Diagnosing Malaria via Microscopy: A Comprehensive Guide

By NIAID - Malaria Parasite Connecting to Human Red Blood Cell, CC BY 2.0, https://commons.wikimedia.org/w/index.php?curid=62117171

Malaria is usually diagnosed by testing a person's blood using methods like looking at the parasites under a microscope, using rapid tests to find parasite proteins, or checking for the parasite’s DNA or the body’s immune response.

Microscopy remains the gold standard for diagnosing malaria, especially in resource-limited settings. It allows not only for detection of Plasmodium parasites but also quantification and species identification—crucial for guiding appropriate treatment.

In this blog post, we walk you through the preparation, staining, and identification of malaria parasites in blood films, and how to quantify parasitemia to assess disease severity.

1. Understanding Blood Stages of Malaria

Malaria diagnosis is based on the erythrocytic stage of the parasite life cycle. This is when the parasite infects red blood cells (RBCs), making it visible under a microscope. Parasites are released into the blood following liver-stage development, known as the exo-erythrocytic or pre-erythrocytic stage.

Inside the RBC, the parasite progresses through several stages:

Ring stage (early trophozoite)
Trophozoite
Schizont (multiple nuclei, which ruptures RBCs, contributing to anemia)
Gametocyte (sexual form, infectious to mosquitoes)

Fig.1: Morphology of the different stages of the Plasmodium falciparum life cycle in thin blood films. Image credit: microbeonline.com

Under stress (e.g., fever or anemia), the parasite may switch to gametocytes, which are then picked up by mosquitoes during a blood meal.

2. Types of Blood Films

Microscopy involves preparing two types of blood smears:

✅ Thick Film

Used for detecting and quantifying parasites.
RBCs are lysed during the washing stage after Giemsa staining, releasing parasites into the film.
Appears grainy; parasites stand out more clearly.
White blood cells (WBCs) remain intact and are used as a reference for quantification.

Fig.2: Rings of P. falciparum in a thick blood smear. Note the absence of red blood cells. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

✅ Thin Film

Used for species identification (speciation).
RBCs remain intact, enabling observation of parasite morphology and cell deformation.
Provides more structural detail of both parasites and infected RBCs.

Fig.4: Rings of P. falciparum in a thin blood smear. Note the presence of red blood cells. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

3. Preparing Blood Films

Blood Collection:

Capillary (finger-prick) or venous blood may be used.
Thick film: ~6 μL blood — smear should be semi-transparent.
Thin film: ~2 μL blood — allows for accurate speciation.

Fixing Thin Films:

Dip or spray with methanol for 1 second to “glue” cells to the slide.
Let dry flat; avoid methanol pooling toward thick film.

Staining:

Use Giemsa stain—a mix of methylene blue (basic) and eosin (acidic).

10% solution for rapid diagnostic work (stain for 10 min.).
3% for higher quality, research-level slides (30–45 min staining).

Slides should be stained within 72 hours to prevent artefacts.
Use buffer at pH 7.2 to balance the stain.

4. Preventing Artefacts

Use clean spreaders for each slide to avoid cross-contamination.
Avoid using hemolysed samples.
Use EDTA blood within 24–48 hours to preserve parasite morphology.

5. Identifying Plasmodium Species

Correct species identification is critical. Here's how to distinguish some common types:

P. falciparum

Infects mature RBCs (cells don’t enlarge).
Often shows double infection.
Features:

Multiple chromatin dots
Nipple-like protrusions
Ring and comma shapes
Gametocyte is banana shaped
Maurer’s dots

Fig.5: P. falciparum trophozoites in a thin blood smear, where Maurer’s clefts are seen. Maurer’s clefts are membranous, disc-shaped structures seen in Plasmodium falciparum-infected red blood cells (iRBCs), particularly during the trophozoite and schizont stages. They are parasite-induced membranous organelles within the host erythrocyte cytoplasm and are critical for trafficking virulence proteins, notably PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1), to the red cell surface. They appear as fine, stippled dots or delicate granulations in the cytoplasm of P. falciparum-infected RBCs. Maurer's clefts are best visualized via transmission electron microscopy (TEM). Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.6: P. falciparum gametocytes in a thin blood smear. Ring-form trophozoites are seen, as well as Maurer's clefts. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.7: A schizont of P. falciparum in a thin smear. Note the presence of trophozoites in the bottom right and top left of the image. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

P. malariae

Characteristic band form, basket form, or bird’s eye form.
Often seen in schizont stage in thick film (6–12 merozoites).
Easier to identify via ring stage in thin film.

Fig.8: Characteristic Bird’s eye trophozoite of P. malariae in a thin smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.9: A ring-form trophozoite in P. malaria as seen on a thin smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.10: Upper left - a developing P. malariae trophozoite. Lower right, a ring-form trophozoite of P. malariae as seen on a thick smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.11: A trophozoite of P. malariae in a thick blood smear, surrounded by three white blood cells (large purple-stained round cells). Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.12: A characteristic band-form trophozoite of P. malariae in a thin smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.13: Another band-form trophozoite of P. malariae in a thin blood smear, to deepen the impression. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.14: A third band-form of P. malariae with slight variation in the orientation of the band. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.15: A basket-form P. malariae trophozoite stage in a thin blood smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.16: A gametocyte developmental stage of P. malariae in a thick smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.17: A gametocyte developmental stage of P. malariae in a thin smear for comparison. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.13: Another image of P. malariae gametocyte in a thin smear to deepen the impression. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.14: A schizont of P. malariae in a thick smear. The merozoites (Usually 8-10) in the schizont form a rosette shape. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.15: Another P. malariae schizont for comparison. This time, it’s a thin smear. I feel like it looks a bit like the thin smear gametocyte stage. Pay attention to the individual clusters of cells to avoid confusion. I notice that there are a lot more merozoites visible in the thin smear prep. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

P. ovale 🐣

Infected cells become oval and display spiky edges (fimbriation).
Displays a red zone appearance in thick films.
Prefers reticulocytes (young RBCs).

