Title: Microbial Limit Test For Material And Product
Objective: Determination of Microbiological limit test for materials and products as per IP/BP/USP.
2. Principle: This test determines whether materials are free from microbial contamination and precautions to be taken to avoid contamination which adversely affect the material or product quality.
- Procedure:
| Total Aerobic Viable Counts (TAVC) | |
| Method : | By Pour Plate Method |
| Solution A (1:10 dilution): | |
| Aseptically transfer 10 g of sample (10 ml in case of liquid) or (number of tablets equivalent to 10 g weight to 90/100 ml of Buffered sodium chloride peptone solution pH 7.0 or Soyabean casein digest medium. A surface active agent such as 0.1% w/v solution of polysorbate 80 may be added to assist the Suspension of poorly wettable substances (Non fatty samples or which are insoluble in water).
If the product is fatty, then homogenise the 10 g sample with 5g polysorbate 80. If necessary, heat to not more than 400C. Mix carefully and add 85 ml of Buffered sodium chloride peptone solution pH 7.0 or any other suitable diluent. Adjust the volume to 100 ml with same diluent. If the product is containing antibiotic properties carry out dilutions as follow. Aseptically transfer 10 g of sample (10 ml in case of liquid/10 g in case of ointment) or (number of tablets equivalent to 10 g weight) to 40 ml of Buffered sodium chloride peptone solution pH 7.0 or soyabean casein digest medium. A surface active agents such as 0.1% w/v solution of polysorbate 80 may be added to assist the suspension of poorly wettable substances. Transfer 5 ml of homogenate and dilute to 10 ml with same solvent. If necessary heat the solution to not more than 40°C with intermittent shaking but not more than 30 mins. |
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| 4 Total Aerobic Microbial Counts (TAMC) | |
| Transfer 1 ml of Solution A i.e 1:10 dilution separately into 2 sterile Petri dishes. Pour about 15 to 20 ml of Soyabean casein digest agar, cooled at about 45°C. Swirl the plate to mix sample and medium. Allow the plates to solidify at room temperature and incubate the plates in inverted position at 30°C to 35°C for 3 to 5 days. After completion of the incubation period count the number of colonies and express the average for the two plates in terms of the number of micro organisms per ml or gram of sample. | |
| Interpretation of Results | |
| If no microbial colonies are recovered from the dishes representing the initial 1:10 dilution of the sample, express the results as “less than 10 CFU per ml or gram of sample”.
Calculation (TAMC) CFU/ml = Average number of colonies x dilution factor. The total aerobic viable 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 a part of TAMC. |
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| Total Yeast & Mould Count (TYMC) Or Total Fungal Count (TFC) | |
| Transfer 1ml of Solution A i.e. 1:10 dilution separately into 2 sterile Petri dishes. Pour about 15 to 20 ml of Sabourauds dextrose agar with or without antibiotic. Cooled at about 45°C. Swirl the plate to mix sample and medium. Allow the plates to solidify at room temperature and incubate the plates in inverted position at 20°C to 25°C for 5 to 7 days. After completion of the incubation period count the number of colonies and express the average for the two plates in terms of the number of microorganisms per ml or gram of sample. | |
| Interpretation of Results | |
| If no microbial colonies are recovered from the dishes representing the 1:10 dilution of the sample, express the results as “less than 10 CFU per ml or gram of sample”.
Calculation (TFC /TYMC) CFU/ml = Average number of colonies x dilution factor. The total Fungal Count (TFC) or Total Yeast & Mould Count (TYMC) is considered to be equal to the number of CFU found using Sabourds Dextrose agar with antibiotic. If colonies of bacteria are detected on Sabourds Dextrose agar without antibiotic medium they are counted as a part of TFC/TYMC. |
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| Test for Specified Microorganisms: | |
| Preparation of Solution B: | |
| Transfer 10 ml of Solution A in 90/100 ml of Sterile Soyabean Casein Digest Medium, mix and incubate at 30°C to 35°C for 18-24 hours (Solution B) | |
| Test for Escherichia coli | |
| Primary Test | |
| Inoculate about 1ml from Solution B to 100 ml of MacConkey broth. Incubate at 42°C to 44°C for
24 to 48 hours. |
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| Selection and
Subculture: |
Take a loopful from MacConkey broth and streak on MacConkey agar.
