Microscopic examination of feces. Intravital diagnosis of helminthiases

Direct methods: detection of the helminths themselves, their fragments, eggs, larvae in feces, urine, duodenal secretion, sputum, nasal and vaginal mucus, the contents of the subungual spaces, biopsied tissue pieces.

When diagnosing, it is impossible to identify by any one method the eggs or larvae of all types of helminths living in digestive system person. Thus, when using the flotation method, trematode eggs and, in some cases, unfertilized roundworm eggs do not float into the surface film (due to the high specific gravity). In feces, it is very rare to find pinworm eggs, teniid oncospheres, which are detected by special research methods: scraping from perianal folds for pinworms and teniids, sedimentation methods for trematodes (opistorch eggs, etc.). Therefore, for a targeted examination of the patient for helminthiases, the doctor in the referral should indicate which helminths should be given the main attention (diagnosis), which will allow the laboratory assistant to choose the appropriate technique for identifying this type of helminth. Feces taken from different places feces in an amount of at least 50 grams (teaspoon) in a clean glass dish should be sent to the laboratory no later than one day after defecation and examined on the day of admission.

If necessary, save feces until next day it is placed in a cold place (0-4°C) or filled with one of the preservatives.

Before the study, the feces are mixed with a stick so that the helminth eggs are evenly distributed in the total mass.

If any helminth eggs are found in the preparation, the viewing is not stopped, because. may be double or triple invasion.

Monitoring the effectiveness of the treatment of helminthiasis is carried out by examining feces for helminth eggs in 2-3 weeks or 2-3 months after treatment, depending on the detected helminth.

Macroscopic methods are used to detect whole sexually mature helminths or their fragments in the feces with the naked eye or with a hand-held magnifying glass.

Often on the surface of the feces after defecation, you can see actively crawling pinworms; excreted with feces roundworm; sometimes people themselves notice the discharge of helminths. In patients with diphyllobothriasis, fragments of the strobili of the tapeworm (in the form of "noodles") can stand out, and in those infested with teniids (pork or bovine tapeworm), segments of helminths often leave with feces (in the form of "white cuts") or they actively crawl out of anus.

The macroscopic method is the main one for the differential diagnosis of teniadosis and teniarhynchosis (in combination with a survey).

Of the special macroscopic methods, the method of sequential washing of feces is used.

Feces are mixed in water to obtain a uniform suspension, after which, when good lighting they are carefully examined in separate small portions in black photographic cuvettes or on dark background in Petri dishes. With tweezers or a dissecting needle, all suspicious white particles, large formations suspicious of fragments of helminths are removed and examined under a magnifying glass between two glass slides. Small helminths or heads of cestodes are examined under a magnifying glass in a drop of glycerin or under a microscope.

When using this method for the diagnosis of segments of the pork, bovine tapeworm, wide tapeworm, the washed segments are placed between two glasses and, looking at the light under a magnifying glass or a low magnification of the microscope, the species is determined by the structure of the uterus (in a mature segment of the pork tapeworm, 8- 12 side branches, and bull tapeworm 18-32, more often 28-32, in a wide tapeworm the segments are wider and the uterus is in the center in the form of a "rosette"). If the uterus is poorly visible, then it can first be held for some time in a 50% glycerin solution, after which even the deserted uterine trunks are clearly visible.

When determining these cestodes by the structure of the detached heads, they are carefully placed with a neck in a drop of glycerol between glass slides (or covered with a coverslip) and, without squeezing, are examined under a microscope at low magnification.

Microscopic methods divided into simple, complex and special.

Simple methods include native smear, native smear with Lugol's solution, thick smear under cellophane according to Kato, twisting (according to Shulman) and perianal scraping.

Complex Methods are more effective and are based on the concentration of eggs in the preparations. They include pre-treatment of faeces with liquid reagents, as a result of which helminth eggs either precipitate or float to the surface of the liquid.

Complex methods include enrichment methods:

a) flotation (when specific gravity eggs less specific gravity saline solution and eggs float to the surface film);

b) sedimentation (when the specific gravity of the eggs is greater than the specific gravity of salt solutions and the eggs settle into sediment).

Special methods for detecting eggs and larvae of helminths, cysts and vegetative forms of protozoa are the methods of scraping, flotation, sedimentation, larvoscopy, protozooscopy, the study of bile and the methods of staining smears of feces, sputum, etc.

Simple Methods faecal studies

native smear. A small particle of excrement is taken with a wooden stick from different parts of the delivered portion, rubbed well on a glass slide in a drop of 50% glycerin solution and a thin smear is made on 2-3 glass slides. At least 3 preparations are examined under a microscope. Disadvantage of the method: a small amount of material is viewed, therefore it is not used as an independent method.

Twisting method(Shulman). 2.0-3.0 g of feces are placed in a glass, thoroughly stirred with a glass rod with a 5-fold volume of saline or distilled water, making quick movements for 2-3 minutes, and quickly remove the stick from the mixture. A drop of the mixture at the end of the stick is transferred to a glass slide and microscoped. The principle of centrifugal force causes the accumulation of eggs and larvae on a stick.

Kato thick smear method with cellophane. Chemical reagents: 100% glycerol, 6% phenol solution (100 ml water + 6 g phenol), 3% malachite green solution (2.5 ml distilled water + 75 ml malachite green).

Preparation of working solution: 100 ml of 6% phenol solution + 100 ml of pure glycerin + 1.2 ml of 3% malachite green solution.

Preparation of cellophane strips: cellophane strips are cut, the size of which corresponds to the glass slide. Cellophane must be hydrophilic (cellophane that burns is suitable; if it melts, then it is unsuitable). Up to 5 thousand strips can be processed in the working solution. The exposure time of cellophane strips until ready for use in the working solution is at least 24 hours.

Research progress. 50 mg of feces (the size of a large pea) is applied to a glass slide, rubbed with an individual stick (glass, wooden), covered with a cellophane strip and rubbed on top with a rubber stopper until a uniform thick smear is obtained. The drug is dried at room temperature for an hour or in a thermostat at 40°C for 20-30 minutes and microscopically (the exposure time can be increased at room temperature to 5-6 hours or more).

In terms of efficiency, this method approaches the flotation method, but reveals intense and medium intensity invasion.

It is used as an independent diagnostic method and is recommended for mass examination of the population.

In clinical diagnostic laboratories, it is used as a unified method for diagnosing helminths in the absence of specific diagnoses in the directions of doctors.

Methods for perianal scraping and native smear with Lugol's solution are described in the section on special methods.

Sophisticated faecal enrichment methods

The enrichment methods are based on the difference in the specific gravity of helminth eggs and the salt solution used.

When applying the flotation method, the following salt solutions can be used:

1. A solution of lead nitrate PbNO3 (lead nitrate) with a density of 1.5. Prepared at the rate of 650 g of the substance per 1 liter of water. Salt is dissolved in portions in hot water in an enamel bowl, heated on the stove and constantly stirring until completely dissolved. It is not necessary to filter the solution. The solution is prepared on the day of the study, since over time it gives a precipitate, and its density begins to fall after 24 hours. If the solution is prepared in large quantities, then in the following days before the study it is heated, stirring the precipitate. The preparation of the solution is carried out in a fume hood, since lead nitrate is a heavy metal salt.

2. A solution of ammonium nitrate NH4NO3 (granular or ordinary ammonium nitrate) with a density of 1.3 is prepared in the same way as the previous one, but at the rate of 1500 g of the substance per 1 liter hot water.

3. A solution of sodium nitrate NaNO3 or sodium nitrate with a density of 1.38-1.4 is prepared at the rate of 1000 g of the substance per 1 liter of hot water.

