Giemsa Stain: Principle, Procedure, Results

Giemsa stain is a type of Romanowsky stain named after Gustav Giemsa, a German chemist who created a dye solution. It was primarily designed for the demonstration of malarial parasites in blood smears, but it is also employed in histology for routine examination of blood smears.

Principle of Giemsa Stain

Giemsa stain is a differential stain and contains a mixture of azure, methylene blue, and eosin dye. It is specific for the phosphate groups of DNA and attaches itself to where there are high amounts of adenine-thymine bonding.

Azure and eosin are acidic dye that variably stains the basic components of the cells like the cytoplasm, granules, etc.

Methylene blue acts as the basic dye, which stains the acidic components, especially the nucleus of the cell.

Methanol act as a fixative as well as a cellular stain. The fixative does not allow a further change in the cells and makes them adhere to the glass slide.

Preparation of Giemsa Stain

Giemsa is the most commonly used stain for staining blood films for malaria diagnosis. It is available commercially as a ready-to-use product, but the quality varies according to the source. By following simple rules, laboratories can prepare a stock solution of Giemsa stain using Giemsa stain powder, thus ensuring the use of consistent, high-quality stain.

Composition

The essential ingredients of Giemsa stain are the same; however, dilutions can be made depending on their use.

Ingredients	Gm/L
Giemsa powder	7.6
Glycerol	500 ml
Methanol	500 ml

Supplies, Materials, and equipment  

1. Giemsa powder or stain, 7.6 g (preferably Biological Stain Commission grade, to ensure a very good product of standard quality;
2. absolute methanol, pure, high-grade, acetone-free, 500 mL;
3. glycerol, high-grade, pure, 500 mL;
4. methanol-cleaned solid glass beads, 3-5 mm in diameter, 50-100 pieces;
5. a spatula or measuring spoon;
6. weighing paper;
7. a graduated cylinder;
8. a glass or plastic funnel;
9. a screw-capped, dark or amber glass bottle, clean and dry, 500-ml capacity (If not available, a chemically clean, dry, clear hard glass or polyethylene bottle of suitable size may be used, but should be wrapped in dark paper);
10. an analytical balance capable of weighing to 0.01 g; and
11. a shaker, if available.

Note:

• The person preparing the Giemsa stain should follow universal precautions, including the use of relevant personal protective equipment (PPEs) such as gloves, safety glasses, and a laboratory gown.
• Avoid contact and inhalation of methanol and Giemsa stain. Methanol and Giemsa stain are inflammable and highly toxic if inhaled or swallowed. Keep both chemicals in a locked cabinet or cupboard when they are not in use.

Preparation of Giemsa Stock Solution

Working Solution of Giemsa Stain

Working solution of Giemsa stain should be freshly prepared from Giemsa stock solution. Depending upon the method of staining used to stain malaria blood films, the Giemsa working solution is either 10% (for the rapid method) or 3% (for the slow method).

A rapid method is used in outpatient clinics and busy laboratories where a quick diagnosis is essential for patient management, whereas a slow method is used for staining a large number of slides collected during epidemiological or field.

Rapid (10% working solution) method

1. Commonest method for staining 1-15 slides at a time.
2. Used in outpatient clinics and busy laboratories
3. Efficient method but costly (as more stain is consumed)

Slow (3% working solution) method

1. Used for staining a larger number of slides (>20)
2. Ideal for staining blood films collected during cross-sectional or epidemiological surveys, field research, or for preparing batches of slides for teaching
3. Time-consuming method, so less appropriate when a quick result is needed
4. Less expensive compared to the rapid method as it requires much less stain.

Materials and Supplies

• Giemsa stain, transferred and filtered from the stock solution into a 25-or 50-ml bottle;
• buffered water, pH 7.2;
• a beaker or tube, clean, 5-10-ml capacity;
• a Pasteur pipette and
• Whatman filter paper, grade #1.

Preparation of Giemsa Working Solution

Prepare either 10% or 3% Giemsa working solution, depending on your need. About 3 mL of stain is required for each slide with a blood film.

1. Place 90 mL of prepared buffered water, pH 7.2, into a clean beaker or tube.
2. Filter the Giemsa stock solution through paper Whatman #1 and transfer it to a 25 to 50 mL container.
3. Add 10 mL of Giemsa stock solution using a clean, dry pipette. Do not take the aliquot from the large bottle containing the Giemsa stock solution to avoid contaminating it.
4. Prepare the Giemsa working solution just before staining the blood film(s), and use it within 15 minutes of preparation. Discard any unused stain.

To prepare 3% Giemsa working solution, follow the procedure mentioned above, but mix 97 mL of buffered water with 3 mL of Giemsa stock solution.

Staining of the Slides

For Thin blood smears

1. Fix air-dried film in absolute methanol by dipping the film briefly (two dips) in a Coplin jar containing absolute methanol.
2. Remove and let air dry.
3. Stain with a working solution of Giemsa stain
4. Wash by briefly dipping the slide in and out of a Coplin jar of buffered water (one or two dips).
   Note: Excessive washing will decolorize the film.
5. Let air dry in a vertical position. Observe under the microscope first at 40X and then using an oil immersion lens

For Thick blood smears

1. Allow the film to air dry thoroughly for several hours or overnight. Do not dry films in an incubator or by heat, because this will fix the blood and interfere with the lysing of the RBCs.
   Note: If a rapid diagnosis of malaria is needed, thick films can be made slightly thinner than usual, allowed to dry for 1 hour, and then stained.
2. DO NOT FIX.
3. Stain with diluted Giemsa stain
4. Wash by placing the film in buffered water for 3 to 5 min.
5. Let air dry in a vertical position, observe under the microscope at 40X, and then use an oil immersion lens.

