An opsonization assay was performed with serum collected from immunized mice to evaluate the bactericidal efficacy. Clinical isolates were provided from Princess Alexandra Hospital that includes (i) ACM-2002 (Royal Brisbane Hospital, human abscess–lymph gland), (ii) ACM-5199 (ATCC 12344, NCIB 11841, scarlet fever), (iii) ACM-5203 (ATCC 19615, pharynx of child followed by episode of sore throat), (iv) GC2 203 (wound swab), (v) D3840 (nasopharynx swabs), (vi) D2612 (nasopharynx swabs). The bacteria were prepared by streaking on THB agar supplemented with 5% yeast extract and incubated (37°C, 24 hours). A single colony from the bacterium was transferred to THB (5 ml) supplemented with 5% yeast extract and incubated for 24 hours at 37°C to give approximately 4.6 × 106 colony-forming units (CFU)/ml. The culture was serially diluted to 10-2 in PBS, and an aliquot (10 μl) was mixed with heat-inactivated sera (10 μl) and horse blood (80 μl). The inactivated sera were prepared by heating in a 50°C water bath for 30 min. The bacteria incubation was conducted in the presence of sera in a 96-well plate at 37°C for 3 hours. Ten microliters of this suspension was plated on Todd-Hewitt agar plates supplemented with 5% yeast extract and 5% horse blood. The plates were incubated at 37°C for 24 hours. The bacteria survival rate was analyzed on the basis of the CFU enumerated from the incubated Todd-Hewitt agar plates. The assay was performed in triplicate from three independent cultures.

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