Microbiological Analysis of Water – Procedure

Standard Operating Procedure for Microbiological Analysis of Water used for manufacturing of various drug product in pharmaceutical industry.

Procedure for Microbiological Analysis of Water

1.0   Objective :

• • To lay down a Procedure for microbiological analysis of Water.

2.0   Scope :

• • This SOP is applicable in Microbiology for microbiological analysis of water.

3.0   Procedure – Microbiological Analysis of Water:

• • Microbiologist shall collect the sample of water as per sampling plan following SOP – Sampling of Water for Testing.

• • Total Aerobic Microbial Count:

• • ON receipt of water sample for microbiological analysis, Microbiologist shall enter details in annexure.

• • Allocate A.R. No to received water samples as per SOP.

• • Prepare the sufficient media as per SOP for Media Preparation.

• • Sample Preparation for purified water:

• • Connect autoclaved filtration assembly with vacuum flask which is connected with vacuum pump.

• • Place the 0.45mm sterile filter membrane aseptically on the supporting disc of autoclaved filtration assembly & fix the sterilized funnel on the membrane filter holder aseptically.

• • Take 1ml of water sample from the bottle and add in 100ml peptone water, mix well and filter though sterile membrane filter, hold in a sterile filtration assembly in duplicate manner for each purified water individually .

Note: Use sterile support disc & sterile funnel separately for each sample.

• • Incubation:

• • After filtration remove the membrane filter aseptically using sterilized forceps and Place it on a pre-incubated sterile R₂A media plate.

• • Incubate the R₂A Petri dish for 5 days at 30 – 35°C and record the observations in annexure-II.

• • Sample Preparation for potable water:

• • Take 01 ml of potable water sample, separately in duplicate sterilize Petri plates.

• • Then add 15 to 20ml of Soyabean Casein Digest Agar in two petri plates

• • Allow it to solidify, after solidifying. Keep the plate in inverted position and Incubate at 30⁰C to35⁰C for 05 days.

• • Acceptance Criteria:

• • Pathogens detection in water samples:

• • The sample shall be tested for the following four specific pathogens:

• • • Salmonella species.

• • • Escherichia coli.

• • • Pseudomonas aeruginosa.

• • • Staphylococcus aureus.

• • • Shigella

• • Preparation of Sample:

• • Take 90ml of pre incubated Soyabean casein digest medium and add 10 ml of purified/ potable water.

• • After inoculation incubate at 30-35°C for 18- 24 hrs.

• • Test for Salmonella species:

• • Use 0.1 ml inoculated broth (SCDM) to 10 ml preincubated Rappaport Vassiliadis Salmonella enrichment broth incubates at 30-35°C for 18-24 Hrs.

• • The water sample complies with the test if Rappaport Vassiliadis Salmonella enrichment broth tube does not show turbidity,

• • If there is any turbidity or any doubt for poor microbial growth then takes the loopful of test sample and Sub culture on pre incubated Xylose- Lysine Deoxycholate agar plate.

• • Incubate at 30°C to 35°C for 18 to 48 hours.

• • The water sample complies with the test, if above described colonies are not present

• • If colonies are found confirming to the above descriptions identification shall be performed by identification tests as follow:

• • Identification Tests I:

  • Individually  transfer  the  suspected  colony  by first  Sub culturing  on the pre incubated slope of  slant,  of  Triple Sugar-Iron  Agar  with inoculating loop  and 

• • Then stabbing  with inoculating straight wire  well  in the butt. Incubate at 30-35° C for 24-48 hours.

• • After incubation, examine the tube of Triple Sugar Iron Agar Medium for the presence of microbial growth and for the following Physical characteristics:

• • • Color changes from red slant to yellow butt, with or without concomitant blackening of butt due to H₂S production.

• • If the butt, slant of Triple Sugar Iron Agar shows growth and physical characteristics confirming to the above descriptions the presence of Salmonella species is indicated.

• • The water sample complies with the test if the confirmatory identification tests are negative.

• • Record the observations in annexure II.

• • Test for Escherichia coli:

• • After incubation of soyabean casein digest medium, add 01 ml SCDM to 100ml pre incubated MacConkey’s broth incubate it at 42 to 44°C for 24 to 48 hours.

• • The water sample complies with the test if tube does not show any turbidity.

• • If present, take loop full from MacConkey’s broth & Sub culture on preincubated MacConkey’s Agar plate & incubate at 30-35°C for 18 to 72 hour.

