procedure for microbial analysis of finished product (API)

1.0 Purpose:

 To lay down the procedure for microbial analysis of finished product (API).

2.0 Scope:

 This procedure applicable to test the total aerobic microbial count and total combined yeast and molds count and to test for the presence of pathogens in any finished product (API).

3.0 Responsibility: 

 The responsibility to carry out the test for total aerobic microbial count, total combined yeast and molds and pathogens lies with the Microbiologist.

4.0  Definition/Abbreviation: 

4.1 Pre incubation: Prepare the media before 48 hours of use.

5.0  Procedure:

5.1   Sample preparation (USP/EP): Take 10 g of sample in 100 ml diluent i.e sterile Phosphate buffer pH 7. 2 / sterile (Buffered) Sodium chloride-peptone broth pH 7.0 / sterile Soya bean casein digest broth. This is 1:10 dilution. If required sample size and diluent can be increase or decrease. If     product has antimicrobial property then add inactivating agent that is neutralizing agent such as polysorbate or lecithin or thiosulphate or glycine or sodium hydrogen sulphite or increase the dilution volume, to the above mentioned diluent and sterilized it, then only add required amount of sample. Check the pH of sample preparation by taking 10 ml, it should be 6to 8, if necessary adjust it with 1N NaoH or 1N Hcl (0.2micron filtered).

5.2   Preparation of media: Prepare respective media as per SOP ‘Procedure for handling of media in Microbiology’

5.3   Testing procedure for the Total Aerobic Microbial Count and Total Combined Molds and Yeast count by Membrane Filtration (USP/EP):

5.3.1    Filter 10ml or required volume of above sample preparation (equivalent to 1 gm) through 0.45µ filter membrane. Rinse the filter membrane with 100ml of diluent for 3 times. Transfer the membrane aseptically over the surface of a pre incubated soya bean-casein digest agar plate for bacterial growth in duplicates and incubates the plate in inverted position at 30–35°C for 3 – 5 days. Each 10 ml of prepared sample represents 1g of sample.

5.3.2 Take another 10ml or required volume of above sample preparation (equivalent to 1 gm) through 0.45µ filter membrane. Rinse the filter membrane with 100ml of diluent for 3 times. Transfer the membrane aseptically over the surface of a pre incubated Sabouraud dextrose agar plates for fungal growth in duplicates and incubate the plate in inverted position at 20 -25 °C for 5 – 7 days.

5.3.3 Following incubation, examine the plates for the growth of CFU and express the average CFU for two plates will give the number of CFU per gram of the sample.

5.3.4    Record the result and prepare the report in the form (or) LIMS.

5.3.5  Growth promotion test for media: Pipette out required (1 ml / 0.1 ml) volume of any one of the (Bioball) viable  culture suspension of 10-100 CFU cells of E. coli / S. aureus / P. aeruginosa / S. abony / B. Subtilis and filter it through 0.45µfilter membrane and wash with 100 ml of sterile diluent. Transfer the membrane aseptically over the surface of a pre incubated soya bean-casein digest agar plate for growth. Pipette out required (1 ml / 0.1 ml) volume of C. albicans (Bioball) viable culture suspension of 10-100 CFU cells on the 0.45µfilter membrane and wash with 100 ml of sterile diluent. Transfer the membrane aseptically over the surface of a preincubated sabourauds dextrose agar plate for growth.

5.3.6 Incubate soya bean-casein digest agar plate at 30-35°c for not more than 3 days and sabourauds dextrose agar plate at 20-25°c for not more than 5days in inverted position.

5.3.7   Following incubation examine the plates for growth of colonies and count the colonies it should not be more than the 2 factor value of original (Bioball) viable culture suspension value.

5.3.8  Negative control: Place 0.45µfilter membrane without any diluent on pre incubated soya bean casein digest agar plate and sabourauds dextrose agar plate respectively. Incubate soya bean-casein digest agar plate at 30-35°c for not more than 3 days and sabourauds dextrose agar plate at 20-25°c for not more than 5 days in inverted position. Negative control plate should not show any growth.

5.3.9  Sterility of the diluent: Transfer each 100 ml of sterile diluent on the 0.45µ filter membrane and filter it transfer aseptically over the surfaces of preincubated soyabean-casein digest agar plate and sabourauds dextrose agar plate respectively. Incubate all the plates in inverted position at 30–35°C for not more than 3 days and 20-25 °C for not more than 5 days respectively. Diluent should not show any growth of colonies.

5.4       Testing procedure for the Total Aerobic Microbial Count and Total Combined Molds and Yeast count by Pour plate count method (USP/EP):

5.4.1    Sample preparation & Preparation of media: Follow the procedure as in step 5.1 & 5.2.

5.4.2   Pipette out 1ml each from above sample preparation in two sterile petridishes. Promptly add to each dish 15 to 20 ml of sterile Soyabean Casein Digest agar (duplicates) which has been sterilized and cooled to approximately 45°C. Cover the petridishes, mix the sample with the agar by rotating the dishes and allow the contents to solidify at room temperature. Invert the petridishes and incubate at 30-35°C for 3 – 5 days for bacterial growth.

