Microbiological examination of Non sterile products
This Document describes (Standard Operating procedure) Sop for Microbiological examination of Non sterile products
Sop for Microbiological examination of Non sterile products
I. Purpose & Scope:
- The purpose of this SOP is to lay down the procedure for Microbiological examination of Non sterile products.
- This standard operating procedure is applicable for Microbiological examination of non sterile products.
II. Responsibilities:
- All Quality Control and Quality Assurance personnel’s shall be responsible to follow and implement this SOP.
III : Introduction and Procedural Part :
Precautions shall be taken during Microbiological examination of Non sterile products :
- Before and after the each analysis, Laminar Air Flow (LAF) workplace should be clean with 70% IPA.
- Analysis and negative controls shall be performed in LAF of Microbiology Testing Area. Positive controls and culture handling related activities shall be performed in LAF of Culture Handling Area. Preferably perform the analysis of samples earlier and then perform the positive controls & culture related activities to avoid cross- contamination.
- Positive control shall be performed for each sterilized media lot to be use for analysis.
- After usage of the pipette tips, put them into 2.5% Dettol or 2.5% Savlon for decontamination purpose.
- After completion of Incubation and observation, plates or contaminated media should be kept in dedicated SS bin/SS Trolley for decontamination purpose and shall be destructed as per procedure given in SOP.
Preparation of sample for microbiological examination of non sterile products :
- The method for sample preparation depends on the physical characteristics of the product or material to be tested.
- On the day of analysis, before starting the analysis, make entry of sample in MLT analysis register. Refer Annexure- I.
- Unless until specified, analysis should be carried out on 10 grams or 10 ml of the sample.
- Take required number of tablets or capsules to add specified amount of sample into an appropriate diluent. For tablets and capsules, weight of sample shall be done by considering average weight of given product.
- For creams and gels, weigh the sample on analytical balance in the sterile bottles. These bottles shall be transferred to Microbiology testing area, where required sterile diluent shall be added into these bottles aseptically.
- Unless until specified, sample solution shall be prepared in a sterile diluent and ratio should be of 1 in 10 dilutions.
- If required prepare media with surfactants as supplements to facilitates homogenization of sample solution. Use the neutralizers as supplements in media, in case of samples having antimicrobial properties.
- Depending upon nature of sample use appropriate medium for sample solution preparation.
- For water-soluble products: Dissolve or suspend (usually1 in 10 dilution) the product to be examined in Buffered Sodium Chloride Peptone solution pH 7.0 or Soybean Casein Digest Medium (SCDM) or if antimicrobial activity present in product use Soybean Casein Digest Medium with Tween 80 and Lecithin (SCDMTL). If necessary adjust the pH 6 to 8.
- For products insoluble in water (non fatty): Suspend the product to be examined (usually 1 in 10 dilution) in Buffered Sodium Chloride Peptone solution pH 7.0 or Soybean casein digest medium containing a suitable surface-active agent such as 1g/lit of Polysorbate80. If necessary, homogenize the suspension mechanically. If necessary adjust the pH 6 to 8.
- For fatty products insoluble in water: Suspend the product to be examined (usually 1 in 10 dilution) in Soybean Casein Digest Medium with minimum 3%w/w of Polysorbate80 or other suitable surface active agent.
- Give minimum about 10 minutes contact time to product and medium.
- In case of capsules and creams if required, prepared dilution of sample solution should be kept at 40 to 45°C for about 30 minutes, for proper mixing of sample.
Microbiological examination of Non sterile products ( Enumeration Method) :
Membrane Filtration Method :
-
-
- For Membrane filtration method, use sterile filtration assembly, sterile membrane filter paper having nominal pore size not greater than 0.45µm.The type of filter paper material is chosen in such a way that bacteria retaining efficiency is not affected by component of the filter.
- Transfer suitable quantity of the sample prepared, as described under preparation of the sample, to the filtration cup, filter immediately. Rinse the membrane with appropriate volume of diluent.
- For determination of Total Aerobic Microbial Count, transfer membrane filter to the surface of Soybean casein digest agar (SCDA) Plate. For determination of Total combined Yeast & Mould Count, transfer membrane filter to the surface of Sabouraud Dextrose agar (SDA)/ Sabouraud Chloramphenicol Agar (SCA).
