Helminthological methods. Testing for enterobiasis and taeniasis
To detect fragments of helminths, feces are examined with the naked eye, then mixed with water and examined in small portions in a Petri dish against a dark background. All suspicious particles are placed on a glass slide in a drop of water and examined under a magnifying glass. You can place a daily portion in a cylinder with the addition of 5-10 times the amount of water. After stirring, the vessel is left until the suspended particles settle completely. Surface layer The liquids are drained and clean water is poured. The washed sediment is examined in small portions with the naked eye or under a magnifying glass. Microscopic examination methods are used to detect eggs.
Native smear method. Not a large number of feces from different places The test portion is ground on a glass slide in a drop of 50% glycerol solution, isotonic sodium chloride solution or water. The mixture is covered with a coverslip and viewed under a microscope.
Fulleborn floating method. One part of the feces is mixed with 20 parts of a saturated sodium chloride solution ( specific gravity 1.18), added in small portions. Large particles that float to the surface are immediately removed, and the mixture is left for 45 minutes. During this time, helminth eggs, having a lower specific gravity than the sodium chloride solution, float to the surface. The surface film is removed with a wire loop about 1 cm in diameter and transferred to a glass slide for examination under a microscope.
Kalantaryan method. The effectiveness of the floating method increases when replacing sodium chloride with a saturated solution of sodium nitrate. In this case, the mixture is kept for 10-15 minutes.
The surface film formed after settling a mixture of feces with a solution of sodium chloride or sodium nitrate can also be removed with a glass slide. For this purpose, a jar filled to the brim with a mixture of feces and a salt solution is covered with a glass slide so that its lower surface is in contact with the liquid. After settling, the glass is removed and, quickly turning upward with the surface on which the film is located, is examined under a microscope.
Scraping of sperianal folds (to identify pinworm eggs and oncospheres bovine tapeworm) do in the morning before going to the toilet. Using a wooden spatula soaked in water or a 50% glycerin solution, scrape around the anus. The resulting material is transferred to a glass slide in a drop of water or 50% glycerol solution and viewed under a microscope. The spatula can be replaced with a damp cotton swab, which is used to wipe the perianal area, then rinse well in water. The water is centrifuged and the sediment is examined under a microscope.
Behrmann method (for identifying larvae). A metal mesh with 5-6 g of feces applied to it is fixed on a glass funnel inserted into a tripod. A rubber tube with a clamp is placed on the lower end of the funnel. The funnel is filled with water heated to t° 50°, so that Bottom part the mesh containing feces came into contact with the water. The larvae actively move into the water and accumulate in the lower part of the rubber tube. After 4 hours, the liquid is drained into centrifuge tubes, centrifuged, and the sediment is examined under a microscope.
Analysis of sputum, nasal mucus and vaginal discharge to identify eggs of the pulmonary trematode paragonimus, roundworm and hookworm larvae, pinworm eggs, and fragments of echinococcal bladder. The portion of mucus (discharge) being examined is smeared onto glass and viewed macroscopically against a black and white background and then under a microscope. You can add a 25% antiformin solution to the test material, shake thoroughly and leave for 1-1.5 hours to dissolve the mucus. The mixture is centrifuged and the precipitate is examined under a microscope.
Analysis of duodenal and gastric juice to detect eggs of liver flukes, hookworms, and Strongyloides larvae. All three portions of duodenal contents obtained with are centrifuged and the sediment is examined under a microscope. They are also researching.
Tissue research. To identify Trichinella larvae, pieces of biopsied muscle are carefully split into fibers, squeezed between compressor glasses (thick glasses with screws) and examined under a microscope with a darkened light. To identify cysticerci, the muscles are dissected with dissecting needles, the isolated vesicle is cleared of surrounding tissue, squeezed between two glass slides and examined under a magnifying glass.
Blood test (to detect filaria larvae). Examine the hanging drop on a cover glass edged with Vaseline. You can mix 0.3 ml of blood with 10 times the amount of 3% solution. The mixture is centrifuged and the precipitate is examined under a microscope. For enrichment of drugs to 1 ml venous blood add 3 ml of 2% formalin solution or 5 times the amount of liquid consisting of 95 ml of 5% formalin solution, 5 ml acetic acid and 2 ml of concentrated alcohol solution of hematoxylin. The mixture is centrifuged, the precipitate is washed with distilled water and examined under a microscope. For differentiation different types filariae are examined by smears stained using the Giemsa-Romanovsky method.
Methods immunological diagnostics. Allergic diagnostic tests (see) with the corresponding type of helminth are also used (agglutination, complement fixation).
Helminthological methods research. Rice. Helminth eggs. 1-10 - eggs roundworms(nematodes): 1 - 3 - roundworms (1 - fertilized egg, 2 - fertilized egg without albumen, 3 - unfertilized egg); 4 - cat roundworms; 5 - carnivorous roundworms; 5 - pinworms; 7 - whipworm; 8 - tominx; 9 - hookworm; 10 - trichostrongylid. 11-15 - eggs tapeworms(cestodes): 11 - bovine tapeworm; 12 - dwarf tapeworm; 13 - rat tapeworm; 14 - pumpkin tapeworm; 15 wide tape. 16 - 24 - eggs of flukes (trematodes): 16 - trematodes (schistosomes) Japanese; 17 - trematodes (schistosomes) urine - sexual; 18 - trematodes (schistosomes) Munson; 19 - trematodes (parogonymus) pulmonary; 20 - trematodes (opisthorchis) Siberian (feline); 21 - trematodes (clonorchis) chinensis; 22 - intestinal trematodes (metagonimus); 23 - trematodes (fasciolas) of the liver; 24 - trematodes (dicrocelium) lanceolate.
