SU1004469A1 - Method for determining concentration of live cells of microbial biomass - Google Patents

Description

This invention relates to a microbiological assay, in particular to methods for determining the concentration of viable microbial cells.

The known method of determining the concentration of viable cells by sieving on nutrient medium

The closest to the invention of the technical solution to the technical essence and the achieved effect is the method of determining the concentration of viable cells of microbial biomass in the process of cultivation of microorganisms on the nutrient medium Tr.

The disadvantage of this method is its duration (114 days) and low accuracy of the analysis (at least ± 30%).

The purpose of the invention is to accelerate the method and improve its accuracy.

The goal is achieved by the method of determining the concentration of viable cells of microbial biomass during the cultivation of microorganisms on a nutrient medium to measure the viscosity of the biomass obtained at the temperature of cultivation of microorganisms, and the concentration of viable cells is determined by the formula

T (a + b-Jnl)) 10,

T - concentration of viable cells, mg, L) - kinematic viscosity

biomass, bst,

ten

a, b are empirical coefficients determined by the nature of the biomass.

In this case, before measuring the viscosity of the biomass is filtered.

15

The essence of the proposed method for determining the concentration of viable cells is that for a number of microbial cultures (spore-forming culture, you culture Ml us dendrollmus you.

20 thuringlensis, vegetative culture (Rhizolium pjsum) experimentally established Mengoiy interconnection with the viscosity of biomass and the number of viable cells per unit volume

25 (titer) in the form of

T- (a + b InV) 10,

(one)

T - titer (concentration microb

Claims (2)

30 cells)) cells / ml; t) - kinematic viscosity of b omass, cSt; a, b are empirical coefficients determined by the nature of biomass. The viscosity of the biomass is measured by a capillary viscometer, the diameter of which is chosen taking into account the nature of the test liquid. If there are residual nutrient media in the liquid, liquid is filtered through a cut filter with a cell size that does not exceed the viscose diameter of the capillary to prevent clogging of the viscometer. The viscometer filled with liquid is thermostatic in a device that provides visual observation of fluid outflow through a capillary at a temperature not higher than the cultivation temperature of the biomass (28–30 s) and after 10–15 minutes a measurement of the outflow time of the fluid through a capillary is performed using a stopwatch. The calculation of viscosity is made according to the formula (Passport to the VSG viscometer), 1 98077 where g is time, s; g is the acceleration of gravity C4981, cm / s, k is the constant of the viscometer. The calculation of the average viscosity of the test liquid is made on the basis of 5-7 measurements. In parallel with the measurement of viscosity, a microbiological analysis of a sample of the test liquid was carried out in order to determine the titer (cells / ml) according to a standard method by the method of successive dilutions of dosing on a solid nutrient medium. As a result of statistical processing of experimental data, an analytical dependence of the titer of the biomass under study on viscosity is obtained (1 The drawing €. Shows the dependence of the titer of the biomass under study on viscosity. The straight line, constructed on the basis of this dependence, is a calibration graph for determining the biomass tHTpa (drawing) Example: In laboratory conditions, the dependence of viscosity on the concentration of viable cells (titer) of culture fluid of Bacillus thurIng tenstS strain 507 and cell suspension of the same strain is measured. water on VSW-1 capillary visco zometer (, 03033) at a constant temperature of 20 ° C with a capillary diameter of 0.86. There is no need to filter the liquid. The viscometer filled with liquid is placed in a thermostatic device, which provides a vigorous observation of the outflow of liquid through The capillary tube is held for 15 minutes and the time taken for the fluid to flow through the capillary of the viscometer is measured. Then, the viscosity of the test liquid is calculated using formula (2). The first stage of the study is the construction of the calibration graph T f (). The titer of the cell suspension and the culture fluid is determined by a standard procedure. The titer is in the range of (0-2) -10 cells / mp, the viscosity value varies from 1-1.8 cSt. In the statistical processing of the obtained data, the T f (V) dependence is established in the form (1) with coefficients a -3 and b +10, which corresponds to the graph (drawing; section 1. Then, according to this Curve, the titer of the sample of the cultural fluid is determined, for which the value of the titer from the calibration graph is obtained; 48.5 s and 1.47 cSt. T 1,1 109 cells / ml Example 2: A change in culture liquid (biomass) of a W. dendrolimus culture from a titer is determined. s of the culture fluid of strain 49-8 on a VPZH-1 capillary viscometer with a capillary diameter of 0.86 mm (K 0.02743) at ambient temperature. To do this, 40 ml of the liquid sample is filtered through a filter with a cell size of 0.7-0. 8 mm and poured into a viscometer.The viscometer filled with the test liquid is installed in a thermostatic device, similar to example 1, and held for 15 minutes, then the viscosity of the fluid outflow through the capillary is measured with the help of a stopwatch. The calculation of the average value of viscosity is made on the basis of data of 5-7 measurements of the sample of liquid under study. Build a calibration graph. To do this, microbiological analysis of samples is carried out in parallel with the measurement of viscosity according to the standard procedure, similarly to Example 1. Then, after statistical processing of experimental data in the viscosity range of 2-3 cSt and titer 2-6-10 cells / ml, dependence (1) is obtained with those the same as in example 1, the coefficients and b IO (drawing, section P). A semi-graph is used to determine the titer of samples of the culture fluid. Thus, for a sample of culture fluid, T 88.2 s is obtained, 2.42 cSt. From the graph, 3.10 cells / ml are found. EXAMPLE 3 The change in viscosity of the paste (biomass) of the culture You is determined. thuringiensIS strain 844 from titer. The paste is obtained by concentrating the culture liquid by separation and differs from the culture liquid by a lower moisture content and, consequently, by a significantly higher concentration of biomass. About 30 paste samples are tested on a VPZH-2 capillary viscometer with a capillary diameter of 1.31 mm. (to 0,3399) at ambient temperature. viscosity measurement technique (paste filtering, viscometer filling, temperature control, time measurement, viscosity calculation) as in example 2; Microbiological analysis of samples according to standard methods is carried out similarly to examples 1 and 2. The decal plot is made after statistical processing of experimental data (range viscosity 3-27 cSt, titer - (b-30) -10 cells / ml in the form of dependence (1) (line I and I). With such factors as for plots and II direct. the curve serves to determine the titer of the paste samples. k, for a paste sample, 66.2 s, 22.5 cSt are obtained. From the graph T 28 is found, cells / ml. Thus, the advantages of the proposed method in comparison with the known are: reduction in the time of determination of the microbial biomass titer (15-20 min), which makes it possible to use the method as an expresscontrol under production conditions and to increase control accuracy by at least 2 times. Formula of the invention The method for determining the concentration of viable cells of microbial biomass during the cultivation of microorganisms on a nutrient medium, is schiys in that in order to accelerate the method and its accuracy povyleni, measured in the bone of a biomass-derived, at. the cultivation temperature of the microorganisms, and the concentration of viable cells is determined by the formula. T () w9. where t is the concentration of viable cells, ml, t is the kinematic viscosity of biomass, cSt, and Ojb are empirical coefficients determined by the nature of the biomass. 2. The method according to claim 1, wherein the biomass is filtered before measuring the viscosity. Sources of information taken into account in the examination 1. Zadan to practical lessons in microbiology. L., LSU, .1974. 2. A large workshop on microbiology. Ed. prof. Propulsion L. Seliber. M., Higher School, 1962.