Page 206 - The Veterinary Laboratory and Field Manual 3rd Edition

Parasitology 175

immersion. This is especially important when for example, the microfilaria of the canine
examining brain smears or smears for trypano- heart worm, Dirofilaria immitis. The parasite is
somes. transmitted by mosquitoes (Aedes sp.). The fol-
lowing technique is also useful for detecting
trypanosomes in fresh blood.
Stain preparation
concEntratInG MIcroFILarIaE and
GIEMSa StaIn
Preparation of a stock solution: Add 1g of tryPanoSoMES:
Giemsa powder to 66 ml Glycerol and mix 1 Take about 3–5 ml of fresh blood and add it
thoroughly. Heat to 56°C for 90 minutes to 0.5 ml of 3.8% sodium citrate to stop
and add 66 ml methanol. Label the stock it clotting. Solid sequestrene can also be used
solution and mix thoroughly. Leave to stand as an anticoagulant, but a liquid anticoagu-
for 7 days before filtering and storage in lant solution is better.
glass-stoppered bottles. 2 Centrifuge the blood sample for 10 min. If
To prepare the buffer, add pH tablets to looking for trypanosomes, centrifuge at fast
distilled water. pH 7.2 is usually recom- speed. If looking for microfilariae centrifuge
mended but some laboratories have found the blood at about 1000 rpm. The ‘buffy’ coat
that the stain will work in the range of of a prepared and centrifuged microhaema-
pH 6.8–7.2. tocrit can be examined for the presence of
trypanosomes.
3 After centrifuging there will be three layers –
LEISHMan StaIn: PrEParatIon oF a
a layer of plasma, a layer of white cells (buffy
Stock SoLutIon:
Add 0.15 g Leishman powder to 100 ml of coat) and a layer of red cells.
methanol. Dissolve by grinding and stirring 4 Using a Pasteur pipette take off the super-
for 2 h. Keep the stock solution in a dark natant plasma to the white cell layer or, if a
glass dropper bottle. haematocrit tube has been used, break off the
To prepare the buffer, add pH tablets to haematocrit tube at the white cell layer.
distilled water. pH 7.2 is usually satisfac- 5 Take as much of the white cell layer as pos-
tory for staining blood smears and some sible and prepare a smear on a slide. Examine
protozoa. under a coverslip. If looking for microfilariae
Fix air dried smears in Leishman stain take some of the red cell layer as well and
for 2 min. Add an equal amount of buffered examine this under the coverslip.
distilled water. Mix by gently rocking the 6 Scan the entire coverslip using the low power
slide to allow even distribution. Leave for (10× or 20×) objectives for microfilariae and
10–15 min. Rinse in buffer and immerse a high power (100×) objective for trypano-
until the smear appears pink (1–2 min). somes (see Figure 3.35).
Blot or air dry.
concEntratInG MIcroFILarIaE
(ModIFIEd knott’S tESt)
1 Measure 1 ml of anticoagulated (EDTA/heparin
A note on the examination of blood
smears for the presence of microfilariae treated) whole blood in a 15 ml centrifuge tube.
2 Add 9 ml of 2% formalin.
Microfilariae are the pre-larval forms of hel- 3 Cap the centrifuge tube with rubber stopper
minth parasites which may occur in the blood, and mix by inversion.

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