RU2113467C1 - Method of bacterium identification - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: method involves determination of photoelectrocolorimetric curve of process of diffusional distribution of bacterial cells in concentration gradient. Bacterium species is identified by gradient concentration disappearance time. Method can be used in laboratory diagnosis of infectious diseases and microbiological investigation of environment. EFFECT: improved method of identification. 1 tbl, 2 dwg

Description

 The invention can be used in microbiological practice, and specifically in laboratory diagnosis of infectious diseases, microbiological examination of environmental objects.

 A known method of biochemical identification of bacteria (Handbook of microbiological and virological research methods. / Ed. Birger M. O., M., 1982), which consists in studying the enzymatic properties of microbes (the ability to ferment carbohydrates and break down protein products). To detect these properties of bacteria, they are sown on differential diagnostic media (liquid and solid) containing substances against which microbes are able to show their enzymatic activity. The basis of such media is the Gissa medium - 1% peptone water, to which an indicator and the substance that should decompose the enzymes of the studied microbe are added. Such substances are carbohydrates (glucose, maltose, mannitol, lactose, sucrose, xylose, arabinose, dulcite, rhamnose and others), deoxyribonucleic acid (DNA), amino acids, urea and other substances. Changes in the reaction of the medium resulting from the activity of the enzyme systems of microbes are expressed in a change in the color of the medium after 24 hours of cultivation of crops.

The disadvantages of this method include:
- a large duration of research, up to 24 hours;
- a large range of drugs used in the study;
- the high cost of materials used in the study;
- great complexity.

 A known method of serological identification of bacteria (Handbook of microbiological and virological methods of research. / Under the editorship of Birger M.O.M., 1982). It is carried out with a pure culture and diagnostic sera in agglutination, precipitation and other reactions. However, this method of identifying bacteria is very laborious and expensive, since it requires the manufacture of a large number of diagnostic sera of various serological variants. In addition, serological identification is not acceptable for microorganisms such as staphylococci due to the abundance of its serovariants.

 There is a method of phage identification of bacteria (Handbook of microbiological and virological methods of research. / Ed. By Birger M.O.M., 1982), which is based on the ability of the studied microbe to lyse by the corresponding phage. To do this, in two test tubes with broth, a culture of determined microbes is seeded, in one of which phage from 1 to 10 drops is added, depending on the lytic power of the phage. After 20-24 hours of culturing the crops in the thermostat, the full or partial effect of the phage on the bacteria is taken into account in the growth density (turbidity) as compared to growth in the control tube.

The disadvantages of this method include:
- long duration of the study;
- the need to maintain a museum of phages;
- the high cost of research.

 Closest to the achieved technical result to the claimed method is a method for the identification of bacteria (Vinas LDM, Loren JG, Bermudes J. Analysis of microcalorimetric curves for bacterial identification. - Can. J. Microbiol., 1987, 33, N 1, p. 6- eleven). A method for identifying bacteria is the digital processing of microcolorimetric curves of bacterial growth using an automated identification system, which is based on the cultivation of bacteria under the same conditions. The identification coefficient is calculated using a computer according to the results of correlation assessment and analysis of the similarity of the parameters of the micro-colorimetric curves of identifiable bacteria and comparison curves belonging to known microorganisms.

The disadvantages of this method include:
- long duration of the identification process;
- the use of expensive equipment;
- the use of a large number of culture media.

 The aim of the invention is to reduce the duration and cost of a method for identifying bacteria.

 This goal is achieved by the fact that in the proposed method, a photoelectrocolorimetric curve of the process of diffuse distribution of bacterial cells in a closed volume of a liquid medium is recorded in the presence of a concentration gradient. According to the invention, the time of disappearance of the concentration gradient at a given volume of the liquid medium and the inoculum introduced into the medium is used as an identification criterion.

 The claimed technical solution differs from the prototype in that, as an identification criterion, the time of disappearance of the concentration gradient of bacterial cells in a liquid medium is used.

 These differences allow us to conclude that the claimed technical solution meets the criteria of the invention of "novelty." The features that distinguish the claimed technical solution from the prototype are not identified in other technical solutions when studying this and related areas of technology and, therefore, provide the claimed technical solution with the patentability condition "inventive step".

 The implementation of the proposed method is performed using a photoelectric colorimeter type KFK-2MP.

 The technique for measuring the diffuse distribution of bacteria in a liquid medium is as follows.

Before the study, according to the instructions for use of the device, the light flux is calibrated by selecting cuvettes and filters. The control and experimental cuvettes are filled with meat and peptone broth to the mark on the lateral surface, placed in the cuvette compartment of the photoelectrocolorimeter, and kept in the closed compartment for some time necessary to establish a certain temperature in the cuvette compartment and in the volume of the nutrient medium. The temperature is controlled by a thermometer placed in the cell compartment of the photoelectric colorimeter. Studies are carried out in this case at 30 ^o C. After reaching the required temperature, a standard suspension in a physiological solution of 0.2 ml of the bacteria culture broth washed from the vital products of the studied bacteria is introduced into the experimental cuvette onto the surface of the nutrient medium without mixing. 0.2 ml of physiological saline solution is added to the control cuvette. The cuvettes are sterilized with a Nikiforov mixture for 30 minutes immediately before filling them with meat-pentone broth, thoroughly wiped from the remains of the mixture with a sterile swab. The optical density of the solution is determined every 5 s for 15-60 minutes at a wavelength of light flux of 540 nm. Based on the obtained digital data, a curve is constructed of the diffuse distribution of bacterial cells in a liquid medium and the diffusion coefficient is calculated. According to Fick's II law, the diffusion coefficient is related to distance and time.

