Microbial Limit Test For Pharmaceutical Products
v Clean the outer Surface of the sample with 70% IPA and Actuate 10 ml sample under sterile
condition in sterile class A volumetric measuring cylinder or Approx. 10gm sample weighing on calibrated weighing balance. Exact volume of sample transfer to 90 ml sterile
Tryptone Soya Broth with 0.5% Soya lecithin & 4% Tween 20 in screw caped Glass Bottle.
v Mix well to obtain a uniform 1:10 (v/v or w/v) dilution.
v If required, make further dilution with concern to specification limit.
Note: If required add exact 10 ml or 10 gm sample to 10 ml tween 80 for neutralization, Add this solution to 80 ml sterile Tryptone Soya Broth with 0.5% Soya lecithin & 4% Tween 20 in screw caped Glass Bottle.
Determination of Total Aerobic Microbial Count by (Pour Plate Method)
v Pour 1 ml or required quantity from prepared dilution of sample using sterile calibrated Micropipette to sterile duplicate Petri plates.
v Pour 20-25ml of sterile Soybean Casein Digest Agar/Tryptone Soya Agar (cooled up to
450 C) in both plates.
v Mix the content of petri plates by rotating clock and anticlock wise the plate, and allow
medium to solidify.
v Incubate the plates in an inverted position at 30 to 35o C for 3-5 days.
v Determination of Total Yeast & Mold Count by (Pour Plate Method)
v Aseptically transfer 1ml or required quantity from the prepared dilution into two sterile duplicate Petri plates.
v Add 20-25 ml of sterile Sabouraud Dextrose Agar in both petri dishes.
v Mix the contents of petri plates by rotating clock and anticlock wise the plate and allow
medium to solidify.
v Incubate all the plates at 20 to 25oC for 5-7 days in an inverted position.
v After incubation period count the number of colonies from each plate and find out the
average.
v Express the result as Colony Forming Unit (cfu) per gm/ml divided by volume taken by multiplying average number of cfu/plate with dilution factor. If no colonies are observed express the result as number of colonies less than dilution factor.
v If counting of colonies is not possible due to high count or merged colonies total count
test shall be repeated after further diluting solution/suspension.
v If total count observed in retest are not conforming to specified limits the material will be
considered failing in the tests.
Positive Control
Carry out a control test by adding 1 ml of Microbial Culture Suspension of B. subtilis containing 10-100 cfu/ml and pour SCDA/TSA media & Incubate 30-35° C for 3-5 days and C. albicans containing 10-100 cfu/ml and pour 20-25 ml SDA Media & after solidify Incubate at 20-25°C for 5-7 days.
Negative Control
Perform negative control using 1 ml diluent and pour SCDA Media & after solidify Incubate at 30-35°C for 3-5 days & SDA media at 20-25°C for 5-7 days.
Determination of Total Aerobic Microbial Count by (Filtration Method)
Procedure: microbial limit test for pharmaceutical products pdf
Arrange sterile membrane filtration assembly. Add 10 ml sample (from 4.1) to the 100 ml
sterile purified water with the help of Micropipette and mix thoroughly. filter the solution
through NMT 0.45μm sterile filter and Rinse the filter with 200 ml Sterile purified water.
Transfer the filter paper with sterile forceps on SCDA/TSA plate and Incubate in inverted
condition at 30-35oC for 3- 5 days. After incubation period counts the number of colonies.
v Express the result as Colony Forming Unit (cfu) per gm/ml, If no colonies are observed
express the result as number of colonies less than dilution factor.
v If counting of colonies is not possible due to high count or merged colonies total count
test shall be repeated after further diluting (1:10) solution/suspension.
Positive Control
v Take 1 ml of Culture Suspension containing 10-100 cfu/ml of B. subtilis and add to 100 ml
Sterile purified water and mix thoroughly. filter the solution through 0.45μm sterile filter and Rinse the filter with 200 ml Sterile purified water. Transfer the filter paper by sterile
forceps on SCDA/TSA plate and Incubate in inverted condition in incubator at 30-35oC for
3-5 days. After incubation period counts the number of colonies.
Negative Control
v Take 10 ml of Diluent in 100 ml Sterile purified water and filter through 0.45μm sterile
Filter and Rinse the filter with 200 ml Sterile purified. Transfer the filter paper by sterile
forceps on SCDA/TSA Plate and Incubate in inverted condition in incubator at 30-35oC
for 3-5 days. After incubation period counts the number of colonies.
v Determination of Total Yeast & Mold Count by (Filtration Method)
Procedure: microbial limit test for pharmaceutical products pdf
v Arrange sterile membrane filtration assembly. Add 10 ml sample (from 4.1) to the 100 ml
sterile purified water with the help of Micropipette and mix thoroughly. filter the solution
through NMT 0.45μm sterile filter and Rinse the filter with 200 ml Sterile purified water.
