RU2178171C1 - Method of isolation of lactobacillus genus bacterium from clinical material - Google Patents

Abstract

FIELD: medicinal microbiology. SUBSTANCE: accumulation of bacteria of genus Lactobacillus from clinical material is performed at 39 C in selective liquid nutrient enrichment medium in the following ratio of medium components, g/l: dry enzymatic hydrolyzate of defatted milk, 30.0 ± 3.0; glacial acetic acid, 3.4 ± 0.1; concentrated yeast autolysate, 110.0 ± 10.0; agar, 0.8; distilled water, up to 1 l; 20% sodium hydroxide solution, to pH value of medium 5.4 ± 0.1. Then lactobacilli are isolated in solid selective nutrient medium in the following ratio of medium components, g/l: dry enzymatic hydrolyzate of defatted milk, 30.0 ± 3.0; glacial acetic acid, 3.4 ± 0.1; concentrated yeast autolysate, 110.0 ± 10; agar, 22.5 ± 2.5; distilled water, up to 1 l; 20% sodium hydroxide solution, to pH value of medium 5.4 ± 0.1. Invention can be used for selective isolation of bacteria of genus Lactobacillus from clinical material (feces, vaginal secretion). EFFECT: enhanced effectiveness, simplified method, improved diagnosis of gastroenteric diseases. 1 cl, 1 ex

Description

 The invention relates to medical microbiology and can be used for the selective isolation of bacteria of the genus Lactobacillus from clinical material (feces, vaginal discharge).

 Bacteria of the genus Lactobacillus are widespread in nature. They are typical representatives of the normal microflora of the human and animal body, found in many foods (dairy, meat, vegetable).

 Bacteria of the genus Lactobacillus are known to be the dominant flora of the human vagina. There is a close relationship between the state of the microflora of the birth canal of pregnant women and the formation of eubiotic microflora of the intestines of the child in the neonatal period. In the event of a number of obstetric and gynecological diseases of both infectious and non-infectious nature, pronounced disorders of resident vaginal microflora are formed. Bacterial vaginosis, accompanied by abrupt changes in lactoflora, is currently regarded as vaginal dysbiosis. In the diagnosis of intestinal and vaginal dysbiosis, one of the criteria for dysbiosis is the content of bacteria of the genus Lactobacillus. When conducting bacteriotherapy of dysbiosis, the content of bacteria of the genus Lactobacillus serves as an indicator of the normalization of microflora.

 Due to the fact that before conducting antibiotic therapy, the attending physician must know whether the drug chosen for treatment affects the patient’s indigenous lactoflora, it is necessary to determine the sensitivity of the indigenous bacteria of the genus Lactobacillus to antibiotics, and this requires a way to isolate them from the clinical material.

 In addition, in cases where the content of indigenous bacteria of the genus Lactobacillus in a patient is below normal (Dysbacteriosis of the intestine and methods of its laboratory diagnosis. - Method. Recommendations. - Khabarovsk. - 1987), he is given bacterial replacement therapy with eubiotic strains of bacteria of the genus Lactobacillus. However, the use of eubiotic strains is not always effective. It is known that exogenous bacteria of the genus Lactobacillus do not always take root in the host organism. This is due to the fact that interspecific and even intraspecific antagonism is widespread among bacteria of the genus Lactobacillus, they can have a damaging effect on indigenous microflora, or vice versa, autoflora can inhibit the development of eubiotics, which reduces its therapeutic effect (Korshunov V.M., Smeyanov V V., Efimov B. A. Rational approaches to the problem of correction of intestinal microflora // News. Ros. AMN, - 1996. - 2. - P. 60-65). In this regard, before each bacteriotherapy, each patient must determine the biocompatibility of eubiotic bacteria of the genus Lactobacillus with autolactoflora, and this requires a simple and accessible method for isolating bacteria of the genus Lactobacillus from clinical material in bacteriological laboratories.

 Isolation of a pure culture of bacteria of the genus Lactobacillus from clinical material (feces, vaginal discharge) is also necessary to obtain an auto-strain, on the basis of which a bacterial preparation is then made to restore the indigenous lactoflora of a particular patient. It is believed that the use of auto-strains is the most optimal way to restore indigenous microflora after antibiotic therapy.

 Currently, there are many nutrient media used for the cultivation of lactobacilli, but not all of them are suitable for the isolation of lactobacilli from clinical material containing concomitant bacterial flora. Mostly, known nutrient media are intended for the accumulation of biomass of lactobacilli and bifidobacteria.