4. A solution of sodium thiosulfate Na2S2O3×5H2O (sodium hyposulfite) with a density of 1.4 is prepared at the rate of 1750 g of the substance per 1 liter of hot water.

5. A solution of sodium sulfate Na2SO4 (epsom salt) with a density of 1.26-1.28 is prepared at the rate of 920 g of the substance per 1 liter of hot water.

6. A solution of zinc chloride ZnCl2 (zinc chloride) with a density of 1.82 is prepared at the rate of 2000 g of the substance per 1 liter of hot water. The cooled solution does not crystallize.

7. Saturated chloride solution sodium NaCl(table salt) with a density of 1.18-1.2. On the! l of water, add 400-420 g of salt in portions to an enameled bucket of boiling water, stirring constantly until completely dissolved. As the solution cools, crystals of sodium chloride precipitate.

The specific gravity of flotation solutions is measured with an aerometer only after the solution has completely cooled down at room temperature.

Flotation methods are most effective for detecting eggs of pygmy tapeworm, whipworm, hookworm, ascaris, and broad tapeworm.

The surface film can be removed with a wire loop or glass slide.

in saturated solution table salt the film can be examined after 30-40 minutes, in ammonium nitrate solution - after 10-20-30 minutes after settling.

When removing the film with a wire loop, at least 8 drops are examined.

Slides remove more eggs from the film than wire loops. The glass must be in contact with the flotation solution liquid, which is added to the beaker with a pipette. After settling, the glass is removed, put the wetted surface up on the glass bigger size and examined under a microscope. Slides must be degreased before use.

For research, you must have: glass slides, chemical cups, wire loops, cuvettes, Petri dishes, pipettes, pears, glass or wooden sticks.

Research progress. 5 g of feces is poured with 10 times the amount of flotation solution (preferably with a specific gravity of 1.38-1.40), thoroughly stirred, non-dissolving large particles are removed from above and the suspension is left for 10-15 minutes. The film is then removed either with a loop on a glass slide or with a glass slide. To dilute feces, it is better to take cups with a capacity of 30-50 ml, pour the solution flush with the edges (or underfill 2-3 mm) and cover the mixture with a slide, and then add the flotation solution with a pipette until it comes into contact with the slide. After 10-20 minutes, the glass is quickly removed and the film remaining on it is microscopically scanned without a cover glass.

Settling-sedimentation methods

Sedimentation methods are used to detect eggs of geohelminths, biohelminths in the feces and as special research methods for opisthorchiasis.

Goryachev-Zolotukhin method(simplified method of Goryachev). About 1.5 g of feces is stirred in a chemical glass in 3-4 ml of water. The resulting suspension is filtered through two layers of gauze into a centrifuge tube, layering carefully on top of the 4-5 ml of a saturated sodium chloride solution present in it. Test tubes are placed in a rack for 15-20 hours. During this time, heavy trematode eggs settle. Get two clearly demarcated layers. The sediment is microscopically examined.

Ether-formalin method. It is used to diagnose all intestinal invasions and as a special method for protozoa and opisthorchia eggs.

Equipment: centrifuge at 3000 rpm; centrifuge graduated tubes, funnels; metal strainer (tea) or two-layer bandage; glass slides and coverslips; wooden (or glass) sticks; cotton, bandage.

Chemical reagents: 10% formalin solution (1 part pharmaceutical formalin solution and 4 parts distilled water); ethyl ether (medical).

7 ml of a 10% formalin solution are poured into centrifuge tubes and 1 g of feces is placed (such an amount of feces that the solution in the test tube rises to 8 ml). Feces are mixed with formalin until a homogeneous mixture is formed, and then poured through a metal strainer (or two-layer gauze, bandage) into another centrifuge tube (if feces remain on the strainer, then the strainer must be rinsed with formalin). Add 2 ml of ether to this centrifuge tube, stopper and shake vigorously for 30 sec.

The mixture is centrifuged at 3000 rpm for one minute (possible for two minutes at 1500 rpm). Due to the reaction of ether-formalin, coagulation of proteins occurs in the form of a cork at the top of the test tube, and helminth eggs precipitate. The coagulant layer is removed, the supernatant is drained, the precipitate is applied to a glass slide directly from the test tube or with a Pasteur pipette, covered with a cover slip and viewed under a microscope.

Ether-acetic precipitation method. The principle of ether-acetic precipitation of helminth eggs is the sequential treatment of fecal samples with a 10% aqueous solution acetic acid and ether. Acetic acid is better than others chemical compounds emulsifies a fecal sample. It penetrates into undigested particles, consisting mainly of fiber, which, when great content interfere with research by precipitating after centrifugation. The subsequent addition of ether to the tube and stirring leads to the extraction of acetic acid from the contents of the tube along with fecal particles impregnated with it. Since the specific gravity of the mixture of ether and acetic acid is less than the specific gravity of water, stool samples treated with these substances float up, and helminth eggs, which have a large specific gravity, settle.

The amount of precipitate obtained after ether-acetic precipitation is 3-4 times less than after ether-formalin precipitation. This greatly facilitates the detection of helminth eggs in it and allows you to study the sediment as a whole with a sample of 0.5-1 g. The toxicity of the ether-acetic method is 5 times lower.

Research progress. 7 ml of a 10% solution of acetic acid is poured into a graduated centrifuge tube and a 1 g sample of feces is added (i.e., such an amount of feces at which the acetic acid solution rises to 8 ml). Stir thoroughly with a glass or wooden stick. Strain through a funnel with two layers of gauze into another centrifuge tube. 2 ml is added to the emulsate ethyl ether(i.e. up to 10 ml). The test tubes are closed with a rubber stopper (it is possible from a penicillin vial) and shaken for 15 seconds. After removing the stopper, the tubes are centrifuged for one minute at 3000 rpm for 2 minutes. The supernatant is discarded from the tube. In some cases, the formed fecal plug interferes with the discharge of the supernatant. In this case, the cork is separated from the walls of the test tube with a glass or wooden rod. The whole precipitate is pipetted onto a glass slide and microscoped under a coverslip at low magnification. The precipitate is usually small and colorless. Helminth eggs, especially small fluke eggs, are well detected.

Chemical sedimentation method. The principle of the study is based on the sedimentation of eggs from a faecal sample in a saline solution with a specific gravity of 1.15. The essence of the method lies in the direct centrifugation of a test tube in which a stool plug emulsified in a 1% solution of acetic acid is layered on a solution of sodium nitrate (sp. weight 1.16). The high specific gravity of the saline solution and the bubbles released by the chemical reaction, penetrating the layer of homogenized feces, prevent its sedimentation. Helminth eggs precipitate with a small amount of fiber. The sediment can be cleaned of remaining detritus by treating it with 10% acetic acid and ether. In this case, only one helminth egg remains, which greatly facilitates their identification.

Research progress. 6 ml of sodium nitrate solution with a specific gravity of 1.15 is poured into a graduated centrifuge tube. Pour 7 ml of 1% acetic acid solution into another test tube. A sample of feces is introduced (up to the mark of 7.5 ml for a sample of 0.5 g and up to 8 ml for a sample of 1.0 g). Thoroughly mix the sample with a glass rod. Strain through a funnel with one layer of gauze, layering on a solution of sodium nitrate. The tube with the layered filtrate is centrifuged at 1500-2000 rpm for 5 minutes. Discard the supernatant by rapidly inverting the tube. Add 3-4 ml of a 10% solution of acetic acid and 0.5 ml of ether to a test tube with a precipitate, close with a rubber stopper and shake. Repeat centrifugation for 1 min. Discard the supernatant. The precipitate is transferred to a glass slide, covered with a coverslip and examined under a microscope.

