Page 205 - The Veterinary Laboratory and Field Manual 3rd Edition

174 Susan C. Cork and Mani Lejeune

cells. This method is not sensitive and it is also Stains and staining
hard to differentiate parasite species. Protozoa in Stains and staining techniques vary according
faeces and Trichomonas sp. in vaginal mucus can to individual preference. The stain usually used
also be identified by direct examination.
is Giemsa diluted 1 : 15 and the usual stain-
ing technique is as follows. (Note that timing,
braIn cruSH SMEar dilution and pH may vary slightly with different
Cut a small piece of brain tissue containing cap- batches of stain.)
illaries. Put this on a clean slide and then crush
between two slides. Slide these together gen- 1 Using a pipette, measure 6 ml of concen-
tly and then move the slides apart. Allow the trated (stock) Giemsa stain into a clear empty
smear to dry in air. Rapid fixation is absolutely bottle (the stock stain may need to be passed
essential. through a filter to remove particles).
2 Add 90 ml of water at pH 7.2 (the ideal pH
LyMPH nodE SMEar may vary with batches of stain).
Locate the position of the lymph node of inter- 3 Mix well and pour into a staining jar contain-
est and hold it firmly. With sterile precautions, ing the smears.
and by means of a syringe-needle pierce the 4 Allow to stain for 20–30 min (in some cases
gland and move the needle tip around. Pull the 10 min may be sufficient).
plunger of the syringe to aspirate a sample of 5 Wash the smears in distilled water or tap
the contents of the gland and withdraw the nee- water.
dle. Expel the contents on to a clean slide and 6 Dry in air and examine.
make a smear (as for a thick and/or thin blood
film) and stain. This method is good for detect- The Leishman stain may also be used (see below).
ing stages of Theileria sp. in lymphocytes. Other stains are available in kit test form to
allow quick staining for fieldwork (for example,
SPLEEn SMEar Diff Quick™ ). Most of the stains are based on
6
At necropsy, cut open the spleen using a knife. Romanowsky stains (see Chapter 5).
With the edge of a slide scrape the cut surface
and spread the material across the slide using PurPoSE oF StaInInG
a spreader to draw the spleen tissue behind it. Staining helps in the differentiation of cells.
Allow to dry in air. It is important to protect Cellular constituents stained by the basic com-
prepared smears from dust and rain by covering ponents of the stain appear blue, purple or
them or putting them into a box immediately violet in colour and are said to be basophilic;
after they are prepared. while those constituents stained by the acidic
components of the stain appear red, pink or
FIxatIon orange in colour and are said to be acidophilic or
Fixation of smears should be carried out as soon eosinophilic. Those cellular constituents stain-
as possible after smear is air dried and is a pre- ing between the two extremes in colour are said
liminary step before staining can be carried out. to be neutrophilic (see also Chapter 5).
The fixative usually used is methanol. Smears
5
are fixed for at least 1 min (could be more, but MIcroScoPIc ExaMInatIon
not less) and again air dried. It is good technique to examine smears using
low magnification before going on to examine
specific areas under high magnification using oil

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