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Contents
Escherichia Coli Coli forms Sulphur Reducing Anaerobes Pseudomonas Aeroginasa Aerobic Microbial Count Aerobic Microbial Count Yeast & Mould

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ESCHERICHIA COLI PRINCIPLE: It shall be absent in any 250ml sample when tested in accordance with the method. APPARATUS: Laminar Airflow Petri dish Conical flask Single Pan Balance Auto clove Bottom Flask Vacuum Pump Incubator Forceps Filter Paper 0.45m Bunsen Burner Water bath

Chemicals: Ethanol Distilled water TTC Solution

Hi Media: Tergitol-7-Agar Tryptophan broth Tryptone Soy Agar

Ethanol: It shall be mainly used for the Disinfection. Media Preparation: 3.313gms of Tergital-7-Agar is mixed in 100 ml of Distilled water. Sterilized at 121 Degree for 15 Minutes.

Plate Preparation: After sterilization the media. is allowed to cool (50 degree). Then add 0.3 Ml of TTC solution, Mixed gently without air bubbles. Immediately Poured into the Sterilized Petri plates. (20 ml). After solidification (45 minutes) the plate is stored in Refrigerator.

PROCEDURE: 250ml of Product Water Sample Collected to Sterile Screw Cap Bottle

Filter through in 0.45m membrane Paper

Filter paper placed in Petriplate with Tergitol-7-Agar

Incubation at 37 degree for 48 hrs (or) 2 days Confirmation Test for E.coli: Yellow Colour with Pink Spot

Sub Culture into Tryptone Soy Agar

Oxidase test

Not appeared Dark Purple Blue Colour

Confirmation Test for E.coli: Yellow Colour with Pink Spot

Sub Culture into Tryptophane Broth

Indole test

Cherry Red Colour

Oxides Negative Indole Positive

E.Coli

Oxides Negative Iodole Negative

Coli forms

Tryptophan broth Preparation:

Tryptic digest of Casein L-Tryptophan Sodium Chloride Distilled water

- 10g - 1g - 5g - 1litre

Dissolve the ingredients in water by heating. Dispense 3 ml per test tube. Close the tubes with cotton plugs, plastic or metal caps. Autoclave for 15mins at (121+3)0C. The pH of the ready to use medium should be 7, 250C. Kovacs Reagent for Indole Test, Standard test:
P

-Dimethylamino benzaldehyde

5g 75ml 25ml

Amyl or butyl alcohol Hydrochloric acid

Dissolve the aldehyde in the alcohol. Add the concentrated acid with care. Product from light and store at (5+3)0C. Indole reagent Test:
P

-Dimethylamino benzaldehyde

0.5g 100ml

Hydrochloric acid Oxidase Test:

Tetramethyl-p-phenylenediamine hydrochloride Distilled water

0.1g 10ml

This reagent is not stable and shall be freshly prepared each time it is needed. Result: Not Observed Yellow colour colonies = E.coli & coli forms Absent in 250 ml Observed Yellow colour Colonies, Oxidase +ive, Indole test not necessary = E. coli & Coli forms Absent in 250 ml.

COLIFORM PRINCIPLE: It shall be absent in any 250ml sample when tested in accordance with the method. APPARATUS: Laminar Airflow Petri dish Conical flask Single Pan Balance Auto clove Bottom Flask Vacuum Pump Incubator Forceps Filter Paper 0.45m Bunsen Burner

Chemicals: Ethanol Distilled water

Hi Media:

Violet red bile Agar

Ethanol: It shall be mainly used for the Disinfection Process. Media Preparation: 100ml of Distilled water + 2.0765gms of Violet Red Bile Agar mixed it. Sterilization at 121 C for 15 minutes. Avoid Storing & reusing .

Plate Preparation:

After sterilization the media. is allowed to cool (50 degree). Mixed gently without air bubbles. Immediately Poured into the Sterilized Petri plates. (20 ml). After solidification (45 minutes) the plate is stored in Refrigerator.

PROCEDURE: 250ml of Product Water Sample Collected to Sterile Screw Cap Bottle

Filter through in 0.45m membrane Paper

Filter paper placed in Petriplate with Violet Red Bile Agar Plate

Incubation at 37 for 48hrs (or) 2 days

Result: Not observed Purple Red Colonies.

SULPHITE REDUCING ANAEROBES PRINCIPLE: It shall be absent in any 250ml sample when tested in accordance with the method. APPARATUS: Laminar Airflow Screw Cap Bottle Conical flask Single Pan Balance Auto clove Bottom Flask Vacuum Pump Incubator Forceps Filter Paper 0.22 Iron Wire Bunsen Burner

Chemicals:

Ethanol Distilled water Sodium Sulphite Ferric Citrate

Hi Media: Differential Reinforced Clostridial Broth

Ethanol: It shall be mainly used for the Disinfection Process.

Broth Preparation: Single Strength Broth Preparation(SS Broth): 100ml of Distilled water + 4.35gms of Differential Reinforced Clostridial Broth are mixed it. Double Strength Broth Preparation (DS Broth): 100ml of Distilled water + 8.7gms of Differential Reinforced Clostridial Broth are mixed it. Chemical Preparation: Sodium Sulphite Solution Preparation: 100ml of Distilled water + 4gms of Sodium Sulphite thoroughly mixed it. Sterilization Through 0.22m filter. (Freshly Prepared for every 14 days). Ferric Citrate Solution Preparation: 100ml of Distilled water add in the 7gms of Ferric Citrate thoroughly mixed it. Sterilization. Sterilization Through 0.22m filter. (Freshly Prepared for every 14 days). Mixer Solution Preparation:

Equal volume of Sodium Sulphite Solution & Equal volume of Ferric Citrate solution is mixed thoroughly.( Freshly prepared before the test being carried ) Take 2ml of this Mixer into Every 50 Ml of Single Strength DRCM broth.