Incubate at 30°C to 35°C for 18 to 72 hours. |
| Interpretation: | Growth of colonies indicates the possible presence of E. coli. Confirm the results by performing the identification tests or Confirmatory test.
(Pink, non – mucoid colonies indicates the presence of E. coli.) |
| Identification Tests / Confirmatory test: | |
| Indole test: | Inoculate the few colonies obtained on MacConkey agar in 5 ml of peptone Water and incubate at 42.0°C to 44.0°C for 24 hours. Add 0.5 ml of Kovac’s reagent into peptone water tubes. Shake well and allow to stand for one minute. If red colour ring is produced in reagent layer, Indole is present Confirms the presence of E. coli. |
| Acid and Gas
Production: |
Inoculate the few colonies grown on MacConkey agar in 5ml of MacConkey broth containing inverted durhams tube and incubate at 42.0°C to 44.0°C for 24 hours. Change in color of the broth from purple to yellow and presence of gas
in Durhams tube confirms the presence of E. coli. |
| Test for Salmonella | |
| Preparation of Solution C | |
| Aseptically transfer 10 g of sample (10 ml in case of liquid/10 g in case of ointment) or (number
of tablets equivalent to 10 g weight) to 90/100 ml of Soyabean casein digest medium (Solution C). A surface active agents such as 0.1% w/v solution of polysorbate 80 may be added to assist the Suspension of poorly wettable substances. Mix and incubate at 30°C to 35°C for 18 to24 hours. |
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| Primary Test | |
| Inoculate about 0.1 ml from Solution C to 10 ml of Rappaport vassilliadis salmonella Enrichment broth. Incubate at 30°C to 35°C for 18 to 24 hours. | |
| Selection and
Subculture: |
Take a loopful from Rappaport vassilliadis salmonella enrichment broth and streak on a Xylose Lysine Deoxycholate agar. Incubate at 30°C to 35°C for 18 to
48 hours. |
| Interpretation: | Growth of colonies indicates the possible presence of Salmonella. Confirm the
results by performing the identification tests or confirmatory test. (Well developed Red colonies with or without black center indicates the presence of salmonella.) |
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| Identification Tests / Confirmatory test | ||||||
| Inoculate the few colonies to triple sugar iron agar by inoculating surface of slope first and then
Stabbing butt using deep inoculation and incubate at 30°C to 35°C for 24 to 48 hours. If tubes have alkaline (pink) slants and acidic (yellow) butts with or without production of hydrogen sulfide gas confirms the presence of Salmonella. |
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| Test for Shigella boydii | ||||||
| Preparation of Solution C( Use same sample as used for Salmonella) | ||||||
| Aseptically transfer 10 g of sample (10 ml in case of liquid/10 g in case of ointment) or (number
of tablets equivalent to 10 g weight) to 90/100 ml of Soyabean casein digest medium (Solution C). A surface active agents such as 0.1% w/v solution of polysorbate 80 may be added to assist the Suspension of poorly wettable substances. Mix and incubate at 30°C to 35°C for 18 to 24 hours. |
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| Primary Test | ||||||
| Inoculate about 0.1 ml from Solution C to 10 ml of GN Broth (Medium 11). Incubate at 30°C to 35°C for 18 to 24 hours. | ||||||
| Selection and
Subculture: |
Take a loopful from GN Broth (Medium 11) and streak on a Xylose Lysine Deoxycholate agar. Incubate at 30°C to 35°C for 18 to 48 hours. | |||||
| Interpretation: | Growth of colonies indicates the possible presence of Shigella. Confirm the
results by performing the identification tests or confirmatory test. (Well developed Red colonies without black center indicates the presence of shigella.) |
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| Identification Tests / Confirmatory test | ||||||
| Inoculate few colonies to Triple sugar iron agar by inoculating surface of slope first and then
Stabbing butt using deep inoculation and incubate at 30°C to 35°C for 24 to 48 hours. If tubes have alkaline (pink) slants and acidic (yellow) butts without production of hydrogen sulfide gas confirms the presence of Shigella boydii. |
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| Test for Pseudomonas aeruginosa | ||||||
| Selection and
Subculture: |
Streak a loopful of Solution B on Cetrimide agar. Incubate the plates between
30°C to 35oC for 18 to 72 hours. |
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| Interpretation | Growth of colonies indicates the possible presence of Psuedomonas aeruginosa. Confirm the results by performing the identification tests.