HELMINTHOLOGICAL STUDIES OF BILLE, DUODENAL CONTENT, Sputum, BLOOD, URINE AND MUSCLES

Detection of eggs and larvae of helminths in duodenal contents and bile. The study of bile and duodenal contents is performed with suspicion of helminthiases of the liver and gallbladder (opisthorchiasis, clonorchiasis, dicroceliasis) and duodenum(strongyloidiasis).

Research progress. Duodenal contents and bile (portions A, B, C) are obtained in the usual way using probing. For portion B, it is recommended to obtain a gallbladder reflex by introducing a 33% solution through a probe magnesium sulfate. From the investigated liquid, flakes floating in it are selected and viewed under a microscope, and then it is mixed with an equal amount of sulfuric ether. The mixture is thoroughly shaken and centrifuged. The supernatant is discarded and the entire precipitate is examined under a microscope.

In the absence of pus and mucus, bile and duodenal contents are centrifuged without mixing with ether.

Sputum examination. In helminthological practice, sputum examination is carried out for the purpose of laboratory diagnosis of paragonimiasis. Sometimes, schistosome eggs, ascaris larvae, elements of the echinococcal bladder are detected.

A native smear is prepared from sputum on a glass slide, which is examined under a microscope.

With an abundant content of pus in the sputum, it is mixed with a 0.5% solution of caustic soda or caustic potassium, shaken for 5 minutes and centrifuged. The sediment is microscopically examined.

Blood study. Blood is examined if a patient is suspected of having filariasis.

Due to the fact that with some types of filariasis (loaosis, wuchereriosis caused by a subperiodic strain), larvae (microfilariae) are in the peripheral blood only during the day, and with some (brugiasis, wuchereriosis caused by a periodic strain) - only at night, respectively, at this time take blood for analysis.

The technique of blood sampling, preparation of preparations (thin smear and thick drop), their staining (according to Romanovsky) and the study are similar to those in the laboratory diagnosis of malaria.

Microfilariae of different species are distinguished by length, width, body curvature, the presence or absence of a cap, the shape of the caudal end, the location of the nuclear substance in the body and the caudal region of the larvae.

Research progress. Urine is defended for at least 30 minutes, then upper layer drain, leaving 10-15 ml of sediment, which are poured into centrifuge tubes and centrifuged for 1-2 minutes. After draining the supernatant, the precipitate is transferred to a glass slide and examined under a microscope.

MUSCLE BIOPTY STUDY

Trichinoscopy method. The need for the study of biopsy specimens of the patient's muscles arises when trichinosis is suspected.

A biopsy is performed according to the general rules. The deltoid muscle is usually biopsied. With a pathoanatomical examination, it is possible to do a biopsy of the muscles of the diaphragm, esophagus, tongue, masticatory and intercostal muscles, limb flexors, muscles eyeball, as they are most intensively affected by Trichinella larvae.

In the laboratory, a biopsied piece of muscle is cut into very small pieces (microtome), which are placed between two glasses, crushing the muscle fibers, and examined under a microscope. Currently, special microscopes, trichinelloscopes, are widely used for this purpose.

In the case of trichinosis, trichinoscopy along the muscle fibers reveals sharply prominent oval shape(similar to lemon) trichinosis capsules. average value capsules in humans is 0.4 x 0.26 mm. The capsule, as a rule, contains one trichinella spirally folded into 2.5 turns. With a high intensity of invasion, one capsule may contain 2 or 3 larvae. Muscle fibers adjacent to the capsule lose their transverse striation and take on a homogeneous appearance.

In the same way, the meat or meat products that caused the infection are examined.

Muscle Digestion Method. The method is more efficient.

Research progress. The studied muscles are finely crushed and filled with artificial gastric juice in a ratio of 1:15-20. The resulting mixture is placed in a thermostat at 37°C for 12-16 hours. After this period, the sediment is microscoped, in which free Trichinella larvae are found among the mass of remains of digested muscle fibers.

Artificial gastric juice can be purchased at a pharmacy or prepared in a laboratory. To do this, add 10 ml of concentrated hydrochloric acid to 1 liter of distilled water; before use, 30 g of pepsin is added to 1 liter of diluted acid.

QUANTITATIVE RESEARCH METHODS

Quantitative Methods studies are used in determining the intensity of invasion, evaluating the effectiveness of various anthelmintic drugs, determining the quality of deworming, monitoring ongoing mass therapeutic and preventive measures, etc.

The quantitative determination of helminth eggs is carried out by two methods: the Stoll method and the method of Krasilnikov and Volkova (1974).

Stoll method. To conduct the study, you must have a microscope, a glass flask with a mark of 56 and 60 ml, a measuring cylinder, glass beads, a rubber stopper for the flask, graduated pipettes, glass slides and 0.4% sodium hydroxide solution.

Research progress. Pour decinormal sodium hydroxide solution (approximately 0.4% concentration) into the flask with a measuring cylinder to the 56 ml mark and add feces until the liquid level reaches 60 ml (i.e. 4 ml of feces). The mixture is thoroughly shaken with glass beads for 1 minute, closing the vessel with a rubber stopper (you can also mix with a stick). Immediately after shaking, 0.075 ml of the mixture is collected with a graduated pipette (it contains 0.005 ml of feces), transferred to a glass slide and the number of eggs in the preparation is counted under a microscope. To determine the number of eggs in 1 g of feces, the number found is multiplied by 200.

Comparison of the number of eggs in the preparation, found in the patient before and after treatment, allows us to judge the effectiveness of deworming.

Stoll's method is simple, gives comparable results with all helminthiases, the pathogens of which systematically secrete eggs into the intestines of the patient. However, the disadvantage of this method is its relatively low sensitivity, especially at low intensity of invasion.

Krasilnikov-Volkova method. When examining this method, at least 1 g of feces is mixed in a glass flask or a large test tube with 1% Lotus solution (or 1.5% Extra solution) in a ratio of 1:10. The suspension is thoroughly shaken until a homogeneous suspension is formed, then 0.1 ml of the suspension (equivalent to 0.01 g of feces) is quickly collected with a graduated pipette and transferred to a glass slide. The preparation is covered with a cover glass or cellophane plate (20 x 30 mm), aged for at least one day in 50% aqueous solution glycerin.

Count the number of eggs in the entire preparation. To calculate the number of eggs in 1 g of feces, the resulting number must be multiplied by 100.

This method has a number of advantages over the Stoll method. Firstly, it is more sensitive and allows you to detect helminths when low degree invasions. Secondly, it is very convenient for mass examinations, since detergent solutions, being preservatives for helminth eggs, make it possible to conduct studies of not quite fresh material. However prerequisite this is the collection of faeces directly into the detergent solution.

For quantitative research any of the described unified qualitative methods based on the principle of floating eggs can be used. But in this case, the same amount of feces, the same volume of flotation solution should be taken for analysis. The calculation of the degree of invasion can be made, knowing the number of eggs in 1 g of feces, according to the table below.

Intensity of invasion depending on the number of helminth eggs

in 1 g of faeces

SEROLOGICAL DIAGNOSIS

These methods, based on the detection of specific antibodies in blood serum, are used for diagnostic and screening purposes.

Serological methods include:

Ring precipitation reaction (RCP) (trichinosis, cysticercosis);

Ring precipitation reaction in test tubes in the cold (trichinosis, cysticercosis);

Microprecipitation reaction on live larvae (trichinosis, ascariasis);

The reaction of indirect hemagglutination (RIHA) (trichinosis, echinococcosis, alveococcosis, cysticercosis, etc.);

Latex agglutination reaction (RAL) (echinococcosis, alveococcosis, trichinosis, teniarinhoz, etc.);

Complement fixation reaction (RCC) (trichinosis, echinococcosis, cysticercosis);

The reaction of enzyme-labeled antibodies (REMA) (echinococcosis, onchocerciasis, schistosomiasis, trichinosis, toxocariasis);

Linked immunosorbent assay(ELISA) (trichinosis, opisthorchiasis, toxocariasis, toxoplasmosis, etc.).

