RU2593343C1 - Method for staining the histological cuts in diagnosing trichinosis - Google Patents

Abstract

FIELD: biology.

SUBSTANCE: invention relates to microbiology and concerns method for staining histological sections in diagnosing trichinosis. Proposed method consists in staining histologic cuts with Ehrlich hematoxylin, by adding 2-3 drops of 10 % dimethyl sulphoxide, washed in water to blueing of cut. Then poured thereon 2 drops of eosin solution, washed cut with water. Poured alcohol, added antireflection agent.

EFFECT: use of method enables to obtain stable cuts of preparations.

1 cl, 4 ex

Description

The invention relates to veterinary medicine and medicine, in particular to a method for staining histological sections in the diagnosis of trichinosis, and can be used in histology for morphological studies in pathological anatomy, cytology, and forensic medicine.

There is a method of staining blood smears according to Romanowski-Giemsa, which consists in a single placement of blood smears in a dye solution with further washing with distilled water and drying in the open air (see G. Merkulov “Pathology and Histology Technique Course.” Publishing House Medicine - Leningrad Branch, 1969 , p. 423).

The disadvantages of this method are:

- the working solution does not retain its dye properties for a long time, deteriorates quickly and cannot be used for re-staining;

- the quality and speed of staining depends on the ambient temperature, i.e., the lower the temperature, the longer the staining.

A known method for staining histological sections with hematoxylin and eosin, including the use of the main dye of hematoxylin staining the basophilic cell structures with bright blue, and the alcoholic acid stain of eosin Υ staining the eosinophilic structures of the cell with red-pink color, while the basophilic structures, as a rule, are those that contain nucleic acids (DNA and RNA): the cell nucleus, ribosomes and RNA-rich sections of the cytoplasm (see Sapozhnikov A.G., Doroshevich A.E. Histological and microscopic t hnika Smolensk. ACS, 2000, p 190-191)..

The disadvantages of this method is that the dye solution is applied to the histological section containing the calcareous capsule, inside which there are trichinella, the colorant solution does not penetrate the calcareous capsule, which can cause poor-colored preparations, in which case the calcareous capsule is decalcified by acting 5 -8% solutions of organic acids: hydrochloric, or nitric, or acetic, and others. In the process of decalcification, the dye solution penetrates the trichinella, but the acids destroy the tissues surrounding the capsule, cause structural changes in trichinella, which complicates the differential diagnosis of trichinosis and other parasites, for example, animal sarcocystosis and an objective assessment of the level of histomorphological changes in the localization of parasites (in tissues, surrounding a capsule with parasites, trichinella).

There is also known a method for staining histological preparations, which consists in staining histological sections with methyl green and pyronin, in addition, use a 2% aqueous solution of a salt of strong blue BB, which is pre-etched before applying the dye mixture, after 5 min the solution is drained and applied to the section 2 drops each of a 2% aqueous solution of a salt of a strong blue BB and a dye mixture of the following composition: 0.6 g of methyl green, 0.4 g of pyronin, 20 ml of glycerin and 80 ml of distilled water; for the differentiation of nuclei using 96 ° ethanol (see US Pat. RU No. 2202776, IPC G01N 1/30, publ. 04/20/2003).

The disadvantage of this method is the unstable staining of cells and cell structures.

The closest in technical essence and the achieved positive effect and accepted by the authors for the prototype is a method for staining hematoxylin-eosin, which includes removing paraffin from sections in ortho-xylene or toluene, carrying out a downward concentration on alcohols, bringing to water (two portions of xylene or toluene - 3 -5 minutes, 96 ° ethanol - 3 minutes, 80 ° ethanol - 3 minutes, 70 ° ethanol - 3 minutes, distilled water - 5 minutes), hematoxylin staining for 7-10 minutes (depending on the maturity of the dye), washing in distilled water - 5 minutes, differentiation in 1% hydrochloric acid at 70 ° ethanol before drilling sections, washing with distilled water and then a weak (0.5%) ammonia solution until the sections turn blue, staining with an aqueous eosin solution for 0.5-1 minute (depending on the desired color) washing in three portions of distilled water to remove excess eosin, removing water from the slices in one portion of 70 ° ethanol, two portions of 96 ° ethanol, exposure in each portion of alcohol - 2 minutes, clarification of the slices in two portions of carbolxylene (a mixture of molten phenol and xylene or tol Wal in the ratio 1: 4 or 1: 5) - 1 minute, then the sections are finally dehydrated in two portions of xylene or toluene, the sections are left for 2 minutes, followed by the conclusion of the sections in Canadian balsam or synthetic medium for the conclusion of histological sections (see Big Medical Encyclopedia).

