RU2061045C1 - Method of quantitative determination of biological liquid sample microbial dissemination - Google Patents

Abstract

FIELD: medicine, sanitary, microbiology. SUBSTANCE: to liquid biological sample of analyte containing 10000 cells/ml (not less) sodium periodate solution in Triton X-100 is added and obtained mixture is kept for 1-3 min. Then glucose solution (concentration is 50-100 mM) is added to the mixture, incubated for 5-20 min and aliquot taken from the mixture is transferred to dimethylsulfoxide at the volume ratio mixture:dimethylsulfoxide = 1: (4-9) and in the obtained solution the concentration of microbial ATP is determined by bioluminescent method. At the parental microbe cell content in the sample less 10000 cells/ml the sample is incubated preliminary in nutrient or selective medium (at coli-form assay); then microbe cells were concentrated by filtration of the obtained microbe suspension through the diaphragm filter under excess pressure, and glucose is added on the filter and kept its for 5-20 min. Filter is treated with dimethylsulfoxide and obtained solution of microbial ATP is used for its concentration determination by bioluminescent method using internal standard procedure. Recording parameter - maximum of luminosity intensity. Method is used for determination of the total microbial dissemination or coli-form content in foodstuffs, biological fluids, washing off. EFFECT: improved method of determination. 3 cl, 6 tbl

Description

 The invention relates to sanitation and medicine and can be used in determining the total microbial contamination or coli content in food products, biological fluids, washes from technological equipment using bioluminescence.

Currently, to control the microbial purity of products in the food, pharmaceutical, cosmetic industries, as well as in medicine, the use of “fast” microbiology methods is promising. One of such methods is the bioluminescent method for determining the microbial contamination of the analyzed sample by the number of intracellular microbial ATP, which differs from the traditional method of counting colonies on plates with expressivity, ease of execution, and high reproducibility. The method for determining intracellular ATP is based on the ability of the enzyme firefly luciferase [1] to catalyze the oxidation of D-luciferin in the presence of a magnesium salt, O ₂ and ATP to oxyluciferin with the release of a quantum of light. The luminescence intensity at a fixed content of D-luciferin and magnesium salt in the reaction mixture is proportional to the ATP content in the analyzed sample, i.e. depends on the content of viable cells of microorganisms in it [2]
In work [3] adopted as an analogue, a bioluminescent method for the quantitative determination of microorganisms in liquid samples (water, milk, blood, urine) was proposed. According to this method, to the sample, initially containing 1000-5000 cells / ml, add a mixture of 0.1% Triton X-100 and potato apyrase (0.01-0.1 units / ml) and incubated for 5-20 minutes, after which NRB reagent (0.05-2% aqueous solution of a mixture of ethoxylated amines and their quaternary ammonium salts) contribute the same cuvette, or an aqueous acid solution, or an aqueous solution of the base from 1 to 10 volumes.

 In the resulting solution, the concentration of microbial ATP is determined using a bioluminescent reagent based on soluble firefly luciferase. If the initial content of microbial cells in the sample is at the level of 1-100 cells / ml, 1-2 volumes of culture medium are added to the sample and incubated for 0.2-16 hours or 0.5-15 days (when determining specific microflora). To reduce the incubation time to 4-6 hours, the sample is preconcentrated by filtration under vacuum through a membrane filter with a pore diameter of 0.2-0.5 μm, after which a nutrient medium is applied to the filter. The resulting suspension of microorganisms is processed according to the above scheme. The initial number of microbial cells in the sample is determined by the measured ATP concentration and incubation time, taking into account the multiplicity factor.

The disadvantages of the method described in [3] include:
the use of aqueous solutions of acids, bases or NRB reagent, also a mixture of dilute aqueous solutions of ethoxylated amines and their quaternary ammonium salts;
the use of an enzyme for the degradation of non-microbial ATP, apyrase from potatoes;
the duration of incubation in the nutrient medium of the sample, initially containing 1-100 cells / ml, after its concentration on the membrane filter;
filtering the sample under vacuum while concentrating microbial cells on a membrane filter;
the use of a bioluminescent reagent based on soluble luciferase.

In the work [4] adopted as a prototype, a method for the quantitative determination of microorganisms in tissues in the diagnosis of purulent and wound infections is proposed. To determine the concentration of microorganisms, according to this method, a sample of the tissue is homogenized in saline, and the resulting homogenate containing initially 10 ³ -10 ⁵ cells / ml were incubated in an air oven at ³⁷ ° C for 5 hours. The homogenate was treated with Triton X solutions -100 at a concentration of 0.05-0.08% and sodium periodate at a concentration of 1-5 mm, incubated for 1-3 minutes, then dimethyl sulfoxide is added at a volume ratio of 1: (4-9). Next, in the resulting solution, the ATP concentration is determined using a bioluminescent reagent based on immobilized firefly luciferase. The magnitude of the intensity of bioluminescence is judged on the number of microorganisms in the sample.

