SU1231077A1 - Method of estimating concentration of bacteria in suspension - Google Patents

Description

The invention relates to microbiology, in particular medical microbiology, as well as the microbiological industry, and can be used to determine the amount of bacteria in preparations.

The purpose of the invention is to improve the accuracy and sensitivity of the analysis.

The proposed method is explained in FIG. 1-7.

The essence of the method is the preparation of a bacterial suspension; ZIN in distilled water, which is introduced into the solution (mp 1 "-HCl, 100 mmol, pH 7.2-7.5), solutions of Triton X-100 are added (to a concentration of 0, 03 - 0.05%), ethylenediaminetetraacetate-tris (EDTAT), pH 7.2-7.5 (to a concentration of 0.3-3.0 j41) and ethium bromide (2-, 7-diamine- 10-ethyl-9-phenyl-phenanthiridium bromide to a concentration of 4-7 μmol). After 5 minutes of incubation, the fluorescence intensity of ethidium bromide in bacteria suspension and in buffer is measured. The difference between the first and second values characterizes the concentration of bacteria in the suspension.

The method is based on the fact that the fluorescent dye ethidium bromide is capable of selectively interacting with nucleic acids, and the quantum yield of its fluorescence increases. The increase in fluorescence of the dye is directly proportional to the amount of nucleic acids. To ensure membrane permeability of gram-negative bacteria for fluorochrome, cells are treated with a mixture of a detergent and a chelator (Triton X-100 with ethylenediaminetetraacetate). Registration of the increase in ethidium bromide fluorescence when interacting with nucleic acids uniquely reflects the amount of bacteria in the preparation.

Two options are possible for the quantitative characterization of bacterial preparations by ethidium bromide fluorescence. According to the first, a calibration graph is constructed of the relationship between the increase in fluorescence of ethidium bromide in a suspension of bacteria after multiple dilutions and the number of cells counted under a microscope in a counting chamber. The second option involves taking into account the number of bacteria in the CONDITIONAL units of increase in fluorescence 31077J

dye in the presence of bacteria in relation to its fluorescence without bacteria. The latter option is useful for those

5 types of bacteria, which cannot be counted under the microscope.

Example 1. 3 ml of a buffer solution containing

to 100 mmol of Tris-HCHE, pH 7.2, ethidium bromide to a concentration of 0.1 µmol, and the intensity, fluorescence at 600 nm with a wavelength of exciting light of 322 nm was measured on a Perkin-Elmer spectrophotometer, model 1000. These operations are repeated at different dye concentrations. The measurements made (Fig. 1) indicate that to study the fluorescence of a dye, it is advisable to use its concentrations not exceeding 8 µmol, when the linear relationship between its concentration and the fluorescence intensity remains.

Example 2. 3 ml of the buffer specified in Example 1, ethidium bromide to a concentration of 6 µmol were successively introduced into the optical cell.

30 EDT, pH 7.2 and the fluorescence intensity is recorded as described in Example 1. This procedure is repeated at different concentrations of EDTAT. The results of FIG. 2) mean

35 that the fluorescence intensity of ethidium bromide does not depend on the concentration of EDTAT in a wide range.

In the same manner, the effect of triton X-100 on the fluorescence of these 40 bromide dummies was examined. The results of the study (Fig. 3) show that Triton X-100 at concentrations greater than 0.05% causes an increase in ethidium bromide fluorescence intensity of more than 45 than 5%. Therefore, the use of higher concentrations of detergent during the analysis is impractical.

The culture of Escherichia coli K-12 is grown on glucose-mineral

50 medium to the stationary growth phase. The cells are precipitated by centrifugation and washed once with distilled water. 3 ml of the buffer of example 1 and ethidium are added to the optical cell.

55 bromide to final concentration

6 μmol. Measure the fluorescence intensity of the dye as described in Example 1. In the same cuvette, add 3

suspension of E. coli bacteria to a final concentration corresponding to the optical density of the sample at 600 nm 0.01 - 0.5 units. The fluorescence is measured again. The results of the analysis are shown in FIG. 4, where the activity of cells in the system has almost no effect on ethidium bromide fluorescence, since the dye does not penetrate through the cell wall to nucleic acids.

Buffer, ethidium bromide and a suspension of bacteria in the indicated concentrations are added to the optical cell, a solution of EDTAT is added to a final concentration of 0.3 mmol, and the increase in dye fluorescence over time is recorded. Analysis of the results (Fig. 4, curve b) shows that the maximum fluorescence increase of the dye (maximum binding to nucleic acids) occurs after 30 minutes of incubation.

