SU1382848A1 - Method of measuring concentration of microorganisms in suspension - Google Patents

Description

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1CE

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This invention relates to microbiology and can be used in immunofluorescent analysis of microorganisms.

The aim of the invention is to increase the reliability of the method and reduce the time of determination.

The method consists in adding a specific luminescent immunoglobulin to the cell suspension, introducing the resulting mixture into the gel, applying the microvolume of the mixture to a glass slide, allowing it to stand before the polymerisation, cover it with a cover glass, and allow it to solidify without forming voids and count the number of microorganisms (cells / ml) according to the formula

S-, M and

N

So. 25-v

-1 e

flp

where s

Ok

cover glass area

mm,

area of the ocular grid, reduced to

lens mm

year

etc

but

I 25 is the number of fields of view; M is the number of cells in 25 fields of view of each sample; At „„ - microvolume of mixture for one sample, mm; the number of diluted suspension;

Result of 10 samples.

Example 1. Determination of the concentration of BiSubfills cells in the daily broth culture.

A daily batch of B.SU bf i1 is grown in 3 m septon broth with a volume of 3 MP is placed in a cuvette (1 cm thick) of the spectrophotometer and the density is measured at a wavelength of 600 nm, the value of which is more than 3 optical density. The culture was diluted with physiological saline (0.85% NaCl) 20 times and the optical density was measured, which is 0.36 units. optical density. To 3 ml of the resulting suspension, 0.375 ml of a luminescent immunoglobulin specific to B is added, sub-fllis is diluted 1: 8. After conducting an immunofluorescent reaction (15 minutes), 0.3 ml of deoxycholate (0.3 mg / ml) is added to the resulting mixture, and the closed tube is inverted several times without foaming.

0.3 ml of the resulting mixture was added to 2.7 ml of gel. Then, two drops of a mixture of 3 µl are applied to a defatted glass slide, after 3 minutes of incubation, each drop is covered with a cover glass of 0.15 ml measuring 16x16 mm. The cells were counted on a microscope in 25 fields of view on five smears: 90.0, 89, 87, 100, 82. The average number of cells is (in 25 fields of view) M 91 + 3. Concentration B, subfi1fs

So "25-V,

± e (7.5 + 0.2)

etc

x10 cells / ml.

The analysis time is 30-40 minutes.

Example 2. Counting of E. coli cells Ml 7 in the preparation Bifikol.

4.5 ml of undiluted suspension of Bifikol containing cells of E. coli Ml 7 ibifidobacteria is combined with 0.6 ml of luminescent immunoglobulin specific to the 1: 8 dilution stick. After carrying out the immunofluorescent reaction for 15 minutes, 0.45 ml of deoxycholate (0.3 mg / ml) is added to the mixture, the tube is inverted several times (hermetically sealed), without foaming, 4.2 ml of the suspension obtained is added to polyacrylamide gel instead of distilled water. Then 3 drops of the mixture were applied to the defatted glass, 3 µl each, and after 3 minutes of incubation, each drop was covered with a cover glass with a thickness of 0.15 mm, under which, after 7 minutes, a vitrified sample was taken that occupied the chamber 16x16x0, OPO mm. Cell counting was performed on a microscope in 25 fields of view on six smears: 15, 20, 17, 16, 13, 16. The average number of cells in 25 fields of view of smears M is 15 ± 2. Concentration of chopstick Ml 7 in Bifikol

N

 ± 1.7-0.2

So. 10 cells / MP

The analysis time is 30-40 minutes.

The cells of bifidobacteria do not interfere with the quantitative evaluation of the cells of Escherichia coli due to the specific staining of only the cells of Escherichia coli.

Example 3. Counting the total number of microorganisms in the preparation Bifikol using nonspecific staining of microorganism cells.

4.5 ml of undiluted suspension of the drug Bifikol is mixed with 0.5 ml of FITZ (fluorescein isothiocyanate isomer). FITC stock solution of 1 MI-ml pH 8.0 is incubated for 10 minutes. To the mixture obtained, 0.45 ml of deoxycholate (0.3 mg / ml) is added. 4.2 of the resulting suspension is added to the polyacrylamide gel used in this case instead of distilled water. Then, 2 drops of the mixture of 3 µl are applied to the defatted glass. After 3 minutes of incubation, each drop is covered with a cover glass with a thickness of 0.15 mm and a size of 16x16 mm. Cell counting was performed on a microscope in 25 fields of view on six smears: 86, 95, 87, 101, 91. The average number of cells in 25 fields of view of smears M

  ± f (1.02 ± wok / J y „,

Etc

 91 + 4. N

+ 0.04) -10 cells / ml.

The analysis time is 30-40 minutes.

The gel used must satisfy the following conditions: do not have its own luminescence when excited in the wavelength range of 400-500 nm, do not extinguish the luminescence of the dye, form a transparent vitreous body after polymerization, must not contain extraneous inclusions, the formation of the glassy body should end up 7-10 min.

For example, polyacrylamide gel, acrylic acid amide (CH2 CHCONH), ammonium persulfate (NN4)) 1 dodecyl sulfuric acid, sodium salt DCC (CH3 (CH2) t050eMa N, N-methylene bis-acrylamide (OH 2 (NHOCCH CH) N, N, NN -tetramethylethylenediamine ((CHj) (CH), tris- (hydroxymethyl) aminomethane (NH CCCH-gOH))), as well as tris- (hydroxymethyl) aminomethane hydrochloride (NH2C (CH20H) jHCl.

Solution A is prepared for operation: 1.85 g of Tris-HC1, 7.7 g of Tris-OH, 0.2 g of SDA are dissolved in 25 ml of distilled water, the volume is brought to 50 ml after dissolving; solution B: in 50 ml of distilled water dilated

five

0

five

0

five

0

five

0

five

14.6 g of acrylamide, 0.4 g of bis-acrylamide are stolen; Solution C: TEMED ready solution (N, N, M, N-tetramethyl-ethylenediamine); solution D: 200 mg of ammonium persulfate are dissolved in 2 ml of distilled water.

To prepare the gel, mix solutions A, B, Ci D in the following order: 2.5 ml of solution A, 3.3 ml of solution B, bring up to 10 MP with distilled water, 5 µl of solution C, 50 µl of solution D.

Cell concentration after total dilution Incomplete suspension

Zia

PP ,,

where P is the dilution of the initial suspension with phosphate buffer to 0, „„ 0.2-0.5;

P - dilution luminescent immunoglobulin; dilution with deoxycholate;

Pz P - breeding gel

should be 5 40, ... 10 cells / ml.

The invention can be used in standardizing devices intended for counting cells in suspensions, concentrators, devices used for monitoring environmental pollution by microbiological plants. In addition, the compound can be used to determine the quantitative and qualitative compositions of cell suspensions in ready-made forms of vaccines and drugs.

Claims (1)

Invention Formula The method of determining the concentration of microorganisms in suspension, which includes staining it, diluting, fixing the cells to the medium, applying the suspension to a glass slide, followed by microscopic examination, characterized in that, in order to verify the accuracy of the method and reduce the time of determination, the suspension of cells is diluted with saline, containing 0.85% sodium chloride, to a concentration of 10–10 cells / ml, a luminescent immunoglobulin specific to this microorganism is added, and the mixture obtained removing immunoflyuorestsentvoy until completion of the reaction, is added thereto deoxycholate 513828D86 and polymer gel, and before the polymerization microscope, cover the mixture applied on the base glass with a coating and incubate until the glass has hardened, maintain until the beginning of the development.