The method of preparation supnik, tularemia, brucellosis microbes to determine concentrations of counting in hemocytometers cameras


G01N1/28 - INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (separating components of materials in general B01D, B01J, B03, B07; apparatus fully provided for in a single other subclass, see the relevant subclass, e.g. B01L; measuring or testing processes other than immunoassay, involving enzymes or micro-organisms C12M, C12Q; investigation of foundation soil in situE02D0001000000; monitoring or diagnostic devices for exhaust-gas treatment apparatus F01N0011000000; sensing humidity changes for compensating measurements of other variables or for compensating readings of instruments for variations in humidity, seeG01D; or the relevant subclass for the variable measuredtesting or determining the properties of structures G01M; measuring or investigating electric or magnetic properties of materials G01R; systems in general for determining distance, velocity or presence by use of propagation effects, e.g. Doppler effect, propagation time, of reflected or reradiated radio waves, analogous arrangements using other waves G01S; determining sensitivity, graininess, or density of photographic materials G03C0005020000; testing component parts of nuclear reactors G21C0017000000)

 

(57) Abstract:

The invention is intended for standardization of microbial cultures in the production of diagnostic and therapeutic drugs. Analyzed the microbial suspension is treated first by caustic soda. Then prepare from them a tenfold dilution. Received cultivation process within 3-5 min sequentially with solutions of tannin and 0.1 M solution of hydrochloric acid with the addition of 4% lactic acid. The treated microbial suspension fill hemocytometers the camera. Stand her in pairs formalin for at least 1 h For supnik, tularemia and brucellosis microbes use 0,05 respectively; 0,001; 0,01 M solutions of sodium hydroxide and 0,15; 0,2; 0,5% solution of tannin. The invention improves accuracy and provides expressnet counting of microbial cells. 3 C.p. f-crystals, 3 tables.

The invention relates to methods test organisms microscopic methods, in particular to methods for determining the total concentration (living and dead) of microbes by counting under the microscope, and can be used in the production of diagnostic and therapeutic bacterial preparations, and when standartizaciyi suspensions for counting in hemocytometer Goryaeva, Tom and others, consisting in the breeding of the investigated suspension with saline or distilled water to a final dilution introduced into the chamber (B. A., fichman. Optical standardization of bacterial preparations. - M., Office of scientific information, 1960).

In common with the claimed method is the stage of preparation of the investigated suspensions prior cultivation required multiplicity.

The disadvantages of this method include low accuracy and poor repeatability of the results. Source of errors are low academist on the bottom of the camera cells of many species of bacteria associated with high sedimentation stability of their suspensions in distilled water or physiological solution. Inherent in such microbes mobility in the environment of the suspension, due to Brownian motion or the presence of cells flagella, causes them to move from one field of view to another, which prevents the calculation and significantly reduces its accuracy.

A serious disadvantage of this method is the risk of infection in the counting process, since many pathogenic microbes capable of long-term viable in physiological Rastatt or respirator "Lepestok-200". The breath of the operator through these remedies causes fogging of the eyepiece, to correct which you want to interrupt the work and clean the lens. All this is not conducive to expressnet and accuracy of analysis.

There are also known methods of preparation of microbial suspensions for calculating, based on the use of dispersion of the cells of colloidal solutions, capable of solidification to move in the gel state, for example 4% aqueous solution of polyvinyl alcohol (B. A., fichman. The new environment for microscopic account of bacteria in the camera. - Journal. microbiol., Epidemiol. and immunobiol. - 1962. N 2. -S. 55 - 56).

In common with the claimed method is the pre-breeding analyzed the suspension to the desired concentration of cells in suspension.

The disadvantages of this method include the complexity and the complexity of the calculation, since the microbes are fixed the formed gel on the entire depth of hemocytometer equal to 100 μm. As a result, only part of the cells into the field of view of the microscope. For the calculation of the remaining cells is required to repeatedly refocus the microscope and view each field of view throughout the depth of hemocytometer with the exception counted microbe is the first way is to preserve the viability of many pathogenic bacteria in 4% solution of polyvinyl alcohol, which leads to the above-described consequences.

There are also known ways of preparing suspensions of bacteria for calculating, based on the use of mixtures of chemical compounds able to reduce sedimentation stability of microbial suspensions. For example, liquids, consisting of a 10 cm3alcohol, 30 cm3glycerol, 60 cm3distilled water and 10 cm3an alcoholic solution of gentian violet (D. Horvath. Ein eifaches Verfahres fur Bakterien - Zahlung. - Zbl. Bakteriol., parariterl., Jnfektions Krankh. 1. Abt. Jrig., 1930, Bd 1, H 3/4, S. 238-239).

