SU1138409A1 - Strain bacillus thuringiensis var. galleriae 114869/7 for use at test object for indication of bacteriophages - Google Patents

Abstract

The strain Bacillus thuringiensis var. Galleriae 1148/69/7 /, CIPMB-2605 (Central Museum of Industrial Micro- / Roorganisms of the Institute of Scientific Research Institute of Genetics), used as a test object to indicate bacteriophages.

Description

with

00 4 O CO 1 The invention relates to agricultural microbiology and is related to the indicator strain Bacillus thuringiensis, which is used to detect mild bacteriophages secreted by lysogenic cultures of the same species as producers of insecticides. Analogs in the patent and research literature are not found. The purpose of the invention is a strain of Bacillu thuringiensis, which can be used as a test object for indicating bacteriophages of production strains of the species Bacillus thuringiensis. This goal is achieved by the strain Bacillus thuringiensis var. galleriae 1148/69/7 /. The strain obtained from the culture of Bacillus thuringiensis var. galleriae 69/6 by sequential treatment of the vegetative culture with nitrosoguanidine and methyl bromide and selection of an asporrgenic clone resistant to antibiotics, rifampicin and Streptomycin. Strain of Bacillus thuringiensis var. The galleriae 1148/69/7 / is kept by the Central Museum of Industrial Microorganisms of the Institute of Scientific Research Institute of Genetics under the number of CMPM B-2605 and is characterized by the following features. Cultural and morphological with naki. On the medium, MPA forms flat colonies of light gray color, with a smooth or slightly rugged edge, 3–5 m in diameter, easily removable from agar. On DPS medium (yeast 30 g, corn flour 15 g, agar 20 g, water 1 forms flat colonies, gray, dull, 3 to 5 mm in diameter. On medium No. BT containing 8 g / l nutrient medium, 3 g / l of yeast extract, 20 g / l of agar (all components of the Difco company), with a pH of 7.3, colonies are flat, translucent, 5 mm in diameter, soft consistency. On semi-synthetic media containing, g / l: MgSO 0.2 ; CaCl2 0.0 agar 20; NlO 1 l and as an organic component peptone 20 g / l or tryptone 20 g / l or casein 20 g / s pH 7.4 colonies are flat, dull, rounded 1 to 5 mm in diameter. 9 On semi-synthetics the growth of the culture is uniform throughout the volume of the medium, continuous without strips or flakes. Optimal growth parameters at pH 7.2 - 7.4 and t 28c. The cell shape is rod-shaped, the cells are single or in chains, stained The gram is positive, according to the H-antigen, the culture belongs to the V serovariant. Absorbs glucose, mannose, maltose, cellobiose. Active in relation to salicin and esculin. Nitrates do not restore. The strain is sensitive to virulent bacteriophages of Bacillus thuringiensis (26 bacteriophages) and to moderate bacteriophages of the strains; Bacillus thuringiensis 1,111, IV and in serovariants. The use of the strain to indicate bacteriophages spontaneously taken up by production cultures of Ba .cillus thuringiensis is shown in Examples 1-3. Example 1. Investigated and, indicator cultures are transplanted from a Petri dish in m septonous broth and incubated on a rocking chair at 220 rpm for 4-6 hours. 25 ml of MPA medium (2% agar) containing 50 µg is poured into Petri dishes. / ml of rifampicin, after the agar has hardened, the cups are dried for 3-4 hours. The test tubes are poured into 3 ml of MPA medium (0.7% agar), kept in a water bath, and 0.2 ml of 4 to 6 hour cultures are added. the test and indicator strains, the contents of the tubes are poured into Petri dishes on the surface of MPA medium with an antibiotic. The experiment was carried out with two controls: 0.2 ml of 4 g of a 6-hour indicator culture was poured into a tube of melted and cooled to 45 ° C soft agar, the contents were poured into a Petri dish on the surface of MPA with rifampicin; 0.2 ml of 4 6-hour test culture is introduced into a test tube with soft agar melted and cooled to 45 ° C. The contents are drunk in a Petri dish on the surface of MPA with rifampicin.

The test and control bowls are incubated at 16 h. A bacterial lawn is formed in the control culture dish. In the control plate with the inoculum of the test strain, the medium remains transparent. Negative colonies of bacteriophage are visible on the test cup against the background of a lawn indicator culture.

Example 2. The experiment was carried out analogously to example 1, replacing the antibiotic rifampicin with streptomycin (250 units / ml).

Example 3. The experiment was carried out as in Example 1, but without the use of an antibiotic. The culture of the studied strain is centrifuged at 8000 rpm for 15 minutes and the supernatant is titrated. Records of results are carried out analogously to example 1.

The use of the strain will allow one to control the titer of phage particles under various conditions of growth, to determine the stability of the lysogenic state of the strains.