SOP for microbial limit test for Raw Material and Finished Product is described in this post which you can follow in the section of microbiological lab.

Objective:

This procedure has been established for microbial limit test for raw material and finished products.

Scope:   

This procedure is applicable to microbiology lab.

Responsibility:                          

Microbiologist.                             

Accountability:

Quality Control Manager.            

Procedure:

Microbial Enumeration Test

Dissolve or dilute (a 1 in 10 dilution) the product/ sample to be examined in sterile buffered sodium chloride peptone solution pH 7.0 or Soybean casein digest medium prepared as per SOP for media preparation. 

If further dilution is required prepare with the same diluents used as above.

Microbial Enumeration Test: Determination of total aerobic microbial count (TAMC) and total combined yeasts and molds count (TYMC) by the Pour-plate method.

Aseptically transfer 1 ml from the prepared solution/suspension

into each of four sterile Petri plates. 

Add approximately 15- 20 ml of sterile Soybean Casein Digest Agar, prepared as per SOP (cooled approximately up to 45°C) in two Petri plates for TAMC, add 15- 20 ml of sterile Sabouraud Dextrose Agar (cooled approximately up to 45°C) prepared as per SOP in another two Petri plates for TYMC. 

Mix the contents of Petri plates by gently swirling the plate to proper mixing of the sample, and allow medium to solidify, taking the precaution of not spilling the media from the plate. 

Simultaneously perform the negative control by plating 1 ml of diluent used instead of the sample for each medium. 

Incubate the plates of Soybean Casein Digest Agar at 30°C to 35°C for 5 days for TAMC and plates of Sabouraud Dextrose Agar at 20°C to 25°C for 5 days for TYMC in an inverted position. 

Microbial Enumeration Test: 

Determination of total aerobic microbial count and total combined yeasts and molds count by Filtration method. 

Use membrane filter having a nominal pore size not greater than 0.45 ┬Ám. Cellulose nitrate filters may be used for aqueous, oily and weakly alcoholic solutions and cellulose acetate filters may be used for a strongly alcoholic solution or any other suitable membranes like PVDF. 

Aseptically transfer the aliquot (equivalent to 1 g or 1 ml of the sample/as determined during the validation) from the prepared solution/suspension (Prepared in point No.5.1.1) into each of two membrane filters and filter immediately. 

Wash each filter with three portions of about 100 ml of 0.1% Peptone Water or as determined in validation. 

Transfer one membrane filter on the surface of pre-incubated Soybean Casein Digest Agar plate, prepared as per SOP for TAMC and the other membrane filter on the surface of Sabouraud Dextrose Agar plate prepared as per SOP for TYMC.

Simultaneously perform the negative control by filtering 100 ml of diluent used instead of the sample for each medium. 

Incubate the plates of Soybean Casein Digest Agar and Sabouraud Dextrose Agar at 30°C to 35°C for 5 days for TAMC and at 20°C to 25°C for 5 days for TYMC in an upright position. Note: When the TYMC is expected to exceed the acceptance criteria due to the bacterial growth on Sabouraud Dextrose Agar containing antibiotics may be used. 

Observation and Expression of Results 

After completion of incubation period observe the plates and express the result as Colony Forming Unit (CFU) per g/ml, by multiplying an average number of CFU/plate with dilution factor. If no colonies are observed express the result as a number of colonies less than dilution factor. Note down the result. 

If colonies of fungi are detected on Soybean casein digest agar, they are counted as part of TAMC and If colonies of bacteria are detected on Sabouraud dextrose Agar they are counted as part of TYMC. 

If the counting of colonies is not possible due to high count or merged colonies, total count test shall be repeated with further dilution steps (Example. 1:10. and so on) to the solution/ suspension.

The negative control plates should not show any growth and any failed negative control result should be investigated. 

If TAMC and TYMC observed is not conforming to specified limits, follow the SOP for Handling of out of specification. 

Sample Preparation for Test for Specified microorganisms 

Prepare the media as per SOP. 