где C_o - концентрация бактерий в объеме инокулята;
C_{x, t} - концентрация бактерий, зависящая от координаты;
x - расстояние, пройденное бактериальной клеткой от поверхности среды в направлении уменьшения градиента концентрации;
t - время исчезновения градиента концентрации;
D - коэффициент диффузии.

where C _o is the concentration of bacteria in the volume of the inoculum;
C _{x, t} is the concentration of bacteria, depending on the coordinate;
x is the distance traveled by the bacterial cell from the surface of the medium in the direction of decreasing the concentration gradient;
t is the time of disappearance of the concentration gradient;
D is the diffusion coefficient.

 Since the diffusion coefficient is rather difficult to determine using modern methods, therefore, as the criterion for identifying bacteria, the time of the disappearance of the concentration gradient, i.e., the time to establish a constant optical density of the test solution, was used.

 The proposed identification method is based on the mechanism of diffuse distribution of particles in a closed volume of a liquid medium.

 The essence of the proposed method for the identification of bacteria is as follows.

 From the classical point of view, the mechanism of diffuse particle distribution is feasible if two conditions are met: 1 - the presence of a concentration gradient, 2 - the presence of thermal motion.

 A killed microbial cell is an object close to a solid, that is, it is a particle with only thermal motion. Each type of microorganism has its own morphological features, such as the size of a bacterial cell, the shape that determines the nature of the environmental resistance during thermal movement of microbial cells. In other words, each microorganism must have its own individual rate of thermal displacement in the volume of a liquid medium in the presence of a concentration gradient. Consequently, the time of disappearance of the concentration gradient is an individual sign for each type of microorganism and can serve as a criterion for their identification if studies are carried out with the same parameters as the composition and uniformity of the medium, pH, temperature, age of the inoculum of the studied culture, dose of the inoculum. The fulfillment of these conditions from a technical point of view is a fairly easy task.

 As for living microorganisms, they can also be taken as an inanimate object in the initial period of their development, which is described in the literature as a period of diffuse distribution of bacterial cells (Kolpakova S.D., Kolpakov A.I. Investigation of the features of the development of bacteria in lag phase .-- Bull. expert of biology and medicine, 1994, N 3, pp. 331-336). However, living microorganisms, in addition to thermal movement, can have an individual mechanism of their movement in the nutrient medium even in the absence of a concentration gradient, due to the presence of their movement organs - flagella, cilia, axial threads, etc., which determine the individual nature of their movement. The nature and speed of movement of a bacterial cell in a nutrient medium in this case is determined by the number of flagella, the location and nature of their location. In this regard, living living microorganisms with a high individual speed of movement, it is not always possible to register the period of diffuse distribution, and therefore, to determine the time of disappearance of the concentration gradient. But with the appropriate equipment, the proposed identification method will become acceptable for this group of microorganisms.

 The following is a specific example of the implementation of the proposed method.

 Museum strains - Escherichia coli, Enterobacter aerogenes, Salmonella choleraesuis were used as the studied bacteria.

 The research results are presented in FIG. 12.

 Due to the lack of a device that implements the prototype method, a practical comparison of the bacteria identification results by the proposed method was carried out with a more accessible method - an analogue - biochemical identification, as the most common among the group of bacteria mentioned.

 The results of the biochemical identification of the studied bacteria, carried out using test systems API - 20, Enterotest and Giss environments, are presented in the table.

 In FIG. 1 shows the curves of the process of diffuse distribution of the slain, and in FIG. 2 - live microbial cells of E. coli (curve 1), E. aerogenes (curve 2), S. choleraesuis (curve 3).

 From the analysis of the curves shown in FIG. 1, it is seen that, firstly, the nature of the diffuse distribution in different types of bacteria is different. Secondly, the time of the disappearance of the concentration gradient in the volume of the working solution is different, that is, the time it takes to establish a constant optical density. For example, for E. coli, the time to establish a constant optical density is 40 minutes, for E.aerogenes - 60 minutes, for S. choleraesuis - 30 minutes.

 As for live bacteria of the same species, the analysis presented in FIG. 2 curves showed that the time of disappearance of the concentration gradient is also different for each of these species, although it is significantly reduced. For example, the disappearance of the concentration gradient for E. coli is 12 minutes, for E. aerogenes - 8 minutes, for S. choleraesuis - 5 minutes.

 The measurement of the diffuse distribution of bacteria of each species in a liquid medium was determined 20 times. The experimental error is 5-7%.

From the analysis of the comparison results it is seen that the proposed method for identifying bacteria differs from the prototype and analogues:
1. A short duration of the identification process (15-60 minutes).

 2. It is economically more profitable, since it does not require the use of expensive equipment, a large range of culture media, and the maintenance of a phage museum.

Claims (1)

 A method for identifying bacteria, including recording a photoelectrocolorimetric curve of the process of diffuse distribution of bacterial cells in a liquid medium in the presence of a concentration gradient, by which bacteria are identified, characterized in that the concentration gradient for a given volume of liquid medium and the inoculum introduced into the medium is used as an identification criterion.

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