sop for microbiological testing of water

Sop for microbiological testing of water

1 OBJECTIVE
To describe the procedure for analysis of water sample for estimation of the number of viable aerobic micro-organisms present & for the detection of Pathogenic microbial species.

2 SCOPE
This procedure is applicable to the analysis of water for microbial load in microbiology lab

3 RESPONSIBILITY
Microbiologist.

4 ACCOUNTABILITY
Head – Q. C

5 ABBREVIATIONS
SOP : Standard Operating Procedure
Q .A. : Quality Assurance
Q. C. : Quality Control
NO. : Number
MLT : Microbial limit test
SCDM : Soyabean Casein Digest Medium.
HLAF : Horizontal laminar air flow
DM : De-mineralized.
WFI : Water for Injection.

6 PRECAUTIONS
6.1 Water sampling should be done in sterile screw cap bottles following the SOP No.
6.2 Sample the water aseptically in sampling bottle.
6.3 Media should be fresh for use in test.

7 MATERIALS REQUIRED
Soyabean Casein Digest Medium, R-2A media, Peptone water, Sterile Petri plates, test tubes, Micropipette (1ml), Micro tips (1ml), Filtration assemblies, Sterile Gridded nitrocellulose Filter paper (0.45 μ),
Sterile forceps, Sterile scissor, Filtered 70%IPA, Selective media as per requirement.

8 EQUIPMENTS REQUIRED
HLAF, Autoclave, Incubators, Burner, Colony counter, Inoculating loop.

9 PROCEDURE
9.1 Total Aerobic Microbial Count
9.1.1. Plate count method: (Potable Water)
9.1.1.1 Take 1.0 ml of the sample in sterilized Petri plate.
9.1.1.2 Pour 15.0 to 20.0 ml of autoclaved and cooled to 45 to 48°C of R2A agar medium.
9.1.1.3 Mix the contents of the Petri plate by rotating it gently and allow the media to solidify.
9.1.1.4 Invert the Petri plates and incubate in BOD incubator at 30˚C to 35˚C for 5 days for bacterial
and fungal count.
9.1.1.5 After incubation examine the Petri plates for growth and count the number of colonies by using
Colony counter.
9.1.1.6 01ml Sample in 09ml Soyabean casein Digest Medium mix well and keep at 20 to 25ºC for about 2 to5
hours to resuscitate the organism.
9.1.1.7 01ml inoculated prepared sample inoculate in 09ml Enterobacteria Enrichment Broth-Mossel and
incubate at 30 to 35ºC for 24 to 48 hours and subculture Violet Red Bile Glucose Agar incubate at 30
to 35ºC for 18 to 24 hours. Sample is passed if there is no growth on media plates.
9.1.2. Membrane filtration method (WFI and Purified Water)
9.1.2.1 Filtered 200ml of WFI & 1ml Purified water sample inoculate to100ml sterile water/peptone, from
water sampling bottle.
9.1.2.2 Remove butter paper from filtration assembly and silicon tubing under HLAF.
9.1.2.3 Connect filtration assembly to the filtration flask and vacuum pump with the help of silicon tubing.
9.1.2.4 Take out the sterile 0.45μ membrane filter from the individual pack and place the filter paper
sandwiched between filtration cup & filtration receptacle with the help of sterile forcep.
9.1.2.5 Open the lid of filtration cup filter the approx 15-20ml sterile peptone water.
9.1.2.6 Take water sample collected in sampling bottle, open the lid carefully without touching.
9.1.2. 7 Switch “ON” the vacuum pump. Open the lid of filtration cup & pour the content of sampling bottle
into the filtration cup.
9.1.2.8 Switch off the vacuum pump and remove the SS receptacle from the holder base aseptically.
9.1.2.9 By means of sterile forcep transfer the filter paper on the R2A agar plate aseptically.
9.1.2.10 Incubate the plate at 30˚C to 35˚C for 5 days.
9.1.2.11 Complete incubation period, observe the colonies and count by using colony Counter.
9.2. Pathogens testing
9.2.1 Prepare 100ml SCDM tube and all selective media as per SOP for media preparation
(EH/MIC/SOP/018)

9.2.2. Test for E.coli
9.2.2.1 Enrichment:
Filter the 100ml of water sample and transfer the filter paper very carefully in 100ml Soybean Casein
Digest Medium, shake gently and incubate at 30°C -35°C for 18 to 24 hrs. After incubation examine the tube. If growth is present mix by gentle shaking. Further proceed for the specified test.

