Method of selective isolation of Acinetobacter baylyi from river water

The aim of the study is to increase the selectivity of isolating Acinetobacter baylyi from river water. For developing a selective culture medium, we used 5 strains of A. baylyi, 4 of which were isolated from the water of the river Neva, 1 — from clinical material. A. baylyi strains were identified by a combination of phenotypic features and/or using the mass spectrometric method (MALDI-ToF MS). In preliminary studies we found that the bacteria A. baylyi utilizes no L-phenylalanine as a single source of carbon and nitrogen, as well as the only source of carbon in a medium with mineral nitrogen sources, but utilizes L-phenylalanine as the only source of nitrogen only if it is supplemented with ethanol as the only source of carbon. Based on this A. baylyi property, a selective medium was developed for the isolation of these bacteria from river water. Composition and preparation of liquid selective medium were as follows: 1 liter of distilled water added with L-phenylalanine (CAS 63-91-2) 2.0 (g/l); NaCI 5.0; Na₂SO₄ 2.0; KH₂PO₄ 1.0; K₂HPO₄ 2.5; MgSO₄ 0.1; dissolved while heating, boiled for 5 minutes, 4 ml of 96-degree ethanol was added to a warm medium, pH 7.2±0.2 assessed; 40 ml were poured into sterile vials. Dense selective medium: the same ingredients and extra 15.0 g of agar are added to 1 liter of distilled water; with similar preparation technique, the medium is poured into sterile Petri dishes. Method of application of selective nutrient medium for isolation of A. baylyi from river water: water from the river Neva is seeded in a volume of 10 ml into a vial with 40 ml of liquid selective medium, incubated in aerobic conditions at 28°С for 48 h, then the enriched material from the flask is placed with a bacteriological loop onto the surface of same dense selective medium in two flasks; incubated in aerobic conditions at 28°С for 48 h. 10 isolated colonies are selected for subsequent identification, they are transplanted into sectors of nutrient agar, incubated at 28°С 24 h and isolates are identified by a complex of phenotypic characteristic of A. baylyi, and/or by the MALDI-ToF MS method. In this case, 19 isolates from 20 were identified as A. baylyi (95% of isolates). A study using this technique of three more water samples from the river Neva showed the results of the same narrowly selective orientation — 90–95% of the isolates were bacteria A. baylyi.