RU2619834C2 - Method research on feces dysbacteriosis of the intestine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: smear for study is collected on tampon by dipping it into feces alternately in several places. Then, tampon is immersed in a test tube with Cary Blair transport medium in the amount of 2 ml of Fecal Swab, Copan, Italy transport system and is shaked until a homogeneous compound is obtained. True weight of feces in the test tube (X) is determined by subtracting the weight of tubes with tampon from the weight of those test tubes with the sample, the degree of feces dilution (N) is calculated according to the formula N=2/X+1. Then, sample solutions are prepared in dilutions of 10^-2 to 10^-9, inoculations are made for various liquid and dense nutrient mediums, are incubated at a temperature of 35…37°C for 24-48 hours, and the number of bacteria colonies (Y) grown is counted, the results are quantified on 1 gram of feces according to the formula: Z=Y*10*N*M, where Z is the quantitative evaluation of one type of bacteria on 1 gram of studies material, Y is the number of bacteria grown, 10 is the conversion factor from the inoculating dose calculation of 100 μl, N is the dilution rate of feces in liquid transport medium, and M is the dilution rate in test tube from which the bacterial count test is taken.

EFFECT: use of method makes it possible to increase the accuracy of study for detection of dysbiosis in human intestine by increasing the preanalytical stage to 48 hours.

2 tbl, 1 dwg

Description

The invention relates to the field of microbiology, namely to bacteriological laboratory research.

Persons belonging to risk groups for the development of intestinal dysbiosis are subject to examination:

1. Newborns born prematurely; who underwent resuscitation at birth; Stayed in a maternity hospital for a long time; with late attachment to the chest; from mothers who had purulent-inflammatory diseases during pregnancy; with signs of primary immunodeficiency.

2. Infants and young children in cases of adverse course of the neonatal period, early artificial feeding, with signs of dyspeptic disorders, frequent acute respiratory infections, malnutrition, anemia, rickets, allergic reactions.

3. Children of other ages in the presence of allergic reactions, frequent acute respiratory infections, reduced immunity.

4. Adults undergoing long-term hormone therapy or antimicrobial treatment; persons with unbalanced nutrition, occupational hazards, suffering from long-term intestinal infections.

A limitation of the application of the procedure is: the use of antimicrobial agents, the use of rectal suppositories, R-studies using barium, the study of frozen samples, samples without a transport medium.

A known method for the diagnosis of dysbiosis of the colon, including the study of feces by direct electrochemical method using a pH meter (RU 2324191 C2, 05/10/2008).

A well-known method of bacteriological research, including grinding a sample of feces and diluting it in physiological saline, preparing dilutions and culture on nutrient media with the number and percentage of lactose-negative (colorless) colonies in relation to the total number of colonies grown (Bacteriological diagnosis of intestinal dysbiosis. Guidelines (approved) .Ministry of Health of the RSFSR 04/14/1977)).

The disadvantages of the known methods and techniques are: short delivery time of the collected sample to the laboratory for research, low viability of bacteria during the period of delivery and storage of the sample in a conventional container, which negatively affects the accuracy of the results of bacteriological studies.

The proposed invention eliminates the above disadvantages.

The aim of the invention is:

- ensuring the safety of bacteria detected in the study on intestinal dysbiosis, using a modified medium of Cair Blair in the Fecal Swab transport system (Copan, Italy);

- providing surveyed people from remote regions of Russia with a new laboratory service in order to improve the quality of their examination.

The technical result of the invention is to increase the accuracy of the study to detect intestinal dysbiosis in the subject, achieved by: conducting research at a higher level with an increase in the preanalytical stage to 48 hours; excellent preservation of the viability of bacteria during the delivery of a sample of feces for research in the specified transport system and the conversion of the number of microorganisms per 1 gram of feces according to the above formula, taking into account the dilution of feces in the transport system.

