Buffer solution for staining for malaria parasites

A phosphate buffer solution of pH 7.2 is essential for accurate Giemsa microscopy. Buffer tablets can be
used but are expensive and spoil easily in the tropics.

A buffer solution for daily use

1. Dissolve 1.0 g anhydrous hydrogen phosphate (Na2PHO4) and 0.7 g potassium dihydrogen
phosphate (KH2PO4) in 11 distilled or deionized water.

2. Check the pH with a pH meter, color-based indicator or narrow-range pH test strips

3. If it is below pH 7.2, adjust with small quantities of 2% Na2HPO4 ; if it is above pH 7.2, add 2%
KH2PO4.

4. When the buffer is balanced to pH 7.2, transfer if to a tightly stoppered, dark-glass bottle. Store
away from sunlight.

The solution is good for some weeks, but it should be checked regularly for growths and moulds by
shaking the bottle and replaced when cloudy.

A concentrated stock solution for field trips or for dispatch to remote locations

1. Dissolve 3.0 g anhydrous Na2HPO4 and 2.1 g KH2PO4 in 25 ml distilled or deionized water.

2. If the pH is below 7.2, add small amount of 2% Na2HPO4; if it is above 7.2, add 2% KH2PO4.

3. Store in a dark-glass bottle; the solution remains good for some weeks; check regularly for
moulds

4. For a working solution, dilute 1 mL of the concentrate with 20 mL of distilled or deionized water.

Effect of pH on staining

For consistently reliable diagnosis by microscopy, the water used to dilute the stain must be buffered to
pH 7.2. The water used should be deionized or distilled; rainwater is too acid, and the water may be
contaminated with bacteria and other microscopic forms. If potentially contaminated water has to be
used, it should first be boiled, cooled and filtered. These com- ments also apply to the water used to
flush off the stain at the end of staining. If the water used to rinse newly stained slides is acidic, it will
have a de-staining effect. Perfectly good stain- ing can be spoiled if, for example, the delicately stained
chimeff (Schuff) ner stippling fades away as it is 'over-rinsed' with acidic water. It is strongly
recommended that the water used to rinse stain from slides be at pH 7.0 or above. In limestone areas,
tap water commonly at about pH 7.2 and can be used to flush slides with confidence.
Accurate diagnosis of parasite stage and species is based on combinations of colours, shapes, size and
the presence of pigment and parasite forms in the thick film or, later, in the thin film if diagnosis in the
thick film proves too difficult; when staining is like a black-and-white photo, usually due to variance from
the standard pH 7.2 errors in diagnosis are made.

The four photomicrographs of P. vivax shown below illustrate the effect of pH on Giemsa staining of
malaria parasites and blood elements. In (a), note the markedly greenish-blue staining of the
erythrocytes at pH 7.6. A trophozoite is recognizable, but the Schüffner stippling in the erythrocyte
cytoplasm faint. At pH 7.4 (b), the erythrocyte containing a trophozoite has a greenish-blue tinge, and
again Schüffner stippling in the cytoplasm is pale and barely recognizable. The pH of the buffered water
used to stain the parasites shown in (c) is about 7.0: the chromatin and cytoplasm stain well, with clear
Schüffner stippling, facilitating the diagnosis of P. vivax, especially when linked to enlargement of the
infected red cells. When a blood film is made from anticoagulated blood, the blood pH may be changed,
and the pH of the buffer water might have to be adjusted, by trial and error. At the more ideal pH value
of 7.2, the red blood cells have a pinkish appearance, the trophozoites stain well and Schüffier is stippling
is prominent (d).

Plasmodium vivax thick film

Gambar

P. vivax ring forms are typically larger than those of P. falciparum (a-c, with a double chromatin in c,
arrow) and contain the pinkish “ghosts” of the (dehaemoglobinized) enlarged red cells (b and c).
Ttrophozoites in this species vary considerably in size and shape; when the staining is of a good standard,
the Schuffner stippling clearly delineates the area occupied by each infected red cell (e and f). Parasites
in thick films may strain densely, and P. vivax stages can appear surprisingly large (d and e). The presence
of various stages in the blood film usually indicated P. vivax, but the examiner must always remain alert
to signs of a mixed infection.

Gambar

Even immature schizonts (g.h) are large than in other species, while mature schizonts (i) finally contain
12-24 merozoites. Mature micro (k) and macro (l) gametocytes exhibit the usual differences in
coloration, although it is often difficult for an examiner to differentiate between immature gametocytes
and mature trophozoites (j, arrow). This, however, is of little importance in routine malaria microscopy.