GENERAL TEST PROCEDURE (GTP) Page 1 of 8

Title Detection of Pathogens in Water Samples Effective Date 04/01/05

GTP Number GTP 114-02 Review Date 03/01/07

REVISION SUMMARY

GTP Number Date Revised Reason for Revision

GTP 053-00 01/07/2002 First Issue
GTP114-01 16/06/2003 Renumbering of GTP and Format Changed
GTP 114-02 04/01/2005 The format of the GTP is changed as per SOP QA
030-00, the procedure is revised and title of the
GTP is changed

1.0 PURPOSE
1.1 To lay down the procedure for Detecting of pathogens in water samples.
Prepared By Checked By Approved By
Name G.S.R.K.RAJU N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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 GENERAL TEST PROCEDURE (GTP) Page 2 of 8

Title Detection of Pathogens in Water Samples Effective Date 04/01/05

GTP Number GTP 114-02 Review Date 03/01/07

This test is applicable to all the samples for detection of following pathogen.
1) Salmonella species
2) Pseudomonas aeruginosa
3) Staphylococcus aureus
4) Escherichia coli

2.0 APPARATUS
2.1 Petri Plates (90mm diameter)
2.2 Bottles
2.3 Test tubes
2.4 Inoculating loop

3.0 REAGENTS:
3.1 Soya bean casein digest medium,
3.2 Mac Conkey broth purple,
3.3 Mac Conkey agar,
3.4 EMB agar,
3.5 Vogel Johnson agar,
3.6 Mannitol salt agar,
3.7 Cetrimide agar,
3.8 Selenite F broth,
3.9 Tetrathionate broth,
3.10 Brilliant green agar,
3.11 Bismuth sulphite agar,
3.12 Xylose lysine deoxychollate agar.
3.13 Gram staining Kit.
4.0 PROCEDURE
4.1 TEST FOR Escherichia coli
Prepared By Checked By Approved By
Name G.S.R.K.RAJU N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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 GENERAL TEST PROCEDURE (GTP) Page 3 of 8

Title Detection of Pathogens in Water Samples Effective Date 04/01/05

GTP Number GTP 114-02 Review Date 03/01/07

4.1.1.1 Prepare and sterilize the media Soya bean casein digest medium,
Mac Conkey Broth, Mac Conkey Agar and Eosin Methylene Agar.
4.1.1.2 For Mac Conkey Broth transfer 10 ml medium into test tubes
(Size 25 x 100 mm).
4.1.1.3 After sterilization of Mac Conkey agar and Eosin Methylene blue
agar, pour approximately 20ml of the medium into the Petriplates at 45-50°C
and allow it to solidify.

4.2 INOCULATION
4.2.1 Add 10ml of water sample into 100ml of SCD medium tube for enrichment.
Incubate the tubes at 30-35°C for 24 Hrs.
4.2.2 Transfer 1.0 ml of the sample from the SCD medium tube into the tube containing
10 ml of Mac Conkey Broth medium and mix gently.
4.2.3 Incubate one un-inoculated tube as negative control from each autoclaved lot of
medium.
4.2.4 Incubate the tubes at 30 to 35°C for 24 hours.
4.2.5 Observe the sample tubes after 24 hours for growth.
4.2.6 If growth is present transfer one loopful from the Mac Conkey broth medium and
streak on to the Mac Conkey agar plate.
4.2.7 Keep the Mac Conkey agar plate for incubation at 30 to 35°C for 48 hrs.
4.2.8 Further if growth is present in the plate go for confirmatory test by streaking on
the Eosine Methylene blue agar plate.

