RU2481394C2 - Nutrient medium for extraction, cultivation and determination of haemolytic properties of bacteria from clinical material - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: nutrient medium contains HMF - agar, erythrocytic mass from donor's human blood, serum of cattle and yeast extract at the specified ratio.

EFFECT: invention makes it possible to increase sensitivity of medium to extracted microorganisms, to improve growth properties of nutrient medium and to simplify method of its preparation.

Description

The invention relates to microbiology, in particular to nutrient media, can be used in research and practical work for bacteriological diagnosis of bacteria, including bronchopulmonary pathogens, such as: Streptococcus pneumoniae, Moraxella (Branchamella) catarrhalis, Streptococcus pyogenes, β-hemolytic Streptococcus spp., Staphylococcus aureus.

According to official statistics, respiratory diseases steadily occupy the first place in the structure of the overall incidence of children and adolescents in our country. Many clinical forms of respiratory pathology determine the incidence of childhood morbidity and infant mortality. Often, bronchopulmonary diseases, starting in children, continue into adulthood, leading patients to disability, and sometimes to dramatic outcomes [10]. Therefore, it is important to improve the laboratory diagnosis of inflammatory diseases of the respiratory system and ENT organs, including bacterial etiology in children.

The choice of culture media for inoculating clinical material from patients with inflammatory diseases of the respiratory system and ENT organs is difficult due to the many pathogens and the variety of clinical material for this pathology.

Known culture medium for sowing and isolation of Streptococcus pneumoniae, the recipe of which was developed by Katosova L.K. [1, 2], for which erythritol agar, dry nutrient agar, Timing agar No. 1 (containing pancreatic hydrolyzate of fish meal, dry), Timing agar (dry), AGV (Givental-Vedminoy agar can be used as agar base) ) with an optimal pH of the medium - 7.2 ± 0.2. After sterilization, the agar is cooled to a temperature of 45 ° C, 10% inactivated horse serum, 3% erythrocyte mass, or 5% defibrinated blood are added.

In the finished base, which can be used erythritol agar, dry nutrient agar, timing agar No. 1, timing agar, Giventhal-Vedminoy agar, prepared according to the recipe indicated on the label, pH 7.2 ± 0.2, cooled to 45 ° С, add:

1) 3% of erythrocyte mass (per 100 ml of the base - 3 ml) or 5% of defibrated blood;

2) 10% inactivated horse serum (per 100 ml of the base - 10 ml);

3) pour into Petri dishes.

The disadvantages of this nutrient medium include good growth properties in relation to only Streptococcus pneumoniae; the use of horse serum for most practical laboratories in clinical microbiology, especially in the field, is difficult, which ultimately increases the cost of the final product.

A nutrient medium of 5% blood agar is known for the primary isolation of microorganisms and the determination of their hemolytic properties, which is recommended by Order of the Ministry of Health of the USSR No. 535 of April 22, 1985 "On the unification of microbiological (bacteriological) research methods used in clinical diagnostic laboratories of medical institutions "[9], as the main nutrient medium for inoculation, of the separated airways p.119 (separated pharynx and nose; sputum; bronchial contents obtained with bronchus Oscopy or with suction through a tracheostomy (in patients who are on a breathing apparatus); exudates; resected tissues, etc.) and ENT organs. To prepare this medium, according to order No. 535, dry nutrient agar is used as the basis. According to the recipe indicated on the label, 2% agar is prepared, pH 7.4-7.6. 5% (5 ml of blood per 100 ml of culture medium) defibrinated lamb, horse or rabbit blood, citrated or defibrinated human blood without antibacterial drugs is added to the agar, which is molten and cooled to 45 ° C, following aseptic rules. The mixture is thoroughly mixed so that foam does not form, and poured into sterile Petri dishes, preheated in a thermostat, with a layer of 3-4 mm. The agar layer should be uniformly colored red. Store no more than two weeks in cellophane bags at 4-6 ° C.