5.4.3   Pipette out 1 ml each from above sample preparation in two sterile petridishes. Promptly add to each dish 15 to 20 ml of sterile Sabouraud dextrose agar and incubate the plates at 20-25°C for 5 – 7 days for fungal growth.

5.4.4   Following incubation, examine the plates for the growth of CFU and record the average for the two plates and calculate number of CFU as follows. 

Number of CFU / g = Average of two plates x 10

           *10 is dilution factor.

5.4.5  Growth promotion test for media: Pipette out required (1 ml / 0.1 ml) volume of any one of the (Bioball) viable culture suspension of 10-100 CFU of E.coli / S.aureus / P.aeruginosa / S.abony / B. Subtilis in a sterile petriplate then add 15 to 20 mL of soyabean-casein digest agar and mix it by rotating. Cool it at room temperature and incubate at 30-35°c for not more than 3 days in inverted position. Pipette out required (1 ml / 0.1 ml) volume (Bioball) viable culture suspension of 10-100 CFU of C. albicans in a sterile petriplates then add 15 to 20 mL of sabourauds dextrose agar and mix it by rotating. Cool it at room temperature and incubate it at 20-25°c for not more than 5 days in inverted position.

5.4.6  Following the incubation examine the plates for growth of colonies and count the colonies it should not be more than the 2 factor value of original viable culture suspension value.

5.4.7 Negative control: Pour 15 to 20 mL soyabean casein digest agar in sterile plate without adding any diluent, cool at room temperature and incubate it in inverted position at 30–35°C for not more than 3 days. Pour 15 to 20 mL sabourauds dextrose agar in sterile plate without adding any diluent, cool at room temperature and incubate it in inverted position at 20-25°C for not more than 5 days.

5.4.8    Negative control should not show any growth of colonies.

5.4.9  Sterility of the diluent: Transfer the 1ml of sterile diluent in a sterile petriplates and add 15 to 20 mL of sterile soyabean-casein digest agar, cool it in room temperature and incubate in inverted position at 30–35°C for not more than 3 days. Transfer the 1ml of sterile diluent in a sterile petriplates and add 15 to 20 mL of sterile sabourauds dextrose agar, cool it in room temperature and incubate in inverted position at 20-25°C for not more than 5 days. Diluent should not show any growth of colonies.

5.5.5       Detection of Pathogens (USP/EP):

5.5.1    Preliminary Test (for E.coli, S. aureus and P. aeruginosa):

Test: Add 10 ml of above sample preparation to 100 ml of sterile Soyabean Casein Digest broth. 

Growth Promotion Test: Add required volume (1 ml / 0.1 ml) of any one of the (Bioball) viable culture suspension containing 10-100 CFU of E. coli / S. aureus / P. aeruginosa in 100 ml of Soyabean Casein Digest broth. It shows turbidity.

Negative control: 100 ml of sterile Soyabean-Casein Digest broth without any viable culture. It shows no turbidity.

5.5.2  Incubate all of them above at 30–35°C for 24 hours.

5.5.3    For Escherichia Coli:

5.5.3.1    Transfer 1ml of preliminary test into 100ml of sterile Macconkey broth and incubate at 42 - 44°C for 24 – 48 hours.

5.5.3.2    By means of an inoculating loop, take a loop full of inoculum from the above broth and streak on the surface of sterile Macconkey agar plate (15-20ml).

5.5.3.3 Incubate the plates at 30–35°C for 18 - 72 hours.

5.5.3.4    Growth of colonies indicates the possible presence of E.coli. This is confirmed by identification tests.

5.5.3.5 The product complies with the test if no colonies are present or if the identification tests are negative.

5.5.3.6    Identification tests are Grams Nature - negative rods, Motility-motile, Indole- positive, Methyl red - positive, Voges-Proskauer-negative, Citrate-negative and Oxidase- negative.

5.5.4      For Pseudomonas aeruginosa:

5.5.4.1    Take one loop full of inoculum from the preliminary test, streak on the surface of sterile Cetrimide agar plate (15-20 ml) and incubate the plates at 30–35°c for 18 - 72 hours.

5.5.4.2    Growth of colonies indicates the possible presence of P.aeruginosa. This is confirmed by   identification tests.

5.5.4.3    The product complies with the test if colonies are not present or if the confirmatory identification tests are negative.

5.5.4.4    Identification tests are Grams Nature - Negative rods, Motility - Motile, Oxidase test Positive.

5.5.5      For Staphylococcus aureus:

5.5.5.1    Take one loop full of the inoculum from the preliminary test, streak on the surface of Mannitol Salt agar plate (15-20 ml) and incubate them at 30–35°C for 18 – 72 hours.

