SOP for Microbial Limit Test (MLT) of Non sterile products in pharmaceuticals
1.0 Purpose:
To lay down a procedure for Microbial Limit Test of non sterile products.
2.0 Scope:
This procedure is applicable for Microbial Limit Test of non sterile products in microbiology Laboratory.
3.0 Responsibilities:
Microbiologist : To perform the procedure of sample analysis and observe the results as per SOP.
Head-QC : To review and implementation and ensure compliance of SOP.
Head-QA : To approve, implement and ensure compliance of SOP.
4.0 Procedure:
4.1 Sample preparation:
Dissolve 10 gm or 10 ml of the product in 90 ml sterile phosphate buffer pH 7.2 / sterile buffered sodium chloride peptone solution pH 7.0 / soyabean casein digest medium (usually 1 in 10 dilution). Mix well and allow standing for 10-15 minutes at room temperature.
4.1.2 Water insoluble Non Fatty products:
Dissolve 10 gm or 10 ml of the product in 90 ml Sterile Phosphate buffer pH 7.2 / Sterile Buffered Sodium Chloride Peptone Solution pH 7.0 / Soyabean Casein Digest Medium with Polysorbate 20 (4.0%) (usually 1 in 10 dilution). Mix well and allow standing for 10-15 minutes at room temperature.
4.1.3 Fatty Products:
Homogenize 10 gm or 10 ml of the product in 90 ml Sterile Phosphate buffer pH 7.2 / Sterile Buffered Sodium Chloride Peptone Solution pH 7.0 / Soyabean Casein Digest Medium with Polysorbate 20 (4.0%) and mix well (usually 1 in 10 dilution). If necessary Sterile Buffered Sodium Chloride Peptone Solution pH 7.0 / Soyabean Casein Digest Medium subject the contents of the tube for warming to a temperature not exceeding 45°C.
4.1.4 Fluids or solids in aerosol form:
Aseptically transfer the product into a membrane filter apparatus or a sterile container for further sampling. Use either the total contents or a defined number of metered doses from each of the containers tested.
4.1.5 Transdermal patches:
Remove the protective cover sheets ('release liners') of the transdermal patches and place them, adhesive side upwards, on sterile glass or plastic trays. Cover the adhesive surface with a sterile porous material, for example sterile gauze, to prevent the patches from sticking together, and transfer the patches to a suitable volume of the chosen diluent containing inactivators such as polysorbate 80 and/or lecithin. Shake the preparation vigorously for at least 30 min.
4.2 Test for Total Aerobic Microbial count (TAMC) and Total Yeast and Mould count (TYMC):
4.2.1 Pour-plate method:
4.2.1.1 Aseptically transfer 1ml of pretreated sample into four different petridish (2 of SCDA and 2 of SDA), which is previously labeled and kept under Laminar Air Flow.
4.2.1.2 Pour approximately 15-20 ml of SCDA (in 2 plates) and SDA (in 2 plates) media into the respective plates and rotate the plates clockwise and anti clockwise to mix the contents and allow to solidify.
4.2.1.3 Incubate the SCDA plates at 30-35oC for 3-5 days & SDA plates at 20–25oC for 5-7 days.
4.2.1.4 Positive Control: Add known volume of cells (10-100 CFU/ml) from one of the mention organisms S. aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Bacillus subtilis ATCC 6633, in each plate & pour 15-20 ml SCDA. Gently rotate the plates for the even distribution of cultures and allow solidifying. Incubate the plates of bacterial cultures at 30-35oC for 2-3 days.Add known volume of cells 10-100 CFU/ml of Candida albicans ATCC 1023 and Aspergillus brasiliensis ATCC 16404 in each plate & pour 15-20 ml SDA. Gently rotate the plates for the even distribution of cultures and allow solidifying. Incubate the plates at 20–25oC for 3-5 days.
4.2.1.5 Negative Control: Pour the SCDA / SDA Media in pre-sterilized and labeled plates without sample and allow solidifying.
4.2.1.6 The incubated plates must be monitored every 24 hours. Record the Observation.
4.2.1.7 Interpretation:Count the colonies produced and report the results as CFU/total volume of sample tested. Negative Control plate must not show any CFU. Positive Control plate must show luxuriant growth within 48-72 hours of incubation for bacteria and 72-120 hours for fungi. The mean of the two plates calculated with dilution factor and quantity of sample finally expressed as CFU/ gm of Sample.CFU/gm/ml = Average No. of Colonies X Dilution Factor Volume of sample tested.The total aerobic microbial count (TAMC) is considered to be equal to the number of CFU found using soybean casein digest agar (SCDA); if colonies of fungi are detected on this medium, they are counted as part of the TAMC. The total combined yeasts/mould count (TYMC) is considered to be equal to the number of CFU found using Sabouraud dextrose agar (SDA); if colonies of bacteria are detected on this medium, they are counted as part of the TYMC. When the TYMC is expected to exceed the acceptance criterion due to the bacterial growth, Sabouraud dextrose agar containing antibiotics may be used.