9.2.2.2 Primary Test:
Shake the tube, transfer 1.0 ml of soyabean casein digest broth into bottle containing 100ml of
MacConkey broth and incubate at 42 C to 44˚C for 24 to 48 hrs.
9.2.2.3 Secondary Test:
Subculture on the surface of MacConkey agar plate and incubate at 30°C to 35°C for 18 to 72 hrs. If
Appearance of pinkish red color colonies on Mac Conkey agar indicates the presence of E.coli.
9.2.2.4 Confirmatory Test: Indole Test:
Inoculate the suspected colonies to test tube containing 5.0 ml peptone water. Incubate the tube at
30°C -35°C for 24 hrs, for indole production. Add 0.5 ml Kovac’s reagent shake well and allow to stand
for one minute if red colour ring is observed in the reagent layer indole is present.
MORPHOLOGICAL CHARACTERITICS OF E.coli SPECIES ON
SELECTIVE CULTURE MEDIA TABLE- I

MEDIUM

DESCRIPTION OFCOLONY

Mac Conkey agar

Mac Conkey broth

Kovac’s reagent

Pinkish red colour colonies may have surrounding zone of precipitated bile

Growth present

Red ring formation

9.2.3 Test for Salmonella
9.2.3.1 Enrichment
Filter the 100ml of water sample and transfer the filter paper very carefully in 100ml Soybean Casein
Digest Medium, shake gently and incubate at 30°C to 35°C 18 to 24 hrs. After incubation examine
the tube. If growth is present mix by gentle shaking. Further proceed for the specified test.
9.2.3.2 Primary Test:
Add 0.1 ml of enrichment culture transfer test tube containing 10 ml Rappaport Vassiliadis
Salmonella Enrichment Broth and incubate at 30˚C to 35˚C for 24 to 48 hrs. After incubation
the tube subculture on the Xylose Lysine Deoxycholate Agar and incubate at 30˚C to 35˚C for 24 to
48 hrs. If red colonies with or without black centers possibly of salmonella.
9.2.3.3 Secondary Test:
Subculture those colonies which show the characteristics as given in table II. Subculture the triple
sugar iron agar slants by stabbing the wire well beneath the surface. Incubate the stabbed slants at
30°C to 35°C for 24 hrs. The formation of acid and gas in the stab culture (with or without Concomit –
-ant blackening) confirms the presence of Salmonella while its absence confirms the specimen or
sample to be free from Salmonella.
MORPHOLOGICAL CHARACTERITICS OF SALMONELLA SPECIES ON
SELECTIVE CULTURE MEDIA TABLE-II

MEDIUM	DESCRIPTION OF COLONY
Xylose Lysine Deoxycholate Agar	Red with or without black centers

9.2.4 Test for Pseudomonas aeruginosa
9.2.4.1 Enrichment: Filter the 100ml of water sample and transfer the filter paper very carefully in 100ml.Soybean Casein Digest Medium, shake gently and incubate at 30°C to 35°C 18 to 24 hrs. After incubation examine the tube. If growth is present mix by gentle shaking. Further proceed for the specified test.
9.2.4.2 Primary Test: Streak one loop full of the enrichment culture on the surface of Cetrimide agar plate, and incubate the plates in inverted position at 30 to 35 ˚C for 18 to 72 hrs. Greenish color colonies indicate the possibility of Pseudomonas aeruginosa
9.2.4.3 Oxidase test: Place 2 or 3 drops of Oxidase reagent (Tetra methyl para phenylene diammonium dihydro-Chloride) on a piece of filter paper (WhatmanNo.1) and transfer the suspected colony making smear on the paper if a pink colour is produced changing to purple within 5 –10 seconds, the test confirms the presence of P. aeruginosa.

MORPHOLOGICAL CHARACTERITICS OF SALMONELLA SPECIES ON
SELECTIVE CULTURE MEDIA Table – III

Medium	Characteristics colonial morphology	Fluorescence in UV light
Cetrimide agar	Generally greenish	Greenish

9.2.5 Test for Staphylococcus aureus 9.2.5.1Enrichment: Filter the 100ml of water sample and transfer the filter paper very carefully in 100ml
Soybean Casei Digest Medium, shake gently and incubate at 30°C to 35°C for 18 to 24 hrs. After
incubation examine the tube. If growth is present mix by gentle shaking. Further proceed for the
specified test.
9.2.5.2 Primary Test:Streak one loop full of the enrichment culture on the surface of Mannitol Salt agar incubate the plates inverted position at 30°C to 35°C for 18 to 72 hrs. Yellow or white colonies with yellow zones indicate the possibility of presence of Staphylococcus aureus. Proceed further for confirmatory test.
9.2.5.3 Confirmatory Test Coagulase Test:
With the help of inoculating loop transfer representative suspected colonies from the agar surface of
the Vogel Johnson agar or Mannitol salt agar to individual tube containing 0.5 ml mammalian plasma
preferably rabbit or horse plasma with or without suitable additive. Incubate in a water bath at 30°C to
35°C and three hours and subsequently at suitable intervals up to 24hrs. along with test positive and
negative control simultaneously. If no coagulation in any degree is observed, the specimen meets the
requirements of the test of absence of Staphylococcus aureus

