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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5512_Библиотеки_им_академика_М_И_Перельмана.pdf
Morphology and Life Cycles of Human Parasites
Laboratory Examination For ParasitEs
Preservation and Shipment of Specimens to be Examined for
Trophic and Encysted Protozoa
PVA (Polyvinyl Alcohol) Method of Brooke and Goldman
Excellent for amoeba, especially if permanent slides are required. For collection of eld specimens and
for mass surveys, the merthiolate–iodine–formalin (MIF) method is recommended.
1. Reagent: Modied Schaudinn’s solution is prepared by mixing 5 ml of glacial acetic acid, 1.5 ml
of glycerol and 93.5 ml of Schaudinn’s solution (2 parts saturated aqueous mercuric chloride and
1 part 95% ethyl alcohol).
Heat the above solution to 75°C; while stirring, slowly add 5 gm polyvinyl alcohol (PVA) powder.
This nal solution should be clear and free of lumps after cooling. It is used at room temperature,
and lasts several months.
2. Preparation of specimens
a.. One specimen should be sent without above xative to be used for detection of protozoan
cysts and helminthic ova. This specimen can be used for temporary or permanent smears or
for concentration procedures.
b. Second specimen is prepared by thoroughly mixing with 3 or more parts of xative in a
small vial. To prepare slides, a small amount of this faecal mixture (recent or months old)
is spread thinly over about one-third of the slide. After drying for 3 or more hours at 37°C
or overnight at room temperature, the smear is stained by the iron haematoxylin method.
This procedure is particularly suitable for the preservation and staining of the trophic forms
of intestinal protozoa. To obtain satisfactory stained faecal smears containing cystic stages,
the excess clear PVA solution is decanted from the vial and a small amount of the remaining
xed faecal material is placed on a piece of facial tissue or toilet paper. The excess PVA
solution is allowed to absorb for 5–10 minutes, leaving a moist faecal residue. Gently scrape
up a small amount of the residue with the sharp edge of a broken applicator stick and smear
with gentle brushing strokes on a slide. Drop smear immediately into 70% alcohol to which
iodine has been added to produce a portwine colour, and stain by the iron haematoxylin
method. To ensure satisfactory preparation of both types smears are recommended.
xxi
MIF (Merthiolate–Iodine–Formalin) Method of
Sapero and Lawless
This is now the standard method for mass surveys, collection of bulk material, or parasitologic eld
studies. Faecal specimens are xed and stained immediately, and can be examined at any time within
several months of collection. This method is particularly good for protozoa.
1. Reagents: MIF stock solution is made from 250 ml distilled water, 200 ml of tincture of
merthiolate, 25 ml of formaldehyde, 5 ml of glycerol. Store in brown bottle. Lugol’s solution
(5% iodine in 10% potassium iodide in distilled water), not over 1-week old, forms the second
solution.
2. Preparation of specimens: For each specimen to be collected, have ready 2.35 ml MIF stock
solution in a Kahn test tube with cork stopper and 0.15 ml Lugol’s solution in a second Kahn tube
with a rubber stopper. Combine the 2 solutions just before adding the faecal specimen. Break
up about 0.25 gm faeces into the combined solution, mix thoroughly, and stopper well (may be
Medical Laboratory Technology: Volume 2
xxii
routinE stooL Examination and ConCEntration mEthods havE
bEEn dEaLt with ELsEwhErE (PrEvious ChaPtEr)
examined immediately on a slide, 1 drop faecal preparation and 1 drop distilled water; or stored in
a well-stoppered tube, where the stain will be retained for several months).
Negative Stain Direct Faecal Smear Examination
Prepare normal faecal smear in saline to which 1–2 drops of 1% isotonic eosin are added. Background
material and dead parasites turn uniformly pink. Stain will not penetrate living trophozoites, causing
them to stand out markedly as clear, translucent organisms against a pink background. A rapid and useful
procedure. Isotonic eosin, 1%, and brilliant cresyl blue, 0.2%, makes a good vital stain, causing living
material to appear as shiny pale blue-green objects on the pink background.