The method described in [4] has a limited scope only for samples of infected tissues with a high initial seed rate of 10 ³ -10 ⁵ cells / ml. It cannot be used to determine microorganisms in a sample with a content of 1-100 cells / ml, as well as in liquid samples.

 The proposed bioluminescent method for the quantitative determination of microbial contamination in biological fluids, products, and washes from technological equipment consists in adding a 1-5 mM sodium periodate solution of 0 to a sample containing at least 10,000 cells / ml to remove non-microbial and dissolved ATP. 05-0.08% Tritone X-100 and incubated for 1-3 minutes. Then, 50-100 mM glucose solution is added to the resulting mixture and incubated for 5-20 minutes. An aliquot of the mixture is then transferred to dimethyl sulfoxide (DMSO) at a volume ratio of mixture: DMSO 1: (4-9), and the concentration of microbial ATP is determined by the bioluminescent method in the resulting solution.

 If the initial sample contains less than 10,000 cells / ml of microorganisms, the sample is pre-incubated in a general or selective (when determining coli-forms) nutrient medium for 3-6 hours, after which the microbial cells are concentrated by filtering the sample under overpressure through a membrane filter, and glucose is applied to the filter and kept for 5-20 minutes.

This operation is best carried out by slowly pumping through a membrane filter 5-10 ml of saline containing 1-5 mm sodium periodate and 0.05-0.08% Triton X-100, and then 5-10 ml of saline. In the laminated filter 0.05-0.1 ml 50-100 mM glucose, incubated for 5-20 min at ³⁷ ° C and then applied to the filter 0.4-0.9 ml of dimethyl sulfoxide. The resulting solution of microbial ATP is used for bioluminescent determination of its concentration according to the internal standard method. The recorded parameter is the maximum intensity of the glow.

 As a bioluminescent reagent, a reagent based on immobilized firefly luciferase is used, containing D-luciferin, magnesium salt, stabilizers, components of a buffer solution with a solution: reagent volume ratio of 1: 9. The initial content of microorganisms in the sample is determined using a calibration table depending on the time of incubation of the sample and the measured concentration of microbial ATP.

 Distinctive features of the invention are the introduction of glucose into the solution after the destruction of non-microbial ATP, as well as preliminary incubation of the sample if the initial number of microbial cells is less than 10,000 cells / ml. The addition of glucose, on the one hand, stops the destruction of ATP under the influence of sodium periodate, on the other hand, leads to an increase in the content of intracellular ATP by 1.5-2 times and thereby increases the sensitivity of the bioluminescent method.

 Another hallmark of the invention is the incubation of the sample, if the cell content is less than 10,000 in 1 ml, in a nutrient medium before filtering it, as well as filtering through a membrane filter under overpressure. This sequence of sample processing allows reducing the incubation time to 4-6 h, and filtering under excessive pressure has a less traumatic effect on microbial cells compared to the above-described vacuum filtration method.

 The expected effect of the proposed method is improving the accuracy, reliability of determining microbial ATP and reducing analysis time in the case of a low initial content of microbial cells in the sample.

 Example 1. Obtaining a calibration table for determining the number of cells by the level of intracellular ATP without incubation and filtering of the sample.

A suspension of E. coli cells of various concentrations from 10 ⁴ to 10 ⁹ cells / ml is prepared. 0.1 ml from each sample is taken into separate tubes, to which 0.01 ml of a solution of 10 mM sodium periodate in 0.5% Triton X-100 is added, stirred on a magnetic stirrer, after 1 min 0.1 ml is added 0.1 M glucose solution, incubated for 5 min at 37 ^about C, then add 0.8 ml of DMSO. The resulting extract is used to determine the concentration of ATP. To do this, 0.9 ml of Immolum ATP reagent is placed in a 2 ml transparent tube. Register the background glow (I _background ), add 0.1 ml of the extract and register the glow of the sample (I _sample ). 0.01 ml of the ATP standard solution is introduced into the same test tube and the increment of the glow intensity (I _st ) is recorded.

• [АТФ]_ст
где [АТФ]_ст концентрация АТФ-стандарта.The concentration of ATP in the sample is calculated by the formula
[ATP] _arr =

• [ATP] _st
where [ATP] _{st is the} concentration of the ATP standard.