An effective and convenient method of helminthological research is study of thick smear according to Kato, the essence of which is to detect helminth eggs in a thick smear of feces, cleared with glycerin and tinted with malachite green. Composition of Kato mixture - 6 ml 3% aqueous solution malachite green, 500 ml glycerin, 500 ml 6% phenol solution. The solution is stable and can be stored at room temperature. To prepare the preparations, pieces of feces the size of a pea are applied to a glass slide, covered with a film of hydrophilic cellophane kept in the Kato mixture for 24 hours, and pressed onto the glass to uniform distribution material. The smears cleared for 40-60 minutes are examined under a microscope. The detected helminth eggs are counted and morphological characteristics determine their species. The method allows you to identify eggs of roundworms, whipworms, cestodes, trematodes, and, to a lesser extent, hookworms and dwarf tapeworms.
Also widely used enrichment methods. The principle of flotation methods is to suspend feces in a saline solution, which has a higher relative density compared to helminth eggs, as a result of which they float to the surface. The contents of the surface film are examined under a microscope. The following solutions are used as enrichment mixtures: table salt - 400 g in 1 liter of water (relative density according to Fulleborn -1.18); sodium nitrate - 1 kg in 1 liter of water (relative density according to "Kalantaryan-1.38); sodium nitrate - 900 g and potassium nitrate - 400 g in 1 liter of water (relative density according to Brudastov and Krasnonos-1.48). Prepared the solutions are boiled and cooled.
For research, add 5-10 g of feces into a glass or earthenware glass, add 100-200 ml to it saline solution and stir thoroughly. Then the floating large particles are removed with a wooden spatula or a scoop made of paper or cardboard and immediately a wide slide is applied to the surface film so that the saline solution and the slide are in complete contact. After allowing the mixture to sit for 30-40 minutes, the slide is removed, placed under the microscope with the film facing up, and the entire surface is carefully examined. To avoid drying out, you can add 2-3 drops of a 50% glycerin solution. The surface film can also be removed with a wire loop. The efficiency of flotation methods increases as the relative density of the brine solutions increases. Using these methods, it is possible to detect eggs of nematodes, cestodes and trematodes.
To detect eggs in feces, the Krasilnikov sedimentation method is used. Feces are mixed with a 1% detergent solution ( washing powder“Lotus”, etc.) in a ratio of 1:10 until a suspension is formed. Under the influence of the detergent, various components of feces (proteins, fats, tissue elements) dissolve. After 30 minutes, the contents of the tube are shaken for 1-2 minutes, after which they are centrifuged for 5 minutes. Preparations are prepared from the sediment and examined under a microscope.
IN field conditions, as well as during mass examinations of the population for helminth infestation, it is convenient to use the Kato thick smear method. IN inpatient conditions When examining patients, it is preferable to use the most effective flotation methods. When using these methods during microscopy, it is advisable to count the eggs found in the preparation. If the sample or volume taken for the study of feces is standardized (for example, 1 teaspoon or tablespoon), and a constant uniform volume of saline solutions, one can roughly judge the intensity of invasions. This quantitative accounting can be useful in justifying prescribed treatment and assessing the effectiveness of deworming. In addition, other more accurate quantitative methods, in particular the Stoll method, can be used to establish the intensity of infection.
For the diagnosis of teniarhynchosis and enterobiasis The method of studying perianal-rectal scrapings is used. Using a wooden spatula soaked in a 50% glycerin solution, scrape the perianal folds in the morning before defecation in the circumference anus And lower sections rectum. The resulting material, cleaned from a spatula with the edge of a cover glass, is placed on a glass slide in a drop of 50% glycerol solution, covered with a cover glass and examined under a microscope. You can also examine a strip of cellulose tape, which is first pressed with the adhesive side to the perinanal folds, then placed on a glass slide and examined microscopically.
Detection of nematode larvae in feces using the Berman method. The method is based on the thermotropic property of larvae. For research, take 1 tablespoon of feces and place it in a metal mesh or a mesh of several layers of gauze on a wire frame. The mesh is installed in a funnel mounted on a tripod. A rubber tube with a clamp is attached to the funnel. Having lifted the mesh, the funnel is filled with water (temperature +40°...+50°C) so that the lower part of the mesh is immersed in water. Larvae from feces actively migrate to warm water and, settling, accumulate in the lower part of the funnel. After 2-4 hours, the clamp is opened, the water is drained into a centrifuge tube and centrifuged for 2-3 minutes. Then the supernatant liquid is drained, the sediment is transferred to a glass slide and examined under a microscope, where mobile larvae of the causative agent of strongyloidiasis are found.
