SU1130605A1 - Method for detecting ecdisterone in cultur medium - Google Patents

Abstract

A METHOD FOR DETERMINING EXDTERERON IN A CULTURAL MEDIUM, which involves the extraction of salivary projectile from the last larval Drosophila virilis larva of the last larval age, its preparation in a test culture medium of the age of 20-22 ° C, and a sample application of a creator in a sample application. that is, (in order to reduce the definition, the analysis of the range of puffs is carried out in indicator regions of chromosomes with a primary reaction and ecdysteron and according to the presence. the reaction in sections XA-6, З-С-1 , 3-0-2, 4-С-4 judges the presence of ecdystero a in the medium.

Description

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The invention relates to agriculture and biology and can be used for practical purposes as a rapid test system for biologically active substances.

There is a method of determining ecdysterone in culture medium by puffing chromosomes of salivary selez Drosophila virilis. The larvae (30–40 pcs.) Are selected from mass cultures at the time of the 2nd molt in the 48th hour of the 3rd century, the salivary glands are made of them and transferred into the culture medium C-50 of a chemically determined composition, the glands are cultured in 0.5–24 hours in tubes or penicellin bottles containing 1 ml of C-50 medium without hormone or with insect hormones. Then the glands are transferred to a 1% solution of oceine ocein for 2 hours at 22 ° C or 12-14 hours at 10-14 ° C, after staining with iron, the crushed preparations are prepared in a routine manner and carried out analysis of the distal part of the gland (10 pcs.), viewing the expression of the spectrum of puffs in ten or more glands of glands. On each of the terms, an average of 12,200 chromosome regions in the C-50 medium, 12,400 regions in the medium with juvenile hormone, and 12,200 in the medium with ecdysterone are analyzed. A total of 36,800 chromosome regions are analyzed on average.

the last one is carried out in indicator areas of chromosomes with a primary reaction to ecdysterone and by the presence of re-, actions in areas XA-6, 2-D-3, 3-C-1, 3-G-2, 4-C-4 judge on the presence of ecdysterone in the environment.

PRI im p The larvae of Drosophila virilis are selected during the 2nd molt. The salivary glands are isolated from the 48th hour larvae of the PG of their age in 0.6% NaCl solution. Transferred to the cups (diameter 25 mm, height 25 mm) with 1 ml of medium C-50 formulations (concentration of substances in mg / 100 ml three times

The disadvantage of this method is that its karyological analysis is long, complex and laborious, as it includes a range of puffs of the entire genome, most of which react to ecdysterone non-specifically, i.e. sensitive to both ecdysterone and the jusenyl hormone. The purpose of the invention is to reduce the determination time. This goal is achieved by the fact that according to the method of determining ecdysterone in a culture medium that involves the larval Drosophila virilis larvae of the last larval return of the salivary gland, it is kept in the test culture medium at 20-22 ° C, preparation of a crushed acetocene-newgogram of this gland with subsequent analysis of the spectrum of chromos puffs, Glucose 300.0 Fetal bovine serum 5% pH 6.9 + 1 mg / ml ecdysterone Serva, HGF. The glands are cultured for 30 minutes at 20-22c under hermetic conditions. Stained in a 1% solution of acetoorcein for 2 hours at 20-22 ° C or 12-14 hours at 10-14 ° C. Crushed gland preparations are prepared, for which the iron is transferred from the paint onto a glass slide, the paint is removed ; put in a drop of a saturated solution of chloral hydrate (5 g in 2 ml of water); byutro (after 3-4 s) wash it off with 45% acetic acid; put the iron in a drop of 50% lactic acid; cover with cover glass, clamping it to a glass slide.

A karyological analysis is carried out on five indicator puffs in areas XA-6, 2-DZ, 3-C-t, Z-a-2, 4-C-4, the expression of which indicates the presence of ecdysterone in the culture medium. In an environment without ecdysterone, indicator puffs are not formed.

Thus, the proposed method for determining ecdysterone in culture medium by puffing indicator areas in the polythene chromosomes of Drosophila virilis

technically simple, reduces the duration of the analysis by 15 times (the analysis by the proposed method lasts 1 day, by the known - 15 days). The volume of the analyzed material is reduced by a factor of 73 (the proposed method analyzes 500 plots of chromosomes, 36800 plots of the known).

The developed method for the determination of ecdysterone in the culture medium allows the use of indicator areas per ecdysterone of all insects with tissues with a polythenic karyotype.

Claims (2)

METHOD FOR DETERMINING ECDISTERON IN A CULTURAL ENVIRONMENT, which involves injecting the salivary gland of the last larval age of Drosophila virilis larva, its incubation in the test culture medium at 20-22 ° C, preparation of a pressured aceto-orcein preparation of this gland, followed by analysis of the spectrum of chromosome puffs. I mean that, in order to reduce the time of determination, the analysis of the spectrum of ipuffs is carried out in the indicator regions of the chromosomes with a primary reaction to ecdysterone and by presence. reactions in areas XA-6 ,. 2-D-Z, Z-S-1, 3-Q-2, 4-S-4 judge the presence of ecdysterone in the medium. 1 1130605