MORPHOLOGICAL CHARACTERITICS OF SALMONELLA SPECIES ON
SELECTIVE CULTURE MEDIA TABLE- IV

MEDIUM	DESCRIPTION OF COLONY
Mannitol Salt agar	Yellow colonies with yellow zones

9.2.6 Test for bile tolerant gram negative bacteria test:
9.2.6.1 Enrichment: 01 ml sample in 09 ml Soyabean casein digest Medium mix well and keep at 20 to 25ºc for about 2 to 5 hours.
9.2.6.2 Primary test: 01 ml inoculated prepared sample inoculate in 09 ml Enterobacteria Broth Mossel incubate at 30 to 35ºC for 24 to 48 hours. Sub culture on violet Red Bile Glucose agar incubate 30 to 35ºC for 18 to 24 hours. Sample is passed if there is no growth on media pltes.
9.2.6.3 Secondary Test:(if growth observed on media plates then carry out the quantitative test)
01 ml sample in 09 ml soyabean casein digest medium mix well and keep at 20 to 25ºC for about 2 to 5 hours. Take volume corresponding to 0.1 ml,0.01 ml,0.001ml of the sample suitable quantity in Enterobacteria Broth Mossel incubate at 30 to 35ºC for 24 to 48 hours. Sub culture on violet Red Bile Glucose agar incubate 30 to 35ºC for 18 to 24 hours. Sample quantity determined as per the table.

Result for each quantity of sample

Number of Bacteria per ml of Sample

0.1ml

0.01ml

0.001ml

More than 10³

Less than 10³ and more than 10²

Less than 10² and more than 10

Less than 10

–

WATER LIMIT:

	Alert Limit	Action Limit	Specification Limit
RAW WATER	300cfu/ml	400 cfu/ml	500 cfu/ml
PURIFIED WATER	50 cfu/ml	80 cfu/ml	100 cfu/ml
WATER FOR INJECTION	05 cfu/100ml	08 cfu/100ml	10 cfu/100ml
PATHOGENS	Should be absent/100 ml
Bile Tolerant Gram negative Bacteria	Should be absent/ ml

Remark: Alert Limit:- If the alert limit is crossed then immediately inform to QA department and water generation department. Increase the sampling frequency, monitor the trend until proper preventive action is taken.
Action Limit :- If action limit is crossed then immediately inform to QA department and water generation department. Immediately stop the production and drain water from water storage tank. Sanitize the water for injection storage tank, investigate the cause, after sanitization, and collect the sample for testing, until satisfactory result are obtained (sanitize the complete water system if required) After that, intimate to the water generation department to produce the water for regular production.

10. FREQUENCY
Daily

11. ENCLOSURES
ANNEXURE -1 Raw /Treated /Purified Water Analysis Report
ANNEXURE -2 Water For Injection Analysis Report
ANNEXURE -3 Change History Log Format

12. REFERENCE DOCUMENTS
Pharmacopoeias (IP/BP/USP)

ANNEXURE -1

QUALITY CONTROL DEPARTMENT (MICROBIOLOGY)

RAW/PURIFIED WATER ANALYSIS REPORT

1- TOTAL VIABLE AEROBIC COUNT:
Purified Water: 1.0 ml of water sample inoculate to100 ml sterile water/ Peptone water shake well and filter
Through 0.45μm filter paper and put on the R2A Agar plates and incubate at 30°C to 35°C for 5 Days.

