Agar

EVALUATION OF HICROME COLIFORM AGAR FOR THE DETECTION AND ENUMERATION TOTAL COLIFORM AND E.coli FROM POULTRY FEED AND WATER SAMPLES

Thulasi Rajkumar, Sharon MacDonald, R. Kannan, R. Suresh Kumar and CN.Ramchand

Customer Laboratory Services

Kemin Nutritional Technologies (I) Pvt. Ltd.,

No: 39, The Trapezium, Second Floor, Nelson Manickam Road,

Chennai-600 029 Tamil Nadu, INDIA

ABSTRACT

The purpose of this study was to examine the use of Hicrome Coliform Agar medium for isolation and enumeration of Enterobactericae from poultry samples, to compare it to EMB agar and to evaluate its usefulness as a possible alternative selective medium in poultry studies. The medium was shown to be effective in identifying E.coli and coliforms in feed without the need for extensive accompanying biochemical tests for confirmation of identity. This study shows that Hicrome Coliform Agar is an effective replacement for EMB agar in poultry feed and water studies and has the advantage of differentiating E. coli from other coliforms.

Key words: Hicrome Coliform Agar, Coliforms, E.coli, Enumeration, Enterobactericae, Poultry feed and water.

INTRODUCTION

Coliforms are historically used as indicator microorganisms to serve as a measure of fecal contamination Although coliform bacteria themselves are not pathogenic, their presence indicates possible fecal contamination. Within the coliforms Escherichia coli is of particular interest since, its presence in the feed and water indicates that recent fecal contamination has occurred with the possibility of accompanying enteric pathogens. Conventional methods for detecting E.coli and coliforms by MacConkey and EMB agar are still practiced in many laboratories. These techniques will take 24 to 48 hours to enumerate and need further confirmatory biochemical tests for identification that will cost time and money. The major disadvantage of using traditional selective/ differential agars such as EMB Agar is that often these media become overgrown with non-E.coli, making it virtually impossible to isolate the E.coli colonies and also giving false negative results. On EMB agar, E.coli is generally observed as green metallic sheen, which may disappear after 48 hours giving false negative results. The development of chromogenic culture media, although initially used for isolating pathogens from clinical specimens, is revolutionizing pathogen detection in food, feed, water and other environmental samples. Chromogenic substrates, when added to primary selective and nonselective media, permit enumeration, detection, presumptive identification and, in some media, identification directly from the isolation plate. Chromogenic substrates are colorless and, when cleaved by specific enzymes, are oxidized, producing a characteristic color. The detection of a specific enzyme or enzymes can be used to identify genus, species, or groups of organisms. Colonies of organisms with the enzyme produce a distinct color, providing easy recognition. This novel technology has been shown to provide improved accuracy and faster detection than other traditional primary culture media.

HiCrome Coliform Agar is a selective chromogenic medium recommended for simultaneous detection of Escherichia coli and total coliforms in water and food samples. Sodium lauryl sulfate inhibits Gram-positive organisms. The chromogenic mixture contains two chromogenic substrates, Salmon-GAL and X-glucuronide. The enzyme Ð"Ñž-D-galactosidase produced by coliforms cleaves Salmon-GAL, resulting in the salmon-to-red coloration of coliform colonies. The enzyme a-D-glucuronidase produced by E. coli cleaves X-glucuronide. E. coli forms dark blue-to-violet colored colonies due to cleavage of both Salmon-GAL and X-glucuronide. The addition of tryptophan improves the indole reaction, thereby increasing detection reliability in combination with the two chromogens. To confirm E. coli, add a drop of Kovac's reagent on the dark blue-to-violet colony. Formation of cherry-red color indicates the positive reaction thereby increasing the reliability of identification of E. coli.

The purpose of this study was to examine the use of Hicrome Coliform Agar medium for isolation and enumeration of Total coliform and E.coli from poultry Feed and Water samples to increase the reliability of results and to evaluate its use as a possible alternative selective medium to Eosin methelene blue (EMB) agar and MacConkey Agar.

MATERIALS AND METHODS

Feed and Water sample were collected randomly from the poultry farms and analyzed using EMB , MacConkey and Hicrome coliform agar for total coliforms and E.Coli. E.Coli and other coliform colonies were examined for their morphology. Colonies were then subjected to biochemical tests for confirmation and identification.

Enumeration of E.coli

Water and feed samples were evaluated for E.coli and total coliforms using EMB, MacConkey and Hicrome coliform agar.

For the enumeration