SU1451598A1 - Method of determining the wholeness of nuclei in biological tissue sections on cytological apparatus - Google Patents

Abstract

The invention relates to biology, in particular to cytology, and concerns the identification of the integrity of the nuclei in the section. The invention is mainly intended to determine the size of the nuclei and the phytophotometric study of the DNA of the nuclei in sections. The aim of the invention is to improve the accuracy of the method and simplify it by identifying the integrity of the core. To do this, a sample of the test tissue is taken, fixed, dehydrated, enclosed in paraffin, microtome media are prepared, stained with Felgen, and then microscopically examined in phase contrast and its integrity is judged by the green color of the core. W

Description

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1451598

The invention relates to biology, in particular to cytology, and concerns the identification of the integrity of the nuclei in sections of biological tissue. Advantageously, the invention is intended to determine the size of the nuclei and the cytophotometric study of the DNA of the nuclei in sections.

The aim of the invention is to improve the accuracy of the method and simplify it by identifying the integrity of the core.

The method is illustrated by the following examples.

Example 1. Pieces of leaves of wheat germ varieties Emmer and Balladi 116 are fixed according to Carnoy, after which they are dehydrated, they conclude

wheat. Maximum Il, 50i3,80, minimal. When used then (10 µm), such a cut should not be seen only, which is a measure of the cut kernels of the whole. For the wheat leaf pop, the dry method, using a p-micrometer, is whole and separately (99%). Average times 15 (9,41-10,48) had a cut (7, 2 1

The data obtained are the basis of no change in the amount

ten

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cut into paraffin and prepare sections with a thickness of 20 DNA and a size of 10 μm in thickness by the conventional method. The dewaxed sections are stained by Felgen and examined under a LUMAM-I-1 microscope in transmitted light from a incandescent lamp. All cores 25 have the same color - lilac-red. When viewing the same cores in phase contrast, some of them are green, others are yellow and orange-yellow. The use of other acidic and neutral fixatives, usually used for DNA testing by means of the Feelgen reaction, does not affect the result.

By the method of layer-by-layer reconstruction, it was possible to establish that whole cores have green color, and yellow or orange are cut. From 87 der identi-; 35 as whole and 52 as sliced.

Two more methodological approaches are used to prove the established pattern.

Protoplasts are isolated from the mesophyll of the wheat germ leaves, where all the nuclei are obviously intact. After staining the Felgen isolated protoplasts in suspension and preparing smears, they are examined under a microscope. In the passage, in light, all the cores in the brushstrokes, as in the sections, have a violet-red light, and in the phase contrast all the cores are green. Since in the smear all the cores are obviously whole, this is confirmed by the connection of the green color of the core in phase contrast with its integrity.

With the help of the screw eye of the p-micrometer in smears, the average sizes of the leaf mesophyll dermal cells are measured.

50

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wheat germ, by tele stem rust

Example 2. Fresh sous are placed in 10% 4c, crushed by 6 mm, postfixed ice at 3: 1 acetic solu tion for lead, conclude prepare sections with 5 μm of commonly prepared Finlen sections and look through the LUMAM-I- cluster 1 in incandescent bulbs of the same color Ny. Cytoplasm color color. When viewing the phase contrast, they are green, yellow. Cytoplasm acquiring a pale same that does not interfere clearly both the whole and the cut

For evidence, the color in the liver phase slices of the liver as well as wheat has whole Felgen cores in the whole piece of the pech of this piece is prepared, where the cores have one color red and the cytoplasm of the phase contrast, and cytoplasm n vogo.

wheat. The maximum diameter of the core: Il, 50i 3.80, the minimum 5.40 + 0.01. With the slice thickness used (10 μm), a nucleus of this size can be cut only into two parts. Obviously, in this case sredm: e sizes of cut kernels are smaller than the average sizes of integers. On the cross sections of wheat leaves, using the proposed method, the whole and separate cut kernels (P 99%) are measured with the aid of a p-micrometer screw eye. The average sizes of the whole core (9.41 and 10.48) turned out to be larger than the cut (7,, 2 1).

The data obtained in the Experiment serve as a basis for studying the change in the amount of labile

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20 DNA and sizes 25 der AOR

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wheatgrass affected by stem rust pathogen.

Example 2. Fresh white rat liver is placed in 10% formalin at 4c, crushed into 6 mm pieces, postfixed in a mixture of ethanol and acetic acid in a ratio of 3: 1 for 2 hours. Then dehydrated, enclosed in paraffin and Prepare sections of 10 and 5 microns in thickness by the conventional method. Deparated sections are stained by Felgen and examined under a LUMAM-I-1 microscope in transmitted light from a incandescent lamp. All cores have the same color lilac-red. Cytoplasm is colored pink. When viewing the same cores in phase contrast, most of them are green, a smaller part of yellow. The cytoplasm is modified by acquiring a pale yellow hue, which does not interfere with a clear distinction between cores as well as slices.

In order to prove that the green color in phase contrast in liver sections, as well as in wheat leaves, has whole cores, stains with the Felgen core directly in the whole liver piece, and then a crushed preparation is prepared from this piece, where the cores are known to be In such a cytological specimen under a microscope in the passage of 1 light, all the cores have one color - lilac-red, and the cytoplasm is pink. In phase contrast, the nuclei are green, and the cytoplasm is no-r; piv. pink.

In order to prove that the cutting cores in the liver mediums in phase contrast have a yellow color, the number of cut kernels in sections 10 and 5 µm thick is determined. Obviously, in a thinner cut, there should be more cut cuts, therefore, in phase contrast, most of the cuts have a yellow color. The number of green and yellow

The cores are determined using a 12-key counter by drug, choosing several random fields of view on five fields of slices 10 μm thick, on average 24.5 yellow nuclei, and on sections 5 μm thick 41.8. Thus, a decrease in the slice thickness by a factor of 2 approximately the same time increased the number of cut kernels, hence cut cores.

Claims (1)

liver in phase contrast are yellow. Invention Formula The method of determining the integrity of the nuclei in sections of biological tissue on a cytological preparation, including tacos, selection of tissue samples, fixation, dehydration and its embedding in paraffin, preparation of microtome sections, their staining according to Felgen with subsequent determination under a light microscope, distinguished by in order to improve the accuracy of the method and its simplification by identifying the integrity of the core directly in one cut, the determination is carried out in phase contrast and the integrity of the core is judged by the green color of the core.