The specified technical result is achieved due to the fact that a smear collected on a tampon by immersion in feces alternately in several places and placed in a test tube with modified Saga Blair medium of the Fecal Swab transport system, Copan, Italy, which increases the duration of the preanalytical stage, is used for the study; determine the true weight of feces in the test tube (X) by subtracting the weight of the tubes with the swab of the indicated transport system from the weight of these test tubes with the sample, calculate the degree of dilution of feces (N) in 2 ml of the liquid transport medium as N = 2 / X + 1, before examining the sample mix thoroughly and prepare sample solutions in dilutions from 10 ^-2 to 10 ^-9 in separate tubes, after preparing the dilutions, inoculate solid and liquid nutrient media, then evaluate the results and count the number of grown bacteria (Y), then a qualitative record of the results per 1 gram of feces (Z) according to the formula:

Z = Y * 10 * N * M,

where Ζ is a quantitative assessment of the type of bacteria per 1 gram of the test material,

Y is the number of bacteria grown

10 - conversion factor based on the inoculum dose of 100 μl,

N is the degree of dilution of feces in a liquid transport medium,

M - the degree of dilution in vitro, from which a sample was taken to count bacteria.

The investigated material is feces obtained naturally. For the study according to the invention, a swab collected on a tampon is used, which is part of the transport system of Fecal Swab, Copan, Italy, with 2 ml of modified CaryBlair medium.

In FIG. 1 shows a stool sample dilution scheme. Sample preparation for research.

Feces are collected in a disposable plastic container. Immediately after collection, open the packaging from the Fecal Swab transport system (cat. No. 470CE) and remove the applicator with the swab. The swab is immersed in feces alternately in several places, after which it is placed in a test tube with 2 ml of Cary Blair modified transport medium. The excess applicator breaks off on a conditional line. The cap is tightly screwed. Pieces of feces are not placed in a test tube. The sample is labeled.

Storage and transportation of feces sample is carried out until 48 hours at a temperature of + 2 ... + 8 ° C.

