GIEMSA STAIN: RATIONALE, MATERIALS, TECHNIQUE AND USES - BIOLOGY - 2024

The Giemsa stain is a type of coloring clinical samples, based on the mixture of acid and basic dyes. Its creation was inspired by the work done by Romanowsky, where Gustav Giemsa, a chemist and bacteriologist originally from Germany, perfected it by adding glycerol to stabilize the compounds.

The changes generated to the original Romanowsky technique allowed to considerably improve the microscopic observations, therefore the technique was baptized with the name of Giemsa stain.

Various samples stained with Giemsa stain. A. Trypanosoma evansi in peripheral blood. B. Normal blood cells. C. Borrelia theileri in peripheral blood. D. Burkitt's lymphoma.

Because it is a simple technique to perform, highly functional and economical, it is currently widely used in the clinical laboratory for hematological smears, bone marrow samples and tissue sections.

The Giemsa staining technique is very useful for cytological studies, as it allows the observation of specific cell structures. This technique stains the cytoplasms, nuclei, nucleoli, vacuoles and granules of cells, being able to distinguish even fine traces of chromatin.

In addition, significant changes can be detected in the size, shape or coloration of the nucleus, where it is possible to visualize the loss of the nucleus-cytoplasm relationship.

On the other hand, it allows to identify immature cells in bone marrow and peripheral blood, being important for the diagnosis of serious diseases such as leukemia. It is also possible to detect hemoparasites, extra and intracellular bacteria, fungi, among others.

In cytogenetics it is widely used, since it is possible to study the mitosis of cells.

Basis of Giemsa staining

The Romanowsky type dyes are based on using a contrast between acidic and basic dyes, to achieve staining the basic and acid structures respectively. As can be seen, there is an affinity of acid dyes to stain basic structures and vice versa.

The basic dye used is methylene blue and its oxidized derivatives (Azure A and Azure B), while the acid dye is eosin.

The acid structures of the cells are the nucleic acids, the granules of the segmented basophils, among others, therefore they will be stained with methylene blue.

In this same sense, the basic structures of cells are hemoglobin and some granules such as those contained in segmented eosinophils, among others; these will be stained with eosin.

On the other hand, because methylene blue and azure are characterized by being metachromatic colorants, they can provide a variable hue to the different structures according to the load of polyanions they possess.

This is how the strategic combination of basic and acid dyes manages to develop a wide spectrum of colors, according to the biochemical characteristics of each structure, walking through pale blue, dark blue, lilac and purple hues in the case of acid structures.

While the coloration provided by eosin is more stable, generating colors between reddish-orange and salmon.

materials

Materials for preparing the stock solution

Preparation of the stock solution requires weighing 600 mg of powdered Giemsa stain, measuring 500 cc of acetone-free methyl alcohol and 50 cc of neutral glycerin.

How to prepare the stock solution

Place the heavy Giemsa powder in a mortar. If there are lumps they should be sprayed. Subsequently add an appreciable amount of the measured glycerin and mix very well. The mixture obtained is poured into a very clean amber bottle.

The rest of the glycerin is placed in the mortar. Mix again to clean the rest of the colorant that has stuck to the walls of the mortar and pour into the same jar.

The bottle is capped and placed in a water bath at 55ºC for 2 hours. While it is in a water bath, gently shake the mixture every half hour or so.

Subsequently, the mixture is allowed to cool to place the alcohol. Previously, a part of the measured alcohol is placed in the mortar to finish washing the remaining colorant and then it is added to the mixture along with the rest of the alcohol.

This preparation should be left to mature for at least 2 weeks. The used portion of the stock solution should be filtered.

To avoid contamination of the preparation, it is recommended to transfer the portion that will be in constant use to a small amber bottle with a dropper. Refill every time the reagent runs out.

Materials to prepare the Buffer solution

On the other hand, a buffer solution at pH 7.2 is prepared as follows:

6.77 g of sodium phosphate (anhydrous) (NaHPO ₄), 2.59 g of potassium dihydrogen phosphate (KH ₂ PO ₄) and distilled water are weighed up to 1000 cc.

Final preparation of the colorant

For the preparation of the final staining solution, 2 ml of the filtered stock solution are measured and mixed with 6 ml of the buffer solution. Stir the mixture.