The disadvantages of this nutrient medium include: low growth properties for fastidious microorganisms, in particular for Streptococcus pneumoniae, Streptococcus pyogenes, β-hemolytic Streptococcus spp.

The prototype of the claimed medium was the nutrient medium according to Katosova L.K., where the basis is common - dry nutrient agar and the addition of red blood cells. And the distinguishing features are: adding to our proposed environment of cattle serum and yeast extract instead of 10% inactivated horse serum. The prototype - one should be for the preparation of the claims, perhaps the closest analogue (prototype) is a nutrient medium according to the formulation of Katosova L.K. And if you agree with this, then please indicate the Common features and Distinctive features of your environment and the environment of the prototype. And also need a prototype for the method.

To determine the hemolytic activity of bacteria, a number of authors recommend heart-drawn agar (with the addition of blood, HiMedia, Heart Infusion Agar) [11], which is used to isolate and cultivate a large number of various microorganisms, in particular, as a basis for the preparation of blood agar with 5% defibrated blood of a rabbit, horse or ram to determine the hemolytic activity of S. pneumoniae, S. pyogenes. Composition, g / l, includes:

Beef heart hood 500.0

Tryptose 10.0

Sodium Chloride 5.0

Agar 15.0

pH 7.4 ± 0.2

Another nutrient medium for detecting the hemolytic activity of demanding microorganisms is also known - the basis of blood agar No. 2 (HiMedia, Blood Agar No. 2) [11]. Composition, g / l: proteose peptone 15.0; hepatic extract 2.5; yeast extract 5.0; sodium chloride 5.0; agar 15.0; pH 7.4 ± 0.2. After sterilization by autoclaving for 15 minutes at a temperature of 121 ° C, it is cooled to 45-50 ° C and 7% sterile blood of a ram, rabbit, horse or human is added sterile (without preservatives).

A number of media for isolation and cultivation of pneumococcus are known, such as tryptic meat digest according to Hottinger [11], which contains: 70-75% meat hydrolyzate according to Hottinger or casein (1.8-2.0 g / l of amine nitrogen); 20-25% bovine heart hydrolyzate (1.4-1.5 g / l amine nitrogen); 1.7-2.0% agar; distilled water 1000.0 ml; pH 7.6 ± 0.1. This base is sterilized at 121 ° C for 20 minutes. Before use, 0.5% feed yeast extract (together with serum or blood) is added to the medium melted and cooled to 45 ° C.

The disadvantages of the above nutrient media for determining the hemolytic activity of bacteria, isolation and cultivation of pneumococcus include their high cost and the use of scarce components in the preparation.

The technical result is to increase the growth properties of the nutrient medium while reducing its cost, as well as to ensure the availability of its preparation for practical laboratories of clinical microbiology. Due to the fact that the most common bacterial pathogens of inflammatory diseases of the respiratory system and ENT organs of a community-acquired etiology are Streptococcus pneumoniae, Moraxella (Branchamella) catarrhalis, Streptococcus pyogenes, β-hemolytic Streptococcus spp., Staphylococcus aureus, Haemophilus 4 defluen 8 , 14], we propose the following nutrient medium, blood serum agar, for the isolation, cultivation, and determination of the hemolytic properties of bacteria from clinical material.

The specified technical result is achieved by the fact that the following is proposed.

Nutrient medium for isolation, cultivation and determination of the hemolytic properties of bacteria from clinical material, containing GMF - agar, erythrocyte mass from human blood, cattle serum and yeast extract in the following quantitative ratio of components:

GMF - Agar - 100 ml erythrocyte mass from human blood - 3 ml cattle serum - 5 ml yeast extract - 3 ml

A method of preparing a nutrient medium for the isolation, cultivation and determination of hemolytic properties of bacteria from clinical material includes 3 stages:

1) stage - preparation of yeast extract: 1 kg of baker's yeast is dissolved in 2 liters of distilled water, boiled for 1 hour after boiling. Then it is necessary to pour 1 liter in glass volumetric flasks, cover with a napkin and let stand at room temperature until morning. The next day, the supernatant is transferred using sterile pipettes into 100 ml sterile volumetric bottles with a cotton-gauze plug (a total of 1.5 l of yeast extract is obtained) and sterilized by autoclaving at 110.8 ° C (0.5 kg / cm ² [ 6]) 30 min. After cooling completely, the bottles with yeast extract can be stored in the refrigerator + 2- + 8 ° С for up to 1 month.

