The direct microscopic visualization of the malarial parasite on the thick and/or thin blood smears has been the “gold standard” for malaria diagnosis.
Thick Blood smear
- Using the corner of a clean slide, spread the drop of blood in a circle the size of a dime (diameter 1-2 cm).
- Do not make the smear too thick or it will fall off the slide. (You should be able to read newsprint through it.)
- Allow the smear to dry thoroughly. Insufficiently dried smears (and/or smears that are too thick) can detach from the slides during staining. You can accelerate the drying by using a fan or hair dryer.
- #Do not fix thick smears with methanol or heat.
- If there will be a delay in staining smears, dip the thick smear briefly in water to hemolyse the RBCs.
Visually, the smear should appear as a round to oval smear of blood about 2 cm in diameter. It should be of such thickness that newsprint can barely be seen through the wet or dry smear.
Limitation of Thick Smear
- Making a species identification of malarial parasites may be difficult to impossible, even for experienced technicians.
- A thin film should always be examined if a definitive identification based on morphology is required.
- Smears must be prepared from anticoagulated blood within one hour after venipuncture. The morphology of parasitic forms and the erythrocytes become atypical after that time from direct action of the anticoagulant.
Thin Blood Smear:
Thin smears consist of blood spread in a layer such that the thickness decreases progressively toward monolayer. It allows optimal assessment of the morphology of any parasitic forms that may be present. Thin blood film is prepared similarly to that of the differential white-cell count.
Making Thin Blood Smear:
- Bring a clean spreader slide, held at a 45° angle, toward the drop of blood on the specimen slide.
- Wait until the blood spreads along the entire width of the spreader slide.
- While holding the spreader slide at the same angle, push it forward rapidly and smoothly.
- Wait until the thin films are completely dry before staining.
- Fix the thin film with methanol (100% or absolute) for 15-30 second and let it dry completely before staining.
- Note: fixation time for ethanol is 20 minutes
Limitation of thin smear
- Parasitic forms may be missed in light infections. In such instances, a thick film must be examined.
- Smears must be prepared from anticoagulated blood within 1 hour after venipuncture. The morphology of parasitic forms and the RBC become atypical after that time from direct action of the anticoagulant.
Staining of the thick/thin smear with Giemsa Stain:
- Place slides into the working Giemsa stain (2.5%) for 45-60 minutes.
- Remove thin smear slides and rinse by dipping 3-4 times in the Giemsa buffer. Thick smears should be
left in buffer for 5 minutes.
- For perfect malaria staining the pH of the buffer should be 7.2
- Dry the slides upright in a rack.
Note: As alternates to this 45-60 minutes in 2.5% Giemsa stain, the smears could be stained for shorter times
in more concentrated stains. One alternate is 10 minutes in 10% Giemsa; the shorter stains yield faster results,
but use more stain and might be of less predictable quality
First screen the thick/thin smear at a low magnification (10× or 20× objective lens), to detect large parasites(microfilaria) then examine the smear using oil immersion objective. NCCLS recommend examination of at least 300 oil immersion fields for the determination of “No Parasite Seen”.
Diagnostic Points for Plasmodium falciparum
- Red cells are not enlarged.
- Rings (trophozoite ring stage) appear fine and delicate and there may be several in one cell.
- Some rings may have two chromatin dots.
- Presence of marginal or applique/accole forms.
- It is unusual to see developing forms in peripheral blood films.
- Gametocytes have a characteristic crescent shape appearance. However, they do not usually appear in the blood for the first four weeks of infection.
- Maurer’s dots may be present.
- Red cells containing parasites are usually enlarged.
- Schuffner’s dots are frequently present in the red cells as shown above.
- The mature ring forms tend to be large and coarse.
- Developing forms are frequently present.