The disadvantage of this method is that some structures are poorly stained with hematoxylin and eosin (usually hydrophobic) and require other staining methods, for example, cell sections rich in lipids and myelin remain unpainted: adipocytes, myelin sheath of axons of neurons, Golgi apparatus membrane, etc. .

The objective of the invention is to develop a method for staining histological sections in the diagnosis of trichinosis with stable staining of cells and cell structures, eliminating the need for decalcification of the calcareous capsule around trichinella, damage to trichinella and the creation of prerequisites for an objective diagnosis.

The technical result that can be obtained using the present invention is reduced to the stability of staining of cells and cell structures, eliminating the need for decalcification of the calcareous capsule around the Trichinella, damage to Trichinella and creating the prerequisites for an objective diagnosis.

The technical result is achieved using the method of staining histological sections in the diagnosis of trichinosis.

Thus, for example, in laboratories of veterinary and sanitary expertise in the markets, compressor trichinoscopy is used to diagnose trichinosis (see Guidelines MUK 4.2.2747-10 (approved by the Federal Service for Supervision of Consumer Rights Protection and Human Well-Being on October 11, 2010 ), however, in frozen meat of animals (for example, a bear, a badger) it is difficult to detect trichinella, since when it is preserved at low temperatures, the water from the parasite capsules is frozen, and after defrosting they are filled with meat juice. clear juice has the color of meat, so trichinella become invisible, and with the help of the proposed method it is by adding dimethyl sulfoxide that decalcification of the lime capsule around the trichinella is eliminated, damage to the trichinella is created and the preconditions for an objective diagnosis are created, while dimethyl sulfoxide (DMSO) is a viscous, colorless, almost odorless liquid , when mixed with water, it is very hot and is an important bipolar aprotic solvent, less toxic than other representatives of this groups, such as dimethylformamide, dimethylacetamide, N-methylpyrrolidone, due to their strong dissolving ability, DMSO are often used as a solvent in chemical reactions involving inorganic salts, in particular in nucleophilic substitution reactions, the acid properties of DMSO are weakly expressed, therefore it has become an important solvent in chemistry of carboanions, because of the high boiling point, DMSO evaporates extremely slowly at normal atmospheric pressure, which makes it a very convenient solvent for carrying out reactions at Revani, while relatively high melting temperature limits its use at low temperatures.

Specification for DMSO: content of the basic substance DMSO not less than,%: 99.90; crystallization temperature, ° C: 18.20; acidity, mg / g: 0.02; DMSO transmittance in a cuvette 400 mm,%: 96; refractive index at 20 ° C: 1.4778-1.4790.

The essence of the method for staining histological sections in the diagnosis of trichinosis is as follows.

Histological sections are prepared, then staining is carried out with two dyes, which are taken from Ehrlich hematoxylin and eosin - sodium, while first the section is stained with hematoxylin, the stained sections are washed and placed on a glass slide, then stained with an eosin solution, eosin drained, rinsed with tap water water, positioning on a glass slide, removing water with subsequent placement in 96 ° alcohol, holding, draining the alcohol, adding an enlightening agent, washing with xylene, s inclusion in a balm coated with a coverslip, while staining with hematoxylin, additionally add 2-3 drops of 10% dimethyl sulfoxide, and hematoxylin use profiled alum in the amount of 5-7 drops, staining with hematoxylin for 1-5 minutes, and rinsing in water - for 3-5 minutes until the slice turns blue, then the slice is placed on a glass slide and stained with an eosin solution in an amount of 2-3 drops for 1-3 min, the eosin solution is drained and rinsed with tap water for 1 min, exposure alcohol p they are bled for 3-5 minutes, the bleach is poured on the wet slice, the enlightening agent is poured on it, exposure to the bleach is carried out for 1-5 minutes until the slice is completely transparent, then xylene washing is carried out 2-3 times for 1 minute, its residues are removed Put 1 drop of balm and cover with a coverslip.

Examples of specific performance of the method for staining histological sections in the diagnosis of trichinosis.