Raw Water: Take1.0ml of water sample and pour in to sterile Petri plate. Pour 15-20ml sterilized R2A (temp.
Should not more than 50oC), mix by gentle rotating and incubate after media get solidified.
Medium : R2A Agar Autoclave Lot. No.:
Batch No. : Peptone Autoclave lot No.:
Incubation : 30°C to 35°C for 5 Days
Observation: Sampling point No. _________cfu/ml
Sampling point No. _________cfu/ml
Sampling point No. _________cfu/ml Sign:
Negative Control: Growth observed/not observed. Date:
2- PATHOGEN TESTING:
Filter_____ml water sample and transfer filter paper carefully in ______ml Soyabean Casein Digest Medium (Soln A) Incubate at 30°C to 35°C for 24 hrs.
Test for Escherichia coli:-
a) Primary Test:1ml of soln A transfer in 100 ml MacConkey Broth Incubate at 42°C to 44°C for 24 to 48 hrs.
i) MacConkey Broth: Autoclave Lot. No.
Observation:
Positive Control: Growth observed Observed/not observed
Negative Control: Growth observed Observed/not observed
Sampling Point No.: Growth observed Observed/not observed
Sampling Point No.: Growth observed Observed/not observed
Sampling Point No.: Growth observed Observed/not observed
b)Secondary Test : Sign: Date:
Subculture on the surface of MacConkey agar plate and incubate at 30°C to 35°C for 18 to 72 hrs.
MacConkey agar: Autoclave Lot. No.
Observation:
Positive Control: Pinkish red colour colonies with zone of precipitated bile Observed/not observed
Negative Control Pinkish red colour colonies with zone of precipitated bile Observed/not observed
Sampling Point No Pinkish red colour colonies with zone of precipitated bile Observed/not observed
Sampling Point No Pinkish red colour colonies with zone of precipitated bile Observed/not observed
Sampling Point No Pinkish red colour colonies with zone of precipitated bile Observed/not observed
Sign: Date:
c) Confirmatory Test If positive characters observed then carry out the confirmatory test)
Inoculate the suspected colonies to 5 ml Peptone Water. Incubate at 40-45°C for 24 hrs. After Incubation
add 3-5 drops of Kovac’s, reagent. Carry the positive control test using E.coli culture to both tubes.
Observation Positive Control: Red color ring Observed/ not observed
Negative Control: Red color ring Observed/ not observed
Sampling Point No.: ______ Red color ring Observed/ not observed
Sampling Point No.: ______ Red color ring Observed/ not observed
Sampling Point No.: ______ Red color ring Observed/ not observed
Test for Salmonella Species Sign: Date:
a) Primary test: 0.1 ml of Soln A to 10 ml Rappaport Vassiliadis Salmonella Enrichment Broth and incubate
at 30°C to 35°C for 24 to 48 hrs. After incubation subculture on the surface of Xylose lysine deoxycholate agar and incubate the Petri plates at 30°C to 35°C for 24 to 48hrs.
i) Rappaport Vassiliadis Salmonella Enrichment Broth: Autoclave Lot. No.
Observation: Positive Control: Growth Observed/ not observed
Negative Control: Growth Observed/ not observed
Sampling Point No.: ______ Growth Observed/ not observed
Sampling Point No.: ______ Growth Observed/ not observed
Sampling Point No.: ______ Growth Observed/ not observed Sign: Date:
ii) Xylose Lysine Deoxycholate Agar: Autoclave Lot. No.
Observation: Positive Control:Red with or without black centered colonies Observed/not observed
Negative Control: Red with or without black centered colonies Observed/not observed
Sampling Point No.: ______ Red with or without black centered colonies Observed/not observed
Sampling Point No.: ______ Red with or without black centered colonies Observed/not observed
Sampling Point No.: ______ Red with or without black centered colonies Observed/not observed
Sign: Date:
b) Secondary Test: (If positive characters observed then carry out the secondary test)
If typical growth observed then subculture on TSI slant and incubate at 30-35°C for 24 hrs.
Triple Sugar Iron (TSI) Agar Slant: Autoclave Lot. No.
Observation:
Positive Control: Pink slants with yellow butt and gas Observed/not observed
Negative Control: Pink slants with yellow butt and gas Observed/not observed
Sampling Point No.:______ Pink slants with yellow butt and gas Observed/not observed
Sampling Point No.:______ Pink slants with yellow butt and gas Observed/not observed Sign:
Sampling Point No.:______ Pink slants with yellow butt and gas Observed/not observed Date:

Test for Pseudomonas aeruginosa:
a) Primary Test:
If turbidity is observed in the (Sol. A), streak a loopfull from soln. A on cetrimide agar & Pseudomonas
Agar plate & incubate at 30°C to 35°C for 18 to72 hrs.
i) Cetrimide Agar Plate: Autoclave Lot. No.:
Observation
Positive Control: greenish colonies/ Greenish under UV light Observed/not observed
Negative Control: greenish colonies/ Greenish under UV light Observed/not observed
Sampling Point No.:______ greenish colonies/ Greenish under UV light Observed/not observed
Sampling Point No.:______ greenish colonies/ Greenish under UV light Observed/not observed
Sampling Point No.: ______ greenish colonies/ Greenish under UV light Observed/not observed
Sign: Date: b) Secondary Test:( If positive characters observed then carry out the secondary test)
Oxidase Test :
Positive Control: Purple colour Observed/ not observed
Sampling Point No.:______ Purple colour Observed/ not observed
Sampling Point No.: ______ Purple colour Observed/ not observed
Sampling Point No.: ______ Purple colour Observed/ not observed

