RU2360969C1 - Method of erythrocyte analysis for bacteria fixing activity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: mixed suspensions of microbes and erythrocytes are incubated at 37-39°C on a shake-flask propagator during 30 mines, centrifuged to erythrocyte sedimentation and with using photoelectrocolorimeter tested for optical density of the supernatant fluid. bacteria fixing activity of erythrocytes - "БФАЭ" (%) is derived from the difference between D_C (optical density of the supernatant fluid in control system) and total D_T (optical density of the supernatant fluid in test system) and D_T* (optical density of the supernatant fluid in the system containing erythrocyte suspension and physiological solution of sodium chloride instead of microbial suspension and being in the same environment) divided by D_C and multiplied by 100 %.

EFFECT: application of the method allows for erythrocyte analysis for their ability to fix on their surface various microorganisms, including agents of bacterial infections and makes it possible to study adhesive properties of microbes.

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Description

The invention relates to medical and veterinary microbiology and can be used to study the properties of red blood cells to fix various microorganisms on their surface, including pathogens of bacterial infections of humans and farm animals, which can contribute to the study of the pathogenesis of a number of bacterial diseases.

Much attention in the study of the pathogenesis of infectious diseases is given to the study of the adhesive properties of microbes in relation to macroorganism cells [1, 3]. There are various methods for determining the fixing properties of microbes to red blood cells. However, they have some disadvantages, such as the complexity of research, a limited number of studied strains of microorganisms, lack of information, and sometimes the need to use complex and expensive equipment [3].

There is a method of simultaneously determining the adhesion of microbial colonies of primary culture against red blood cells [2], which consists in the fact that isolated colonies of primary culture grown on a solid nutrient medium, apply 3-5% suspension of red blood cells and then after 2-5 minutes count the number of colonies around which a halo forms of red blood cells attached to their edges. Such colonies are considered adhesive-active.

The method makes it possible to study the adhesion of only individual isolated colonies selected by the researcher, which significantly devalues the results. In addition, using this method, it is possible to observe only the movement of red blood cells to the colonies and their fixation along their edges, and it is rather difficult to talk about direct fixation of microbes to red blood cells.

There is a method for determining the adhesive properties of microbial cells [1], which consists in the fact that 0.5 ml of microbial and erythrocyte suspensions at concentrations of 10 ⁹ cells / ml and 100 million / ml, respectively, are added to the test tube. The mixtures are incubated at 37 ° C on a shaker for 30 minutes, after which a smear is prepared on a glass slide, it is dried, stained and examined under a microscope. Adhesive properties are evaluated by 3 indicators: average adhesion index (SPA), adhesion coefficient (CA), microorganism adhesion index (IAM). In this case, SPA is the average number of microbes attached to 1 red blood cell, when counting at least 25 red blood cells. CA is the percentage of red blood cells that have fixed microbes on their surface. And IAM is the average number of microbial cells in one erythrocyte participating in the adhesive process.

The disadvantages of this method include the complexity and relative subjectivity when counting red blood cells and microbes on their surface, as well as a limited number of red blood cells studied. In addition, with smear microscopy, it is quite difficult to separate the fixation of microbes on red blood cells and their spontaneous location near them.

The essence of the proposed method lies in the fact that to determine the fixing activity of red blood cells against microbes, the mixture is first incubated with a suspension of microbes and red blood cells in a test tube at 37-39 ° C on a shaker for 30 minutes, and then, unlike the prototype, the mixture is centrifuged until erythrocyte sedimentation, after which the optical density (OD) of the supernatant in a test tube is determined using a photoelectrocolorimeter.

For control, the OD of the supernatant is determined in a test tube containing a microbial suspension and saline of sodium chloride instead of a suspension of red blood cells and under the same conditions.

In the case of fixing a certain number of microbes on red blood cells, the OD of the supernatant in the experimental system in comparison with the control after centrifugation is reduced.

