1. APPARATUS
1.1. Autoclave
1.2. BOD - Incubator
1.3. Bacteriological Incubator
1.4. pH – meter
1.5. Hot air Oven
1.6. Laminar Air flow
1.7. Analytical Balance
1.8. Spirit Lamp / Bunsen Burner
1.9. Micropipette
1.10. Petri dishes
1.11. Test tubes
1.12. Media bottles
2. REAGENTS AND MEDIAS
2.1. Peptone Water
(Buffered Sodium Chloride - Peptone Solution pH 7.0)
Potassium Dihydrogen Orthophosphate 3.60 g
Disodium Hydrogen Orthophosphate 7.20 g
Sodium Chloride 4.30 g
Peptone(meat and Casein) 1.0 g
Water 1000 ml
0.1 to 1.0 % w/v Polysorbate 20 or Polysorbate 80 may be added. Sterilize by heating in an autoclave at 121–124OC for about 30 min.
2.2. Fluid Lactose Medium
Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
Beef Extract 3.0 g
Pancreatic Digest of Gelatin 5.0 g
Lactose 5.0 g
Water 1000 ml
Final pH after sterilization 6.9 ± 0.2
Mix, and dispense as desired and sterilize in an autoclave at 121–1240C for about 30 min.
Cool as quickly as possible after sterilization.
2.3. MacConkey’s Agar
Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
Pancreatic digest of gelatin 17.0 g
Pancreatic digest of casein 1.5 g
Peptic digest of animal tissue 1.5 g
Lactose 10.0 g
Bile salts mixture 1.5 g
Sodium Chloride 5.0 g
Agar 13.5 g
Neutral red 30.0 mg
Crystal red 1.0 mg
Water 1000 ml
Final pH after sterilization 7.1 ± 0.2
Boil the mixture of solids and water for 1 minute to effect solution. Mix, and dispense as desired and sterilize in an autoclave at 121–1240C for about 30 min.
2.4. MacConkey Broth
Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
Pancreatic digest of gelatin 17.0 g
Pancreatic digest of casein 1.5 g
Peptic digest of animal tissue 1.5 g
Lactose 10.0 g
Bile salts mixture 1.5 g
Sodium Chloride 5.0 g
Neutral red 30.0 mg
Crystal red 1.0 mg
Water 1000 ml
Final pH after sterilization 7.1 ± 0.2
Boil the mixture of solids and water for 1 minute to effect solution. Mix, and dispense as desired and sterilize in an autoclave at 121–1240C for about 30 min.
2.5. Soyabean Casein Digest Medium (SCM)
Prepare media as per the current version of Respective GTP.
If the dehydrated medium is not available then prepare the medium as follows.
Pancreatic Digest of Casein 17.0 g
Papacy Digest of Soybean Meal 3.0 g
Sodium Chloride 5.0 g
Dibasic Potassium Phosphate 2.5 g
Dextrose (C6H12O6. H2O) 2.5 g
Distilled Water 1000 ml
Final pH after Sterilization 7.3 ± 0.2
Dissolve the solids in the water, warming slightly to effect solution. Cool to room temperature and add, if necessary, sufficient 0.1 N Sodium Hydroxide to give a final pH after Sterilization between 7.1 and 7.5. Filter, if necessary, to clarify, distribute into suitable containers and sterilize in an autoclave at 121–124OC for about 30 min.
All the above media should be incubated for 48 hours at 32.50C ? 2.50C before use. Any contaminated media should be discarded.
3. PROCEDURE
3.1. PRE-TREATMENT OF THE PREPARATION BEING EXAMINED
3.1.1. Water - Soluble Products :
3.1.1.1. Dissolve or dilute 10 g or 10 ml of the preparation being examined, unless otherwise prescribed, in SCM solution or Peptone water or another suitable medium shown not to have anti-microbial activity under the conditions of the test and adjust the volume to 100 ml with the same medium
3.1.1.2. If necessary, adjust the pH to about 7.0 ± 0.2.
3.2. Enrichment
3.2.1. Take 10 ml or 10 g, the quantity corresponding to 1.0 ml or 1.0 g ( or as per the specification) of the pretreated sample in a suitable vessel, add to a volume of Fluid Soyabean casein digest medium to make 100 ml and incubate at 30.00C to 35.00C for 18 - 48 hours.
3.3. Primary Test
3.3.1. Transfer 1 ml of enriched medium into 100 ml of Macconkey broth and incubated at 43.00 to 45.00C for 18 to 24 hours.
3.3.2. By means of an inoculating loop, streak a portion from Fluid Soyabean casein digest Medium on the surface of MacConkey agar medium.
3.3.3. Cover and invert the plate and incubate at 30.0C to 35.0C for 18 to 72 hours.Upon examination, if none of the colonies conforms to the description given in the Table below for this medium, specimen meets the requirements of the test for absence of Escherichia coli.
3.3.4. If colonies matching the description in Table given below are found, proceed with further identification i.e. Biochemical tests ( Indole production test )
Morphological Characteristics of Escherichia coli
On MacConkey Agar Medium
Characteristic Colonial Morphology Gram stain
Growth of Brick red, non mucoid colonies Negative rods (Cocco-bacilli)
3.4. Secondary Test ( Indole production Test )
3.4.1. Inoculate the Suspected sample into 5 ml of 1% tryptone broth and incubated 32.50 ? 2.50C for 48 hours.
3.4.2. Add 1 ml of Kovac’s reagent and shake the tube gently with regular intervals for 10 to 15 minutes.
3.4.3. Allow the tube to stand to permit the regent to come to the top.
3.4.4. Development of deep red colour in the top of the layer is a positive test for indole production i.e. confirm as presence of E. coli
3.4.5. Absence of red colour indicates absence of E. coli.
3.5. Note :
3.5.1. Carry out control test by repeating the primary and secondary tests using 1 mL of the enrichment culture and a volume of broth containing 10 to 50 Escherichia coli prepared from a 24 h culture in Nutrient broth, for the inoculation of tubes.
3.5.2. The test is invalid if the results do not indicate that the control contains Escherichia coli.
autoclave, bod - incubator, bacteriological incubator, general testing procedure
