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Development of method to quantify extracellular carbohydrate complexes produced by Escherichia coli O157:H7
Author(s) -
Ryu J.H.,
Beuchat L.R.
Publication year - 2003
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1046/j.1365-2672.2003.02103.x
Subject(s) - centrifugation , escherichia coli , agar , turbidity , chromatography , extracellular , chemistry , agar plate , food science , bacteria , microbiology and biotechnology , biology , biochemistry , ecology , genetics , gene
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 ( μ g 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.