RU2032374C1 - Method for diagnosing balantidiosis in swine - Google Patents

Abstract

FIELD: veterinary. SUBSTANCE: essence of this method resides in making histologic slice, fixing and pouring preparation into either paraffin or celloidine, coloring matter with hematoxyline, subjecting matter to differentiation, neutralization and coloring with iodine-eosin solution taken in ratio of 1:1 during one minute. Disclosed method further prescribes passing whole through alcohol of 70, 80, 96 and 100-percent concentration for compacting section. EFFECT: higher accuracy of diagnosis.

Description

 The invention relates to veterinary medicine, in particular to laboratory methods for the study of pathogenic protozoa and can be used for histological (microscopic) diagnosis of swine balantidiosis.

 In sick pigs, balantidia penetrate the walls of the colon into the tissue. To detect them, histological sections are made, special staining methods are used.

 A known method for diagnosing swine balantidiosis is to fix pieces of the intestinal wall in censer formol fluids, Carnoy fixative, formalin and others, followed by pouring it into paraffin or celloidin and studying them as histological objects (see Laboratory methods for studying pathogenic protozoa. M. Medgiz, 1957, p. 260).

 The disadvantages of this method are that these fixatives significantly compact the tissue of the intestinal wall, as a result of which, when histosections are stained with hematoxylin-eosin and other paints, the individual structural elements of balantidia (membrane, digestive vacuoles, etc.) are poorly stained.

A known method of studying the microstructure of balantidia on histological sections stained with iron hematoxylin according to Heidenhain (see Veterinary laboratory practice. M. Selkhozizdat, 1963, v. 2, p. 279). In this case, the processing of pieces of tissue of the intestinal wall with parasites is carried out according to the general rules of the histological technique. The two-stage method of staining with Heidengain’s iron hematoxylin is based on the formation of iron-hematoxylin varnish. For mordants, use a 2-5% aqueous solution of ferric ammonia ("iron") alum. To prepare the paint, dissolve 1 g of hematoxylin in 10 ml of 96 ^about alcohol and add 90 ml of distilled water. For differentiation, alum is used weaker than for mordant.

 This method has the following disadvantages. For mordant, exclusively fresh, unused and completely clear solutions should be used, otherwise irreparable precipitation may appear on the preparation. When staining, the maturity of hematoxylin matters, the older the solution, the stronger the accumulation of higher hematoxylin oxides in it, the color gives more and more black tones, and the preparations differentiate with great difficulty. Old solutions compared to more recent ones are much less reliable in the sense of the possible identification of stained elements (see Laboratory methods for the study of pathogenic protozoa. M. Medgiz, 1957. p. 77-81).

 There is a method of studying balantidia by preparing histological sections, which are fixed with 10-20% formalin and stained with hematoxylin and eosin (see Veterinary laboratory practice. M. Selkhozizdat, 1963, v. 1, p. 263).

 However, this method is quite complicated, takes a lot of time and does not always provide a good quality of coloring of balantidia. So, when stained with hematoxylin-eosin on a glass slide, satisfactory results are obtained only if hematoxylin is not repainted, otherwise the preparations will be coarse, with heavily colored nuclei. Even in the case of control under the microscope, there is some staining of the interstitial tissue with hematoxylin, which, with subsequent layering of eosin, leads to a violet background. When stained with differentiation, an unnecessarily long delay in cutting sections in hydrochloric acid leads to complete extraction of the paint. With this method, thorough washing in water from alkali is required (for 20-30 minutes or more up to 24 hours), otherwise the eosin is poorly perceived by the slice. When staining with differentiation, special attention should be paid to eosin since it is difficult to determine the exact exposure due to the fact that eosins are very different in their coloring properties and therefore it is necessary to adapt to each of them. With strong repainting with eosin, differentiation with alcohol also does poorly. The color of paraffin sections also has a number of disadvantages that affect the clarity, contrast of the drug (see Merkulov G.A. Course of histopathological technology. L. Medgiz, 1961, p.129-137).

 The aim of the invention is to simplify the method and improve the accuracy of diagnosis of balantidiosis.

This goal is achieved by the fact that in the diagnostic method by examining balantidia in histological sections, in which the drug is stained with hematoxylin and eosin, in the existing staining scheme at the stage of staining with eosin, a solution of Lugol and eosin in a ratio of 1: 1 for 1 min is used and to compact the slice sequentially carried out through alcohols of ascending concentration, transferring the sections each time for 25-30 s through alcohol 70, 80, 96 and 100 ^{about the} concentration.

 This allows you to get a clear contrast drug. In this case, the eyelashes of vegetative forms and the double-shell of cysts, digestive vacuoles and nuclei of the parasites are stained in dark blue or pale blue, are clearly distinguishable, in their cytoplasm erythrocytes, leukocytes and intestinal epithelial cells are stained in pale pink. The resulting clear contrast drug allows us to judge the parasitism of balantidia.

A comparative analysis of the proposed solution with the prototype shows that the proposed method differs from the known one in that at the stage of eosin staining, a solution of Lugol and eosin is used simultaneously in a ratio of 1: 1 for 1 min and in that it is sequentially carried out for compaction of the slice for 25- 30 s through 70, 80, 96 and 100 ^o alcohols. Thus, the proposed method meets the criteria of the invention of "novelty."

