RU2530574C2 - Method for accelerated staining of histological specimen for identifying tuberculosis mycobacteria - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and represents a method for accelerated staining of histological specimen for identifying tuberculosis mycobacteria involving the preparation of the histological specimen according to a common technique, the Ziehl-Nelsen staining differing by the fact that the standard preparation of the specimen for staining is followed by a de-waxing procedure by placing them successfully into xylol and alcohols of the decreasing concentration for 5 minutes in a thermostat at 54°C and then placing into water, then in a 1% periodic acid for 2 minutes and washing in flowing water for 10 seconds; thereafter, Ziehl carbol-fuxine is poured on a section coated with a filter paper and heated on an alcohol burner lamp until evaporating for 2 minutes; the stain is left on the section after the heating procedure is completed, for 3-5 minutes; the filter paper is removed, and the sections are washed in water, differentiated in a 1% acid buffer, washed in main water for 1 minute; thereafter Leffler methylene blue is placed on the section and heated on the alcohol burner lamp until evaporating, washed in water, differentiated in a 1% acid buffer and washed with a plenty of water, de-watered in alcohols of the increasing concentration for 1 minute, placed in xylol and balm.

EFFECT: developing the method for the accelerated stain of the histological specimen.

1 ex

Description

The invention relates to medicine, in particular to pathological anatomy and phthisiology, and relates to a method for an accelerated method of staining histological preparations for the detection of mycobacterium tuberculosis.

A large number of staining methods are known to detect mycobacterium tuberculosis. The closest analogue: this is the method described by G.G. Avtandilov (Fundamentals of pathological practice. - Moscow - 1994 - P.383). Coloring procedure: sections are dewaxed and adjusted to alcohol. Carbol-fuchsin is poured onto a slice covered with filter paper and heated over a flame of an alcohol lamp until vapors appear. Then the heating is carried out for 1-2 minutes, then the paint is left on the cut after stopping heating for another 20-25 minutes. The paint is poured off, filter paper is removed. Differentiate in 1% hydrochloric acid (70 °) until a pale pink tone appears. Washed in tap water for 1-2 minutes. Sections were stained with Lefler's methylene blue for 0.5-0.6 minutes. Stain slightly, then differentiate in 0.5-1% hydrochloric acid and washed well in water. Quickly carried out through alcohols, xylene and enclosed in a balm.

However, the proposed method is inconvenient in that the coloring of the preparations takes a large amount of time. Therefore, it is necessary to improve this method for the reliability and objectification of the post-mortem pathological study of tuberculosis in order to more quickly diagnose it.

The present invention is the development of an accelerated method of staining, allowing for a shorter period of time reliably posthumous diagnosis of pulmonary tuberculosis.

The essence of the invention lies in the fact that in the method of accelerated staining of histological preparations for the detection of mycobacterium tuberculosis, including the preparation of histological preparations according to the generally accepted method, staining them according to Ziehl-Nielson, after standard preparation of preparations for staining, the sections undergo a dewaxing procedure by sequentially placing them in xylene and descending alcohols for 5 minutes in a thermostat at a temperature of 54 ° C, followed by placing them in water, then in a 1% solution of iodic acid per 2 m in and rinsing in running water for 10 seconds, after which Zil carbol-fuchsin is poured onto a slice covered with filter paper and heated over the flame of an alcohol lamp until vapors appear for 2 minutes, paint is left on the cut after stopping heating for 3-5 minutes filter paper is removed and the sections are rinsed in water, differentiated with 1% acid buffer, rinsed in tap water for 1 minute, after which the solution of mitylene blue according to Lefleur is placed on the section and heated over the flame of an alcohol lamp until vapors are washed in water, differentiated in 1% acid buffer and washed well with water, dehydrated in alcohols of ascending strength for 1 minute, placed in xylene and enclosed in balm, and clarification in carbol-xylene is not permissible, as this may lead to to wash out the dye.

The task is achieved as follows.

According to the generally accepted technique, tissue of the lungs, lymph nodes, and bronchi are removed by autopsy. They are placed in a container filled with a 10% solution of neutral formalin. After 12-24 hours, longitudinal pieces of test tissues with a thickness of up to 05, -1.0 cm are cut out of the material and again immersed in a container with a 10% solution of neutral formalin. Then, 12 hours after thorough washing for 5 hours in running water, the material is dehydrated in alcohols of rising strength (50% (3 hours), 60% (3 hours), 70% (12 hours), 80% (12 hours), 96 % (12 hours), 100% (12 hours)), followed by immersion in castor oil for 12-24 hours.

