Biological assay (viable spore count) of lactic acid bacillus

1.0 OBJECTIVE
This SOP describes the procedure for biological assay (viable spore count) of Lactic Acid Bacillus (in house specification)
2.0 PURPOSE
It is the policy of Analytical Division that a written procedure shall be followed for biological assay

(viable spore count) of Lactic Acid Bacillus (in house specification).

3.0 SCOPE

This SOP shall be applicable for Microbiology Laboratory Analytical Division
4.0 RESPONSIBILITY
Persons along with their responsibilities are given below

Sr. No Designation Responsibility
01 Microbiologist Preparation & revision of SOP.
02 Officer- QA Distribution, Training and retrieval of SOP.
03 Quality Manager Approval & Implementation of SOP.

5.0 IDENTIFICATION

5.1 PROCEDURE: A
5.1.1 Select the colony with a platinum wire loop and transfer aseptically, on a microscopic slide and mix well with a drop of sterile distilled water
5.1.2 Air dries the film and fix with minimal heating.
5.1.3 Flood the slide with chromium trioxide (1 in 20) for 2 minutes.
5.1.4 Wash the slide with running water thoroughly and blot and dry.
5.1.5 Flood the slide with phenol fuchsin, heat with a flaming Alcohol swab until steam rises. Do not boil.
5.1.6 Wash the slide with running water thoroughly, blot and dry.
5.1.7 Pour sulphuric acid (3 in 100 ml) to decolorizing the fuchsin in the cell wall for few seconds.
5.1.8 Wash thoroughly with tap water.
5.1.9 Stain the slide by methylene blue solution for 30-60 seconds.
5.1.10 Wash with water and dry in air.

Test Interpretation
Examine the wet mount using a microscope.
Blue coloured rods and red coloured spores are seen under the microscope.

5.2 PROCEDURE: B
5.2.1 Select the colonies with a platinum wire loop and transfer aseptically to tube containing 20 ml of previously sterilized and

cooled glucose yeast extract liquid medium. Incubate the tube at 37° C for 48 hours and then vortex for 10 minutes.
5.2.2 Transfer the clear supernatant liquid to a seperatory funnel and exactly by adding 5 ml of diluted sulphuric acid (10%) and 5 ml of ether.
5.2.3 Collect the ether layer, evaporate in a water bath carefully to dryness and dissolve the residue in 5 ml of water.
5.2.4 Add the solution drop wise to uffelman’s reagent, prepared by adding two drops of 1 N ferric chloride to 10 ml of 10% phenol solution.

The colour of the solution from bluish violet to yellow, indicating the presence of lactic acid.
5.3 Phenol -Fuchsin Solution:
5.3.1 Dissolve 11.0 gm of fuchsine in 100 ml of dehydrated alcohol. Add 100 ml of phenol solution (1 in 100) stir well and filter through whatmann no.11 paper.
5.4 Methylene Blue Solution:
5.4.1 Dissolve 5.0 gm of Methylene blue in 100 ml of dehydrated alcohol, and filter if necessary.
6.0 ASSAY (Viable Spore Count): Not Less Than Label Claim
6.1 Procedure
A) Weigh 1.0 gm of sample, transfer in to 250 ml (for 9000 msg /10000 msg / 12000 msg /15000 msg) sterile standard volumetric

flask and add approximately 150 ml of sterile isotonic saline solution (0.9% w/v) and sonicate for 10 minutes.
B) Weigh 1.0 gm of sample, transfer in to 500 ml (for 6000 msg/ 25000 msg/ 30000 msg/ 50000 msg/ 100000 msg/ 150000 msg/ 200000 msg/ 250000 msg/ 300000 msg/ 320000 msg) sterile standard volumetric flask and add approximately 300 ml of sterile isotonic saline solution (0.9% w/v) and sonicate for 10 minutes.
After completion of sonication dilute up to the mark with same sterile isotonic saline solution. Shake well and use it for further serial dilution (Stock Solution).
Transfer 1 ml of stock solution into 9.0 ml of sterile isotonic saline solution in a sterile test tube and mix thoroughly.
Continue serial dilution to get colonies between 30-300 cfu in test solution.
7.0 Heat Shock
7.1 Allow the final dilution to stand in the water bath at 75°C for 30 minutes.
7.2 Cool the test tubes to about 45°C.
8.0 Plating
8.1 Set out five sterile petriplates and add 1 ml of heat treated final dilution into each petri plate and then pour 15 ml

(temperature around 45°C) of PNY molten medium or Lactobacillus MRS agar medium in to each of the petri plat and mix thoroughly.
8.2 The solidified plates are incubated in an inverted position at 37° C for 48 hours.
9.0 Counting
9.1 Count the no of colonies in each petri plate and calculate by taking the average number of colonies.
10.0 Calculation

11.0 Preparation of sterile isotonic sodium chloride solution:
Weigh accurately about 9.0 gm of sodium chloride in 1000 ml of distilled water. The solutions are sterilized with steam at 1.5 kg/cm pressure at 121°C for 20 minutes and then cool.
12.0 CROSS REFERENCES : SOP FOR SOP

13.0 ABBREVIATIONS & DEFINITION

QA Quality Assurance
SOP Standard Operating Procedure
SS Stainless Steel
IPA Isopropyl Alcohol
QM Quality Microbiology
ml Millilitre
gm Gram
msg Million Spore Per Gram

Biological assay (viable spore count) of lactic acid bacillus

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