OBJECTIVE:
To lay down the procedure for Microbial Enumeration Test for water samples.
SCOPE:
This SOP is applicable for the procedure for Microbial Enumeration Test for water samples at {Company Name} {Location}.
RESPONSIBILITY:
In charge- Microbiology- is responsible to ensure compliance as per SOP.
Head/Designee Quality Control – Shall be responsible for ensuring compliance as per SOP.
ACCOUNTABILITY:
QA Head shall be Accountable for implementation of SOP.
ABOUT MICROBIAL ENUMERATION:
Microbial enumeration tests for water samples are essential for assessing water quality and ensuring safety for consumption and use. These tests involve measuring the number of microorganisms, such as bacteria, fungi, and protozoa, present in a water sample. Common methods include the plate count technique, membrane filtration, and most probable number (MPN) method. Each approach helps estimate microbial load by either growing colonies on nutrient media or detecting metabolic activity. Regular microbial enumeration is crucial in industries like drinking water production, wastewater treatment, and food processing. It helps prevent waterborne diseases, ensures compliance with regulatory standards, and safeguards public health.
PROCEDURE:
Glassware / Chemical Requirements:
Required glassware (Sterile petridishes, test tubes, etc)
Test tube stands
Sterile membranes / Presterilized membranes (0.45 m)
Micropipettes and Glass pipettes
R2A agar
Casein Soya bean digest broth
Casein Soya bean digest agar
Normal saline / Peptone water (0.1%) for dilution.
Selective – Media for pathogens.
70% v/v Isopropyl Alcohol / Sterillium.
Manifold, Filter Holders, Suction flask.
Vacuum pump
Milliflex Plus Pump apparatus/device
Sterile Forceps.
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Testing:
Total microbial count: membrane filtration technique for purified water:
Sterilize the filter kit and 0.45m membranes and transfer aseptically to the Laminar air flow.
Transfer the pre incubated R2A / SCDA plates into the Laminar Airflow.
Arrange the manifold and the filter holder, filter 10 mL of sample through 0.45 m membrane filter and wash the filter with 100 mL of 0.1%Peptone solution.
After the completion of filtration, carefully remove the membrane using sterile forceps and place it over the R2A/ SCD agar surface.
Similarly prepare negative control by filtering 100 mL of 0.1 % peptone solution and place it over the R2A/ SCD agar surface.
Incubate the plates in upright position at 30-35°C for 5 days to enumerate the viable count.
There must be no growth of microorganisms in negative control.
Calculate the number of colony forming units per mL and record the results in the worksheet.
Total microbial count: pour plate method:
Dilute 10 mL of sample with 90 mL of sterile SCDM or buffered sodium chloride – peptone pH7.0 and mix (1:10).
Aseptically transfer 1mL of dilution into sterile petri dishes in duplicate and add 15 mL to 20 mL of medium SCDA / R2A.
Prepare negative control.
Incubate the plates in inverted position at 30°C to 35°C for 5 days.
There must be no growth of microorganism in negative control.
Calculate the number of colony forming units per mL and record the results in the worksheet.
Total Microbial Count: Milliflex Plus Pump Apparatus:
Wherever applicable follow the membrane filtration method by Milliflex plus pump apparatus.
Transfer the pre incubated R2A / SCDA agar plates into the Laminar Airflow.
Select any option (Auto Sampling, Dilution, Manual) in milliflex plus pump and filter approximately 10 mL of sample through the sterile 0.45 m filter membrane filter and wash the filter with 100 mL of pre- incubated 0.1% Peptone solution.
The sample volume range shall be between 8 mL-12 mL. and the diluent range shall be between 95 mL-105 mL.
After the completion of filtration, carefully remove the membrane using sterile forceps and place it over the R2A/ SCD agar surface.
Attach the printout to worksheet as per the Format-I of Preparation of Manual work sheet and manual COA.
Incase printer is not available, write manually the sample volume taken as per Display on Milliflex plus pump.
Like wise Perform the filtration for the remaining samples.
Similarly prepare a negative control by filtering 0.1 % peptone and place it over the R2A/SCDA agar plate.
Incubate the plates in upright position at 30-35°C for 5 days to enumerate the viable count.
Calculate the number of colony forming units per mL and record the Results in the worksheet as per the Format-I of Preparation of manual work sheet and manual COA preparation as pre SOP.
Calculate the colony forming units/mL as per sample volume taken.
The sample complies with the test if test results are within the respective specification limit.
The sample does not comply with the test, if test results are not within the respective specification limit.
Test For Pathogens/Specified Microorganisms:
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Escherichia coli:
Transfer 10mL of sample into a 100 mL sterile broth medium (SCD medium) and incubate at 30°C to 35°C for 18 to 24 hours.
Shake the container and transfer 1 mL to 100mL of broth medium (MacConkey broth) and incubate at 42° to 44° for 24 to 48 hours.
Subculture (streak) on plates of agar medium (MacConkey agar) and incubate at 30° to 35°C for 18 to 72 hours.
Growth of brick red, non-mucoid colonies of Gram Negative rods indicate the possible presence of E. coli.
The product passes the test if such colonies are not seen or if the confirmatory biochemical tests are negative.
Confirmatory Test: – Add 1 mL of Kovac’s reagent to 24 hours enriched Nutrient broth and mix well. Pink colour in Kovac’s reagent layer confirms the presence of Escherichia coli.