 Analysis of the known technical solutions in this field of science allows us to conclude that a technical solution is known that provides for the staining of bacteria in sections using a Lugol solution according to the Gram-Weigert method. However, in the proposed method, the coloring is carried out using a solution of Lugol and eosin in a ratio of 1: 1. The use of hematoxylin and a solution of Lugol and eosin (1: 1) for staining exhibits a new, unknown property, which determines the achievement of a positive effect. The inventive method is less time-consuming and simple, the staining procedure is reduced, since filter paper and aniline oil are not used to differentiate the structure of parasites in the histosection. The method provides a clear identification of cilia, shell, nucleus, as well as starch, glycogen grains and leukocytes, erythrocytes, intestinal epithelial cells absorbed by balantidia, which is a criterion for their parasitism.

 Thus, although the distinguishing features of the invention are known, however, in comparison with the solutions known in the art, it is characterized by a new set of features exhibiting a new property of clear contrast staining of balantidia, which improves the accuracy of diagnosis of balantidiosis. This allows us to conclude that the proposed solution meets the criteria of the invention "significant differences".

The proposed method is implemented as follows. Cut out pieces from the wall of the large intestine with a size of 0.3-0.5 cm, they are fixed in 8-10% aqueous solution of neutral formalin (can be in 70-80 ^about ethanol). It is washed in running water (under the tap of the sink) for 1-2 hours. Dehydration, degreasing and compaction of intestinal pieces are carried out in ascending alcohols (70, 80, 96, 100 ^o ) for 6 hours, then in alcohol-ether (100 ^o alcohol + ether in half) for 2 hours. Pouring pieces from the intestinal wall is carried out in a liquid (2%) solution of celloidin for 24 hours, then in a thick (10%) solution of celloidine for 1-2 days. Next, the pieces soaked in solutions of celloidin are placed in Petri dishes, poured with a fresh portion of thick celloidin (10%) so that a 1 cm thick layer of celloidine is placed over the pieces, covered with a lid, but not completely tight, so that the alcohol and ether in a 10% celloidine solution gradually evaporated during the day. Then, using pieces of a safety razor in a Petri dish, cut out pieces densified in celloidin and paste them on dry skimmed birch cubes (blocks), with which 6–8 microns thick sections are prepared using a sled microtome and a microtome knife and stained with hematoxylin-iodine-eosin.

Pouring in paraffin. Pouring pieces from the intestinal wall is carried out in a homogenized molten (at a temperature of 50-55 ^about C) paraffin. First, the pieces are transferred to a jar of 100 ^o alcohol in half with chloroform for 1 h (dehydration and degreasing), then to pure chloroform for 30 minutes, then to a molten (at a temperature of 40 ^o C) mixture of chloroform with paraffin (equally) and put in a thermostat for 1 h. The mixture of chloroform with paraffin pieces alternately shift to two cups of molten paraffin placed in a thermostat at a temperature of 54-55 ^{° C} is maintained 1.5-2 h (not to destroy balantidiums, the residence time of the pieces in the oven may be cut in half).

 After that, pieces from the cups with molten paraffin are removed using anatomical tweezers, transferred to cardboard (preferably lead) molds and quickly cooled, immersed in water.

 When the paraffin hardens together with the pieces, the molds are destroyed, and intestinal pieces are cut out from the paraffin block with a razor blade and glued to the birch cubes with molten paraffin. Subsequently, using a toboggan microtome and a microtome knife, histoslices 6-8 microns thick are obtained and stained with hematoxylin-iodine-eosin.

To seal the sections were placed in an alcohol 96 in ^about 2 minutes. Then the slices are placed in water for 2 minutes. Stained with alum hematoxylin for 3 min. After that, the slices are placed in water for 3 minutes. Quickly rinse with hydrochloric acid alcohol (70 ^about ), then quickly rinse with water. To neutralize hydrochloric acid, rinse with a weak solution of ammonia (until the slice turns blue). After that, it is washed well in water and stained with a solution of iodine-eosin (1: 1) for 2 minutes. Washed in water for 2 minutes. For compaction, sections are carried out through alcohols 70, 80, 96 and 100 ^about concentration, 30 days each. Then carbol-xylene for 1 min until enlightenment, then xylene for 30 s. Then imprisonment in Canadian or fir balm, coating the histoslice with a coverslip, microscopy.

 As a result of the implementation of the method, cilia, shell, nucleus, vegetative forms and cysts of balantidia are stained in dark blue, digestive vacuoles in pale bluish color, protoplasm of balantidia in pale pink, and leukocytes, erythrocytes, epithelial cells of the mucous membrane absorbed by balantidia are painted in a pale red color. This allows you to establish an accurate diagnosis of swine balantidiosis and to differentiate similar gastrointestinal diseases of pigs.

Claims (1)

 METHOD FOR DIAGNOSTIC OF PIG BALANTIDIOSIS by detecting balantidia in histological sections, including fixation of the drug, pouring it into paraffin or celloidin, staining with hematoxylin and eosin, differentiation, neutralization, compaction and enlightenment of the section, microscopy of the drug, which is differentiated by neutralization and staining after staining mixing it with a Lugol solution in a ratio of 1 to 1 and treating this mixture for 1 min, and densification is carried out by exposing the sections sequentially to the solution rah ethanol 70-, 80-, 96- and 100% concentration for 25 to 30.