To set up serological reactions, standard antigens are produced or they are prepared independently (for example, from echinococcal bladders of sheep), special test systems for ELISA are produced.

Special Methods research on enterobiosis, taeniarhynchosis, taeniasis

Scraping from perianal folds. To obtain a scraping from the perianal folds, you can use a wooden spatula, cellophane strip, cellulose paper or tape, eye sticks with a special adhesive layer:

a) scraping with a wooden spatula (a spatula is a flattened match or stick) moistened with a 1% solution of glycerin (or a 0.5% solution drinking soda), is performed by light scraping from the surface of the perianal folds around the entire circumference of the anus. The resulting scraping is transferred with the edge of the coverslip from the end of the spatula to a glass slide in a drop of 50% glycerol solution, covered with the same coverslip and microscoped. The disadvantage of the method is the insufficient detection of eggs and irritant effect;

b) according to the Torgushin method, a wash is done with a cotton swab on a wooden or other spatula moistened with a 50% solution of glycerin, and a smear is prepared on a glass slide in a drop of glycerin;

c) according to the Kevorkova method, about 5 ml is poured into a centrifuge tube boiled water, place a spatula (stick) with a cotton swab in it. Before taking the material, the swab is slightly squeezed against the inner wall of the test tube, the perianal folds are wiped with it, and the spatula with the swab is inserted into the test tube. The tampon in the test tube is shaken thoroughly, the wash is centrifuged for 3 minutes, and the resulting precipitate is examined under a microscope;

d) scraping with adhesive tape according to Graham. Part of adhesive tape (transparent polyethylene tape with an adhesive layer for children's creativity, but it is better to use the operating film LPO-1, LPO-2) 8-10 cm long, glued with a sticky layer to the perianal folds of the skin, holding the ends, and then transfer it to the glass slide with a sticky layer down (the ends of the tape that extend beyond the edges of the glass are cut off ), glasses are numbered, and the patient's data and glass number are recorded in the journal. In the laboratory, the tape is peeled off at one end over a long distance, 1-2 drops are dripped under it vaseline oil or glycerin (to eliminate optical defects) and microscopically;

e) scraping with glass eye sticks, the surface of which is coated with a special adhesive composition. Eye sticks are installed in a special tripod. The material is taken by contacting the flat part of the spatula to the skin of the perianal opening. Then the stick is fixed again in a tripod for transportation. Microscopy is carried out directly on the spatula on both sides (without slides and coverslips) with preliminary fastening in cassettes at low magnification (eyepiece x 10, objective x 8). At the end of the work, the sticks are disinfected by boiling in a soapy solution, and the tripod and cassettes are treated with alcohol and washed with a soapy soda solution. Glue composition: cleol - 10 g, Castor oil- 2.5 g, ethyl ether - 5 g, ethanol 96% - 2.5 g.

The spatulas are dipped in a solution of glue, then dried in air at room temperature. The adhesive film formed on the surface remains for several days.

The method is convenient for mass examination of the child population for enterobiosis and the adult population for teniidoses.

In addition to the scraping method for teniarhynchosis and teniasis, the survey method and the macroscopic method described above (when segments are detected) are also used.

Special research methods for strongyloidiasis

The ether-acetic method is used to detect eggs (see above) and the Berman method to detect larvae in stool.

Berman method. Examine fresh feces, better after taking a laxative. A 5 g sample of faeces is placed on a metal sieve (the mesh is lined inside with two layers of gauze) in a glass funnel fixed in a tripod. A rubber tube with a clamp is put on the lower end of the funnel (Berman's apparatus).

The mesh with feces is lifted and water heated to 40-50 ° C is poured into the funnel in such a way that Bottom part the mesh was immersed in water and the feces were completely covered with water. After 2-4 hours, the clamp on the rubber tube is quickly opened, and the liquid descends into the centrifuge tube. After centrifugation (1-2 min), the upper part of the liquid is quickly drained, and the precipitate in the amount of 1 ml is applied in a thin layer on a glass slide and microscopically under a low magnification of the microscope.

IMP and TM method. A portion of feces the size of a nut is placed in a chemical beaker, poured with warm 40 ° C saline so that the feces are covered with a solution, left for 20 minutes. After 20 minutes, the liquid is poured into a Petri dish and viewed under the MBS binocular microscope.

For the diagnosis of strongyloidiasis, especially for monitoring the effectiveness of treatment, it is recommended to combine the Berman method with the study of duodenal contents.

Special research methods for nematodosis

Nematoses

Ascariasis

In the anamnesis, attention is drawn to the attitude to gardening, horticulture. Eating raw vegetables, salads made from these vegetables.

Clinical manifestations early phase ascariasis are caused by allergic changes in the body. The early or migratory larval stage of ascariasis often occurs in the presence of fever with a body temperature of up to 38 ° and above, with a symptom complex of lung damage, and the presence of severe blood eosinophilia. In most cases, in children, the first signs of the disease are malaise, weakness, recurrent headaches, sweating, and sometimes muscle and joint pain. Often there is a profuse urticaria-type rash with severe or moderate itching. The diagnosis of the early phase is confirmed by x-ray studies for the presence of volatile particles in the lungs. eosinophilic infiltrates Leffler. Fresh sputum smears often show eosinophilic cells, red blood cells, Charcot-Leiden crystals, and ascaris larvae.

In the intestinal imaginal stage of ascariasis, there is a combination of manifestations from the gastrointestinal and asthenic syndromes, enterocolitic pain symptoms. In children, there is often a decrease in weight, sometimes quite significantly. Nausea, increased salivation, irritability, delayed psychomotor development, decreased intelligence.

For laboratory diagnosis of intestinal st

The simplest are divided into 4 classes:

During encystation, the microorganism acquires round shape and covered with a protective sheath. In the form of a cyst, protozoa become less susceptible to unfavorable factors environment.

Research may include:

Note:There are a lot of varieties of diagnostics, we will consider those types that are most common in clinical laboratory practice.

Private types of diagnostics

In each specific case, the laboratory assistant is tasked with finding a specific pathogen, sometimes others are found along with the main one.

There are 6 species of this microorganism capable of living in the human intestine. Only dysentery amoeba, which occurs in a vegetative form and in the form of cysts, is of clinical importance.

Additionally, immunological methods are used:

• indirect immunofluorescence;
• indirect agglutination (PHA);
• radial immunodiffusion.

Note: serological methods are uninformative and are used only as an addition to the main ones in doubtful cases.

Diagnosis of ciliary (ciliates)

The pathogenic form of microorganisms of this genus is balantidia. This is a microbe that causes balantidiasis - a disease accompanied by an ulcerative process of the large intestine. The causative agent is found in a native smear in the form of a vegetative form and a cyst. The material for the smear (feces and mucus) is taken during a sigmoidoscopy examination and sown on special media.

Diagnostics of flagellates (leishmania, giardia, trypanosomes, trichomonads)

Leishmania, trypanosoma, giardia, trichomonads are dangerous for humans.

Leishmania- microbes causing leishmaniasis, are examined in blood smears, materials bone marrow, scrapings from skin infiltrates. In some cases, in the diagnosis of Leishmania, sowing on nutrient media is used.

Trypanosomes– pathogens sleeping sickness(American/African trypanosomiasis, or Chagas disease).

The African version is defined in initial period in the study of peripheral blood. Pathological microbes during the progression of the disease are found in the material of punctures of the lymph nodes, in advanced stages - in the cerebrospinal fluid.