5.5.5.2    The possible presence of s.aureus is indicated by the growth of yellow/white colonies surrounded by a yellow zone. This is confirmed by identification tests.

5.5.5.3    The product complies with the test if colonies of the types described are not present or if the confirmatory identification tests are negative.

5.5.5.4    Identification tests are Grams Nature - positive cocci, Motility - Non Motile.

5.5.6  For Salmonella:

5.5.6.1    Preliminary test:

Test: Add 100 ml of prepared sample to 100 ml of sterile Soyabean Casein Digest broth.

 Growth Promotion Test: Add required volume (1ml / 0.1 ml) viable culture suspension containing 10-100 CFU of S. abony in 100 ml of Soyabean Casein Digest broth. It shows turbidity.

Negative control: 100 ml of sterile Soyabean-Casein Digest broth without any viable culture. It shows no turbidity.

5.5.6.2  Incubate above all of them at 30–35°C for 24 hours.

5.5.6.3   Transfer 0.1ml of preliminary test growth of sample into 10ml of sterilized Rappaport     vassiliadis salmonella enrichment broth and incubate at 30 – 35°C for 18 - 24 hours.

5.5.6.4 Take one loop of inoculum from the above enrichment broth streak on the surface of sterile  Xylose-Lysine-Desoxycholate agar plate (15-20 ml) and incubate the plates at 30 – 35°C for18 – 48 hours.

5.5.6.5    The presence of salmonella is indicated by the growth of well developed red colonies with or without black centre.

5.5.6.6   The product complies with the test if colonies of the above mentioned are not present.

5.5.7    For Bile-Tolerant Gram Negative Bacteria (Enterobacteriaceae) – Qualitative Test:

5.5.7.1    Add 10 ml from above sample preparation to 100 ml of sterile Soyabean Casein Digest      broth for Enterobacteriaceae. Incubate the tubes at 20–25°C for 2 hours but not more than 5hours for a time sufficient to revive the bacteria but not sufficient to encourage multiplication of the organisms. 

5.5.7.2    Transfer 10ml from above Soyabean Casein Digest broth to 100 ml of sterile Enterobacteriaceae Enrichment broth mossel (EE-Mossel Broth) and incubate at 30–35°for 24 to 48 hours.

5.5.7.3    Take loop full of sample from EE-mossel broth streak on the sterile Violet Red Bile  Glucose agar plate (15-20 ml) and incubate at 30–35°C for 18- 24 hours.

5.5.7.4    The product complies with the test if there is no growth of colonies (red or reddish colonies).

5.5.7.5    If above mentioned characteristics of colonies are present conclude that presence of Bile-Tolerant gram negative bacteria(Enterobacteriaceae).

5.5.8    For Bile-Tolerant Gram Negative Bacteria (Enterobacteriaceae) – Quantitative Test:

5.5.8.1 0.1 g preparation:

5.5.8.1.1 Add 1 ml of above sample preparation to 10 ml of sterile Soyabean Casein Digest broth. Incubate the tubes at 20–25°C for 2 hours but not more than 5 hours for a time sufficient to revive the bacteria but not sufficient to encourage multiplication of the organisms.

5.5.8.1.2 Transfer 1 ml from above soyabean casein digest broth to 100 mL sterile Enterobacteriaceae Enrichment broth mossel (EE-Mossel Broth) and incubate at 30–35°C for 24 to 48 hours.

5.5.8.1.3 Take loop full of culture from EE-Mossel Broth streak on the surface of sterile violet red bile glucose agar and incubate at 30-35 °C for 18-24 hours.

5.5.8.1.4 Growth of colonies constitutes a positive result (presence of Enterobacteriaceae). Determine the probable number of bacteria from given below table-1.

5.5.8.2 0.01 g preparation:

5.5.8.2.1Add 0.1 ml of above sample preparation to 10 ml of sterile Soyabean Casein Digest broth. Incubate the tubes at 20–25°C for 2 hours but not more than 5 hours for a time sufficient torevive the bacteria but not sufficient to encourage multiplication of the organisms.

5.5.8.2.2 Transfer 1 ml from above soyabean casein digest broth to 100 ml sterile Enterobacteriaceae Enrichment broth mossel (EE-Mossel Broth) and incubate at 30–35°C for 24 to 48 hours.

5.5.8.2.3 Take loop full of culture from EE-Mossel Broth streak on the surface of sterile violet red bile glucose agar plate (15-20 ml) and incubate at 30-35 °C for 18-24 hours.

5.5.8.2.4 Growth of colonies constitutes a positiveresult (presence of Enterobacteriaceae).Determinethe probable number of bacteria from given below table-1.