Harada method and Mori makes it possible to differentiate between hookworm and necator larvae. Hookworm larvae are cultured on filter paper. For this purpose, 0.5 g of fresh feces, taken from the patient no later than 1 hour after defecation, is applied to strips of filter paper measuring 12X1.5 cm, leaving both ends of the strip clean. One end of the strip is immersed in a test tube, a quarter of which is filled with water, and the other is clamped with a stopper. The test tubes are placed in a thermostat at a temperature of +26... + 28°C. The larvae that develop from the eggs descend through the filter paper and settle to the bottom of the test tube. After 5-6 days, the strip of paper is removed, and the liquid remaining in the test tube is examined under a magnifying glass or centrifuged. The precipitate formed during centrifugation is examined under a light microscope. When using tetrahedral glass jars (15xxx7 cm in size) instead of test tubes, with 4 paper strips attached to the walls, the efficiency of analyzes increases (G.M. Maruashvili et al., 1966).
Test methods for schistosomiasis . Examination of feces - a portion of feces is mixed with 250 ml of water, filtered through 3 layers of gauze into a conical vessel, which is filled to the top with water. After 30 minutes, the liquid layer is drained, and a fresh portion of water is added to the sediment. The precipitate is washed until a clear supernatant is obtained and examined under a microscope.
Larvoscopy method - 20-25 g of feces are placed in an Erlenmeyer flask with a glass tube soldered on the side, pointing upward, and washed tap water. Then the flask is covered with dark paper, leaving a soldered glass tube in the light at a temperature of +25...+30°C. The hatched miracidia are concentrated at the meniscus in the lateral tube, where they can be seen with a magnifying glass or with the naked eye. Urine examination - 100 ml of urine collected between 10 a.m. and 2 p.m., or a daily portion, is settled and then centrifuged at 1500 rpm. The resulting sediment is applied* to a glass slide and examined under a microscope. The WHO recommends a method of filtering the entire urine sample. After filtration, the filters are treated with formaldehyde or heated (to kill the eggs) and then moistened with an aqueous solution of ninhydrin. In dried preparations, egg embryos acquire a purple color.
Application of immunological research methods for diagnosing schistosomiasis are associated with difficulties, since adult schistosomes and their eggs contain a large number of antigens that cause immune reactions, which are not species-specific (D. Bradley, 1979).
In our country, for staging immunological reactions for helminthiasis, it is produced whole line standard diagnostics. Practical use have allergies skin test for echinococcosis and alveococcosis, RLA with echinococcal antigen, RSC in the cold, precipitation reaction in the cold in various modifications (ring precipitation, precipitation in test tubes or capillaries, which are placed with trichinosis, cysticercosis and migroascariasis antigens). Standard diagnostic kits for performing the above-mentioned serological reactions are produced by enterprises producing bacterial preparations. The manufacturer includes with the diagnostic kits produced detailed instructions on storage rules, shelf life of the drug and instructions on the use of relevant antigens with the technique of staging the reaction.
IN last years The list of serological reactions used to diagnose helminthiasis has expanded significantly. For these purposes, the following reactions are used: RIGA, indirect immunofluorescence (RIF), immunodiffusion in a gel (RID), counter immunoelectrophoresis (CIEF), CEMA. These reactions can be used for ascariasis, toxocariasis, trichinosis, hookworm disease, filariasis, echinococcosis and alveococcosis, opisthorchiasis, schistosomiasis, paragonimiasis. However, for these reactions in our country, standard diagnostic tests are not issued and the technique for their diagnosis is not regulated. Individual laboratories, mainly scientific ones, independently prepare specific antigens and use them in various modifications. The description of these methods is widely presented in the literature and is not strictly unified. Interpretation of immunological analysis data should be based on studying the dynamics of the immune response, taking into account the level of specificity and sensitivity of each method used. serological reaction. Therefore, when making a diagnosis, as well as during seroepidemiological surveys of the population, it is advisable to use several of the most sensitive reactions. From this point of view, the reactions of VIEF and REMA, which differ, have proven themselves well high sensitivity and quite high specificity (P. Ambroise-Thomas, 1978; I. E. Ballad, 1979; G. M. Negreanu, 1980; A. M. Ponomareva, 1981; A. Ya. Lysenko, 1978, etc.). The effectiveness of REMA has been tested for amoebiasis, leishmaniasis, trypanosomiasis and toxoplasmosis (G. A. Ermolin, 1980).
Chapter III. Diagnosis of helminthiasis and methods of helminthological research
It is necessary to examine for helminthiasis all patients applying for medical care, and especially patients who turn to a pediatrician, therapist and neurologist with complaints of side effects gastrointestinal tract, nervous system and with anemia. If the doctor is not always able to use laboratory research methods, then every medical worker providing care in an outpatient clinic or hospital is required to interview the patient about the isolation of helminths.
If there are clinical indications given in the relevant chapters, the diagnosis should be clarified using laboratory methods studies for helminthiasis.
Due to the predominance intestinal helminthiases greatest practical significance has a stool examination.
Methods for testing stool for helminthiasis
Stool is delivered to the laboratory in a clean glass container (about a quarter cup of stool taken from different places in one portion); During a routine examination, it is allowed to deliver stool to the laboratory in matchboxes or splint boxes.
To control deworming, the entire portion of feces collected after administration is delivered (as prescribed by the doctor). anthelmintic and laxative (in large closed glass jars, buckets).
Microscopic examination of stool is basic in the diagnosis of intestinal helminthiases; it should always be preceded by a general macroscopic examination of feces to detect segments of large cestodes, pinworms, roundworms, etc.
The stool should be fresh or canned (in a 5% formaldehyde solution), since drying dramatically changes the structure of the eggs. In addition, when feces stand, fast development eggs of some helminths (for example, hookworms), which makes diagnosis difficult.