To diagnose trypanosomes in case of suspected Chagas disease, the test material is examined under a microscope at low magnification. In this case, smears and a thick drop are pre-stained.

Trichomonas(intestinal, oral,) are detected by microscopy of materials taken from the affected mucous membranes.

Identification of sporozoans (malarial plasmodium, causative agent of coccidosis, etc.)

The most common and dangerous species for humans is the malarial plasmodium, which has 4 main varieties of the pathogen: pathogen three day malaria, four-day malaria, tropical malaria and malaria oval.

Sexual development of Plasmodium (sporogony) takes place in Anopheles mosquitoes. Asexual (tissue and erythrocyte schizogony) - in the liver tissue and human erythrocytes. These features of the life cycle must be taken into account when diagnosing malarial plasmodium.

So, in the blood of a newly ill patient, germ cells of the sporogony cycle can be found. But at the height of malarial attacks, schizonts appear in large numbers in the blood.

Moreover, in different phases malarial fever appear various forms Plasmodium:

• during the period of chill, the blood is filled with merozoites, a kind of schizont;
• at the height of temperature, ring-shaped trophozoites accumulate in erythrocytes;
• the decrease in temperature is characterized by the predominance of amoeboid trophozoites;
• during periods of normal condition, the blood contains adult forms of schizonts.

The study of the causative agent of malaria (malarial plasmodium) is carried out in a smear and in a thick drop.

Note:the diagnosis of malaria in the study of smears and thick blood drops is sometimes erroneous. Blood platelets in some cases may be erroneously classified as a malarial pathogen. Also, fragments of leukocytes and other cells sometimes simulate plasmodium.

Basic research methods for protozoa

Let's take a brief look at the most common research methods for the presence of protozoa.

Diagnosis of protozoa using a native smear and a smear stained with Lugol's solution (in feces)

The drug is prepared from an emulsion of feces in an isotonic solution. Two drops of sodium chloride and Lugol's solution are applied to a glass slide. The test material is added to both compositions with a wooden stick and, after being covered with glass, is viewed at different resolutions of the microscope.

According to certain signs, the found protozoa are registered. For accuracy, 2-3 preparations are prepared from one material. In doubtful cases, the analysis is repeated several times over 2-3 weeks.

The method can detect vegetative and cystic forms:

• lamblia;
• balantidia;
• dysentery amoeba.

Together with pathogenic forms, non-pathogenic protozoa are also determined. Healthy carriers also have luminal and cystic forms.

Important:research in order to avoid inaccuracies and errors should be carried out repeatedly.

The result of the diagnosis of protozoa by the method of a native and stained smear should contain a description of the form of the pathogen (translucent, cyst, tissue).

Research requirements:

• the material taken for analysis (liquid feces) is examined no later than 30 minutes after defecation;
• formed feces must be diagnosed within 2 hours after defecation;
• the material should not contain impurities ( disinfectants, water, urine);
• only wooden sticks are used to work with the material, glass ones are not suitable due to slipping of the mucus;
• Sticks must be burned immediately after use.

Conservation method (examination of feces) in the diagnosis of protozoa

The study is carried out by fixing the protozoa with a preservative. The difference between this method and the previous one is that preservatives allow you to save the drug for a long period.

Used preservatives:

• Barrow. Contains preservative ingredients: 0.7 ml sodium chloride, 5 ml formalin, 12.5 ml 96% alcohol, 2 g phenol and 100 ml distilled water. Coloring composition: 0.01% solution of thionine (azure).
• Safarliev's solution. Composition: 1.65 g zinc sulfate, 10 ml formalin, 2.5 g crystalline phenol, 5 ml acetic acid, 0.2 g methylene blue, 100 ml water. This preservative is used in cases where the material must be stored for more than a month.

Empty bottles are filled with a preservative, the material is transferred into them, in proportions of 3: 1, then, if necessary, a dye is added. Evaluation of the results is carried out in the study of 2-3 drugs.

Formalin-ether enrichment method (analysis for the presence of protozoa in feces)

This diagnostic method allows you to separate and concentrate protozoan cysts. The following ingredients are needed for analysis: formalin (10 ml), 0.85 g of isotonic solution, distilled water, sulfuric ether, Lugol's solution.

A mixture of biomaterial with the listed liquids is mixed and centrifuged. The precipitate obtained at the bottom of the tube is stained with Lugol's solution and examined for the presence of cysts and vegetative forms.

Leishmania detection method (bone marrow smear)

For the diagnosis of leishmaniasis, reagents are used: a mixture of Nikiforov (sulfuric ether and ethyl alcohol), phosphate buffer, Azur-eosin according to Romanovsky.

The bone marrow substance is very carefully placed on a glass slide after special preparation. A microscope with an immersion system is used.

IN acute period diseases in the punctate found a large number of Leishmania.

Note:Sometimes blood cells may resemble processed leishmania, so it is very important for the laboratory technician to be attentive and have sufficient experience for self-examination.

Method for detecting leishmania in a smear from a skin infiltrate

The required reagents are similar to the previous assay.

The test material is obtained from the existing tubercle or ulcerative contents. Scraping with suspicion of leishmaniasis is done very carefully with a scalpel, without blood. Then the preparation is prepared on glass. For the accuracy of the results obtained, several preparations are simultaneously examined.

In the presence of a disease, among the macrophages, fibroblasts, and lymphoid cells present in the test material, Leishmania is also determined.

Method for isolating a pure culture of Leishmania obtained by scraping pathological tissues

With this method of diagnosing the simplest tissue scrapings are placed in a special nutrient medium in which Leishmania actively reproduces.

Before taking a scraping, the skin is carefully treated with alcohol, then an incision is made in the tubercle, from the bottom of which the contents are removed and placed in a test tube with the medium. The material is taken several times, after which it is placed in different test tubes. Then, in a thermostat at a temperature of 22-24 degrees, cultivation takes place. The results are evaluated under a microscope. This method is used when other, cheaper and faster methods of diagnosing protozoa are ineffective.

You can see how tests for the presence of protozoa are deciphered in practice by a drop of blood by watching a video review:

Lotin Alexander, medical columnist

The stools are examined in two ways:

1. Macroscopic - find helminths, their heads, segments, scraps of strobili. Small portions of feces are mixed with water in a flat bath or Petri dish and viewed in good light against a dark background, using a magnifying glass if necessary. All suspicious formations are transferred with tweezers to another cup of water or onto a glass slide in a drop of diluted glycerin.

With the method upholding the investigated portion of feces is stirred with water in a glass cylinder, after settling, the top layer of water is drained. This is repeated several times. When the liquid becomes transparent, it is drained, and the sediment is viewed in a Petri dish.

2. Microscopic - to detect eggs and larvae of helminths. There are many research methods.

1). native smear - the most common and technically available research method. You can find eggs and larvae of all helminths. However, with a small number of eggs, they are not always found. Therefore, the enrichment method is used.

1). Fülleborg method - this is a method of enrichment, based on the emergence of helminthiasis eggs in a saturated solution of NaCl (1.2 - density; 400 g NaCl per 1 liter of water; 40% NaCl solution). The method is more effective than the native smear. 2-5 g of feces are placed in glass jars and filled with NaCl solution, stirred, and after 45 minutes the formed film is removed with a metal loop, a drop of glycerin is placed on a glass slide. Examine under a microscope. The disadvantage of the method is the delayed emergence of eggs of various helminths, dwarf tapeworm - after 15-20 minutes, roundworm - 1.5 hours, whipworm - 2-3 hours.