In common with the claimed method is the stage of preparation of the investigated suspensions prior cultivation required multiplicity.

The disadvantage of this method is its low efficiency in relation to the causative agents of glanders, tularemia and brucellosis. Consider the fluid provides deposition on the bottom of hemocytometer from 10 to 15% of the cells of these pathogens. Almost half of the germs before the analysis is complete (within 3 hours after cooking mist) maintains viability. All this makes the calculation and leads to unacceptable variability of results.

Closest to the claimed solution is the method of preparation of microbial weighing the liquid Calisson, consisting of 2 cm3hydrochloric acid, 100 cm3duhalista mercury 1:500 with the addition of 1% fuchsin (J. Callison. A diluting fluid for standardization of vaccinnes with the hemacytometr. - J. Med. Res. 1912. V. 27. N 2. P. 225 - 227).

In common with the claimed method is the pre-cultivation of the analyzed suspension solution containing hydrochloric acid.

The disadvantages of this method should be considered bad academist in hemocytometer cells causative agents of glanders, tularemia and brucellosis, suspended in a liquid of Callison. So, 24 hours after injection of the mixture into hemocytometer the vast majority of 80...90% supnik, tularemia and brucellosis microbes remains suspended and is subject to Brownian motion. In addition, at least half of these types of microbes remain viable for 3 hours after suspension in a liquid of Callison. All this is a source of significant errors, unacceptable in the standardization of bacterial preparations, and requires compliance with stringent measures special precautions.

The objective of the invention is the provision of a sufficiently rapid (within 1 hour from the moment of introduction into the camera) deposition on the bottom hemocytometer definition of the observed microscopic picture. All this is necessary for accurate and rapid calculation.

The problem is solved due to the fact that in the method of preparing suspensions of cells of the causative agents of glanders, tularemia and brucellosis to count, including the pre-cultivation of the analyzed materials, provided the following differences:

pre-processing of microbes with a solution of caustic soda;

subsequent treatment of the cells with a solution of tannin;

use for the preparation of the final dilution of the suspension solution containing hydrochloric and lactic acid;

keeping filled hemocytometer in pairs formalin.

These differences are due to the following reasons:

sedimentation stability in dispersive media supnik, tularemia and brucellosis microbes is determined by electrokinetic potential of the cells, they have a powerful solvate shells, due to the special hydrophilic biopolymer microcapsules, and their small size (small mass), which together prevent the action of gravitational forces. Therefore, to ensure deposition of microbes required to eliminate or reduce the effects of these factors. Among these factoryoutlet and salt cotton shell;

to reduce the hydrophilicity of the surface of their cells first treated with caustic soda solution, providing the hydrolysis of biopolymers microcapsules;

a solution of tannin helps to reduce the dimensions of the solvate shell;

hydrochloric and lactic acid to provide a pH environment, close to the isoelectric point of microbes. Cations of hydrogen improves the adsorption of cells on glass;

hydrochloric and lactic acids cause cell death causative agents of glanders, tularemia and brucellosis;

keeping filled hemocytometer in pairs formalin for 1 hour provides disinfection of its surfaces, contaminated which could occur in the process of introducing the suspension into the chamber;

used solutions do not cause lysis of the cells and their excessive aggregation, characterized by transparency, low indices of refraction of light and provide a high contrast image supnik, tularemia and brucellosis microbes in the phase-contrast microscope.

The method includes the following steps:

breeding investigated mist;

introduction prepared the final dilution in hemocytometer;

keeping hemocytometer in the chamber with pairs of formalin for ensuring that what has been created material is diluted in a test tube with sodium hydroxide solution until the concentration the respective optical densities of the standard turbidity GISC name L. A. Tarasevich 10 UNITS. Thus take into account the breeding material (value P). Microbes in the alkaline solution stand for 30 minutes.

Then prepare two more ten-fold dilution.

One - on solution of tannin, and from there to hydrochloric and lactic acids. In test tubes with tannin and acid microbes can withstand up to 5 minutes. The tube is closed with cotton-gauze plugs. The contents of the test tubes thoroughly mixed by periodic shaking.