5.5.8.3 0.001 g preparation:

5.5.8.3.1Add 0.01 ml of above sample preparation to 10 ml of sterile Soyabean Casein Digest broth. Incubate the tubes at 20–25°C for 2 hours but not more than 5 hours for a time sufficient torevive the bacteria but not sufficient to encourage multiplication of the organisms.

5.5.8.3.2Transfer 1 ml from above soyabean casein digest broth to 100 ml sterile Enterobacteriaceae Enrichment broth mossel (EE-Mossel Broth) and incubate at 30–35°C for 24 to 48 hours.

5.5.8.3.3 Take loop full of culture from EE-Mossel Broth streak on the surface of sterile violet red bile glucose agar plate (15-20 ml) and incubate at 30-35 °C for 18-24 hours.

5.5.8.3.4 Growth of colonies constitutes a positive result (presence of Enterobacteriaceae). Determine the probable number of bacteria from given below table-1.

Table - 1

 5.5.9  For Clostridia:

5.5.9.1    Take 10ml each of above sample preparation in 2 sterile test tubes and label it 1 portion and 2 portion. Heat 1^st portion test tube at 80 °C for 10 minutes and cool rapidly. Do not heat the 2^nd portion test tube.

5.5.9.2    Transfer each portion of test tubes in each 100 ml of sterile Reinforced Clostridia medium broth. Incubate the inoculated medium anaerobically at 30 -35°C for 48 hours in an anaerobic chamber.

5.5.9.3    Take loop full of inoculums from the above incubated samples (Portion 1 & 2) and streak individually on the sterile Columbia agar plate (15-20 ml). Incubate the plates under anaerobic conditions at 30 to 35°C for 48-72hours in an anaerobic chamber.

5.5.9.4 The product complies with the test if colonies are not present or if the identification tests are negative.

5.5.9.5    If colonies observed do Endospore staining, the occurrence of anaerobic growth of rods (with or without end spores) and catalase test shows negative. Then indicates the presence of Clostridia. If the above characteristics are observed in any one of the portions 1& 2 samples confirm the presences of clostridia. If above characteristics are absent confirm that clostridia is absent.

5.5.10  Candida Albicans:

5.5.10.1Add 10 ml from the above sample preparation to sterile 100ml of Sabouraud Dextrose broth Incubate at 30 to 35°C for 3 - 5 days.

5.5.10.2Take a loop full of inoculum from above growth streak on the sterile Sabourauds dextrose agar plate (15-20 ml). Incubate the plate at 30 to 35°C for 24 to 48 hours.

5.5.10.3Growth of white colonies may indicate the presence of Candida albicans. This is confirmed by identification tests / use commercial kit.

5.5.10.4The product complies with the test if such colonies are not present.

5.5.11  Growth promotion test and Negative control:

5.5.11.1  Each of the tests (parameters) mentioned above shall be followed with growth promotion    test (positive) of each sterile media with corresponding    microorganisms containing 10-100 CFU viable culture suspension.

5.5.11.2All the sterile agar medias shall be under go for GPT test by adding sufficient amount (1 ml/ 0.1 ml) of respective (Bioball) viable culture suspension containing 10-100 CFU cells and  incubate them at respective conditions. It shows count

5.5.11.3  All the sterile broth medias shall be under go for GPT test by adding sufficient amount (1ml / 0.1 ml) of respective (Bioball) viable culture suspension containing 10-100 CFU cells and incubate them at respective conditions.

5.5.11.4  Acceptance criteria:

5.5.11.4.1 For agar medias the growth of colonies should not be more than ±2 factor from original  viable culture suspension.

5.5.11.4.2 For broth medias it should show turbidity.

5.5.11.5  Negative control of agar and broth with out any viable culture suspensions and it should not show any colonies or turbidity.

5.5.12 Additional test:

5.5.12.1If any additional test is required for the finished product (API), it shall be carried out as per the test procedure mentioned in the Pharmacopoeia.

5.5.13  Record the results of Finished Product Microbial Analysis Report, Form no xxxxx

5.5.14   Finished product (API) shall be tested for Endotoxins {Bacterial Endotoxinsá85ñ} or Pyrogens {Pyrogen test á151ñ} when required by the customer by external agencies as per USP/EP.

5.5.15  Frequency: The microbial analysis (TAMC, TYMC and Pathogens) of finished product shall be carried out on validation batches / as per customer requirement and monthly one batch from each product.

5.5.16  Enter the data of Product name, batch no., sample collection date, sampled by, date of analysis, date of report, A.R. number / Inspection Lot No., sign and remarks in their respective log book.

6.0       Related Documents:

6.1       Procedure for isolation, identification and gram staining of microorganisms, SOP

6.2         Procedure for operation of microscope, SOP.

Microbiology, finished product, microbial analysis, 1n naoh, 1n hcl