2) Kalantaryan method - also an enrichment method, but a saturated solution of NaNO 3 (1.38 density) is used. Most of the eggs float, no examination of the sediment is required. The disadvantage is that the eggs are kept in solution for a long time, which leads to the fact that some eggs begin to swell and settle to the bottom, disappearing from the surface film.

3. Goryachev's method - based on the principle of egg deposition, detection of small trematode eggs. A saturated solution of NaCl is used as a solution and 3-4 ml of feces solution is carefully layered on top. After 15-20 hours, trematode eggs settle to the bottom. The liquid is drained, sediment on a glass slide and under a microscope.

4. Shulman twisting method – to detect helminth larvae in the feces. Examine only freshly isolated feces. 2-3 g is placed in a glass jar and 5 times the amount of water is poured, quickly stirred with a stick, without touching the walls of the jar - 20-30 minutes, then the stick is quickly removed, and a drop of liquid at the end is transferred to a glass slide and microscoped.

5. Berman method - based on the ability of helminth larvae to migrate towards warmth, and serves to identify them in feces.

6. Method of Harada and Mori (method of growing larvae) and is recommended for testing for ankylostomiasis. The method is based on the fact that in heat and on moist filtered paper, hookworm eggs develop into filariform larvae, which can be easily detected. 15 g of feces are applied to the middle of a strip of filtered paper, the paper with feces is placed in a jar, so that the lower end is immersed in water, and the upper end is fixed with a cork. The jar is kept in a thermostat at 28 0 C for 5-6 days. Filariform larvae develop during this time and descend into the water. The liquid is examined under a magnifying glass. If it is difficult to detect, the liquid is centrifuged, after killing the larvae by heating to 60 0. The laboratory technician must wear gloves.

7. Methods for enterobiasis – identification of pinworm eggs and bovine tapeworm.

a) scraping from the perianal folds - with a cotton swab tightly wound on a wooden stick and moistened with a 50% glycerin solution. In the laboratory, the swab is washed off with 1-2 drops of a 50% aqueous solution of glycerol.

b) sticky mite method (Graham method)

The adhesive tape is applied to the perianal folds, then with a sticky layer to the glass slide and microscoped.

C) scraping with the help of eye sticks (Rabinovich's method). For perianal scrapings, glass eye sticks are used, the widest part of which is covered with a special glue, which makes it possible to hold pinworm eggs.

Examination of blood, bile, sputum and muscles

Blood microscopy - filariae larvae are detected.

Sputum examination - paraganim eggs, roundworm larvae, necator, strongyloid, elements of echinococcal bladder.

Examination of the muscles - if trichinosis is suspected, the muscles of the patient or the corpse, as well as the meat that allegedly caused the infection of the person, are examined. For the purpose of trichinoscopy, the muscle is cut into small pieces and placed in compressors, these are two wide, thick glasses that crush the muscles and Trichinella larvae are found in the form of capsules - the compression method.

Digestion method - the muscles are poured with artificial gastric juice (hydrochloric acid solution and pepsin). The muscles are digested and the larvae are easily detected. Determination of the intensity of invasion: the number of larvae up to 200 per 1 g muscle tissue– moderate intensity of invasion; up to 500 - intensive; over 500 - superintensive invasion.

Serological methods

Chapter III. Diagnosis of helminthiases and methods of helminthological research

It is necessary to examine for helminthiases all patients seeking medical help, and especially patients who turn to a pediatrician, internist and neuropathologist with complaints of phenomena from the gastrointestinal tract, nervous system and with anemia. If the doctor cannot always apply laboratory research methods, then every medical worker providing assistance in an outpatient clinic or hospital is obliged to interview the patient about the release of helminths from him.

In the presence of those given in the relevant chapters clinical indications the diagnosis should be clarified using laboratory tests for helminthiasis.

Due to the predominance intestinal helminthiases greatest practical value has a study of bowel movements.

Methods for the study of feces for helminthiases

The feces are delivered to the laboratory in clean glassware (about a quarter cup of feces taken from different places in one serving); during a routine examination, delivery of feces to the laboratory in matchboxes or popular prints is allowed.

To control deworming, the entire portion of feces collected after intake is delivered (as prescribed by a doctor). antihelminthic and laxative (in large closed glass jars, buckets).

Microscopic examination of feces is the main one 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 stools should be fresh or canned (in 5% formalin solution), as drying dramatically changes the structure of the eggs. In addition, when standing feces occurs fast development eggs of some helminths (for example, hookworm), which makes diagnosis difficult.

According to the instructions of the Ministry of Health of the USSR, it is necessary to examine the feces simultaneously using the Fülleborn method and a native smear.

native smear

Native smear: a small piece of feces (the size of a pea), taken with a match, a glass or wooden stick from different places of the delivered portion, is carefully triturated on a glass slide in a drop of 50% glycerol solution or in saline, or in water. Cover with a coverslip, slightly pressing 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 give enrichment of the drug. At least two preparations should be viewed.

In order to detect helminth larvae (as well as their eggs), a native smear is done in the following way(according to Shulman): 2-3 g of feces are thoroughly stirred by “twisting” with a glass rod into an emulsion with five times the amount of pure water or saline. During stirring, the larvae accumulate at the glass rod, therefore, immediately after the end of stirring, a drop of the emulsion is quickly transferred with a glass rod to 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 ascaris eggs. Based on the work of S. D. Lyubchenko, we consider it appropriate to replace the smear method with the twist method.

Fülleborn method

Fülleborn method: 5-10 g of feces taken from different places are placed in a jar with a capacity of 50-100 ml and thoroughly triturated with a glass or wooden rod in a saturated solution of sodium chloride (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 poured in gradually until a uniform suspension is obtained, and the total amount of the poured solution should be approximately 20 times the amount of feces. Fülleborn recommended the use of tea glasses for mixing faeces, but it is more convenient to prepare the suspension in 50-100 ml ointment jars, using two jars for each analysis (or in 100 ml cups).

Immediately after the preparation of 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 residues, 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 not more than 1 cm, bent at a right angle; The film is shaken off on a glass slide and covered with a coverslip. Under each coverslip (18x18 mm) place 3-4 drops. In total, at least 4 preparations should be prepared (one coverslip for each preparation). The loop is calcined on fire and washed with water after each analysis.

According to the Fülleborn 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 study feces for helminth larvae (with strongyloidiasis). This method is as follows: 5 g of faeces on a metal grid (a milk strainer is convenient for this purpose) are placed on a glass funnel attached to a tripod. A rubber tube with a clamp is put on the lower end of the funnel.

The mesh with feces is raised 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 lowered into one or two centrifuge tubes.

After centrifugation for 1-2 minutes, the upper part of the liquid is quickly drained, and the precipitate is applied in drops on glass slides and examined under coverslips or distributed in a thin layer on 2-3 large slides and then examined without coverslips.

The Berman method is also used to examine the soil for the presence of hookworm larvae.

Stoll method

The Stoll method is used to determine the intensity of invasion. A decinormal sodium hydroxide solution is poured into a special glass flask to the mark of 56 cm 3, 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 get the number of eggs contained in 1 cm 3 of feces.

Study of duodenal contents

Duodenal juice and cystic bile, obtained in the usual way by probing (and cystic bile and after a reflex from the gallbladder), are thoroughly mixed with an equal volume of ethyl ether; the mixture is centrifuged, after which the precipitate is examined under a microscope. In addition to sediment microscopic examination flakes floating in the liquid, which may contain helminth eggs, are necessarily exposed. When examining eggs of helminths of gastric juice and vomit, you can use the same technique.

Examination of duodenal juice and stomach contents should be performed if you suspect helminthic diseases liver, gallbladder (opisthorchiasis, fascioliasis, dicroceliosis) and duodenum (strongyloidiasis).

