Method for microorganism adhesive potential rapid test

FIELD: medicine.

SUBSTANCE: method involves the preliminary sorption of a specific ligand in polystyrene tray wells; the above ligand is specified in: haemoglobin, myoglobin, collagen, fibrinogen, fibronectin, immunoglobulin A; that is followed by adding a microorganism cell suspension into the tray wells with the sorbed ligand, incubating the cell suspension in the tray wells for 15 minutes, sampling a suspension aliquot from the well and adding it to wells of another or the same tray containing 0.5% sodium chloride or 0.2 M sodium phosphate; the bacterial cell adhesion is assessed by measuring a decrease of the optical density of the prepared diluted suspensions at wave length 600 nm as compared to the well references free from ligands.

EFFECT: invention is characterised by a high test rate of the microorganism adhesion to ligands of various nature, high reproducibility, using a minimum amount of the microorganism biomass, with no need for hazardous chemicals to be used.

7 tbl, 7 ex

 

The invention relates to Microbiology and can be used in medical bacteriology and medical Mycology to quantify the adhesive properties of conditionally pathogenic microorganisms isolated from human and environmental objects, to characterize their virulent properties in vitro the level of adhesion to ligands of different nature.

There are different approaches to quantify the level of adhesion of cells of microorganisms (bacteria and yeast-like fungi) to ligands of different nature.

There is a method of quantitative determination of cell adhesion of microorganisms to collagen and fibronectin, based on the specific staining of adhered cells dye crystal violet, however, such method requires deficitii cells with glutaraldehyde, the use of additional procedures for cell disruption using the detergent Triton X-100 [1].

A simpler approach is to use membranes that enclose the appropriate ligand (e.g., hemoglobin) [2]. In this case, the cells of the microorganisms associated with membrane carrier ligands, using specific adhesins, located on the surface of their cell walls. Quantitative adhesion level in this case is determined by counting the number of glue is OK microorganisms, adhered on the membrane with ligands, or by reducing the optical density of a suspension of cells of the microorganism after interaction with the membrane. However, this approach requires waste approach in the manufacture of membranes using specific components of nitrocellulose, organic solvents. In practice requires the use of relatively large amounts of membranes (to achieve a large area of the working surface of several square centimeters) and large volume (several milliliters) of a suspension of microorganisms, which is not always possible to use this method with a small number of biomaterial, for example in the presence of one small colonies of microorganisms growing on solid (agar) nutrient medium.

The closest to our invention include a method for determining the adhesion of microorganisms to the ligands adsorbed on the polystyrene surface of a 96-well plate [3]. This approach allows to avoid the use of different, including unsafe substances, as well as to bring the work to the use of micro volumes (about 100 µl), do not require a large amount of source material. However, the described invention has a number of disadvantages. First, to quantify the adhesion of cells of microorganisms are offered either the adding in of the wells (containing the adhered cells) liquid nutrient medium, either selection aliquots of a suspension of microorganisms from wells in the liquid (or culture on solid nutrient medium followed by incubation and growth assessment (counting the resulting colonies) culture of microorganisms, which allows to obtain results only after a significant amount of time (from 6 to 48 h depending on the incubation conditions and the type of microorganism). In addition, in this case, do not take into account the possibility of differences in growth rate among different strains of the same species of microorganism that can artificially increase or dilute the real difference in adhesion potential of specific strains. Secondly, the use of quantitative assessment of adhered cells of microorganisms for determining the activity of bacterial enzymes in adhered cells is practically unusable due to the variation of the activity (number) enzymes in different strains of the same species of microorganism [4] and, consequently, the lack of absolute correlation between the level of enzyme activity and the level of adhesion potential. This approach can also increase or dilute the real difference in adhesion potential of the compared strains.

In this regard, the objective of the claimed invention is to develop a simple way to quickly, less h is m for 1 hour, using the minimum amount of biomass of microorganisms in microvolumes without the use of chemical reagents to quantify the level of adhesion of cells of microorganisms (bacteria and yeast-like fungi) to ligands of different nature.

