RU2518304C2 - TWO-PHASE NUTRITIONAL MEDIUM FOR THIN LAYER CULTIVATION OF Helicobacter pylori AND METHOD OF ITS REALISATION - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to field of microbiology and can be applied in medicine. Method includes preliminary inoculation of analysed material on Columbia agar with addition of 5% of sheep blood. Thermostatting is performed under microaerophilic conditions in CO₂ incubator at 37°C for 3-5 days at higher humidity. Sampling colonies and their re-inoculation on Petri dishes with chocolate agar are performed and thermostatting is carried out under microaerophilic conditions in CO₂ incubator at 37°C for 3-5 days at higher humidity. When thermostatting stage is completed Petri dishes with chocolate agar are poured on the top with liquid nutritional medium based on Schaedler broth, enriched with 10% cattle serum with their further thermostatting in CO₂ incubator at 37°C for 72 hours.

EFFECT: obtaining nutritional medium.

4 cl

Description

The invention relates to the field of microbiology and can be used in medicine.

The discovery of H. pylori in 1982 was the starting point in the formation of a new concept of the etiology of gastroduodenal diseases. Currently, the generally accepted fact is the role of H. pylori in the pathogenesis of chronic gastritis. In 1990, these bacteria were officially included in the international classification as Helicobacter pylori gastritis or gastritis associated with Helicobacteriosis, type B gastritis. About 70-80% of cases of gastritis, more than 70% of cases of gastric ulcer, 90% of peptic ulcer are associated with H. pylori infection duodenum, increased risk of developing cancer of the stomach and B-cell lymphoma. Recent scientific reports actively discuss the relationship of H. pylori with extragastric manifestations (diseases of the intestine, hepatobiliary system, pancreas), as well as lesions of the cardiovascular system, musculoskeletal system, skin, etc.

For the correct and timely diagnosis of Helicobacter pylori, many methods have been developed, grouped using several parameters for classification. Depending on the purpose, material, technique, they are divided into screening and confirming; invasive and non-invasive; direct and indirect. One of the methods of laboratory diagnosis of H. pylori infection is a bacteriological study - isolation, identification, study of the biological properties of the pathogen. The complexity and complexity of isolating the H. pylori culture limit the application of this method in practical healthcare practice, but it remains the only one for the phenotypic determination of the sensitivity of strains to antibacterial drugs.

Various culture media have been proposed for the cultivation of H. pylori, the essential components of which are basic agar, growth additives, and for selective ones, growth inhibitors of concomitant microflora. Most basic agars satisfy the requirements for cultivation of H. pylori, for example, Colombian agar, erythritol agar, cardiac cerebral agar. H. pylori is a very whimsical microorganism that requires additional growth factors (vitamins, trace elements) [1]. An obligatory component of the medium should be the addition of 5-10% of the blood or serum of animals (horses, rams). The use of human blood is limited by the presence of protective antibodies in most adults that can inhibit the growth of H. pylori [2]. In this case, red blood cells can be lysed so that growth substances can be used faster, which is achieved by using chocolate agar. A nutrient medium containing no serum and animal tissue -SATFM (serum - animal tissue - free medium) has been developed, which can be used both for isolation of a pure culture (dense) and for transportation (semi-liquid) with a difference in the percentage of agar [3 ]. N.V.Safonova and A.B.Zhebrun (1995) proposed a medium prepared on the basis of erythritol agar, into which blood, hemin and a selective additive are added ex tempore [4]. M.M. Majidov and colleagues (2000) developed Helicobacter agar containing a selective additive consisting of antibacterial and antifungal drugs and blood introduced ex tempore [5]. AB Zhebrun with a team of authors (2002) recommend using Colombian agar or cardiac cerebral agar supplemented with 5-7% horse serum or 5-7% defibrinated horse blood and 1% isovitalex solution as the basis of the nutrient medium [6]. Closest to the claimed is a method of cultivating H. pylori on chocolate agar prepared on the basis of Columbia agar medium with the addition of 2.5% donated blood. Then, to a nutrient medium cooled to 50 ° C, the authors propose adding 2.5% of hemolized donated blood. The method provides faster cultivation in a cheaper environment, but is not without a number of disadvantages [7]. The disadvantage of the prototype in the opinion of the authors is the use of donor blood, which can reduce the frequency of seeding due to the presence of antibodies to H. pylori in the donor blood, which have an inhibitory effect. Particularly acute is the problem of obtaining a pure culture, when, after reseeding isolated colonies, isolated colonies grow to isolate a pure culture, and biological material is not enough to identify and formulate an antibioticogram.

