:- The glasswares used for testing should be

cleaned with detergent like Lavoline and Sterilized in Hot air Oven at 180 oC

for 3 hours.

3. Sterilization: - Media, distilled water and micro tips should be autoclaved at

1.05 Kg/cm2 (15 psi) pressure for 20 minutes.

4. Inoculation: - Inoculation of test material into sterile media should be carried

out under aseptic conditions using Laminar Air Flow Cabinet.

5. Incubation: - After inoculation all the Perti Plates should be incubated in a

BOD Incubator under florescent illumination at 35 oC ± 2 oC and at 65% ± 5 %

R.H. for 02 days.

Selective media for Bacillus subtilis (Nutrient Agar media)

S. No. Components Quantity

1. Peptone 10 g.

2. Sodium Chloride 05 g.

3. Yeast Extract 03 mg

4. Agar powder 20 ml

5. Distilled Water 1 litre

6. pH 7.0 ± 1

Calculation:-

Number of colony forming unit /plates

Dilution

factor

No. of colony forming units/ plate Average

Y R1 R2 R3 R4 X

CFU/g = average no. of colonies (X)/dilution factor (Y)

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ESTIMATION OF pH

pH (acidity or alkalinity test) of bio-pesticides is conducted as per the IS: 6940-

1982. Take 10g of sample in 50 ml of glass beaker & mixed with 25 ml of distilled

water. pH of the suspension is observed by using the meter.

R1 R2 R3 R4 Average

X

pH of sample = X

(A) Moisture Content:-

Moisture content to be determined as per the method describes in IS; 6940-1982.

Results Moisture Content % by mass = 100V M

OR

Moisture content % by mass:-

Weigh 5 g sample of product and pour into beaker of 50 g. capacity. Beaker

containing weighed sample is kept in hot air oven at 65 OC for 30 minutes. After

30 minutes beaker is taken out from oven and again weigh it for determination of

mass content.

Moisture content per cent = (M-m) x 100 M

Where, M= Initial weight of product M= mass of product found after hot air

Moisture content % bymass : X w/w Mean

R1 R2 R3 X

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PATHOGENIC CONTAMINANTS

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OTHER MICROBIAL CONTAMINANTS

ANTAGONISTIC CAPABILITY

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