This object is achieved through the development of a detection method, which involves a preliminary sorbirovaniya in the wells of polystyrene tablet specific ligand, is added to wells of a suspension of cells of microorganisms, a short incubation of the cell suspension in the wells, the subsequent selection of an aliquot of the suspension from the wells and add it to the wells of another or of the same tablet, containing an aqueous solution of salt (for example, 0.5% sodium chloride or 0.2 M of sodium phosphate), followed by assessment of the level of adhesion of bacterial cells by determining the decrease of optical density obtained with the diluted suspension at a wavelength of 600 nm. On the basis of data obtained from optical density to determine the index of adhesion. The adhesion index (in percent) is calculated by the formula:

IA=100-100×(OP600experience/OP600control)

where

OP600experience the value of optical density of the suspension at 600 nm, selected from the wells containing ligand;

OP600control - the value of optical density is spencie at 600 nm, selected from the wells not containing ligand.

The higher the index of adhesion, the higher the level of adhesion of the microorganism to the selected ligand.

The technical result of the claimed invention is to develop a rapid method for determining the level of adhesion of microorganisms to ligands of different nature, with good reproducibility, a minimum quantity of biomass of microorganisms, using the minimum amount of biomass of microorganisms in microvolumes, there is no need to use dangerous chemicals.

Example 1. The determination of the level of adhesion of Staphylococcus aureus to the hemoglobin. In wells pre-Sorb ligand - human hemoglobin. For this hemoglobin is dissolved in 0.2 M sodium phosphate buffer solution with pH 7.4 rate of 20 μg/ml of the Prepared solution in a volume of 50 μl contribute to the wells, incubated for 24 h at 4°C. the wells washed three times with 0.2 M sodium phosphate buffer solution, pH 7.4, containing 0.05% tween-20, filling 100 ál in each well, then the tablet drained by shaking out the remaining liquid. The thus prepared tablets can be stored for years at 4°C in dry conditions. In the finished wells made in 200 μl of a suspension of Staphylococcus aureus in 0.2 M sodium-fo is Putnam buffer solution at pH 7.4 with an optical density at 600 nm in the range from 0,500 to 0,600. Incubare for 15 minutes, then extracted with 100 μl of the suspension of the pit and move it to another well containing 100 μl of 0.2 M sodium phosphate buffer solution with pH 7.4, stir the diluted suspension and then measure the optical density of the obtained diluted suspensions at 600 nm. The control options are wells that do not contain hemoglobin. The results are presented in table 1.

Example 2. The determination of the level of adhesion of Staphylococcus aureus carried out analogously to example 1, but as a specific ligand adsorbed on the tablet, use the myoglobin. The results are presented in table 2.

Example 3. The determination of the level of adhesion of Staphylococcus aureus carried out analogously to example 1, but as a specific ligand adsorbed on the tablet, use collagen. The results are presented in table 3.

Example 4. The determination of the level of adhesion of Staphylococcus aureus carried out analogously to example 1, but as a specific ligand adsorbed on the tablet, use fibronectin. The results are presented in table 4.

Example 5. The determination of the level of adhesion of Staphylococcus aureus carried out analogously to example 1, but as a specific ligand adsorbed on the tablet, use the fibrinogen. The results are presented in table 5.

Example 6. Determination of the level is desii strains of Staphylococcus aureus carried out analogously to example 1, but as a specific ligand adsorbed on the tablet, using immunoglobulin A (IgA). The results are presented in table 6.

Example 7. Determination of the adhesion level is carried out analogously to example 1, but as the microorganism used yeast-like fungus Candida albicans. The results are presented in table 7.

0,269
Table 1
The determination of the level of adhesion of Staphylococcus aureus cells to hemoglobin
The strain of S. aureus from the skin of a healthy personThe strain of S. aureus with human skin with atopic dermatitis
S. aureus was ATSS 25923
controlhemoglobincontrolhemoglobincontrolhemoglobin
the OD value of 600 nm
0,3000,2800,3100,2850,315
The index of adhesion %
6,78,014,6

Table 2
Determining the level of cell adhesion of Staphylococcus aureus to myoglobin
The strain of S. aureus from the skin of a healthy personThe strain of S. aureus with human skin with atopic dermatitis
S. aureus was ATSS 25923
controlmyoglobincontrolmyoglobincontrolmyoglobin
the OD value of 600 nm
0,3000,2830,310in 0.2880,3150,270
The index of adhesion %
5,67,014,2

