Use of eosin methylene blue agar to differentiate Escherichia coli from other gram-negative mastitis pathogens.

Abstract

Coliform mastitis accounts for 20–80% of acute clinical mastitis cases2 and is a continuous concern for US dairy producers because of its economic consequences.2,3 Coliform mastitis pathogens are gram-negative, usually lactose-fermenting bacilli and include Escherichia coli, Klebsiella spp., and Enterobacter spp.1 Other gram-negative organisms that can be isolated from the mammary gland include species of Serratia, Pasteurella, Proteus, and Pseudomonas.9 Rapid identification of the causative organism is essential for implementing a timely and prudent treatment plan. Coliform bacteria generally grow rapidly when plated on 5% sheep blood agar and, following overnight incubation, usually provide an adequate amount of bacterial growth for follow-up work.7 Escherichia coli can be identified with eosin methylene blue (EMB) agar based on the occurrence of a green-metallic sheen (Fig. 1) that appears on the surface of the bacterial colonies.12 The dyes in EMB agar, eosin Y and methylene blue, are pH indicators and inhibitors of gram-positive bacteria and at an acid pH combine to form a green-metallic precipitate (sheen).12 The food industry has been using various culturing methods to enumerate the numbers of E. coli O157:H7 in meat products and unpasteurized apple cider following several outbreaks of E. coli O157:H7 and Salmonella infection.4,5,8,18 Media evaluated for culturing heator cold-stressed E. coli included EMB agar, violet red bile agar, modified sorbitol MacConkey agar, sorbitol MacConkey agar supplemented with 4-methylumbelliferyl-b-D-glucuronide, and tryptic soy broth.4,5,8,18 MacConkey and EMB agars are used in some mastitis laboratories to identify and differentiate gram-negative mastitis pathogens. The primary goal of this study was to evaluate the use of EMB agar as a method for early differentiation of E. coli from other gram-negative mastitis pathogens. The secondary goal was to determine the culture time needed for the first visible sheen to develop. Frozen milk samples from which gram-negative bacteria had been isolated were received from Maryland, New York, and North Carolina. Gram-negative bacterial isolates from milk samples were received from Georgia, Illinois, Michigan, and Utah. Milk samples were also obtained from the Virginia Tech Dairy Science Complex. One hundred twenty-nine milk samples or isolates from milk samples were received. Fifty microliters of milk were plated on 5% sheep blood agar and incubated aerobically for