The prototype of the method of preparing the yeast extract was the method described in the book "Enterobacteria", 1985, edited by V. Pokrovsky. [12]. Ingredients: 1. Pressed bread yeast (preferably uterine) - 0.5 kg. 2. Distilled water - 1000 ml. The yeast is crushed and boiled over low heat for 1 hour, stirred again, poured into bottles, sterilized at 121 ° C for 30 minutes. The finished extract is placed in the refrigerator for 5-7 days. For the preparation of media using a supernatant. Canned by adding 0.5% chloroform. Canned extract can be stored at 4-10 ° C for several months.

Common to both methods is the dissolution of baker's yeast in distilled water and boiling for 1 h; supernatant is used to prepare further nutrient media.

Distinctive features of our proposed method is: 1) after boiling, the finished extract is poured into volumetric flasks and allowed to stand at room temperature, after which the supernatant is poured into sterile volumetric vials - this is convenient from a practical point of view, since there is no possibility of sediment from adding yeast extract into culture media; 2) autoclaving sterilization at 110.8 ° C (0.5 kg / cm ² [6]) for 30 minutes, as a result of which the harmful effect of the temperature factor on nutrients contained in the yeast extract is minimized; 3) we do not add a preservative - 0.5% chloroform, as a result of which the harmful effect of the preservative on the nutrients contained in the yeast extract is minimized, and subsequently there is no inhibitory effect on the growth of microorganisms on the nutrient medium with the addition of yeast extract.

2) stage - preparation of a base from dry nutrient agar for the cultivation of microorganisms, pH 7.4-7.6 (GMF - agar, composition: enzymatic meat hydrolyzate 15.0 g; sodium chloride 9.0 g; agar 12.0 g Ready-made dry medium, manufacturer of Scientific Research Center of Pharmacotherapy, St. Petersburg), according to the recipe indicated on the label: 36.0 g of GMF agar are mixed in 1 liter of distilled water, boiled for 1-2 minutes until the agar is completely melted. Filter through a cotton-gauze filter, pour into sterile volumetric bottles and sterilize by autoclaving at 121 ° C (1.1 kg / cm ² ) for 15 minutes. The medium is cooled to 45-50 ° C, poured into sterile 100 ml volumetric bottles with a cotton-gauze plug, the pH of the base is 7.4-7.6. After complete cooling, the bottles with yeast extract are stored in the refrigerator + 2- + 8 ° C for up to 1 month.

3) stage - the direct preparation of a nutrient medium for isolation, cultivation and determination of the hemolytic properties of bacteria from clinical material: melt 100 ml of the base in a water bath, then add to the cooled base to 45 ° C, in a sterile box, 5 ml of cattle serum , 3 ml of red blood cells from human blood and 3 ml of yeast extract. The mixture is thoroughly mixed so that foam does not form. Then pour the medium (KSA) with a sterile pipette of 20 ml into sterile Petri dishes (agar thickness should be 3-4 mm). The agar layer should be uniformly colored red. Allow the medium to solidify and store the finished medium in a refrigerator + 2- + 8 ° C for 2 weeks in bags (to prevent drying out).

The quantitative ratio of the components of the nutrient medium and its pH ensure the sensitivity of the medium.

