STANDARD OPERATING PROCEDURE FOR MICROBIAL ANALYSIS OF WATER

1.0 OBJECTIVE:

To lay down the procedure for microbiological analysis of water.

2.0 SCOPE:

This SOP shall be applicable for microbiology analysis of water in Microbiology Laboratory

3.0 RESPONSIBILITY:

In-charge Microbiology shall be responsible for overall compliance of this procedure.

4.0 INTRODCUTION:

 

Bacteriological water analysis is a method of analyzing water to estimate the numbers of bacteria present and, if needed, to find out what sort of bacteria they are. It represents one aspect of water quality. It is a microbiological analytical procedure which uses samples of water and from these samples determines the concentration of bacteria. 

It is then possible to draw inferences about the suitability of the water for use from these concentrations. This process is used, for example, to routinely confirm that water is safe for human consumption or that bathing and recreational waters are safe to use. 

The interpretation and the action trigger levels for different waters vary depending on the use made of the water. Whilst very stringent levels apply to drinking water, more relaxed levels apply to marine bathing waters, where much lower volumes of water are expected to be ingested by users. 

The common feature of all these routine screening procedures is that the primary analysis is for indicator organisms rather than the pathogens that might cause concern. Indicator organisms are bacteria such as non-specific coliforms, Escherichia coli and Pseudomonas aeruginosa that are very commonly found in the human or animal gut and which, if detected, may suggest the presence of sewage. 

Indicator organisms are used because even when a person is infected with a more pathogenic bacteria, they will still be excreting many millions times more indicator organisms than pathogens. It is therefore reasonable to surmise that if indicator organism levels are low, then pathogen levels will be very much lower or absent. 

Judgments as to suitability of water for use are based on very extensive precedents and relate to the probability of any sample population of bacteria being able to be infective at a reasonable statistical level of confidence.

5.0 PROCEDURE:

Sampling of Water:

ü The water sample shall be collected in pre sterilized water sampling bottles after draining (for 1 minute) from all the points of use.

ü Before collection of water samples sampling point shall be sanitized with 70% filtered IPA giving the contact time of 1 minute.

ü Sample of 250 to 300 ml is collected aseptically from individual points of use, into sterile sampling bottles for microbial analysis.

ü After drawing the sample, the analysis shall be carry out with in 2 hrs of sampling. If the analysis is not carried out with in 2 hrs water sample can stored at 2 to 80C for a period of 12 hrs.

· Total Aerobic Microbial Count: By pour plate method

ü Ensure that 1ml of sample shall be take into a sterile Petri plate.

ü Pour aseptically approx. 15 to 20 ml of sterile liquified cooled (450C) SCDA media for bacterial count & SDA or SCA agar media for fungal count in plate. Mix the contents properly by gently rotating the Petri plates clockwise & anticlockwise on the LAF platform.

ü Allow media in Petri plates to solidify under LAF. After solidification of the media, incubate Petri plates containing SCDA agar media incubate at 30 to 350C for 3 to 5 days for Bacterial incubation and Petri plates containing SDA or SCA agar media incubate at 20 to 250C for 5 days for fungal incubation.

ü After completion of incubation, count the number of cfu formed on the plates with the help of colony counter and records the results properly.

· Tests for Specified Microorganisms:

ü Take 100 ml of water sample and filter it through filter membrane having 0.45 µ pour size and 47 mm dia. After filtration transfer the filtered membrane into 100 ml SCDM. (Tube A)

ü Incubate the tube in an incubator at 30 to 350C for 18 to 24 hrs. (Tube A)

· Tests for E. coli Species

ü After completion of incubation period shake the broth and transfer 1 ml (from Tube A) to 100 ml of sterilized MCB & incubate it at 42 to 440C for 24 to 48 hrs.

ü Subculture on a plate of MCA and incubate the plates at 30 to 350C for 18 to 72 hrs.

ü Growth of pink, non mucoid colonies indicates the possible presence of E. coli. This shall be confirmed by identification test.

ü If there is no growth of such type of colonies, the identification test are negative it indicates absence of E. coli and the sample passes the test for E.coli.

Characteristic of E. coli on selective agar

Name of Media	Colony characteristic	Gram Staining
MCB	Acid formation (Color change) &gas formation (bubbles in Durham’s tubes) observed / doesn’t observed	NA
MCA	Growth of pink, non – mucoid colonies observed / doesn’t observe.	Gram Negative Rods
EMB Agar	Growth of green metallic sheen	Gram Negative Rods

Test for Salmonella sp.

ü After incubation shake the SCDM broth and transfer 0.1 mL (from Tube A) to 10 ml of RVSB and incubate at 30 to 350C for 18 to 24 hrs.

ü Subculture on a plate of XLDA and incubate at 30 to 350C for 18 to 48 hrs.

