Preparation of Thick and Thin Blood Film for Malarial Parasite Identification

1. Clean 2 glass slides to remove the grease.

2. Using a lead pencil, label the frosted edge with the patient’s full name, date, time of collection.
3. Perform capillary puncture using the 3rd or 4th finger. Make sure the puncture site is warm. Cleanse the site
with 70% ethyl alcohol and allow to dry.
4. Hold the finger and firmly puncture the side of the ball of the finger.
5. Wipe off the first drop of blood with a dry cotton.
6. Apply moderate pressure, approximately 1 cm behind the site of the puncture to obtain a round a drop of
blood.
7. Release the pressure immediately to allow recirculation of blood.
8. Hold the capillet in a horizontal position and allow it to come in contact with the drop of blood. Allow it to be
filled with blood by capillary action.
9. Using the capillet as a transfer vessel, make 4 drops of blood on each slide.
10. For the thick smear, use the corner of another slide and connect the 3 drops of blood to form a circle using 1
stroke only. Make sure the slide with the blood sample is placed on a flat surface like a table.
11. For the thin smear, use a spreader slide and hold it at an angle of 45 degrees and place the end of the
spreader slide slightly in front of the drop of blood.
12. Draw the spreader slide back toward the drop of blood. As soon as the spreader slide comes in contact with
the drop of blood, the blood will begin to spread to the edge of the spreader slide. If this does not occur,
wiggle the spreader slide a little until it does so.
13. Push the spreader slide firmly against the horizontal slide in a quick, smooth forward direction.
14. Allow both smears to dry.

Procedure for Staining the Blood Films

1. Immerse the thick film in tap water for 2-3 minutes.
2. Fix the thin film with methanol for 2-3 minutes.
3. Dry then stain with Giemsa for 30 minutes.
4. Rinse with tap water and allow to air dry.
5. Observe under the OIO.

Colors using Giemsa Stain:

Plasmodium Cytoplasm - Robin’s egg blue
Nuclear chromatin - Crimson or Violet
Cytoplasm or Infected RBC - Yellowish or Salmon pink
Malarial pigment - Brown or Black
Malarial Parasite Counting
1. Use the thick smear for malarial parasite counting. Make use of a differential tally counter.
For P. vivax, P. malariae, and P. ovale, no need for stage differentiation when counting.
Count:
a. Malarial parasite
b. WBC
For P. falciparum, count:
a. Gametocyte
b. Ring forms
c. WBC
2. Count every other field making sure that there should be at least 10-15 RBCs per field.
3. Determine the count using the formula below:

Malarial parasite counted

______________________________ X 8000 = Malarial Count / cumm or microliter of blood

200 WBCs