(Growth of Greenish colour colonies indicates the presence of P. aeruginosa.) |
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| Identification Tests / Confirmatory test | ||||||
| Oxidase test: | place 2 or 3 drops of freshly prepared 1%w/v solution of N, N, N’, N’, tetraethyl-
4-phenylene-diamine dihydrochloride on filter paper and smear with colonies obtained on cetrimide agar. Pink colour develops which change to purple. |
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| Test for Staphylococcus aureus | ||||||
| Selection and
Subculture: |
Streak a loopful of Solution B on Mannitol Salt Agar. Incubate the plate at 30°C to 35°C for 18 to 72 hours. | |||||
| Interpretation: | Growth of colonies indicates the possible presence of Staphylococcus aureus. Confirm the results by performing the identification tests or confirmatory test
(Yellow or white colonies surrounded by yellow zone indicates the presence of S. aureus. ) |
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| Identification Tests / confirmatory Test | ||||||
| Coagulase test: | Transfer individual colonies to tubes containing 0.5 ml coagulase plasma.
Incubate the tubes in a water bath at 37°C, examining the tubes after 3 hours and subsequently at suitable interval up to 24 hours. The formation of clot confirms the presence of S. aureus. |
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| Test for bile tolerant gram negative bacteria (Quantitative Test) | ||||||
| Transfer 10 g of the sample in 90 ml of sterile soyabean casein digest medium(0.1g/ml),(Solution D)
Homogenize this homogenate and incubate at 20°C to 25OC for 2 to 5 hours but not more than 5 hours to resuscitate the organisms. Transfer 1ml of 0.1g/ml solution in 9 ml of soyabean casein digest medium (0.01g/ml). Further transfer 1ml of 0.01 g/ml Solution in 9 ml of soyabean casein digest medium(0.001g/ml). |
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| Primary test | Inoculate 9 ml of Enterobacteria Enrichment broth–Mossel with each 1ml
Dilutions containing 0.1 g, 0.01 g, 0.001g of the sample. Incubate these tubes at 30°C to 35°C for 24 to 48 hours. |
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| Selection and
Subculture: |
Subculture from each of these three tubes onto violet red bile glucose agar plates and incubate at 30°C to 35°C for 18 to 24 hours. | |||||
| Interpretation: | Growth of red or reddish colonies of gram negative bacteria gives a positive result. Note the smallest quantity of product, which gives a positive result, and
the largest quantity that gives a negative result. Determine the probable number of bacteria per gram of product from the given table. |
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| Results for each quantity of products | Probable number of bacteria / 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 | ||||
| + | + | + | More than 103 | |||
| + | + | – | Less than 103 and more than 102 | |||
| + | – | – | Less than 102 and more than 10 | |||
| – | – | – | Less than 10 | |||
| Controls | ||||||
| Simultaneously perform a negative and positive control. | ||||||
| Refer SOP for Serial dilution preparation of Microbial cultures. | ||||||
| Inoculate NMT 100 CFU/0.1 ml of the specific culture for enumeration media & for Selective media
by using surface spread technique for solid media & observe the colony character & follow as per SOP for Identification and Purity Verification of Master Microbial Cultures. For liquid media inoculate with NMT 100 CFU/0.1 ml of specific culture & check for turbidity or colour change. |
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| Interpretation: | ||||||
| Positive Control | ||||||
| Growth obtained on solid media must not differ by a factor greater than 2 from the calculated value
For standardized inoculums. In case of solid selective media growth of typical colonies should observe. in case of Liquid selective media typical reactions should occur by showing colour change. |
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| Negative Control | ||||||
| Should not show any growth on solid media and turbidity or colour change in case of liquid media. | ||||||