UA150070U - METHOD OF ACCELERATED DETERMINATION OF FUNGICIDAL ACTIVITY OF COLLOID DISPERSIONS OF METAL NANOPARTICLES CONCERNING ASPERGILLUS FUMIGATUS, ASPERGILLUS FLAVUS AND ASPERGILLUS - Google Patents

Abstract

Method of accelerated determination of fungicidal activity of colloidal dispersions of metal nanoparticles on test cultures of mold micromycetes Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger includes cultivation of test cultures, sowing in nutrient media, washing, standardization and incubation. To enrich and accelerate the growth of test cultures add to the nutrient medium agar mixture of salts (Fe (NO3) · 9H2O, ZnSO4 · 7H2O, MnSO4 · 4H2), incubation is carried out at a temperature of (37 ± 0.5) ° C, counting the number of colonies carried out in terms of 1, 3, 5 and 7 days.

Description

The useful model belongs to the field of veterinary medicine, sanitation, microbiology, mycology, in particular, to the determination of the fungicidal activity of colloidal dispersions of metal nanoparticles in relation to AzregouPyv Titidatsv, AzregouyPyv5 Yaami5 and Azregayiz podeg.

It is known that nanoparticles of Argentum and Cuprum exhibit clearly expressed biological activity.

In particular, the bactericidal effect of Argentum and Cuprum nanoparticles on the strains of aerobic and anaerobic microflora EzsPegisnia soii, Reepdotopaz aegidipos, KierzivIa rpeshtopia, Epiegosossiv iaesaiii5, ZpidePa Pehpegi, ZaitopeiPa iurpitigsht, Rgoii5 myidagiv, and earnpuiosossiv, fungicidal activity was established.

There is a way to determine the bactericidal effect of disinfectants on test cultures of bacteria - "Methodical instructions on the procedure for testing new disinfectants for veterinary practice". Moscow, Utv. GUV Gosagroproma USSR 07.01.1987". This method determines the bactericidal concentrations of disinfectants on strains of test cultures EuspPegisnpia soi, Earnuiosossiv agei5, ZGeriosossiv Gaesaiii5, Misobasieitst rpiei, Oozrogaiasiikh, Rzendotopav aegodiposa.

The disadvantage of the known method is the limited scope of application, that is, it can be used only to determine bactericidal properties.

The closest to the claimed method is the method of determining the fungicidal properties of disinfectants on test cultures of the genus Azregaish5 (patent of Ukraine for a utility model Mo 47469 IPC (01 M33/00).

The method is based on the determination of the fungicidal (fungistatic) effect of disinfectants on test cultures of Azregdiyi and Titidatshv5, Avreguyi5 Pamiye and Azregoyi5 pods, standardized by the number of fungal spores in 1 cm3 of water suspension. The method includes: cultivation of test cultures, sowing in nutrient media, washing, standardization, incubation and counting of grown colonies. This solution can be a close analogue.

The disadvantage of the method is that it is long in terms of time (observation and counting of colonies that have grown over 21 days), the seeds are carried out in different nutrient media (wort agar and

Chapek), incubation - at a temperature of (25-27)"C, and the method does not determine the fungicidal activity of colloidal dispersions of metal nanoparticles.

The task of developing a method of accelerated determination is set as the basis of a useful model

From the fungicidal activity of colloidal dispersions of metal nanoparticles on test cultures of mold micromycetes Avzregadiiy5 yitidatv5, Avreguyiv Pamiye ota Avzregdiyy5 a method that includes growing test cultures, sowing in nutrient media, washing, standardization, incubation and counting colonies by adding to the nutrient medium wort agar a mixture of salts (Re(Моз):9Н2гО, 27п504:7Н2гО, Мп5О.4:4Н»г) for enriching and accelerating the growth of test cultures, incubation at a temperature of (37х0.5)С and counting the number of colonies that grew in terms of 1 , 3, 5 and 7 days.

A comparative analysis with a close analogue allows us to conclude that the enrichment of wort agar with a mixture of salts, increasing the incubation temperature leads to a reduction of the incubation period and the counting of colonies of test cultures to 7 days, which meets the "novelty" criterion.

The method is performed as follows.