Transfer the filter paper with sterile forceps on SDA Plate and Incubate in inverted
condition at 20-25oC for 5-7 days. After incubation period counts the number of colonies.
v Express the result as Colony Forming Unit (cfu) per gm/ml, If no colonies are observed
express the result as number of colonies less than dilution factor.
v If counting of colonies is not possible due to high count or merged colonies total count
test shall be repeated after further diluting (1:10) solution/suspension.
Note: - If a high bacterial count observed in Plate of SDA Repeat the test and use Sabouraud
dextrose Agar with 0.5% Chloramphenicol.
Positive Control
v Take 1 ml of Culture Suspension containing 10-100 cfu/ml of C. albicans and add to 100
ml Sterile purified water and mix thoroughly. filter the solution through 0.45μm sterile filter and Rinse the filter with 200 ml Sterile purified water. Transfer the filter paper by sterile forceps on SDA Plate and Incubate in inverted condition in incubator at 20-25oC for 5-7 days. After incubation period counts the number of colonies.
Negative Control
v Take 10 ml of Diluent in 100 ml Sterile purified water and filter through 0.45μm sterile
Filter and Rinse the filter with 200 ml Sterile purified. Transfer the filter paper by sterile forceps on SDA Plate and Incubate in inverted condition in incubator at 20-25oC for 5-7 days. After incubation period counts the number of colonies.
v The maximum countable number on a plate is 250 CFU for TAMC and 50 for TYMC
v TAMC was formerly referred to as Total Mesophilic Count(TMC) and Total Viable Count
(TVC).
v To avoid growth, the Sample preparation should be tested within 1 hour of dilution.
v Interpretation of Results
v TAMC
The TAMC is considered to be Equal to the number of CFU found using TSA; if colonies
of Fungi are detected on this medium, they are counted as part of TAMC.
v TYMC
v The TYMC is considered to be Equal to the number of CFU found using SDA; 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 bacterial growth,
Sabouraud dextrose Agar containing Antibiotic may be used. The organism should be
identified and noted.
v Specified Micro-organisms
Pre-treatment of sample
Transfer 10gm/10 ml of the sample to the 90 ml of sterile Tryptone Soya Broth or
Soyabean Casein Digest Broth with 0.5% Soya lecithin & 4% Tween 20 in screw cap glass
bottle and mix.
Note:
• If required, make further dilution with concern to specification limit.
• If required add exact 10 ml or 10 gm sample to 10 ml tween 80 for neutralization, Add this solution to 80 ml sterile Tryptone Soya Broth with 0.5% Soya lecithin & 4% Tween 20 in screw caped Glass Bottle.
Enrichment of sample
v Transfer 10 ml of pretreatment sample from (4.9.1) in 90 ml Tryptone Soya Broth or
Soyabean Casein Digest Broth with 0.5% Soya lecithin & 4% Tween 20 in screw cap glass
bottle and mix. and Incubate at 30-35oC for 18-24 hrs.
Negative control
Incubate 90 ml of sterile Tryptone Soya Broth or Soyabean Casein Digest Broth with 0.5%
Soya lecithin & 4% Tween 20 in screw cap glass bottle at 30-35° C for 18-24 hrs. as a
Negative Control.
Escherichia coli
Procedure
After incubation shake the broth (under the section enrichment of sample) and transfer
1 ml of enriched sample to 100 ml of MacConkey broth and mix thoroughly. Incubate at
42 to 44oC for 24 to 48 hrs. If growth observed in MacConkey broth medium, streak on a
plate of MacConkey agar and incubate in inverted condition in incubator at 30 to 35oC for
18 to 72 hrs. If pink color, non-mucoid colonies observed on MacConkey agar medium,
it indicates the possible presence of Escherichia coli and carry out biochemical tests.
Biochemical Test
Indole Test
Transfer suspected colonies from MacConkey agar to 10ml of peptone water. Incubate at
30 to 35°C for 24 to 48 hrs. After incubation, add 3 to 4 drops of Kovac’s reagent in
peptone water tube. If red color ring produced in tube. it indicates the presence of
Escherichia coli.
Positive Control
Carry out a control test using 0.1ml of the enrichment culture containing 10 to 50
Escherichia coli (MTCC 452/NCIM 2065). if pink color, non-mucoid colonies not observed
test is invalid.
Perform negative control using Autoclaved media as blank.
Salmonella spp.
Pre-treatment of sample
Clean the outer Surface of the sample with 70% IPA and Actuate 10 ml sample under sterile
condition in sterile class A volumetric measuring Cylinder or Approx 10gm sample weighing
on calibrated weighing balance and transfer to (required dilution) sterile Tryptone Soya Broth with 0.5% Soya lecithin & 4% Tween 20 in screw caped Glass Bottle.
If required add exact 10 ml or 10 gm sample to 10 ml tween 80 for neutralization, Add this solution 180 ml sterile Tryptone Soya Broth with 0.5% Soya lecithin & 4% Tween 20 in screw caped Glass Bottle.
Note: If required, make further dilution with concern to specification limit.
