INTRODUCTION:
Escherichia coli are one of many species of bacteria living in the lower intestines of mammals, known as gut flora. When located in the large intestine, it assists with waste processing, vitamin K production, and food absorption. It was discovered in 1885 by Theodor Escherich, a German pediatrician and bacteriologist. However, the bacteria are not confined to this environment, and specimens have also been located, for example, on the edge of hot springs. The E. coli strain O157:H7 is one of hundreds of strains of the bacterium that causes illness in humans.
Escherichia coli is Gram-negative organisms, E. coli are unable to sporulate. Thus, treatments which kill all active bacteria, such as pasteurization or simple boiling, are effective for their eradication, without requiring the more rigorous sterilization which also deactivates spores. As a result of their adaptation to mammalian intestines, E. coli grows best in vivo or at the higher temperatures characteristic of such an environment, rather than the cooler temperatures found in soil and other environments.
PREPARATION OF CULTURE MEDIA:
Sodium hydroxide 1N: Dissolve 4.0 g of sodium hydroxide in water to make 100 mL.
Hydrochloric Acid 1N: Dissolve 8.5 ml of Hydrochloric Acid in water to make 100 mL.
Soyabean-Casein Digest broth Medium (SCDM)/ MacConkey Broth (MCB)/ MacConkey Agar (MCA): Reconstitute dehydrated media as directed by the manufacturer and sterilize in an autoclave at 121°C for 20 minutes.
PROCEDURE:
Sample Preparation and Pre enrichment:
Aseptically add 1g of specimen if it’s a solid or 1ml accurately measured, if the specimen is a liquid to make 100ml of Soyabean-Casein Digest Medium, or transfer 10 ml (Quantity corresponding to not less than 1 g or 1 mL specimen) of sample from the Total aerobic microbial count sample preparation to make 100ml of Soyabean-Casein Digest broth Medium unless otherwise described in the specific STP.
Negative Control: Take 100 mL of sterile Soyabean-Casein Digest broth Medium without any inoculation.
Positive Control: Add 1mL of <100 cfu/ ml of Escherichia coli culture suspension in to 100ml Soyabean-Casein Digest broth Medium.
Incubate the sample, negative control and positive control 30-35°C for 18 - 24hours
Examine the medium for growth.
Interpretation:
Negative control flasks should not show any growth.
Growth should be present in Positive control.
If growth is present (turbidity observed) in the sample flask, or the media difficult to observe the growth with sample (Likely some powders and capsules can alter the color and the clearness of the media) mix by gentle shaking and proceed to test for Escherichia coli. If there is no growth in the sample flask, that indicates, sample meets the requirement for absence of Escherichia coli.
Note: Incase of water sample add 1ml sample in 9 ml Soyabean-Casein Digest broth Medium for test and negative control with out adding anything and positive control by adding Escherichia coli culture in 10 ml media.
Selective Enrichment:
After shaking the container transfers 1 ml of Soyabean-Casein Digest broth Medium to 100 ml of MacConkey Broth.
Negative Control: Take 100 mL of sterile MacConkey Broth without any inoculation.
Positive Control: Add 1mL of Escherichia coli culture in to 100ml MacConkey Broth.
Incubate the sample, negative control and positive control at 42 - 44°C for 24- 48hours.
Interpretation:
Negative control flasks should not show any growth.
Growth should be present in Positive control.
If growth is present (turbidity observed) in the sample flask, or the media difficult to observe the growth with sample (Likely some powders and capsules can alter the color and the clearness of the media) mix by gentle shaking and proceed to test for Escherichia coli. If there is no growth in the sample flask, that indicates, sample meets the requirement for absence of Escherichia coli.
Note: Incase of water sample add 1 ml sample in 100 ml MacConkey Broth for test and Maintain negative control with out adding anything and positive control by adding Escherichia coli culture in 100 ml media.
Confirmation test:
Steak a loop full of culture from MacConkey Broth to MacConkey Agar plate. Maintain negative control without any inoculum. Maintain positive control by streaking with Escherichia coli
Cover and invert the plates and incubate at 30-35°C for 18 - 72 hours.
After incubating, examine the plates.
Interpretation:
Negative control plate should not show any growth.
If the colonies on the sample plate does not match with the colony characteristic given table-1, that indicates the sample does not meets the requirements for the absence of E.coli. If the colonies on sample plate do not matches to above said characteristics that indicates sample meets the requirements for the absence of E.coli. If require perform identification test.
General testing procedure,