Buffer, ethidium bromide, cell suspension, EDTAT in the indicated concentrations are added to the optical cuvette, Triton X-100 solution is added to a final concentration of 0.03% and the fluorescence increase in dye: l over time is recorded. As can be seen from the presented data (Fig. 4, curve b), the presence of Triton X-100 significantly accelerates the penetration of the dye into E. coli bacteria. Triton X-100 without EDTAT does not promote the penetration of ethidium bromide into E. congeni cells (Fig. 4d). Thus, it is the combination of EDTAT with Triton X-100 that promotes the rapid and complete binding of ethidium bromide with nucleic acids of E. coli bacteria.

Example 3. The cultures of three gram-positive (Bacillus subtilis, Staphylococcus aureus Streptococcus facealis) and three gram-negative species (Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens) of bacteria are grown on a Mammoth agar. Cells are washed from the medium with 0.15 M NaHa solution and suspended. 3 ml of Buffer rice -NCE (100 mmol, pH 7.5), citium bromide (6 µmol) are added to the optical cuvette and the fluorescence intensity of the dye nq of Example 1 is measured. In this sample, one of the microorganisms is suspended in at a concentration corresponding to optical

31077

density at 600 nm is not more than 0.5 units. and at least 0.1 units. After 5-7 minutes, the increase in fluorescence of the dye is recorded. Similar studies 5 (Fig. 5) were carried out after addition to the EDTAT samples (0.3 mmol) together with Triton X-100 (0.03%), the creeping results are presented in Table. 1. From the data table. 1 it follows that

10 gram-positive bacterial species bind ethidium brymide and without EDTAT to Triton X-100. At the same time, these compounds do not adversely affect the binding of the dye to the studied species. For gram-negative bacterial species, treatment with EDTAT with Triton X-100 is a necessary condition for the penetration of ethidium bromide to nucleic acids. Selected

20 concentrations of chelator and detergent are effective for all species studied.

In tab. 1 shows the presence of the pride of fluorescence ethidium bromide in

25 the presence of bacteria depending on the treatment with a detergent chelator.

T a b l and c a I

thirty

35

Note. (+) - presence of increase in fluorescence; (-) - lack of fluorescence growth.

Example 4. E. coli K-12 cells are grown on m septone agar, as well as on liquid glucose-mineral and m. Septone media to a logarithmic and stationary growth phase. Bacteria grown on agar are washed off with distilled water and a suspension is prepared with an optical density at 600 nm of 0.01-0.5 units. Bacteria grown in a liquid medium are diluted with water to the same optic density. Triton X-100 was added to an opt1-gage cuvette with three -HNE buffer (100 mmol, pH 7.5) to a final concentration of 0.03%, EDTAT (pO 7.5) to a final concentration of 1.0 mmol, ethidium bromide to a final concentration of 7 μmol. The fluorescence intensity is measured, as indicated in example 1. For further calculations, it is convenient to take the level of fluorescence in this sample (control) as 0.

The same compounds in the same concentration are added to the optical cell, and then the bacterial suspension is added and the level of fluorescence is measured after 5 minutes. Such operations are performed with bacterial suspensions after multiple dilutions. At the same time, the concentration of bacteria in the samples was counted under a phase-contrast microscope in the chamber of Gorev. Based on the data obtained, a relationship is established (Fig. 6) between the increase in fluorescence of ethidium bromide (iF) and the number of bacteria in 1 ml of suspension (N). This dependence is expressed by the formula

where K - corresponds to the tangent of the slope of the dependence of N RTG.

In the resulting formula, the K coefficient is the same for E. coli bacteria grown on different nutrient media and does not depend on the phase of culture development. The minimum concentration of E. coli bacteria in the sample, which can be determined using this method with an accuracy of 3-5%, is 10 cells / ml, which corresponds to an optical density at 600 nm of 0.001 units.

Similar measurements are made for B. iubtilis, S. aureus, S. saecalis, F. aeruginosa, S. marcescehs. Bo In all cases, the increase in ethidium bromide fluorescence depends linearly on the concentration of bacteria in the samples.

Example 5. The E. coli K-12 culture is grown and prepared for operation as indicated in Example 3. 3 mltris-HC buffer (100 mmol, pH 7.5), ethidium bromide (6 µmol), EDTAT ( 0.3 mmol) Triton X-100 (0.03%) and suspension of bacteria at a concentration of 10 cells / ml. After a 5 minute incubation, the fluorescence increment at 590 nm in the sample is measured with respect to the sample containing no cells. The measurements are carried out at two wavelengths of the exciting light (320 and 520 nm) corresponding to the two main absorption bands of the dye. The results (Fig. 7) indicate a linear dependence of the increase in ethidium bromide fluorescence on the concentration of bacteria at both wavelengths. This makes it possible to determine the concentration of bacteria upon the excitation of the f ;. resorption of ethidium bromide in both main absorption bands.