- Incubates the SCDA Plate at 30 to 35ºC for 3 to 5days and SDA/SCA Plate at 20 to 25 ºC for 5 to 7days.
- Calculate and record the results as cfu/g or cfu/ml.
-
Plate count method ( Pour plate method):
-
-
- Perform the plate count method in duplicate and use the mean count of both plates as a result.
- Add 1ml of the sample solution (Solution A) prepared as described under preparation of the sample, to the sterile petri plates in duplicate.
- Add approximate 15 to 20 ml of sterile molten agar media, Soybean casein digest agar (SCDA) for determination of Total aerobic microbial Count (TAMC) and Sabouraud Dextrose agar (SDA)/ Sabouraud Chloramphenicol agar (SCA) for determination of Total yeast and mould Count (TYMC).
- Temperature of the molten media should not be more than 45ºC. If larger Petri plates are used, the amount of agar medium should increased accordingly.
- Swirl the content of plates carefully in such a way that media should not touch the lid of the plate.
- Prepare negative control plate by using same media but without sample.
- Prepare another plate in same manner for positive control. After solidifying the plate, inoculate with microbial culture by spread plate method.
- Use 10 to 100 cfu of subtilis for inoculation of positive control of SCDA. Use 10 to 100 cfu of C.albicans for inoculation of positive control of SDA/SCA plates.
- After solidifying the plates, Incubate SCDA Plates at 30-35ºC for 3 to 5 days and SDA/SCA Plates at 20-25 ºC for 5-7 days in invert position.
- Examine the plates after incubation, count the number of colonies and calculate result by using dilution factor. Calculate the mean count of colonies from two plates.
-
Tests for specified Micro-organisms :
Enrichment/Pretreatment:
Proceed as described under the Preparation of the sample, this solution should be referred as ‘Solution A’. Transfer quantity corresponds to 1 gram or 1 ml of the product sample from ‘Solution A’ into 90 ml of Soybean Casein Digest Medium, this solution should be referred as Solution B. Then incubate ‘Solution B’ at 30 to 35°C for 18 to 24 hours. After Completion of incubation/enrichment, Solution A can be used for test for Salmonella species. Solution B is to be used for test for Escherichia coli, Pseudomonas aeruginosa , and Staphylococcus aureus.
Tests for Escherichia coli :
- Incubate/enrich the ‘Solution B’ at 30 to 35°C for 18 to 24 hours.
- After incubation, Transfer 1 ml enriched sample from ‘Solution B’ to 100 ml of MacConkey’s broth and incubate at 42 to 44 °C for 24 to 48 hours.
- After incubation, Subculture the enriched MacConkey’s broth by streaking on plates of MacConkey’s Agar. Incubate plates at 30 to 35°C for 18 to 72 hours.
- Interpretation: Growth of colonies (Brick red, non mucoid colonies) indicates the possible presences of Escherichia coli.
- The presence of colonies should be confirmed by identification tests. Transfer suspected colony by means of inoculating loop, to the surface of Levin eosin ethylene blue (EMB) agar medium and incubate at 30-35°C for 24 to 48 hours.
- The product complies with the test if colonies of the type described in Table-I are not present or if the identification tests are negative.
Table-I
| Name of Media | Description of Colony |
| MacConkey’s Agar
(Selective agar) |
Brick red ,non mucoid colonies |
| EMB Agar
(Confirmative test) |
Colonies with green metallic sheen |
- Negative controls: Incubate 100 ml of sterile SCDM or diluent of same media lot used for sample preparation at 30°C to 35°C for 18 to 24 hours. Incubate 100 ml sterile MacConkey’s Broth at 42° C to 44° C for 24 to 48 hours. Incubate an uninoculated plate of MacConkey’s Agar at 30°C to 35°C for 18 to 72 hours. There must be no growth of microorganisms in negative control.
- Positive control: Inoculate not more than 100 cfu Culture of subtilis (ATCC6633) to Soybean Casein Digest Broth; incubate at 30°C to 35°C for 18 to 24 hours. Inoculate not more than 100 cfu of E.coli in MacConkey’s broth and incubate at 42°C to 44°C for 24 to 48 hours, from positive control of MacConkey’s broth streak on MacConkey’s agar and incubate at 30°C to 35°C for 18 to 72 hours.
