TEST FOR TOTAL AEROBIC MICROBIAL COUNT

1.  INTRODUCTION:    
    Total aerobic microbial count is one type of microbiological purity testing, which is used to count the number of colony forming     unit present in an article complying with monograph standards.        

2.  PREPARATION OF CULTURE MEDIA:
    Sodium hydroxide 1N: Dissolve 4.0 g of sodium hydroxide in water to make 100 mL.
    Hydrochloric Acid 1N: Dissolve 8.5 ml of Hydrochloric Acid in water to make 100 mL.
    Soyabean-Casein Digest Broth Medium (SCDM)/ Soyabean-Casein Digest Agar(SCDA)/ Plate Count Agar(PCA): Reconstitute       dehydrated media as directed by the manufacturer and sterilize in an autoclave at 121°C for 20 minutes.
    
3.  PROCEDURE:
    Sample preparation:
    Water soluble products: Aseptically add 10g of specimen if it’s a solid or 10ml accurately measured, if the specimen is a liquid     to make 100ml of sterile Soybean Casein Digest broth Medium unless otherwise specified in the individual standard testing           procedure, shake and mix the sample preparation to achieve a homogeneous solution/suspension or dilute the specimen in a       suitable quantity of diluter unless otherwise specified in the individual standard testing procedure.
    Water insoluble products: Aseptically add 10g of specimen in 100ml of sterile Soybean Casein Digest broth Medium with 0.1ml     of Polysorbate 80, unless otherwise specified in the individual standard testing procedure. Shake and mix the sample     preparation to achieve a homogeneous solution/suspension or dilute the specimen in a suitable quantity of diluter unless otherwise specified in the individual standard testing procedure.
    Capsules: Aseptically add 10g of capsules in 100ml of sterile Soybean Casein Digest broth Medium unless otherwise specified in the individual standard testing procedure, subject the  
    dilution to a water bath at temperature NMT 45 ºC for 15 min. shake every 5 min to achieve a homogeneous solution/suspension or dilute the specimen in a suitable quantity of diluter unless otherwise specified in the individual standard testing procedure.
    POUR PLATE METHOD:
Note: For specimen that is sufficiently soluble or translucent perform Pour Plate Method, otherwise use the Multiple-tube Method or Membrane filtration method.
    Test preparation:
    Pipette 1 mL of the final diluted sample into each of two sterile petri dishes.
    Transfer to each dish 15-20mL of Soyabean -Casein Digest Agar Medium that previously has been melted and cooled to approximately 45°C.
    For water samples add 1ml of sample in to each of two Petri dishes and pour 15-20 mL of Plate Count Agar Medium that previously has been melted and cooled to approximately 45°C.
    Cover the petridishes, mix the agar by tilting or rotating the dishes and allow the contents to solidify at room temperature.
    Negative Control:
     Transfer 15-20mL of Soyabean -Casein Digest Agar Medium or Plate Count Agar Medium (for water samples) without any sample that previously has been melted and cooled to approximately 45°C.
     Cover the petridishes, mix the agar by tilting or rotating the dishes, and allow the contents to solidify at room temperature.
    Positive Control:
    Pipette 1mL of <100 cfu/ ml of Escherichia coli or Staphylococcus aureus culture suspension into a sterile Petri dish.
    Transfer 20mL of Soyabean -Casein Digest Agar Medium or Plate Count Agar Medium (for water samples) without any sample that previously has been melted and cooled to approximately 45°C.
 
    Cover the petridishes, mix the suspension with agar by tilting or rotating the dishes and allow the contents to solidify at room temperature.
    Incase of water samples incubate all the petridishes at 30-35°C for 48-72 hours at inverted position for products incubate at 30-35°C for 3-5 days at inverted position.
    After incubation, examine the plates for growth.
    
    MOST PROBABLE NUMBER METHOD (MPN):
    Prepare three consecutive 1:10 dilutions (1:10, 1:100 & 1:1000) of the sample (as taken from the sample preparation) to 9 ml Soyabean-Casein Digest broth medium, each dilution should have triplicate as mentioned in below.
    Transfer each 1 ml of sample preparation in to three 9 ml of Soyabean-Casein Digest broth medium (1:10 dilution).
    Add each 1 ml of 1:10 dilution in to three 9 ml of Soyabean-Casein Digest medium broth (1:100 dilution).
    Add each 1 ml of 1:100 dilution in to three 9 ml of Soyabean-Casein Digest medium broth (1:1000 dilution).
    Incubate all 9 tubes and one negative control and one positive control tube for 3-5 days at 30-35ºC.
    Record for each level of dilution the number of tubes showing microbial growth.
    If the results is difficult to read, subculture the tube into fresh sterile Soyabean-Casein Digest broth medium or Soyabean-Casein Digest Agar and incubate for 24 to 48 hrs at 30-35ºC.
       Determine the most probable number of micro organisms per gm or per ml from table 1
                                                                             Table-1