Example 6 The E. coli K-12 culture inoculum is divided into two portions. One is grown on a glucose-mineral medium to logarithmic, and the other is grown on the same medium before the stationary growth phase. Cells are pelleted by centrifugation and suspended in. Using the Gorev camera, the concentration of bacteria in preparations from the logarithmic and stationary phases of growth is calculated separately. By dilution with water, the concentration of bacteria in both preparations is adjusted to 4.6 Yu cells / ml. 3 ml of Tris-HCl buffer (100 mmol) and 0.1 ml of the suspension of bacteria from the logarithmic growth phase are introduced into the optical cuvette. The optical density of the suspension was measured at 600 nm on an SF-4 spectrophotometer. The same measurements are made with bacteria from the stationary growth phase.

B, 3 ml of Tris-HC buffer (100 mmol,), ethidium bromide (6 µmol), EDTAT to a concentration of 0.3 v & l (pH 7.5), Triton X-100 (0.03% ), 0.1 ml of the suspension of bacteria and logarial growth phase and after 5 minutes of incubation, the fluorescence growth is measured by comparison with a sample without bacteria, as indicated in Example 5. The same measurements are made with bacteria from the stationary phase (data from these measurements are summarized in Table 2).

From the data table. 2 it follows that bacterial suspensions of the E. coli K-12 culture from the logarithmic and stationary growth phases with the same cell concentration have different turbidity (in Table 2, the data are given in units of optical density). Thus, measurements of the same

71231077 8

concentrations of bacteria from various stagnant 40%. Under the same conditions of the phases of the development of culture, the prototypes have discrepancies in measurement, measurement. According to the proposed method, there are discrepancies of no more than 6

Investigated bacteria

E. coli grown up to the stationary stage. . E. coli grown to logarithmic stage

Note. The concentration of bacteria in all samples is the same 1.5-10® cells / ml.

In tab. A comparison is made of the turbidometric and fluorimetric methods for determining the concentration of bacteria E. coli.

The proposed method for determining the concentration of bacteria in the suspension is of greater accuracy, since the results of the analysis are not affected by the resulting 40%. In the same conditions

measurements According to the proposed method, the differences are no more than 6%.

table 2

.)

Optical density at 600 them (rel. Units)

47 + 2 0.184 + 0.005

50 + 2 0.272 + 0.005

neither the culture development phase, nor the growth conditions of the bacteria, nor their ability to aggregate. The method is more sensitive: it allows one to estimate with an accuracy of 3-6% the concentration of bacteria 100 times less than the method of the prototype allows to do.

AAA

g. 6 in

condchetrii 9dtat, nn fttt.r

a "giz

Koniyitraii mfumoMt x-yuo, / lH J

at

Wren, HUH

.d

gW4Ai4

about ig b m

Komtamfitufia zatlg nn

W №

 ftuentfuu pvak vti / Li Mai

in 1G

KtHuemr nuuu Sanmtpuu, U m / n

D Sannepitu It) ayushlp ML .. L, l

01 g -

Jtamtfuu, and U / N

 Sanmepuu, StipauttttMie and Itazv-nimera my F. Sanmepuu, Korachennye nkepe Jupnom Vuyyznk

d SuKtrafuu, grown on nisopeptonnon agar Fig f

Editor E. Kopcha

Compiled by O. Skorodumova

Tehred I.Popovich Proof-reader I. Erdeyi

Order 2525/31 Circulation 490 Subscription

VNIIPI USSR State Committee

for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5

Production and printing company, Uzhgorod, st. Project, 4

- ueujorAxee "s $ fas" M (w (prix. LOT

+ tol firauiut luyofHeentMuau ri. Zyain Aa.7

Claims (1)

METHOD FOR DETERMINING BACTERIA CONCENTRATION IN SUSPENSION, which involves preparing a bacterial suspension and recording an optical parameter, characterized in that, in order to increase the accuracy and sensitivity of the analysis, Triton X-100 is added to the suspension to 0.03-0.05%, ethylene diamine tetraacetate Tris up to 0.3-3.0 mmol at pH 7.2-7.5, ethicium bromide up to 4.0-7.0 mmol and record the increase in fluorescence intensity in samples with bacteria compared with samples without bacteria in the range _; the fluorescence wavelengths of 540620 nm and the fluorescence excitation wavelength ranges of 260-320 or 420-530 nm, and the concentration of bacteria is inferred from the increase in fluorescence intensity. Fig.)