Development of method to quantify extracellular carbohydrate complexes produced by Escherichia coli O157 : H7

Aims: The aim of this study was to optimize conditions to separate extracellular carbohydrate complexes(ECC) produced by Escherichia coli O157:H7 and to standardize the amount of ECC produced on a per cell basis. Methods and Results: ECC fraction I was removed from E. coli O157: H7 cells produced on tryptic soya agar and lettuce juice agar by centrifugation. To remove ECC fraction II, cells were heated at 100 C for 10 min, then centrifuged. The sum of ECC fractions I and II was considered as the total ECC produced by E. coli O157: H7. A correlation between cell mass and turbidity (O. D.(750nm)) of cell suspensions was determined. Cell mass has a linear relationship (R-2 = 0.93) with turbidity of cell suspensions from which ECC is removed. The amount of ECC produced on a per cell basis was calculated by dividing total amount of ECC (mug ml(-1)) produced by the turbidity (O.D.(750nm)) of heated cell suspension after removal ECC fractions I and II. Conclusions: A method for separating ECC from cells of E. coli O157: H7 has been developed and conditions have been optimized. A standard method to estimate the amount of ECC produced on a per cell basis was also developed. Significance and Impact of the Study: Using these procedures to prepare extract of ECC from E. coli O157: H7 and to standardize values, production of ECC on a per cell basis can be estimated and a comparison of the amount of ECC produced by the pathogen grown under different environmental conditions can be accurately measured.