Procedure
After incubation shake the broth and transfer 0.1 ml of enriched sample to 10 ml of
Rappaport Vassiliadis Salmonella enrichment broth and mix thoroughly. Incubate at
30-35oC for 18 to 24hours. If growth observed in Rappaport Vassiliadis Salmonella
enrichment broth, streak on a plate of Xylose lysine Deoxycholate Agar and incubate in
inverted condition in incubator at 30o to 35o for 24 to 48 hrs. If red colonies with or without
tests.
H2S Production
Inoculate suspect colonies of Salmonella in triple sugar agar from Xylose lysine
Deoxycholate agar, incubate the tubes at 30 to 35°C for 18-24 hrs. If black coloration along
the line of stab inoculation observed (due to H2S production), it indicates the presence of
Salmonella.
Interpretation of the results
If there is no growth of colonies with black center and in 48 hrs. the colonies become
uniformly black surrounded by a dark zone and metallic sheen and identification tests are negative it indicates absence of Salmonella and the sample passes the test.
Positive Control
Carryout a control test using 0.1ml of the enrichment culture containing 10 to 50
Salmonella spp., prepared from 24 hrs. old culture. The test is invalid if the result does not
indicate that the control contains Salmonella spp.
Negative Control
Incubate Sterilized media as blank.
• Pseudomonas aeruginosa
Procedure
Streak loop full pretreated sample on the
surface of Cetrimide Agar and incubate in inverted condition in incubator at 30-35oC for
18-72 hrs. If greenish colored colonies are observed, then carry out
the identification test (oxidase) and pigment test.
Pigment Test
Streak respective suspect colonies from the surface of Cetrimide Agar on the surface of
Pseudomonas Agar Medium for detection of fluorescein and Pseudomonas Agar medium
for detection of Pyrocyanin contained in petri dishes. Cover the plate and incubate in an
inverted position at 30-35oC for 48hrs. Examine the colonies (Table 1).
Oxidase Test
Oxidase disc touch in colonies rapidly colour change in blueish within 20 sec. If there is
no changing to blue, the sample meets the requirements of the test for the absence of
Pseudomonas aeruginosa.
Medium | Colony Characteristics | Fluorescence in UV light | Oxidase Test | Gram Strain |
Cetrimide Agar Base | Generally greenish | Greenish | Positive | Negative Rods |
Pseudomonas Agar Medium for detection of Fluorescein | Generally colorless to yellow | Yellowish | Positive | Negative Rods |
Pseudomonas Agar Medium for detection of Pyrocyanin | Generally greenish | Blue | Positive | Negative Rods |
Interpretation of the results
If there is no growth of such type of colonies (table 1) and the identification tests are
negative, it indicates the absence of Pseudomonas aeruginosa and the sample passes
the test.
Positive Control
Carryout a control test by using 0.1ml of the enrichment culture containing 10 to 50
Pseudomonas aeruginosa (MTCC 1688/NCIM 2200. The test is invalid if the result does
not indicate that the control contains Pseudomonas aeruginosa.
Negative Control
Incubate pre incubated Cetrimide Agar media as blank.
Staphylococcus aureus
Procedure
Streak loop full pretreated sample (under the section enrichment of sample 4.9.2) on the
surface of Mannitol-Salt Agar Medium. Incubate the plates in an inverted position at
30-35°C for 18 to 72 hrs. If upon observation yellow or white colonies with yellow zones
are observed carry out the coagulase test.
Coagulase Test
Transfer representative suspect colonies from the agar surface of Mannitol Salt agar to
tube containing 0.5 ml of mammalian, preferably rabbit or horse, plasma with or without
additives. Incubate at 30-35°C and examining the tubes at three hours and subsequently
at suitable intervals up to 24 hours. If coagulation in any degree not observed, the sample
meets the requirements of the test for the absence of Staphylococcus aureus.
Interpretation of the results
If there is no growth of yellow or white colonies with yellow zones and the identification
tests are negative, it indicates the absence of Staphylococcus aureus and the sample
passes the test.
Positive Control
Carry out a control test using 0.1ml of the enrichment culture containing 10 to 50
Staphylococcus aureus (MTCC 737/NCIM 2079). The test is invalid if the result does not
indicate that the control contains Staphylococcus aureus.
Negative Control
Incubate pre incubated Mannitol Salt Agar media as blank.
Transfer 10 ml of pretreatment sample from (4.9.1) in 90 ml Tryptone Soya Broth or
Soyabean Casein Digest Broth with 0.5% Soya lecithin & 4% Tween 20 in screw cap
Glass bottle and mix thoroughly and Incubate at 30-35oC for 48 hrs.
Procedure
Streak a portion of the pretreated sample from (4.13.1) on the surface of Burkholderia
Agar/ MacConkey agar and Incubate in inverted condition in incubator at 30-35°C for 48
to72 hrs.
Interpretation
Growth of colonies indicates the possible presence of objectionable organisms
(including Pseudomonas species; Ralstonia & Burkholderia cepacia). This is confirmed
by identification test. The sample complies with the test if no colonies are present or if
the identification tests are negative.
Positive Control
Carry out a control test using 0.1ml of the enrichment culture containing 10 to 50
Burkholderia cepacia.
Negative Control
Perform negative control using Autoclaved media as blank.