Tests for Salmonella:
- For testing of salmonella, add the product/sample quantity corresponding to not less than 10g or 10ml into the suitable amount of Soybean Casein Digest Medium. If necessary prepare ‘Solution C’ for test of Salmonella species by using suitable diluent. Incubate at 30 to 35°C for 18 to 24 hours.
- After incubation, transfer 0.1 ml enriched sample in 10ml Rappaport vassiliadis salmonella enrichment broth (RVSEB) and incubate at 30° to 35°C for 18-24 hours.
- After incubation, Subculture on plate of Xylose Lysine Deoxycholate Agar and incubate at 30°C to 35°C for 24 to 48 hours and observe for any growth of colonies.
- Interpretation: Growth of well developed red colonies with or without black centers indicates the possible presence of Salmonella, should be confirmed by identification tests.
- The product complies with the test if, colonies of the types described in Table-II are not present, or if the confirmatory identification tests are negative.
- Negative control: Incubate the uninoculated Rappaport Vassiliadis Salmonella enrichment broth at 30 to 35ºC for 18-24 hours and Xylose Lysine Deoxycholate Agar plate at 30° C to 35°C for 24 to 48 hours. There must be no growth of microorganisms in negative control.
- Positive control: Inoculate not more than 100cfu Culture of Salmonella abony culture to Rappaport vassiliadis salmonella enrichment broth. Incubate at 30° to 35° for 18 to 24 hours. From the positive control of RVSEB, streak on Xylose Lysine Deoxycholate Agar and Incubate at 30° C to 35°C for 24 to 48 hour as positive control.
- If colonies conforming to the description in the table-II are seen, carry out secondary test (Confirmatory/Identification test).
- Subculture any colonies showing the characteristics given in the table-II on Triple Sugar Iron Agar slant by first inoculating the surface and then making a stab culture with the same inoculating needle. Incubate at 30 to 35° C for 24 to 48 hours.
Table-II
| Name of Media | Description of colony |
| Xylose Lysine Deoxycholate Agar
(Selective agar) |
Red colonies with or without black centers. |
| Triple sugar iron agar (Confirmative test) | Red(alkaline) slant and acid( yellow) butt with H2S gas production |
Tests for Pseudomonas aeruginosa :
- Incubate/enrich the ‘Solution B’ at 30 to 35°C for 18 to 24 hours.
- Selection and subculture: After incubating the Solution B, subculture on a plate of Cetrimide agar and incubate at 30° to 35° for 18 to 72 hours.
- Interpretation: Growth of colonies indicates the possible presence of Pseudomonas aeruginosa. This should be confirmed by identification test.
- Negative control:-Incubate 100 ml sterile Soybean Casein Digest Broth at 30° C to 35°C for 18 to 24 hours. Incubate uninoculated plate of Cetrimide agar at 30° C to 35° C for 18 to 72 hours. There must be no growth of microorganisms.
- Positive Control: – Inoculate the Pseudomonas aeruginosa culture on the plate of Cetrimide agar plate. Incubate at 30° C to 35° C for 18 to 72 hours and observe for growth.
- The product complies with the test if, colonies of the types described in Table-III are not present, or if the confirmatory identification tests are negative.
- If any colonies developed on cetrimide agar conforming to the description in the below table-III, carry out the oxidase and pigment test.
- Streak representative suspect colonies from the surface of Cetrimide agar on the surface of Pseudomonas Agar for Fluorescin (PAF) for detection of Fluorescein and Pseudomonas agar for Pyocyanin (PAP) for detection of Pyocyanin.
- Cover and invert the agar plates and incubate at 30°C to 35°C for not less than 3 days.
Table-III
| Name of Media | Description of colony |
| Cetrimide agar | Greenish Colored Colonies. |
| Oxidase disc
(Confirmative test) |
Deep Purple colour observed on disc |
| Psedomonas agar for Flurescin
(Confirmative test) |
Yellowish colonies under UV light |
| Psedomonas agar for Pyocyanin
(Confirmative test) |
Greenish colonies under UV light |
Tests for Staphylococcus aureus :
- Incubate/enrich the ‘Solution B’ at 30 to 35°C for 18 to 24 hours.
- Selection and subculture: After incubating the Solution B, subculture on a plate of Mannitol Salt agar (MSA) and incubate at 30°C to 35°C for 18 to 72 hours.