4.2.2 Membrane Filtration Method:
4.2.2.1 Aseptically transfer 1ml of pretreated sample through 0.45 µm pore size membrane having 47mm diameter. Rinse the membrane with minimum three 100ml portions of 0.1% peptone water. Number of rinses can be increased as per the validation report for the product up to 2 to 3 times.
4.2.2.2 With the help of sterile forceps transfer the membrane to the surface of pre-incubated Soybean Casein Digest Agar (SCDA) plate for determination of total aerobic microbial count (TAMC).
4.2.2.3 Repeat as mentioned above and transfer the membrane to the surface of pre-incubated Sabouraud Dextrose Agar (SDA) for the determination of total combined yeast and mold (TYMC).
4.2.2.4 Incubate the SCDA plate at 30-35°C for 3 to 5 days and SDA plate at 20-25°C for 5 to 7 days.
4.2.2.5 Positive Control: Aseptically filter 100 ml of BSCP/ sterile 0.1% of Peptone water, which previously inoculated with one of mention organism 10 to 100 cells of S. aureusATCC 6538, Pseudomonas aeruginosa ATCC 9027, Bacillus subtilisATCC 6633 and place the filter paper on pre incubated SCDA plates. Incubate the plates of bacterial cultures at 30–35oC for 3 days.
4.2.2.6 Negative Control: Aseptically filter 100 ml quantity of BSCP/ Sterile 0.1% Peptone Water by the same procedure followed for the sample & incubate the SCDA plate at 30 -35oC for 3-5 days & SDA plate at 20-25oC for 5-7 days.
4.2.2.7 The incubated plates must be monitored every 24 hours. Record the observation.
4.2.2.8 Interpretation:Count the colonies produced and report the results as CFU/total volume of sample tested. Negative Control plate must not show any CFU. Positive Control plate must show luxuriant growth within 48-72 hours of incubation for bacteria and 72-120 hours for fungi. The mean of the two plates calculated with dilution factor and quantity of sample finally expressed as CFU/ gm of Sample.CFU/gm/ml = Average No. of Colonies X Dilution Factor Volume of sample tested.
4.3 Tests for Pathogens:
Take 10ml of early prepared sample of product and inoculate it in to 90ml Soybean Casein Digest Medium (SCDM). Incubate at 30-35oC for 18-24 hours.
4.3.2 Test for Escherichia coli:
4.3.2.1 Primary Test:
Observe the above SCDM tube for growth. If growth is present, aseptically pipette 1mlaliquot of the inoculated SCDM into 100ml of MacConkey broth. Incubate at 42-44°C for 24-48 hours. Examine broth for the growth in terms of turbidity. If turbidity found carry out Secondary Test.
4.3.2.2 Secondary Test:
Using a sterile loop, subculture a portion of the inoculated MacConkey broth, onto the surface of a MacConkey agar plate. Incubate at 30-35°C for 18-72 hrs. If upon examination, the plates containing colonies having characteristic brick red in color, have surrounding zone of precipitated bile found then carry out Confirmative Test-Growth characteristics on EMB agar & Indole test.
4.3.2.3 Confirmative Test (Growth on EMB Agar and Indole Test):
· Streak representative colonies from MacConkey agar plate & subculture on the surface of EMB Agar plate.· Same time inoculate 1ml enriched culture from MacConkey Broth inoculate in to 5ml of Peptone Water for indole production.· Incubate EMB plate & Peptone water tube at 30-35°C for 24-48 hrs.· If upon examination, EMB Agar plates containing colonies having Metallic Sheen under reflected light and Blue-Black appearance under transmitted light found, Escherichia coli is present.4.3.2.4 Positive control: Confirm the above tests by carrying out the test simultaneously using 24 hours old broth culture of E.coli. ATCC 8739 or NCIM 2065. The test is invalid if the results do not indicate that the control contains E.coli.
4.3.3 Test for Pseudomonas aeruginosa:
4.3.3.2 Confirmative Test (Oxidase Test):
4.3.3.3 Positive control: Confirm the above tests by carrying out the test simultaneously using 24 hrs old broth culture of Pseudomonas aeruginosa ATCC 9027 or NCIM 2200. The test is invalid if the results do not indicate that the control contains Pseudomonas aeruginosa.