Example 1. Prepare histological sections in an amount of 24, and when investigating outbreaks of trichinosis, it is necessary to increase the number of sections to 72, while the thickness of the sections should not exceed 1.5-2.0 mm. The histological sections (celloidine immediately and frozen after degreasing) are transferred to the bacteriological plates with water, then the best section is taken out on a flat glass in an expanded form, to use the black background of the table for orientation, 2 drops of filtered alum hematoxylin are poured onto the section from the filter with using a funnel, stain for 0.5 min depending on the quality and maturity of hematoxylin, additionally add 1 drop of 10% dimethyl sulfoxide (DMSO), hold the slice on the glass dissected with a needle, the paint is poured back into the working flask, and the preparation is lowered from the glass into a large cup with water, washed in water for 2 minutes until the slice turns blue, while the sections that were just stained with hematoxylin and, therefore, the nuclei of the cells have a reddish-violet tone, which, with sufficient washing in water, due to its alkalinity, turns into blue. The blue nuclei of the cells will be more contrasted when combined with eosin, a well-washed and blue-blue slice is removed again from the water (straightened) on a glass slide and 2 drops of an eosin solution are poured onto it, stained for 0.5 min, depending on the coloring ability or other eosin, eosin is drained, holding the slice with a needle, washed in a large cup with tap water for 0.5 min, the color of the preparation ends, then the slice is removed from the water on a glass slide, all water is removed around it, and if possible os carefully so that the slice does not curl, pour alcohol 96 °, keep it in alcohol for various times, up to about 2 minutes, because, in addition to dehydrating, it also has a differentiating effect on eosin, drain the alcohol, holding the slice with a needle, quickly remove it with a cloth its remnants around the section and, not allowing it to dry, pour an antireflection agent (creosote, essential oil / carbolxylene, turpentine, etc.), kept in the latter until the section is completely transparent for up to 3 minutes, with a slow clarification of the section, filter paper is pressed oh, they control the course of enlightenment on a black background, on which the unenlightened whitish areas stand out well, then they quickly rinse with xylene for 1 minute, pouring and draining it once more, remove the xylene residues around the section, put a drop of balm and cover with a coverslip, and the coverslip the glass is lowered carefully, holding the right rib with its dissecting needle, since air bubbles can get under the glass when quickly applied.

Result: analysis of the data showed insufficient staining of the nuclei and inclusions due to insufficient exposure of the slices over time, therefore, the diagnosis of trichinosis is difficult.

Example 2. Carried out analogously to example 1, but was carried out according to the following parameters. The histological sections are transferred to plates with water, then the best section is removed on a slide in a straightened form, 5 drops of filtered alum hematoxylin are poured onto a section, stained for 1 min, an additional 2 drops of 10% DMSO are added, the section is held on a glass with a dissecting needle , paint is drained, and the preparation is lowered from the glass into a large cup with water, washed in water for 3 minutes until the slice turns blue, while the sections that have just been stained with hematoxylin and, therefore, the nuclei of the cells have a reddish-violet tone, when washed sufficiently in water, due to its alkalinity it turns into blue. The blue nuclei of the cells will be more contrasted when combined with eosin, a well-washed and blue-blue slice is removed again from water on a glass slide and several drops of an eosin solution are poured onto it, stained for 1 min, the eosin is drained, holding the slice with a needle, washed in a large cup with tap water for 1 min, this ends the color of the preparation, then remove the slice from the water on a glass slide, remove, if possible, all the water around it and carefully so that the slice does not curl, pour 96 ° alcohol, keep it in alcohol For a good time, up to about 3 minutes, the alcohol is drained, holding the slice with a needle, quickly remove the rest of it around the slice with a napkin and, not letting it dry, pour antireflective agent, stand in the latter until the slice is completely transparent for up to 3 min, with a slow clearing of the slice, apply pressing with filter paper, the course of enlightenment is monitored on a black background, on which the unenlightened whitish areas stand out well, then quickly washed with xylene for 1 min, pouring and draining it again 2 times, remove the residues silol around the slice, put a drop of balm and cover with a coverslip.

Result: analysis of the data showed sufficient staining of the nuclei and inclusions, as a result of which the diagnosis of trichinosis is not difficult and an objective diagnosis was made.

Example 3. Carried out analogously to example 1. But was carried out according to the following parameters. The histological sections are transferred to plates with water, then the best section is removed on a slide in a straightened form, 7 drops of filtered alum hematoxylin are poured onto the section, stained for 5 minutes depending on the quality and maturity of the hematoxylin, 3 additional drops of 10% are added DMSO, hold the slice on the glass with a dissecting needle, pour the paint back into the working flask, and lower the preparation from the glass into a large cup with water, wash it in water for 10 minutes until the slice turns blue, while the hematomas just painted Ilin sections, and hence the cell nuclei are reddish-purple color, which is washed with sufficient water due to its alkalinity becomes blue. The blue nuclei of the cells will be more contrasted when combined with eosin, a well-washed and bluedish slice is removed again from the water (straightened) on a glass slide and a few drops of eosin solution are poured onto it, stained for 3 min depending on the coloring ability of one or another eosin, eosin is drained, holding the slice with a needle, washed in a large cup with tap water for 1 min, the color of the preparation ends, then the slice is removed from the water on a glass slide, removed around it, if possible, all in ode and carefully so that the slice does not curl, pour alcohol 96 °, keep it in alcohol for various times, up to about 5 minutes, pour the alcohol, holding the slice with a needle, quickly remove the rest of it around the slice with a napkin and, pouring antireflection agent, they are kept in the latter until the cut is completely transparent for up to 5 min, with slow clarification of the cut, press filtering is applied, the course of bleaching is monitored on a black background, on which unlightened whitish areas stand out well, then quickly For 1 min, wash with xylene, pouring and pouring it again 3 times, remove the remaining xylene around the section, put a drop of balm and cover with a coverslip.