 So, the nutrient medium for the cultivation of bifidobacteria and lactobacilli "Epidermate" has the following ratio of components, g / l: 20.0-25.0 enzymatic hydrolyzate for gouging skins of pinnipeds or skins of cattle fruit with a final concentration of amine nitrogen of 100-140 mg%; 9.5-10.0 lactose; 4.5-5.5 sodium chloride; 0.0025-0.0030 tryptophan, 0.65-0.70 agar; water up to 1 liter (Ermishina I.G., Vlasova T.F. RU 2039814; B.I. 20, 07.20.95).

 The disadvantage of this medium is that it does not have inhibitory properties against microorganisms present in the clinical material (enterobacteria, staphylococci) and can only be used to accumulate a pure culture of lactobacilli and bifidobacteria in the food industry and the production of eubiotics.

A known nutrient medium for growing microorganisms, consisting of the following components, g / l: 0.14-0.16 casein pancreatic hydrolyzate; 4.5-5.5 baker's yeast extract; 19-21 lactose; 0.15-0.25 cystine; 0.15-0.25 MgSO ₄ • 7H ₂ O; 0.04-0.06 MnSO ₄ • 4H ₂ O; 0.4-0.6 ascorbic acid; tap water up to 1 liter (Vashurin O.A., Yakshina T.V., Krasnova S.P. et al. RU 2027754; B.I. 3, 01.27.95).

The disadvantage of this nutrient medium is the multicomponent composition, the complexity of preparing pancreatic casein hydrolyzate, which must be subjected to ultrafiltration on the membrane, which requires complex hardware design. The applied concentration of casein pancreatic hydrolyzate (0.14-0.16 g / l) as a source of nutrients is insufficient for the cultivation of lactobacilli, which is offset by a large consumption of lactose (19-21 g / l) and vitamin supplements (baker's yeast extract, cystine , ascorbic acid) and mineral (MgSO ₄ • 7Н ₂ О; MnSO ₄ • 4Н ₂ О).

Closest to the claimed is a method for isolating bacteria of the genus Lactobacillus from clinical material by preparing 10-fold dilutions in a liquid selective nutrient enrichment medium for lactobacilli in the following ratio of components: hydrolyzed skim milk liquid 20 ± 5%; yeast autolysate concentrated 10 ± 2%; technical agar 0.08%; distilled water up to 100%; pH of the medium is 5.4 ± 0.1. Cultivation of crops is carried out at 39 ^o C for 24-48 hours with a high content of carbon dioxide (5%) and a low oxygen concentration (16%), and then lactobacilli are isolated from a liquid selective nutrient culture medium by inoculation into a dense selective nutrient medium in the following ratio components, g / l: 150-250 hydrolyzate of skim milk liquid; 80-120 yeast autolysate concentrated; 20 agar; distilled water up to 1 liter; pH of the medium is 5.3-5.5; cultivation of crops is carried out at 39 ^o C for 24-48 hours with a high content of carbon dioxide (5%) and a low oxygen concentration (16%) (Glushanova N.A. Lactobacilli in the study and correction of resident microflora of a person: Abstract. Candidate of Medical Sciences .-- Chelyabinsk, 1999. - P. 7). The disadvantage of this method is the duration and the complexity of the preparation of pancreatic hydrolyzate of skim milk (Instructions for the preparation of sour milk bifilact in dairy kitchens 11-14 / 6-33. - Approved. 03.27.87. MH USSR. - M, 1987. - P. 11).

 The objective of the invention is the creation of a simpler technology for the preparation of selective nutrient medium for the isolation of bacteria of the genus Lactobacillus from clinical material (feces, vaginal discharge).

The problem is achieved by the fact that they carry out the accumulation of bacteria of the genus Lactobacillus from clinical material at 39 ^o C on a liquid selective nutrient enrichment medium of the following composition, g / l:
The skim milk hydrolyzate is enzymatic dry - 30.0 ± 3.0
Glacial acetic acid - 3.4 ± 0.1
Concentrated yeast autolysate - 110.0 ± 10.0
Agar - 0.8
Distilled water - Up to 1 liter
A solution of sodium hydroxide 20% to a pH of 5.4 ± 0.1,
and then lactobacilli are isolated on a dense selective nutrient medium in the following ratio of medium components, g / l:
The skim milk hydrolyzate is enzymatic dry - 30.0 ± 3.0
Glacial acetic acid - 3.4 ± 0.1
Concentrated yeast autolysate - 110.0 ± 10.0
Agar - 22.5 ± 2.5
Distilled water - Up to 1 liter
A solution of sodium hydroxide 20% to a pH of 5.4 ± 0.1,
and the cultivation of crops is carried out at a temperature of 39 ^o C.