Sputum examination

Sputum is rubbed on a glass plate, tightly covered with another glass plate and examined with the naked eye on a light and black background, as well as under a magnifying glass in transmitted light. Separate pieces of sputum (“rusty” accumulations, scraps of tissue, etc.) are applied in a thin layer on a glass slide, tightly covered with a coverslip and examined at low and high magnification microscope.

a) For the diagnosis of cysticercosis of the skin, subcutaneous tissue or muscles, an aseptically cut piece of the corresponding tissue is examined first with the naked eye. The tissue sections are moved apart with the help of dissecting needles in order to detect a vesicle visible to the naked eye - a cysticercus (photo A); its length is 6-20 mm, width is 5-10 mm. When a bubble is found that is suspicious of cysticercus, 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 halo of hooks (photo B).

Photo. A - cysticerci with scolexes turned outward; B - Head of pork tapeworm.

b) To diagnose trichinosis, an aseptically cut piece of muscle (biceps or gastrocnemius) is carefully crushed in a 50% glycerol solution into the thinnest fibers using dissecting needles. Crushed muscles are squeezed between two glass slides and examined at low magnification of the microscope in a darkened field of view. Examination of muscles for trichinosis is recommended to be performed no earlier than on the 8th day of the disease. Trichinella larvae are in the muscles in a coiled position: they are enclosed in lemon-shaped capsules.

Photo. A - Trichinella larvae in the muscles; B - Calcified capsules of Trichinella.

Fluoroscopy

Most often, fluoroscopy is used to diagnose echinococcosis and, less often, cysticercosis. Cysticerci are detected by fluoroscopy only after calcification (in cases prolonged 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 ascaris larvae (and hookworm) in the lungs, unstable, sometimes multiple inflammatory foci are detected; 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, out of 180 patients with ascariasis identified by fluoroscopy, 54 ascaris eggs were not found in the feces (see).

7.7. Methods for determining the viability of eggs and larvae of helminths

The viability of helminth eggs is determined by their appearance, by staining with vital dyes, by cultivation in optimal conditions and setting up a biological sample.

7.7.1. Determination of the viability of eggs or larvae of helminths by appearance

Helminth eggs are microscoped first at low magnification, then at high magnification. In deformed and dead eggs of helminths, the shell is torn or bent inward, the plasma is cloudy, loosened. In segmented eggs, cleavage balls (blastomeres) are unequal in size, irregular in shape, and often shifted to one pole. Sometimes there are abnormal eggs, which, having external deformities, develop normally. In living larvae of roundworms, fine granularity is present only in the middle part of the body, as they die, it spreads throughout the body, large shiny hyaline vacuoles appear, the so-called "strings of pearls".

To determine the viability of mature eggs of roundworms, whipworms, pinworms, you should call active movements larvae by slightly heating the preparation (to a temperature not exceeding 37 ° C). It is more convenient to observe the viability of ascaris 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 larvae of ascarids, a cap is often seen that has exfoliated at the head end, and in larvae of whipworms that have completed development in the egg, a stylet is found in this place at high magnification. Dead larvae of helminths, regardless of their location (in the egg or outside it), notice the decay of the body. Wherein internal structure the larva becomes lumpy or granular, and the body is cloudy and opaque. Vacuoles are found in the body, and breaks are found on the cuticle.

The viability of teniid oncospheres (bovine, porcine tapeworm, etc.) is determined by the movement of the embryos when they are exposed to digestive enzymes. The eggs are placed on watch glass with dog 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, the living embryos are released from the 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 teniid eggs are placed in a 1% solution of sodium sulfide or a 20% solution of sodium hypochlorite, or in a 1% solution of chlorine water at 36 - 38 ° C, mature and live embryos are released from the shells and do not change during 1 day. Immature and dead oncospheres shrivel or swell and enlarge dramatically and then "dissolve" within 10 minutes to 2 hours. Live embryos of teniids 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 fasciolia adolescariae collected from plants and other objects of water bodies is checked by examining them on a glass slide in saline under a microscope with a heating stage. When heated, the trematode larvae in the cyst begin to move.

To determine the viability of the eggs of the pygmy tapeworm, the method of Ionina N.S. is the simplest: in live eggs, the median pair of embryonic hooks is either parallel to the lateral ones, or the latter form an angle at the base of less than 45 ° with the median. In dead eggs, the lateral pairs form an angle at the base with a median pair of more than 45 °, or the hooks are randomly scattered (their paired arrangement is lost); sometimes there is wrinkling of the embryo, the formation of granularity. 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.

Determining the viability of immature nematode eggs should be studied in a humid chamber (Petri dishes), placing ascaris eggs in a 3% formalin solution prepared in an isotonic sodium chloride solution at a temperature of 24 - 30 ° C, whipworm eggs in a 3% hydrochloric acid solution at a temperature of 30 - 35 ° C; pinworm eggs in isotonic sodium chloride solution at 37 °C. Petri dishes should be opened 1 - 2 times a week for better aeration and re-moisten the filter paper 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, the division of 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, rhabditoid larvae hatch from the eggs, and after 5 - 7 days they become already filariform: the larvae crawl up the walls of the cylinder, where they are visible even to the naked eye.

Trematode eggs that naturally develop in water, such as opisthorchis, diphyllobothriids, fasciols, and others, are placed on a watch glass, a Petri dish, or in another 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 miracidium is formed in live eggs at a temperature of 22–24 ° C after 9–12 days. When microscopy of developing trematode eggs, miracidium movements are clearly visible. Fasciola miracidium emerges from the egg shells only in the light.

Fulleborn method. Hookworm and strongylid larvae are cultivated 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 spread over the agar, leaving behind a path of bacteria.

Method of Harada and Mori. 7 ml of distilled water are added to test tubes placed in a rack. Take 0.5 g of feces with a wooden stick 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 table). Then the strip with the smear is inserted into the tube so that the left end free from the smear reaches the bottom of the tube. Cover the upper end with a piece of cellophane and wrap it tightly with an elastic band. On the test tube write the number, the name of the subject. In this state, the test tubes are stored for 8-10 days at a temperature of 28 °C. To study the larvae, remove and remove the cellophane cover and remove a strip of filter paper with tweezers. Care should be taken in this case, as a small number of infective larvae can move to the upper end of the filter paper or to the wall of the test tube and penetrate under the surface of the cellophane.

The tubes are placed in a hot water bath at 50°C for 15 minutes, after which the contents are shaken and quickly poured into a 15 ml larval sedimentation tube. After centrifugation, the supernatant is removed, and the precipitate is transferred to a glass slide, covered with a coverslip and microscoped under low magnification.

For the differential diagnosis of filariform larvae, it is necessary to use the data in Table 3.

Table 3

DIFFERENTIAL DIAGNOSTICS OF FILARIATED LARVOYS OF A. DUODENALE, N. AMERICANUS, S. STERCORALIS, trICHOStrONGYLUS SP.

Larvae	Dimensions	Characteristic features
A. duodenale	Body length about 660 microns, cap - 720 nm	The striation of the cap is less pronounced, the mouth 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 μm, cap - 660 nm	The sheath is noticeably striated, especially in the caudal part of the body, the mouth appears dark, the anterior end of the body (but not the sheath) is rounded like a narrow end. chicken egg, the anterior part of the intestinal tube of such a diameter as the bulb of the esophagus, the tail end is sharply pointed
S. stercoralis	Body length about 500 µm	Larva without sheath, esophagus is about half the length of the body, tail is blunt or branched
Trichostrongylus sp.	Body length about 750 microns	The intestinal lumen is not straight, but zigzag, the caudal end is rounded and has the shape of a button

7.7.2. Methods for staining eggs and larvae of helminths

Dead tissues in most cases perceive colors faster than living ones. These features are used in helminthology to determine the viability of eggs and larvae of helminths. However, in some cases, some paints are better perceived by living tissues than by dead ones.