The bacteriofixing activity of red blood cells was proposed to be calculated by the formula

,

,

- ОП надосадочной жидкости системы, содержащей взвесь эритроцитов и физраствор хлорида натрия вместо микробной взвеси, и находящейся в тех же условиях.where BFAA% is the bacteriofixing activity of red blood cells, D _to is the OD of the supernatant in the control system, D _about is the OD of the supernatant in the experimental system,

- OD of the supernatant of a system containing a suspension of red blood cells and saline solution of sodium chloride instead of microbial suspension, and under the same conditions.

ввели в формулу для более точного рассчета БФАЭ, чтобы устранить влияние частичного гемолиза эритроцитов при манипуляциях с ними на результаты измерения ОП.Magnitude

introduced into the formula for a more accurate calculation of BPAE to eliminate the effect of partial hemolysis of red blood cells during manipulations with them on the results of measuring OD.

For the experiment, the plague microbe vaccine strain EV and the museum strain Staph.epidermidis culture were used, which were grown for 24 hours in Petri dishes on meat peptone agar (MPA). Using a physiological solution of sodium chloride, washings of grown cultures from the surface of the media were made. The large suspended particles contained in the resulting suspensions were released by centrifugation at 1000 rpm for 1 min to precipitate them. Then the optical density (OD) of microbial suspensions was adjusted to 1.0 units. extinction (to standardize experience). To measure the OD, we used a KFK-2 instrument, a green filter, cuvettes with a working distance between the faces of 5 mm, and a physiological solution of sodium chloride as a suspension solvent.

In the experimental system, 1 ml of a suspension of human erythrocytes, washed three times with physiological saline, at a concentration of 0.6-0.7 billion / ml and 2.5 ml of a suspension of a microbial culture with an OD of 1 unit were introduced into siliconized tubes. The tubes were kept for 30 min in a shaker at a temperature of 37-39 ° C. A 358 S type laboratory shaker was used with a maximum amplitude (25 mm) and a minimum vibration frequency (50 cycles / min). The tubes were then centrifuged at 1000 rpm to precipitate red blood cells and the absorbance of the supernatant was measured. Samples with 1 ml of saline and 2.5 ml of suspension of microbes under similar conditions served as a control.

The results of bacterial fixation to erythrocytes were taken into account by the difference in the measurement of the optical density (OD) of the supernatant in test tubes of the experimental and control systems (table 1).

Table 1
The measurement results of the OD of the supernatant in the experimental and control systems, units extinction, (n = 6) Microbe type OD of the supernatant in the experimental system OD supernatant in the control system Plague Microbe Vaccine Strain EV 0.33 ± 0.05 0.65 ± 0.05 Staph.epidermidis 0.79 ± 0.06 0.57 ± 0.06

This table shows that in the experimental system containing a suspension of the vaccine strain of the plague microbe, there was a decrease in the OD of the supernatant after centrifugation compared with the control system, which indicated the fixation of microbes on red blood cells. In tubes with staphylococcus, there was no decrease in OD in the experimental system compared to the control, i.e. fixation was not observed.

) предложили учитывать при расчете БФАЭ.It was found that in the experimental system during the experiment, insignificant hemolysis of erythrocytes occurs, which affects the result of measuring the OD of the supernatant. During research, 1 ml of a suspension of erythrocytes and 2.5 ml of saline solution were added to 1-2 tubes. They measured the OD of the supernatant in them after cultivation in a shaker and centrifugation and found that it differs from the OD of saline by 0.05-0.13 units. extinction. Given value (

) proposed to take into account when calculating BFAE.

In the next series of experiments, a comparative assessment of human BPAE was carried out in relation to the vaccine strain EV and several museum strains of saprophytic microbes. MPA was used as a nutrient medium; MPA with the addition of gentian violet was used to grow the plague microbe of the vaccine strain. Using the data obtained, according to the proposed formula, human BPAE was determined in relation to these microbes (table 2).

table 2
Determination of BPAE for various microorganisms, (n = 5) Microbe type BFAE,% Plague Microbe Vaccine Strain EV 50 ± 16.4 Ps.aeruginosa 12 ± 4.3 E.coli 9.2 ± 5.6 Pr.vulgaris - Staph.epidermidis -

For a more objective characterization of BFAE in relation to various microbes, a rating scale has been introduced, according to which BFAE less than 10% is considered weak, 11-20% - medium, 21-40% - high, 41% and more - very high.