Table 3
Determining the level of cell adhesion of Staphylococcus aureus to collagen
The strain of S. aureus from the skin of a healthy personThe strain of S. aureus with human skin with atopic dermatitis
S. aureus was ATSS 25923
controlcollagencontrolcollagencontrolcollagen
the OD value of 600 nm
0,3000,2850,3100,2900,3150,279
The index of adhesion %
5,06,411,4

Table 4
Determining the level of cell adhesion of Staphylococcus aureus to fibronectin
The strain of S. aureus from the skin of a healthy personThe strain of S. aureus with human skin with atopic dermatitis
S. aureus was ATSS 25923
controlfibronectincontrolfibronectincontrolfibronectin
the OD value of 600 nm
0,300in 0.2880,3100,2950,3150,287
The index of adhesion %
4,04,88,8

Table 5
Determining the level of cell adhesion of Staphylococcus aureus to fibrinogen
The strain of S. aureus from the skin of a healthy personThe strain of S. aureus with human skin with atopic dermatitis
S. aureus was ATSS 25923
controlfibrinogencontrolfibrinogencontrolfibrinogen
the OD value of 600 nm
0,3000,2850,310in 0.2880,3150,285
The index of adhesion %
5,07,09,5

Table 6
Determination of the level of edge the AI Staphylococcus aureus cells to IgA
The strain of S. aureus from the skin of a healthy personThe strain of S. aureus with human skin with atopic dermatitis
S. aureus ATCC 25923
controlIgAcontrolIgAcontrolIgA
the OD value of 600 nm
0,3000,2910,3100,2970,3150,295
The index of adhesion %
3,04,16,3

Table 7
The determination of the level of adhesion of Candida albicans cells to hemoglobin
The strain of C. albicans from the skin h is Oronogo man The strain of C. albicans with human skin with atopic dermatitis
Candida albicans strain Museum No. 4
controlhemoglobincontrolhemoglobincontrolhemoglobin
the OD value of 600 nm
0,3000,2750,3100,2800,3150,273
The index of adhesion %
8,39,613,3

LITERATURE

1. Pynnonen M., Stephenson, R. E., Schwartz K., M. Hemandez, B. R. Boles / Hemoglobin promotes Staphylococcus aureus nasal colonization // PLoS Pathogens, 2011.

2. Lisovskaya S. A. / the dissertation on competition of a scientific degree of candidate of biological Sciences "a New approach to the assessment of the pathogenic potential of clinical strains of Candida albicans" // Kazan, 2008. 25 C.

3. Tyurin, Y. A., pasahow R. S., Mustafin, I. / the Method to determine the adhesion of Staphylococcus spp.to hemoprotein the m // Patent RU No. 2393229.

4. Bayazitova L. T., Tyurin, Y. A., Kulikov S. N., Dolbin D. A., pasahow R. S. / study of the antibacterial activity of the drug, "Skin cap" against Staphylococcus aureus when local therapy of atopic dermatitis // Practical Medicine, 2009, №3 (35), S. 89-91.

The method for determining the adhesive potential of microorganisms, characterized in that in the wells of polystyrene tablet absorb specific ligand selected from the range: hemoglobin, myoglobin, collagen, fibrinogen, fibronectin, immunoglobulin And add in wells with sorbed ligand cell suspension of the microorganism, spend incubation of the cell suspension in the wells for 15 minutes, take an aliquot of the suspension from the wells and add it to the wells of another or the same tablet containing an aqueous salt solution of 0.5% sodium chloride or 0.2 M of sodium phosphate and assess the level of adhesion of bacterial cells by determining the decrease of optical density obtained with the diluted suspension at a wavelength of 600 nm when compared with the control variant holes, not containing ligand.



 

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2 ex, 3 tbl

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29 cl, 23 dwg, 20 tbl, 23 ex

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2 tbl, 2 cl, 1 ex

FIELD: biotechnology.

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EFFECT: invention enables to reduce the time differentiation of Rhodococcus and to improve the accuracy of the method.