Salmonella sp:
Transfer 10 mL of sample into a 100 mL sterile broth medium (SCD medium) and incubate at 30° to 35°C for 18 to 24 hours.
Transfer 0.1mL of enrichment culture to tube containing 10 mL of broth medium (Rappaport vassiliadis Salmonella enrichment broth) incubate at 30° to 35°C for 18 to 24 hours.
Subculture on Xylose -lysine-deoxycholate agar media and incubate at 30° to 35°C for 18 to 48 hours.
The presence of the salmonella indicated by the growth of Well developed colonies, with or without black centers.
Confirmatory Test – Transfer a few of suspected colonies to agar medium (Triple Sugar Iron agar) by first inoculating the surface of the slope and then making a stab culture.
With the same inoculating needle, and at the same time inoculate a tube of urea broth. Incubate at 30° to 35°C for 18 to 24 hours
The presence of the Salmonella is indicated by the change of colour from red to yellow in deep inoculation and not in the surface and usually formation of gas with or without production of H2S. (Hydrogen sulphide)
The product passes the test if such colonies are not seen or if the confirmatory biochemical are negative.
Pseudomonas aeruginosa:
Transfer 10 mL of sample to 100 mL of broth medium (SCD medium) and incubate at 30° to 35 °C for 18 to 24 hours.
Subculture (streak) on plate of agar medium N (Cetrimide Agar) and incubate at 30° to 35°C for 18 to 72 hours.
The presence of Pseudomonas aeruginosa is indicated by the presence of colonies.
This is confirmed by confirmation test.
Confirmation test: Transfer the suspected colonies to strips of N,N-dimethyl-P-phenylene dihydrochloride.
The change of colour from pink to purple confirms the presence of Pseudomonas.
Staphylococcus aureus:
Transfer 10 mL of sample to 100 mL of broth medium (SCD medium) and incubate at 30° to 35°C for 18 to 24 hours.
Subculture (streak) on a plate of agar medium (Mannitol-salt-agar) and incubate at 30° to 35°C for 18 to 72 hours.
The presence of Yellow colonies with yellow zones indicate the possible presence of Staphylococcus aureus.
Confirmation by coagulase test – Transfer representative suspected colonies from the Mannitol salt agar to tubes containing 0.5 mL of mammalian (preferably rabbit or horse) plasma with or without additives.
Incubate in water bath at 37°C up to 24 hours ( With 3 hours frequency)
Coagulation in the tube confirms the presence of Staphylococcus aureus.
The product passes the test if such colonies are not seen on Mannitol salt agar or if the confirmatory biochemical tests are negative.
Record the Results in Microbiology Work Sheet as per Format 1 of Preparation of manual work sheet and manual COA.
Test Controls:
Negative control:
Negative control shall be performed by using the same lot/batch of media for each set of analysis. The Negative control shall be performed by using the same media and procedure used for test for specified microorganism, except the addition of sample.
There must be no growth of microorganisms observed in the negative control.
If growth occurred in the negative control, the test shall be invalided. A failed negative control results requires an investigation through PNC.
Note: If two different lot numbers/batch numbers of media are used for analysis, perform the negative control for each lot/batch number separately.
Positive control:
Positive control shall be performed by using the same media and procedure used for test for specified microorganism, except the addition of sample and by adding the respective standard microorganism. Inoculate not more than 100 cfu of respective standard microorganism into liquid media and subsequently, streak the enriched culture on to each selective agar media.
The positive control shall be performed once in a day for each specified microorganism.
Growth of microorganisms must be observed in the positive control.
If growth is not observed in the positive control, the test shall be invalided. A failed positive control results requires an investigation through PNC.
Acceptance criteria:
Total Microbial Count / Total Viable Count: Purified water
Specification limit – 100 cfu/mL
Total Microbial Count / Total Viable Count: Raw Water
Specification Limit – 500 cfu/mL
Specified Microorganisms:
Escherichia coli – Should be absent in 10 mL
Salmonellae – Should be absent in 10 mL
Pseudomonas aeruginosa – Should be absent in 10 mL Staphylococcus aureus. – Should be absent in 10 mL
Interpretation of Alert limit and Action limit and action recommended:
If the test result exceeds the Alert limit, alert the Quality assurance Department and Maintenance department.
If the test result exceeds the Action limit, immediately inform the Quality Assurance and Production Department to stop usage of water from the respective point.
Quarantine the products manufactured during the period.
Products shall be subjected to Microbiological testing and Release the Products if they meet the requirement.
If the test result (for raw water) exceeds the specification limit, inform to Quality Assurance Department and Maintenance department for further corrective action.
Investigate thoroughly as per SOP and take corrective action.
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REFERENCES:
Not Applicable
ANNEXURES:
Not Applicable
ENCLOSURES: SOP Training Record.
- DISTRIBUTION:
- Controlled Copy No. 01 : Head Quality Assurance
- Controlled Copy No. 02 : Head Quality Control (Micro.)
- Master Copy : Quality Assurance Department
ABBREVIATIONS:
| No. | : | Number |
| cfu | : | Colony Forming Units |
| SOP | : | Standard Operating Procedure |
| QC | : | Quality Control |
REVISION HISTORY:
CHANGE HISTORY LOG
| Revision No. | Details of Changes | Reason for Change | Effective Date |
| 00 | New SOP | Not Applicable | To Be Written Manual |
Click the link to download word file copy of this document:
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