BFAE was identified as very high with respect to strain EV, moderate with respect to Ps.aeruginosa, and weak with E. coli. For two museum strains of two types of microbes - Pr.vulgaris and Staph.epidermidis, BFAE did not appear.

It was found that BPAE in relation to the same microbes repeatedly passaged through nutrient media sharply decreases, in addition, the composition of a dense nutrient medium for growing the microbe has a significant effect on the manifestation of the adhesive properties of red blood cells in the microbe. For this, an experiment was carried out in which fresh cultures of the vaccine strain EV of the plague microbe were taken, taken from new bottles with the vaccine and grown for 24 hours on MPA and Hottinger digest, and cultures of the same microbe passaged three times through solid nutrient media (MPA). Gentian violet was added to the composition of media for suppressing extraneous microflora. The results of the determination of BFAE in the experiment are presented in table 3.

Table 3
The influence of the composition of the nutrient medium and the number of passages on BFAE in relation to the plague microbe vaccine strain, (n = 6). Differences in obtaining microbial culture BFAE,% Growing on the Hottinger digest after a single pass of re-passage from a fresh bottle with a vaccine 81 ± 8.2 Growing on MPA after a single reseeding from a fresh vaccine vial 54.8 ± 7.54 Growing on MPA after 3-fold passage 35.3 ± 19.7

The table shows that the more optimal growth medium for the manifestation of a vaccine strain of the plague microbe adhesive properties in relation to human red blood cells is Hottinger digest. When cultivating this microbe on MPA, this ability is manifested to a lesser extent, and with repeated passage through a dense nutrient medium is significantly reduced.

Thus, the proposed method for determining BPAE allows one to study the fixing properties of red blood cells in relation to various microorganisms, including bacterial pathogens, to assess their severity, to identify conditions that affect the fixation of microbes on red blood cells. This makes it possible to obtain new, additional information about the nature of the interaction of microbial cells with human red blood cells. The proposed method for determining BPAE does not require the use of complex and expensive equipment, is sensitive enough, and also simple to perform. It allows, unlike the prototype, to simultaneously establish the interaction of millions of red blood cells with tens of millions of bacteria. With its help, it is possible to study the adhesive properties of a wide range of microbes and reveal even their slight changes.

Literature

1. Brilis V.I. The methodology for studying the adhesive process of microorganisms. / V.I. Brilis, T.A. Brilene, H.P. Lenzner, A.A. Lenzner // Laboratory. - 1986. - No. 4. - S.210-212. - prototype.

2. Gizatulina S.S. A method for assessing the state of human intestinal microflora by the number of adhesive-active colonies and the type of adhesins. / S.S. Gizatulina, M.O. Birger, L.I. Kulinich and others // Journal of Microbiology, Epidemiology and Immunology. - 1991. - No. 4a. - S.21-23.

3. Kulikovsky A.V. Change in the adhesive ability of microorganisms. / A.V. Kulikovsky, I. B. Pavlova // Veterinary medicine. - 1993. - No. 7. - p.22.

Claims (1)

A method for determining the bacteriofixing activity of erythrocytes (BFAE), including incubating a mixture of suspended microbes and erythrocytes at 37-39 ° C on a shaker for 30 minutes, characterized in that the mixture is subsequently centrifuged until the erythrocytes precipitate, and then the optical density is determined using a photoelectric colorimeter liquid mixture according to the formula

,
where BFAE (%) is the bacteriofixing activity of red blood cells;
D _to - the optical density of the supernatant in the control system;
D _about - the optical density of the supernatant in the experimental system;

- the optical density of the supernatant of the system containing a suspension of red blood cells and saline sodium chloride instead of microbial suspension and under the same conditions.