Test for Staphylococcus aureus:
a) Primary Test:
If turbidity observed in the (Soln. A), streak a loop full from soln. A on Mannitol Salt Agar Incubate at 30°C to 35°C for 18 – 72 hrs.
i) Mannitol Salt Agar: Autoclave Lot. No.:
Observation:
Positive Control: Yellow colonies with yellow zones Observed/not observed
Negative Control: Yellow colonies with yellow zones Observed/not observed
Sampling Point No.:______ Yellow colonies with yellow zones Observed/not observed
Sampling Point No.:______ Yellow colonies with yellow zones Observed/not observed
Sampling Point No.:______ Yellow colonies with yellow zones Observed/not observed
Sign: Date:
b) Secondary Test: Coagulase Test (If positive characters observed then carry out the secondary test)
Transfer representative suspected colonies from the agar surface of above Medium to the tubes,
each containing 0.5 ml of mammalian plasma (preferably rabbit or horse). Incubate in water bath at
37°C; examine the tubes at 3 hrs and subsequently at suitable interval up to 24 hrs.
Observation:
Positive control: Coagulation observed/not observed
Negative control: Coagulation observed/not observed
Sampling Point No.:______ Coagulation observed/not observed
Sampling Point No.:______ Coagulation observed/not observed
Sampling Point No.:______ Coagulation observed/not observed
Sign: Date:

Test for Bile Tolerant Gram Negative Bacteria test:
a) Primary Test —–ml sample in —–ml Soyabean Casein digest Medium mix and keep at 20 to 25ºC for about 20to 5 hours.
—-ml inoculated prepared sample inoculate in —-ml Enterobacteria Enrichment Broth-Mossel incubate at 30 to 35ºC for 24 to 48 hours.
Sub culture on Violet Red Bile Glucose Agar incubate at 30 to 35ºC for 18 to 24 hours. Sample is passed if there is no growth on media plates.
Observation:
Positive Control: reddish colonies Observed/Not observed
Negative Control: reddish colonies Observed/Not observed
Sampling Point No: reddish colonies Observed/Not observed
Sampling Point No: reddish colonies Observed/Not observed
b) Secondary Test: (If growth observed on media plates then carry out the Quantitative test)
—-ml sample in —ml Soyabean casein digest Medium mix well and keep at 20 to 25ºC for about 2 to 5 hours. Take volume corresponding to 0.1ml, 0.01ml, 0.001ml of the sample suitable quantity Enterobacteria Enrichment Broth-Mossel incubates at 30 to 35ºC for 18 to 24 hours. Sample quantity determine as per the table.

Result for each quantity of sample

Number of Bacteria per ml of Sample

0.1ml

0.01ml

0.001ml

More than 10³

Less than 10³ and more than 10²

Less than 10² and more than 10

Less than 10

–

Observation :
Sampling Point No:————- cfu/ml Observed. Sing:
Sampling Point no:————- cfu/ml Observed Date:

RESULT:

TOTAL VIABLE COUNT

RESULT

REMARKS

SIGN./DATE

Sampling Point………

Bacterial Cfu/ml

Fungal Cfu/ml

Bacterial Cfu/ml

Fungal Cfu/ml

Bacterial Cfu/ml

Fungal Cfu/ml

Complies/does not comply

Sr. No.

PATHOGEN

SampleSpot NO.

Observation

REMARK

SIGN./DATE

E.coli

Detected/ Not detected

Complies/ not comply

Salmonella

Detected/ Not detected

Complies/ not comply

Pseudomonas aeruginosa

Detected/ Not detected

Complies/ not comply

Staphylococcus aureus

Detected/ Not detected

Complies/ not comply

Bile- Tolerant Gram Negative Bacteria

Detected/ Not detected

Complies/ not comply

	Alert Limit	Action Limit	Specification Limit
RAW WATER	300cfu/ml	400 cfu/ml	500 cfu/ml
PURIFIED ( DM ) WATER	50 cfu/ml	80 cfu/ml	100 cfu/ml
PATHOGENS	Should be absent/100 ml
Bile Tolerant Gram negative Bacteria	Should be absent/ ml