Advanced laboratory techniques

in the parasitology laboratory

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 Iron Haematoxylin Solution A/B
Method: preparation of Working Iron Haematoxylin Solution
1. Mix equal volumes of the two solutions and filter.
2. Allow to stand at least two hours
• Parasite stain blue if used immediately after preparation
• Mature stains stain light blue with grey back ground
• If a slide appears cloudy, then dehydration has been
inadequate
• Agitation in the final alcohols can improve the clarity of the
smear

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 Procedure: Iron Haematoxylin Staining
1. Prepare smear using Mayer’s Albumin
2. Treat with 95% ethanol for at least 10 minutes
3. Slides should be brought to water stepwise through 70%
and 25% alcohols.
4. Stain slides for 10 minutes in Haematoxylin stain
5. Wash for 30 seconds in distilled or de-ionised water.
6. Treat for 10 minutes using 50% saturated Picric Acid.
7. Wash slides in running tap water for at least 10 minutes

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 Procedure: Iron Haematoxylin Staining

8. Transfer to alkalinised tap water for 2 minutes.

9. Treat for 10 minutes in alkalinised 70% alcohol).
10.Bring to 100% alcohol stepwise through 95% and
absolute alcohol (twice)
11.Clear in Xylene for at least 10 minutes twice.
12.Mount in DPX or any similar neutral mounting
medium.
13.Examine using Immersion Oil under oil immersion lens

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 Trichrome for Microsporidia
Method
1. Make smears from unconcentrated stool specimens.
2. Fix in methanol for 5 mins.
3. Stain in modified Trichrome stain for 90 mins.
4. Rinse in acid alcohol for 10 sec then in 95% alcohol.
5. Place in 95% alcohol for 5 mins.
7. Place in 100% alcohol for 10 mins.
8. Clear in Xylene for 10 mins.
9. Examine under x 100 oil immersion objective
Result: microsporidial spores are ovoid, refractile and the
spore wall stains bright pinkish-red 5
Enterotest/String test for G.lambilia trophozoites in
duodenal aspirate
• A gelatin capsule attached to a long string (Naylon
yarn coiled within gelatin)
• The end of the string remains outside the mouth
and is taped to your cheek
• The gelatin capsule is then swallowed
• The capsule dissolves in the stomach and the string
passes into the upper part of the small intestine
(duodenum)
• The string is left in place for 4 to 6 hours or
overnight. Then it is withdrawn and the end is
examined under the microscope for parasites that
have become attached
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 Immunological Diagnosis (Immunodiagnosis)

o Pertaining to diagnosis by immune reactions

o Is based on the detection of :

A. Antibody (Ab) from person's serum

• Ab is produced in response to a particular parasitic infection
B. Antigen (Ag) detection

– Ag. is excreted by parasites and can be found in the

serum, urine, CSF, feces or other specimens.
– Antigen tests provide evidence of present infection

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 Molecular diagnosis
 Microscopic examination is still considered the “gold standard” for
the diagnosis of parasitic diseases

 The stool specimen can be analyzed using molecular techniques

such as polymerase chain reaction (PCR)

 PCR amplified fragments can be analyzed by using:

– Restriction fragment length polymorphisms (RFLP) or

– DNA sequencing if further characterization is needed

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 Culture

 Growth medium is provided for a specific parasite

 A specimen is tested for the presence of an infectious agent
able to grow within that medium
 Culture allows identification of infectious organisms by
examining their
– Microscopic features,
– Substances produced by the pathogens, and
– Directly identifying an organism by its genotype
 Relatively few of protozoa and helminths parasites, can be
cultured.
– E. g. E.histolytica, T.vaginalis, T.cruzi and Leishmania species
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Leishmanin or Montenegro test

• Delayed Hypersensitivity Skin

Test
• intradermal inoculation of
leishmanin
• suspension of whole or
killed promastigotes
• preferably from local area
• include negative control
• Positive reaction: when the
area of indurations ± erythema
of 5mm in diameter or more
indurations in 48-72 hours

