CS233440B1 - Agar-eggplant for mycobacteria cultivation - Google Patents

Abstract

The invention relates to an agar-egg medium for the cultivation of mycobacteria. The agar-egg medium according to the invention consists of 0.25% by weight of meat extract, 0.43% by weight of peptone, 1.28% by weight of agar, 0.33% by weight of sodium chloride, 2.57% by weight of glycerol, 81.73% by weight of distilled water, 13.08% by weight of egg yolk, 0.33% by weight of malachite zinc. The agar-egg medium according to the invention is equivalent to other egg mediums used for the cultivation of mycobacteria and does not require imported chemicals. However, its advantage is that during primary cultivation it is less sensitive to non-specific microflora, which increases diagnostic reliability.

Description

233440 2

The present invention relates to agar-egg pride culturing mycobacteria.

Cultivation procedures in mycobacterlology, including the selection and preparation of culture media, are very challenging compared to other methods used in bacteriology. Of the several known nutrient pfids for mycobacteria, solid egg and liquid proteins are most suitable in the presence of a proteinaceous protein (Sula, 1970). Of these, Lowensteln-Jensen's pseudonym has been used. The choice of culture media depends on the methodologies used in that laboratory. The laboratory-specific methodologies in veterinary medicine (1974) determine the cultivation of cycobacteria by adding Petragnani, Stonebrink, and Shula. As a standard, RVHP methods for laboratory diagnostics of tuberculosis (1980) design for mycobacterium cultivation Lowenstein-Jennes pfidu. Laboratory methodologies in veterinary mycobacterlology used in the USA (1975) determine four species pfid for cultivation: LówenstedLn-Jensenova, Heroldova, Ulddlebrookova7 H 10, Stonebrinkova.

Efforts to simplify and improve culture media continue. In our conditions, it is primarily the search for the possibility of replacing expensive import chemicals. For example, the significance of japaraparin "and its replacement in Lowenatein-Jensen is discussed by Slosarek et al. (1981). In mycobacteriological practice, especially when isolating mycobacteria from contaminated material, there are very frequent cases where primary culture is unsuccessful because of nonspecific microflora that contaminates used culture media In such cases, the more severe decontaminating agents are used to devitalize non-specific microflora, thereby killing 70-80% of mycobacteria and thus diagnosis reliability is low, on the other hand, less susceptible to non-specific microflora can be used, but allow mycobacteria to grow better. agar pfids, which are difficult to prepare and contain a lot of import chemicals.

These drawbacks can be substantially eliminated by the agar-egg addition of the invention, which consists of 0.25 wt. of meat extract, 0.43 wt. of peptone, 1.28 wt. agar, 0.33 wt. sodium chloride, 2.57 wt. glycerol, 81.73 wt. distilled water, 13.08 wt. egg yolk, 0.33 wt. 2% malachite green.

The agar-egg suspension according to the invention is opaque, deep green in color with a perfectly smooth and shiny surface, which allows for production control in both incubation and mycobacterial growth. It should be stored in a cool and dark place at + 4 ° C. For a preparation and storage process, it will last for at least 6 weeks without any appreciable influence on its quality.

The agar-egg pride of the invention is equivalent to other egg yeast used for mycobacterial culture. Its advantage, however, is that in primoculture less microflora is less sensitive, especially when isolating mycobacteria from external environment specimens, thereby increasing diagnostic reliability. Example 1 Preparation of Agar-Egg Paste 24 g Nutrient Agar # 1 (SEVAC - ie 3.13 g meat extract, 5.22 g pepton and 15.65 g agar) is perfectly swollen in distilled water for half an hour. with 4 g of sodium chloride and 28 ml of glycerol. After cooling to 60 ° C, the pH is adjusted to 7.5 and autoclaved at 121 ° C for 25 minutes. After cooling to 52 ° C, 8 pieces of egg yolk and 4 ml of 2% malachite green are added. After homogenization, it is added to the bacteriological tubes at an oblique position. POD solidifies after 10 minutes. 3 233440

The subject of the invention was verified and statistically evaluated under experimental conditions. I. Comparison of the proposed agar-egg species with the most widely used mycobacterial solids and statistical evaluation of results. Prior to testing, mycobacterial strains from the IHE collection in Prague were used. 1. M. scrofulaceum 2. M. avium serotype 2 3. M. intracellulare serotype 4 4. M. intracellulare serotype 8 5 '. M. fortuitum 6. M. phlei

Comparing culture media; 1. Suggested agar-egg puddle 2. Stonebrink peada 3. Lowenstein-Jensen p6da 4. Ogaw p8da 5. Herold's paw The day of cultivation was followed for the first time to record strain and strain growth on day 30. The results were statistically processed by the Stud T-test, at a significance level = 0.05 (Table 1). T a b u 1 'k and 1

Comparative values t = 0.05 MH +: Lowenstein-Jensen 0.175 MH +; Stonebrink 0.049 1.960 MH +: Ogaw 0.321 MH +; Herold's 0.22 MH + = proposed agar-egg soil From a comparison, it is evident that there is no statistically significant difference between the test media and thus the agar-egg pdda according to the invention is equally suitable for the cultivation of selected mycobacterial strains as the comparative species. II. Preliminary results of the practical use of the agar-egg soil according to the invention.

Agar-egg p8da (MH) was used to directly cultivate mycobacteria from an external environment in parallel with Herold (H) and Lowenstein-Jensen (LJ) p6da.

35 mycobacterial isolates II., III. and IV. Bunyonorvej group classification from 11 examined water and soil samples from the wetland as well as the liquid, manure of pigs, molluscs and the like. The p8d evaluation was based on monitoring the absolute number of isolates, the number of positive cultures, the number of contaminated p8d non-specific micro flora, the number of p8d without mycobacterial growth (Table 2). The relationship of the number of contaminants and the number of mycobacterial growths was not exceeded by the "Chi-quadrat" test (Table 3).

Claims (1)

Agar-unique pSda for the cultivation of mycobacteria, characterized in that it consists of 0.25% by weight meat extract, 0.43% by weight peptone, 1.28% by weight agar, 0.33% by weight sodium chloride, 2.57% by weight glyoerol 81.73% by weight of distilled water, 13.08% by weight of egg yolk and 0.33% by weight of 2% malachite green.