Sop for Analysis of Raw water Purified water water for injection and pure steam water

1.0 OBJECTIVE
1.1 To lay down 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.

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

3.0 RESPONSIBILITY
3.1 Microbiologist QC

4.0 ACCOUNTABILITY
4.1 Q. C. Manager

5.0 PROCEDURE
5.1 PRECAUTIONS
5.1.1 Water sampling should be done in sterile screw cap bottles following the SOP No.
5.1.2 Sampled the water aseptically in sampling bottle.
5.1.3 Media should be fresh for use in test.

5.2 MATERIALS REQUIRED
5.2.1 Soybean 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.

5.3 EQUIPMENTS REQUIRED
5.3.1 HLAF, Autoclave, Incubators, Colony counter, Inoculating loop.

5.4 Methodology
5.5 Total Aerobic Microbial Count
5.6 Plate count method: (Potable Water)
5.6.1 Take 1.0 ml of the sample in sterilized Petri plate.
5.6.2 Pour 15.0 to 20.0 ml of autoclaved and cooled to 45°C of R2A agar medium.
5.6.3 Mix the contents of the Petri plate by rotating it gently and allow the media to solidify.
5.6.4 Invert the Petri plates and incubate in BOD incubator at 30˚C to 35˚C for 5 days for bacterial
and fungal count.
5.6.5 After incubation examine the Petri plates for growth and count the number of colonies by using
Colony counter.

5.7 Membrane filtration method ( WFI/PSG and Purified Water)
5.7.1 Filtered 100ml of WFI /PSG& 1ml Purified water sample inoculate to100ml sterile peptone, from
water sampling bottle.
5.7.2 Remove butter paper from filtration assembly and silicon tubing under HLAF.
5.7.3 Connect filtration assembly to the filtration flask and vacuum pump with the help of silicon tubing.
5.7.4 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 forceps.
5.7.5 Open the lid of filtration cup filter the approx. 15-20ml sterile peptone water for rinsing of filter paper.
5.7.6 Switch “ON” the vacuum pump. Open the lid of filtration cup & pour the content of sampling bottle
into the filtration cup.
5.7.7 Switch off the vacuum pump and remove the SS receptacle from the holder base aseptically.
5.7.8 By means of sterile forceps transfer the filter paper on the R2A agar plate aseptically.
5.7.9 Incubate the plate at 30˚C to 35˚C for 5 days.
5.7.10 After Completion of incubation period, observe the colonies and count by using colony Counter.
5.8 Pathogens testing
5.8.1 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.
5.9 Test for E.coli
5.9.1 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.
5.9.2 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 MacConkey agar indicates the presence of E.coli.

MORPHOLOGICAL CHARACTERITICS OF E.coli SPECIES ON

MEDIUM

DESCRIPTION OFCOLONY

Mac Conkey agar

Pinkish red colour colonies may have surrounding zone of precipitated bile

Growth present

5.10 Test for Salmonella
5.10.1 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.
5.10.2Primary 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 indicate the presence of salmonella.

MORPHOLOGICAL CHARACTERITICS OF SALMONELLA SPECIES ON

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

5.11 Test for Pseudomonas aeruginosa

5.11.1Filter 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.
5.11.2Primary 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

MORPHOLOGICAL CHARACTERITICS OF SALMONELLA SPECIES ON

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

5.12 Test for Staphylococcus aureus
5.12.1Filter 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 for 18 to 24 hrs. After
incubation examine the tube. If growth is present mix by gentle shaking. Further proceed for the
specified test.
5.12.2Primary 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.
.

MORPHOLOGICAL CHARACTERITICS OF SALMONELLA SPECIES ON

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

5.13 FREQUENCY
Daily

6.0 ABBREVIATIONS

Sr. No.	Abbreviation used	Full form of abbreviation used
1.0	SOP	Standard Operating Procedure
2.0	IPA	Iso Propyl Alcohol
3.0	QA	Quality assurance
4.0	HEPA	High Efficiency Particulate Air
5.0	MLT	Microbial Limit Test
6.0	HLAF	Horizontal Laminar Air Flow
7.0	WFI	Water For Injection
8.0	CFU	Colony Forming Unit

Acceptance criteria
TYPE OF WATER	TAMC	PATHOGEN
Raw water	500cfu/ml	Absent
Purified water	100cfu/ml	Absent
Water for injection/Pure steam	10cfu/100ml	Absent