THE COMPLEX METHOD FOR DETERMINATION OF INTESTINAL MICROFLORA COMPOSITION OF LABORATORY ANIMALS

M. V. Kaminska

Determination of qualitative and each test tube) are prepared to identify individual quantitative composition of intestinal microflora species of enterobacteria. Test tubes with slope of the laboratory animals, namely albino rats is medium can be stored in the refrigerator for 30 important for conduct of the study regarding days. assessment of the impact of probiotic For microscopic examination of the preparations for the restoration of intestinal samples from Blaurock medium and suspicious microbiocenosis of animals under the colonies from other media, set of reagents for experimental dysbiosis. There is no single Gram staining and microscope are used. common method to establish this composition. Selection and preparation of samples. Normative documents establish methods for the Samples of rat intestinal content (commonly quantitative determination of the content of from the rectum) are taken immediately after specific groups of microorganisms [1-4], such as slaughter. For this purpose, a region of intestine, E. coli, staphylococci, proteus, pathogenic the content of which will be investigated is cut enterobacteria. However, the research work with sterile scissors, and placed into a sterile Petri should simultaneously determine different dish. Further, applying aseptic regulations, groups of microorganisms in one sample of the intestine is cut and its content is removed. 1 g of intestinal content to establish the overall picture content is placed in the sterile weighing bottle of infringements or correction of the composition and 9 mL of sterile normal saline solution is of intestinal microbiocenosis of the laboratory added. Thus, ten-fold dilution (10-1) is obtained. animals. It is not infrequent that the intestinal The aim of the work was to develop and region has less content than one gram, especially provide generalizing method for establishment of when working with sick animals. Then maximum qualitative and quantitative composition of possible number of content is weighed, and microflora of the intestinal content in laboratory volume of normal saline solution to be included albino rats. in the weighing bottle is-recalculated for 10-fold Preparation of glassware and selective dilution. For example, when the sample weight is media. Each sample requires preparation of 500 mg, we are adding 4.5 mL of solution, and in sterile weighing bottle (for entering sample of the case of 300 mg – 2.7 mL of solution. intestinal content of 1 g), sterile glass rod, 7 Weighing bottles with samples are not keep, and sterile spatula, 3 sterile test tubes, 1 mL (6 pcs.) immediately used for inoculation of media. and 10 mL (for dilutions) sterile pipettes, 8 sterile Before preparing dilutions, content of the Petri dishes. weighing bottle is thoroughly mixed with sterile All glassware is sterilized in a steam glass rod. sterilizer for 30 minutes at a pressure of 0.75 atm. Preparation and inoculation of and temperature of 120°C. dilutions. Selective media are prepared and To prepare the required dilutions, three sterile sterilized according to the instructions of the test tubes are used in which 9.9 mL of sterile manufacturer and poured into sterile Petri dishes. normal saline solution is added. Therefore, the following is prepared: 3 dishes Sample 0.1 mL of solution from the with Endo medium, 1 dish with bismuth-sulphite weighing bottle with sterile pipette and transfer to medium, 1 dish with Sabouraud medium, 1 dish a test tube No. 1 with 9.9 mL of normal saline with blood agar, 1 dish with Baird-Parker solution, and then shake. Thus, we obtain 10-3 medium. Blaurock medium is prepared as dilution. Transferring 0.1 mL of the solution from semi-liquid in accordance with recipe [5], poured this test tube to the next test tube (tube No. 2), we into 2 test tubes of 4.5 mL and autoclaved with obtain 10-5 dilution, and so on under the scheme cotton stoppers. Sterile slope agarized (Fig. 1). Olkenytskyi and Simons media (10 of each for After preparation of all dilutions,

withdraw 0.1 mL of the solution from each test representatives of staphylococci, streptococci, tube using sterile pipette and place into the Petri and E. coli are predominant. Number of dish with a corresponding selective medium, staphylococci is within 104-106 CFU/g, and the increasing each dilution for another 10 times. The number of pathogenic strains does not exceed solution is rubbed over the surface with a sterile 10% of the total number of coccal forms. The spatula until its complete absorption with agar. most numerous group of microorganisms is Place 0.5 mL of the solution with certain bifidobacteria and lactobacilli, which generally dilutions into the test tubes with Blaurock account for about 99% of the total number of medium, and at the same time dilution degree is microorganisms in the intestine of rats. Their increased ten times. number is 108-1010 CFU/g for bifidobacteria and Thus, according to the scheme (see. Fig. 1), 106-108 CFU/g for lactobacilli. Reduction of their withdraw 0.1 mL of solution from the weighing number by two decades (100 times), at the bottle and place into the Petri dish with Endo background of the growing number of medium and bismuth-sulphite agar, getting a 10-2 opportunistic groups of bacteria indicates the dilution. From the test tube No. 1 place 0.1 mL in occurrence of a serious intestinal dysbiosis. the dishes with Endo medium, blood agar, Number of fungi of the genus Candida ranges Baird-Parker and Sabouraud agar. This resulted within 104-105 CFU/g. Increase in their number in a 10-4 dilution. From the test tube No. 2 place by decade or more suggests occurrence of fungal 0.1 of solution into the dish with Endo medium dysbiosis. Development of Proteaceae dysbiosis and 0.5 mL of solution into the test tube with is evidenced by the rise in the number of proteus Blaurock medium obtaining 10-6 dilution. cells from 102-103 CFU/g to 104-105 CFU/g. Withdraw 0.5 mL of the solution from test tube Total number of other lactose negative No. 3 into the test tube with Blaurock medium opportunistic enterobacteria ranges from 102-103 and obtain 10-8 dilution. CFU/g. Leave inoculated dishes and test tubes on It should also be noted that study of the table for 15 minutes, and then put in a intestinal microflora of laboratory rats, which are thermostat at 37°C. Dishes with Endo medium, fed with probiotic supplements allows to predict blood agar and bismuth-sulphite agar are increase in the total number of bifidobacteria and controlled 24 hours after inoculation, dishes with lactobacilli, therefore, it is required to make Baird-Parker and Sabouraud medium – after 48 additional dilution (test tube No. 4), and inoculate hours, and Blaurock medium undergoes Sabouraud medium with 10-10 dilution. In case of microscopy at day 4 of growth. dysbiosis modelling by administration of Evaluation of results. Determination of antibiotics, we predict decrease in the total qualitative and quantitative composition of number of E. coli or staphylococci (depending on intestinal microflora of laboratory animals makes the type of antibiotic), and bifidobacteria and it possible to judge the health of animal abuse lactobacilli. Therefore, inoculation on selective dysbiotic infringements in the composition of medium for these types of microorganisms intestinal microflora and their correction with should be done at lower dilutions. probiotics. Thus, we have proposed the complex Thus, according to our studies [7-11], method for determination of intestinal normal value of the total number of E. coli in microbiocenosis of laboratory animals, which laboratory rats is within 106-108 CFU/g, while the generally takes 4 days, but makes it possible to number of weak-fermenting strains does not determine the qualitative and quantitative exceed 25%. There are isolated lactose negative composition of microflora content of rat rectum strains. Among haemolytic microorganisms, and diagnose its infringements.