EXERCISE NO.

9
Parasitological Examination of Blood
A. PREPARATION OF A BLOOD SMEAR
Objective: To prepare a good blood smear using wedge-edge method.
Principle:
The blood smear examination is a routine part of the complete blood count. It is
prepared by spreading blood on a glass slide or microscope slide, drying, staining and
viewing it under the microscope. The blood components are microscopically identified,
evaluated, as in the white cell differential count.
The examination of a well-prepared blood smear is valuable in the diagnosis of
leukemia, sickle cell anemia, pernicious anemia and infectious mononucleosis. It is also a
method of choice for the diagnosis of malaria. The morphology or structure of blood cell
components can be studied. The erythrocytes, leukocytes and platelets size, structure,
number and maturity can be evaluated. Thus it is important to prepare and stain the blood
smear properly.
Blood smears can be either on glass slides or cover slip. It can be prepared in three
ways: (1) Erhlichs method or the two-cover slip method. However it is difficult to make
because blood is thick and viscous and the cover slip is fragile; (2) Beacons method or the
glass slide cover slip method; and (3) Wedge method or the two-glass slide method,
commonly employed due to ease of preparation.
The best specimen for a blood smear is capillary blood, with no anticoagulant. The
blood with EDTA anticoagulant could also be used provided the smear is made within two
hours of collection. Other anticoagulant should not be used since they may alter the
morphology or staining characteristics of cells.
Slide to be used must be free of grease and dust. Slides may be washed with soap and
water, rinsed in hot water, then distilled water, dipped in 95% ethyl alcohol and polished with
a clean, lint-free cloth. Handle slides by the edges only.
Wedge method (Two-slides method)
Materials:
Clean glass slides; Blood specimen (capillary or venous), ; EDTA vacutainer tube (or
heparinized capillary tube); venipuncture set (or lancet); tissue paper.
Procedure:
1. Place half-drop of blood about 1-2 cm from one end of the clean glass slide and place
it on a flat surface.
2. Get another slide with an even, and unchipped-edge (and its corners broken off,
preferably so that the width of the spreading edge less than the total slide wedge) to
be used as a spreader.
3. Place the spreader slide to rest at a 30 35 degree angle in front of the drop of blood.
4. Draw backwards the spreader slide edge until it touches the drop of blood and the
blood spreads along the edge of the spreading slide.

5. Push away the spreader from the drop of blood smoothly, evenly, steadily and
quickly. The more rapid the movement, the thicker the smear. The bigger the angle,
the thicker the smear. Clean the end of the spreader slide once it is used again. Keep
the spreader unchipped.
6. Allow the smear to air dry as quickly as possible and then stain. Write the name of
patient and other identification on the slide using diamond marker or lead pencil on
the blood smear.
7. If immediate staining is impossible, immerse the dried blood smear in methanol for
30-60 seconds and stain at a later date.
Characteristics of a good blood smear:
a. gradual transition from thick to thin area
b. smears occupy 1/2 to only of the slide
c. no overlapping of cells
d. smooth appearance with no scratches, ridges and holes
e. has feathery edge
Factors affecting smear quality:
a. size of the drop of blood too large drop, thick smear
b. angle at which the spreader slide is held large angle thick smear
c. too much pressure applied to spreader thin smear
d. faster spreading thin smear
e. high humidity causes slow drying, cells appear abnormal (RBCs appear motheaten)
Activity:
1. Practice preparing blood smears using two-slide method.

RESULTS:
Each student must present to the instructor 1 good blood smears

Drawing: Draw a good blood smear you have prepared.

B. STAINING OF A BLOOD SMEAR

Objective: To stain properly the prepared blood smear using Giemsas or Wrights stain.
Principle:
Blood smears are stained to view and evaluate the formed elements easily. The
stains commonly used for the routine microscopic examination of blood are called
polychromatic. These stains are named because they contain dyes that will stain various

components of cells different colors. Most of these stains contain combinations of methylene
blue and eosin (red-orange stain); and methyl alcohol as fixative. The two most commonly
used bloodstains are Wrights stain and Giemsas stain.
Materials: Wrights stain (commercially prepared), blood smear, staining rack, tissue paper

Procedure:
1.
2.
3.

4.
5.

Two-step staining
Place the dried blood smear on the staining rack.
Cover the smear with undiluted stain and leave for 1-3 minutes.
Dilute with distilled water or buffer of the same quantity as the stain and mix by blowing
gently on the surface until a metallic scum appears. Allow this diluted stain to act for 35minutes.
Rinse with distilled water until the stain appears pink. Remove the slide from the rack.
Allow slides to air-dry and examine using the microscope.

Quick Staining
The blood smear is dipped into two or three solutions quickly, then rinsed and dried. This
method takes less than a minute.
Automatic Staining (Hema-Tek automatic slide stainer)
1. The blood smears are placed on a moving belt, which carries them through various
staining reagents. The other types of stainer is the basket or batch type in which
basket of slides are taken through the staining process stepwise. One basket of slides
is fixed, then dipped into the stain solution, etc. this is continued through the
complete staining process.
2. Automatic stainers are an advantage when large number of slides must be stained or
staining must be performed frequently during a workday. The disadvantage is that if
the stain is bad, the worker may not know until many slides have been processed.
STAIN QUALITY EVALUATION:
1. Stained smear should appear pinkish-purple macroscopically.
2. Microscopically, the red cells should appear orange-pink to rose
Activity:
1. Practice staining blood smears using either two-step or quick step method
2. Submit 1 good stained blood smear
Drawing: Draw a picture of properly stained blood smear below.