The increase in the growth properties of the nutrient medium is due to the presence of 1) yeast extract, which is a source of carbon and nitrogen with high nutritional value, a source of B vitamins, vitamin D and other growth factors - purine and pyrimidine bases. The yeast protein is balanced in amino acid composition and is close to animal protein in this indicator. It is characterized by a high content of lysine, leucine, isoleucine, aspartic and glutamic acids, as well as essential amino acids - arginine, histidine, tryptophan, tyrosine, cysteine, phenylalanine, methionine, threonine [3, 8]; 2) cattle serum, necessary for the growth of Streptococcus pneumoniae and the manifestation of typical morphology and cultural properties: delicate translucent, clearly defined colonies with a diameter of about 1 mm or they can be flat with a depression in the center. In addition, serum stimulates the formation of Streptococcus pneumoniae capsules [5]; 3) the addition of erythrocyte mass, which, firstly, has diagnostic value - determining the hemolytic properties of the microorganism (α, β, γ-hemolysis), and secondly, ensures the effective growth of Streptococcus pneumoniae, Streptococcus pyogenes, β-hemolytic Streptococcus spp., they do not have cytochrome and utilize atmospheric oxygen using a flavoprotein system, resulting in the formation of hydrogen peroxide, which microorganisms are not able to neutralize on their own, since they do not possess catalase [2].

Thus, the obtained data showed that the proposed environment and the method of its preparation allow us to identify the main bronchopulmonary pathogens using the bacteriological method even with a small amount of the pathogen in the pathological material. The proposed environment has better growth properties than in the currently used environments, in addition, the availability of the components of its production does not require scarce preparations, such as horse inactivated serum or defibrinated lamb, horse or rabbit blood, etc. The simplicity of the preparation of the claimed medium allows its use for isolation, cultivation and determination of hemolytic properties of bacteria, including bronchopulmonary pathogens, such as Streptococcus pneumoniae, Moraxella (Branchamella) catarrhalis, Streptococcus pyogenes, β-hemolytic Streptococcus spp., Staphylococcus aureus from clinical material in the laboratory for the diagnosis of inflammatory diseases.

The authors conducted comparative studies of the inventive medium of blood serum agar (nutrient agar for the cultivation of microorganisms, pH 7.4-7.6 (GMF - agar) - 100 ml + red blood cell mass from human blood - 3 ml + cattle serum - 5 ml + yeast extract - 3 ml), nutrient medium according to the formulation Katosova L.K. (100 ml of the base prepared from dry nutrient agar according to the recipe indicated on the label, pH 7.2 ± 0.2 + 10 ml of inactivated horse serum + 3 ml of erythrocyte mass) and 5% blood agar according to order No. 535 (100 ml of the base - 2% agar, pH 7.4-7.6, prepared from dry nutritious agar according to the recipe indicated on the label, + 5 ml of blood of defibrinated blood).

For biological control of culture media, a culture of Streptococcus pneumoniae ATCC 49619 and clinical material from patients with inflammatory diseases of the respiratory system and ENT organs (sputum, posterior pharyngeal and nasopharyngeal smears, bronchoalveolar lavage, tracheal aspirate, punctured nasal sinuses and pleural cavity, obtained from tympanum were used). middle ear from sick children). All crops were cultured at a temperature of 37 ° C in a normal atmosphere for 24-48 hours (up to 48 hours if no microorganism growth was detected during inoculation of clinical material from the patient on the first day).

To determine the sensitivity of the media, a microbial suspension was prepared from a daily culture of Streptococcus pneumoniae ATCC 49619, corresponding to a density of 0.5 according to the McFarland standard (containing approximately 1.5 × 10 ⁸ CFU / ml), a series of serial dilutions 1 was prepared from the obtained microbial suspension: 10. Then, 0.1 ml of a suspension of -5, -6, -7 dilution containing 1 × 10 ³ , 1 × 10 ² , 1 × 10 ¹ CFU / ml, respectively, were sown on prepared plates with the corresponding agar. With good nutritional properties of the medium, growth of microorganisms from -6, -7 dilutions should be noted [7].

The research results showed that the proposed composition of the nutrient medium has high sensitivity - isolation of the minimum amount of Streptococcus pneumoniae ATCC 49619 (up to 10 colonies when plated from -7 dilution), and the proposed method for preparing a complete medium in a practical laboratory is simple and affordable for laboratories of any level .