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 Examination of CSF for trypanosomes
• When there is involvement of CNS in HAT, the following may
be found
– Few trypanosomes
– More than 5 white cells/l
– IgM usually more than 10% total CSF protein
– Raised total protein
• Trypanosomes unable to survive for more than 15-20 minutes
in CSF
– Become inactive and lysed

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 Xenodiagnosis
 in chronic and subacute infections where their
number in the blood is usually very few
• uninfected, susceptible, laboratory reared triatomine
bugs are starved for 2 weeks and
• then fed on the patients blood
– If trypanosomes are ingested they will multiply
and develop into epimastigotes which can be
found 25-30 days later in the faeces or rectum of
the bug

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 Quantitative Buffy Coat (QBC) Test
• New method for identifying the malarial parasite in the
peripheral blood.
• Involves:
– staining of the centrifuged and compressed red
cell layer with acridine orange
– examination under UV light source.
• It is fast, easy and more sensitive than the traditional thick
smear examination
• equipment and special disposable capillary tubes required
for the QBC system is very expensive
• considerable skill and experience is required to process and
examine the tubes correctly and confidently
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 Quantitative Buffy Coat (QBC) Test
• Method:
– Hematocrit tube, pre-coated internally with
acridine orange stain and potassium oxalate.
– Filled with 55-65 µm of blood from a finger, ear or
heel puncture
– Centrifuged at 12,000 rpm for 5 minutes.
– The components of the buffy coat separate
according to their densities, forming discrete
bands.
– The QBC tube is placed on the tube holder and
examined using UV microscope.
– Fluorescing parasites are then observed at the red
blood cell/white blood cell interface
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 Quantitative Buffy Coat (QBC) Test
The key feature of the method
• Centrifugation and thereby concentration of the RBC
in a predictable area of the QBC tube, making
detection easy and fast.
• Red cells containing plasmodia are less dense than
normal ones and concentrate just below the
leukocytes, at the top of the erythrocyte column.
• Parasites contain DNA which takes up the acridine
orange stain, and appear as bright specks of light
among the non-fluorescing red cells.
• Virtually all of the parasites found in the 60 microliter
of blood can be visualized by rotating the tube under
the microscope
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 Concentration procedures
 Separate parasites from fecal debris and increase the
chances of detecting parasitic organisms when these are
in small numbers

– They are divided into flotation techniques and sedimentation

techniques

1. Flotation techniques (most frequently used: zinc

sulfate or Sheather's sugar) use solutions which have
higher specific gravity than the organisms to be floated
so that the organisms rise to the top and the debris
sinks to the bottom
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 Concentration procedures
 The main advantage of this technique is to produce a cleaner
material than the sedimentation technique

 The disadvantages of most flotation techniques are that the walls of

eggs and cysts will often collapse, thus hindering
identification. Also, some parasite eggs do not float

Flotation technique using sodium chloride solution (Willis)

 This method is recommended for the detection of eggs of A.

duodenale and Necator americanus (best method), Ascaris
lumbricoides, Hymenolepis nana, Taenia spp. and Trichuris trichiura

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Flotation technique using sodium chloride solution (Willis)
 It is not suitable for the detection of eggs of flukes and
Schistosoma spp., larvae of Strongyloides stercoralis, or
protozoal cysts or trophozoites
Principle:
• The stool sample is mixed with a saturated solution of sodium
chloride (increasing the specific gravity). The eggs are lighter in
weight and float to the surface where they can be collected
Materials and reagents
• Microscope
• Microscope slides
• Coverslips

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Flotation technique using sodium chloride solution (Willis)

• Wide-mouth bottle, 10ml

• Wooden applicators
• Gauze
• Petri dishes
• 95% Ethanol
• Ether
• Willis solution (reagent no. 64)
• Petroleum jelly
• Wax