For processing supnik microbes using 0.05 M sodium hydroxide solution and 0.15% solution of tannin; for tularemia microbe - 0.001 M solution of sodium hydroxide and 0.2% solution of tannin; for brucellosis microbes - 0.01 M sodium hydroxide solution and 0.5% solution of tannin.

Final processing of the germs of all these types of exercise 0.1 M solution of hydrochloric acid with the addition of 4 % lactic acid (by volume).

The solutions of all reagents prepared with distilled water.

A suspension of cells from a test tube with acids are introduced with a pipette in hemocytometer with ground glass cover slip. Hemocytometer and glass cover has preliminarily">

Filled hemocytometer placed in a Petri dish. Close it with a lid, the bottom of which is placed a disk of filter paper moistened with 2...3 cm3formalin. Petri dishes with cameras kept on the desktop for 1 hour.

For lack of control pollution hemocytometer and counting cells using the optical system for phase-contrast microscope (microscope MBI-3 or MBR, MB-1A, "Biolam L-211", phase-contrast device KF-4, lens x 20F, the binocular attachment AU-12, eyepieces x 10). The configuration of the microscope is carried out according to the description supplied with the kit KF-4.

The microscope focus to the grid Goryaeva, seeking by microvita clear images of microbes. They look as black rods and cocci.

Count the cells that are located in large grid squares Goryaeva diagonally in the camera. First, the cells in the grid square, and then, slowly picking up the tube of the microscope micro-screw, take into account over the same square single microbes, which can adhere to the bottom surface of the cover glass.

The analysis does not consider the cells crossed the bottom and left edges of the square. Accept them for cells lying in Desenchantee microbes in the studied material are calculated according to the formula

< / BR>
where C is the cell concentration,

The P - value breeding material with alkali solution,

H - number of analyzed squares

M - the number of counted cells.

To compare the effectiveness of the proposed method and the prototype was prepared by dilution from the same materials causative agents of glanders strain C-5, tularemia strain Schu and brucellosis strain Br. melitensis M 16. To ensure equal concentrations of cells in the final dilutions introduced into the chamber, the materials in experiments on the prototype of the" bred in the same way as in the proposed method, with the only difference that instead of the proposed solutions of alkali, tannin, hydrochloric and lactic acids used fluid Callison at all stages of cultivation. In each experiment were analyzed in 10 fields of view. Academist microbes characterized by the average number of cells per square. The viability of the microbes to the beginning count was estimated by the method of plating on a selective nutrient medium and bioassays in experimental animals. To control contamination of the surface of hemocytometer did the cotton swabs-gauze swab. The obtained samples were sown on solid nutrient medium and used for zarajenie microbes - on the basis of articular, brutally on blood agar. For the production of bioassay experiments with low glanders, tularemia and brucellosis used accordingly Golden hamsters, white mice and Guinea pigs. Comparative studies show that the proposed method is compared with the prototype, provides significantly more academist germs on the bottom of hemocytometer and their inactivation in preparation for counting (table. 1).

The causal connection between the set of essential features of the claimed object and achievable technical results are shown in table. 2.

The proposed method for the preparation of suspensions of cells of the causative agents of glanders, tularemia and brucellosis for counting has the following advantages:

provides deposition on the bottom of the camera 80...90 % of the cells within one hour after the introduction of the prepared cultivation in hemocytometer. Other microbes adsorbed by the lower surface of the cover glass; leads to the inactivation of microbes these species both in the camera and on the entire surface of hemocytometer that allows you to count without strict protection measures. Samples analyzed outside the fume cupboard, without the use of respirators and VA is used in the optical system, in conjunction with the immobilization of cells on the bottom of the chamber and cover glass provides the conditions necessary for the implementation of the counting under the microscope. The analysis available to the technician with the skills to work with phase-contrast microscopes;

the method provides sufficient for the practical application of the accuracy of the calculation, the error does not exceed 10%, and the discrepancy between the results of the analysis between laboratories - 12%;

the time for determining the total concentration of microbes in a sample by counting under the microscope is 2 hours from the moment of receipt by the laboratory, which allows us to characterize the counting method with the proposed method of preparation of suspensions as Express.

The possibility of implementing the method shown in the following examples.

Example 1. Preparation of suspensions supnik microbes to determine the total concentration of the cells in sapnay vaccine.