For test of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Salmonella, prepare the sample using not less than 1 g or 1 ml of sample or 1 in 10 dilution corresponding to not less than 1 g or 1 ml of sample to be examined and dissolve or dilute in 100 ml sterile Soybean Casein Digest Medium and incubate at 30 to 35°C for 18 to 24 hours. After incubation proceeds for testing individual organisms as described below. 

For test of Bile tolerant gram-negative bacteria, prepare the sample using not less than 1 g or 1 ml of sample or 1 in 10 dilution corresponding to not less than 1 g or 1 ml of sample to be examined and dissolve or dilute in 10 ml sterile Soybean Casein Digest Medium, mix and incubate at 20 to 25°C for a time sufficient to resuscitate the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 hours but not more than 5 hours). After incubation proceeds for testing of Bile tolerant gram-negative as described below. 

For testing of Clostridia prepare the sample using not less than 2 g or 2 ml of sample or 1 in 10 dilutions corresponding to not less than 2 g or 2 ml of sample to be examined and dissolve or dilute in each of 10 ml sterile Soybean Casein Digest Medium. Proceed for testing of Clostridia as described below.

For testing of Candida albicans, prepare the sample using not less than 1 g or 1 ml of sample or 1 in 10 dilution corresponding to not less than 1 g or 1 ml of sample to be examined and transferred to 100ml Sabouraud Dextrose medium and incubate at 30 to 35°C for 3 to 5 days. After incubation proceed for testing of Candida albicans as described below 

Negative Control: During at each stage of testing for each media, perform negative control test simultaneously with sterile SCM instead of sample preparation. 

Test for Staphylococcus aureus 

After incubation of Soybean Casein Digest Medium  mix Soybean Casein Digest Medium tube and streak loopful on a plate of Mannitol salt agar (MSA) using inoculating loop. 

Invert the plate and incubate at 30°C to 35°C for 18 to 72 hours. 

After incubation, examine the plates and the possible presence of S. aureus is indicated by the growth of yellow or white colonies surrounded by a yellow zone.

If colonies are found conforming to the above descriptions, further identification shall be done using Biomeriux as per SOP. 

If there are no colonies of the type described observed or if the identification test is negative, the product complies with the test for Staphylococcus aureus. 5.6.6 Record the results. 5.7 Test for Pseudomonas aeruginosa 

After incubation of Soybean Casein Digest Medium  mix Soybean Casein Digest Medium tube and streak loopful on a plate of Cetrimide Agar using inoculating loop. 

Invert the plate and incubate at 30°C to 35°C for 18 to 72 hours. 

After incubation, examine the plate and growth of colonies indicates the possible presence of Pseudomonas aeruginosa. 

If colonies are found, further identification shall be done using Biomeriux as per SOP. 

If there are no colonies observed or if the identification test are negative, the product complies with the test for Pseudomonas aeruginosa. 

Record the results. 

Test for Salmonella 

After incubation of Soybean Casein Digest Medium  mix the Soybean Casein Digest Medium tube and transfer 0.1 ml using a sterile pipette to 10 ml of Rappaport Vassiliadis Salmonella Enrichment Broth (RSB) and incubate at 30°C to 35°C for 18 to 24 hours.

After incubation, mix the contents of the tube and streak a loopful from RSB media on a plate of Xylose lysine Deoxycholate agar (XLD) media using an inoculating loop. 

Invert the plate and incubate at 30°C to 35°C for 18 to 48 hours. 

After incubation, examine the plate and the possible presence of Salmonella is indicated by the growth of well-developed, red colonies, with or without black centers. 

If colonies are found conforming to the above descriptions, further identification shall be done using Biomeriux as per SOP. 

If there are no colonies of the type described observed or if the identification tests are negative, the product complies with the test for Salmonella. 

Record the results. 

 Record the results. 

Test for Escherichia coli 

After incubation of Soybean Casein Digest Medium  mix the Soybean Casein Digest Medium tube and transfer 1.0 ml using a sterile pipette to a tube containing 100 ml of MacConkey’s broth and Incubate at 42°C to 44°C for 24 to 48 hours. 

After incubation, mix the contents of the tube and streak a loopful from MacConkey’s broth media on a plate of Mac Conkey’s agar media using

an inoculating loop and incubate at 30°C to 35°C for 18-72 hours in an inverted position. 