- Interpretation: Growth of yellow /white colonies surrounded by a yellow zone indicates the possible presence of Staphylococcus aureus, should be confirmed by identification tests.
- This should be confirmed by Coagulase test. In this test with the aid of inoculating loop transfer the suspected colony to mammalian plasma/Coagulase test kit, incubate it at 37°C; examine tube at 3 hour intervals up to 24 hours.
- If coagulation observed then it predicts that above test is positive for Staphylococcus aureus.
- The product complies with the test if colonies of the types described in Table-IV are not present or if the confirmatory identification tests are negative.
- Negative control: Incubate 100 ml sterile medium of Soybean Casein Digest Broth at 30°C to 35° C for 18 to 24 hours. Incubate uninoculated plate of Mannitol Salt Agar at 30° C to 35° C for 18 to 72hours .There must be no growth of microorganisms.
- Positive Control: Inoculate the Staphylococcus aureus culture on the plate of Mannitol salt agar. Incubate at 30° C to 35° C for 18 to 72 hours and observe for growth.
Table-IV
| Name of Media | Description of colony |
| Mannitol Salt agar
(Selective agar) |
Yellow/white colonies surrounded by yellow zone |
| Coagulase test
(Confirmative test) |
Coagulase/agglutination observed in the tube |
Test for Candida albicans:
- Transfer the quantity corresponds to 1 gram or 1 ml of product sample from ‘Solution A’ in 90ml Sabouraud dextrose broth. Incubate this at 30-35°C for 72 hrs.
- After completion of incubation, streak portion from Sabouraud dextrose broth on the surface of Sabouraud dextrose Agar and incubate at 30-35°C for 24 to 48 hrs.
- Growth of white coloured colonies indicates may indicates presence of Candida albicans, shall be confirmed by identification by microscopic observation.
- An uninoculated Sabouraud dextrose broth (SDB) and Sabouraud dextrose Agar (SDA) plate shall be incubated alongwith test as Negative control.
- Sabouraud dextrose broth (SDB) and Sabouraud dextrose Agar (SDA) plate inoculated with albicans shall be incubated alongwith test as positive control.
Test for Bile tolerant Gram negative Bacteria:
- Using a 1 in 10 dilution of not less than 1 g of the product to casein soya bean digest broth as the chosen diluent, mix and incubate at 20-25 °C for a time sufficient to resuscitate the bacteria usually 2 to 5 hours( not more than 5 hours).
- After incubation, add the volume correspond to 1g of product into suitable amount of Enterobacteria enrichment broth Mossel. Incubate at 30-35°C for 24 to 48 hours.
- Subculture on plates of Violet red bile glucose agar. Incubate at 30 to 35°C for 18 to 24 hours.
- An uninoculated Enterobacteria enrichment broth Mossel and Violet red bile glucose agar plate shall be incubated alongwith test as Negative control.
- Enterobacteria enrichment broth Mossel and Violet red bile glucose agar plate inoculated with coli shall be incubated alongwith test as positive control.
- All analysis details and result should be recorded in Annexure II.
Quantitative Test for Bile tolerant Gram negative Bacteria:
- Inoculate the suitable amount of sample into casein soybean digest broth and incubate it to 20 to 25°c for 2 to 5 hours.
- Inoculate the quantity corresponds to 0.1g, 0.01g and 0.001g of sample into each of three separate tubes containing 10 ml of enterobacteria enrichment broth-Mossel(EEB).
- Incubate all the tubes of EEB at 30 to 35oC for 24 to 48 hrs.
- Subculture each of the solution on plates of violet red bile glucose agar.
- Incubate at 30 to 35oC for 18 to 24 hrs.
- Record the results in Annexure-III
- Interpretation: Growth of colonies constitutes a positive result.
Determine the probable number of bacteria by referring the below table:
| Result of each quantity of Product | Probable number of bacteria / g of product | ||
| 0.1 g | 0.01g | 0.001 g | |
| + | + | + | > 103 cfu |
| + | + | – | < 103 and > 102cfu |
| + | – | – | < 102 and > 10cfu |
| – | – | – | < l0 cfu |
IV: Action to be taken if microbial count exceed the limit :
- On observation of out of limit results, in the samples, Microbiologist shall inform to Head of Quality Control and Quality Assurance.
- After Review the results, Start the Out of Specification investigation as per SOP for further investigation.