For Chlamydia trachomatis

Follow the aforementioned steps with the dilute stain of 1:40 dilution (add 0.5 ml stock Giemsa solution to 19.5 ml buffered water) and leave the stain for 90-120 minutes.

Observation

On microscopic observation, cell organelles, bacteria, and parasites are distinguished based on their morphology and color;

Cell Components	The color observed after staining
Red blood cells	Mauve-pink
Neutrophils	Reddish purple nuclei with pink cytoplasm
Eosinophils	Purple nuclei, faintly pink cytoplasm, and red to orange granules.
Basophils	Purple nuclei, blue coarse granules.
Lymphocytes	Dark blue nucleus with light blue cytoplasm.
Monocytes	Pink cytoplasm with a purple color nucleus.
Platelets	Violet to purple color granules.
Nuclei of host cells	Dark purple
Nuclei of WBCs	Dark purple
The cytoplasm of host cells	Pale blue
The cytoplasm of white cells	Pale blue or grey-blue
Melanin granules	Black green
Bacteria	Pale or dark blue
Chlamydia trachomatis inclusion bodies	Blue-mauve to dark purple depending on the stage of development
Borrelia spirochetes	Mauve-purple
Yersinina pestis coccobacilli	Blue with dark stained ends (bipolar staining)
Malaria parasite	Malaria parasites have a red or pink nucleus and blue cytoplasm. If P. vivax is seen, the Schüffner dots are seen as an even carpet of pink dots in the cytoplasm of red blood cells. If P. falciparum is observed, Maurer clefts will be seen as unevenly distributed, coarse bodies in the red cell cytoplasm.

Uses of Giemsa Stain

Wright-Giemsa’s stain is commonly used to demonstrate the cellular elements in peripheral blood and bone marrow smears. Giemsa stain is used to obtain differential white blood cell counts. It is also used to differentiate the nuclear and cytoplasmic morphology of the various blood cells like platelets, RBCs, and WBCs.

In Microbiology, Giemsa stain is used for staining inclusion bodies in Chlamydia trachomatis, Borrelia species, and if Wayson’s stain is not available, to stain Yersinia pestis. Giemsa stain also is used to stain Histoplasma capsulatum, Pneumocystis jiroveci, Klebsiella granulomatis, Talaromyces marneffei (formerly called Penicillium marneffei), and occasionally bacterial capsules.

Cytogenetics also uses this stain to stain the chromosomes and identify chromosomal aberrations. It is commonly used for G-banding (Giemsa-Banding)

Parasitology

In microbiology, this stain is most commonly used in parasitology to detect intraerythrocytic (plasmodia, babesiae) and exoerythrocytic (trypanosomes, microfilaria) parasites. It is also used for the detection of intracellular amastigotes of Leishmania species or Trypanosoma cruzi.

Photomicrograph of a Wright-Giemsa-stained peripheral blood smear illustrating several stages of Plasmodium species. 

Giemsa stain is also used for the laboratory diagnosis of Toxoplasmosis. Tachyzoites of Toxoplasma gondii are best seen in needle aspirates, or impression smears stained with Wright-Giemsa. In Giemsa-stained smears characteristics, bow-shaped or crescent-shaped tachyzoites with the central dark-staining nucleus are seen.

The Wright-Giemsa-stained impression smear illustrates a few background macrophages and numerous tiny 2 to 3 amastigotes of Leishmania. These forms are often difficult to differentiate from the yeast cells of Histoplasma capsulatum. Careful observation, however, will reveal that many of these forms have a small, rod-shaped kinetoplast, characteristics of Leishmania amastigotes.

Bacteriology

Wright-Giemsa stain has little use for staining bacteria, but it can be used for the laboratory diagnosis of various obligate intracellular parasites. 

The diagnosis of Chlamydia trachomatis infection can be made if large numbers of chlamydial inclusion bodies are seen in a sample stained by the Giemsa or Gimenez methods.

The laboratory diagnosis of granuloma inguinale relies on the staining of intracellular bacteria in mononuclear cells and observation of “Donovan bodies” in tissue smears or biopsy specimens examined by Giemsa and Wright stains.

Wright-Giemsa stains of peripheral blood smears of people suffering from bubonic plague reveal the characteristics of bipolar staining typical of Yersinia. Note: bipolar staining “closed safety pin” shaped cells.

In people suffering from Carrion’s disease, Bartonella bacilliformis can be seen in the tissues both intra-and extracellularly. On Giemsa-stained blood films, the organism appears blue-to-purple extraerythrocytic and intraerythrocytic bacilli and coccobacilli.

Mycology

Detect the intracellular yeast forms of Histoplasma capsulatum.

Virology

The stain is also helpful for demonstrating specific intracellular viral inclusions. Herpes simplex virus produces multinucleated giant cells with intranuclear inclusions, which can be visualized after staining with Wright’s stain (or Wright-Giemsa stain).

References

1. Stockert, J. C., Blázquez-Castro, A., & Horobin, R. W. (2014). Identifying different types of chromatin using Giemsa staining. Methods in molecular biology (Clifton, N.J.), 1094, 25–38. https://doi.org/10.1007/978-1-62703-706-8_3 
2. Barcia J. J. (2007). The Giemsa stain: its history and applications. International journal of surgical pathology, 15(3), 292–296. https://doi.org/10.1177/1066896907302239