In addition, in the study of 20 sputum samples and 496 samples of bronchoalveolar lavage from sick children with chronic infectious and inflammatory diseases (HIVZL) in the period from January 2005 to April 2011. S. pneumoniae and M. catarrhalis were isolated in 18.8% and 10.6% of cases, respectively, on the claimed nutrient medium, which can be used for diagnostic purposes. In 49.1% of cases with chronic obstructive pulmonary obstruction, Haemophilus influenzae was isolated on chocolate agar, and its growth was noted on the inventive medium on the 2nd day of incubation at 37 ° C, the usual atmosphere in the form of point colonies without hemolysis.

When examining the posterior pharyngeal and nasopharyngeal smears from children on the claimed medium, Neisseria meningitidis and in one case Corynebacterium diphtheriae were also isolated.

The cost of the claimed medium is 680 rubles / liter.

Literature

1. Isolation, identification and determination of sensitivity to Streptococcus pneumonia antibiotics / L. S. Strachunsky, O. I. Krechikova, R. S. Kozlov, T. M. Bogdanovich, O. U. Stetsyuk, M. M. Suvorov, L. .K.Katosova, L.A. Vishnyakova, M.E. Faustova // Clinical Microbiology and Antimicrobial Chemotherapy. - 2000. - T.2, No. 1. S.88-98.

2. Kozlov R.S. Pneumococci: past, present and future. Smolensk: Smolensk State Medical Academy, 2005. - 128 p.

3. Kozlov Yu.A. Nutrient media in medical microbiology. Guidelines for the production of culture media for microbiological institutes and sanitary-bacteriological laboratories. State Publishing House of Medical Literature MEDGIZ, 1950, Moscow. - 252 p.

4. Murray P.R., Shay I.R. Clinical Microbiology. Quick Guide: Per. from English M .: Mir, 2006 .-- 425 p.

5. Medical microbiology. Edited by V.I. Pokrovsky. - 3rd ed. M .: GEOTAR-Media, 2005 .-- 768 p.

6. Guidelines for monitoring the operation of steam and air sterilizers, approved. Ministry of Health of the USSR of 28.02.91, N 15 / 6-5.

7. MUK 4.2.1890-04 "Determination of the sensitivity of microorganisms to antibacterial drugs." - M.: Federal Center for Sanitary Inspection of the Ministry of Health of Russia, 2004. - 91 p.

8. Polyak M.S., Sukharevich V.I., Sukharevich M.E. Nutrient media for medical and sanitary microbiology. SPb .: ELBI-SPb. - 2008 .-- 352 p.

9. Order of the Ministry of Health of the USSR No. 535 dated April 22, 1985 “On the Unification of Microbiological (Bacteriological) Research Methods Used in Clinical and Diagnostic Laboratories of Medical Institutions”

10. Pulmonology of childhood: problems and solutions. Ed. Misernitsky Yu.L., Tsaregorodtseva A.D. Issue 4. Moscow, 2004. - 257 p.

11. Private medical microbiology with the technique of microbiological research: a training manual. Ed. Labinskaya A.S., Blinkova L.P., Yeschina A.S. - M .: OJSC "Publishing house" Medicine ", 2005. - 600 p.

12. Enterobacteria: A guide for doctors / I.V. Golubeva, V.A. Kileso, B. S. Kiseleva and others. Ed. V.I. Pokrovsky. - M .: Medicine, 1985 .-- 321 p.

13. Essential procedures for clinical microbiology. Editor in chief, Henry D. Isenberg. Washington, DC 20005, 1998. 841 P.

14. Manual of clinical microbiology. Editor in chief, Patrick R. Murray. 7 ^th ed. Washington, DC 20005, 1999. 1773 P.

Claims (1)

Nutrient medium for isolation, cultivation and determination of the hemolytic properties of bacteria from clinical material, containing GMF agar, erythrocyte mass from human blood, cattle serum and yeast extract in the following quantitative ratio of components, ml:
GMF agar one hundred erythrocyte mass from human blood 3 cattle serum 5 yeast extract 3