In order to ensure convenient for counting the number of cells in one square hemocytometer (about 30...80 individuals) pre-determine the optical concentration of cells in the vaccine according to the turbidity standard gisk named after. L. A. Tarasevich 10 units turbidity. In the studied series it is equal to five times. To do this in test tubes pour 4.0 cm3the alkali solution and contribute 1.0 cm3vaccine graduated pipette 1.0 cm3. The amount of dilution of the vaccine "P" is 5.

The tube is closed with cotton-gauze plugs. Contents periodically stirred by shaking. After 30 minutes of test tubes with alkali prepare two rows of two consecutive ten-fold dilutions. The first of them is prepared 0.15% solution of tannin, and the second in 0.1 M hydrochloric acid solution, adding thereto 4% lactic acid. A solution of tannin and acid are poured into a test tube 4.5 cm3. Graduated pipette from the middle part of the tube with the alkali selected based on 0.5 cm3content and transferred into a test tube with tannin. It is covered with a cotton-gauze tube, the contents mixed. This tube is similarly transferred to 0.5 cm3the suspension of cells in a test tube with acids. The tube is closed with cotton-gauze plugs. The contents are mixed by shaking.

Thus, for the investigational vaccine prepared two series of test tubes containing three parallel tubes.

After shaking of the tubes with acids carry droplets suspended in hemocytometer using pipedreaming paper, soaked in formalin. After 1 hour shall count.

The cell concentration calculated according to the formula presented in the description, based on the number of counted cells, the number of analyzed squares and the magnitude of the dilution of the vaccine. From averaging the results of two parallel determinations establish the total concentration of cells in the vaccine.

Example 2. Preparation of suspensions of tularemia microbe to determine their total concentrations in the culture method of calculation.

Pre-determine the optical concentration of cells in culture using standard gisk named after. L. A. Tarasevich 10 units turbidity, to ensure the subsequent dilution of culture is useful for counting the number of cells in one square camera (30...80 individuals). Optical concentration was equal to 15 billion cells/cm3. Based on these data, the culture is diluted in parallel in two test tubes, 0.001 M sodium hydroxide solution 15 times. To do this in test tubes pour 7.0 cm3alkali and contribute 0.5 cm3the studied culture. The value of breeding a culture of "P" is 15. Subsequent stages of the method of preparation of the suspensions are similar to those shown in example 1, with the only rashtovka suspensions brucellosis microbes to determine their total concentrations in the culture method of calculation.

In the same manner as in examples 1 and 2, set the optical cell concentration in the culture. It was equal to 20 billion cells/cm3.

Based on these data, the culture is diluted in parallel in two tubes of 0.01 M sodium hydroxide solution in 20 times. This tube is poured into 1.9 cm3alkali and contribute to 0.1 cm3analyze culture. The value of cultivation culture is 20.

Subsequent ten-fold dilution is prepared in the same manner as in example 1, with the only difference that for the cultivation of Brucella bacteria using 0.5% solution of tannin.

In table. 3 presents the results of determining the total concentration of cells by counting and number of living microbes in the cultures of P. mallei, F. tularensis, and Br. melitensis seeding of the Cup with nutritionally dense environments. Suspension of microbes before filling hemocytometer prepared by the present method.

From table. 3 shows that after treatment by the present method capnia, tularemia and brucellosis microbes become available for counting under a microscope. The set values of the total concentration (living and dead) bacteria exceed the data plating method on colocasiae, which resulted in the formation of some of the colonies is not from a single cell, and from their units, as well as the presence in the studied cultures not only the living but the dead animals. Living microbes in the preparation of suspensions of the proposed method are inactivated, so when calculating morphologically they can't be separated from dead animals, the source available in the investigated material.

1. The method of preparation supnik, tularemia, brucellosis microbes to determine concentrations of counting in hemocytometers cameras, including a preliminary dilution of the investigated material and filling hemocytometer camera, characterized in that the microbes initially treated for 25 - 35 minutes by caustic soda, and then using a tenfold dilution process within 3 to 5 minutes sequentially with solutions of tannin and 0.1 M solution of hydrochloric acid with the addition of 4% lactic acid, and filled hemocytometers the camera is kept in pairs formalin for at least 1 h

2. The method according to p. 1, characterized in that sapne microbes treated with 0.05 M sodium hydroxide solution, and then 0,15% solution of tannin.

3. The method according to p. 1, characterized in that toposar under item 1, characterized in that brucellosis microbes treated with 0.01 M sodium hydroxide solution and then with 0.5% solution of tannin.

 

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