IDENTIFICATION OF ESCHERICHIA COLI

OBJECTIVE
The test for Escherichia coli is provided to determine compliance with the requirements given in individual monograph / specifications.
PRINCIPLE
This includes identification of Escherichia coli, a bacteria pathogenic to human body and causes infection of stomach. It is detected by using specific, differential media which supports growth of Escherichia coli.
REAGENTS AND MEDIA
1. Kovac’s Reagent
Dissolve 5 g of 4-Dimethylaminobenzaldehyde in 75 mL Amyl alcohol by warming on a water bath at 50 - 55 OC , cool and add 25 ml of Conc. Hydrochloric acid. Store between 2 - 10 OC protected from light or use readymade reagent.
2. Nutrient Broth
Beef Extract 10.0 g
Peptone 10.0 g
Sodium chloride 5.0 g
Water 1000 mL
Dissolve with the aid of heat. Adjust to pH 8.0 - 8.4 with 5M Sodium Hydroxide and boil for 10 min. Filter, adjust to pH 7.2 - 7.4 and sterilize by maintaining at 115 OC for 30 min.
3. MacConkey’s Broth
Peptone 20.0 g
Sodium Chloride 5.0 g
Sodium Taurocholate 5.0 g
Bromocresol purple 10.0 mg
Lactose 10.0 g
Water 1000 mL
Dissolve the peptone, the sodium chloride and the sodium taurocholate in the water with the aid of heat. Adjust to pH 8.0 and boil for 20 min. Cool, filter and adjust to pH 7.4, Add the lactose and the indicator solution, mix and distribute in tubes containing inverted Durham’s tubes. Sterilize by maintaining at 115 OC for 30 min.
4. Peptone Water ( Buffered sodium chloride-peptone solution pH 7.0)
Potassium dihydrogen orthophosphate 3.56 g
Disodium hydrogen orthophosphate 7.23 g
Sodium chloride 4.30 g
Peptone (meat or casein) 1.0 g
Water 1000 mL
0.1 to 1.0% w/v of Polysorbate 20 or Polysorbate 80 may be added. Sterilize by heating in an autoclave at 121 OC for 15 min.
5. Lactose Broth
Beef Extract 3.0 g
Pancreatic digest of gelatin 5.0 g
Lactose 5.0 g
Water 1000 mL
Adjust the pH so that after sterilization it is 6.7 to 7.1. Sterilize by heating in an autoclave at 121 OC for 15 min. and cool immediately.
6. Levine Eosin-Methylene Blue Agar Medium
Pancreatic digest of Gelatin 10.0 g
Dibasic potassium phosphate 2.0 g
Agar 15.0 g
Lactose 10.0 g
Eosin Y 400 mg
Methylene blue 65 mg
Water 1000 mL
PH 7.1 ± 0.2
Dissolve the pancreatic digest of Gelatin, the Dibasic potassium thiosulphate and the agar in the water, with warming and allow to cool. Just prior to use, liquefy the gelled agar solution, add the remaining ingredients, as solutions, in the following amounts, and mix; for each 100 mL of the liquefied agar solution 5 mL of Lactose solution (1 in 5) , 2 mL of the Eosin Y solution (1 in 50) , and 2 mL of Methylene blue solution (1 in 30) . The finished medium may not be clear.
All the above media should be incubated for 24 h at 37 Oc before use. Any contaminated media should be discarded. Prepare media by using different ingredients as above or use readymade dehydrated media of Hi media / Difco make.and use required quantity as per instructions on the bottle label,. dispense in required quantities and sterilize at 15 lbs and 121 OC for 15 min.
PRE-TREATMENT OF THE PREPARATION BEING EXAMINED
Water - Soluble Products :
1. 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
2. If necessary, adjust the pH to about 7.
Non - Fatty Products Insoluble in Water :
1. Suspend 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 dilute to 100 mL with the same medium.
2. If necessary divide the preparation being examined and homogenize the suspension mechanically.
3. A suitable surface-active agent such as 0.1 % w/v Polysorbate 80 may be added to assist the suspension of poorly wettable substances. If necessary, adjust the pH of the suspension to about 7.
Fatty Products :
1. Homogenize 10 g or 10 mL of the preparation being examined, unless otherwise prescribed with 5 g of Polysorbate 20 or Polysorbate 80. If necessary, heat to not more than 40 OC.