To remove alcohols, the pieces are immersed in chloroform (3 portions with a change after 1.5 hours), then to remove chloroform, they are placed alternately in three portions of molten paraffin for 1.5 hours each. After all this, pieces are poured with molten paraffin into special paper boxes with the labeling of the material. The resulting blocks are glued onto a plastic cassette and histological sections with a thickness of 5 μm are made using a microtome. And glued onto a glass slide with subsequent staining according to Zil-Nielson. First, the sections undergo a dewaxing procedure by sequentially placing them in xylene and descending alcohols for 5 minutes in a thermostat at a temperature of 54 ° C, followed by placement in water. Then the sections are placed in a 1% solution of iodic acid for 2 minutes. Washed in running water for 10 seconds. Zilya carbol-fuchsin is poured onto a slice covered with filter paper and heated over the flame of an alcohol lamp until vapors appear for 2 minutes, then paint is left on the slice after heating is stopped for 3-5 minutes. Next, filter paper is removed and the sections are rinsed in water, differentiated with 1% acid buffer, rinsed in tap water for 1 minute. Next, a Leffler solution of methylene blue is placed on a slice and heated over a flame of an alcohol lamp until vapors appear, washed in tap water, differentiated in 1% acid buffer and washed well with water. Dehydrated in alcohols of ascending strength for 1 minute, placed in xylene and enclosed in a balm. Clarification in carbol-xylene is not permissible, since this can lead to leaching of the dye. The total staining time by this method is 20-25 minutes, and by the proposed methods of other authors more than one hour.

An example of using the proposed method.

The proposed method stained the tissue of the lungs, lymph nodes and bronchi in 105 deceased patients with drug-resistant tuberculosis aged 21 to 60 years. The studied material was extracted, fixed, washed, dehydrated and embedded in paraffin. Further, coloring was carried out according to Tsil-Nielson. First, the sections undergo a dewaxing procedure by sequentially placing them in xylene (5 minutes in a thermostat at 54 ° C) and descending alcohols (5 minutes in a thermostat at 54 ° C), followed by placement in water. Then the sections are placed in a 1% solution of iodic acid for 2 minutes. Washed in running water for 10 seconds. Zilya carbol-fuchsin is poured onto a slice covered with filter paper and heated over the flame of an alcohol lamp until vapors appear for 2 minutes, then paint is left on the slice after heating is stopped for 3-5 minutes. Remove the filter paper and rinse the sections in water, differentiate with 1% acid buffer, rinse in tap water for 1 minute. Next, a Leffler solution of methylene blue is placed on a slice and heated over a flame of an alcohol lamp until vapors appear, washed with water, differentiated in 1% acid buffer and washed well with water. Dehydrated in alcohols of ascending strength for 1 minute, placed in xylene and enclosed in a balm. Clarification in carbol-xylene is not permissible, since this can lead to leaching of the dye. The total staining time by this method is 20-25 minutes, and by the proposed methods of other authors more than one hour.

The technical result of using the invention:

1. Reliability and objectification of the post-mortem pathological diagnosis of tuberculosis.

2. By heating the solutions in the thermostat and above the flame of the spirit lamp, the color quality of the histological sections is improved.

3. The study time is reduced, which allows us to conclude earlier, to reduce the toxic effect of reagents on medical personnel.

4. This method can be used for differential diagnosis of various granulomatous formations.

5. The method can be used for urgent intraoperative diagnosis (by making histological sections on a freezing microtome).

Literature

1. Avtandilov G.G. Fundamentals of pathological practice. - Moscow, 1994, - p. 383.

2. Volkova OV Yeletsky Yu.K. Fundamentals of histology and histological technology. Moscow. "The medicine". 1982.

3. Greenberg L. M. Baranova E.Yu., Kondratov D.L. Histobacterioscopy in the morphological diagnosis of pulmonary tuberculosis: A manual for doctors. - Ural Research Institute of Phthisiopulmonology. - Ekaterinburg, 2004.

4. Tsinserling A.V. Processing and staining of smears and sections to identify microorganisms // Pathology Archive 1992. No. 5, - p. 35-40.

5. Khmelnitsky O.K., Belyanin V.L. Identification of pathogens of infectious diseases during morphological examination: A manual for doctors. - St. Petersburg: SPbMAPO, 1996, - p. 62.

Claims (1)

The method of accelerated staining of histological preparations for the detection of tuberculosis mycobacteria, including the preparation of histological preparations according to the generally accepted method, staining them according to Ziehl-Nielson, characterized in that after standard preparation of preparations for staining, the sections undergo a dewaxing procedure by sequentially placing them in xylene and descending concentration alcohols according to 5 minutes in a thermostat at a temperature of 54 ° C, followed by placing them in water, then in a 1% solution of iodic acid for 2 minutes and washing in a fine water for 10 seconds, after which Zil carbol-fuchsin is poured onto a slice covered with filter paper and heated over the flame of an alcohol lamp until vapors appear for 2 minutes, paint is left on the cut after stopping heating for 3-5 minutes, and filter paper is removed and rinse the sections in water, differentiate them with 1% acid buffer, rinse in tap water for 1 minute, after which the solution of mitylene blue according to Lefleur is placed on the section and heated over the flame of the spirit lamp until vapors appear, washed in water, they are differentiated in 1% acid buffer and washed well with water, dehydrated in alcohols of ascending strength for 1 minute, placed in xylene and enclosed in balsam, and clarification in carbol-xylene is not permissible, since this can lead to leaching of the dye.