ü Well developed red colonies with or without black centers indicates possibility of Salmonella spp.

ü If any colonies as described above are produced. Then proceed for the secondary test.

ü Secondary Test

ü Using sterile inoculation loop, transfer the suspected colonies on to a sterile TSI slant by first streaking the surface of the slant and the stabbing the loop well beneath into the surface. At the same time inoculate a tube of Urea Broth.

ü Incubate in an incubator at 35 ± 20C for 18 to 24 hrs

ü If examination shows evidence of having alkaline (red) slants and acid (yellow)butts with (or) without concomitant blackening of the butt from the hydrogen sulphide, together with the absence of a red color in the UB that indicates the presence of Salmonellae sp.

ü This shall be confirmed by identification test.

ü If there is no growth of such type of colonies, or the identification test is negative it indicates absence of salmonella and the sample passes the test for Salmonella spp.



Characteristic of Salmonella species on selective agar

Name of Media	Colony characteristic	Gram Staining
RVSB	Growth in terms of turbidity observed	NA
BGA	Small,transparent and colourless or opaque,pimkish or white surrounded by pink	Gram Negative Rods
XLDA	Red colonies with or without black centers observed	Gram Negative Rods

· Test for P. aeruginosa:

ü After completion of incubation period Tube A Streak a portion of the medium from tube A on the surface of CA and incubate the plates at 30 to 350C for 18 to 72 hrs

ü A greenish color colony indicates the possibility of presence of P.aeruginosa.

ü If any colonies as described above are produced. Gram’s staining and oxidize test must be carried out with colonies suspected of P. aeruginosa.

ü Oxidase test

Smear a portion of colony with the help of sterile inoculation loop across on oxidase disk kept in sterile Petri dishes.

ü If there is no development of pink color, changing to purple, the specimen meets the requirement of the test for absence of P. aeruginosa.

Characteristic of P.aeruginosa on selective agar

Selective Media	Morphology	Oxidize test	Gram’s stain
CA	Generally greenish Colonies observed	Positive	Negative rods

 


ü If none of the plates shows colonies having the characteristics described above, the sample meets the requirements for freedom from P. aeruginosa.

· Test for S.aureus:

ü After completion of incubation period of tube A Streak a portion of the medium from tube A on the surface of MSA surface and incubate the plates at 30 to 350C for 18 to 72 hrs.

ü Examine the plates for the typical growth of S.aureus colonies on the selective agar medium colonies, which shows the following characteristics.

Characteristic of S.aureus on selective agar

Selective Media	Morphology	Oxidize test	Gram’s stain
CA	Generally greenish Colonies observed	Positive	Negative rods

ü If none of the plates contains colonies having the characteristic expressed above, the sample meets the requirement for freedom from S.aureus

ü If any colonies are positive for S.aureus perform gram’s staining and coagulase test using single typical colony from selective agar media.

ü Secondary test

ü Coagulase test

ü With a sterile inoculation loop, transfer suspect colonies from selective agar media to a tube containing 0.5 ml of rabbit or horse plasma.

ü Keep negative control without addition of culture to 0.5 ml of rabbit or horse plasma.

ü Keep positive control by transferring S.aureus culture with the help of sterile inoculation loop in 0.5 ml of rabbit or horse plasma.

ü Incubate in an incubator at 35 to 370C for 24 hrs and observe for coagulation at 3 hrs intervals.

ü If no coagulation is observed in the test sample meets the requirements for absence of S.aureus.

ü Frequencies: As per sampling schedule.

ü Limits/ Acceptance Criteria

Type of water	Alert Limit	Action Limit	Standard Limit
Raw water	100 cfu/ml	300 cfu/ml	500 cfu/ml
Pretreated	50 cfu/ml	100 cfu/ml	250 cfu/ml
Purified	30 cfu/ml	50 cfu/ml	100 cfu/ml

ü Fungi & Pathogen should be absent.

· Action Plan

ü If the Alert limit is exceeded or pathogen is present in water, immediately inform to Production department & Maintenance department to carry out cleaning and sanitization of water system.

ü After sanitization, again conduct the microbiological analysis to verify the cfu is within limits and absence of pathogens.

6.0 PRECAUTIONS:
Protective laboratory coats, gowns, smocks, gloves or uniforms designated for lab use will be worn while in the laboratory.
All infectious agent cultures, stocks, animal carcasses and/or tissues, sharps used to inoculate animals, etc. and bedding from infected animals will be disposed of as hazardous waste (i.e. put in an appropriately labeled red biohazard bag, and decontaminated By autoclaving.



7.0 REFERENCES:
API, Part-I, Vol.-IX (Extracts); Appendices-3; pp 124

8.0 ABBREVATIONS:

SOP: Standard Operating Procedure

cfu: colony forming unit

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