A relevant fact that must be taken into account is that the coloring preparation techniques can change depending on the commercial company.

Additional materials needed to perform the coloring

Apart from the materials described, you must have coloring bridges, t-shirts with water or buffer for washing, object slides or covers for objects, a stopwatch to control the coloring times and blotting paper or some material that can be used to dry (gauze or cotton).

Technique

Staining process

1) Prior to staining, the smear of the sample must be ready on a clean slide.

The samples can be blood, bone marrow, histological tissue sections or cervico-vaginal samples. It is recommended that the spreads be thin and have 1 or 2 hours of drying before coloring.

2) On a coloring bridge, place all the sheets that have to be colored. You always work in the same order and each sheet is well identified.

3) Place a few drops of 100% methyl alcohol (methanol) on the smear and allow it to act for 3 to 5 minutes, in order to fix and dehydrate the sample.

4) Discard the methanol present on the sheet and allow to air dry.

5) Once dry, place the final staining solution with a dropper until the entire sheet is covered. Leave to act for 15 minutes. Some authors recommend up to 25 min. It depends on the business house.

6) Drain the stain and wash the smear with distilled water or with a 7.2 buffer solution.

7) On a blotting paper, let the sheets dry in the open air, arranged vertically with the help of a support.

8) Clean the back of the slide with an alcohol swab or cotton swab to remove any traces of stain.

Utilities

The Giemsa staining technique is used in various areas, including: hematology, mycology, bacteriology, parasitology, cytology and cytogenetics.

Hematology

It is the most frequent use given to this stain. With it, each and every one of the cells present in bone marrow or peripheral blood samples can be identified. As well as estimating the number of each series, being able to detect leukocytosis or leukopenia, thrombocytopenia, etc.

Because it is sensitive in identifying immature cells, it is relevant in the diagnosis of acute or chronic leukemias. It is also possible to make the diagnosis of anemia, such as sickle cell anemia, sickle cell, among others.

Mycology

In this area, its use is common to search for Histoplasma capsulatum (intracellular dimorphic fungus) in tissue samples.

Bacteriology

In hematological smears stained with Giemsa, it is possible to detect Borrelias sp in patients who present with the disease called recurrent fever. Spirochetes are abundant among erythrocytes, in samples taken at the peak of fever.

It is also possible to visualize intracellular bacteria such as Rickettsia sp and Chlamydia trachomatis in infected cells.

parasitology

In the field of parasitology, Giemsa staining has made it possible to diagnose parasitic diseases such as malaria, Chagas disease and leishmaniasis.

In the first two, Plasmodium sp and Trypanosoma cruzi parasites respectively can be seen in the peripheral blood of infected patients, they can be found in different stages depending on the stage of the disease.

To improve the search for parasites in blood, it is recommended to use the Giemsa stain mixed with the May-Grünwald stain.

Likewise, cutaneous leishmaniasis can be diagnosed by evaluating Giemsa-stained skin biopsy samples where the parasite is found.

Cytology

Giemsa staining is also used for the cytological study of endocervical samples, although it is not the most frequently used technique for this purpose.

But in cases of scarcity of resources it can be used, having a similar functionality to that offered by the Papanicolaou technique and at a lower cost. However, it requires expertise on the part of the examiner.

Cytogenetics

A relevant feature of Giemsa staining is its ability to bind strongly to adenine and thymine rich regions of DNA. This allows DNA to be visualized during cell mitosis, in different states of condensation.

These studies are necessary to detect chromatic aberrations such as duplications, deletions or translocations of the different regions of the chromosomes.

Research demonstrating the efficacy of Giemsa stain

Cannova et al (2016), compared 3 staining techniques for the diagnosis of cutaneous leishmaniasis.

For this, they used samples obtained from an experimental animal (Mesocrisetus auratus) experimentally inoculated with Leishmanias.

The authors demonstrated that the Giemsa stain was better than the Pap-mart® and Gaffney stain. Therefore, they considered the Giemsa stain to be ideal for diagnosing cutaneous leishmaniasis.