Fig.16: Plasmodium ovale ring-form trophozoite parasite as seen on a thick smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.17: A close-up of an ovale ring-form trophozoite next to a white blood cell, in a thick smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.18: P. ovale can have multiple ring-form trophozoites in a single red blood cell (right hand cell). This can be viewed via a thin blood smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.19: Another image of ovale ring-form trophozoite. Here, it reminds of the P. falciparum ring stage, with the exception that the cytoplasm is better defined? What do you think? I would love to know in the comments.

Fig.19: I couldn’t resist showing this P. ovale ring stage trophozoites with the multiply infected red blood cell. In two (right hand side) of the three infected red blood cells, you can see the pinched or jagged edges (fimbriation) seen in ovale infections. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.20: A characteristic fimbriation appearance of a P. ovale infected red blood in a thin smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig.21: Another fimbriation example in P. ovale infected red blood cell. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 22: P. ovale development in a thick smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 23: P. ovale schizont morphology in another thick smear. The schizonts of P. ovale tend to be smaller relative to the other species discussed so far. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 24: P. ovale schizont development in a thin smear. Schizonts of P. ovale tend to be smaller relative to the other species discussed above. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 25: P. ovale schizont stage of development in a thin smear. The schizonts of P. ovale tend to be smaller relative to the other species discussed so far. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 26: The gametocytes of P. ovale are large, similar to P. malariae. However, with the fimbriation, it helps to distinguish it from the P. malariae species. This form is noted to be a macrogametocyte in a thin smear. See below for a microgametocyte. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 27: A microgametocyte of P. ovale in a thin smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 28: A thick smear of P. ovale for comparison. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 29: A thick smear image of P. ovale for comparison. The arrow in the image indicates a gametocyte. I think it looks surprisingly like a schizont Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

P. vivax 🍼

Enlarged, ameboid-shaped cells.
Schizonts are large and visible in thin film.
Gametocytes are round, unlike falciparum's banana-shape.
Also infects reticulocytes, leading to cell enlargement.

Fig. 30: P. vivax ring-form trophozoites in a thick smear. I seriously doubt that I would know that these are vivax if I saw it in a smear. What about you? Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 31: Additional P. vivax ring-form trophozoites in a thick smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 32: An example of P. vivax ring-form trophozoites in a thin smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 33: Another example of P. vivax ring-form trophozoites in thin smear. This image has more ring-forms in view, than the previous image. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 34: The amoeboid appearance of P. vivax trophozoites in a thick smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 35: The amoeboid appearance of P. vivax trophozoites in a thin smear. Image credit: CDC (https://www.cdc.gov/dpdx/malaria/index.html)

Fig. 36: Plasmodium vivax schizonts in a thin blood smear. On the right is the mature form, and on the left, the immature form. Image credit: Dr. mae Melvin/CDC

Note: P. ovale and P. vivax are not typically found in Ghana, more common in Southeast Asia.

6. Counting Malaria Parasites

Quantifying parasitemia helps determine disease severity and treatment efficacy.

Thick Film Counting

Count parasites relative to 200 WBCs.
Use two tally counters: one for parasites, one for WBCs.
If <100 parasites found after 200 WBCs, extend count to 500 WBCs.
Use estimated WBC count: 8000 WBCs/μL (unless actual count available).

Formula (IN THICK FILM):

If more than 100 Parasites counted, = parasites counted / 200 WBCs X 8000 WBC

If less than 100 WBCs parasites counted (count more than 200 WBCs until you get 500 WBCs), use Parasites counted / 500 WBCs X 8000 WBCs

Thin Film Counting

Count 2000 total RBCs and number of parasite infected cells.

Formula:

Parasites infected RBCs/ 2000 total RBC x 5,000,000 (RBC per ul of blood).

Use thin films to quantitate when thick films show >100 parasites per field.

Before declaring a slide as negative, you must read at least 100 fields!

7. Parasite Culture Monitoring (Lab-based)

When culturing parasites:

Count at least 250 RBCs per field, minimum 1000 RBCs total.
Use results to determine percent parasitemia.

Conclusion

Microscopy offers a robust, low-cost, and effective way to diagnose malaria and monitor treatment success. Mastery of smear preparation, staining, and species identification is essential for accurate diagnosis and public health monitoring.

Need a refresher to deepen the impression? Watch the video here:

Bibliography

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June 17, 2025 Tags : blood tests , malaria diagnosis , microscopy , Plasmodium identification , thick and thin blood smears

Tuesday, June 17, 2025

Spotting Malaria: A Step-by-Step Guide to Microscopy-Based Diagnosis

1. Understanding Blood Stages of Malaria