Growth of colonies indicates the possible presence of Escherichia coli. 

If colonies are found, further identification shall be done using Biomeriux as per SOP. 

If there are no colonies observed or if the identification tests are negative, the product complies with the test for Escherichia coli. 

Record the results. 5.10 Test for Bile tolerant gram-negative bacteria  

Proceed for testing of Bile tolerant gram-negative bacteria from the step

Mix Soybean Casein Digest Medium after incubation. 

Test for absence: 

Use the volume corresponding to 1 gram or 1 ml of the product to inoculate 100 ml EEB Mossel with the preparation mentioned in point 

Incubate at 30°C to 35°C for 24 to 48 hours in an inverted position. 

After incubation mix the contents of tube and Subculture on a plate of Violet red bile glucose agar to obtain selective isolation. Incubate the plates at 30°C to 35°C for 18 to 24 hours. 

If there are no colonies observed, the product complies with the test of Bile tolerant gram-negative bacteria. 

Quantitative test 

Inoculate suitable quantities of EEB Mossel with the preparation mentioned in point 

containing respectively 0.1 g, 0.01 g and 0.001 g (or 0.1 ml, 0.01 ml, 0.001 ml) inoculate to the product to be examined. Incubate at 30°C to 35°C for 24 to 48 hours. 

Subculture each of the dilution on a plate of Violet red bile agar with glucose to obtain selective isolation. Incubate the plates at 30°C to 35°C for 18 to 24 hours.

Interpretation: Growth of colonies constitutes a positive result. Note the smallest quantity of the product that gives a positive result and the largest quantity that gives a negative result. Determine the the probable number of bacteria as per following table. 

Test for Clostridia 

Prepare the product to be examined as described in the step 

take two equal portions corresponding to 1 g or 1 ml of the product to be examined. 

Heat one portion to 80°C for 10 minutes and cool rapidly, Do not heat the other portion. 

Transfer the 10 ml (or quantity corresponding to 1g or ml) of each of the homogenized portions to two tubes containing 100 ml of Reinforced Medium for Clostridia.

Incubate under an anaerobic condition at 30°C to 35°C for 48 hours. 

After incubation make sub-culture from each tube on Columbia agar to which Gentamicin has been added. 

Incubate under anaerobic conditions at 30°C to 35°C for 48 to 72 hours. 

If the anaerobic growth of microorganisms is observed then perform Gram Staining and Catalase test. 

Perform Gram Staining as per SOP for Microbial Staining Procedures. 

For catalase test place a drop of hydrogen peroxide on a clean slide and take a portion of suspected colony with the help of inoculating loop (Do not use nichrome loop) & rub with drop of hydrogen peroxide then it will generate effervescence of oxygen bubble, it indicates the positive catalase test and if there is no effervescence of oxygen bubble observed then it indicates negative catalase test.

The occurrence of anaerobic growth of rods (with or without endospores) giving a negative catalase reaction indicates the presence of Clostridia.  

If no anaerobic growth of microorganisms is detected on Columbia agar or catalase test is positive, the product complies with the test for Clostridia.  

Test for Candida albicans 

After completion of incubation of Sabouraud Dextrose Medium , mix the Sabouraud Dextrose Medium tube, and streak a loopful from Sabouraud Dextrose Medium on plates of Sabouraud Dextrose Agar media using an inoculating loop and incubate at 30°C to 35°C for 24-48 hours. 

After incubation, if the plates show White colonies, indicate the presence of Candida albicans. 

If colonies are found conforming to the above descriptions, further identification shall be done using Biomeriux as per SOP. 

If there are no colonies of the type described or if the identification tests are negative, the product complies with the test of Candida albicans

Negative Control: During at each stage of testing for each media, perform negative control test simultaneously. 

Interpretation of Results 

If no characteristic growth is observed or if the identification tests are negative, then the product complies with the test for absence of specified organisms. 

The negative controls should not show any growth and any failed negative control result should be investigated. 

In case of non-conformance of results follow the SOP. “Handling of out of specification results in microbiological testing.”