The excellent results obtained by the authors are due to the fact that the combination of dyes that make up the Giemsa mixture presents the necessary conditions to create a favorable contrast, allowing the structures of amastigotes to be clearly distinguished, both intracellularly and extracellularly.

The other techniques (Pap-mart® and Gaffney) did it too, but in a weaker way and therefore more difficult to visualize. That is why the Giemsa stain is recommended for the parasitological diagnosis of leishmaniasis.

Likewise, a study by Ramírez et al (1994) evaluated the validity of Giemsa and Lendrum stains in conjunctival smears for the identification of Chlamydia trachomatis.

The authors determined that Giemsa and Ledrum stains have equal specificity, but Giemsa was found to be more sensitive.

This explains why Giemsa stain is currently the most frequently used for the diagnosis of chlamydial infections, especially if there are few resources.

Source: PanReac Applichem ITW Reagents. Giemsa stain. Version 2: JMBJUL17 CEIVD10ES. Castellar del Vallés, Spain.

Recommendations for good staining

The drying of the sheets should not be accelerated. A reasonable amount of time must be expected to dry it in the open air. Approximately 2 hours.

Color immediately after 2 hours for best results.

For the smears to fix and stain better, the sample must be distributed on the slide in such a way that a thin and uniform layer remains.

The preferred blood sample is capillary, since the smear is made directly from the drop of blood and therefore the sample does not contain any additives, which favors the maintenance of cellular structures.

However, if venous blood is used, EDTA should be used as an anticoagulant and not heparin, since heparin generally deforms cells.

Common mistakes in Giemsa staining

In the practice of this coloring mistakes can be made. They are evidenced by sudden changes in the tonalities of the structures.

Extremely blue coloring

It may be due to:

• Very thick smears
• Exceeding staining time
• Wash insufficiently.
• Use of reagents well above neutral (alkaline) pH.

Under these conditions the colors of the following structures are distorted, in such a way that the erythrocytes instead of staining salmon-pink will appear green, the granules of the eosinophils that must be stained brick red will turn bluish or gray and so on there will be deviation in the usual tones.

Excessively pink coloring

It may be due to:

• Insufficient staining time.
• Prolonged or excessive washing.
• Bad drying.
• Use of highly acidic reagents.

In this particular case, structures that normally stain blue will not be nearly visible, while structures that stain pink will have greatly exaggerated hues.

Example: Red blood cells will turn bright red or bright orange, nuclear chromatin will appear pale pink, and eosinophil granules will stain deep bright red.

Presence of precipitates in the smear

The causes can be:

• Use dirty or poorly washed films.
• Do not allow the smear to dry well.
• Leaving the fixing solution for too long.
• Inadequate washing at the end of staining.
• Inadequate filtration or no filtration of the colorant being used.

Presence of morphological artifacts

Morphological artifacts may appear in smears, making it difficult to visualize and interpret the structures present. This is due to:

• Type of anticoagulant used, such as heparin.
• Use of dirty, deteriorated or greasy films.

Storage mode

After preparation, the dye must be kept at room temperature (15 - 25 ° C), to prevent the dye from precipitating. It should be stored in a tightly closed amber container.

References

1. Cannova D, Brito E and Simons M. Evaluation of staining techniques for the diagnosis of cutaneous Leishmaniasis. Salus. 2016; 20 (2): 24-29.
2. PanReac Applichem ITW Reagents. Giemsa stain. Version 2: JMBJUL17 CEIVD10ES. Castellar del Vallés, Spain.
3. Clark G. Staining procedures (1981), 4thed. Williams & Willkins.
4. Applied Clinical Chemistry. Giemsa stain for in vitro diagnostics. Distributor: cromakit.es
5. Ramírez I, Mejía M, García de la Riva J, Hermes F and Grazioso C. Validity of Giemsa and Lendrum stains in conjunctival smears for the identification of Chlamydia trachomatis. Bol of Sanit Panam. 1994; 116 (3): 212-216.
6. Casas-Rincón G. General Mycology. 1994. 2nd Ed. Central University of Venezuela, Library Editions. Venezuela Caracas.
7. "Giemsa stain." Wikipedia, The Free Encyclopedia. 1 Sep 2017, 01:02 UTC. Dec 6, 2018, es.wikipedia.org.