Result: analysis of the data showed sufficient staining of the nuclei and inclusions, as a result of which the diagnosis of trichinosis is not difficult and an objective diagnosis was made.

Example 4. Carried out analogously to example 1. But was carried out according to the following parameters. The histological sections are transferred to plates with water, then the best section is taken out on a flat glass in a straightened form, 8 drops of filtered alum hematoxylin are poured onto a section, stained for 5.5 minutes depending on the quality and maturity of the hematoxylin, an additional 4 drops of 10% are added DMSO, hold the slice on the glass with a dissecting needle, pour the paint back into the working flask, and lower the preparation from the glass into a large cup with water, rinse it in water for 11 minutes until the slice turns blue, while the hemato Silinya sections, and hence the cell nuclei are reddish-purple color, which is washed with sufficient water due to its alkalinity becomes blue. The blue nuclei of the cells will be more contrasted when combined with eosin, a well-washed and bluedish slice is removed again from water on a glass slide and a few drops of an eosin solution are poured onto it, stained for 3.5 minutes depending on the coloring ability of a particular eosin, drained eosin, holding the slice with a needle, is washed in a large container with tap water for 1 min, the color of the preparation ends, then the slice is removed from the water on a glass slide, all water, if possible, is removed around it and carefully so that the slice does not curl, pour alcohol 96 °, stand in alcohol for various times, up to about 5.5 minutes, pour the alcohol, holding the slice with a needle, quickly remove the rest of it around the slice with a napkin, and pour antireflective agent into it, hold it in the latter until the cut is completely transparent up to 6 min, with a slow clarification of the cut, press filtering is applied, the progress of the bleaching on a black background is monitored, on which unlightened whitish areas stand out well, then I rinse quickly for 1 min xylene, again 4 times its loading and unloading, xylene residues removed around the cut, put a drop of balm and covered with a coverslip.

Result: analysis of the data showed sufficient staining of the nuclei and inclusions, as a result of which the diagnosis of trichinosis is not difficult, but the time frame has increased and economic costs as well.

Thus, the most optimal are examples 2 and 3 of the method for staining histological sections in the diagnosis of trichinosis.

Thus, the addition of DMSO eliminates the need for decalcification of the calcareous capsule around trichinella, does not damage trichinella, and creates favorable conditions for an objective diagnosis. An analysis of the data obtained allowed us to conclude that the proposed method for staining histological sections for the diagnosis of trichinosis exceeds, for example, the most commonly used according to Romanowski-Giemsa, and allows to obtain qualitatively stained histological sections in a shorter time of 6-9 minutes, depending on the exposure time of the solution hematoxylin.

The invention in comparison with the prototype and other known technical solutions has the following advantages:

- stability of staining of cells and cell structures;

- eliminating the need for decalcification of the calcareous capsule around the Trichinella and damage to Trichinella;

- Creation of prerequisites for conducting objective diagnostics;

- reduction of staining time;

- makes it possible to reuse working solutions of dyes;

- does not require special laboratory equipment;

- allows employees of veterinary and medical laboratories to save time and material resources.

Claims (1)

A method of staining histological sections for the diagnosis of trichinosis, including the preparation of histological sections, staining with two dyes, which take Ehrlich hematoxylin and eosin sodium, first stained with hematoxylin, washed stained sections and placed on a glass slide, then stained with an eosin solution , drainage of eosin, rinsing with tap water, location on a glass slide, removal of water, followed by placement in 96 ° alcohol, aging, drainage of alcohol, add the appearance of a bleaching agent, washing with xylene, closing in a balm coated with a coverslip, characterized in that when hematoxylin staining is additionally added 2-3 drops of 10% dimethyl sulfoxide, and hematoxylin is used profiled alum in the amount of 5-7 drops, staining with hematoxylin for 1-5 minutes, and washing in water - for 3-5 minutes until the slice turns blue, then the slice is placed on a glass slide and stained with an eosin solution in an amount of 2-3 drops for 1-3 minutes, draining the eosin solution and rinsing with tap water for 1 min, aging in alcohol is carried out for 3-5 minutes, it is drained and a bleach is poured onto a wet slice, soaking in the bleach is carried out for 1-5 minutes until the slice is completely transparent, then 2- 3 times for 1 min washing with xylene, remove its residues, put 1 drop of balm and cover with a coverslip.