K. OXYTOCA BACTERIOPHAGES ISOLATION METHODS IMPROVEMENT

Abstract

The article presents the results of a study related to increasing the efficiency of phage isolation of bacteria of the species K. oxytoca, by developing the optimal composition of the medium used in the work. In scientific research, in almost all methods associated with the isolation of bacteriophages, meat-peptone broth and meat-peptone agar are used as the nutrient basis. The peculiarities of growth and cultivation of microorganisms create certain difficulties for the isolation of phages active against bacteria of the species K. oxytoca. The selection of components and the creation of an environment that would ensure the optimal growth of both the bacterial culture and the reproduction of the virus makes it possible to facilitate the isolation of bacteriophages. The number of bacterial strains used in the work was 7. All strains of cultures were obtained from the Museum of the Department of Microbiology, Virology, Epizootology and Veterinary and Sanitary Expertise of the Federal State Budget Educational Institution of Higher Education “Ulyanovsk State Agrarian University named after P.A. Stolypin”. The studies included 2 main stages. The first stage consisted in isolation of bacteriophages by the method of isolation from the external environment by the method of Adelson L.I., Lyashenko E.A. The material for the studies were samples: soil, sewage sample, fecal samples (2). Only 4 samples. According to the chosen method, the sowing of the putative phagolysate was carried out on meat-peptone agar (1.5%) and the agar for isolating bacteriophages (Aph) (1.5%). A positive result was the presence on the environment of negative colonies, clearly visible on the matt background of deep growth of bacteria. A negative result is a continuous growth (“lawn”) of bacterial culture. As a control, the culture of the microorganism studied was used for the media. In the course of the conducted studies for the first stage, 2 bacteriophages were isolated, active against the desired species of microorganism. The second stage consisted in a comparative analysis of the indices of cultivation of the isolated phages on standard meat-peptone agar and the proposed medium for isolation of bacteriophages. The main biological properties of the phages studied were also determined: morphology of negative colonies, lytic activity, specificity. The negative colonies formed on the compared media were classified as one type. The lytic activity of the studied phages on meat-peptone agar and Aph medium was the same. According to Grazia 2 × 107 BOE/ml for bacteriophage KO-3 UGSHA, (3–4) × 108BOE/ml for bacteriophage KO-8724 UGSHA. The isolated bacteriophages showed no lytic activity against strains of heterogeneous crops (Escherichia spp., Citrobacter spp., Serratia spp., Hafnia spp., Yersinia spp.).