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Microbial Limit Test (MLT) of Non Sterile Product

Guideline (SOP) for quantitative enumeration of mesophilic bacteria & fungi that may grow under aerobic conditions and for detecting the presence of specified microorganisms in pharmaceutical raw materials and finished products (Microbial Limit Test – MLT).

Microbial Limit Test (MLT) procedure

1.0   Purpose

    • The purpose of this SOP is To lay down the procedure for quantitative enumeration “Microbial Limit Test (MLT)”of mesophilic bacteria & fungi that may grow under aerobic conditions and for detecting the presence of specified microorganisms in pharmaceutical raw materials and finished products.

2.0   Scope

    • The scope of this SOP is applicable for Microbial Limit Test (MLT) in Microbiology Department.

3.0   Responsibility

    • Microbiologist/Officer Microbiology shall be responsible for performing Microbial Limit Test (MLT) as per this SOP.
    • Executive Microbiology/Designee shall be responsible for ensuring the Procedure for Microbial Limit Test (MLT) as per this SOP.
    • Head QC/Designee shall be responsible for ensuring the adherence to this SOP.
    • Head QA/Designee shall be responsible for approval and compliance of this SOP.

4.0   Procedure for Microbial Limit Test (MLT)

    • Precautions during Microbial Limit Test (MLT):

    • During testing, the Microbiologist shall wear sterile garments (Head gear, booties and boiler suit) to prevent the risk of contamination.Microbial Limit Test
    • All glassware being used shall be properly sterilized.
    • Before and after handling LAF surface shall be sanitized with 70% IPA.
    • During sample preparation after reconstitution of the sample in SCDM, transfer 1 ml in respective plates within 5 minutes.
    • Carry out the determination under conditions designed to avoid extrinsic microbial contamination of the product to be examined.
    • Due precautions should be taken to avoid contamination must be such that they do not affect any microorganisms that are to be revealed in the test.
    • If the product to be examined has antimicrobial activity this is so far as possible removed or neutralized.
    • When neutralizers are used for this purpose their efficacy and their absence of toxicity for microorganisms must be demonstrated.
    • If Surface-active substances are used for sample preparation, their absence of toxicity for microorganisms and their compatibility with any neutralizers used must be demonstrated.

    • Sample Preparation for Microbial Limit Test (MLT):

    • 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 10 gm or 10 ml of product in 90 ml Buffered Sodium Chloride Peptone Solution pH 7.0 / Phosphate Buffer Solution pH 7.2 /Soyabean Casein Digest Broth. If necessary, adjust to a pH of 6.0 to 8.0.
      • Further dilutions where necessary are prepared with the same diluent.

    • Non-fatty products insoluble in water:

      • Suspend 10 gm or 10 ml of the product to be examined in 90 ml Buffered Sodium Chloride Peptone Solution pH 7.0 / Phosphate Buffer Solution pH 7.2 / Soyabean Casein Digest Broth.
      • A suitable surface active agent such as 1gm per liter L of Polysorbate 80 may be added to assist the suspension of poorly wetted substances.
      • If the product is known to have antimicrobial activity, an inactivating agent may be added to the diluents.
      • If necessary, adjust to about pH 6.0 to 8.0 and further dilutions where necessary are prepared with the same diluent.

    • 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°C or, in exceptional cases, to not more than 45°C. Mix carefully and if necessary maintain the temperature in a water bath.
      • Add a sufficient quantity of the pre-warmed chosen diluent to make a 1 in 10 dilution of the original product.
      • Mix carefully, while maintaining the temperature for the shortest time necessary for the formation of an emulsion.
      • Further serial 10-fold dilutions may be prepared using the chosen diluents containing a suitable concentration of sterile polysorbate 80 or another non inhibitory sterile surface active reagent.

    • Total Aerobic Microbial Count:

