RU2275422C2 - Method for reversion of mycobacterial l-forms - Google Patents

Abstract

FIELD: veterinary microbiology.

SUBSTANCE: the suggested method deals with reinoculation of mycobacterial L-forms onto dense nutritive medium Fast-3L for it additionally contains growth factor - native cattle or equine blood serum at the dosage of about 0.1-0.2 ml/5 ml medium. The innovation enables to increase sensitivity of the method and shorten the terms for obtaining revertant cultures.

EFFECT: higher efficiency.

2 ex, 2 tbl

Description

The invention relates to veterinary microbiology and can be used in bacteriological diagnosis of tuberculosis.

There is a method of accelerated reversal of L-forms of mycobacteria on 7-day-old chicken embryos, when chicken embryos are infected with the resulting L-culture, followed by seeding on a dense Levenshtein-Jensen nutrient medium, which is repeated for 3-4 passages until a reversion is obtained.

However, the known method is time-consuming and expensive (1).

There is a method of reversing the L-forms of mycobacteria by passaging selected cultures on dense egg media of Levenshtein - Jensen or Fin-2 (3-5 or more passages, the duration of one passage is 1-1.5 months). The disadvantage of this method is the duration of the revertants (2).

The objective of the invention is to increase the sensitivity of the method and reduce the time for producing culture-revertants, which is achieved by sowing the L-forms of mycobacteria isolated from biomaterial on a solid Fast-3L nutrient medium, which additionally contains native cattle or horse serum. The Fast-3L nutrient medium has high growth qualities due to the presence of a saturated hydrocarbon with a C ₁₄ -C ₁₇ chain length in it. It actively affects the metabolic processes in microorganisms, being for them a source of energy and plastic material. The enrichment of the environment with native cattle or horse serum contributes to the creation of favorable conditions for the cultivation of L-forms. Due to its buffering properties, serum neutralizes the action of certain growth inhibitors of L-forms contained in nutrient media, stimulates nutrient intake, and accelerates cell division rate.

Example 1

The effect of various doses of native blood serum of cattle or horses on the rate of production of revertant cultures was tested. To do this, L-forms cultures isolated from pathological material were transferred to Fast-3L solid nutrient medium, into which immediately before sowing, native blood serum of cattle or horses was added at 0.05, 0.1, 0.2, 0.5 ml per tube under sterile conditions. Then, L-form cultures were reseeded with a semi-liquid medium to a dense one. Crops were incubated in a thermostat at t 37 ° C for 3-4 weeks, scanning daily with the appearance of tuberculous or atypical mycobacteria, a smear was prepared, stained with Ziehl-Nielsen and examined under a light microscope.

In the study of 97 different biomaterials, 54 L-forms of mycobacteria were isolated, of which 23 revertant cultures were obtained. 1 culture is assigned to M. bovis, 3 cultures are assigned to the 3rd group according to the Runon classification and 19 to fast-growing. The table shows that with the addition of 0.05 ml of serum to the growth medium of cultures of bovine revertants was not observed, atypical cultures of 3-4 groups in small quantities (8-17%) appeared after 14-28 days. On media supplemented with 0.1 and 0.2 ml of serum, growth of bovine species was observed on days 21–28, and atypical ones mainly grew on days 7–14 (21–43%).

The results of the study are presented in table 1.

Table 1.
The results of the passivation of L-forms of mycobacteria on the Fast-3L nutrient medium with the addition of various doses of serum Test material Number of test samples Number of selected L-forms Blood serum content in ml Dates of emergence of revertant cultures (number of cultures) n revertants 7 days 14 days 21 days 28 days 1. Lymph nodes from cattle with tuberculosis lesions

31

6

2 0.05
0.1
0.2
0.5 -
-
-
- -
-
-
- -
one
one
one -
one
one
one 2. Lymph nodes from cattle without tuberculosis lesions

52

42

17 0.05
0.1
0.2
0.5 -
four
four
3 one
8
6
6 2
2
four
2 3
3
3
6 3. Biomaterial from guinea pigs infected with atypical mycobacteria

fourteen

6

four 0.05
0.1
0.2
0.5 -
2
one
2 one
2
3
2 one
-
-
- one
-
-
- Total 97 54 23 0.05
0.1
0.2
0.5 -
6
5
5 2
10
9
8 3
3
5
3 four
four
four
7

When serum was added at a dose of 0.5 ml, similar results were obtained.

Example 2

The experiment was carried out as described in example 1. For the test, Levenshtein-Jensen, Fast-3L culture media without native serum and Fast-3L culture media with native cattle or horse serum were taken.

The test results of the Fast-3L nutrient medium supplemented with 0.1-0.2 ml of native blood serum of cattle or horses for reversing various L-forms of mycobacteria are presented in table 2.

table 2
The rate and growth rate of L-forms of mycobacteria on standard and test nutrient media Test material Culture media Levenshtein-Jensen Fast 3l Fast-3L + blood serum Speed (days) Intensity (points) Speed (days) Intensity (points) Speed (days) Intensity (points) L-forms M.bovis 32-45 one 30-38 1-2 25-35 2-3 L-forms of atypical mycobacteria 17-24 2-3 10-16 6-15 6-15 3-4

A comparative analysis of the data showed that the use of Fast-3L medium with the addition of native cattle or horse serum in a dose of 0.1-0.2 ml per 5 ml of medium allows reducing the reversal time by 7-10 days.

Thus, the proposed method of reversing the L-forms of mycobacteria is more sensitive and shortens the time for obtaining culture-revertants by 7-10 days.

The combination of distinctive features meets the criteria of "novelty" and "inventive step".

Literature

1. B.C. Fedoseev, I.N. Rubtsova, N.T. Kirilenko: Isolation of L-forms of Mycobacterium tuberculosis from animals. Method. recommendations, 1989.

2. I.R. Dorozhkova, Z.N. Kochemasova, M.M. Breathing: Isolation of L-forms of Mycobacterium tuberculosis from pathological material. Method. recommendations. M., Ministry of Health of the USSR, 1984.

Claims (1)

The method of reversing the L-forms of mycobacteria by reseeding them on a solid nutrient medium, characterized in that Fast-3L nutrient medium is used as a dense nutrient medium, which additionally contains a growth factor - native blood serum of cattle or horses at a dose of 0.1- 0.2 ml per 5 ml of medium.