4.3.4 Test for Staphylococcus aureus:
4.3.4.1 Primary Test:Observe the above SCDM tube for growth, if growth is present, streak a portion of the medium on the surface of Mannitol salt agar medium aseptically. Cover the Petri plates and incubate at 30-35°C for 48 hrs. If upon examination, the plates containing colonies having Yellow color with yellow zones found carry out Confirmative Test (Coagulase test).4.3.4.2 Confirmatory Test (Coagulase Test):
· Transfer representative suspected colonies from the agar surface of the media to individual tube.· Each containing 0.5ml of mammalian, preferably rabbit or horse plasma with or without additives.· Incubate the tube in water bath at 37°C. Examine tubes after 3 hrs to suitable intervals up 48 hrs.· If no coagulation occurs, confirm the absence of Staphylococcus aureus.· The entire test are positive, indicate the presence of Staphylococcus aureus.4.3.4.3 Positive control: Confirm the above tests by carrying out the test simultaneously using 24 hrs old broth culture of Staphylococcus aureus ATCC-6538. The test is invalid if the results do not indicate that the control contains Staphylococcus aureus.
4.3.5 Test for Salmonella abony:
4.3.5.2 Secondary Test (Growth on XLD agar):
· Secondary test is done on Xylose Lysine Deoxycholate (XLD) agar. XLD should not be autoclaved. It should be heated with frequently agitation until medium boil. Transfer immediately to a water bath at 50°C & after that pours the plates.· Streak a portion of the medium (RVSEB) on the surface of XLD agar medium aseptically.· Incubate all the plates at 30-35°C for 18-24 hrs. If the specific colonies having Red, with or without black center found, carry out Confirmative Test.4.3.5.3 Confirmative Test:
· Subculture any colonies from XLD agar in Triple Sugar Iron (TSI) by first streaking the surface of the slope & making a stab culture with the same inoculating needle & incubate at 30-35°C for 18 to 24 hour.· Forming alkaline (Red) slant & acid (Yellow) butt with or without blackening of butt (H2S) production, indicate the presence of Salmonella.· If the entire tests are positive, indicate the present of Salmonella.4.3.5.4 Positive control: Confirm the above tests by carrying out the test simultaneously using 24 hrs old broth culture of Salmonella abony NCTC 6017 or NCIM 2257. The test is invalid if the result does not indicate that the control contains Salmonella.
4.3.6 Test for Bile-Tolerant Gram-Negative Bacteria:
4.3.6.2 Qualitative test:· After incubation transfer 10ml from it into 100ml Enterobacteria Enrichment Broth –Mosel & incubate at 30-35oC for 24-48 hours.
4.3.6.3 Quantitative test:· Take three tubes containing 100mL of sterile Enterobacteria enrichment broth-Mossel. Mark the tubes with A, B, and C.
Result of each quantity of Product
|
Probable number of Bacteria per gram or ml of Product
| ||
0.1g / 0.1ml
|
0.01 g/
0.01 ml |
0.001g/ 0.001ml
| |
+
|
+
|
+
|
> 103
|
+
|
+
|
-
|
< 103 and > 102
|
+
|
-
|
-
|
< 102 > 10
|
-
|
-
|
-
|
<10
|
4.3.7 Test for Clostridia sporogenes:
4.3.7.1 After preparing sample (see sample preparation step), take 10ml of sample into two sterile tubes. Heat one tube at 80°C for 10 minutes & cool rapidly. Do not heat other tube.4.3.7.2 Inoculate separately both tube into separate 100ml reinforced medium & incubate under anaerobic conditions at 30-35°C for 48 hours. After incubation streak it on preincubated Columbia agar and incubate under anaerobic conditions at 30-35°C for 48-72 hours.
4.3.7.3 The occurrence of anaerobic growth (with or without endospore) giving a negative catalase reaction indicates presence of clostridia. This is confirmed by further biochemical identification tests.
4.3.7.4 The product passes the test if colonies of the described type are not present or if the confirmatory identification test is negative.
4.3.7.5 Confirmatory Test (Catalase Test): Transfer the colony from the plate to the glass slide. Add a drop of dilute hydrogen peroxide solution. The formation of gas bubbles indicates a positive catalase reaction.
4.3.8 Test for Candida albicans:
4.3.8.1 After preparing sample (see Sample preparation step), aseptically transfer 10 ml of sample into 100 ml of Sabouraud Dextrose Broth and mix well. Incubate it at 30-35°C for 3-5 days.4.3.8.2 After incubation streak on the surface of preincubated Sabouraud Dextrose Agar and incubate at 30-35°C for 24-48 hours.
4.3.8.3 Growth of white colonies may indicate the presence of C. albicans.
4.3.8.4 Confirmatory Test: Streak loopful colony from SDA plate and streak on Bi.G.G.Y agar. This gives chocolate brown color colony of C. albicans.
4.3.8.5 The product passes the test if such colonies are not present or if the confirmatory test is negative.
4.3.9 Interpretation of Pathogen Test:If all pathogenic test are negative then it shows the absence of Bile tolerant gram-negative bacteria, E.coli, Salmonella, S. aureus, P. aeruginosa, Clostridia & C. albicans then the test of pathogens expressed as Absence of pathogen per gm or ml of product. (For salmonella, absent per 10gm/10ml)
5.0 Abbreviation:ATCC- American Type Culture CollectionNCTC-National Culture Type Collection
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