2. Mix carefully while maintaining the temperature in a water bath or in an oven.
3. Add 85 mL of Lactose broth or another suitable medium shown not to have anti-microbial activity in the conditions of the test, heated to not ore than 40 OC if necessary.
4. Maintain this temperature for the shortest time necessary for formation of an emulsion and in any case for not more than 30 min.
5. If necessary, adjust the pH of emulsion to about 7.
For a Fluid Specimen in Aerosol Form :
1. Chill the container in an Alcohol-dry ice mixture for approx. 1 h., cut open the container, allow it to reach room temperature, permit the propellant to escape, or warm to drive off the propellant if feasible and transfer the quantity of test material required for the procedures as appropriate.
2. Where 10 g or 10 mL of the specimen, whichever is applicable, cannot be obtained from 10 containers in aerosol form, transfer the entire contents from 10 chilled containers to the culture medium, permit the propellant to escape, and proceed with the test on the residue.
PROCEDURE
As per IP
A. Enrichment
1. Transfer aseptically 10 g or 10 mL of sample to 100 Nutrient broth and incubate at 37 OC for 24 h.
2. After incubation, examine the tube for growth, and if present, mix by gentle shaking.
B. Primary Test
1. Pipette 1 mL of the enrichment culture into tubes containing 5 mL MacConkey’s broth and incubate at 37 OC for 48 h.
2. If the contents show acid and gas carry out the secondary test.
C. Secondary Test
1. After incubation, if the tube shows presence of acid and gas transfer 0.1 mL from the tube to each of two tubes containing
i. 5 mL MacConkey’ s broth
ii. 5 mL Peptone water.
2. Incubate both tubes in a water bath at 43.5 - 44.5 OC for 24 h. After incubation examine tube (a) for acid and gas and (b) for Indole.
3. To test for Indole production add 0.5 mL of Kovac’s reagent, shake well and allow to stand for one min. , if a red color is observed in the reagent layer, indole is present.
4. The presence of acid and gas and of Indole indicates presence of Escherichia coli.
As per BP
A. Enrichment
Transfer a quantity of the homogenate in Lactose broth containing 1 g or 1 mL of the preparation being examined, prepared and incubated as of the test for Enterobacteriaceae and certain other Gram-negative bacteria, to 10 mL of MacConkey’s broth and incubate at 43 - 45OC for 18 - 24 h.
B. Primary Test
1. Sub-culture on a plate of MacConkey agar and incubate at 43 - 45 Oc for 18 - 24 h.
2. Growth of red, generally non-mucoid colonies of Gram negative rods, sometimes surrounded by a reddish precipitation zone, indicates the possible presence of Escherichia coli.
C. Secondary Test
1. This may be confirmed by the formation of Indole at 43.5 - 44.5 OC and by other biochemical reactions.
2. The preparation being examined passes the test if such colonies are not seen or if the confirmatory biochemical reactions are negative.
As per USP
A. Enrichment
1. to the specimen contained in a suitable vessel, add a volume of Fluid Lactose medium to make 100 mL and incubate at 37 OC for 48 h.
2. Examine the medium for growth, if growth is present mix by gentle shaking.
B. Primary Test
1. by means of an inoculating loop, streak a portion from the remaining Fluid Lactose Medium on the surface o f MacConkey agar medium.
2. Cover and invert the plate and incubate at 37 OC for 48 h.
3. 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.
If colonies matching the description in Table given below are found, proceed with further identification.
Morphological Characteristics of Escherichia coli
On MacConkey Agar Medium
Characteristic Colonial Morphology-Brick-red; may have surrounding zone of precipitated bile
Brick-red; may have surrounding zone of precipitated bile-Negative rods
(Cocco-bacilli)
C. Secondary Test
1. Transfer the suspect colonies individually, by means of an inoculating loop, to the surface 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 37 OC for 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 thet est for the absence of Escherichia coli.
5. The presence of Escherichia coli may be confirmed by further suitable cultural and biochemical tests.
NOTE:
Carry out a control test by repeating the primary and secondary tests adding 1 mL of the enrichment and culture a volume of broth containing 10 to 50 Escherichia coli (NCTC 9002), prepared from a 24 h, culture in Nutrient broth, to 5 mL of MacConkey’s broth. The test is invalid if the results do not indicate that the control contains Escherichia coli.
NOTE: Use the method as per the pharmacopoeial status (grade) of the material. In case of In-house specificaiton, follow the method as per IP or as specified.