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


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 degrees 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. 750 nm) of cell suspensions was determined. Cell mass has a linear relationship (R2 = 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 (microgram ml-1) produced by the turbidity (O.D. 750 nm) 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.


CONCLUSIONS
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.
Ryu
J-H
JH
Center for Food Safety and Department of Food Science and Technology, University of Georgia, Griffin, GA, USA.
Beuchat
L R
LR
D001426:Bacterial Proteins / Q000032:analysis; D001431:Bacteriological Techniques / Q000379:methods; D018533:Biomass; D003470:Culture Media; D019453:Escherichia coli O157 / Q000254:growth & development / Q000378:metabolism*; D005516:Food Microbiology; D006358:Hot Temperature; D006801:Humans; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D001426:Bacterial Proteins; D003470:Culture Media; D011135:Polysaccharides, Bacterial