Total Aerobic Microbial Count

1.  APPARATUS

1. Autoclave
2. BOD - Incubator
3. Bacteriological Incubator
4. pH – meter
5. Hot air Oven
6. Laminar Air flow
7. Analytical Balance
8. Micropipette
9. Petri dishes
10. Test tubes
11. Media bottles   

2. REAGENTS  AND  MEDIAS

1. pH 7.2 Phosphate Buffer
2. Stock Solution  : Dissolve 34 g of Monobasic Potassium Phosphate in about 500 ml of water contained in a 1000 ml volumetric flask. Adjust to pH 7.2 ± 0.1 by the addition of 4 % w/v aqueous solution of Sodium Hydroxide (about 175 ml), add water to volume, and mix.  Dispense and sterilize an Autoclave at 121–124⁰C, 1.1psi pressure for about 30 min.  Store under refrigeration.
3. For use :  Dilute the Stock Solution with Sterile water in the ration of 1 to 800, and sterilize in an autoclave at 121-124⁰C,  for about  30 min.
4. Soyabean Casein Digest Medium

Prepare media as per the current version of Respective GTP

1. Fluid Casein Digest-Soy Lecithin-Polysorbate 20 Medium

Weigh, prepare and prepare the media as per Manufacture instruction.

1. Soyabean Casein digest agar

Prepare the media as per the current version of Respective GTP

Sabouraud Dextrose Agar Medium

Prepare media as per the current version of Respective GTP

Peptone Water 

(Buffered Sodium Chloride - Peptone Solution pH 7.0)

 Potassium Dihydrogen Orthophosphate                   3.56 g

 Disodium Hydrogen Orthophosphate                       7.23 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 121–124^OC for about 30 min.

1. Potato Dextrose Agar Medium

Prepare media as per the current version of Respective GTP.                    

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. 

1. PROCEDURE    
   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 the same,

            b.Incorporating a sufficient quantity of a suitable inactivating agent in the diluentsor by,

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

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.

Effectiveness of Culture Media and Validity of the Counting Methods:

When necessary, operate as follows.  Grow the following test strains separately in tubes containing soybean casein digest broth at 30  - 35⁰C for 18 - 24 hours or, for Candida albicans, at 20 -25⁰C for 48 hours.

Staphylococcus aureus such as NCIMB 8625 (ATCC 6538, CIP 530156) or NCIMB 9518 (ATCC 6538, CIP 4038)

Bacillus Subtilis, such as NCIMB 8054 (ATCC 6633, CIP 52.62)

Candida albicans, such as ATCC 2091 (CIP 1180.79) or ATCC 10231 (NCPF 3179, CIP 48.72)

Dilute portions of each of the cultures using buffered sodium chloride - peptone solution pH 7.0 to make test suspensions containing about 100 viable microorganisms per milliliter.  Use the suspension of each of the microorganisms separately as a control of the counting methods in the presence and absence of the preparation being examined if necessary.

When testing the method, a count for any of the test organisms differing by not more than a factor of 10 from the calculated value for the inoculum should be obtained.  To test the sterility of the medium and of the diligent and the aseptic performance of the test, carry out the total aerobic microbial count method using sterile buffered sodium chloride - peptone solution pH 7.0 as the test preparation.  There should be no growth of microorganisms.

1. Method
   1. For specimens that are sufficiently soluble or translucent to permit use of the Plate Method.
   2. In Plate method, first dissolve or suspend 10 g of the specimen if it is a solid, or 10 ml, accurately measured if the specimen is a liquid, in pH 7.2 Phosphate Buffer, Fluid Soyabean-Casein Digest Medium, or Fluid Casein Digest-soy Lecithin - Polysorbate 20 Medium to make 100 ml
   3. For viscous specimens that cannot be pipetted at this initial 1:10 dilution, dilute the specimen until a suspension is obtained i.e., 1:50 or 1: 100 etc. that can be pipetted.
   4. Perform the test for absence of inhibitory (anti-microbial) properties as described under Preparatory Testing before the determination of Total Aerobic Microbial Count.
   5. Add the specimen to the medium not more than 1 hr after preparing the appropriate dilutions for inoculation.
   6. Plate Method
      1. Dilute further, if necessary, the fluid so that 1 ml will be expected to yield between 30 and 300 colonies.
      2. Pipette 1 ml of the final dilution (Pretreated Sample) onto each of two sterile petri dishes.
      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 or Potato Dextrose Agar medium for molds and yeasts that previously has been melted and cooled to approx. 45⁰C.
      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.
      5. Invert the petri dishes, and incubate for 48 - 72 hours at 32.5⁰C±2.5⁰C for Bacteria and incubate for 5 - 7 days at 20 – 25⁰C for Yeast and Moulds.
      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 gm or per ml of specimen.
      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 gm or per ml of specimen.”
      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.

Aerobic microbial count, general testing procedure, incubated