V: Annexure :
Annexure I: Microbiological Limit test analysis register
Annexure II : Microbiological examination of non sterile products
Annexure III: Quantitative test for Bile tolerant Gram negative bacteria
ANNEXURE I
Microbiological limit test analysis register
| Date | Name Of Product | Batch
No. |
A.R.
No. |
Mfg.
Date |
Total viable aerobic count | Pathogen test | Completed
On. |
Analysed
By |
Checked By | ||||||
| Total Bacterial count | Total fungal count | E.coli | Salmonella
Sp. |
Staphylococcus aureus | Pseudomonas
aeruginosa |
Entero bacteria | C.
albicans |
||||||||
Annexure II
Microbiological examination of non sterile products
| Type of Sample | Batch. No. | A.R. Number | |||
| Analysis started on | Analysis completed on | Analyzed By |
- A) Sample Preparation:
Added gm /ml of sample in ml of _______________________(Solution A )
Added ml of Solution A in ml of_________________________(Solution B )
Added gm /ml of sample in ml of _______________________(Solution C )
- B) Total Aerobic Microbial count:
| Name of Media | Incubation condition | 30-35ºC/ 3-5 Days | ||||
|
Performed By |
Sign:
|
Date: |
Time: |
|||
| Pour plate method (cfu/gm)
Dilution Factor: |
Filtration Method | |||||
| Test | Plate1 | Plate2 | Mean | Plate | Negative
control |
Positive control |
| Observation of Test (cfu/gm)
|
||||||
| Observed By/Date/Time | Sign:
|
Date: | Time: | |||
- C) Total Yeast and Mould Count :
| Name of Media | Incubation condition | 20-25ºC/ 5-7 Days | ||||
|
Performed By |
Sign:
|
Date: |
Time: |
|||
| Pour plate method (cfu/gm)
Dilution Factor: |
Filtration Method | |||||
| Test | Plate1 | Plate2 | Mean | Plate | Negative
control |
Positive control |
| Observation of Test (cfu/gm)
|
||||||
| Observed By/Date/Time | Sign:
|
Date: | Time: | |||
- D) Test for specified microorganism:
1) Escherichia coli :
| Test | Enrichment
|
Sub culturing-Primary test | Confirmation Test | ||||||
| Name of media | MCB | MCA | EMB | ||||||
| Incubation condition | 42-44 ºC for 24-48 hrs | 30-35 ºC for 18-72 hrs | 30-35 ºC for 24-48 hrs | ||||||
| Negative control | MCB | MCA plate | EMB Plate | ||||||
| Positive control | MCB | E. coli on MCA plate | E. coli on EMB plate | ||||||
| Performed By/Date/Time | |||||||||
| Observed By/Date/Time | |||||||||
Tick correct option.
| Media | MCB | MCA | EMB |
| Test observation | – | Brick red colour colonies
¨ Not observed ¨ Observed |
Colonies with Metallic sheen
¨Not observed ¨ Observed |
| Positive control | Growth in Turbidity
¨ Growth observed ¨ No growth |
Brick red colour colonies
¨ Observed ¨ Not observed |
Colonies with Metallic sheen
¨ Observed ¨ Not observed |
| Negative control | No growth
¨No growth ¨ Growth observed |
No growth
¨No growth ¨ Growth observed |
No growth
¨No growth ¨ Growth observed |
2) Staphylococcus aureus :
| Test | Sub culturing-Primary test | Confirmation Test | ||||
| Name of media | MSA | Coagulase test | ||||
| Incubation condition | 30-35 ºC for 18-72 hrs | 30-35 ºC for 3-24 hrs | ||||
| Negative control | MSA plate | – | ||||
| Positive control | S. aureus on MSA plate | S. aureus in test kit | ||||
| Performed By/Date/Time | ||||||
| Observed By/Date/Time | ||||||
| MSA | Coagulase test | |
| Test observation | Yellow/white colonies surrounded by yellow zone
¨ Not observed ¨ Observed |
Coagulase/agglutination in tube
¨Not observed ¨ Observed |
| Positive control | Yellow/white colonies surrounded by yellow zone
¨ Observed ¨ Not observed |
Coagulase/agglutination in tube
¨ Observed ¨ Not observed |
| Negative control | No growth