The novelty of the proposed method lies in the fact that the isolation of bacteria of the genus Lactobacillus is carried out in two stages with the combination of several factors: low pH (5.3-5.5), optimal ratio of the components of the nutrient medium, increased incubation temperature (39 ^o C), cultivation of crops in an atmosphere containing 5% carbon dioxide, 16% oxygen, which ensure the accelerated growth of bacteria of the genus Lactobacillus that live in the human body and at the same time inhibit the development of concomitant microflora adapted to a neutral pH value (7 , 0), a temperature of 37 ^o C and aerobic conditions. The development of concomitant obligate anaerobic microflora in such conditions is impossible.

The essence of the method is as follows:
Isolation of bacteria of the genus Lactobacillus from clinical material (feces, vaginal discharge) is carried out in two stages. The first stage consists in preparing a series of 10-fold dilutions of the test material in a liquid selective nutrient enrichment medium for the accumulation of lactobacilli, of the following composition:
The skim milk hydrolyzate is enzymatic dry - 30.0 ± 3.0
Glacial acetic acid - 3.4 ± 0.1
Concentrated yeast autolysate - 110.04 ± 10.0
Agar - 0.8
Distilled water - Up to 1 liter
A solution of caustic soda 20% to a pH of 5.4 - 0.1
Dilutions are made in tubes containing 4.5 ml of liquid selective nutrient enrichment medium using an automatic dispenser with removable tips, by transferring 0.5 ml from the tube to the tube. Titration of 0.5 ml is carried out up to 10 ^-8 -10 ^-10 . After titration, the crops are incubated for 24 hours at a high content of carbon dioxide (5%) and a low oxygen concentration (16%) in the cultivation atmosphere at a temperature of 39 ^o C. The result is taken into account visually, by the presence of a general turbidity of the medium or the growth of isolated colonies. Then, smears are prepared from tubes with growth, stained according to Gram. The last breeding is noted, where the growth of gram-positive rods characteristic of morphology was detected. The titer of lactobacilli (the number of bacteria of the genus Lactobacillus in 1 g of clinical material) is determined by the maximum dilution in which typical gram-positive bacilli are found. Tubes with dilutions of clinical material were noted in which abundant growth of bacteria of the genus Lactobacillus was detected.

Then, the second stage is carried out - the isolation of lactobacilli in a dense selective nutrient medium. To do this, from tubes with dilutions of clinical material in which abundant growth of bacteria of the genus Lactobacillus was detected, a bacteriological loop is sown on the surface of a dense selective nutrient medium to obtain isolated colonies. Dense selective nutrient medium has the following composition, g / l:
The skim milk hydrolyzate is enzymatic dry - 30.0 ± 3.0
Glacial acetic acid - 3.4 ± 0.1
Concentrated yeast autolysate - 110.0 ± 10.0
Agar - 22.5 ± 2.5
Distilled water - Up to 1 liter
A solution of caustic soda 20% to a pH of 5.4 - 0.1
Cultivation of crops is carried out at 39 ^o C in an atmosphere containing 5% carbon dioxide, 16% oxygen, for 24-48 hours.

In the conditions of the first stage of cultivation on a liquid selective medium, the accumulation of bacteria of the genus Lactobacillus occurs due to their accelerated growth and inhibition of the growth of concomitant microflora when cultured in an atmosphere containing 5% carbon dioxide and 16% oxygen for 24 hours at a temperature of 39 ^o C. Lactobacilli, living in the human body as part of indigenous microflora, are thermophilic and grow well at 39 ^o C. Bacteria of the genus Lactobacillus are facultative anaerobes, therefore, grow in an atmosphere containing 5% carbon dioxide logo of gas, 16% oxygen. To obtain a cultivation atmosphere containing 5% carbon dioxide and 16% oxygen, the crops are placed in a hermetically sealed container (anaerostat, desiccator), a candle is lit and tightly closed.

 The skim milk milk hydrolyzate is enzymatic (Nizhny Novgorod state enterprise for the production of bacterial preparations "ImBio", the content of amine nitrogen 1485 mg%) in an amount of 27.0-33.0 g / l is a source of nitrogen. Glacial acetic acid (GOST 61-75) in an amount of 3.3-3.5 g / l provides suppression of concomitant microflora, without interfering with the reproduction of lactobacilli. Concentrated yeast autolysate (amine nitrogen content 580-600 mg%) in an amount of 100.0-120.0 g per 1 liter of medium serves as a growth promoter and a source of plastic substances that promote the regeneration of lactobacilli damaged by selective factors (pH 5.4 ± 0 , 1, acetic acid).