      • Dissolve/suspend 10 gm or 10 ml of product in 90 ml of suitable diluents as mentioned above (Solution A).
      • If necessary homogenize the solution ( by using sterile Polysorbate 80 or another non-inhibitory sterile surface active reagent) and heat to not more than 40ºC with intermittent shaking for not more than 30 minutes.
      • For Total Aerobic Microbial Count aseptically pipette out in duplicate 1.0 ml of sample from solution A into two sterile Petri plates of 90 mm diameter.
      • Pour 20-25 ml of molten Soyabean Casein Digest Agar and mix the solution gently by rotating the plate clockwise and anticlockwise.
      • Allow the plate to solidify and incubate the plates at 30-35°C for 3 to 5 days.
      • After incubation count the number of colonies in each plate. Calculate the mean and multiply it with dilution factor.
      • Negative control: Pour 20-25ml of sterile SCDA media into the 90 mm plate without the test sample and incubate along with the test plates.
      • Positive control: GPT of the SCDA will be considered as a positive control.
      • Record the observation as per format 1.
    • Total Yeast and Mold Count (Microbial Limit Test) :
      • For Total Yeast and Mold Count aseptically pipette out in duplicate 1.0 ml of sample from solution A into two sterile Petri plates of 90 mm diameter.
      • Pour 20-25 ml molten Sabouraud Dextrose Agar/Sabouraud Chloramphenicol Agar and mix the solution gently by rotating the plate clockwise and anticlockwise.
      • Allow the plates to solidify and incubate them at 20-25ºC for 5 to 7 days.
      • After incubation count the number of colonies in each plate. Calculate the mean and multiply it with dilution factor.
      • Negative control: Put the 20-25 ml of SDA media into the 90 mm plate without the test sample and incubate along with the test plates.
      • Positive control: GPT of the SDA/SCA will be considered as a positive control.

    • Testing of Products for Specified Pathogens:

      • Sample Preparation for Pathogen testing:
      • Take 10 ml sample from solution A with help of micropipettes and add into 90ml of preincubated Soyabean Casein Digest Medium, mix and incubate at 30-35ºC for 18-24 hrs (Solution B).
      • Bile- Tolerant Gram –Negative Bacteria:
      • After pipetting out of the required solution for TAMC and TYMC, rest of the sample (Solution A) is incubated at 20 to 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).
      • Test for Absence:
      • Take 10 ml or equal volume to 1 g or ml of the product from the above Solution A and transfer to suitable amount of volume in pre incubated Enterobacteria Enrichment Broth Mossel.
      • Incubate at 30°C to 35°C for 24 to 48 hours. After completion of incubation period, subculture on plates of pre incubated Violet Red Bile Glucose Agar and incubate at 30°C to 35°C for 18 to 24 hours. After incubation period check the plates for growth.
      • The product complies with the test if there is no growth of colonies.
      • Test Negative control: Perform a negative control as test sample, using the chosen pre incubated diluent in place of the test preparation. There must be no growth of microorganisms. Failed negative control needs investigation.
      • Quantitative Test: Selection and Subculture:
      • If qualitative test fails than proceed for quantitative test.
      • Transfer 10 ml sample from the solution A (Freshly prepared) to 90 ml of the pre incubated Enterobacteria Enrichment Broth Mossel.
      • Take three test tubes each tube containing 10 ml pre incubated Enterobacteria Enrichment Broth Mossel.
      • For the first tube add 10 ml (equal to 0.1 g or 0.1 ml of the product to be examined) from the solution A.
      • The second tube add 1 ml (equal to 0.01 g or 0.01 ml of the product to be examined) from the solution A.
      • Third tube add 0.1 ml (equal to 0.001 g or 0.001 ml of the product to be examined) from the solution A.
      • Incubate at 30°C to 35°C for 24 to 48 hours.
      • After incubation period subculture each of the cultures on an each plate of Violet Red Bile Glucose Agar.
      • Incubate at 30°C to 35°C for 18 to 24 hours.
      • Test Negative control:
      • Perform a negative control as test sample, using the chosen pre incubated diluent in place of the test preparation.
      • There must be no growth of microorganisms.
      • Failed negative control needs investigation.
      • Interpretation:
      • Growth of well developed reddish colonies of gram negative bacteria constitutes a positive result.
      • Note the smallest quantity of the product that gives a positive result and the largest quantity that gives a negative result.
      • Determine from Table 1 the probable number of bacteria.

Table 1 – Interpretation of Results (Microbial Limit Test)

Results for Each Quantity of Product Probable Number
of Bacteria
per g or ml of Product
0.1 g or
0.1 ml		 0.01 g or
0.01 ml		 0.001 g or
0.001 ml
+ + + > 103
+ + – < 103 and more than 102
+ – – less than 102 and more than 10
– – – less than 10

 

    • Identification Test:
    • Biochemical test or identification by automated methods can be used for confirmatory identification.
    • The product complies with the test if colonies of the types described are not present or if the confirmatory or identification tests are negative.

