METHOD OF ESTIMATION OF ADHESIVE PROPERTIES OF CHOLERA VIBRIOS Vibrio cholerae El Tor AND Vibrio cholerae O139 ON CELL CULTURE HuTu-80 Russian patent published in 2016 - IPC C12Q1/02 C12N1/20 

FIELD: medicine.

SUBSTANCE: invention relates to method of estimation of adhesive properties of cholera vibrios. Disclosed method involves following steps: a) cell monolayer HuTu-80 is prepared by growing in plastic bottles with volume of 50 ml at 100-150 thousand at 1 ml with subsequent trypsinization of culture and introduction into pen flasks with disks (disks 12.0 X 0.11 SPL Lifesciences) at 50 thousand cells in volume of 1 ml; b) preparing a bacterial suspension strains of Vibrio cholerae El Tor and Vibrio cholerae O139, which is grown on Martin agar (pH 7.7) for 18 hours, then by loop is inoculated into broth LB (pH 7.7) and maintained in a thermostat at 37 °C for three hours; c) experiment is carried out in pen flask with discs, on which formed sparse monolayer HuTu-80, added 10 mcl of bacterial suspension and 1 ml of DMEM medium with 5 % of bovine embryo serum up to concentration of cholera vibrios of 10⁶ mln. cells/ml, followed by incubation for 90 minutes at 37 °C and removal of nutrient medium, twice washed monolayer with Hanks solution with pH of 7.4, fixed with 5 % formalin for 1 hour, then dried and stained discs according to Romanovsky-Giemsa 1:5 for 40 minutes; d) by coloured discs count associated bacterial cells count is conducted, for this purpose, discs are washed with Hanks solution with pH of 7.4, removed from vials, dried, examined under microscope and adhesive properties of cholera vibrios are determined by number of vibrios, bound with 100 cells of HuTu-80, and adhesion index is calculated by formula $$AI=\frac{\sum KB}{100K},$$ where AI is adhesion index, ΣKB is total number of cells of bacteria, attached to one cell of HuTu-80 line, 100K - 100 cells line HuTu-80, results are evaluated as per obtained quantitative adhesion index, if it is more than 20, then analysed strain is highly adhesive, from 10 to 20 is mildly adhesive strain, less than 10 is low adhesive strain.

EFFECT: invention can be used in laboratory diagnostics for determination of adhesion index of cholera vibrios Vibrio cholerae El Tor and Vibrio cholerae O139 (Bengal).

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