Cultivation of test cultures AzreguyShivye Titidaish5, Avzregodiyy5 Pamie and Azregoyiv5 podeg is carried out on a dense nutrient medium used for isolation and cultivation of mushrooms - wort agar.

To prepare agar wort, non-hoppy beer wort, which contains 12-1495 sugar, is filtered through a thin layer of cotton wool and diluted with distilled water (in a ratio of 1:2 or 1:3) to (3-7) C according to the Balling hydrometer, add (1 ,8-2.0)96 agar-agar and a mixture of salts (Re(MOz):9NgO, 2p505 7NgO, Mp5O. 4H") in the amount of 2.0 cm per 1.0 dm" of the medium. The finished agar is poured into test tubes or another container and sterilized in an autoclave at a temperature (110-11237C, pressure - 0.5 kgf/cm" for 20 minutes.

Before seeding, test tubes with strains of museum test cultures, after storage in the refrigerator at a temperature of (4-8)"C, kept at room temperature (18-20)"C for at least an hour, sown on slanted wort agar, 4 tubes for each strain and kept in a thermostat for 7 days. Spores of each 7-day test culture (Azregaiiy5 Titidagshv5, Avregoaiy5 PChamiv or Avregaiiv pede") are washed with 5.0 cm of 0.595 Tween-80 solution and combined in a separate sterile vial. Is the suspension of the test culture used in the experiment standardized by the number of spores in 1 cm? in Goryaev's cell. Spores are suspended from each dilution, 1 drop of each dilution is taken with separate Pasteur pipettes, and the number of spores is counted under a microscope (when magnifying the lens x90 and the eyepiece x10). The working dilution of the spore suspension is considered to contain 120 spores in 1/5 mm3, i.e. 600,000 in 1 cm3.

In the experiments, positive and negative controls are prepared from the tested cultures at different time intervals. A working dilution of the spore suspension of test cultures of museum strains (120 spores of 1/5 mmu) not treated with colloidal dispersions of metal nanoparticles and added to the nutrient medium with antibiotics (penicillin - 50 thousand

IU, streptomycin - 100 thousand IU per 1 dm") to suppress accompanying bacterial flora.

The negative control is prepared with the introduction of the well-known fungicide nystatin into the nutrient medium (30 mg of nystatin per 100 cm" of the medium). Incubation of the seeds is carried out at a temperature of (370.5)"C and in terms of 1, 3, 5 and 7 days the number of colonies is counted that grew up

Processing of analysis results.

To determine the average result of the number of fungal colony growth from the determination of the fungicidal activity of colloidal dispersions of metal nanoparticles on test cultures of mold micromycetes AzregoPivitidati5, Avreguyi5 Pamy5 and Azregdiyni5 podeg, colonies are counted in at least three repetitions. Based on the results obtained at different times, the indicator of the average number of colonies is calculated, the variation series is written out and the median is determined.

The average result of the number of mushroom colonies that grew for each term using a certain concentration range of colloidal dispersions of metal nanoparticles is determined by running the DUMA of all colonies per number of Petri dishes of all repetitions:

M with where X is the average number of colonies grown in Petri dishes;

X is the number of colonies in the first, second, third cups, etc

M - the number of experiments (repetitions).

Example 1. Determination of fungicidal activity of colloidal dispersions of nanoparticles

Argentum, Kuprum regarding the museum strain of the test culture A 5regoiiv5 Tit i dai i5.

For determining the fungicidal activity of colloidal dispersions of Argentum nanoparticles,

Kuprum with regard to the museum strain of the test culture AvzregaiShy5 and yitidayb5 found that fungistatic properties were manifested by MRAd and MRSi at a concentration of 150 μg/cm3 after exposure for 60 minutes. and 180 min. in accordance.

In particular, the average number of Azregaishv5 Tshtidaiishv5 colonies that grew, in the case of exposure in

From a solution of MRAd with a concentration of 100 μg/cm3 and an exposure of 60 min. the average indicator of mushroom colonies was 22.0, and at 150 μg/cm? and 200 µg/cm" for metal - 17.0 and 19.0 CFU, respectively, against continuous growth of micromycete spores in the "positive" control.