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Flotation technique using sodium chloride solution (Willis)
Method:
1. Place approximately 0.5g of stool in a wide-mouth bottle. Fill the bottle
to the 2.5-ml mark with Willis solution.
2. Using an applicator, crush the portion of stool and mix it well with the
solution. Then fill the bottle to the top with Willis solution; the
suspension should be completely uniform.
3. Place a coverslip carefully over the mouth of the bottle.
4. Check that the coverslip is in contact with the liquid, with no air bubbles.
Leave for 10 minutes
5. Remove the coverslip with care; a drop of liquid should remain on it.
Place the coverslip on a slide and examine under the microscope (using
the x10 objective) at once because the preparation dries very quickly.
Otherwise seal the coverslip with petroleum jelly and wax
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 Sedimentation techniques
2. Sedimentation techniques use solutions of lower specific
gravity than the parasitic organisms, thus concentrating the
latter in the sediment.

• Sedimentation techniques are recommended for general

diagnostic laboratories because they are easier to perform
and less prone to technical errors.

Sedimentation techniques include:

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 Formalin-Ethyl Acetate Sedimentation

1. Mix the specimen well.

2. Strain 5 ml of the fecal suspension (more or less depending
on its consistency) through wetted cheesecloth-type gauze
placed over a disposable paper funnel into a 15 ml conical
centrifuge tube. (Conical paper cups with the tips cut off are
sufficient.)
3. Add 0.85% saline or 10% formalin through the debris on the
gauze to bring the volume in the centrifuge tube to 15
ml. Distilled water may be used; however, Blastocystis
hominis may be deformed or destroyed
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 Formalin-Ethyl…
4. Centrifuge at 500 × g for 10 minutes.
5. Decant supernatant. Add 10 ml of 10% formalin to the sediment
and mix thoroughly with wooden applicator sticks.
6. Add 4 ml of ethyl acetate, stopper the tube, and shake vigorously
in an inverted position for 30 seconds. Carefully remove the
stopper.
7. Centrifuge at 500 × g for 10 minutes.
8. Free the plug of debris from the top of the tube by ringing the
sides with an applicator stick. Decant the top layers of supernatant
9. Use a cotton-tipped applicator to remove debris from sides of the
centrifuge tube.
10. Add several drops of 10% formalin to resuspend the concentrated
specimen. Proceed with applicable testing
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Sedimentation technique for larvae of Strongyloides
stercoralis (Harada–Mori)

Principle:

• A strip of filter-paper is partially submerged in a test-tube

containing water.

• Any larvae of Strongyloides stercoralis present in the

specimen migrate against the current of water that rises by
capillary action and accumulate at the bottom of the tube

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 Harada Mori…
Materials and reagents
• Microscope
• Cellophane tape
• Test-tubes
• Test-tube rack
• Strips of filter-paper (30mm x 150mm)
• Spatula
• Lugol iodine, 0.5% solution

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 Harada Mori…
Method

1. Use the spatula to spread a small quantity of the faecal

specimen along a strip of filter-paper (previously folded
lengthwise to keep it straight), but leave the last 4 or 5cm
clean to be put into water.

2. Put the strip of filter-paper, clean end first, into a test-tube

containing filtered or boiled water 2.5–3.0cm deep; fold the
strip at the top so that the bottom does not touch the
bottom of the tube

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 Harada Mori…

3. Record the serial number or name of the patient

indelibly on the tube.
4. Plug the tube with cotton wool or, preferably, seal with
cellophane tape and keep for 7–8 days at room
temperature.
5. Look for the larvae at the bottom of the tube. Stain
with iodine solution for 1 minute and then examine under
the microscope, using the x 10 objective

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 Harada Mori…

• The larvae usually seen in fresh stool specimens are the

rhabditiform (first-stage) larvae of S. stercoralis.