The disadvantage of cultivation methods on solid nutrient media is:

- low sowing efficiency of Helicobacter pylori during primary isolation,

- with further reseeding to obtain pure cultures, the strains are lost, germination is weakened and lost,

- the scarcity of growth of pure cultures leads to the inability to determine the sensitivity of the selected strain to antibiotics and chemotherapy.

The problem of culture accumulation can be solved by using liquid media. A composition similar to solid media (foundations, growth and selective additives) is also used in liquid media, although the cultivation process is more time-consuming and has not been widely used in practical laboratories [8]. Various bases of liquid media have been proposed: Brucella - broth, cardiac broth, soy broth with growth additives (serum, yeast extract, cyclodextrin and others) and antimicrobial agents (vancomycin, nalidixic acid, amphoterium [9, 10]. A medium having the constant composition of vitamins and minerals without the addition of serum is serum-free Ham's F-12 [11]. When grown on liquid nutrient media, bacteria have a typical tortuous shape, are mobile and biochemically active, are fully antigenic [12]. H. pylori grows in microaerophile atmosphere consisting of 5% O ₂ , 5-10% CO ₂ , 85-90% N _2. Various systems can be used to create microaerophilic conditions, among them a microaerobic box or an incubator in which an adequate atmosphere is maintained automatically. the atmosphere can be created in a BBL type GasPac 100 or GasPac 150 Anaerobic Systems microanaerostat using BBL or Oxoid type gas generator bags, which begin to produce gas mixtures when water is added, which also creates the humidity needed for H. pylori growth. But when cultured in a liquid medium, the creation and maintenance of microaerophilic conditions can be difficult. This problem can be solved by constant monitoring of the microaerobic atmosphere in test tubes by mixing on special platforms at an angle of 20 ° with a rotation speed of 120 revolutions per minute [13] or culturing in bottles with openings for accessing gases, for which mini-bioreactors for forced injection into the atmosphere of carbon dioxide cultivation through tubes immersed in bottles with a liquid nutrient medium [14].

The disadvantages of the methods of cultivation in liquid nutrient media:

- the complexity of creating and maintaining microaerophilic conditions in liquid nutrient media,

- the need to use additional equipment (shakers, rotary platforms, bioreactors, etc.) for the forced introduction of carbon dioxide.

The objective of the invention is to develop a method for thin-layer cultivation of Helicobacter pylori using a two-phase nutrient medium available for bacteriological laboratories that are not equipped with special equipment. The technical result achieved is the absence of the need to use additional equipment to maintain microaerophilic conditions in the liquid phase of the medium, the use of standard nutrient media (Colombia agar and Shedler broth) and the accumulation of pure H. pylori culture in an amount sufficient for morphological, biochemical identification and formulation of antibiograms.

Some microorganisms can hardly be grown on solid or liquid nutrient media, but they grow and multiply much more actively in two-phase nutrient media consisting of a combination of solid and liquid phases. A possible explanation may be that such nutrient media create conditions of growth and reproduction that are close to natural, when part of the microbial life cycle requires a solid nutrient medium, and other stages of the life cycle take place in a liquid nutrient medium. This, in particular, is characteristic of epithelial pathogens, which include H. pylori, which partially reproduce to the cells of the mucous membranes, and partially - on the surface of the mucosa in the lumen of the organ. Moreover, the composition of the components of the liquid and solid phases of the nutrient medium is determined by the biology of a particular microorganism.

So, RF patent No. 2272834 (15) protects a nutrient two-phase medium for isolation of unicellular microorganisms - Trichomonas. RF patent No. 2412991 (16) protects a two-phase nutrient medium for the cultivation of cryptosporidia, and RF patent No. 2346052 (17) provides a two-phase nutrient medium for the isolation of brucella.

The authors of the claimed invention offer the following composition of the nutrient medium. A two-phase nutrient medium consists of two phases: solid and liquid. The solid phase is chocolate agar prepared on the basis of Columbia agar with 5% ram blood.

