How to test the quality of Bacillus subtilis Bio control agent formulation

  I. MORPHOLOGICAL CHARACTERISTICS: Colony Morphology on growth medium 

: Irregular, rough, opaque and off-white.

 Microscopic view: Staining-Gram positive rod shaped.

 II. PHYSICAL AND CHEMICAL PROPERTIES a. Physical state (form and appearance): Fifty ml of the sample was taken into three 100 ml covered glass beakers and observed the physical state of the samples.

 b. Colour :About 50ml of the sample is taken in a clear glass beaker and tested for its colour by viewing against a white opaque background in direct sunlight.

 c. pH: Ten mL of the sample is taken into 100ml of glass beaker and the pH is measured by using pH meter.

 III. ESTIMATION OF COLONY FORMING UNITS (CFU's) : 

 1. Chemicals: All the chemicals used in the studies are of Analytical Grade (A.R) quality. Glass distilled water is used throughout the studies. 

 2. Glassware: Only Borosil glassware are used for all the studies. 

 3. Cleaning and sterilization: The glassware used in the present studies were cleaned with cleaning solution and sterilized in hot air oven at 180°C for 3 hours. All nutrients media were autoclaved at 1.05 Kg/cm2 pressure for 20 min. 

 4. Composition of cleaning solution for glassware: Potassium dichromate - 60 g Distilled water - 100 ml Cool to room temperature and add - 60 ml Concentrated sulphuric acid with constant stirring 

 5. Medium composition Ingredients Gms / Litre Peptone - 5.00g Sodium chloride - 5.00g Yeast extract -1.50g Agar -15.00g Final pH (at 25°C) 7.4±0.2

6.Method of inoculation - The Spread Plate Technique was adopted for the quality analysis of Bacillus subtilis. Ten mL of the sample has to  transferred into a 250mL conical flask containing 90 ml of sterile 0.85% sodium chloride solution (10-1 dilution) and thoroughly mixed using cyclo mixer. One ml of the suspension from 10-1 dilution further diluted into a test tube containing 9 ml of sterile sterile 0.85% sodium chloride solution (10-2 dilution) and serially diluted up to 10-10 dilutions. Pipetted out 0.1ml of the solution from each diluent and transferred into the labeled petri dish containing agar medium that has been poured and solidified in the sterile petri dish and gently place the sterile L –shaped glass rod over the agar surface and spin the petriplate on its own axis 10–15 times. Three replicates were maintained for each dilution factor. The analytical procedure is carried out aseptically by using laminar air flow chamber. 

 7. Incubation After inoculation, all the petri dishes are incubated in a B.O.D. incubator illumination at 28±2°C for 48 hours.

 8. Test Condition Type of culture medium : Solid Test duration : 48 hours Culturing instrument : B.O.D. incubator Temperature : 28±2°C Handling : The test culture is handled with necessary protective measures. 

 9. Counting of colonies After incubation all the colonies are observed and counted using a colony counter. 

                        No. of colonies (average 3 replication) x Dilution factor of the product

                                                         Volume of sample added