According to the instructions of the USSR Ministry of Health, it is necessary to examine stool simultaneously using the Fulleborn method and the native smear.
Native smear
Native smear: a small piece of feces (about the size of a pea), taken with a match, glass or wooden stick from different places in the delivered portion, is thoroughly ground on a glass slide in a drop of 50% glycerol solution or in saline solution or in water. Cover with a coverslip, pressing lightly on the latter (with a dissecting needle). The smear should be thin, transparent and uniform. It is used only as an addition to other methods that provide enrichment of the drug. At least two drugs should be reviewed.
In order to detect helminth larvae (as well as their eggs), a native smear is made as follows (according to Shulman): 2-3 g of feces are thoroughly mixed by “twisting” a glass rod into an emulsion with a five-fold amount of clean water or saline. During stirring, the larvae accumulate near the glass rod, so immediately after the end of stirring, a drop of the emulsion is quickly transferred with a glass rod onto a glass slide, covered with a coverslip and examined. S. D. Lyubchenko (1936) proved that the twisting method is more effective than the smear method, especially with regard to roundworm eggs. Based on the work of S. D. Lyubchenko, we consider it advisable to replace the smear method with the twisting method.
Fulleborn method
Fulleborn method: 5-10 g of feces taken from different places are placed in a jar with a capacity of 50-100 ml and thoroughly rubbed with a glass or wooden rod in a saturated solution table salt(400 g of this salt is dissolved in 1 liter of water, heated to a boil and filtered through a layer of cotton wool or gauze; the solution is used cold: specific gravity 1.2). The solution is added gradually until a uniform suspension is obtained, and the total amount of solution added should be approximately 20 times more quantity feces. To mix feces, Fulleborn recommended using tea glasses, but it is more convenient to prepare a suspension in ointment jars with a capacity of 50-100 ml, using two jars for each analysis (or in cups with a capacity of 100 ml).
Immediately after preparing the suspension, large particles that have floated to the surface are removed from the surface with a spatula, a metal scoop or a piece of clean paper ( plant formations, undigested food remains, etc.), after which the mixture is left to stand for 1-1.5 hours. After this time, the entire film is removed from the surface of the mixture by touching a wire or platinum loop (flat) with a diameter of no more than 1 cm, bent at a right angle; The film is shaken onto a glass slide and covered with a coverslip. Place 3-4 drops under each cover slip (18x18 mm). In total, at least 4 preparations should be prepared (one cover glass for each preparation). The loop is heated over a fire and washed with water after each analysis.
Using the Fulleborn method, eggs of all nematodes (with the exception of unfertilized roundworm eggs) and dwarf tapeworm eggs are quickly and easily detected.
The Berman method is used to examine feces for helminth larvae (for strongyloidiasis). This method is as follows: 5 g of feces on a metal mesh (a milk strainer is convenient for this purpose) is placed on a glass funnel attached to a tripod. A rubber tube with a clamp is placed on the lower end of the funnel.
The mesh with feces is lifted and water heated to approximately 50° is poured into the funnel so that the lower part of the mesh with feces is immersed in water. The larvae actively move into the water and accumulate in the lower part of the rubber tube. After 2-4 hours, the clamp is opened and the liquid is drained into one or two centrifuge tubes.
After centrifugation for 1-2 minutes top part the liquid is quickly drained, and the sediment is placed in drops on glass slides and examined under coverslips or spread thin layer on 2-3 large glasses and then examine without cover glasses.
The Berman method is also used to test soil for the presence of hookworm larvae.
Stoll method
The Stoll method is used to determine the intensity of invasion. A decinormal solution of caustic soda is poured into a special glass flask to the 56 cm 3 mark, and then feces are added until the liquid level reaches 60 cm 3, i.e. 4 cm 3. After shaking with glass beads, 0.075 ml of the mixture is taken for examination and examined under one or two ordinary coverslips. The resulting amount is multiplied by 200 to obtain the number of eggs contained in 1 cm 3 of feces.
Study of duodenal contents
Duodenal juice and bladder bile, obtained in the usual way using probing (and bladder bile and after a reflex from the gallbladder), are thoroughly mixed with an equal volume ethyl ether; the mixture is centrifuged, after which the sediment is examined under a microscope. In addition to the sediment, microscopic examination flakes floating in the liquid, which may contain helminth eggs, are necessarily exposed. When testing gastric juice and vomit for helminth eggs, you can use the same technique.
Examination of duodenal juice and stomach contents should be carried out if there is a suspicion of helminthic diseases liver, gall bladder (opisthorchiasis, fascioliasis, dicroceliosis) and duodenum(strongyloidiasis).
