|
 Identification
of parasites
 Worm eggs and larvae in saline mounts
Eggs may be easily detected and identified in saline
mounts. They should not be stained (stains may interfere with
identification). Most of the eggs are large enough to be recognized with the
low-power 10x objective, but a few small eggs will require a high-power dry
lens. In saline mounts, larvae of Strongyloides stercoralis may be seen. Hookworm larvae are not usually
present if the sample is fresh, but it may be necessary to distinguish
between these two species if an old sample is examined
Protozoa in wet mounts
Saline wet mounts
In saline mounts, trophozoites and cysts of amoebae (cysts of Isospora belli) and flagellates
may be seen. Cysts will appear as round or oval, refractile
structures; the trophozoites of amoebae may be
round or irregular; the trophozoites of flagellates
are usually pyriform (elongated, pear-shaped). In
freshly passed faeces (the stool must not be more
than an hour old), motile trophozoites may be seen.
Motility can be very helpful in identifying species, especially in the case
of flagellates.
Organisms may be detected with
the low-power 10x objective, but a high-power, dry objective will be
necessary to reliably identify the structure as a cyst or trophozoite.
With the high-power, dry objective, you can see motility, inclusions like
erythrocytes and yeast in amoebic trophozoites, chromatoid bodies in amoebic cysts, and the shape and
structural details (e.g. sucking discs, spiral grooves, or filaments) of
flagellate trophozoites and cysts. Oocysts of Isospora belli are ellipsoidal, 22-33 x 12-15 µ m size, containing
two sporocysts each. You will not be able to see
any detail in the nucleus in saline mounts. However, it is necessary to
regulate the microscope illumination carefully so that the objects appear
clearly. Too much or too little light will interfere with your observations.
It is also necessary to focus up and down to see all the layers (levels) of
the specimen. Remember to examine the whole coverslip
area in a systematic manner to reduce the chances of overlooking organisms.
BMB mount
The
nucleus and inclusions will stain dark blue and the cytoplasm will stain
light blue. Look for peripheral nuclear granules around the nucleus for Entamoeba species.
Iodine
wet mount
Iodine mounts are examined for
protozoa and coccidian cysts. They can be detected with the 10x objective,
but they are not as refractile as in saline mounts.
High-power dry magnification must be used to see the characteristics of the
cysts and they must be measured to ensure correct identification. In the
iodine mount, cytoplasm of the cysts will stain yellow or light brown and
nuclei will stain dark brown.
In iodine-stained cysts of Entamoeba, the arrangement of the peripheral chromatin
and the position of the karyosome can be seen. (If
the peripheral chromatin is not present, the cyst is not Entamoeba
species.) These peripheral chromatoid bodies stain
light yellow and may not be very clear. Sometimes, young cysts contain
glycogen; this stains dark brown with iodine.
In iodine-stained flagellate
cysts, the fibrils (filaments) can be seen.
In iodine-stained coccidian
cysts, a central undivided mass of protoplasm can be seen.
Specific identification of
amoebic and flagellate cysts can usually be made from iodine wet mounts.
However, occasionally a definite identification cannot be made, and it may be
necessary to use permanent stains.
Modified
Ziehl - Neelsen Stain
When stained by Ziehl -Neelsen technique, Cryptosporidium
oocysts appear as bright rose-pink spherules
generally 4-6 µm in diameter, in a pale green background. The color may be
unevenly distributed due to variable carbol fuchsin uptake by the oocyst
wall, especially in rapidly shed young with a less mature wall.
Yeast cells and faecal debris assume the color of the blue or green
counter stain. Although sometimes sporozoites can
be clearly seen within the oocysts, size is
important in differentiating oocysts from other
organisms with similar staining properties. Cyclospora
spp. resemble
Cryptosporidium parvum but are roughly twice the
size, i.e. 8-10 µ m in diameter. As this is unsporulated,
they show a spherical cytoplasmic mass.
The large oocysts
of Isospora belli contain
two sporocysts measuring 12-14 x 7-9 µm each with
four sporozoites of 20-23 x 11-19 µm in size
elongated or ellipsoidal in shape with no residuum (in freshly passed stool,
only one sporocyst may be seen).