PROCEDURE: 50ml of Product Water Sample Collected to Sterile Screw Cap Bottle

Heated in the water bath @ 72+/-5degree C

50ml of Double Strength Broth Pour in the Screw Cap Bottle

Then Added in the 2ml of Mixer Solution

Heat the Iron Wire by the Fire till it gets the redess in Color

Dip the Iron wire with broth and then pour 50 ml Heated sample

Fill the Gap in Screw Cap Bottle With SS Broth Neck full

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Then Cover With Sterile Paraffin Wax Screw Cap Bottle are fully in closed

Incubation at 37 Deg. C for 48 hours (or) 2 days Result: Black Colours can not be formed in the bottom of Screw Cap Bottle.

PSEUDOMONOS AERUGINOSA

PRINCIPLE: It shall be absent in any 250ml sample when tested in accordance with the method. APPARATUS: Laminar Airflow Screw Cap Bottle Conical flask Single Pan Balance Auto clove Bottom Flask Vacuum Pump Incubator Forceps Filter Paper 0.22m Bunsen Burner

Chemicals: Ethanol Distilled water

Hi Media: Asparagine Proline Broth 11

Ethanol: It shall be mainly used for the Disinfection Process.

Broth Preparation: Double Strength Broth Preparation: 100ml of Distilled water add in the 2.9gms of Double Strength Asparagine Proline Broth is mixed. Sterilization at 121 C for 15 minutes. PROCEDURE: 250ml of Water Sample Collected

Filter through in 0.22m membrane Paper

50ml of Double Strength Asparagine Proline Broth Pour in the Screw Cap Bottle

Then Added in the 4ml Sterile Ethanol

Filter paper are Dipped in the Asparagine Proline Broth

Screw Cap Bottle are fully in closed

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Incubation at 37 deg. for 48hour (or) 2 days Result: Colours can not be formed on the Screw Cap Bottle.

AEROBIC MICROBIAL COUNT PRINCIPLE: The total viable colony count shall not exceed 100 per ml at 200C to 220C in 72hrs and 20 per ml at 370C in 24hrs. When tested in accordance with the method. APPARATUS: Laminar Airflow Petri dish Conical flask Single Pan Balance Auto clove Bottom Flask Vacuum Pump Incubator Pipette Bunsen Burner

Chemicals: Ethanol Distilled water

Hi Media: Plate Count Agar

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Ethanol: It shall be mainly used for the Disinfection Process. Media Preparation: 100ml of Distilled water add in the 2.35gms of Plate Count Agar mixed it. Sterilization at 121 C for 15 minutes. After 1 hour cool the media.

PROCEDURE: 1ml of Product Water Sample Collected to Sterile Screw Cap Bottle

Pour 1 ml of Sample in to the Petri dish

Pour 20ml of Plate Count Agar in to the Petri dish

Shake well to the Petri dish

Incubation at 220C for 72 hrs (or) 3 day

Result:

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Colonies can not formed on the Petri Plate.

AEROBIC MICROBIAL COUNT PRINCIPLE: The total viable colony count shall not exceed 20per ml at 370C in 24hrs. When tested in accordance with the method. APPARATUS: Laminar Airflow Petri dish Conical flask Single Pan Balance Auto clove Bottom Flask Vacuum Pump Incubator Pipette Bunsen Burner

Chemicals: Ethanol Distilled water

Hi Media: Plate Count Agar 15

Ethanol: It shall be mainly used in the Disinfection Process. Media Preparation: 100ml of Distilled water add in the 2.35gms of Plate Count Agar mixed it. Sterilization at 121 C for 15 minutes. After 1 hour cool the media.

PROCEDURE: 1 ml of Water Sample Collected

Pour 1 ml of Sample in to the Petri dish

Pour 20ml of Plate Count Agar in to the Petri dish

Shake well to the Petri dish

Incubation at 370C for 24 hrs (or) 1 day

Result: Colonies can not formed on the Petri Plate.

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YEAST & MOULD PRINCIPLE: It shall be absent in any 250ml of water sample when tested in accordance with the method. APPARATUS: Laminar Airflow Petri dish Conical flask Single Pan Balance Auto clove Bottom Flask Vacuum Pump Incubator Forceps Filter Paper 0.45m

Chemicals: Ethanol Distilled water

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Hi Media: Chloramphenicol Yeast Glucose Agar

Ethanol: It shall be mainly used in the Disinfection. Media Preparation: 100ml of Distilled water add in the 2.45gms of Chloramphenicol Yeast Glucose Agar mixed it. . Sterilization at 121 C for 15 minutes. After 1 hour cool the media.

Plate Preparation: After sterilization the media is allowed to cool (50 degree). Mixed gently without air bubbles. Immediately Poured into the Sterilized Petri plates. (20 ml). After solidification (45 minutes) the plate is stored in Refrigerator.

PROCEDURE: 250ml of Product Water Sample Collected to Sterile Screw Cap Bottle

Filter through in 0.45m membrane Paper

Filter paper placed in Petriplate with Chloramphenicol Yeast Glucose Agar Plate

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Incubation at 120 hrs (or) 5 days

Result: Colonies can not formed on the Petri Plate.

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