 The indicated amounts of skim milk hydrolyzate of dry enzymatic and yeast autolysate make it possible to obtain a concentration of amine nitrogen in the nutrient medium from 100 to 130 mg%, which ensures the sensitivity (germination) of the medium.

 Using in the inventive method 0.8 g / l agar increases the viscosity of the medium, which allows to obtain the growth of isolated colonies of bacteria of the genus Lactobacillus in the column of the medium, and also reduces the solubility of oxygen in the thickness of the medium. At the same time, 0.8 g / l of agar in the medium does not complicate the preparation of dilutions.

Setting the pH of the medium in the range of 5.3-5.5 in combination with the claimed composition ensures the suppression of concomitant microflora, without inhibiting the growth of seeded lactobacilli. The cultivation of crops at 39 ^o C in an atmosphere containing 5% carbon dioxide, 16% oxygen provides accelerated growth of colonies of lactobacilli, while delaying the growth of concomitant microflora in the study of clinical material. The atmosphere of such a gas composition can be obtained using an anaerostat and a candle. The concomitant indigenous anaerobic microflora does not grow under such conditions; among representatives of aerobic microflora, under these conditions, the growth of enterococci and fungi of the genus Candida is possible.

 The introduction of 20.0-25.0 g of agar per liter into the medium makes it possible to obtain a dense selective nutrient medium for isolation of isolated colonies of lactobacilli, which makes it possible to isolate a pure culture of bacteria of the genus Lactobacillus for further study.

Example
Isolation of bacteria of the genus Lactobacillus from feces with preliminary accumulation on a liquid selective nutrient enrichment medium.

In a flask with a capacity of 1 liter, 120.0 g of concentrated yeast autolysate (amine nitrogen content 583 mg%), 33.0 g skim milk hydrolyzate, enzymatic skim milk powder (amine nitrogen content 1485 mg%), 0.8 g agar, 3.5 g glacial acetic acid, distilled water to 1 liter, 2.5 ml of a 20% sodium hydroxide solution to pH 5.5, heated until the agar melts, sterilized in an autoclave at a temperature of (121 ± 1) ^o C for 15 minutes. The medium is quickly cooled by placing the flask in a water bath with a temperature of 15-20 ^o C, periodically stirred to accelerate cooling and uniform distribution of agar in the medium. After sterilization, the medium can be stored in the refrigerator for up to 2 months.

Aseptic liquid selective nutrient enrichment medium is poured into 4.5 ml tubes. Test tubes can be stored in the refrigerator for up to 4 weeks by being placed in a sealed plastic bag to prevent drying out. Before use, the tubes must be heated in a thermostat or in a water bath to 37-39 ^o C.

From the main dilution of feces 10 ^-1 (in sterile physiological saline) using a dispenser, a series of 10-fold serial dilutions in 4.5 ml of a liquid selective nutrient enrichment medium is prepared (titration of 0.5 ml, up to 10 ^-10 ). Crops are incubated at 39 ^o C for 24 hours in an atmosphere containing 5% carbon dioxide and 16% oxygen. The result is taken into account visually, by the presence of a general turbidity of the medium or the growth of isolated colonies. Then, smears are prepared from tubes with growth, stained according to Gram. The last breeding is noted, where the growth of gram-positive rods characteristic of morphology was detected. The titer (the number of bacteria of the genus Lactobacillus in 1 g of feces) is determined by the maximum dilution in which typical gram-positive bacilli are found. In feces, in addition to lactobacilli, enterococci can grow under these conditions, which are easily distinguishable by morphology in Gram smears. Test tubes with faecal dilutions are noted in which abundant growth of bacteria of the genus Lactobacillus without enterococci is detected, or with a minimal presence of enterococci, and then from these test tubes a bacteriological loop is used to seed a dense selective nutrient medium to obtain isolated colonies.

