CS233440B1 - Agar-egg soil for cultivation of mycobacteriums - Google Patents

Abstract

The invention relates to agar-egg yeast culturing mycobacteria. The agar egg composition of the present invention from 0.25% by weight of meat extract, 0.43% by weight of peptone, 1.28% % agar, 0.33 wt% sodium chloride, 2.57% by weight of glycarol, 81.73% distilled water, 13.08% weight egg yolk, 0.33% w / w Salsa malachite zetane. The agar-egg pbd of the invention is and other egg products used for cultivating mycobacteria and increasing does not require import chemicals. Its an advantage However, there is less for primary cultivation susceptible to nonspecific mlkroflore, whereby increases diagnostic reliability.

Description

The invention relates to an agar-egg supplement for the cultivation of mycobacteria.

Cultivation procedures in mycobacteriology, including the selection and preparation of culture supplements, are very demanding compared to other methodologies used in bacteriology. Among the many mycobactar nutrients known today, solid egg and liquid proteins containing a fat protein are most suitable (Sula, 1970). Of these, Lowenstein-Jensen addition is the most important. The choice of culture media depends on the methodologies used in each laboratory. Laboratory investigative methodologies in veterinary medicine (1974) determine the addition of Petragnani, Stonebrink and Sula for the cultivation of mycobacteria. Standard RVHP methods for laboratory diagnosis of tuberculosis (1980) suggest Lowenstein-Jensen addition for mycobacterial culture. Laboratory methods in veterinary mycobacteriology used in the USA (1975) determine four types of pfid for cultivation: LówenstedLn-Jensen, Herold, Middlebrook 7 H 10, Stonebrink.

Efforts to simplify and improve the culture media are ongoing. In our conditions, we are mainly looking for the possibility of replacing expensive chemicals from imports. For example, the importance of jasparagine * and its replacement in Lowenstein-Jensen adds has been discussed by Šlosarek et al. (1981).

In mycobacteriological practice, in particular, in isolating mycobacteria from contaminated material, there are very often cases that primocultivation is unsuccessful due to a non-specific microflora that contaminates the culture media used. In such cases, stronger decontamination agents are used to devitalize the nonspecific microflora, but killing 70 to 80% of the mycobacteria, and hence diagnostic reliability is low. On the other hand, additives less sensitive to non-specific microflora but allowing good growth of mycobacteria may be used. These properties have especially agar additives, which are, however, difficult to prepare and contain a number of imported chemicals.

The above-mentioned drawbacks can be substantially eliminated by the agar-egg additive according to the invention, which consists in that it consists of 0.25 wt. meat extract,

0.43% wt. peptone, 1.28 wt. % agar, 0.33 wt. sodium chloride, 2.57 wt. glycerol, 81.73 wt. distilled water, 13.08 wt. sieve, 0.33% wt. 2% malachite green.

The agar-egg addition of the invention is an opaque, deep green in color with a perfectly smooth and shiny surface, allowing production control both during incubation and subtraction of mycobacterial growth. Store in cold and dark at +4 ° C. In the preparation and storage preparation process it lasts for at least 6 weeks without any apparent impact on its quality.

The agar-egg additive of the invention is equivalent to other egg additions used for cultivating mycobacteria. However, it has the advantage that it is less sensitive to nonspecific microflora in primo-cultures, especially when isolating mycobacteria from environmental samples, thereby increasing diagnostic reliability.

EXAMPLE

Preparation of agar-egg additive g Nutrient agar no. 1 (SEVAC - i.e. 3.13 g meat extract, 5.22 g peptone and 15.65 g agar) after swelling for half an hour in distilled water to a total volume of 1000 ml, boil thoroughly with 4 g sodium chloride and 28 ml glycerol. After cooling to 60 ° C to Up, the pH is adjusted to 7.5 and autoclaved at 121 ° C for 25 minutes. After cooling to 52 ° C, 8 pieces of egg yellow and 4 ml of 2% malachite green are added. After thorough homogenization, the add is dispensed into bacteriological tubes at an inclined position. If it stiffens after 10 minutes.

The object of the invention has been verified and statistically evaluated under experimental conditions.

I. Comparison of the proposed agar-egg soil with the most commonly used solid soils for mycobacteria and Statistical evaluation of results.

Mycobacterial strains from the IHE collection in Prague were used for soil testing.

1. M. scrofulaceum

2. M. avium serotype 2

3. M. intracellulare serotype 4

4. M. intracellulare serotype 8

5 '. M. fortuitum

6. M. phlei

Culture media to be compared:

1. Proposed agar-egg p6da

2. Stonebrink p6da

3. Lowensteln-Jensen p6da

4. Ogawa p8da

5. Herold p6da

During the cultivation, the day on which the growth of the strain and the amount of the grown mass on the 30th day was recorded for the first time was observed. The results were statistically processed by Student's T-test, at a significance level = 0.05 (Table 1).

T a b u 1 'k a 1

Comparing soil t value = 0.05 MH ⁺ : Lowenstell-Jensen 0,175 MH ⁺ : Stonebrink 0,049 1,960 MH ⁺ : Ogawa 0,321 MH ⁺ : Herold 0.22

MH ⁺ = proposed agar-egg soil

It is apparent from the comparison of the values that there is no statistically significant difference between the test media and thus the agar-egg soil according to the invention is as suitable for the cultivation of the selected mycobacterial strains as the reference soil.

II. Preliminary results of the practical use of the agar-egg soil according to the invention.

Agar-egg soil (MH) was used to primo-cultivate mycobacteria from environmental samples in parallel with Herold (H) and Lowenstein-Jensen (LJ) soil.

A total of 35 mycobacterial isolates II, III were isolated. and IV. of the Runyon classification of 11 water and soil samples examined from the marsh site as well as liquid, shed manure, macaques and the like. The p8d score was based on the follow-up of the absolute number of isolates, the number of positive cultures, the number of contaminated p8d by non-specific microflora, the number of p8d without mycobacterial growth (Table 2). The relationship of contamination counts and p8d counts without mycobacterial growth was evaluated by the Chi-square test (Table 3).

I and b u Γ k a 2

If the number is based on the number. number of positives- number of counts counted.

Cultivation of contaminated cultures without growth of mycobacteria isolator

LJ 33 12 36,4 « 3 9.09 » 21 63.6% 3 MH 33 2 6.06% 16 48,5 » 16 48,5% 20 H 33 3 0.09% 12 36,4 « 19 57.6% 12

Table 3

Comparative additions X

LJ: MH 15.9

H, MH 0.512

Critical X ² for 0.05 = 3.8

The evaluation shows that the Herold and HH agar-egg soils are more suitable for isolating atypical mycobacteria from the environment than the Lowenstein-Jensen soil, which is more susceptible to contamination.

Another advantage is that the composition of the agar-egg soil does not require import chemicals, while the ingredients - agar, peptoa and meat extract - are already commercially produced in the semi-product, i.e., in the Czech Republic. Vol. nutrient agar no. 1 n. p. Imuna Saris IfichaXany. It is also advantageous in terms of its ease of preparation, which does not require special precipitation apparatus, such as e.g. AmoldOv precipitation machine.

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.