  • Test for Escherichia coli:

    • Selection and Subculture:
    • Add 1 ml sample from Solution B into 100 ml of pre incubated MacConkey broth and incubate at 42-44°C for 24-48 hrs. After incubation subculture on a plate of pre incubated MacConkey agar and incubate at 30-35°C for 18-72 hrs.
    • Interpretation:
    • Growth of brick red colonies indicates the possible presence of Escherichia coli.
    • This is confirmed by identification tests.
    • The product complies with the test, if colonies of the types described are not present or if the confirmatory identification tests are negative.
    • Test Negative control:
    • Perform a negative control as test sample, using the chosen pre incubated diluent in place of the test preparation.
    • There must be no growth of microorganisms. Failed negative control needs investigation.
    • Identification test:
    • Take well-isolated suspected colonies from MacConkey agar plate in 10 ml (0.1%) of peptone tube, incubate at 30-35ºC for 24 hrs.
    • Add 1 ml of kovac’s reagent to 0.1% peptone tube for the confirmation of indole.
    • Allow to stand for 1 minute; if a red colour is produced in the reagent layer, indole is present.
    • It indicates the presence of Escherichia coli.
    • Positive control:
    • Confirm the above test by carrying out test simultaneously using 10-100 cfu culture suspension of E .coli.
    • The test is invalid if the results do not indicate that the control contains E .coli.

  • Test for Salmonella – Microbial Limit Test (MLT):

    • Sample preparation:
    • Dissolve or dilute to not less than 10 gm or 10 ml of product in 90 ml of Soyabean Casein Digest Broth, mix and incubate at 30-35ºC for 18-24 hrs. (Solution C).
    • Selection and subculture:
    • After completion of incubation period transfer 0.1 ml of Solution C to 10 ml of Rappaport Vassiliadis Salmonella Enrichment Broth and incubate at 30-35°C for 18-24 hrs.
    • After incubation subculture on plates of Xylose Lysine Deoxycholate Agar and incubate at 30-35°C for 24-48 hrs.
    • Interpretation:
    • The possible presence of Salmonella 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 are not present or if the confirmatory identification tests are negative.
    • Test Negative control:
    • Perform a negative control as test sample, using the chosen pre incubated diluent in place of the test preparation. There must be no any growth of microorganisms. Failed negative control needs investigation.
    • Identification Test:
    • If colonies with characteristics described for XLDA is present, the suspected colonies are transferred to a slant of Triple Sugar Iron Agar (TSI) using an inoculating loop, by first streaking the surface of the slant, and then stabbing the wire well beneath the surface. Incubate at 30-35°C for 24 to 48 hrs.
    • If the tubes do not have red alkaline slants and yellow acid butts, with or without concomitant blackening of the butts from hydrogen sulphide production, the test specimen meets the requirement for the absence of Salmonella.
    • Positive control:
    • Confirm the above test by carrying out test simultaneously using 10-100 cfu culture suspension of Salmonella.
    • The test is invalid if the results do not indicate that the control contains Salmonella.

  • Test for Pseudomonas aeruginosa – Microbial Limit Test (MLT)

    • Selection and subculture:
    • Subculture from Solution B on a plate of Cetrimide Agar and incubate at 30-35°C for 18-72 hrs.
    • Interpretation:
    • Growth of greenish colonies indicates the possible presence of aeruginosa.
    • This is confirmed by identification tests.
    • The product complies with the test, if colonies are not present or if the confirmatory identification tests are negative.
    • Test Negative control:
    • Perform a negative control as test sample, using the chosen pre incubated diluent in place of the test preparation.
    • There must be no any growth of microorganisms. Failed negative control needs investigation.
    • Identification Test: Oxidase test:
    • Put a portion of suspected colonies present on Cetrimide agar plates on Oxidase disc and wait for a few minutes, observe the disc if white colour disc is converted into purple colour, the test is positive.
    • If it is not converted into purple colour, the test is negative.
    • Positive control:
    • Confirm the above test by carrying out test simultaneously using 10-100 cfu culture suspension of Pseudomonas aeruginosa.
    • The test is invalid if the results do not indicate that the control contains Pseudomonas aeruginosa.

  • Test for Staphylococcus aureus – Microbial Limit Test (MLT):

    • Selection and subculture:
    • Subculture from Solution B on a plate of Mannitol Salt Agar and incubate at 30-35°C for 18-72 hrs.
    • Interpretation:
    • The possible presence of aureus is indicated by the growth of yellow/white colonies surrounded by a yellow zone.
    • This is confirmed by identification tests.
    • The product complies with the test, if colonies are not present or if the confirmatory identification tests are negative.
    • Test Negative control:
    • Perform a negative control as test sample, using the chosen pre incubated diluent in place of the test preparation. There must be no any growth of microorganisms.
    • Failed negative control needs investigation
    • Identification test: Coagulase Test:
    • If characteristic colonies are present, perform coagulase test as follows.
    • Transfer representative colonies to a separate tubes containing 0.5 ml of Rabbit Plasma or Horse Plasma, or any other Mammalian Plasma.
    • Incubate in a water bath at 37ºC. Examine for coagulation after 3 hrs. of incubation and at suitable intervals up to 24 hrs.
    • Comparing with positive and negative controls, the absence of a Coagulase reaction indicates the absence of Staphylococcus aureus.
    • Positive control:
    • Confirm the above test by carrying out test simultaneously using 10-100 cfu culture suspension of Staphylococcus aureus.
    • The test is invalid if the result does not indicate that the control contains Staphylococcus aureus.