• • The growth of brick red, may have surrounded zone of precipitated bile colonies indicates the possible presence of Escherichia coli.

• • The water sample complies with the test if colonies are not present.

• • If colonies are found, identification shall be performed by identification tests as follow:

• • Identification Tests I: Indole test

• • Aseptically prepare culture suspension from the suspected colonies which are appears on MacConkey agar in a sterilized tube.

• • Add 0.1 ml of the prepared suspension to the tube containing 10 ml of peptone water; incubate the tube at 30°C to 35°C for 18 – 24 hours.

• • From incubated peptone water tube perform Indole test as follow:

• • Add 0.5 ml of Kovac’s reagent to peptone water tube; allow to stand for one minute.

• • The presence of cherry red colour ring along the side of test tube indicates the positive test (the presence of Escherichia coli).

• • Presence of Escherichia coli shall be confirmed by Gram staining (Gram-ve rods).

• • The water sample complies with the test if the confirmatory identification tests are negative.

• • Record the observation in annexure II.

• Test for Pseudomonas aeruginosa – Microbiological Analysis of Water:

• • Take a loop full from SCDM & subculture on pre incubated Cetrimide agar & incubate at 30-35°C for 18 to 72 hour.

• • Interpretation:

• • The growth of colonies indicates the possible presence of P. aeruginosa.

• • The water sample complies with the test if colonies are not present.

• • If colonies are found, identification shall be performed by identification tests as follow.

• • Identification Tests I (Oxidase test):

  • With the aid of an inoculating loop, transfer suspected colonies to strip or discs of filter paper impregnated with N, N-dimethyl-p-phenylenediamine dihydrochloride.

• • If a purple colour is produced within five to ten seconds, the presence of Pseudomonas aeruginosa is confirmed, record the results in annexure II.

• • The water sample complies with the test if the confirmatory identification tests are negative.

• • Record the observation in annexure II.

• Test for Staphylococcus aureus – Microbiological Analysis of Water:

• • Take loop full from SCDM & subculture on, Pre incubated Mannitol Salt agar plate & incubate at 30-35°C for 18 to 72 hour.

• • Interpretation:

• • The possible presence of S. aureus is indicated by the growth of yellow or white colonies surrounded by yellow zone.

• • The water sample complies with the test if colonies are not present.

• • If colonies are found confirming to the above descriptions identification shall be performed by identification tests as follow.

• • Identification Tests I (Coagulase test):

• • With the aid of an inoculating loop, individually transfer suspected colonies to separate tubes containing 0.5 ml of mammalian plasma (preferably rabbit or horse).

• • Incubate in a water-bath at 37°C for 3 to 24 hours, in parallel with positive control using known strain of Staphylococcus aureus and negative control using Plasma alone.

• • Examine after 3 hours and at suitable intervals thereafter for the presence of coagulation

• • If coagulation in any degree is observed, the presence of Staphylococcus aureus is indicated. Record the results.

• • The water sample complies with the test if colonies of types described are not present or if the confirmatory identification tests are negative.

• Test for Shigella

• • After incubation of SCDM transfer 1 ml to 100 ml Pre incubated GN Broth and incubate at 30º to 35º C for 24 to 48 hours.

• • Further subculture on a pre incubated plate of Xylose Lysine Deoxycholate Agar and incubates at 30º to 35º C for 24 to 48 hours.

• • If red colored translucent colony without black center appears on the medium, indicates possible presence of Shigella.

• • Then proceed for further confirmation tests.

• • If such characteristic growth doesn’t appear then sample passes the test for absence of Record the results in annexure-I.

• • Further confirmation can be done by sub culturing the suspected colonies on Modified SS agar.

• • If the characteristic growth than confirms the presence of Shigella in the sample under examination, sample fails the test for absence of Shigella.

• Testing frequency:-

  • Pathogen testing of all purified water samples shall be done fortnightly except storage tank and return loop.

• • Pathogen testing of storage and return loop shall be performed daily.

• Trend frequency:-

• • The trend of all sampling points shall be prepared on quarterly basis.

4.0   Reference :

• • IP  : General chapter

• • BP : General Notices, Purified Water

• • USP  : Chapter No. 1231, Water for Pharmaceutical purposes

• • Revised Schedule M

5.0   Annexure (S)

Annexure I : Test Data Sheet of water analysis for specified organism.

Annexure II : Microbial Analysis Report of Water

Click to open the word file : Microbial Analysis Report of Water