RU2232805C2 - Strain of bacterium of species acinetobacter calcoaceticus varietas iwoffii 12 r used as indicator strain in biotesting drinking water (r-resistant strain) - Google Patents

Abstract

FIELD: ecology, microbiology.

SUBSTANCE: invention relates to control for environment pollution, namely, - aqueous sources. The strain Acinetobacter calcoaceticus var. Iwoffii 12 R (R-resistant) is isolated from lake water and deposited at number GISK named for L. A. Tarasevitch № 266. The strain survives in any ecologically pure water and can be used as indicator in biotesting drinking water. Invention provides evaluating the quality of analyzed water by ability of indicator strain to survive after contact with it.

EFFECT: valuable properties of strain.

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Description

The present invention relates to ecology, in particular to assessing the degree of environmental pollution, specifically water.

Using modern chemical-analytical methods for monitoring the quality of drinking water, the Sanitary and Epidemiological Service monitors the presence of three to four dozen substances harmful to human health (GOST, 1998), while thousands fall into their bodies of water.

In this regard, various methods of biotesting water are of particular importance. The most difficult in bioassay is the selection of test organisms (bioassays, bioindicators) of the desired sensitivity. Various plants, aquatic invertebrates, mold fungi, microorganisms, etc. can be used as indicators of toxicity. It is even proposed to use fish, namely their larvae, as the most sensitive in the ontogenesis of fish to toxic substances (Gelashvili DB, “Ecological monitoring”, N Novgorod, 2000).

Biotesting methods are actively being introduced into the practice of supervision abroad. So, in France, the assessment of water quality by toxicological indicators is mandatory in the “Freshwater Quality Control System” at the interagency level. A multi-test system of biological analysis of water toxicity was introduced in the USA (Zakharchenko MP et al., “Gig. And San.” - 1994. - No. 9. - C.3-4).

The most indicative, reliable and fast-reacting bioindicators are microorganisms (Zhurkov BC et al., “Gig. And San.” - 1997. - No. 1. - C.11-13; Tumanov A.A. et al., “J. analyte Chemistry. ”- 1998. - T.53, No. 12. - S.1252-1260), but they are most often used to indicate individual toxicants or related chemical groups. The content of toxic impurities is judged by growth, morphological, enzymatic and other reactions (Tumanov A.A. et al., J. Analytical Chemistry. - 2000. - T55, No. 2. - S.208-211). This approach involves the use of a significant number of bioassays that are sensitive to individual toxicants, therefore it looks no less complicated and cumbersome than traditional physicochemical analysis. He also does not give an idea about the full effect of the pollutants present in water, especially about their combined harmfulness to living organisms.

The most important task is the selection of such bio-indicators, which would make it possible to judge the integral (total) toxicity of drinking water. Developed for these purposes, the method of biotesting using daphnia gives not very reproducible results (Naboka MV, “Gig. And San.” - 1993. - No. 6. - P.75-76).

The purpose of the invention is the isolation of a bacterial strain that is sensitive enough to the total effect of pollutants to assess the integral toxicity of drinking water: it is a bacterial culture of a certain sensitivity that allows you to track the reaction to harmful impurities simultaneously on millions of individual cells (individuals) and therefore provides the most reliable results.

The proposed strain of Acinetobacter calcoaceticus var. Iwoffii 12 R (R-resistant) is isolated from a lake located in the vicinity of N. Novgorod; deposited in the GISK them. L.A. Tarasovich at number 266 and is characterized by the following features:

Cultural and morphological properties: gram-negative coccobacilli, motionless, have no dispute; aerobe.

Growth on solid nutrient media: on meat-peptone agar (MPA) (pH 7.0-7.4), after 18 h of growth at 37 ° C, they form small, opaque, whitish, round, with a straight edge colony, there are no specific needs for growth.