A dense selective nutrient medium is prepared as follows:
100.0 g of concentrated yeast autolysate (amine nitrogen content 583 mg%), 27.0 g skimmed milk powder hydrolyzate (amine nitrogen content 1485 mg%), 20.0 g agar, 3.3 g are placed in a flask with a capacity of 1 liter glacial acetic acid, distilled water to 1 liter, 2.5 ml of a 20% sodium hydroxide solution to pH 5.5, heated until the agar melts, sterilized in an autoclave at a temperature of (121 ± 1) ^o C for 15 minutes. The medium is cooled to a temperature of 45-50 ^o C, poured under aseptic conditions, 20 ml in sterile Petri dishes, before sowing the cups are dried in an oven at 37-39 ^o C until the droplets of condensate disappear on the surface of the medium.

The incubation of crops in a dense selective nutrient medium is carried out in an atmosphere containing 5% carbon dioxide and 16% oxygen, with the lid down, at 39 ^o C for 24-48 hours.

 Colonies of bacteria of the genus Lactobacillus grow in different sizes, from punctate (dewdrops) to 2-3 mm in diameter, often flat, rough, slightly opalescent, transparent, sometimes with an elevation in the center and a roller along the edge, rarely white. In a Gram smear, gram-positive small or large, capsule-free, non-spore-forming sticks located separately or in chains. When sowing feces, along with colonies of bacteria of the genus Lactobacillus, enterococcus colonies are found. Enterococcal colonies are white, round, convex, shiny, smooth, rarely rough. From bacteria of the genus Lactobacillus, they are differentiated in Gram smears.

To accumulate a pure culture of bacteria of the genus Lactobacillus, after microscopy, the remains of the colony are seeded in 5 ml of milky-yeast liquid accumulation medium. The incubation of crops on the accumulation medium is carried out at 39 ^o C for 16-20 hours, the purity of the culture is checked and used for further study (determination of biocompatibility with eubiotic strains of bacteria of the genus Lactobacillus, preparation of lactobacterin based on the auto-strain of bacteria of the genus Lactobacillus, determination of the sensitivity of the auto-strain of bacteria of the genus Lactobacillus to antibiotics).

To justify the belonging of the selected bacteria to the genus Lactobacillus, the following characteristics are used. The very fact of the development of culture on a selective nutrient medium in an atmosphere containing 5% carbon dioxide and 16% oxygen suggests the development of a culture of lactobacilli. In addition, morphological studies are carried out: a rod-shaped form and a positive Gram stain, as well as the identification of metachromatic inclusions when stained with methylene blue, the lack of ability to spore and capsule formation. Confirmation of the belonging of the culture to the genus of lactobacilli is the lack of mobility (especially from a liquid medium) under phase contrast microscopy, the inability to reduce nitrates and methylene blue, negative oxidase and catalase samples, and the absence of pigmentation. The fact that the culture belongs to the group of anaerobic and facultative anaerobic microorganisms is indicated by the inability to grow on a non-selective solid medium for culturing lactobacilli at 39 ^° C under aerobic conditions, the lack of growth at 39 ^° C on simple agar in an atmosphere containing 5% carbon dioxide and 16% oxygen.

 Thus, the obtained results prove that the inventive method for isolating bacteria of the genus Lactobacillus from clinical material can not only significantly improve the diagnosis of gastrointestinal diseases, but also significantly improve the quality of bacteriotherapy and antibiotic therapy.

Claims (1)

A method of isolating bacteria of the genus Lactobacillus from clinical material (feces, vaginal discharge), which consists in preparing a series of 10-fold dilutions of the test material in a liquid selective nutrient enrichment medium, seeding from dilutions on the surface of a dense selective nutrient medium, culturing crops at 39 ^o C for 24-48 hours with a high content of carbon dioxide (5%) and a low concentration of oxygen (16%) in the atmosphere of cultivation, characterized in that the accumulation of bacteria of the genus Lactobacillu s from clinical material on a liquid selective nutrient enrichment medium in the following ratio of medium components, g / l:
The skim milk hydrolyzate is enzymatic dry - 30.0 ± 3.0
Glacial acetic acid - 3.4 ± 0.1
Concentrated yeast autolysate - 110.0 ± 10.0
Agar - 0.8
Distilled water - Up to 1 L
A solution of caustic soda 20% to a pH of 5.4 - 0.1
and then lactobacilli are isolated on a dense selective nutrient medium in the following ratio of medium components, g / l:
The skim milk hydrolyzate is enzymatic dry - 30.0 ± 3.0
Glacial acetic acid - 3.4 ± 0.1
Concentrated yeast autolysate - 110.0 ± 10.0
Agar - 22.5 ± 2.5
Distilled water - Up to 1 L
A solution of caustic soda 20% to a pH of 5.4 - 0.1