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

  1. Place about 100 methanol-cleaned glass beads into a dark or amber bottle.

  2. Weigh 7.6 g of Giemsa stain powder on an analytical balance, and pour it into the bottle containing the beads through a funnel.

  3. Gently pour about 200 mL of methanol, ensuring that all dry stain is washed into the bottle.

  4. Tighten the screw cap on the bottle and shake it in a circular motion for 2-3 minutes to start dissolving the stain crystals.

  5. Add 500 mL glycerol to the mixture through the funnel, and shake again for 3-5 minutes.

  6. Add the remaining 300 mL of methanol to the mixture through the funnel, ensuring that the last of the methanol washes the last of the glycerol from the funnel into the stain mixture.

  7. Tighten the screw-cap on the bottle.

    The bottle should be tightly capped at all times to prevent absorption of water vapor and to avoid evaporation and oxidation of the stain by high humidity. If the bottle is tightly stoppered and free of moisture, the Giemsa stain is stable at room temperature for longer.

  8. About six times on the first day, continue shaking for 2-3 minutes each.

  9. Shake for at least seven days every day for 2-3 minutes, about six times each. A shaker may be used, if available.

  10. Label the bottle clearly with the batch number, the name of the person who prepared the stock, date of preparation and date of expiry, and document in the quality control log-book.

    Giemsa stock solution
    Batch No.: 2022-01 Prepared by: First name Last name
    Date prepared: 17 Aug 2022
    Expiry date: 17 Aug 2024
    #2022-01 indicates the year prepared and the stock number.

  11. Tighten the screw-cap on the bottle to prevent absorption of water vapor from the air, and store it in a cool place away from direct sunlight.

    Do NOT contaminate the stock Giemsa solution with water; even the smallest amount of water will cause the stain to deteriorate, making staining progressively ineffective. Store in a dark glass bottle in a cool, dry, shady place, away from direct sunlight. If a clear stock bottle is used, wrap it in thick dark paper to avoid light penetration.

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. 

Tachyzoites of Toxoplasma
Tachyzoites

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. 

Giemsa Staining Photograph showing epithelial cells of conjunctiva containing intra-cytoplasmic inclusions “draped” around nucleus (source)

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.

Picture of Giemsa-stained blood smear from a patient with Oroya fever, showing parasitism of all erythrocytes, with bacillary and coccoid forms of B. bacilliformis.
(Courtesy of P. Ventosilla and M. Montes, Universidad Peruana Cayetano Heredia, Lima, Peru)

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.

Histoplasma capsulatum within histiocytes seen on bone marrow biopsy staining: (A) Wright–Giemsa and (B) Giemsa

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).

Nisha Rijal

I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.

4 thoughts on “Giemsa Stain: Principle, Procedure, Results

  1. i have try to prepare the giemsa stock solution as per the SOP which is same as above mention statement.

    but i final, when i try to run the QC, the blood film macroscopically reveal bit dark purple color and the RBCs are bit draker in coluor

    any suggestion

  2. If methylene blue stains nucleus and eosin stains cytoplasm of the cell, Why nucleus of malarial parasite looks pink and cytoplasm blue when staining with giemsa ? Ideally it should be opposite.

  3. please can anybody solve my problem..i have to stain fat fed liver cells by giemsa and i am not able to distinguish the nuclei…can anybody share his procedure of giemsa staining…

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