  • Test for Candida albicans – Microbial Limit Test (MLT):

    • Sample preparation:
    • Add 10ml of sample from Solution A into 100 ml of Sabouraud Dextrose Broth and incubate at 30-35°C for 72-120 hrs. (Solution D).
    • Selection and subculture:
    • Subculture from Sabouraud dextrose broth on a plate of Sabouraud dextrose agar and Incubate at 30-35°C for 24-48 hrs.
    • Interpretation:
    • Growth of white/cream colored colonies may indicate the possible presence of albicans.
    • This is confirmed by identification tests.
    • The product complies with the test if such colonies are not present or if the confirmatory identification tests are negative.
    • Test Negative control:
    • Perform a negative control as test sample, using the chosen pre incubated diluent in place of the test preparation.
    • There must be no any growth of microorganisms. Failed negative control needs investigation.
    • Identification test:
    • Biochemical test or identification by automated methods can be used for confirmatory identification.
    • Positive control:
    • Confirm the above test by carrying out test simultaneously using 10-100 cfu culture suspension of Candida albicans.
    • The test is invalid if the result does not indicate that the control contains Candida albicans.
  • Test for Clostridia – Microbial Limit Test (MLT) :
    • Sample preparation:
    • Take 10 ml in 2 equal portions from Solution A, heat 1 portion at 80ºC for 10 min and cool rapidly, do not heat the other portion.
    • Selection and subculture:
    • Transfer 10 ml of each portion into 2 containers containing 100 ml of Reinforced Medium for Clostridia and incubate under anaerobic conditions (using anaerobic Jar at 30-35°C for 48 hrs. After incubation, subculture from each tube on Columbia Agar and incubate under anaerobic conditions at 30-35°C for 48 to 72 hrs.
    • Interpretation:
    • The occurrence of anaerobic growth of rods (with or without endospores) giving a negative catalase reaction indicates the possible presence of clostridia.
    • This is confirmed by identification tests.
    • The product complies with the test if such colonies are not present or if the confirmatory identification tests are negative.
    • Test Negative control:
    • Perform a negative control as test sample, using the chosen pre incubated diluent in place of the test preparation.
    • There must be no any growth of microorganisms. Failed negative control needs investigation.
    • Identification test:
    • Biochemical test or identification by automated methods can be used for confirmatory identification.
    • Positive control:
    • Confirm the above test by carrying out test simultaneously using 10-100 cfu culture suspension of clostridia.
    • The test is invalid if the results does not indicate that the control contains Clostridia.

  • Test for Shigella – Microbial Limit Test (MLT):

    • Sample preparation:
    • After incubation of Solution C shake and transfer 1ml of Solution C into 100ml of GN Broth and incubate at 30-35°C for 18-24 hrs.
    • Selection and subculture:
    • After incubation subculture on a plate of Xylose Lysine deoxycholate agar medium and incubate at 30-35°C for 24-48 hrs.
    • Interpretation:
    • The possible presence of Shigella is indicated by the growth of red coloured translucent colonies without black centre.
    • This is confirmed by identification tests.
    • The product complies with the test, if colonies are not present or if the confirmatory identification tests are negative.
    • Test Negative control:
    • Perform a negative control as test sample, using the chosen pre incubated diluent in place of the test preparation.
    • There must be no growth of microorganisms.
    • Failed negative control needs investigation.
    • Identification test:
    • Biochemical test or identification by automated methods can be used for confirmatory identification.
    • Positive control:
    • Confirm the above test by carrying out test simultaneously using 10-100 cfu of culture suspension of Shigella.
    • The test is invalid if the results does not indicate that the control contains.

5.0   Abbreviation and Definition

    • MLT                               : Microbial Limit Test 
    • IPA                                : Isopropyl Alcohol
    • LAF                               : Laminar Air Flow
    • °C                                  : Degree Centigrade
    • SCDA                             : Soyabean Casein Digest Agar
    • SDA                               : Sabouraud Dextrose Agar
    • SCA                               : Sabouraud Chloramphenicol Agar
    • XLDA                              : Xylose Lysine Deoxycholate Agar
    • TSI                                 : Triple Sugar Iron Agar
    • gm                                  : Gram
    • ml                                   : Milliliter
    • %                                    : Percentage
    • CFU                                : Colony Forming Unit
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Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

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