1.0       OBJECTIVE

To lay down a standard procedure for Microbial analysis of drain water in the production area.

2.0       SCOPE

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

this SOP.



6.0       PROCEDURE

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     Perform the test in duplicate     Take 1 ml sample with the help of sterile micropipette and dispense into sterile petridishes   aseptically for bacterial and fungal count.      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).      Cover the Petri dish, mix the sample with agar by tilting or rotating the dishs and allow solidifying   the media at room temperature.      Seal the petriplates with parafilm and invert the petri plates and incubate at 30-35 °C for 5 days      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.      For detection of yeast & mold use sabouraud Dextrose agar & incubate the plates at 20 to 25 for 5  to 7 days      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      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.      Streak a loopful portion of enriched broth onto surface of MacConkey agar plate and incubate at

30 to 35 for 24 to 48 hours.      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.      Further confirmation is done by gram’s staining.

6.3.2         Detection of Salmonella:      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.      Streak above media on the surface of Xylose lysine deoxylate agar and incubate at 30-35 C for 24- 48  hours.     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      Further confirmation is done by gram’s staining.

6.3.3         Detection of Pseudomonas aeruginosa:      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.      A greenish colored colony indicates the possibility of presence of P.aeruginosa.      Carry out the further confirmation by pigment and oxidase tests & gram’s staining.

6.3.4         Detection of Staphylococcus aureus      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.      Examine the plates after incubation.      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.      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.      If characteristics colonies are present, perform coagulation test as follow.      Transfer representative colony to separate tubes containing 0.5 ml of rabbit plasma, horse plasma,

or any other mammalian plasma.      Incubate in a water bath at 37 °C. Examine for Coagulation after 3 hours of incubation and at

suitable intervals up to 24 hours.      Further confirm by gram’s staining.

 6.3            Frequency

  • 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

7.0           REFERENCES



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


Quality Assurance Department

Quality Control Department


Version Number Effective Date Reason for Revision





Microbiology Labs Documents