Method description

Materials and equipment

1. Equipment

2. Culture media

3. Reagents

4. Consumables

Equipment

1. Fume hood, Russia;

2. Vortex, ELMI Sky Line V3, Latvia;

3. Analytical balance with measurement accuracy up to 0.01, Scout Pro, OHAUS, USA;

4. Water thermostat, ULTRATHERM, BWT-U, BIOSAN, Latvia;

5. Looper, BACTI-CINERATOR, McCornick Scientific, USA;

6. Incubator with a normal atm., BINDER, Germany or a thermal room (to create the desired temperature conditions);

7. Incubator with CO ₂ atm., NUARE, USA;

8. MALDI-TOF mass spectrometer microflex LT company BRUKER, Germany;

9. Microscope, Axiostar, Carl Zeiss, Germany.

Culture media

1. Bifidum, dry medium, Obolensk, Russia, lot C242;

2. Sulfite agar for clostridia, modification 1, Obolensk, Russia;

3. Bismuth sulfite agar (ICA) d / exc. Salmon., / ICA, FGUN, Obolensk;

4. Levin medium with eosinmethylene blue, FGUN, Obolensk;

5. Selenite medium dry Obolensk, Russia, cat. 11724;

6. MRS / MPC, Scharlau, cat. No. 01-135;

7. Entrococcosel Agar / Enterococcosel, BD, Cat. No. 2112205;

8. MacConkey Agar / MacConkey Medium, MCV, Bio-Rad, Cat. No. 69084;

9. Columbia Agar base BD / Colombian agar supplemented with 5% mutton blood, cat. No. 211124;

10. Mannitol-Salt Agar (MSA) / Manitol-Salt Agar; BD, cat. No. 211407;

11. Sabouraud Chloramphenicol Agar / Agar Saburo with chloramphenicol, BioMerieux, cat. No. 51021;

12. Clostridium reinforced medium, BD, cat. 218081;

13. Kligler’s double-sugar agar with iron, Obolensk, Russia, cat. No. 17349;

14. Chromogenic agar for the identification of Candida albicans, HiMedia, cat. M1297;

15. Simple nutrient agar, Obolensk, Russia.

Reagents

1. Gram dyes, set BBL Becton Dickinson, USA;

2. Immersion oil, ApexLab, Russia;

3. Sodium chloride (NaCl) for saline solution, Russia;

4. Acetonitrile, Cat. No.221881.1611C, Panreac, Spain;

5. Trifluoroacetic acid, cat. No. 363317.1608, Panreac, Spain;

6. Matrix reagent, Bruker, Germany;

7. Deionized water obtained in laboratory conditions at the Median-Filter installation, Russia.

Disposable supplies

1. Plastic tubes;

2. Plastic petri dishes;

3. Tips with a filter of 5-200 μl; 50-1000 μl;

4. Slides, ApexLab;

5. Pasteur pipettes per 1 ml, art. 12006605, Minimed;

6. Spatulas, Copan, Italy, Cat. 174CS05.

Reusable Consumables

1. Glass test tubes;

2. Glass pipettes;

3. Test tube racks;

4. Corks for test tubes;

5. Automatic pipettes;

6. Microbiological loops calibrated or non-calibrated with holders. Preparatory procedures:

1. Weigh the weight of (1) swabs in the Fecal Swab, Copan, Italy transport system (without samples) in a series of 10 pcs. The average value is calculated, which is subsequently taken as a constant value.

2. Prepare tubes with 2.7 ml of sterile saline for the preparation of dilutions and label them: 10 ^-2 , 10 ^-3 , 10 ^-4 ... 10 ^-10 .

3. Disrupt liquid nutrient media (Bifidum, Sulphite agar) in 10 ml tubes.

4. Pour 4 ml of selenite broth into tubes.

Study progress:

Upon receipt of samples in test tubes with a transport medium, the weight (2) of each on an analytical balance is determined with an accuracy of up to 0.01 g.

The true weight of feces in vitro (X) is determined by subtracting from weight (2) weight (1):

X = 2-1.

Then calculate the degree of dilution of feces (N) in 2 ml of liquid transport medium Cary Blair according to the formula: N = 2 / X + 1.

Before starting the study, the sample is thoroughly mixed on a vortex for 15 seconds, with a rotation speed of 3 PRMX1000.

0.3 ml of the contents is taken from a test tube with Cary Blair medium using a sterile filter tip and an automatic pipette and transferred to a test tube with sterile saline solution and labeled 10 ^-2 . Mix thoroughly and 0.3 ml is taken from this tube for the next dilution of 10 ^-3 and up to 10 ^-9 (final dilution of 10 ^-10 is used for children under 1 year old). The dilution scheme is shown in FIG. one.

The working dilutions in the following description for use in the formulas will be indicated as (M). After preparing the dilutions, sowing on solid and liquid nutrient media is immediately done:

1. In order to detect pathogenic intestinal bacteria, the material is applied to the surface of Levin’s medium using a loop of 10 μl from a Fecal Swab tube, inoculation is done with a dashed zigzag over the entire surface of the medium.

2. On the surface of the MacConkey Agar medium, in order to detect gram-negative bacteria, an automatic pipette with a sterile tip with a filter is applied, 100 μl of 10 ^-5 and 10 ^-7 dilutions per Petri dish, inoculation is done with a spatula, evenly distributing the liquid over the entire surface until completely absorbed .

3. On the surface of the medium 5% blood agar in order to identify the hemolytic properties of bacteria (mainly E. coli), an automatic pipette with a sterile tip with a filter is applied 100 μl of ^10-5 dilutions, inoculation is done with a spatula, evenly distributing the liquid over the entire surface until completely absorbed .

4. On the surface of the MRS Agar medium, in order to detect lactobacilli, an automatic pipette with a sterile tip with a filter is applied, 100 μl of 10 ^-2 and 10 ^-4 dilutions per Petri dish, inoculation is done with a spatula, evenly distributing the liquid over the entire surface until completely absorbed.

5. On the surface of the Entrococcosel Agar medium, in order to detect enterococci, an automatic pipette with a sterile tip with a filter is applied 100 μl of a 10 ^-4 dilution, inoculation is done with a spatula, evenly distributing the liquid over the entire surface until completely absorbed.