Composition of Colombia agar:

Ingredients gram / liter P Biopepton 20.00 Tryptic digest of beef heart 3.00 Corn starch 1.00 Sodium chloride 5.00 Agar agar 15.00 Final pH (at 25 ° C) 7.3 ± 0.2

Cooking:

Stir 44.0 g of powder in 1000 ml of distilled water. Boil to completely dissolve the particles. Sterilize by autoclaving at 1.1 atm (121 ° C) for 15 minutes. Cool to 50 ° C and aseptically introduce sterile defibrinated sheep blood (5% of the total volume). The agar is slightly heated over low heat, not boiling. Mix thoroughly and pour the medium into sterile 14 ml Petri dishes. When preparing chocolate agar, special attention should be paid to proper heating: correctly prepared agar has a light brown color similar to the color of milk chocolate. If the heating is too weak, the agar will have a brick red color, and if it is strong, it will be dark brown.

The advantages of the used solid phase

Colombia agar is characterized by a variety of protein nutrition sources included in the formulation of the medium. The combination of peptones provides fast and plentiful growth of very fastidious microorganisms on it. As a result of the preparation of chocolate agar, hemin, NAD are extracted from red blood cells, and growth factors are used faster by hemophilic and whimsical bacteria, including H. pylori.

The liquid phase is a Shedler broth enriched with 10% cattle serum.

The composition of the Shedler broth:

Ingredients gram / liter P Casein Hydrolyzate 5.67 Proteozopeptone 5.00 Papain digest of soy flour 1.00 Yeast extract 5.00 Glucose 5.83 Sodium chloride 1,67

Potassium hydrogen phosphate 0.83 Tris (hydroxymethylaminomethane) 3.00 L-Cystine 0.40 Gemin 0.01 Final pH (at 25 ° C) 7.6 ± 0.2

Cooking:

Stir 28.41 g of powder in 1000 ml of distilled water. Boil with frequent stirring to completely dissolve the particles. Sterilize by autoclaving at 1.1 atm (121 ° C) for 15 minutes. Cool and aseptically add up to 10% of the total volume of sterile cattle serum. Stir the medium thoroughly before bottling. Avoid overheating of the medium or its oxidation in the light, as this leads to a delay in the growth of bacteria.

The advantages of the used liquid phase:

This medium is an excellent basis for adding blood or other enrichment supplements to the selection of fastidious anaerobic bacteria, including capnophilic bacteria. The combination of casein hydrolyzate, proteozopeptone, papain digest of soy flour, yeast extract and L-cystine provides the presence of nitrogenous nutrients, vitamins and other factors necessary for the growth of microorganisms. Glucose is a source of energy. Gemin and vitamin K stimulate the growth of whimsical microorganisms.

The authors propose the following method of thin-layer cultivation of H. pylori on a two-phase nutrient medium.

Stage 1. The primary inoculation of the test material (biopsy specimens of the gastric mucosa) is performed on Colombian agar with the addition of 5% ram blood. Thermostating under microaerophilic conditions (CO ₂ incubator) at 37 ° C for 3-5 days at high humidity.

2 stage. Assessment of the results of sowing. Detection of suspicious colonies (small, transparent with a diameter of 1-2 mm similar to "dew drops"). Reseeding the selected colony on chocolate agar to obtain a pure culture. Incubation under microaerophilic conditions at 37 ° C for 3-5 days.

3 stage. The accumulation of pure culture. In the case of sparse growth, single colonies are scattered by a loop on the surface of a dense medium and poured with a liquid nutrient medium prepared on the basis of Shedler broth enriched with 10% cattle serum. The optimal amount of liquid medium poured into a Petri dish with a diameter of 90 mm is 3.5 ml (the ratio of solid and liquid phases is 4: 1). Incubation under microaerophilic conditions of 37 ° C for 3 days.

4th stage. Identification of isolated culture grown in the liquid phase.

The study of morphological properties. Fixed smears are prepared from the culture fluid, stained according to Gram and the drug “crushed” or “hanging drop” to determine mobility.

The study of biochemical properties. Testing for catalase, oxidase, urease.

Setting antibiogram.

Thus, the inventive method of thin-layer cultivation of Helicobacter pylori involves the advantage of the liquid phase compared with the solid phase for culture accumulation, and its thin-layer distribution supports microaerophilic conditions in the thickness of the liquid without additional equipment.

Literature

1. Jiang X., Doyle M.P. Growth supplements for Helicobacter pylori. // J Clin Microbiol. - 2000. - Vol. 38. - P.1984-1987.