Kato Cellophane Thick Smear Technique (Kato and Miura,
1954)
This method permits rapid examination of a large number of samples (up to 70/hour) for eggs of the
common helminths. It is not suitable for protozoa or minute helminths or for highly brous or gaseous
samples.
Materials
1. Wettable cellophane of medium thickness (40–50 μm), cut in strips 22 × 30 mm.
2. Glycerine-malachite green solution: 100 ml pure glycerine, 100 ml water, 1 ml 3% aqueous
malachite green.
The cellophane strips should be soaked in the glycerine mixture for at least 24 hours before use.
Procedure
1. Place 50–60 mg faeces (4 mm cube) on a clean slide.
2. Cover with a glycerine-soaked cellophane strip, press to spread faeces in an even layer. Faeces
need not be spread to all areas of cellophane; a circumference equal to the width of the strip is
sucient.
3. Allow to stand at room temperature for 1 hour (or 20–30 minutes at 40oC in a dry incubator).
This dries and clears the specimen. (Do not over dry, as gas bubbles will form and air cells will
surround the eggs).
4. Examine the entire lm under low poor magnication.
Heidenhain’s Iron Haematoxylin Staining Method for
Intestinal Protozoa
Iron haematoxylin is generally accepted as the most reliable stain for nuclear detail in amoebae and
for accurate laboratory diagnosis. It also provides a permanent stain preparation. However, it requires
critical care and individual slide destaining (to be done carefully), a major handicap in routine handling
of specimens by clinical laboratories. The trichrome stain has therefore superseded iron haematoxylin
staining for routine diagnosis, though for critical denition, iron haematoxylin is still unsurpassed. Both
iron haematoxylin and trichrome can be used for either PVA-xed or nonpreserved material.
Morphology and Life Cycles of Human Parasites
Staining Solutions
1. Schaudinn’s xative: Described earlier.
2. Haematoxylin stain (stock solution): Dissolve 100 gm haematoxylin powder in 100 ml absolute
ethanol. Let stand several weeks for maturation. For use, 5 ml of ripened hematoxylin is added to
95 ml distilled water.
3. Mordant: Dissolve 5 gm ferric ammonium sulphate in 10 ml distilled water and lter.
Procedure
1. Make thin faecal smear on a clean glass slide with toothpick, applicator, or sti haired paste brush.
2. Before drying occurs, immerse slide in Schaudinn’s uid with acetic acid added, heated to 45°C.
Fix for 5–15 minutes at this temperature or for 30 minutes at room temperature (omit this step for
PVA-xed specimens).
3. Staining in Coplin jars—
70% alcohol 15 minutes
70% iodine alcohol 5 minutes
70% alcohol 2 minutes
50% alcohol 2 minutes
Tap water (running) 2 minutes
Distilled water Rinse
5% aqueous iron-alum (mordant) 5 minutes at 30°C
Distilled water (2 changes) Rinse
0.5 % haematoxylin 10 minutes
Dierentiate in 1% aqueous iron-alum Usually 3–5 minutes
Tap water (running) 15–20 minutes
50% alcohol 2 minutes
70% alcohol 2 minutes
95% alcohol 5 minutes
Isopropyl alcohol 2 changes of 5 minutes
Carbol-xylol 5 minutes
Xylol (2 changes) 5 minutes each
Mount in xylol-balsam, xylol-Damar or DPX mountant with a cover glass of No. l thickness.
xxiii
Gomori’s Trichrome Stain
Preferred permanent stain for routine diagnosis.
Staining Solution
To 100 ml distilled water, add 0.6 gm chromotrope 2 R, 0.3 gm light green SF, 0.7 gm phosphotungstic
acid, and 1 ml glacial acetic acid.
Procedure
Prepare slides as for haematoxylin staining. (Omit steps 1 and 2 for PVA-xed specimens).