Modified
gram chromotrope stain for Microsporidia
When
stained by Gram chromotrope staining technique, Microsporidia spores are ovoid and refractile.
They measure approximately 0.9-1.5 µ m in diameter. The spore wall stains
bright pinkish red. The cellular content of some spores appears transparent
and some spores show a distinct pinkish-red-stained belt-like stripe that
girds the spores diagonally or equatorially. Although other faecal elements, such as yeast, sometimes stain reddish,
they can be distinguished from Microsporidia spores
by their large size and more intense staining. Most bacteria and background
debris counterstain faint green.
Reporting
procedure
Reporting
should include appearance of specimen:
 Consistency
Blood, mucus, pus
Worms
or worm fragments
The
written report should include WBCs, erythrocytes,
and organisms detected. The written report should be submitted within 24-72
hours. In urgent cases the result should be reported immediately by telephone
(if available) or personally.
Preservation
of specimens
Materials
and reagents
1. Adhesive
tape
2. Applicator
sticks, wooden
3. Bottles,
1000 ml
4. Labels
5. Pen
or marker for labeling
6. Vials,
20 ml, with tight-fitting screw-caps
7. 10%
formalin (formaldehyde)
Technique
1. Label two 20-ml vials with patient’s name or
number. Write F in the upper right-hand corner of the label on one vial.
2. Fill
the "F" vial about half full with 10% formalin.
3. With
an applicator stick, pick up a portion of the stool to include areas from the
inside and edges of the sample and mix with the 10% formalin. Be sure to mix
very well; break up lumps. Use enough, but not too much stool so that the
mixture will occupy about 2/3 to 3/4 of the vial.
4. Screw
the caps of the vials securely. Wrap a piece of adhesive tape around the top
of each vial to prevent leaking.
5. Pack
the vials carefully in a box or shipping container and send to the reference
laboratory. Be sure that the vials are surrounded by absorbent materials
(e.g. cotton wool, newspaper) and are packed so they will not break.
6. Be
sure to include the necessary information: patient’s name or number, date of
shipping, organisms you found.
For
long-term preservation, faecal samples may be
stored in 2.5% (weight/volume) potassium dichromate in which oocysts remain for as long as six months.
Disposal
of specimens
1. If stools are collected in paper boxes, the
way to dispose of them is by burning the entire container. If they cannot be
burnt, or if the stool was collected in a metal or glass container, add
enough 10% formalin to cover the stool left in the container. This will kill
any parasites that might be present. Allow to stand for an hour or more
before discarding or washing (if the container is glass).
2. Slides
used for wet mounts should be put in a pan of disinfectant (e.g., sodium
hypochlorite) for at least an hour before washing. Use an applicator stick to
push the coverslip off into a breaker or small pan
of disinfectant and then put the slide into another pan of disinfectant.
3. Coverslips break easily, and if put in with the slides,
they may break and cut the hands of the person washing them.
4. Funnels,
stoppers, and centrifuge tubes should also be put into disinfectant for an
hour before washing.
5. Applicator
sticks and gauze squares should be burned. If burning is not possible, they
can be discarded after soaking in disinfectant.
Quality
assurance for faecal examination
To
ensure accurate and reliable results, good laboratory practices must be
applied to laboratory procedure for diagnosing parasitic infections. Quality
assurance must apply to collection of specimens, preparation of reagents,
performance of the techniques, examination of the preparations and reporting.
National level laboratories may be motivated to organize National External
Quality Assessment Schemes for strengthening the quality assurance programme
especially Internal Quality Control for the regional/peripheral laboratories.
Diagnostic
procedures to be performed at different levels
Level 1: Peripheral level
Smear examination for intestinal protozoa and Helminths
Smear examination for P. carinii
Smear examination for Cryptosporidia
Level 2: Intermediate level (regional
hospital, provincial hospital /university)
Histopathology for P. carinii,
T. gondii
Antigen demonstration
Special stains for Microsporidia,
Cryptosporidia,
Cyclospora, Isospora, P. carinii and T. gondii
Level 3: Central level (reference laboratories
and centres of excellence)
Demonstration of antigen by ELISA, PCR and
probes
Strain differentiation
References
WHO Library Cataloging in Publication, Basic
Laboratory Methods in Medical Parasitology
(1991)
Cheesbrough M.