¨No growth ¨ Growth observed |
No Coagulation/agglutination
¨No coagulation ¨ Coagulation |
3) Pseudomonas aeruginosa :
| Test | Sub culturing-Primary test | Confirmation Test | Confirmation Test | ||||||
| Name of media | CA | PAP & PAF | Oxidase Disc | ||||||
| Incubation condition | 30-35 ºC for 18-72 hrs | 30-35 ºC for 72 hrs | Room temperature | ||||||
| Negative control | CA plate | PAP & PAF plate | – | ||||||
| Positive control | Ps.aeruginosa on CA plate | Ps.aeruginosa on PAP & PAF plate | Ps.aeruginosa on Oxidase disc | ||||||
| Performed By/Date/Time | |||||||||
| Observed By/Date/Time | |||||||||
| Media | CA | PAF ( Under U.V) | PAP (Under U.V) | Oxidase disc |
| Test observation | Greenish colonies
¨ Not observed ¨ Observed |
Yellowish colonies
¨ Not observed ¨ Observed |
Greenish colonies
¨ Not observed ¨ Observed |
Deep purple blue colour
¨Not observed ¨ Observed |
| Positive control | Greenish colonies
¨ Growth observed ¨ Not observed |
Yellowish colonies
¨ Observed ¨ Not observed |
Greenish colonies
¨ Observed ¨ Not observed |
Deep purple blue colour
¨ Observed ¨ Not observed |
| Negative control | No growth
¨No growth ¨ Growth observed |
No growth
¨No growth ¨ Growth observed |
No growth
¨No growth ¨ Growth observed |
– |
4) Salmonella spp.:
| Test | Enrichment | Sub culturing-Primary test | Confirmation Test | ||||||
| Name of media | RVSEB | XLDA | TSI | ||||||
| Incubation condition | 30-35 ºC for 18-24 hrs | 30-35 ºC for 24-48 hrs | 30-35 ºC for 24-48 hrs | ||||||
| Negative control | RVSEB | XLDA | TSI slant | ||||||
| Positive control | RVSEB | Salmonella on XLDA plate | Salmonella on TSI slant | ||||||
| Performed By/Date/Time | |||||||||
| Observed By/Date/Time | |||||||||
| Media | RVSEB | XLDA | TSI |
| Test observation | – | Red colonies with or without black centers
¨ Not observed ¨ Observed |
Red(alkaline)slant and yellow (acid) butt with H2S production
¨Not observed ¨ Observed |
| Positive control | Growth in Turbidity
¨ Growth observed ¨ No growth |
Red colonies with or without black centers
¨ Observed ¨ Not observed |
Red(alkaline)slant and yellow (acid) butt with H2S production
¨ Observed ¨ Not observed |
| Negative control | No growth
¨No growth ¨ Growth observed |
No growth
¨No growth ¨ Growth observed |
No growth
¨No growth ¨ Growth observed |
5) Candida albicans :
| Test | C.albicans test | ||
| Name of media | SDB | ||
| Incubation condition | 30-35 ºC for 72 hrs | ||
| Name of media | SDA | ||
| Incubation condition | 30-35 ºC for 24-48 hrs | ||
| Negative control | SDB broth/SDA plate | ||
| Positive control | C.albicans on SDB broth/SDA plate | ||
| Performed By/Date/Time | |||
| Observed By/Date/Time | |||
| Media | SDB | SDA | Confirmation by Microscopic observation |
| Test observation | Growth in Turbidity
¨ Not observed ¨ Observed |
White colonies
¨ Not observed ¨ Observed |
Oval/budding shape
¨Not observed ¨ Observed |
| Positive control | Growth in Turbidity
¨ Observed ¨ Not observed |
White colonies
¨ Observed ¨ Not observed |
Oval/budding shape
¨ Observed ¨ Not observed |
| Negative control | No growth
¨No growth ¨ Growth observed |
No growth
¨No growth ¨ Growth observed |
– |
6) Bile tolerant Gram negative organism :
| Test | Primary Enrichment | Secondory enrichment | Sub culturing-Primary test | ||||||
| Name of media | SCDM | EEB | VRBGA | ||||||
| Incubation condition | 20-25 ºC for 2-5 hrs | 30-35 ºC for 24-48 hrs | 30-35 ºC for 18-24 hrs | ||||||
| Negative control | – | EEB | VRBGA plate | ||||||
| Positive control | – | E.coli in EEB flask | E.coli on VRBGA | ||||||