Sputum examination
The sputum is ground on a glass plate, tightly covered with another glass plate and examined with the naked eye against a light and black background, as well as under a magnifying glass in transmitted light. Individual pieces of sputum (“rusty” accumulations, tissue scraps, etc.) are applied in a thin layer to a glass slide, tightly covered with a coverslip and examined at low and high magnification microscope
a) To diagnose cutaneous cysticercosis, subcutaneous tissue or muscle, an aseptically excised piece of the relevant tissue is examined first with the naked eye. Areas of tissue are moved apart using dissecting needles to reveal visible with the naked eye vesicle - cysticercus (photo A); its length is 6-20 mm, width 5-10 mm. When a vesicle suspicious for cysticercus is detected, it is crushed between two glass slides and examined under a microscope. Cysticercus (Cistycercus cellulosae) is determined by the presence of a scolex with four suckers and a corolla of hooks (photo B).
| Photo. A - cysticerci with scolex turned outward; B - Head of a pork tapeworm. | |
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b) To diagnose trichinosis, an aseptically excised piece of muscle (biceps or gastrocnemius) is carefully crushed in a 50% glycerin solution into the finest fibers using dissecting needles. The crushed muscles are compressed between two glass slides and examined under a low-power microscope in a darkened field of view. It is recommended to test muscles for trichinosis no earlier than on the 8th day of the disease. Trichinella larvae are found in the muscles in a coiled position: they are enclosed in lemon-shaped capsules.
| Photo. A - Trichinella larvae in muscles; B — Calcified capsules of Trichinella. | |
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X-ray
Most often, fluoroscopy is used to diagnose echinococcosis and, less commonly, cysticercosis. Cysticerci are detected by fluoroscopy only after calcification (in cases long-term illness). In recent years, fluoroscopy has also been used to diagnose ascariasis both in the early larval stage and, partly, in the intestinal stage.
During the period of migration of roundworm (and hookworm) larvae, unstable, sometimes multiple inflammatory foci are detected in the lungs; at the same time, significant eosinophilia appears in the blood.
Sexually mature roundworms are clearly visible on fluoroscopy of the intestines of affected individuals. This method, despite its complexity and cumbersomeness, should be used as an additional method for diagnosing ascariasis in cases with a negative scatological analysis. According to E. S. Geselevich, of 180 patients with ascariasis identified by fluoroscopy, 54 had no ascaris eggs found in their stool (see).
Helminthological research methods. Methods for diagnosing helminth infections are divided into direct ones, based on the direct detection of helminths themselves or their fragments, as well as helminth larvae and eggs (methods for examining feces, urine, bile and duodenal contents, sputum, blood and tissues, material obtained by scraping from the perianal area and subungual spaces), and indirect, with the help of which they reveal secondary changes, arising in the human body as a result of the vital activity of helminths (studies of the morphological composition of blood, immunological methods diagnostics of helminthiasis, X-ray examinations, etc.). Of the direct methods, the most common are scatological ones, which are divided into macro- and microhelminthoscopic. In some cases, special methods are used.
Macrohelmitoscopic research methods aimed at searching for helminths or their fragments (scolex, segments, parts of the strobila of cestodes). They are used to diagnose those helminthiasis in which eggs are not excreted in the patient’s excrement or are excreted in small quantities and not always (for example, with enterobiasis, pinworms are found in the feces, with taeniasis - segments).
To detect pinworms or cestode segments in feces, the feces are examined with the naked eye. For differential diagnosis Taeniasis, it is recommended to view feces diluted with water in separate small portions in black photographic cuvettes or in Petri dishes against a dark background. Large formations suspicious for fragments of helminths are examined under a magnifying glass between two slides. If according to clinical indications suggest the detection of small helminths or cestode heads after treatment, then suspicious particles are examined under a magnifying glass in a drop of glycerin, and, if necessary, under a microscope.
Microhelminthoscopic research methods(qualitative) are aimed at identifying helminth eggs and larvae. The thick smear method with a cellophane cover plate according to Kato is used. Kato mixture consists of 6 ml 3% aqueous solution of malachite green, 500 ml glycerin and 500 ml 6% phenol solution. Kato plates (hydrophilic cellophane is cut into pieces measuring 20´ 40 mm) immersed for 24 h into the Kato mixture so that they are adjacent to each other (3-5 ml Kato solution per 100 plates). 100 mg feces are applied to a glass slide, covered with a cellophane cover plate according to Kato and pressed down so that the feces are smeared on the glass slide within the limits of the cellophane plate. The smear is left at room temperature to lighten by 40-50 min, and then examined under a microscope. In the hot season, to prevent the preparation from drying out, place a damp sponge on the plate of the prepared preparation.
For complete detection of helminths of all types, the Kato thick smear method with a cellophane cover plate must be used in combination with one of the enrichment methods. The most common of them are the Kalantaryan method and the Fulleborn method.
Kalantaryan method: in wide-mouth bottles with a volume of 100 ml stir thoroughly with a glass rod 5 G feces, gradually adding saturated sodium nitrate solution (1 kg sodium nitrate per 1 l water when boiling) to the edge of the glass. Large particles that float to the surface are removed with a paper scoop. A glass slide is applied to the surface of the saline solution (the saline solution is added until the mixture comes into complete contact with the glass slide). In 20-30 min the slide is removed and the film is examined under a microscope. In the absence of this salt, you can use a saturated solution of table salt according to Fholleborn (400 G salt in 1 l boiling water).
Due to the fact that eggs with a large specific gravity (unfertilized eggs of roundworms, eggs of trematodes and large cestodes) do not float, in addition to examining the surface layer of the liquid, when using the Fulleborn method, it is necessary to examine 2-4 preparations from the sediment under a microscope.
Special laboratory methods for testing various helminthiases.
7.7. Methods for determining the viability of helminth eggs and larvae
The viability of helminth eggs is determined by appearance, by staining with vital paints, cultivation in optimal conditions and setting up a biological sample.