(1991) Medical Laboratory Manual for Tropical Countries, Volume I.
Forbes BA, Sahm DF, Weissfield AS Diagnostic Microbiology, 1998, 10th
edition.
Moura H et al. Gram-chromotrope
(1996): A new technique that enhances detection of microsporidial
spores in clinical sample. J Eukaryot Microbiol; 43 (5): 94S-95S.
Appendices
Reagents
and Solutions
Lugol’s
iodine (Stock 5% Solution)
Iodine 5 g
Potassium iodide (KI) 10 g
Distilled water up to 100 ml
Dissolve the potassium iodide
in about 30 ml of the water. Add the iodine and mix until dissolved. Add a
further 70 ml of water and mix well. Store in a brown bottle.
Lugol’s iodine (1%
Solution for wet mounts)
Lugol’s
iodine (stock, 5% solution) 5 ml
Saline solution, isotonic 20 ml
Measure the isotonic saline
into a dispensing or dropping bottle. Add the 5% Lugol’s
iodine stock solution. Mix thoroughly. This will give a 1% iodine solution
which will satisfactorily stain cysts.
Buffered
methylene blue stain
Methylene
blue powder 1 g
Disodium
hydrogen phosphate 3 g
Potassium dihydrogen
phosphate 1 g
Distilled water 300 ml
Weigh out the methylene blue powder and put in a clean dry mortar. Add
the disodium hydrogen phosphate and potassium dihydrogen phosphate. With a pestle, grind the dye and
phosphate powders together and mix thoroughly. Weigh 1 g portions of the
mixture and put in a small well-stoppered
vials.
Preparations
of stain
Put 1 g
of the mixture in a 500 ml flask. Add the distilled water and shake the flask
or stir to dissolve the dye mixture. Filter through filter paper into a 500
ml clean, dry, glass-stoppered bottle. This stain
will remain will remain good for two years or more.
PVA-fixative
preparation
Glycerol 1.5 ml
Polyvinyl alcohol (PVA) powder
(low viscosity) 5.0 g
Distilled water 62.5 ml
In a small beaker, add the
glycerol to the PVA powder and mix thoroughly with a glass rod until all
particles appear coated with the glycerol. Scrape the mixture into a 125 ml
flask. Add the distilled water, stopper, and leave at room temperature for
three hours or overnight. Swirl mixture occasionally to mix.
Carbol-fuchsin
solution
Basic fuchsin
10 g
Ethanol, absolute, technical
grade 100 ml
Phenol 50 g
Distilled water 1 l
Preparation
Weigh
the basic fuchsin powder and transfer it into a 1.5
litre bottle. Add 100 ml of absolute ethanol and
dissolve the dye completely. Weigh the phenol in a beaker and dissolve in a
small volume of distilled water. Add the aqueous phenol solution to the dye
solution and mix well. Add the rest of the water, mix well and label the
bottle. The dye solution will be stable indefinitely.
Hydrochloric
acid-ethanol solution
Hydrochloric acid
(concentrated) 1 ml
Ethanol 95% 100 ml
Put 100 ml of 95% ethanol into
a clean 250 ml bottle with glass stopper. Add 1 ml of concentrated
hydrochloric acid and then mix.
Glycerol-malachite
green solution (or methylene blue) solution
Glycerol 100 ml
3% Aqueous malachite green, or
3% aquous methylene blue
1ml
Distilled water 100 ml
Grind some malachite green or methylene blue powder with a pestle in a clean, dry
mortar. Weigh out 3 g of the powder, pour it into a bottle and add distilled
water to give 100 ml. Seal and label the bottle 3% aqueous malachite green or
3% methylene blue . Store
in the cabinet away from light.
To prepare the solution, add 1
ml of 3% aqueous solution into a 250 ml bottle. Add 100 ml of glycerol and
100 ml of distilled water and seal the bottle and mix thoroughly before use.
Hydrochloric
acid-methanol solution
Hydrochloric acid
(concentrated) 3 ml
Methanol, absolute 100 ml
Measure 100 ml of absolute
methanol and pour into a clean 250 ml bottle with glass stopper. Add 3 ml of
concentrated acid and mix.
|