| Performed By/Date/Time | |||||||||
| Observed By/Date/Time | |||||||||
| Media | EEB | VRBGA |
| Test observation | – | Pink to red colour colonies
¨ Not observed ¨ Observed |
| Positive control | Growth in Turbidity
¨ Growth observed ¨ No growth |
Pink to red colour colonies
¨ Observed ¨ Not observed |
| Negative control | No growth
¨No growth ¨ Growth observed |
No growth
¨No growth ¨ Growth observed |
Media and Incubation Equipment Details
| Sr. No. | Name of the Medium
|
In-house lot No. | Incubator ID. | Date & Sign |
| 1 | Soybean casein digest medium (SCDM)/ Soybean casein digest medium with Polysorbate 80/Soybean casein digest medium with Tween 80 and Lecithin/ Buffer peptone solution | |||
| 2 | Sabouraud Dextrose broth (SDB) | |||
| 3 | Soybean casein digest agar (SCDA) | |||
| 4 | Sabouraud Dextrose agar (SDA) | |||
| 5 | Sabouraud chloramphenicol agar (SCA) | |||
| 6 | Soybean casein digest medium (SCDM) | |||
| 7 | MacConkey broth (MCB) | |||
| 8 | MacConkey agar (MCA) | |||
| 9 | Mannitol salt agar (MSA) | |||
| 10 | Cetrimide agar (CA) | |||
| 11 | Rappaport Vassiliadis salmonella enrichment broth (RVSEB) | |||
| 12 | Xylose lysine deoxycholate agar (XLDA) | |||
| 13 | Enterobacteria enrichment broth (EEB) | |||
| 14 | Voilet red bile Glucose agar (VRBGA) | |||
| 15 | Eosine methylene blue agar (EMB) | |||
| 16 | Pseudomonas agar for Flurescin (PAF) | |||
| 17 | Pseudomonas agar for Pyocyanin (PAP) | |||
| 18 | Triple sugar iron agar (TSI) |
Remark: The given sample complies/does not comply as per given specification.
Analysed by : Checked by : Approved by :
Date : Date : Date :
Annexure III
Quantitative test for Bile Tolerant Gram Negative Bacteria
Page 01 of 02
| Name of Product/ Material | |||||
| Type of Product Batch. No. | Batch. No. | A.R. Number
of product |
|||
| Analysis started on | Analysis completed on | Analyzed By | |||
- A) Sample Preparation:
Added gm /ml of Product in ml of _______________________________ (Solution A )
Incubated Solution A at 20-25°c for _____ hours( 2 to 5 hours)
Quantitative Evaluation –
Enterobacteriaceae enrichment broth – mossel is Incubated with solution A
containing specific quantity of sample(0.1g, 0.01g and 0.001g).
Incubation started at :
Incubated at 30-35°C for 24-48 hours. Date and Sign : Incubation completed at :
Date and Sign :
Above enriched EE broths subcultured on Violet Red Bile Glucose Agar
Incubated at 30-35°C for 18-24 hours. Incubation started at :
Date and Sign :
Incubation completed at :
Date and Sign :
Observations –
| Key
|
Solution containing 0.1 g of sample | Solution containing 0.01g of sample | Solution containing 0.001 g of sample |
| Colonies :
+Present – Absent |
No. of bile tolerant gram negative bacteria per g in product/material is ____________
Annexure III
Quantitative test for Bile Tolerant Gram Negative Bacteria
Page 02 of 02
Media and Incubation Equipment Details
| Sr. No. | Name of the Medium
|
In-house lot No. | Incubator ID. | Date & Sign |
| 1 | Soybean casein digest medium (SCDM)/ Soybean casein digest medium with Polysorbate 80/Soybean casein digest medium with Tween 80 and Lecithin/ Buffer peptone solution | |||
| 2 | Soybean casein digest medium (SCDM) | |||
| 3 | Enterobacteria enrichment broth (EEB) | |||
| 4 | Voilet red bile Glucose agar (VRBGA) |
Remark: The given sample complies/does not comply as per given specification.
Analysed by : Checked by : Approved by :
Date : Date : Date :
Note : Microbiological examination of Non sterile products shall be proceed as per standard operating procedure.