7.7.1. Determining the viability of helminth eggs or larvae by appearanceHelminth eggs are microscoped first at low, then at high magnification. In deformed and dead helminth eggs, the shell is torn or bent inward, the plasma is cloudy and loosened. Segmented eggs have crushing balls (blastomeres) of unequal size, irregular shape, often shifted to one pole. Sometimes there are abnormal eggs that, despite external deformities, develop normally. In living roundworm larvae, fine granularity is present only in the middle part of the body; as they die, it spreads throughout the body, large shiny hyaline vacuoles, the so-called “strings of pearls,” appear.
To determine the viability of mature eggs of roundworms, whipworms, and pinworms, you should call active movements larvae by lightly heating the preparation (to a temperature not exceeding 37 °C). It is more convenient to observe the viability of roundworm and whipworm larvae after they are isolated from the egg shell by pressing on the cover glass of the preparation with a dissecting needle or tweezers.
In invasive roundworm larvae, a sheath is often seen peeling off at the head end, and in whipworm larvae that have completed development in the egg, a stylet is found in this place at high magnification. In dead helminth larvae, regardless of their location (in the egg or outside it), body decay is noticed. In this case, the internal structure of the larva becomes blocky or granular, and the body becomes cloudy and opaque. Vacuoles are found in the body, and breaks are found on the cuticle.
The viability of taeniid oncospheres (bovine, pork tapeworm, etc.) is determined by the movement of the embryos when exposed to them digestive enzymes. The eggs are placed on a watch glass with the dog's gastric juice or artificial duodenal juice. The composition of the latter: pancreatin - 0.5 g, sodium bicarbonate - 0.09 g, distilled water - 5 ml. Watch glasses with eggs are placed in a thermostat at 36 - 38 ° C for 4 hours. In this case, living embryos are freed from their membranes. The shells of living oncospheres also dissolve in acidified pepsin and in alkaline solution trypsin after 6 - 8 hours in a thermostat at 38 ° C.
If you place teniid eggs in a 1% solution of sodium sulfide or a 20% solution of sodium hypochloride, or in a 1% solution of chlorine water at 36 - 38 ° C, mature and living embryos are released from the membranes and do not change during 1 day. Immature and dead oncospheres shrink or swell and enlarge sharply, and then “dissolve” within 10 minutes to 2 hours. Living taeniid embryos also actively move in a mixture of 1% sodium chloride solution, 0.5% sodium bicarbonate solution and bile at 36 - 38 °C.
The viability of Adolescaria fascioli collected on plants and other objects of water bodies is checked by examining them on a glass slide in physiological solution under a microscope with a heating stage. When the trematode larvae are heated, they begin to move.
To determine the viability of dwarf tapeworm eggs, the simplest method is by N.S. Ionina: in living eggs, the median pair of embryonic hooks is either parallel to the lateral ones, or the latter form an angle with the median at the base of less than 45°. In dead eggs, the lateral pairs form an angle with the median pair at the base of more than 45°, or the hooks are randomly scattered (their paired arrangement is lost); Sometimes the embryo shrinks and granules form. A more accurate method is based on the appearance of movements of the oncosphere during a sharp change in temperature: from 5 - 10 ° to 38 - 40 ° C.
Determination of the viability of immature nematode eggs should be studied in a humid chamber (Petri dishes), placing roundworm eggs in a 3% formaldehyde solution prepared in an isotonic sodium chloride solution at a temperature of 24 - 30 ° C, whipworm eggs in a 3% solution of hydrochloric acid at a temperature of 30 - 35 ° C; pinworm eggs in an isotonic sodium chloride solution at a temperature of 37 °C. Petri dishes should be opened 1 - 2 times a week for better aeration and the filter paper should be re-wetted clean water.
Observations of the development of helminth eggs are carried out at least 2 times a week. The absence of signs of development within 2 - 3 months indicates their non-viability. Signs of the development of helminth eggs are first the stages of crushing, dividing the contents of the egg into separate blastomeres. During the first days, up to 16 blastomeres develop, which pass into the second stage - morula, etc.
Hookworm eggs are cultured in a glass cylinder (50 cm high and 7 cm in diameter) closed with a stopper. A mixture of equal volumes sterile sand, charcoal and feces with hookworm eggs, diluted with water to a semi-liquid consistency, are carefully poured onto the bottom of the cylinder using a glass tube. During 1 - 2 days of settling in the dark at a temperature of 25 - 30 ° C, rhabditiform larvae hatch from the eggs, and after 5 - 7 days they become filariform: the larvae crawl up the walls of the cylinder, where they are visible even to the naked eye.
Eggs of trematodes that naturally develop in water, for example opisthorchis, diphyllobothriidae, fasciolae and others, are placed on a watch glass, Petri dish or other vessel, and a small layer of ordinary water is poured. When cultivating Fasciola eggs, it should be taken into account that they develop faster in the dark, while in living eggs at a temperature of 22 - 24 ° C, miracidium is formed in 9 - 12 days. When microscopying developing trematode eggs, the movements of the miracidium are clearly visible. Miracidium fasciola emerges from the egg shell only in the light.
Fulleborn method. Hookworm and strongylid larvae are cultured on agar in a Petri dish with animal charcoal. After keeping in a thermostat at a temperature of 25 - 30 ° C for 5 - 6 hours, the larvae crawl across the agar, leaving behind a path of bacteria.
