Detection of Escherichia coli

1.  APPARATUS

1. Autoclave
2. BOD - Incubator
3. Bacteriological Incubator
4. pH – meter
5. Hot air Oven
6. Laminar Air flow
7. Analytical Balance
8. Spirit Lamp / Bunsen Burner
9. Micropipette
10. Petri dishes
11. Test tubes
12. Media bottles   

2.   REAGENTS   AND  MEDIAS

2.1. Fluid Lactose Medium

Prepare media as per the current version of Respective GTP.

2.2. MacConkey’s Agar

Prepare media as per the current version of Respective GTP

2.3.Levine Eosin-Methylene Blue Agar Medium

2.4.Prepare media as per the current version of Respective GTP

All the above media should be incubated for 48 hours at 32.5⁰C ± 2.5⁰C before use. Any contaminated media should be discarded.         

1. PROCEDURE    
   1. Pre-Treatment Of The Preparation Being Examined
   2. Water - Soluble Products :
   3. Dissolve or dilute 10 g or 10 ml of the preparation being examined, unless otherwise prescribed, in Lactose broth 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
   4. If necessary, adjust the pH to about 7.0 ± 0.2.
2. Enrichment
3. Take 10 ml or 10 g, the quantity corresponding to 1.0g or 1.0ml (as per the specification)  of the pretreated sample in a suitable vessel, add a volume of Fluid Lactose medium to make 100 ml and incubate at 32.5.0⁰C ± 2.5⁰C for 24 to 48 hours.
4. Examine the medium for growth, if growth is present mix by gentle shaking.
5. By means of an inoculating loop, streak a portion from the remaining Fluid Lactose Medium on the surface of MacConkey agar medium.
6. Cover and invert the plate and incubate at 32.5⁰C ± 2.5⁰C for 24 to 48 hours.
7. 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.
8. If colonies matching the description in Table given below are found, proceed with further identification.

Morphological Characteristics of Escherichia  coli

On MacConkey Agar Medium

1. Transfer the suspect colonies individually, by means of an inoculating loop, to the surface of Sterile Levine Eosin-Methylene blue agar medium, plated on petridishes.
2. If numerous colonies are to be transferred, divide the surface of each plate into quadrants, each of which may be seeded from a separate colony.
3. Cover and invert the plates and incubate at 32.5⁰C ± 2.5⁰C for 24 to 48 h.
4. Upon examination, if none of the colonies exhibits both a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light, the specimen meets the requirements of the test for the absence of Escherichia coli.
5. The presences of Escherichia coli may be confirmed by further suitable staining and biochemical tests .
6. Note :
   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.
   2. The test is invalid if the results do not indicate that the control contains 

Blue agar , eosin-methylene , general testing procedure,