For the differential determination of live and dead eggs and larvae, the following paints and methods are used.

Methylene blue leucobase is often used to stain living and dead tissue. living cell or the tissue reduces methylene blue to a colorless leucobase, dead tissue does not have this ability and therefore acquires color.

The criterion for the state of the egg is the staining of the embryo, but not the shell. This ability is related to the conditions of egg death. In those cases where the fibrous shell in the dead egg does not lose its semi-permeable properties, it will not pass dyes, therefore, the dead embryo will not be stained. A colored embryo always indicates the death of the egg.

For coloring Ascaris eggs, you can use methylene blue in a solution of lactic acid with caustic alkali (methylene blue 0.05 g, caustic soda 0.5 g, lactic acid - 15 ml). Live eggs do not perceive color; are painted in Blue colour embryos of dead eggs. Ascaris larvae are stained with a basic solution of brilliant-cresyl blue paint at a concentration of 1:10,000 as follows: a drop of liquid with ascaris 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 against the glass slide with light tapping with a dissecting needle. Under the microscope, the number of hatched larvae and the degree of their staining are observed; after which the same drug is reviewed again after 2 to 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 stain when the shell breaks (partially or completely).

When determining the viability of eggs of ascaridia birds, it is possible to stain preparations with 5% alcohol solution iodine. When it is applied to the drug, the embryos of dead ascarid eggs for 1 - 3 seconds. are dyed orange.

Dead eggs of opisthorchis and oncospheres of bovine tapeworm are stained with a solution of toluidine blue (1:1000), and dead oncospheres of bovine tapeworm are stained with a solution of brilliant-cresyl blue (1:10000). At the same time, the embryos and shells of both dead and live eggs acquire color. Therefore, after staining, eggs and oncospheres are washed in clean water and additionally stain them with safranin (in a dilution of 1:10,000 alcohol 10 ° C). Alcohol removes the dye from the shells, and safranin stains red. As a result, live eggs turn red; eggs with dead embryos - in blue, and the shell remains red. Dead embryos of oncospheres of bovine tapeworm quickly, within a few minutes, are stained bright red or pink with safranin or blue with brilliant-cresyl blue at a dilution of 1:4000, or with indigo carmine at a dilution of 1:1000 - 1:2000. Living embryos do not change under the influence of these colors even after 2 - 7 hours.

To determine the viability of pygmy tapeworm eggs, it is recommended to use the following paints:

1. Brilliant creasyl blue (1:8000) - after 1 hour, the oncosphere of dead eggs is especially brightly stained, which stands out sharply against the pale or colorless background of the rest of the egg.

2. Safranin (1:8000 for 2 hours and 1:5000 for 3 to 5 hours).

3. 50% solution of pyrogallic acid in a 1:2 dilution - when exposed for 1 hour at a temperature of 29 - 30 ° C (the lower the temperature, the longer the staining process).

7.7.3. Luminescent method for the study of eggs and larvae of helminths

Fluorescent microscopy makes it possible to differentiate living and dead objects without damaging the egg. Not used for fluorescence ultra-violet rays, and the blue-violet part of visible light, with an ordinary microscope and glass slides; a special set of color filters is added to the OI-18 illuminator.

Live and dead eggs of roundworms, pinworms, pygmy tapeworms, bovine tapeworms, tapeworms and other helminths luminesce differently. This phenomenon is observed both during primary luminescence without the use of dyes, and when stained with fluorochromes (acridine orange, corifosphine, primulin, auroline, berlerin sulfate, tripaflavin, rivanol, quinacrine, etc.).

Unstained, live non-segmented roundworm eggs glow bright green with a yellowish tint; in dead eggs, the shell radiates green light much brighter than the dark green germ part; in roundworm eggs with a larva, only the shell appears, while in the dead, both the shell and the larva are bright yellow.

Non-pigmented and non-segmented live eggs of pinworms and dwarf tapeworms emit a greenish-yellow light; in dead eggs, the shell intensely luminesces against the background of a dark green embryonic mass.

With secondary luminescence (when staining acridine orange at a dilution of 1:10000 and 1:50000 from 30 minutes to 2 hours), the shell of living and dead nematodes, trematodes and cestodes luminesces differently.

The shell of live and dead eggs of ascarids, toxocar, pinworms, pygmy tapeworms, rat tapeworms, bull tapeworms, tapeworms turns orange-red. The embryos of living eggs of ascaris, toxascaris, rat tapeworm, wide tapeworm and oncosphere of bovine tapeworm luminesce with a dull dark green or grey-green. Dead embryos of eggs of these helminths emit a "burning" orange-red color. Live pinworm larvae and toxocars (egg shells) emit a dull grey-green light, when they die, the color changes from the head end to a "burning" light green, then yellow, orange, and finally to bright orange.

When stained with fluorochromes - coryphosphyllum, primulin, dead eggs of ascarids and whipworms show a glow from lilac-yellow to copper-red. Viable eggs do not luminesce, but turn dark green.

Live eggs of trematodes (Paragonimus and Clonorchis) do not luminesce after staining with acridine orange, and a yellowish-green color comes from dead eggs.

The luminescence method can also be used to determine the viability of helminth larvae. So, fluorochromized with a solution of acridine orange (1:2000) larvae of strongylate, rhabdita glow: live - green (with a tint), dead - bright orange light.

Living miracidia that have emerged 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 as a bright "burning" light green, and then orange-red light.

7.7.4. biological assay method

For example, to determine the viability of ascaris eggs (ascaris pigs, humans, toxocara, toxascaris, etc.) per animal (guinea pigs, mice), at least 100 - 300 eggs with a developed larva are needed. Ascaris eggs in isotonic sodium chloride solution are pipetted through the mouth of a mouse or guinea pig. After 6-7 days, the animal is slaughtered, opened and its liver and lungs are examined separately for the presence of ascaris larvae. To do this, the liver and lungs are cut into small pieces with scissors and examined according to the Berman or Supryaga method (section 6.1.2).

If the animals were infected with live invasive eggs, then at autopsy in the liver and lungs, migratory ascaris larvae are found.

In case of infection, Fasciola eggs in the faeces of laboratory animals can be detected in rabbits after 2 months, in guinea pigs- after 50 days, in mice - after 35 - 40 days.

For a faster response, laboratory animals are opened after 20-30 days and the liver is examined for the presence of young fasciols.

To determine the viability of pygmy tapeworm eggs, it is also recommended to feed them to previously uninfected white mice, followed by autopsy of the animals after 92–96 hours and the detection of 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., Beer 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 lid of the egg and the active release of miracidium under experimental conditions.

A 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 a suspension containing 100-150 eggs is added to a centrifuge tube. The test tube is placed in a tripod for 5-10 minutes. During this time, all the eggs have time to sink to the bottom. Then, with a strip of filter paper, the excess water is carefully sucked off and 2 drops of a special medium are added to the test tube. The medium is prepared in 0.005 M Tris-HCl buffer; 12 - 13% ethanol solution and dye (magenta, safranin, eosin, methylene blue, etc.) are added to the buffer. The test tube is shaken, its contents are transferred with a pipette to a glass slide and left for 10 minutes, shaking slightly. Then add 2 drops of the indicated medium. The preparation is ready for microscopy under a conventional light microscope at 20x magnification.

During this time, the lid of the viable larvae opens, and the miracidium actively enters the indicated medium. Due to the presence of ethanol in it, they are immobilized after 2-5 minutes and then stained with a dye. They can be easily detected and counted under microscopy.