2 cl, 3 tbl, 4 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. The method comprises collecting soil samples, adding metsulfuron-methyl (MSM) to the sample in a given amount, followed by incubation and determining specific metabolic activity of the saprophytic soil complex by multisubstrate testing and counting the number of colony-forming units on a diluted agarised medium. The level of the detoxification activity is estimated based on the relative increase in specific metabolic activity of the saprophytic microbial community of the soil when MSM in comparison with characteristics of the soil without adding MSM (Kd factor).

EFFECT: improved method.

4 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: group of inventions relates to biotechnology. Claimed is a method of growing colonies of microbial cells on a surface of a porous plate. The method includes supply of a nutrient solution from bottom to top through the porous plate into zones of growth of colonies of the microbial cells on its upper surface, supply of a suspension of the microbial cells onto the upper surface of the porous plate, creation of controlled conditions for the colony growth, performing observation of the colony growth, separation of the grown colonies of the microbial cells from the zones of growth and their transfer into external means of identification. The nutrient solution is supplied into the zones of growth of the colonies of the microbial cells by creation of a pressure difference between the hole input and output. Holes are made in the plate from an anode aluminium oxide orthogonally to its large plane and are topologically coded. The said zones of growth are formed in them in the form of porous membranes. The porous membranes are located at the same level as the upper surface of the plate or with formation of a hollow and do not pass the microbial cells. After supply of the nutritional solution, the suspension of the microbial cells of a specified concentration is supplied onto the upper surface of the plate until their homogenous distribution is achieved. Between the zones of growth on the surface of the plate a film, preventing attachment of the microbial cells, is formed. Separation of the grown microcolonies from the zones of growth is performed by hydroblow. A hydroblow is directed from the side of the input of cylindrical holes of the plate and spreads along them and farther through the pores of the porous membranes with force, which does not destroy the microcolonies but is sufficient for their separation from the growth zones. Also claimed is a device for growing the colonies of the microbial cells by the claimed method.

EFFECT: providing conditions of automation of processes of the nutrient solution supply and processes of separation and transfer of the grown colonies, possibility of integration into miniature portable devices, and application in laboratories on a chip and provision of the device portability.

6 cl, 14 dwg, 4 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to microbiology and can be used in monitoring environmental-microbiological investigation of the quality of sea water to determine the amount of oil-oxidising microorganisms. The method involves preparing a mineral medium - bases containing NH4NO3, K2HPO4, KH2PO4, MgSO4, CaCl2, FeCl2, a concentrated solution, agar and distilled water in a given ratio, followed by addition of an oil product in a given amount, said product being bunker oil. Seeding sea water on the surface of the culture medium and incubating the seed for 3-4 hours enables to detect colonies of oil-oxidising bacteria.

EFFECT: invention increases precision of the method when detecting oil and hydrocarbon oxidising bacteria when carrying out environmental monitoring.

2 tbl, 3 dwg, 5 ex

FIELD: biotechnology.

SUBSTANCE: method of toxicity assessment of products from polymer and textile materials is proposed. The method comprises the use of biosensor based on oxygen electrode, immobilisation of whole cells of bacteria E.coli K-12 on the surface of the oxygen electrode. The immobilisation is carried out using a semipermeable membrane. After immobilisation the respiratory activity of microorganisms is measured in the presence of the sample and standard samples of positive and negative control. Then the toxicity index is calculated and the sample toxicity is evaluated based on the value of the toxicity index.

EFFECT: simplifying assessment of toxicity and improvement of reliability of the results of the sanitary-epidemiological expertise.

2 dwg, 1 tbl, 3 ex

FIELD: biology.

SUBSTANCE: invention is designated for biotesting water and aqueous extract samples. Daphnia are immersed firstly in solution or water for culturing to be tested and then daphnia are transferred into lightproof chamber with outlet hole for culturing and this chamber is immersed into water. Time passing out daphnia from the testing chamber to light is measured and toxicity of analyzed solution is estimated by difference time in daphnia passing out. Method allows carrying out the rapid control of water and aqueous extracts toxicity in laboratory and field conditions.

EFFECT: improved method for biotesting.

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