6. On the surface of the Mannitol-Salt Agar medium, in order to detect staphylococci, an automatic pipette with a sterile tip with a filter is applied 100 μl of 10 ^-2 dilutions, inoculation is done with a spatula, evenly distributing the liquid over the entire surface until completely absorbed.

7. On the surface of the Sabouraud Chloramphenicol Agar medium, in order to detect fungi, an automatic pipette with a sterile tip with a filter is applied 100 μl of 10 ^-2 dilutions, inoculation is done with a spatula, evenly distributing the liquid over the entire surface until completely absorbed.

8. In test tubes with liquid media do deep plating using an automatic pipette with a sterile tip with a filter. Pipette 100 μl into each tube.

In order to detect bifidobacteria, the material from 10 ^-4 , 10 ^-6 , 10 ^-9 dilutions for the age group from 1 to 60 years, from 10 ^-6 , 10 ^-9 , 10 ^{-10, is} introduced into the depths of the Bifidum medium using an automatic pipette with a sterile tip with a filter dilutions for age groups up to 1 year, and more than 60 years.

Deep into the medium, Sulfite agar, in order to detect clostridia using an automatic pipette with a sterile tip with a filter, add material from dilutions 10 ^-3 and 10 ^-5 .

Deep into the medium, Selenite broth (an enrichment medium for better Salmonella growth) is introduced from a test tube containing Cary Blair medium.

чашки Петри. Инкубацию всех посевов проводят в течение 24-48 часов при температуре +35…+37°C в инкубаторе с обычной атмосферой или термальной комнате, за исключением чашек со средой MRS Agar, которые инкубируют в СО₂ инкубаторе.9. The next day, from a selenite broth, a loop of 10 μl is sown on Bismuth-sulfite agar in order to identify pathogenic intestinal bacteria (Salmonella). Sowing is done by dashed zigzag over the entire surface of the medium on

petri dishes. Incubation of all crops is carried out for 24-48 hours at a temperature of + 35 ... + 37 ° C in an incubator with a normal atmosphere or a thermal room, with the exception of plates with MRS Agar medium, which are incubated in a CO ₂ incubator.

At the end of the incubation, the bacteriologist evaluates the results and counts the number of bacteria grown (Y).

Quantitative accounting of the result per 1 gram of feces (Z) is calculated by the formula:

Z = Y * 10 * N * M,

 where Ζ - a quantitative assessment of the grown bacteria per 1 gram of the investigated material;

Y is the number of grown colonies on 1 Petri dish with a nutrient medium;

N - the main dilution of feces in vitro with Cary Blair medium;

M - the degree of dilution in vitro, from which the sample was taken for inoculation;

10 - conversion factor based on the inoculum dose of 100 μl.

All colonies of diagnostic value are identified on a MALDI-TOF microflex LT mass spectrometer from BRUKER, Germany, or using alternative methods using a Vitek 2 com analyzer manufactured by bioMerieux, France.

Pathogenic intestinal bacteria (salmonella, shigella) are typed using polyvalent diagnostic sera from the Microgen production association.

When pathogenic intestinal bacteria are detected, sensitivity in antimicrobials and bacteriophages is determined. Validation of the method is carried out in 2 stages.

1. Using control strains of bacteria in the laboratory collection (to assess the survival of the studied bacteria in the transport medium for 48 hours and storage at a temperature of + 2 ... 8 ° C).

2. Using stool samples received for the study of intestinal dysbiosis, by comparing the results obtained during the standard methodology, and in the experiment.

Stage 1

During the tests, daily bacterial cultures were used, listed in table 1.

Study progress:

1. Prepared daily culture of the following bacteria.

2. Prepared microbial suspension - inoculum - at a concentration of 0.5 units. by Mac Farland.

3. A series of 10-fold dilutions was prepared from the inoculum - 10 ^-7 , 10 ^-6 , 10 ^-5 , 10 ^-4 , 10 ^-3 (tubes with 4.5 ml of sterile saline solution were prepared on the day of the study), for each dilutions used a new tip, mixing was performed on a vortex.