1. Immerse in Schaudinn’s xative 30 minutes at room temperature
2. 70% alcohol (wash) 15 minutes
3. 70% iodine alcohol 10 minutes
4. 70% alcohol (wash) 2 changes of 5 minutes
5. Trichrome stain 20 minutes
Medical Laboratory Technology: Volume 2
xxiv
6. Acidied 90% alcohol (1% acetic acid) 2 dips
7. 95% alcohol 2 changes of 5 minutes
8. Carbol-xylene 2 minutes
9. Xylene 2 minutes
10. Mount immediately as for haematoxylin preparations.
Cultivation of Intestinal Protozoa
Numerous types of special media have been developed for the cultivation of the intestinal amoebae
and agellates as well as for the ciliate Balantidium coli. Among these are Boeck-Drbohlav-Lockeegg serum medium, Nelson’s egg yolk infusion-liver extract medium, and Dobelle’s medium which
contains serum, egg albumin and starch. The parasite (E. histolytica) grows in 24 to 36 hours at 37°C.
The morphological character of the colony is quite typical of the species.
The cultivation of free-living juveniles of ancylostoma duodenale and strongyloides stercoralis is
done on sterilised sand or charcoal paste. Samples of soil or faeces containing ova are mixed with equal
quantity of sterilised ne sand or animal charcoal and water to make a thick paste. The paste is kept on a
lter paper in a Petri dish and covered with lid. It is kept at 25 to 30°C for several days. The free-living
juveniles collect in the water of condensation.
Urine
Collect urine in a clean, dry container, avoiding faecal contamination. Study centrifuged sediment.
Trophozoites of Trichomonas vaginalis, unhatched eggs of Schistosoma haematobium, and intact
scoleces or hooklets of Echinococcus granulosus may appear in the urine. Viable S. haematobium eggs
will hatch only after dilution of urine.
Sputum
Examine for parasites as a direct smear under a cover glass. If the sputum is thick, bloody, or pusladen, mix
with equal volume 1–2% sodium hydroxide. Stir, let settle, and study sediment. Larvae of Strongyloides
stercoralis, scoleces of Echinococcus granulosus, eggs of P. westermani, or migrating nematode larvae
may be present in the sputum. Charcot-Leyden crystals and eosinophils can also be observed in wet
mounts or after wet xation and staining.
Gastric Washings
For night-swallowed sputum will often yield Paragonimus eggs or migrating nematode larvae better
than will sputum or faeces (owing to less detritus).
Duodenal Aspirates
These are useful for nematode eggs and are particularly helpful for eggs of Clonorchis and other bile-
dwelling parasites.
Spinal Fluid
Examine centrifugate directly under the microscope. Make smears, stain with Giemsa’s stain and
examine. Culture some of the sediment, as for blood. Inoculate guinea pigs or mice if necessary.
Morphology and Life Cycles of Human Parasites
xxv
to cause a few drops of blood to ow freely. On one end of a meticulously clean slide free of ngerprints or oil
Vaginal Secretions
Wet preparation may be examined directly for Trichomonas vaginalis. Cultures can also be made.
Graham Cellulose Tape Technique for Diagnosis of
Enterobiasis
With the help of a tongue depressor, press the adhesive side of a small strip or loop of cellulose tape
(e.g. Scotch tape) over the anal and perianal surfaces, preferably at night. Then place tape with adhesive
side down in a drop of toluene on a microscopic slide. Examine for eggs or worms which have adhered
to the tape.
Blood
Combined Thin and Thick Films
Making Films
Cleanse nger, ear lobe, heel, or toe (of children) thoroughly with spirit and allow to dry. Prick skin deeply
lm, make a thin smear as for a blood count. For the preparation of the thick lm, deposit a large drop of
blood at the other end of the slide and spread it out evenly with the corner of another slide to a diameter
of about 20 mm. The lm should not be too thick since it may crack and peel when dry. Dry the slide in a
at position so that the distribution of blood will be even. Protect from dust and insects, avoid excessive
heat. Allow to dry in air for at least 8–12 hours or for 2 hours in an incubator at 37°C. Keep free from
dust or contamination with excreta of ies or roaches. Stain as soon as practicable, freshly stained
material gives distinctly superior results.