When a spore suspension is kept in a solution of MRSA with a concentration of 150 μg/cm3 and 200 μg/cm3 at an exposure of 180 min. the average number of fungal colonies was 11 and 19 CFU of the culture, respectively, compared to the continuous growth of microscopic fungi in the "positive" control.

On the 7th day of recording the total number of colonies of the AzregouyShiv Ttidashv test culture, the fungicidal properties of MRAd were recorded at concentrations of 100, 150 and 200 μg/cm3 for exposure of 60 minutes.

Example 2 Determination of fungicidal activity of colloidal dispersions of Argentum nanoparticles,

Kuprumu regarding the museum strain of the test culture Azregoish5 Pamiv.

For determining the fungicidal activity of colloidal dispersions of Argentum nanoparticles,

Cuprum in relation to the museum strain of the test culture A5regoya5 Pama5, fungistatic properties were shown by MRAYA and MRSYi at a concentration of 150 μg/cm3 after exposure for 60 minutes. and 180 min. in accordance.

In particular, the average number of colonies of Azregdyia5 Pama5 that grew, in the case of exposure in a solution of MRAd with a concentration of 100 μg/cm3 and exposure for 60 minutes, the average number of fungal colonies was 9.0, and at 150 μg/cm? and 200 μg/cm3 for metal - 11.0 and 15.0 CFU, respectively, against continuous growth of micromycete spores in the "positive" control.

When a spore suspension is kept in a solution of MRSA with a concentration of 150 μg/cm3 and 200 μg/cm3 at an exposure of 180 min. the average number of fungal colonies was 11 and 17 CFU of the culture, respectively, compared to the continuous growth of microscopic fungi in the "positive" control.

On the 7th day of recording the total number of colonies of the Azregoi test culture, 5 PYAamivs recorded the fungicidal properties of MRAd at concentrations of 100, 150 and 200 μg/cm3 for exposure of 60 minutes.

Example C Determination of the fungicidal activity of colloidal dispersions of Argentum nanoparticles,

Kuprum in relation to the museum strain of the test culture Azregaiiy5 podeg.

For determining the fungicidal activity of colloidal dispersions of Argentum nanoparticles,

Cuprum in relation to the museum strain of the test culture Avzregadiyya5, fungistatic properties were shown by MRAZ and MRSY at a concentration of 150 μg/cm3 after exposure for 60 min and 180 min, respectively.

In particular, the average number of colonies of Azregdysha5 will grow, in the case of exposure to a solution of MRAd with a concentration of 100 μg/cm7 and exposure for 60 minutes. the average indicator of fungal colonies was 12.0, and at 150 μg/cm3 and 200 μg/cm? by metal - 15.0 and 18.0 CFU, respectively, against continuous growth of micromycete spores in the "positive" control.

When a spore suspension is kept in a solution of MRSA with a concentration of 150 μg/cmU and 200 μg/cm3 with an exposure of 180 min. the average number of fungal colonies was 10 and 16 CFU of the culture, respectively, compared to the continuous growth of microscopic fungi in the "positive" control.

Already on the 7th day of recording the total number of colonies of the test culture AzregoyShiv podeg, the fungicidal properties of MRAd were recorded at concentrations of 100, 150 and 200 μg/cm3 and exposure for 60 minutes.

This method of determining the fungicidal activity of colloidal dispersions of metal nanoparticles against Azregadiya5 Titidasa5, A5regodiya5 Pama5 and Azregadiya5 podega will find wide implementation and effective use for controlling the degree of contamination by mold fungi after the application of nano-containing products.

Claims (1)

USEFUL MODEL FORMULA The method of accelerated determination of the fungicidal activity of colloidal dispersions of metal nanoparticles on test cultures of mold micromycetes Azregai5 Titiday5, AzreguiShy5 Pamiv and AzregadiiiPy5 podega, which includes the cultivation of test cultures, sowing in nutrient media, washing, standardization, incubation and counting of colonies, which is distinguished by the fact that to enrich and accelerate the growth of test cultures, a wort agar mixture of salts (Be(MOz): 9H2O, 7p5O4y: 7H2O, My5O.: 4H»g) is added to the nutrient medium, incubation is carried out at a temperature of (370.5) "С, calculation number of colonies is carried out in terms of 1, 3, 5 and 7 days.