2. Westblom T.U., Madan E., Riff B.R. Improved growth of Helicobacter pylori using a liquid medium supplemented with human serum. // Ital. J. Gastroenterol. - 1991 .-- Vol.29 (Suppl. 2). - P. 48.

3. Dierikx C. M., Martodihardjo J., Kuipers E.J. et al. Serum and animal tissue free medium for transport and growth of Helicobacter pylori. // Immunol. Med. Microbiol. - 2007. - Vol.50. - P.239-243.

4. Safonova N.V., Zhebrun A.B. Gastritis, peptic ulcer and helicobacteriosis. - Recom. for doctors. - SPb. - 1995.

5. Mejidov M.M., Temirkhanova Z.U., Aliyeva H.M. et al. Assessment of culture medium for isolation and cultivation of Helicobacter pylori. // Journal. microbiol., epidemiol., immunol. - 2000. - No. 2. - S.25-29.

6. Zhebrun A.B., Aleksandrova V.A., Goncharova L.B. Diagnosis, prevention and treatment of diseases associated with Helicobacter pylori infection. (Manual for doctors). St. Petersburg, 2002.

7. Chervynets V.M., Chervinets L.F. Patent for the invention of the Russian Federation No. 2145975 "Method for the cultivation of Helicobacter pylori". - 2000.

8. Shahamat M., Mai U.E., Paszko-Kolva C. et al. Evaluation of liquid media for growth of Helicobacter pylori. // J. Clin. Microbiol. - 1991 .-- Vol.29. - P.2835-2837.

9. Morshed M.G., Karita M., Konishi H. et al. Growth medium containing cyclodextrin and low concentration of horse serum for cultivation of Helicobacter pylori. // Microbiol. Immunol. - 1994 .-- Vol. 38 (11). - P.897-900.

10. Murano A., Miyake M., Kato J. et al. Enhancement of the growth of Helicobacter pylori in Brucella broth by hydrogen peroxide. // Microbiol. Immunol. - 1999 .-- 43 (11). - P.1009-15.

11. Testerman T.L., McGee D.J., Mobley H.L.T. Helicobacter pylori growth and urease detection in the chemically defined medium Ham's F-12 nutrient mixture. // J. Clin. Microbiol. - 2001. - Vol. 39. - P.3842-3850.

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15. RF patent No. 2272834.

16. RF patent No. 2412991.

17. RF patent No. 2346052.

Claims (4)

1. Two-phase culture medium for thin-layer cultivation of Helicobacter pylori, containing a solid phase represented by chocolate agar made on the basis of Columbia agar with 5% sheep blood, and a liquid phase, which is a Shedler broth enriched with 10% cattle serum, characterized in that the ratio of solid phase to liquid is 4: 1 (14 ml of agar and 3.5 ml of broth). 2. Two-phase culture medium for thin-layer cultivation of Helicobacter pylori according to claim 1, characterized in that Colombia agar contains the following ingredients in g / liter:
Ingredients gram / liter Biopepton 20.00 Tryptic digest of beef heart 3.00 Corn starch 1.00 Sodium chloride 5.00 Agar agar 15.00 Final pH (at 25 ° C) 7.3 ± 0.2 3. Two-phase culture medium for thin-layer cultivation of Helicobacter pylori according to claim 2, characterized in that the Shedler broth contains the following ingredients in g / liter:
Ingredients gram / liter Casein Hydrolyzate 5.67 Proteozopeptone 5.00 Papain digest of soy flour 1.00 Yeast extract 5.00 Glucose 5.83 Sodium chloride 1,67 Potassium hydrogen phosphate 0.83 Tris (hydroxymethylaminomethane) 3.00 L-Cystine 0.40 Gemin 0.01 Final pH (at 25 ° C) 7.6 ± 0.2 4. The method of thin-layer cultivation of Helicobacter pylori using a two-phase nutrient medium according to claim 1, which provides for primary seeding of the test material on Columbia agar with the addition of 5% ram blood, thermostating under microaerophilic conditions in a CO ₂ incubator at 37 ° C for 3-5 days at high humidity, reseeding selected Petri dish with colonies chocolate agar plates in microaerophilic subsequent thermostating conditions in a CO ₂ incubator at 37 ^° C for 3-5 days at high humidity, characterized in that after t rmostatirovaniya petri dishes chocolate agar is poured on top of the liquid nutrient medium based on Schaedler broth supplemented with 10% bovine serum according to claim 1 and their finally incubated in a CO ₂ incubator at 37 ^° C for 72 hours.