Staining Films
Fix thin-lmed end of slide in methanol for 2–3 minutes. The thick lms must not be xed. Immerse slides
for 30 minutes in a mixture of 1 drop of concentrated Giemsa’s stain to each ml of phosphate-buered
distilled water (pH 7.2). Wash o with buered water and dry in air. Examine with oil immersion
objective. Haema- toxylin or methylene blue stain is preferred for microlariae since nuclear detail and
sheaths (if present) stain more distinctly.
Concentration of Microfilariae
Haemolyse 10 ml or more of citrated blood with 50 ml of a 2% solution of a saponin in physiologic
saline. Centrifuge and examine the sediment. Chylous urine and scrotal aspirates should always be
centrifuged to attempt recovery of microlariae.
Bone Marrow Smear
For a proper diagnosis of kala-azar, bone marrow smear obtained by sternal puncture is essential. The
Leishman’s stain or Giemsa’s stain are used for this purpose. Microscopic examination under oil
immersion show the presence of intracellular oval bodies containing blue-stained cytoplasm and pinkstained rod-like kinetoplast.
Medical Laboratory Technology: Volume 2
xxvi
Serological Tests
Serum samples obtained from the patients are used for following tests in certain parasitic infections:
1. Precipitation: It is done for trichinelliasis, hydatid disease and cysticercosis.
2. Flocculation test: Slide occulation tests are useful for trichinelliasis and schistosomiasis.
3. Complement xation test: This test is used in several parasitic infections for detecting complement
xing antibodies, but this is of special value in cases of schistosomiasis, trichinelliasis, hydatid
disease, nonpulmonary paragonimiasis, and toxoplasmosis.
4. Fluorescent antibody test (FAT): This test is applied to demonstrate the presence of parasites
or their specic antibodies in tissue or in the blood of the patient, e.g. Toxoplasma gondii and
Entamoeba histolytica.
5. ELISA tests now are available for most parasitic infestations.
6. Immunochromatography technique based kits.
Intradermal Tests
These are done to demonstrate allergic state in certain parasitic diseases. The antigen of the suspected
parasite is injected intradermally and in positive cases, wheal or induration results. These tests are useful
for hydatid disease (Casoni’s test), trichinelliasis, lariasis, chronic schistosomiasis and toxoplasmosis.
Casoni’s Test
It was rst developed by Casoni in 1911. Intradermal injection of 0.2 ml on exor aspect of right arm of a
fresh sterile hydatid uid (sterilised by Seitz lter) produces within half an hour in all positive cases a
large wheal (5 cm in diameter) with multiple pseudopodia, it fades in an hour.
A delayed reaction appears after 18 to 24 hours characterised by oedema and induration 5–6 cm
surrounding the site of injection. A negative reaction does not exclude echinococcal infection. The test
usually becomes positive 8–12 weeks after infection and remains positive after surgical removal of cyst
from the patient.
Hydatid uid from human cases (removed operatively) or from animals (obtained from a slaughter
house) is used as an antigen. While 0.2 ml of antigen is injected to one arm, sterile normal saline 0.2 ml
is injected in the other arm for control.
Muscle Biopsy for Trichinella spiralis
Small pieces of deltoid, biceps, or gastrocnemius muscle are removed from the vicinity of their tendinous
attachment under local anaesthesia.
1. Microscopically examine small pieces of muscle compressed between 2 glass slides for encysting
or encysted larvae.
2. Digest muscle in articial gastric juice (pepsin and hydrochloric acid) and examine sediment for
motile larvae.
Section
Serology
5
'
Chapter
Introduction to Immunology and Principles of Serodiagnosis
Asho Khanwalr and Raima Daa
23
130 I
Medical Laborato Technology: Volume 2
The Immune Process of the Body
First Le of Defence