Harada and Mori method. Add 7 ml of distilled water to test tubes placed in a rack. Using a wooden stick, take 0.5 g of feces and make a smear on filter paper (15 x 150 mm) 5 cm from the left edge (this operation is carried out on a sheet of paper to protect the surface of the laboratory bench). Then the strip with the smear is inserted into the test tube so that the left end free of the smear reaches the bottom of the test tube. Cover the upper end with a piece of cellophane and wrap it tightly with an elastic band. The number and surname of the person being examined is written on the test tube. In this state, the tubes are stored for 8 - 10 days at a temperature of 28 °C. To study the larvae, remove the cellophane cover and remove a strip of filter paper with tweezers. Care should be taken when doing this as small numbers of infective larvae may move to the top end of the filter paper or to the side of the tube and penetrate under the surface of the cellophane.
Test tubes are placed in hot water bath at a temperature of 50 ° C for 15 minutes, after which their contents are shaken and quickly poured into a 15 ml test tube to sediment the larvae. After centrifugation, the supernatant is removed, and the sediment is transferred to a glass slide, covered with a coverslip, and examined microscopically under low magnification.
For differential diagnosis filariform larvae, you must use the data in Table 3.
Table 3
DIFFERENTIAL DIAGNOSTICS OF FILARIOID LARVAES OF A. DUODENALE, N. AMERICANUS, S. STERCORALIS, TRICHOStrONGYLUS SP.
| Larvae | Dimensions | Characteristic signs |
| A. duodenale | Body length about 660 µm, sheath length - 720 nm | The striation of the cap is less pronounced, the oral protrusion is less noticeable, the anterior end of the body (but not the cap) is blunt, the diameter of the intestinal tube is smaller than the esophageal bulb, the caudal end is blunt |
| N. americanus | Body length about 590 microns, sheath length - 660 nm | The cap is noticeably striated, especially in the caudal part of the body, the oral protuberance appears dark, the anterior end of the body (but not the cap) is rounded like a narrow end chicken egg, the anterior part of the intestinal tube has the same diameter as the esophageal bulb, the caudal end is sharply pointed |
| S. stercoralis | Body length about 500 microns | The larva is without a sheath, the esophagus is about half the length of the body, the tail is blunt or branched |
| Trichostrongylus sp. | Body length about 750 µm | The intestinal lumen is not straight, but zigzag, the tail end is rounded and shaped like a button |
Dead tissues in most cases perceive paints faster than living ones. These features are used in helminthology to determine the viability of helminth eggs and larvae. However, in some cases, some paints are better perceived by living tissues than by dead ones.
To differentiate between living and dead eggs and larvae, the following paints and methods are used.
Leukobase methylene blue is often used to stain living and dead tissues. Living cell or the tissue reduces methylene blue into colorless leukobase, dead tissue does not have this ability, so it acquires color.
The criterion for the condition of an egg is the coloring of the embryo, but not the shell. This ability is associated with the conditions under which the egg dies. In cases where the fibrous membrane in a dead egg does not lose its semi-permeable properties, it will not allow dyes to pass through, and therefore the dead embryo will not be stained. A colored embryo always indicates the death of the egg.
To color roundworm eggs, you can use methylene blue in a solution of lactic acid with caustic alkali (0.05 g methylene blue, 0.5 g sodium hydroxide, 15 ml lactic acid). Living eggs do not perceive color; are painted in Blue colour embryos of dead eggs. Staining of roundworm larvae with a basic solution of brilliantcresyl blue paint at a concentration of 1:10000 is carried out as follows: a drop of liquid with roundworm eggs and a drop of the basic paint solution are applied to a glass slide. The preparation is covered with a coverslip, which is pressed tightly to the slide while lightly tapping with a dissecting needle. The number of emerging larvae and the degree of their staining are observed under a microscope; after which the same drug is examined again after 2 - 3 hours. Only undeformed larvae that have not stained for 2 hours are considered alive. Dead larvae either do not emerge from the eggs, or become colored when the shell breaks (partially or completely).
When determining the viability of avian roundworm eggs, it is possible to stain the preparations with 5% alcohol solution Yoda. When it is applied to the preparation, the germs of dead Ascaridia eggs occur within 1 - 3 seconds. are painted orange.
Dead opisthorchis eggs and bovine tapeworm oncospheres are stained with a solution of toluidine blue (1:1000), and dead bovine tapeworm oncospheres with a solution of brilliant cresyl blue (1:10000). At the same time, the embryos and shells of both dead and living eggs acquire color. Therefore, after staining, eggs and oncospheres are washed in clean water and additionally stain them with safranin (diluted 1:10000 in alcohol 10 °C). Alcohol removes paint from the shells, and safranin turns it red. As a result, live eggs turn red; eggs with dead embryos turn blue, but the shell remains red. Dead embryos of bovine tapeworm oncospheres quickly, within a few minutes, turn bright red or pink color safranin or blue brilliant cresyl blue at a dilution of 1:4000, or indigo carmine at a dilution of 1:1000 - 1:2000. Living embryos do not change under the influence of these paints even after 2 - 7 hours.
To determine the viability of dwarf tapeworm eggs, it is recommended to use the following paints:
1. Brilliantcreasyl blue (1:8000) - after 1 hour, the oncosphere of dead eggs becomes especially brightly colored, which stands out sharply against the pale or colorless background of the rest of the egg.
2. Safranin (1:8000 when exposed for 2 hours and 1:5000 for 3 - 5 hours).
3. 50% solution of pyrogallic acid in a dilution of 1:2 - when exposed for 1 hour at a temperature of 29 - 30 ° C (the lower the temperature, the longer the dyeing process).
