Russian Journal of Infection and ImmunityRussian Journal of Infection and Immunity2220-76192313-7398SPb RAACI211210.15789/2220-7619-MOS-2112Research ArticleMethod of selective isolation of <i>Acinetobacter baylyi</i> from river waterSivolodskiiEvgeny P.<p>DSc (Medicine), Professor of the Department of Microbiology; Senior Researcher, Laboratory of Molecular and Biological Technologies</p>lykraeva@yandex.ruKraevaLyudmila A.<p>DSc (Medicine), Head of the Laboratory of Medical Bacteriology; Professor of the Department of Microbiology</p>lykraeva@yandex.ruMelnikovaElena V.<p>Bacteriologist, Laboratory of Bacteriology of the Central Clinical Diagnostic Laboratory</p>lykraeva@yandex.ruMilitary Medical Academy named after S.M. KirovSt.Petersburg Pasteur Institute010420231311911960401202312022023Copyright © 2023, Sivolodskii E.P., Kraeva L.A., Melnikova E.V.2023<p>The aim of the study is to increase the selectivity of isolating <em>Acinetobacter baylyi</em> from river water. For developing a selective culture medium, we used 5 strains of <em>A. baylyi</em>, 4 of which were isolated from the water of the river Neva, 1 from clinical material. <em>A. baylyi</em> 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 <em>A. baylyi</em> 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 <em>A. baylyi</em> 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; Na<sub>2</sub>SO<sub>4</sub> 2.0; KH<sub>2</sub>PO<sub>4</sub> 1.0; K<sub>2</sub>HPO<sub>4</sub> 2.5; MgSO<sub>4</sub> 0.1; dissolved while heating, boiled for 5 minutes, 4 ml of 96-degree ethanol was added to a warm medium, pH 7.20.2 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 <em>A. baylyi</em> 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 28С 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 28С for 48 h. 10 isolated colonies are selected for subsequent identification, they are transplanted into sectors of nutrient agar, incubated at 28С 24 h and isolates are identified by a complex of phenotypic characteristic of <em>A. baylyi</em>, and/or by the MALDI-ToF MS method. In this case, 19 isolates from 20 were identified as <em>A. baylyi</em> (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 9095% of the isolates were bacteria <em>A. baylyi</em>.</p>Acinetobacter baylyiselective synthetic mediumisolation from waterL-phenylalanineethanolecology of bacteriaAcinetobacter baylyiселективная синтетическая средавыделение из речной водыL-фенилаланинэтанолэкология бактерий[Патент № 2769434 Российская Федерация. МПК C12N 1/20 (2006.01), C12Q 1/04 (2006.01), C12R 1/01 (2006). Способ выделения бактерий вида Acinetobacter baylyi из речной воды; № 2021130073, заявлено 2021.10.14, опубликовано 2022.03.31 / Сиволодский Е.П. Патентообладатель: Сиволодский Евгений Петрович. 6 с. [Patent No 2769434 Russian Federation, Int.Cl. C12N 1/20 (2006.01), C12Q 1/04 (2006.01), C12R 1/01 (2006/01). Method for isolation of Acinetobacter baylyi from river water; № 2021130073, application 2021.10.14; date of publication 2022.03.31 / Sivolodskij E.P. 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