To lay down the procedure for carrying out the microbiological analysis of purified water.

2.0)    PROCEDURE:

2.1)    Collection of water sample:

  • Sample for microbiological evaluation should be collected in pre-sterilized screw caped glass bottles.
  • Note: Sampling bottle should be autoclaved and wrapped with aluminium foil.
  • Wear the sterile hand gloves and unwrap bottles just prior collecting the sample.
  • Label the sampling bottle with individual sampling point, sampling volume, sampling date and sampled by.
  • Clean the external area of outlet with 70% IPA and open the sampling/user Point tap until there is a steady stream of water.
  • Drain for approximately 1 minute, remove the cap of pre-sterilized bottle and collect the water.
  • Fill the bottle without overflowing the water from the bottle.
  • Remove the bottle from the sample stream and place the cap on the bottle as soon as possible.
  • Tighten the cap securely.
  • Turn off the tap.
  • Collect all samples and proceed for analysis.
  • It is best to test the samples as soon as possible after being collected. If it is not possible to test the sample within about 2 hours of collection, the sample should be held in refrigerator at temperature 2 to 8°C.
  • Ensure that analysis is initiated within 24 hours after sampling.


3.1)   Preparation of Culture media:

  • Weigh the required quantity of dehydrated Soyabean Casein Digest Medium (Tryptone Soya Broth), Soyabean Casein Digest Agar/ R2A agar (for bacteria) and Sabouraud Dextrose agar (for fungus) and add required quantity of freshly collected distilled water in each sterilized flasks.
  • Mix well and heat the media to dissolve properly and check the pH of media before sterilization.
  • Autoclave the media for sterilization at 121ºC temperature and 15psi pressure for 15 minutes.


            A.) Total microbial count       B.) Pathogen detection


     Membrane Filtration Technique

  • Assemble the filtration unit with filtration cap & vacuum pump.
  • Remove the cap of sterile filtration unit having 0.45micron membrane filter (diameter 47mm) and transfer 100 ml of water into the filtration unit Start the vacuum pump & allow the sample to flow through the membrane filter.
  • Wash the membrane by filtering through it about 100ml sterile 0.1% peptone water and switch off the vacuum pump.
  • Remove the membrane filter using sterile forceps from the apparatus & Place it on the surface of sterile solidified SCDA plate in such a way that no air bubble form between the fitter and the culture medium.
  • Invert & incubate the plates at 30°C to 35°C for 5 days.
  • After 5days incubation examine all the membranes. Count the total no. of colony forming units on each membrane filter.
  • For determination of yeast & mold use Sabouraud dextrose agar instead of R2A/SCDA. Follow similar procedure like total bacterial count. Incubate the plates at 20°C to 25°C for 5-7 days.
  • Examine the plates for the absence of growth.

              Pour plate method:

  • Dispense aseptically 1ml of water sample with the help of pipette into sterile Petriplates.
  • Pour approximately 15-20ml of sterile R2A/ SCDA.(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 dishes and allow solidifying the media at room temperature
  • Seal the petriplates with parafilm and invert the petriplates 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°C for 5 to 7 days.
  • After incubation examine the plates for the absence of growth.
  • Filter 100 ml 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.

                Detection of Escherichia coli:

  • From pre-incubated SCDM, pipette 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°C 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 coli.
  • Further confirmation is done by gram’s staining

        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 30-35for 24-48 hours.
  • Streak above media on the surface of Xylose lysine deoxycholate 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
  • Further confirmation is done by gram’s staining.

                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 30-35for 24-72 hours.
  • A greenish colored colony indicates the possibility of presence of aeruginosa.
  • Carry out the further confirmation by pigment and oxidase tests & gram’s staining.

                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 30-35for 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.

           6.0   PARAMETERS OF TEST


1.        Description Clear, colorless, odorless and tasteless liquid
2.        pH Between 5.0 to 7.0
3.        Microbiological limits
      Total bacterial count        Not more than 100 CFU/ml
    Alert limit   25 CFU/ml
   Action limit   50 CFU/ml
      Total Fungal count      Not more than 10 CFU/ml
     Escherichia coli    Should be absent
     Salmonella     Should be absent
    Staphylococcus aureus     Should be absent
Pseudomonas aeruginosa Should be absent





Microbiology Labs Documents