Thick and Thin Blood Films Procedure

Purpose:

Thick and thin blood films stained with Giemsa hematological stains permit the detection of blood parasites including malarial parasites, trypanosomes, and microfilariae.

Principle:

The thick blood film permits the examination of a large amount of blood for the presence of parasites. Since the smear is not fixed with methanol, the red blood cells (RBCs) lyse, permitting better visualization of the organisms. The thin film allows for the observation of RBC morphology, inclusions, and intracellular and extracellular parasites. A larger area of the slide must be examined in the thin smear, although there is less distortion when compared with the thick smear.

Procedure:

Giemsa Stain Preparation

1. Giemsa stain may be purchased as a concentrated stock or prepared by grinding 1 g powdered Giemsa with 5- 10 ml glycerol in a mortar. Add additional glycerol to a final total volume of 66ml, and heat to 55 deg Celsius in a water bath until stain is dissolved. Cool and add 66 ml absolute methanol. Allow to stand for 2-3 weeks. Filter and store in a brown bottle protected from the light.
2. Prepare working Giemsa stain by mixing one part stock Giemsa stain to 10- 50 parts triton- buffered water. The dilution chosen will determine the staining time. (e.g. 1:50 dilution, stain for 50 minutes.)
3. To prepare buffer 1, add 9.5 g 0.067 M Na2HPO4 (disodium phosphate) to 1000ml distilled water.
4. To prepare buffer 2, add 9.2 g 0.067 M NaH2PO4; H2O to 1000 ml distilled water.
5.  Working buffer is prepared weekly and filtered before each use. Prepare the working buffer by adding 61.1 ml stock buffer 1 and 38.9 ml stock buffer 2 to 900 ml distilled water.

Thick Film Procedure

1.  Prepare thick blood film by applying a free-flowing drop of blood, preferably without an anticoagulant, to a microscope slide cleaned with alcohol to remove any oil present.
2.  Using the corner of  a second slide, spread the film in a circle about the size of a dime. Avoid the formation of fibrin strands by spreading rapidly in a circular motion.
3. Allow to air dry overnight.
4. Place smears in a 1: 50 dilution of stock Giemsa stain for 50 minutes. The lack of methanol fixation allows lysis or laking or the RBCs.
5.  Rinse gently with triton- buffered water three times.
6. Drain slides in a vertical position and air- dry.

Thin Film Procedure

1. Clean a microscope slide with alcohol to remove any oil.
2. Touch the slide to 1 drop free flowing blood toward one end of the slide.
3. Holding a second slide at 30 degree angle to the first slide, draw back into the drop, and allow it to spread into the edge of the spreader slide. Then quickly and evenly push the spreader slide forward, allowing the blood to spread out. There should be a feathered edge “ at the end of the smear and no holes, and the smear should cover one- half to two- thirds the slide.
4. Allow the slide to air dry.
5.  Fix the thin smear in absolute methanol for 30 seconds.
6. Place in working Giemsa stain for 50 minutes if using a 1:50 dilution. Staining can be reduced by reducing the dilution of the stain.
7.  Rinse gently with buffer for 2 minute.
8. Dry vertically and examine under the 100x objective.

Microscopic Examination:

• Thick smear: examine 200-300 oil immersion fields for the presence of blood parasites.
• Thin smear: examine the slide for a minimum of 30 minutes, and examine a minimum of 200 fields under oil immersion objective.
• Giemsa stains blood components as follows:

          Erythrocytes: pale gray- blue

          White Blood Cells: Nuclei- purple

          Cytoplasm: pale purple

          Granules- eosinophils: bright red- purple

          Neutrophils: dark pink- purple

          Parasites: blue to purple with red nuclei