Key words: verocytotoxin-producing Escherichia coli, enterohemorrhagic Escherichia coli, Beutin' s blood agar
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1 Key words: verocytotoxin-producing Escherichia coli, enterohemorrhagic Escherichia coli, Beutin' s blood agar
2 Number of strains used in each experiment *hemolysis on Beutin' s blood agar plate List of verocytotoxin-producing E. coli strains used in experiments and hemolysis on5%washed sheep blood agar plate * basal medium: Soybean-Casein-Digest agar 'Eiken'
3 List of verocytotoxin-nonproducing E. coli strains used in experiments and hemolysis on 5% washed sheep blood agar plate * not hemolysed on Beutin' s blood agar**, but hemolysed on another blood agar **basal medium: Soybean-Casein-Digest agar 'Eiken' Beutin' s washed sheep blood agar Tryptose blood agar base (Difco)* supplemented with10mm CaCl2and 5% defibrinated sheep blood (Oxoid: Statens Seruminstitut) washed three times in phosphate-buffered saline, ph 7.2 *Soybean-Casein-Digest agar
4 Influence of blood washing times on the hemolysis on Beutin's blood agar plate *after overnight incubation at37 Ž **basal medium: Soybean -Casein-Digest agar `Eiken' ***one of the7strains of0157: H7/VT1, 2
5 Influence of sheep blood concentration on the hemolysis on Beutin' s washed blood agar plate* *basal medium: Soybean-Casein-Digest agar Tiken' sheep blood: 5times washed with PBS, ph7.2 **after overnight incubation at37 Ž Influence of divalent metal ion on the hemolysis on Beutin' s blood agar plate *after overnight incubation at37 Ž, - `++,?(doubtful) **basal medium: Soybean-Casein-Digest agar Tiken' Supplement: 4%5times washed sheep blood ***() shows that the margin of hemolysed ring is diffuse.
6 Influence of basal medium on the hemolysis on Beutin' s blood agar plat *basal medium: SCD (Soybean-Casein-Digest agar), NA (Nutrient agar) HI (Heart Infusion agar), BHI (Brain Heart Infusion agar) supplement: 4% 5times washed sheep blood and 10mM CaCl2 **() shows that the margin of hemolysed ring is diffuse ***after overnight incubation at37 Ž.
7 Influence of quality of basal medium on the hemolysis on Beutin' s blood agar plate *basal medium supplement: 4% 5times washed sheep blood and10mm CaCl2 **after overnight incubation at37 Ž
8
9 4) Orskov F: On the occurrence of E. coli belonging to O-group26in cases of infantile diarrhoea and white scours. Acta Path Microbiol Scand1951; 29: ) Gronross JA: Investigations on certain Escherichia coli serotypes. Ann Med Exp Biol Fenn 1954; 32suppl4: ) Charter R : Escherichia coli type O114isolated from infantile diarrhoea and calf scours. J Path Bact1956; 72: ) Cavalieri SJ, Bohach GA, Snyder IS: Characteris- tics and probable role in pathogenicity. Microbiol Rev1984; 48: ) Beutin L, Montenegro M, Zimmerman S, Stephan R: Characterization of hemolytic strains of Escherichia coli belonging to classical enteropatho- genic O-serogroups. Zbl Bact Hyg1986; A261: ) Beutin L, Prada J, Zimmerman S, Orskov I, Orskov F: Enterohemolysin, a new type of hemo- lysin produced by some strains of enteropathogenic E. coli (EPEC). Zbl Bakt Hyg1988; A267: ) Konowalchuk J, Speirs J, Stavric S: Vero response to a cytotoxin of Escherichia coil. Infect Im- mun1977; 18: ) Springer W, Goebel W: Synthesis and secretion 1) Giles C, Sangster G: An outbreak of infantile of hemolysin by Escherichia coli. Bact1980; gastro-enteritis in Aberdeen. J Hyg1948; 46: ) Beutin L, Montenegro MA, Orskov I et al.: Close association of verotoxin (Shiga-like toxin) production with entero hemolysin production in strains of Escherichia coli.j Clin Microbiol1989; 27: : ) Beutin L, Zimmerman S, Gleier K : Rapid detection and isolation of Shiga-like toxin (Verotoxin)- producing Escherichia coli by direct testing of individual enterohemolytic colonies from washed sheep blood agar plates in the VTEC-RPLA assay. J Clin Microbial1996; 34:
10 Basic Study of Beutin's Washed Sheep Blood Agar Plate Used for Selective Screening of Verocytotoxin-producing/Enterohemorrhagic Escherichia coli (VTEC/EHEC) Nobuaki KIMURA, Akiko KOZAKI, Tomiko SASAKI & Akira KOMATSUBARA Ishikawa Health Service Association Enterohemolysin production on Beutin's washed sheep blood agar plate has been used as an epidemiological marker for selective screening of Verocytotoxin-producing/Enterohemorrhagic Escherichia coli (VTEC/EHEC). In this study, we examined about the component of Beutin' s washed sheep blood agar for further improvement of hemolysin production media. The following items were studied: the numbers of washings of sheep blood with phosphate buffered saline (PBS)(pH7.2), concentration of sheep blood, kind and concentration of divalent metal ion (Ca2+, Mg2+) and basal medium. Twenty-seven strains of VT-producing E. coli of 7 different O-serotypes, 74strains of VT-nonproducing E. coli of 24 different O-serotypes and one strain of O157 coded Escherichia hermanii were used for this basic study. In comparison of washing times of sheep blood with PBS, 5 times washing was better than 3 times, the original. In sheep blood concentration, supplement with 4% sheep blood was best for hemolysis observation. In experiment of addition of 2 divalent metal ions, Ca2+and Mg2+, supplement with Ca2+was more suitable than Mg2+for hemolysis, and the supplement with 10 mm CaCl2, the original, was the best concentration. On the basal medium used in Beutin's sheep washed blood agar, 4 kinds of media were compared. In addition to Soybean-Casein Digest (SCD) agar, the original, Nutrient agar, Heart Infusion (HI) agar and Brain Heart Infusion (BHI) agar were examined, HI agar was the best blood agar base in the four media. As the above experimental results, the composition of the better Beutin's washed sheep blood agar may be summarize as follows: Heart Infusion agar 'Eiken'(R) used for blood agar base, supplemented with 10mM CaCl2 and 4% defibrinated sheep blood (Japan Biosupp. Center) washed five times in phosphate-buffered saline, ph7.2.
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