Detection of Total Aerobic Microbial Count as per EP

1.    APPARATUS
1.1.    Autoclave
1.2.    BOD - Incubator
1.3.    Bacteriological Incubator
1.4.    pH – meter
1.5.    Hot air Oven
1.6.    Laminar Air flow
1.7.    Analytical Balance
1.8.    Micropipette
1.9.    Petri dishes 
1.10.    Test tubes
1.11.    Media bottles    
2.    REAGENTS  AND  MEDIAS
2.1.    Soyabean Casein Digest Medium
2.1.1.    Prepare media as per the current version of Respective GTP.
2.1.2.    If the dehydrated medium is not available then prepare the medium as follows.
    Pancreatic Digest of Casein             17.0    g
    Papacy Digest of Soybean Meal       3.0    g
    Sodium Chloride                              5.0    g
    Dibasic Potassium Phosphate          2.5    g
    Dextrose (C6H12O6. H2O)               2.5     g
    Purified Water                                 1000     ml
    Final pH after Sterilization                7.3 ± 0.2
Dissolve the solids in the water, warming slightly to effect solution.  Cool to room temperature and add, if necessary, sufficient 0.1 N Sodium Hydroxide to give a final pH after Sterilization between 7.1 to 7.5. Filter, if necessary, to clarify, distribute into suitable containers and sterilize in an autoclave at   about 121–124 0C for about 30 min.
2.2.    Soyabean Casein digest agar 
2.2.1.    Prepare the media as per the current version of Respective GTP.
2.2.2.    If the dehydrated medium is not available then prepare the medium as follows.
    Pancreatic Digest of Casein                15.0 g
    Papacy Digest of Soybean Meal          5.0 g
    Sodium Chloride                                 5.0 g
    Agar                                                  15.0 g
    Water                                                1000 ml
Final pH after Sterilization                       7.3 ± 0.2
Mix, and boil to effect solution. Sterilize by heating in an autoclave at 121–124 0C for about 30 min.
2.3.    Sabouraud Dextrose Agar Medium
2.3.1.    Prepare media as per the current version of Respective GTP.
2.3.2.    If the dehydrated medium is not available then prepare the medium as follows.
                                  Dextrose                         40.0 g
Mixture of equal parts of peptic digest of 
Animal tissue and pancreatic digest of casein      10.0 g
                                 Agar                                15.0 g
                                 Water                              1000 ml
Mix, and boil to effect solution. Sterilize by heating in an autoclave at 121–124 0C for about 30 min. After sterilization pH shall be 5.6 ± 0.2.
2.4.    Peptone Water
    (Buffered Sodium Chloride - Peptone Solution pH 7.0)
    Potassium Dihydrogen Orthophosphate        3.60 g
    Disodium Hydrogen Orthophosphate            7.20 g
    Sodium Chloride                                        4.30 g
    Peptone(meat and Casein)                         1.0 g
    Water                                                       1000 ml
0.1 to 1.0 % w/v Polysorbate 20 or Polysorbate 80 may be added.  Sterilize by heating in an autoclave at 121–124OC for about 30 min.

2.5.    Sabouraud Chloramphenicol  Agar Medium
2.5.1.    Prepare media as per the current version of Respective GTP.
2.5.2.    If the dehydrated medium is not available then prepare the medium as follows.
    Dextrose                                                          40.0 g
    Mixture of equal parts of peptic digest of 
    Animal tissue and pancreatic digest of casein     10.0 g
    Agar                                                                 15.0 g
    Water                                                               1000 ml
Mix, and boil to effect solution. Sterilize by heating in an autoclave at 121–124 0C for about 30 min. After sterilization pH shall be 5.6 ± 0.2.

All the above liquid media should be incubated for 48 hours at 32.5 ? 2.5 0C before use. Any contaminated media should be discarded.    

3.    PROCEDURE     
3.1.    Preliminary Testing
The methods given herein are invalid unless it is demonstrated that the test specimens to which they are applied to not, of themselves, inhibit the multiplication, under the test conditions, of microorganisms that may be present.  Therefore, prior to doing the tests, inoculate diluted specimens of the substance being examined, with separate viable cultures of Escherichia coli, B. subtilis and Staphylococcus aureus.  Add 1 ml of not less than 10 -3 dilution of a 24-hour broth culture of the microorganism to the first dilution (in buffer solution pH 7.2, Fluid Soybean-Casein Digest medium, of the test material and following the test procedure.  If the organisms fail to grow in the relevant medium the procedure should be modified by 
a.    Increasing the volume of diligent, the quantity of test material remaining thesame,
b.    Incorporating a sufficient quantity of a suitable inactivating agent in the diluents or by,

c.    Combining the afore-mentioned modifications so as to permit growth in the media.

If inhibitory substances are present in the sample 0.5 % of soy lecithin and 4 % of the Polysorbate 20 may be added to the culture medium.  Alternatively, repeat the test as described in the previous paragraph, using fluid casein digest - soy lecithin - polysorbate 20 medium to demonstrate neutralization of preservatives or other anti-microbial agents in the   test material.

PRE - TREATMENT OF THE PREPARATION BEING EXAMINED

Use separate 10 ml or 10 g specimens for testing.

Water - Soluble Products:

Dissolve or dilute 10 g or 10 ml of the preparation being examined, unless otherwise prescribed, in Peptone Water or another suitable medium shown not to have anti-microbial activity under the conditions of the test and adjust the volume to 100 ml with the same medium.  If necessary, adjust the pH to about 7.0 ± 0.2.

3.1.1.    Plate Method
3.1.1.1.    Dilute further, if necessary, the fluid so that 1 ml will be expected to yield between 30 and 300 colonies.
3.1.1.2.    Pipette 1 ml of the final dilution (Pretreated Sample) onto each of two sterile petri dishes.
3.1.1.3.    Promptly add to each dish 15 - 20 ml of Soyabean Casein Digest Agar Medium when testing for bacteria and add 15 - 20 ml of Sabouraud dextrose agar medium with antibiotics for molds and yeasts that previously has been melted and cooled to approx. 450C.
3.1.1.4.    Cover the petri dishes, mix the sample with the agar by tilting or rotating the dishes, and allow the contents to solidify at room temperature.
3.1.1.5.    Invert the petri dishes, and incubate for 5 days at 300C to 350C for Bacteria and incubate for 5 - 7 days at 200 – 250C for Yeast and Moulds.
3.1.1.6.    Following incubation, examine the plates for growth, count the number of colonies, and express the average for the two plates in terms of the number of microorganisms per g or per ml of specimen.
3.1.1.7.    If no microbial colonies are recovered from the dishes representing the initial 1: 10 dilution of the specimen, express the results as “less than 10 micro-organisms per g or per ml of specimen.”
3.1.1.8.    Follow all the steps as above along with the Sample, without Sample as a Negative control and 1 ml of 24 hours old broth culture of not less than 10-3 dilution in place of Sample as a Positive Control.

Autoclave, general testing procedure, laminar air flow, bod - incubator