tap water is not neutral, wash the slide with
distilled water. The stain and the metallic
film must be floated off to prevent streak-
ing, so keep the slide flat and horizontal
in the stream of water. If the slide is tilted,
the metallic film will settle to the surface
of the smear and remain there.
4. Wipe the stain from the bottom of the
slide. Let it air-dry. A good smear should
be thin and evenly distributed, and it must
be dry before staining. Always make at
least two smears for each patient, as the
additional smear should be examined to
verify any abnormal findings.
Staining times will vary with different batches
of Wrights stain. One batch may give best results
with 3 minutes of staining and 2 minutes of buf-
fering, another may give better results with 2
minutes of staining and 3 minutes of buffering.
The only way to determine the best time interval
for a particular stain is by experimentation.
Next to incorrect time intervals, the most com-
mon cause of poor results with Wrights stain is
incorrect pH of the staining fluid. If the stain is
too acid, the red cells in the smear will stain a
bright pink or may even be colorless, while an
alkaline stain will cause the red cells to appear
blue-gray, with poor color definition. In either
case, the pH of the buffering solution should be
Technique for Differential Count
1. Place the stained slide on the microscope
with a drop of immersion oil and adjust
the ocular lenses.
2. Using the oil immersion lens (red) (highest
power), scan the fields for areas where the
red cells just touch. In this area the blood
smear is thinner, and consequently, the
white cells are easier to identify.
3. Count 100 consecutive white cells (see
figure 6-10 for identification), pressing the
correct key on the cell counter for each type
of white cell identified.
4. If you count 20 lymphocytes among the 100
cells, then the patient has 20 percent lymph-
ocytes. If an absolute count has been re-
quested, the total leukocyte count is
multiplied by individual percentages.
Patient has a white count of 8,000.
Differential count shows 20 percent
20 percent x 8,000 = 1,600
The patient has 1,600 lymphocytes per cubic
millimeter of blood.
The ability to identify the different types of
white cells is not difficult to develop, but it does
a thorough knowledge of staining
characteristics and morphology that can be gained
only by extensive, supervised practice. The begin-
ner is likely to encounter some difficulty in learn-
ing to differentiate between monocytes and large
lymphocytes, or between monocytes and atypical
(not typical) lymphocytes. It is possible also to
confuse eosinophils with basophils, since an
alkaline stain may cause the orange granules in
an eosinophil to appear deep blue or purple. If
this happens, the reddish cast in the granules can
often be detected by judicious use of the minor
focusing knob. Also, the granulations of an
eosinophil are generally much finer than those of
a basophil, and they tend to concentrate in the
The neutrophils are subclassified according to
age, and the age is indicated by the nucleus. If
there is any doubt as to the identity of the
neutrophil, always classify it with the older stage.
For instance, if a single irregularity in a horseshoe-
shaped nucleus appears as a break or a concen-
tration of color, classify it as a segmented
neutrophil, not as a neutrophilic band. Practice
is the key. And remembereven experienced
technicians often disagree as to the identity of a
If it is desirable to save a smear for re-
examination, remove the immersion oil by plac-
ing a piece of lens tissue over the slide and
moistening the tissue with xylol. Draw the damp
tissue across the slide and dry the smear with
another piece of lens paper.
A good smear (thin and evenly distributed) is
essential for accurate identification and counting.
The cytoplasm of an eosinophil contains
numerous coarse, reddish-orange granules, which
are lighter colored than the nucleus.
Scattered large, dark-blue granules, which are
darker than the nucleus, characterize the cell as
a basophil. Granules may overlay the nucleus as
well as the cytoplasm.