4.3 TEST FOR Salmonella sps:

4.3.1 Prepare and sterilize the following media.

Prepared By Checked By Approved By

Name G.S.R.K.RAJU N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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Format No.: QA030-F02-00
 GENERAL TEST PROCEDURE (GTP) Page 4 of 8

Title Detection of Pathogens in Water Samples Effective Date 04/01/05

GTP Number GTP 114-02 Review Date 03/01/07

4.3.2 Soya bean casein digest medium, Selenite F broth and Tetrathionate Both and
Bismuth Sulphite Agar, Brilliant Green agar, and Xylose lysine deoxychollate
agar.
4.3.3 Boil the Selenite F broth and Tetrathionate broth and pour 10 ml of the broth in
presterilized test tubes.
4.3.4 Boil the Bismuth Sulphite agar and brilliant green agar till the agar totally melts at
100°C and pour approximately 20ml of the media into Petriplates at 45-50°C.
4.3.5 Allow the plates to solidify.

4.4 INOCULATION
4.4.1 Incubate one un-inoculated tube and plate as negative control from each
autoclaved lot of medium
4.4.2 Transfer 1.0 ml of the water sample into the tube containing 10 ml of Soyabean
casein digest medium and mix gently.
4.4.3 Incubate the tubes at 30 to 35°C for 24 hours.
4.4.4 After incubation observe the tubes for growth. If growth is not observed don’t go
for further testing.
4.4.5 If growth is observed inoculate 1.0 ml of the Fluid lactose medium into 10 ml of
Selenite and Tetrathionate broth tubes.
4.4.6 Incubate the tubes at 30 to 35°C for 24 hrs.
4.4.7 After incubation if there is any change in the color with respect of the negative
control in Selenite Broth and if there is any turbidity in the Tetrathionate broth
tube, streak a loopful of the sample from both the Selenite and Tetrathionate broth
tube into Bismuth sulphite agar plates, Brilliant green agar plate and Xylose lysine
deoxychollate agar plate.
4.4.8 Incubate the plates at 30 to 35°C for 48 hrs.
4.4.9 After 48 hrs of incubation observe the plates.

Prepared By Checked By Approved By

Name G.S.R.K.RAJU N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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 GENERAL TEST PROCEDURE (GTP) Page 5 of 8

Title Detection of Pathogens in Water Samples Effective Date 04/01/05

GTP Number GTP 114-02 Review Date 03/01/07

4.4.10 After incubation if there are colonies with morphological characteristics as given
in the Table No:01it means presence of Salmonella species.
4.4.11 Confirm the presence of Salmonella species by performing Gram Staining.

Table No:01
S.No Selective Medium Typical growth Gram Staining
Characteristics
Black or Green Color
01 Bismuth Sulphite Agar Gram-ve Rods
Colonies
Xylose Lysine Deoxychollate Red with or With out
02 Gram-ve Rods
Agar Black Centers
Small, Transparent,
03 Brilliant Green Agar colorless or pink to Gram-ve Rods
white opaque

4.5 TEST FOR Staphylococcus aureus

4.5.1 Prepare and sterilize the following media.
4.5.2 Soya bean casein digest medium, Vogel Johnson Agar or Mannitol Salt Agar.
4.5.3 After sterilization of media, pour approximately 20ml the Vogel Johnson Agar / Mannitol
Salt Agar / Baird Parker Agar in petri plates and allow it to solidify.

4.6 INOCULATION
4.6.1 Incubate one un-inoculated plate as negative control from each autoclaved lot of medium
Add 1ml of water sample to 10ml of Soyabean casein digest medium and incubate the
tubes at 30 to 35°C for 24 Hrs.
4.6.2 After incubation take out the tubes for growth. If growth is not present don’t go for
further testing.
4.6.3 If growth is present take loopful of sample from the soyabean casein digest medium and
streak it on any of these medium Vogel Johnson Agar / Mannitol Salt Agar / Baird Parker
Agar and Incubate the plates at 30 to 35°C for 48 hrs .
Prepared By Checked By Approved By
Name G.S.R.K.RAJU N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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 GENERAL TEST PROCEDURE (GTP) Page 6 of 8

Title Detection of Pathogens in Water Samples Effective Date 04/01/05

GTP Number GTP 114-02 Review Date 03/01/07

4.6.4 After 48 hrs of incubation observe the plates.

4.6.5 If the colonies are as per the characters mentioned in the given Table No:02 below
Staphylococcus aureus may be present.
4.6.6 Confirm the presence of Staphylococcus aureus by performing the gram staining
4.6.7 If gram positive Cocci in clusters are observed Staphylococcus aureus is confirmed.

