General Test Procedure for Detection of Pathogens in Water Samples

PURPOSE

To lay down the procedure for Detecting of pathogens in water samples.

This test is applicable to all the samples for detection of the following pathogen.

1. Salmonella species
2. Pseudomonas aeruginosa
3. Staphylococcus aureus
4. Escherichia coli

APPARATUS

Petri Plates (90mm diameter)

Bottles

Test tubes

Inoculating loop

REAGENTS

Soya bean casein digest medium,

Mac Conkey broth purple,

Mac Conkey agar,

EMB agar,

Vogel Johnson agar,

Mannitol salt agar,

Cetrimide agar,

Selenite F broth,

Tetrathionate broth,

Brilliant green agar,

Bismuth sulfite agar,

Xylose lysine deoxycholate agar.

Gram staining Kit.

PROCEDURE

Test for Escherichia coli

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

Inoculation

• Add 10ml of water sample into 100ml of SCD medium tube for enrichment. Incubate the tubes at 30-35°C for 24 Hrs.
• 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.
• Incubate one un-inoculated tube as negative control from each autoclaved lot of medium.
• Incubate the tubes at 30 to 35°C for 24 hours.
• Observe the sample tubes after 24 hours for growth.
• If growth is present transfer one loopful from the Mac Conkey broth medium and streak onto the Mac Conkey agar plate.
• Keep the Mac Conkey agar plate for incubation at 30 to 35°C for 48 hrs.
• Further, if growth is present in the plate go for a confirmatory test by streaking on the Eosine Methylene blue agar plate.

Test for Salmonella sps

• Prepare and sterilize the following media.
• Soya bean casein digest medium, Selenite F broth and Tetrathionate Both and Bismuth Sulphite Agar, Brilliant Green agar, and Xylose lysine deoxycholate agar.
• Boil the Selenite F broth and Tetrathionate broth and pour 10 ml of the broth into pre-sterilized test tubes.
• 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 Petri plates at 45-50°C.
• Allow the plates to solidify.

Inoculation

• Incubate one un-inoculated tube and plate as negative control from each autoclaved lot of medium.
• Transfer 1.0 ml of the water sample into the tube containing 10 ml of Soyabean casein digest medium and mix gently.
• Incubate the tubes at 30 to 35°C for 24 hours.
• After incubation observes the tubes for growth. If growth is not observed don’t go for further testing.
• If growth is observed inoculate 1.0 ml of the Fluid lactose medium into 10 ml of Selenite and Tetrathionate broth tubes.
• Incubate the tubes at 30 to 35°C for 24 hrs.
• 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 sulfite agar plates, Brilliant green agar plate, and Xylose lysine deoxycholate agar plate.
• Incubate the plates at 30 to 35°C for 48 hrs.
• After 48 hrs of incubation observe the plates.
• After incubation, if there are colonies with morphological characteristics as given in Table No:01, it means the presence of Salmonella species.
• Confirm the presence of Salmonella species by performing Gram Staining.

Table No: 01

Sr. No	Selective Medium	Typical growth Characteristics	Gram Staining
01	Bismuth Sulfite Agar	Black or Green Color Colonies	Gram-ve Rods
02	Xylose Lysine Deoxycholate Agar	Red with or Without Black Centers	Gram-ve Rods
03	Brilliant Green Agar	Small, Transparent, colorless or pink to white opaque	Gram-ve Rods

Test for Staphylococcus Aureus

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

Inoculation

• 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.
• After incubation take out the tubes for growth. If growth is not present don’t go for further testing.
• If growth is present take a 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.
• After 48 hrs of incubation observe the plates.
• If the colonies are as per the characters mentioned in the given Table No: 02 below Staphylococcus aureus may be present.
• Confirm the presence of Staphylococcus aureus by performing the gram staining
• 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

Test for Pseudomonas Aeruginosa

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

Inoculation

• Incubate one un-inoculated plate as negative control from each autoclaved lot of medium.
• 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.
• After incubation observes the tubes for growth. If there is no growth don’t go for further testing.
• If growth is present, take one loopful of culture from the Soya bean casein digest medium and streak it on the Cetrimide agar plates.
• Incubate the plates at 30 to 35°C for 48 hrs.
• After 24 hrs of incubation observe the plates.
• If green color colonies are observed Pseudomonas may be present.
• Confirm the presence of Pseudomonas by performing the gram staining.
• If gram-negative colonies in small rods are observed Pseudomonas is confirmed.

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

REFERENCE

United States Pharmacopoeia

European Pharmacopoeia

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