The tests described hereafter will allow quantitative enumeration of mesophilic bacteria and fungi that may grow under aerobic conditions. If the product to be examined has antimicrobial activity, this is, insofar as possible, removed or neutralized.
Acceptance criterion for microbiological quality is prescribed it is interpreted as follows

• 10¹ CFU: max. acceptable count =20;
• 10² CFU: max. acceptable count = 200;
• 10³ CFU: max. acceptable count = 2000, and so forth

Criteria for microbiological quality of nonsterile substances for pharmaceutical use:-

• Entries for total aerobic microbial counts (TAMC)
• Entries for total combined yeasts/molds count (TYMC)
• 
  • •  Preparation of the Sample:- The method for sample preparation depends on the physical characteristics of the product to be tested. If none of the procedures described below can be demonstrated to be satisfactory, a suitable alternative procedure must be developed:-
      • Water-Soluble Products– Dissolve or dilute (usually a 1 in 10 dilution is prepared) the product to be examined in Soybean–Casein Digest Broth.
      • Non fatty Products Insoluble in Water—Suspend the product to be examined (usually a 1 in 10 dilution is prepared) in Soybean–Casein Digest Broth containing 4 % Polysorbate 20.
      • Fatty Products—Dissolve in isopropyl myristate sterilized by filtration, or mix the product to be examined with the minimum necessary quantity of sterile polysorbate 80 or another non inhibitory sterile surface-active reagent heated, if necessary, to not more than 40° or, in exceptional cases, to not more than 45°.

      The method of sample preparation depends on the physical characteristics of the product to be tested. Unless otherwise directed, Aseptically transfer 10g or 10mL of product /sample  to be examined to make 100mL (1 in 10 dilution) of Soyabean Casein Digest medium containing 4 % Polysorbate 20 (Solution-A). If the product is known to have antimicrobial activity, an inactivating agent such as Polysorbate 80 may be added to Soyabean Casein Digest broth. Mix and keep the solution for to dissolve and disperse.

      Pour-Plate Method: – Add 1.0 ml of Solution A containing product in duplicate into sterile Petri dishes. Prepare for each medium at least two Petri dishes.  Pour l5mL to 20 mL of liquefied Soyabean-Casein Digest Agar (SCDA) suitable for the cultivation of bacteria and 15 mL to 20 ml of a liquefied Sabouraud Dextrose Agar (SDA) suitable for the cultivation of yeast and molds at not more than 45°C. Cover the Petri dishes, mix the sample with the media by tilting or rotating the dishes and allow the contents to solidify at room temperature. Incubate the plates of Soyabean-Casein Digest Agar at 30°C to 35°C for 3 to 5 days and the plates of Sabouraud Dextrose Agar at 20°C to 25°C for 5 to 7days.
      Along with sample test parallely carry out the Negative control test (to verify testing conditions using the chosen diluent in place of the test preparation). There must be no growth of microorganisms in negative control test.
      Positive control test of media used: – Positive control must show growth of microorganisms on media.
      Interpretation of the results:- The Total Aerobic Microbial Count (TAMC) is considered to be equal to the number of cfu found using Soyabean-Casein Digest Agar. If colonies of fungi are detected on this medium, they are counted as part of TAMC.
      The Total Combined Yeast and Mold Count (TYMC) is considered to be equal to the number of cfu found using Sabouraud Dextrose Agar. If colonies of bacteria are detected on this medium, they are counted as part of TYMC. When the TYMC is expected to exceed the acceptance criterion due to the bacterial growth, Sabouraud Dextrose Agar containing antibiotics may be used.

      TESTS FOR SPECIFIED MICRO-ORGANISMS 
      Sample preparation and pre-incubation: – Unless otherwise prescribed in specification, prepare a sample using a 1 in 10 dilution of quantity corresponding not less than to1g /1mL of the product/sample to be examined or aseptically transfer 10mL of Solution-A to make 100mL of Soybean-Casein Digest Broth and mix. Incubate at 30-35°C for 18 to 24 hours.
       

      1. Test For Escherichia coli
      • Selection and subculture: – After incubation of Soybean-Casein Digest Broth, shake the container and aseptically transfer 1 mL of Soybean-Casein Digest Broth to 100 mL of MacConkey broth and incubate at 42-44 °C for 24-48 hours. After incubation, subculture on a plate of MacConkey agar and incubate the plates at 30-35 °C for 18-72 hrs.
      • Interpretation: – Growth of gram negative rods with brick red colonies surrounded by bile precipitate indicates the possible presence of coli. This is confirmed by identification tests.The product complies with the test if no colonies are present or if the identification tests are negative.
      • Identification tests: – If the colonies of Gram negative rods are matching with the description above, streak a loopful of colonies from the surface of MacConkey Agar medium on to the surface of Levine Eosin-Methylene Blue agar medium, cover and invert the plates and incubate at 30°C-35°C for 48 hours.

