Lab Activity #1
Uploaded by
Charmaine RosalesLab Activity #1
Uploaded by
Charmaine RosalesName: ______________________________ Student No.
: ____________
Year/Section/Cluster: __________________ Group No.: _____________
Laboratory Exercise # 1
PERIPHERAL BLOOD SMEAR
Learning Objectives:
This laboratory exercise will enable the students to:
1. Execute the proper way of preparing blood smears
2. Demonstrate the different methods of smear preparation
3. Identify the characteristics of a good smear
Materials:
Compound light microscope Eosin
Anticoagulated blood Methylene Blue
Glass slides Buffer solution/aged distilled water
Pasteur pipette/Capillary tube Cedar wood oil
Methanol
Procedures:
I. Two-Slide or Wedge Method
1. Place a drop of blood in the centre of a clean glass slide 1 to 2 cm from one end.
2. Position the spreader slide before the drop of blood at an angle of about 30 ⁰-45 ⁰. Move
the spreader backward so that it makes contact with drop of blood and allow the blood to
spread evenly on the vertex.
3. Move the spreader smoothly and rapidly over the length of the slide at a constant angle.
Be sure to keep the spreader firm to the slide.
4. Air-dry.
Criteria of a Good Smear
1. There should be an even transition from the thick area to the thin area.
2. The smear must cover 2/3 or ¾ of the slide.
3. It must have a smooth and an even surface free from ridges, waves or holes.
4. It must have a feathery edge.
Other Methods of Smear Preparation
A. Two-Cover Slip or Ehrlich’s Two-Coverglass Method
Procedure:
1. Get two cover glasses. Hold on cover glass on its adjacent corners with the thumb and
index finger.
2. Place one small of drop of blood on top of held coverglass.
3. Position the other coverglass on top of the coverglass with blood. As this is done,
blood begins to spread.
4. Before the blood completely spreads, separate the coverglass by doing a rapid, even,
horizontal, and lateral pull. Avoid squeezing the coverglasses together.
5. Air-dry the coverglass.
B. Spin Method
The spin method is done using an instrument such as a cytospin centrifuge.
Procedure:
1. Position a clean glass on a platen.
2. Place 3-4 drops of blood in the middle of the slide.
3. Close the instrument. The platen spins at a high speed. Excess blood is expelled into a
catch basin.
4. The resulting slide is completely covered with a thin monolayer of cells.
C. Automated Smear Preparation
II. Wright Staining
Staining Jar or “Dip” Method
Procedure:
1. Dip in solution 1 (methanol, the fixative) for 1 minute.
2. Dip in solution 2 (eosin, the acidic dye) for 10 seconds.
3. Dip in solution 3 (methylene blue, the basic dye) for 3 seconds.
4. Dip in buffer solution/aged distilled water for 45 seconds.
Other methods of Staining
a) Staining dish method – involves placing the blood smear on a rack position on a dish.
b) Automated method
Types of Stains Used in Peripheral Blood Smears
1. Romanowsky stains
Romanowsky stain contains methylene blue or its oxidative product, such as azure B.
It also contains eosin B or eosin Y. The dyes produce multiple colors when used on cells
and cellular components. This is the reason why the stains are considered polychromatic.
Main components of a Romanowsky Stain:
Cationic or basic dye - has affinity for acidic component of the cell (i.e. nucleus)
Anionic or acidic dye - has affinity for basic component of cell (i.e. cytoplasm)
a. Wright’s stain – most satisfactory in general routine hematologic studies.
Composition:
Oxidized methylene blue
Eosin azures
b. Giemsa stain – excellent stain for the demonstration of inclusion bodies and
intracellular parasites as well as for staining WBCs
Composition:
Eosin Y with azure blue
Methylene blue in methanol with glycerin
c. Leishman, Jenner, and May-Grunwald – similar to Wright’s stain except for the
method used to oxidize methylene blue.
2. Panoptic Stains
Panoptic stains consist of Romanowsky stain and another dye.
a. Wright’s-Giemsa
b. Jenner-Giemsa
c. May-Grunwald-Giemsa
Name: ______________________________ Student No.: ____________
Year/Section/Cluster: __________________ Group No.: _____________
POST LABORATORY QUESTIONS
1. Why is it important to smear the blood as soon as the drop is placed on the slide?
2. What 3 things determine the thickness of the smear?
3. Explain how to adjust the thickness of the smear for:
A. High hematocrit
B. Low hematocrit
4. Enumerate the common causes of too thick and too thin blood smears.
5. How would you obtain a good smear if the patient has a cold agglutinin?
6. a) Where do lymphocytes tend to accumulate on a wedge smear? b) Where do the larger cells
tend to accumulate on a wedge smear?
7. Describe the optimal area for evaluating and enumerating blood cells on a well-stained smear.