• However, if the stool was passed more than 12 hours

earlier, the larvae may have hatched into filariform
(infective-stage) larvae.

• These must be differentiated from larvae of

Ancylostoma duodenale and Necator americanus
(hookworm), which may also hatch in stools 12–24
hours after passage 28
Cellophane fecal thick – smear for diagnosis of
intestinal Schistosomiasis (Kato – Katz technique)

• The cellophane fecal thick – smear examination

technique has proven to be an efficient means of
diagnosis of intestinal schistosomiasis and intestinal
helminthes.
• Cellophane thick – smear slides can be prepared in the
field, stored in microscopic slide boxes, and shipped
great distances, for examination at a central laboratory
if required

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 Cellophane…
• The technique is not suitable for examining larvae, cysts, or
eggs from certain intestinal parasites

Materials and reagents:

– Applicator sticks, wooden

– Screen, nylon or plastic (60 – 105 mesh)

– Template, stainless steel, plastic or cardboard

– Microscope slides

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 Cellophane…
– Cellophane, 40 – 50 m thick, strips 25 x 30 or 25
x 35 mm

– Flat bottomed jar

– Forceps

– Toilet paper or absorbent tissue

– Newspaper
– Glycerol – malachite green (or methylene blue) solution

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 Cellophane…
• Technique
– Care must be taken during collection of stool
specimens. Always wear gloves to avoid
contamination of the fingers
1. Soak the cellophane strips in the 50% glycerol –
malachite green (or methylene blue) solution for at
least 24 hours before use
2. Transfer a small amount of feces on to a piece of
scrap paper (news paper is ideal)

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 Cellophane…
3. Press the screen on top of the faecal sample
4. Using a flat – sided applicator stick, scrape across
the upper surface of the screen to sieve the fecal
sample
5. Place a template on a clean microscope slide
6. Transfer a small amount of sieved fecal material
into the hole of the template and carefully fill the
hole. Level with the applicator stick

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 Cellophane…

7. Remove the template carefully so that all the fecal

material is left on the slide and none is left sticking
to the template

8. Cover the fecal sample on the slide with a glycerol –

soaked cellophane strip

9. If an excess of glycerol is present on the upper

surface of the cellophane, wipe off the excess with a
small piece of toilet paper or absorbent tissue

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 Cellophane…

10.Invert the microscope slide and press the fecal

sample against the cellophane on a smooth
surface ( a piece of tile or flat stone is ideal) to
spread the sample evenly

11.Do not lift the slide straight up. The cellophane

may separate. Gently slide the microscope slide
sideways holding the cellophane

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 Fecal egg count by the McMaster Method
 The McMaster technique uses a counting chamber which
enables a known volume of fecal suspension to be examined
microscopically
 Thus, if a known weight of feces and a known volume of
flotation fluid are used to prepare the suspension, then the
number of eggs per gram of feces (FEC) can be calculated
 The McMaster chamber has two compartments, each with a
grid etched onto the upper surface
 When filled with a suspension of feces in flotation fluid, much
of the debris will sink while eggs float to the surface

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 Fecal egg count by the McMaster Method
Procedure
1. Weigh 4 grams of feces in a plastic beaker (100ml)
2. Add 60ml of a saturated salt + sucrose solution
(NaCl 333g, sucrose 200g in 1L of distilled water)
3. Homogenize and pour the fecal suspension 3 times
through a tea strainer to withhold the large debris
4. Homogenize the filtrate by pouring it 10 times from
one beaker to another and fill up one side of a
regular McMaster counting chamber by using a
Pasteur pipette

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 Fecal egg count by the McMaster Method

5. Repeat for the other side

6. Allow the counting chamber to stand for 2 minutes,
place under a light microscope and examine using a 10x
magnification
7. All the eggs under the two separate grids are
counted
8. The number of eggs per gram of feces is obtained by
multiplying the total number of eggs or oocysts under
the two grids by 50

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