Growth on liquid nutrient media: on meat and peptone broth (MPB) and mineral medium No. 9 form a uniform turbidity when tested for 18-20 hours

Biochemical activity: oxidative metabolism, does not utilize glucose, utilizes maltose, sorbitol, inositol and sodium malonate; does not form oxidase, urease, phenylalanine deaminase, lysine and ornithine decarboxylase; gives negative reactions to lactose, sucrose, mannitol, sodium citrate, indole and hydrogen sulfide, as well as the Voges-Proskauer test, has catalase.

Nutritional needs of the strain: chemoorganotroph, not picky on a nutrient substrate; tolerates up to 15% NaCl, 2% MgCl ₂ , 2% BaCl ₂ , 3% urea.

Genetic features of the strain: to a varying degree sensitive to a wide range of antibiotics, especially chloramphenicol, norfloxacin, moxalactam; resistant to penicillin, oxacillin, aztreonam, cefoperazone.

The nucleotide composition of DNA = 39% GC, plasmids by agarose gel electrophoresis were not detected.

Products synthesized by the strain, virulent properties: non-pathogenic, saprophyte.

Strain activity: the comparative agent is not inhibited by high concentrations of ions of various brooms, disinfectants, detergents (MIC = 10-20 mg / l).

Ways to determine the activity of the strain: resistance to a set of salts of heavy metals (Cd, Pb, Zn, Cu) was studied by the generally accepted method of serial dilutions (Miller D. Experiments in molecular genetics. - M., 1976. - S. 436).

The method, conditions and composition of the media for storage of the strain: stored at a temperature of +4 (± 1) (C on MPA with reseeding 1 time in 4 months or in a lyophilized state.

Conditions for the propagation of the strain: the strain is propagated in test tubes with MPB (pH 7.0-7.4) at 37 ° C for 18-24 hours or 1.3% MPA.

Conditions of use: after the strain is kept in the studied water sample, dosed seeding on solid nutrient medium (MPA) is done and its survival is judged by the number of colonies grown.

Biotesting Example

Samples of the test water boiled and boiled for 10 minutes are poured into sterile standard 100-ml glass bottles (“weave”) of 50 ml, 0.1 ml of one-billionth bacterial suspension of the indicator strain is added. The same number of cells contribute to saline, used as a control of the viability of this culture. Suspension of the indicator strain is not added to the “blind” controls to determine whether there is contamination of water samples with its own microflora. The contents of the “hundred parts”, closed with stoppers, are thoroughly mixed and placed in a thermostat at 37 ° C.

After 20 hours of keeping the samples in the thermostat after effective mixing, sterile inoculation of each bottle of 0.1 ml on agar “jambs” is done: beveled 1.3% MPA (5 ml) in glass tubes (15 × 1.5 cm) under one angle. The fluid is distributed smoothly over the entire surface of the “jamb”. “Shoals” are incubated for 20-24 hours in an incubator at the optimum growth temperature of the indicator strain, after which the results are read - analysis of the growth of colonies on the surface of the agar.

The quality of the test water is evaluated by the ability of the indicator strain to survive after aging in it.

Strain Acinetobacter calcoaceticus var. Iwoffii 12 R (R-resistant) survives after soaking for 20 hours in water, not only from environmentally friendly natural sources remote from industrial pollutants, but also in boiled (for at least 10 minutes) tap water (except for the period of spring flood) ), as well as in bottled water (such as Sarov, Vita, etc.), including mineral water, since it (strain) tolerates a rather high content of ions of some harmless salts (Na, Mg, Ca). Water that kills the bacteria of this strain should be considered harmful to human health, and before using it for drinking, additional cleaning in appropriate filtering devices is recommended.

Claims (1)

The bacterial strain Acinetobacter calcoaceticus var. Iwoffii 12 R (R-resistant), deposited under No. 266 in the Collection of the State Research Institute for Standardization and Control of Medical Biological Preparations L.A. Tarasevich, is used for bio-testing of drinking water.