4. 100 µl of dilutions 10 ^-4 and 10 ^-5 (in 3 replicas) were added to FecalSwab

5. The prepared samples were seeded with a loop of 10 μl exhausting method on Petri dishes with 5% blood agar (prepared on the basis of Columbia agar BD, lot 3232498, in the laboratory).

6. Crops were made on the day of preparation (without storage), after 24 and 48 hours (after storage in the refrigerator at + 2 ... + 8 ° С). Each time before sowing, all tubes were thoroughly mixed on a vortex.

7. Crops were incubated for 24-48 hours at 37 ° C (viewing every 24 hours, final recording of results after 48 hours).

8. The following results were obtained:

2 stage

The basis is a comparison of the results obtained with the standard performance of the study on intestinal dysbiosis, and obtained during the experiment from the same samples of feces.

The implementation of the method of a modified study of intestinal dysbiosis is described in detail on pages 3-5.

For statistical reliability, 5 stool samples were examined in three replicas (repeats) immediately after dilution, as well as after 24 and 48 hours. The techniques were carried out simultaneously and under the same conditions:

D - dysbiosis as standard;

E - experimental data.

During the research, to optimize the technique, corrective actions were taken:

1. Reinforced Clostridial Medium was replaced with Sulphite agar when clostridia was detected due to the impossibility of a reliable record of results.

2. Seeding on Wednesday McConkey made of 10 ^-7 and 10 ^-5 dilutions.

3. The sowing dose was increased from 10 μl to 100 μl and the seeds were made from the same dilutions as in the study on dysbiosis.

The study of the last three samples of feces was carried out under conditions close to performing a routine study on dysbiosis, and showed the best results by coincidence.

Performance

In a comparative study of two methods - the modified and standard, used in the daily practice of the laboratory, comparable results were obtained in a group of 5 samples, when setting the method in three replicas and repeats after 0, 24, 48 hours, which allowed us to conclude that the implementation of this method is acceptable into the lab’s working practice.

The invention provides:

1. The longer viability of the bacteria studied in the study on intestinal dysbiosis when using the transport medium Fecal Swab, Copan, Italy compared with the biomaterial (feces) collected in a regular container. Up to 48 hours from the time of capture to the start of the study.

2. The ability to receive material for research at various times of the day, during the entire working time of the laboratory (removing delivery restrictions in a short time).

3. The ability to receive material from remote regions of Russia, where there are no well-equipped laboratories with established transport logistics. Improving the quality of the provision of diagnostic laboratory assistance to the population from remote regions of the country.

4. Improving the processing conditions of the material (easier homogenization during primary processing, etc.) and conducting research at a higher level.

Claims (1)

A method of examining human feces for intestinal dysbiosis, which consists in collecting a smear for examination on a tampon by immersing it in feces in turns at several places, immersing the tampon in a test tube with Cary Blair transport medium in an amount of 2 ml of the Fecal Swab transport system, Copan, Italy shake until a homogeneous composition is obtained, determine the true weight of feces in a test tube (X) by subtracting the weight of test tubes with a swab from the weight of these test tubes with a sample, calculate the degree of dilution of feces (N) using the formula N = 2 / X + 1, then prepare a solution sample dilutions of 10 ^-2 to 10 ^-9 make crops on various liquid and solid nutrient media are incubated at a temperature of +35 ... + 37 ^{° C} for 24-48 hours, is carried out to count the number of colonies of bacteria (Y), is carried out quantitative accounting of results per 1 gram of feces according to the formula: Z = Y * 10 * N * M, where Z is the quantitative assessment of one type of bacteria per 1 gram of the studied material, Y is the number of bacteria grown, 10 is the conversion factor from the calculation of the inoculative dose of 100 μl , N - the degree of dilution of feces in a liquid transport medium, M - the degree of dilution in samples ERC, from which a sample is taken for counting bacteria.

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