Nguyen Thi My Thanh et al.
Laboratory of Enteric Bacteriology
A microtitration method was developed at the Laboratory of Enteric Bacteriology to activate and confirm EPEC strains isolated from stools of children having diarrhoea. Many of them had weak and delayed agglutination with antisera on glass slides. From a subculture on Trypticase soy agar slant incubated at 37°C/18–20 hours, the strain was transferred to Trypticase soy broth, incubated at 37°C/4–6 hours, boiled in water-bath 100°C/1 hour, diluted with 0.5% formalinized normal saline to get the turbidity of Mac Farland No: 1 - that was the antigen suspension - The same antiserum with that used in slide agglutination was successively diluted with 0.6% bovine serum albumin PBS on 96well- microtest plate, starting with the dilution 1:100. The equal volumes of the antigen suspension were added to the wells, then the plate was incubated overnight in water-bath at 48°C. The agglutination reaction was positive if the titer attained 1:1600 or more.
Of 59 strains showing weak and delayed agglutination on slide, 12 strains were activated and confirmed as really positive on microtest plate, 28 were negative and 19 were autoagglutinated. All of 8 strains showing strong reaction on slide were positive on microplate, all of 10 negative on slide were negative on microplate, and all of 3 agglutinated with normal saline on slide were also autoagglutinative on plate.
This method is accurate, antiserum-saving and can be used for other groups of E.coli antisera: ETEC, EHEC, EIEC …