 In the table. Figure 1 shows the dependence of the ATP content in the initial sample on the number of cells.

 Table 1 can be used to determine the number of cells in the test sample, if their number is not lower than 10,000 cells / ml.

 PRI me R 2. Determination of the number of cells in raw milk without prior filtration and incubation of the sample.

To 0.1 ml of raw milk add 0.05 ml of a solution of 3 mm sodium periodate in 0.15% Triton X-100 (which when diluted gives a concentration of 1 mm sodium periodate and Triton 0.05%). Stirred, after 1 min add 0.05 ml of 0.2 M glucose solution and incubate for 20 min at 37 ^about C. Then add 0.8 ml of DMSO. ATP in the extract is measured as described in example 1. The number of cells is determined according to table 1. In parallel, the number of cells in raw milk is determined by colony counting. The data obtained are presented in table.2.

 PRI me R 3. Determination of the number of microbial cells in ice cream with preliminary incubation of the sample.

To 1 g of sample (cream) are added 9 ml of medium was placed on a shaker and incubated at ³⁷ ° C, samples of 1 ml at 4 and 6 hours of incubation. 0.01 ml of 0.1 M sodium periodate in 5% Triton X-100 is added to 1 ml of sample, kept at room temperature for 1 min. The resulting mixture is taken and 0.1 ml was added 0.1 ml of 0.1 M glucose solution and incubated 10 minutes at 37 ° ^C. Then there is introduced 0,8 ml of DMSO. The obtained extract is used to determine the concentration of ATP, as described in example 1. The data obtained are shown in table.3.

 PRI me R 4. Determination of the number of microbial cells in the blood with preliminary incubation of the sample.

To 0.1 ml of blood was added 2.4 ml of sterile distilled water, incubated for 30 minutes, 2.5 ml of culture medium and incubated at 37 ° ^C. Sampling, processing and measurement of ATP is carried out as in Example 3.

 In the table. Figure 4 shows the dependence of ATP concentration on the content of microorganisms in the initial blood sample.

 PRI me R 5. Determination of the number of coli-form cells in a suspension of various microorganisms with preliminary incubation of the sample in a specific nutrient medium and filtering.

Suspensions of E.coli microorganisms with concentrations of 10, 100 and 1000 cells / ml and Bacillus subtilis with a concentration of 100 thousand cells / ml are prepared. To 0.1 ml suspension was added a mixture of 9.9 ml of selective medium coli-forms were incubated at ³⁷ ° C 4 or 6 hours, filtered through a nitrocellulose filter with a pore diameter of 0.45 microns with a syringe and the filter cell (Millipore). The cells on the filter are washed with 1 ml of saline containing 1 mm sodium periodate and 0.05% Triton X-100, then 5 ml of saline. The filter of the filter cell is transferred in a glass weighing bottle, is laminated on filter with 0.1 ml of 50 mM glucose solution was incubated in a thermostat at ³⁷ ° C for 15 min. Then, 0.4 ml of DMSO is applied to the filter and the ATP concentration in the extract is determined as described in Example 1. Table 5 shows the dependence of ATP concentration on the content of E. coli cells in the sample against the background of a large excess of Bac.Subtilis cells.

 PRI me R 6. Determination of the number of cells of coli-forms in smears taken from the surface of the equipment.

 A smear test sample is taken using a cotton swab. After sampling, the tampon is immersed in a selective medium for coli-forms and the contents are washed into the solution from the surface. Incubation of the mixture, sampling and processing of the results are, as in example 3. The data obtained are presented in table.6.

Claims (3)

 1. A method for the quantitative determination of microbial contamination of a biological liquid sample, involving extraction of a non-microbial ATP from a sample with Triton X-100, destruction of the latter using sodium periodate and extraction of microbial ATP from the resulting mixture with dimethyl sulfoxide with a dimethyl sulfoxide mixture ratio of 1 4 9 followed by measurement the intensity of bioluminescence in the resulting solution using an ATP reagent containing immobilized firefly luciferase, and the number ennym determining microbial contamination of biological liquid sample from the dependence of the measured value of the concentration of microbial ATP, characterized in that after the destruction of non-microbial ATP in the resultant solution is introduced into a glucose concentration of 50 100 mM and the mixture incubated for 5 20 minutes.  2. The method according to claim 1, characterized in that when the content of microorganisms in the sample is less than 10,000 cells / ml, the sample is pre-incubated in general or selective nutrient media for 4 to 6 hours  3. The method according to claim 2, characterized in that the sample after incubation is concentrated by filtration through a membrane filter under excessive pressure, and glucose is applied to the filter and kept for 5 to 20 minutes.

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