7.7.3. Luminescent method for studying helminth eggs and larvaeFluorescent microscopy makes it possible to differentiate between living and dead objects without damaging the egg. Not used for fluorescence ultra-violet rays, and the blue-violet part of visible light, with a conventional microscope and glass slides; added to the OI-18 illuminator special set color filters.
Live and dead eggs of roundworms, pinworms, dwarf tapeworms, bovine tapeworms, broad tapeworms and other helminths fluoresce differently. This phenomenon is observed both during primary luminescence without the use of dyes and when stained with fluorochromes (acridine orange, coryphosphine, primulin, auroline, berlerin sulfate, trypaflavin, rivanol, acrhin, etc.).
Uncolored, live, unsegmented roundworm eggs glow bright green with a yellowish tint; in dead eggs the shell radiates green light much brighter than the dark green embryonic part; In roundworm eggs with a larva, only the shell appears, and in dead eggs, both the shell and the larva are bright yellow.
Unpigmented and unsegmented live eggs of pinworms and dwarf tapeworms emit a greenish-yellow light; the shell of dead eggs intensively luminesces against the background of a dark green embryonic mass.
With secondary luminescence (when stained with acridine orange at a dilution of 1:10,000 and 1:50,000 from 30 minutes to 2 hours), the shell of living and dead nematodes, trematodes and cestodes luminesces differently.
The shells of live and dead eggs of roundworms, toxocara, pinworms, dwarf tapeworms, rat tapeworms, bull tapeworms, and tapeworms are colored orange-red. The embryos of living eggs of roundworms, toxascaris, rat tapeworms, broad tapeworms and oncospheres of bovine tapeworms fluoresce dull dark green or gray-green color. The dead embryonic eggs of these helminths emit a “burning” orange-red color. Living pinworm and toxocara larvae (egg shells freed) emit a dull gray-green light; when they die, the color changes from the head end to a “burning” light green, then yellow, orange and finally bright orange.
When stained with fluorochromes - coryphosphylum, primulin, dead eggs of roundworms and whipworms exhibit a glow from lilac-yellow to copper-red. Viable eggs do not luminesce, but are colored dark green color.
Live eggs of trematodes (paragonimus and clonorchis) do not luminesce when stained with acridine orange, but dead eggs produce a yellowish-green color.
The luminescence method can also be used to determine the viability of helminth larvae. Thus, strongylate and rhabditatus larvae fluorochromed with a solution of acridine orange (1:2000) glow: living ones - green (with a tint), dead ones - with a bright orange light.
Living miracidia emerging from the shell emit a dim bluish light with a barely noticeable light yellow corolla of cilia, but 10 - 15 minutes after death they appear with a bright “burning” light green, and then orange-red light.
7.7.4. Biological sample methodFor example, to determine the viability of ascarid eggs (pig roundworm, human roundworm, Toxocara, Toxascaris, etc.) per animal (guinea pigs, mice) you need at least 100 - 300 eggs with developed larvae. Ascaris eggs in an isotonic sodium chloride solution are pipetted through the mouth of a mouse or guinea pig. After 6-7 days, the animal is slaughtered, its liver and lungs are opened and examined separately for the presence of ascaridate larvae. To do this, the liver and lungs are chopped into small pieces with scissors and examined using the Berman or Supryaga method (section 6.1.2).
If animals were infected with live invasive eggs, then upon autopsy, migrating ascarid larvae are found in the liver and lungs.
In case of infection, Fasciola eggs in the feces of laboratory animals can be detected in rabbits after 2 months, in guinea pigs- after 50 days, in mice - after 35 - 40 days.
To more quickly obtain an answer, laboratory animals are opened after 20 - 30 days and the liver is examined for the presence of young fascioli.
To determine the viability of dwarf tapeworm eggs, it is also recommended to feed them to previously uninfected white mice, followed by opening the animals after 92-96 hours and identifying cysticercoids in the intestinal villi or cestodes in the intestinal lumen.
To determine the viability of opisthorchis eggs, a method is recommended (German S.M., Baer S.A., 1984), based on the physicochemical activation of the miracidium hatching gland and stimulation motor activity larvae, which leads to the opening of the egg cap and the active release of miracidium under experimental conditions.
The suspension of opisthorchis eggs in water is pre-cooled to 10 - 12 °C (all subsequent operations are carried out at room temperature 19 - 20 °C). 1 drop of suspension containing 100 - 150 eggs is added to a centrifuge tube. The test tube is placed in a stand for 5 - 10 minutes. During this time, all the eggs manage to sink to the bottom. Then the excess water is carefully sucked out with a strip of filter paper and 2 drops of a special medium are added to the test tube. The medium is prepared in 0.005 M Tris-HCl buffer; a 12 - 13% ethanol solution and a dye (fuchsin, safranin, eosin, methylene blue, etc.) are added to the buffer. The test tube is shaken, its contents are pipetted onto a glass slide and left for 10 minutes, shaking slightly. Then add 2 drops of the specified medium. The preparation is ready for microscopy under a conventional light microscope at 20x magnification.
During this time, the lid of viable larvae opens, and the miracidium actively exits into the specified environment. Thanks to the presence of ethanol in it, they are immobilized after 2 - 5 minutes and then painted with a dye. They can be easily detected and counted by microscopy.