                                                                                                                                        Indole test-Transfer 1.0ml from MacConkey Broth to 5ml Peptone water (1%). Incubate at 30-35°C for 24 hrs. After incubation period add 0.5 ml Kovac’s reagent, shake well, allow to stand for 1minute. The product complies with the test if no Red colour ring is produced in reagent layer.

    • Observation and Results: – Upon examination, if none of the colonies exhibits both a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light, then the specimen meets the requirements for the absence of Escherichia coli. Along with sample test parallely Carry out the Negative control test and Positive control test of media used.
    2. Test for Salmonella species
    • Sample preparation and pre-incubation: – Unless otherwise prescribed in specification, prepare a sample using a 1 in 10 dilution of sample quantity corresponding to not less than to10g /10mL of the product/sample to be examined to make 100mL of Soybean-Casein Digest Broth containing 4% Polysorbate 20 and mix. Incubate at 30-35°C for 18 to 24 hours.
    • Selection and subculture: – After incubation of Soybean-Casein Digest Broth containing 4% Polysorbate 20, transfer 0.1 mL of Soybean-Casein Digest Broth to 10mL of Rappaport Vassiliadis Salmonella Enrichment Broth and incubate at 30-35 °C for 18-24 hours. Subculture on plates of Xylose Lysine Deoxycholate Agar and incubate at 30-35°C for 18-48 hours.
    • Interpretation:- The possible presence of Salmonella species is indicated by the growth of well-developed red colonies with or without black centers. This is confirmed by identification tests.The product complies with the test if colonies of the types described below are not present or if the confirmatory identification tests are negative.

    Medium	Characteristic colonial morphology
    Xylose Lysine Deoxycholate Agar	well-developed, red colonies, with or without black centres

    • Identification tests:- If colonies of Gram negative rods matching the description in above mentioned table then further subculture on to triple sugar iron agar butt-slant, first streaking the surface of slope and then make a stab culture with the same inoculating needle.Incubate the slant at 30-35°C for 24-48 hrs. If upon examination, there is no evidence of tubes having alkaline (red) slants and acid (yellow) butts (with or without blackening of the butt i.e. Hydrogen Sulfide production), the product meets the requirements of the test for the absence of Salmonella species along with sample test parallely carry out the Negative control test and Positive control test of media used.
    3. Test for Pseudomonas aeruginosa:
    • Selection and subculture:- After incubation of Soybean-Casein Digest Broth, streak a loopful of enriched culture on a plate of Cetrimide agar. Incubate the plates at 30-35^oC for 18 – 72 hours.
    • Interpretation:- Growth of green colonies indicates the possible presence of aeruginosa. This is confirmed by identification tests.The product complies with the test if the colonies are not present or if the confirmatory identification tests are negative.
    • Identification tests:– Subculture any colony showing greenish colony on to the surface of Pseudomonas Agar medium for detection of Pyocyanin and Pseudomonas Agar medium for detection of  fluorescin, cover and invert the plates, and incubate at 35-37⁰C for not less than three days.
    • Observation and Result:– Examine the streaked surfaces under UV light to determine whether the colonies are having characteristics given below.
    • Morphological characteristics:- Morphological characteristics of Pseudomonas Agar medium for detection of Fluorescin shows generally colourless to yellowish colonies and yellowish Fluorescence in UV light. Morphological characteristics of Pseudomonas Agar medium for detection of Pyocyanin show generally greenish colonies and Bluish Fluorescence in UV light.

    Medium	Characteristic Colonial Morphology	Fluorescence in UV light
    Pseudomonas Agar medium for detection of Fluorescin	Generally, colourless to yellowish colonies	Yellowish
    Pseudomonas Agar medium for detection of Pyocyanin	Generally greenish colonies	Blue

    Confirm any suspect colonial growth by means of oxidase test.

