To lay down a standard procedure for Microbial analysis of drain water in the production area.
This Standard Operating Procedure is applicable to Microbiological section in Quality Control
Microbiologist shall be responsible to follow the procedure mention in this SOP.
Department Head & QA Head shall be accountable for effective implementation and compliance of
6.1 Sampling procedure
6.1.1 Collect approximately 100 ml of drain water sample ( Before and after sanitization) in a sterile
container from the drainage using a sterile pipette.
6.1.2 Use sucking bulb and pipette for the collection of the sample.
6.2 Testing procedure for Total Microbial Count.
- Prepare and sterilize the media for analysis of water as per SOP for media preparation and sterilization. (SOP SOP/ML/005)
- Enter in the MLT room as per SOP for entry and exit in MLT room. (SOP No. SOP/ML/001)
- Operate the LAF as per SOP operation and cleaning of LAF (SOP No. SOP/Ml/003)
6.2.1 Pour Plate Method
220.127.116.11 Perform the test in duplicate
18.104.22.168 Take 1 ml sample with the help of sterile micropipette and dispense into sterile petridishes aseptically for bacterial and fungal count.
22.214.171.124 Pour approximately 15-20ml of sterile Soyabean Casein Digest Agar (cooled at approximately 45°C (feel on the dorsal side of the hand, it should be bearable).
126.96.36.199 Cover the Petri dish, mix the sample with agar by tilting or rotating the dishs and allow solidifying the media at room temperature.
188.8.131.52 Seal the petriplates with parafilm and invert the petri plates and incubate at 30-35 °C for 5 days
184.108.40.206 After incubation, examine the plates for the total bacterial count and express the average for the two plates in terms of the number of colony forming units per ml.
220.127.116.11 For detection of yeast & mold use sabouraud Dextrose agar & incubate the plates at 20 to 25 for 5 to 7 days
18.104.22.168 After incubation examine the plates for the absence of growth.
6.3 Pathogens detection
- Filter 100ml of water sample through 0.45 um membrane filter. Transfer membrane filter to 100 ml SCDM and incubate at 30-35 C for 24 hours.
6.3.1 Detection of Escherichia coli
22.214.171.124 From pre-incubated SCDM, pippet 1ml aliquot into a test tube containing 10 ml of MacConkey
broth, mix, and incubate at 42 to 44 C for 24 to 48 hours.
126.96.36.199 Streak a loopful portion of enriched broth onto surface of MacConkey agar plate and incubate at
30 to 35 for 24 to 48 hours.
188.8.131.52 After incubation examine the plates, presence of brick red colonies may/may not have surrounding
zones of precipitated bile on MacConkey agar indicate the presence of E.coli.
184.108.40.206 Further confirmation is done by gram’s staining.
6.3.2 Detection of Salmonella:
220.127.116.11 Transfer 0.1 ml of enriched Soyabean Casein Digest medium to 10 ml of Rappaport Vassiliadis Salmonella enrichment broth and incubate at 30-35 for 24-48 hours.
18.104.22.168 Streak above media on the surface of Xylose lysine deoxylate agar and incubate at 30-35 C for 24- 48 hours.
22.214.171.124 Growth of red colonies with or without blank centre and in 48 hours the colonies become uniformly black colonies surrounded by a dark zone and metallic sheen indicates the possibility of
presence of Salmonella
126.96.36.199 Further confirmation is done by gram’s staining.
6.3.3 Detection of Pseudomonas aeruginosa:
188.8.131.52 Streak a loopful portion of the enriched Soyabean Casein Digest Medium on the surface of Cetrimide Agar medium and incubate at 30-35 for 24-72 hours.
184.108.40.206 A greenish colored colony indicates the possibility of presence of P.aeruginosa.
220.127.116.11 Carry out the further confirmation by pigment and oxidase tests & gram’s staining.
6.3.4 Detection of Staphylococcus aureus
18.104.22.168 Streak a loopful culture of enriched fluid Soyabean Casein Digest medium on the surface of
Mannitol Salt Agar plates. Incubate the plates at 30-35 for 24-72 hours.
22.214.171.124 Examine the plates after incubation.
126.96.36.199 If upon examination of incubated plates none of them contains yellow colonies with yellow zones
the sample meets the requirements for the absence of Staphylococcus aureus.
188.8.131.52 If growth occurs further confirm by streak the colonies on the surface of Baired Parker agar plates
black, shinny colonies surrounded by clear zones indicates the presence of Staphylococcus aureus.
184.108.40.206 If characteristics colonies are present, perform coagulation test as follow.
220.127.116.11 Transfer representative colony to separate tubes containing 0.5 ml of rabbit plasma, horse plasma,
or any other mammalian plasma.
18.104.22.168 Incubate in a water bath at 37 °C. Examine for Coagulation after 3 hours of incubation and at
suitable intervals up to 24 hours.
22.214.171.124 Further confirm by gram’s staining.
- Once in a week
6.6 Acceptance criteria:
- Total bacterial count: NMT 1000 CFU/ml
- Total fungal count : NMT 100 CFU/ml
- Specified pathogens : should be absent
SOP: Standard operating procedure
NMT : Not more than
°C : Degree Celsius
CFU: Colony forming unit
MLT: Microbial limit test
I.P.A.: Isopropyl Alcohol
QA : Quality Assurance
QC: Quality Control
9.0 DISTRIBUTION LIST
Quality Assurance Department
Quality Control Department
10.0 HISTORY OF REVISION
|Version Number||Effective Date||Reason for Revision|