Table No:02
Sr. No. Selective Medium Typical growth Characteristics
01 Vogel Johnson Agar Black surrounded with yellow colonies.
02 Mannitol Salt Agar Yellow Colonies with yellow zones
03 Baird Parker Agar Black shiny surrounded by clear zones of 2.0 mm to 5.0 mm

4.7 TEST FOR Pseudomonas aeruginosa:

4.7.1 Prepare and sterilize the Soyabean casein digest medium and Cetrimide agar.
4.7.2 After sterilization of the Cetrimide agar pour, approximately 20ml of the media into petri
plates and allow it to solidify.

4.8 INOCULATION
4.8.1 Incubate one un-inoculated plate as negative control from each autoclaved lot of medium.
4.8.2 Inoculate 1ml of water sample into 10ml of Soya bean casein digest medium and
incubate the tubes at 30 to 35°C for 24 hrs.
4.8.3 After incubation observe the tubes for growth. If there is no growth don’t go for further
testing.
4.8.4 If growth is present, take one loopful of culture from the Soya bean casein digest medium
and streak it on the Cetrimide agar plates.
4.8.5 Incubate the plates at 30 to 35°C for 48 hrs.
4.8.6 After 24 hrs of incubation observe the plates.

Prepared By Checked By Approved By

Name G.S.R.K.RAJU N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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 GENERAL TEST PROCEDURE (GTP) Page 7 of 8

Title Detection of Pathogens in Water Samples Effective Date 04/01/05

GTP Number GTP 114-02 Review Date 03/01/07

4.8.7 If green color colonies are observed Pseudomonas may be present.

4.8.8 Confirm the presence of Pseudomonas by performing the gram staining.
4.8.9 If gram-negative colonies in small rods are observed Pseudomonas is confirmed.

4.9 Positive Control: Keep positive control for each and every test with the known organism on
every plate used in this procedure.

FLOW CHART FOR PATHOGEN TESTING

Enrichment of media with 10ml water sample to be tested
SCDM (100ml)

Incubate for 30-35 0C for 24 hrs

Prepared By Checked By Approved By

Name G.S.R.K.RAJU N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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Format No.: QA030-F02-00
 GENERAL TEST PROCEDURE (GTP) Page 8 of 8

Title Detection of Pathogens in Water Samples Effective Date 04/01/05

GTP Number GTP 114-02 Review Date 03/01/07

Inoculate 1ml to each 10ml of Streak on Vogel John son agar (S.aureus)
 Mac Conkeys broth (for lactose fermentors.) CentrimideAgar( P.aeruginosa)
 Selenite F broth ( Salmonella spp)
 Tetrathionate broth (Salmonella spp)

Incubate for 30-35 0C for 24 hrs Incubate for 30-35 0C for 48 hrs

 Streak on MCA plate from Mac Conkeys broth Observe results(compare with
(for the detection of E.coli) conformation by EMB Agar) positive controls)
 Streak on XLDA& BSA plate from Selenite F broth
 Streak on XLDA& BSA plate from Tetrathionate broth
(for the detection of Salmonella spp)

If respective organism observed follow the confirmative test

Observe results(compare with positive controls )
USE API IDENTIFICATON SYSTEM FOR CONFORMATORY TEST
5.0 REFERENCE
5.1 United States Pharmacopoeia
5.2 European Pharmacopoeia
END OF DOCUMENT

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Name G.S.R.K.RAJU N. RAJENDRA KUMAR SANJAY SHAH K. R. PRAKASH

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Format No.: QA030-F02-00