    • Oxidase test: If growth of suspect colonies occurs, smear the suspected colony on oxidase disc. If there is no development of purple colour within 30 seconds, sample meets the requirement for the test for the absence of Pseudomonas aeruginosa. Along with sample test parallely carry out the Negative control test and Positive control test of media used.
    4. Test for Staphylococcus aureus:
    • Selection and subculture:- After incubation of Soybean-Casein Digest Broth, streak a loopful of enriched culture on a plate of Mannitol salt agar Incubate the plates at 30-35^oC for 18 – 72 hours.
    • Interpretation:- The possible presence of S.aureus is indicated by the growth of yellow or white colonies surrounded by a yellow zone. This is confirmed by identification tests. The product complies with the test if the colonies of the types described are not present or if the confirmatory identification tests are negative.
    • Identification tests:- In case the colonies characteristics matches with the description as mentioned above, then confirm the presence of Gram positive cocci (in clusters) by gram staining.Along with sample test parallely Carry out the Negative control test & Positive control test of media used.
    5. Test For Bile-Tolerant Gram-Negative Bacteria (Qualitative and Quantitative evaluation):- Prepare a sample using a 1 in 10 dilution of quantity corresponding not less than to1g /1mL of the product/sample to be examined in Soyabean Casein Digest Medium containing 4% Polysorbate 20 or incubate Solution-A at 20°C – 25°C for a time sufficient to resuscitate the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 hours but not more than 5 hours)                                                                                                                                                               –Qualitative evaluation (Test for Absence):- Use the volume corresponding to 1.0 gm of product to inoculate 100 ml of Enterobacteria Enrichment Broth Mossel. Incubate at 30 – 35 °C for 24 -48 hours. Subculture on plates of Violet Red Bile Glucose agar. Incubate the plates at 30 – 35 °C for 18 -24 hours.

        

       • Interpretation:- The product complies with the test if there is no growth of red or reddish colonies.

       -Quantitative evaluation  

       • Selection and subculture:- Shake the tube and inoculate 1.0 mL, 0.1 mL and 0.01mL of the enrichment culture to separate tubes each containing 100 mL of Enterobacteria Enrichment broth-Mossel. Incubate these tubes at 30°C-35°C for 24-48 hours. Subculture each of the cultures on a plate of Violet Red Bile Glucose Agar. Incubate at30°C -35°C for 18-24 hours.

        

       • Interpretation:- Growth of colonies generally red or reddish having gram-negative character gives a positive result. Note the smallest quantity of product that gives a positive result and the largest quantity that gives a negative result. Determine the probable number of bacteria per gram or ml of product as per below table.

       Results for each quantity of product			Probable number of bacteria per g or mL of product
       0.1g or 0.1 mL	0.01g or 0.01 mL	0.001g or 0.001 mL
       +	+	+	More than 103
       +	+	–	Less than 103 and more than 102
       +	–	–	Less than 102 and more than 10
       –	–	–	Less than 10

       Along with sample test parallely carry out the Negative control test & Positive control test of media used.

       6. Test for Clostridia
       • Sample preparation and heat treatment:- Take two equal portions from solution A (10 mL each) or corresponding to not less than 1g or mL of the product/sample to be examined in Soyabean Casein Digest Broth. Heat one portion at 80°C for 10 minutes and cool rapidly. Do not heat the other portion.
       • Selection and subculture:- Transfer10 ml of each of the homogenised portions to two containers containing 100 ml of Reinforced medium for Clostridia. Incubate under anaerobic conditions at 30-35 °C for 48 hours. After incubation, make subcultures from each container on Columbia Agar and incubate under anaerobic conditions at 30-35°C for 48 to 72 hours.
       • Interpretation:- The occurrence of anaerobic growth of rods (with or without endospores) giving a negative catalase reaction indicates the presence of If no anaerobic growth of microorganism is detected on Columbia Agar or the identification test is negative, the product complies with the test. Along with sample test parallely carry out the Negative control test & Positive control test of media used.
       7. Test for Candida albicans

       Aseptically transfer 10 ml of Solution-A or prepare a sample using a 1 in 10 dilutions of quantity corresponding not less than to1g or 1mL of the product/sample to be examined to inoculate 100mL of Sabouraud Dextrose Broth and mix. Incubate at 30°C -35°C for 3-5 days.

       • Selection and subculture:- Subculture on a plate of Sabouraud Dextrose Agar. Incubate at 30°C-35°C for 24-48 hours.
       • Interpretation:- Growth of white colonies indicate the presence of albicans. The product complies with the test if such colonies are not present. Along with sample test parallely carry out Negative control test & Positive control test of media used.

       FIND MORE AT…
       Reference links
       https://hmc.usp.org/sites/default/files/documents/HMC/GCs-Pdfs/c61.pdf
       http://www.who.int/medicines/publications/pharmacopoeia/MicrobialEnumerationTests_QAS11-409_FINAL_MODIFIEDMarch2012.pdf?ua=1

        Quantitative evaluation