Giemsa staining is a technique used in microbiology and cytogenetics to stain cells and tissues, allowing the visualization of specific structures. Developed by Gustav Giemsa in 1904, Giemsa staining is one of the most widely used techniques in diagnostic laboratories to identify microorganisms, parasites, and chromosomes.
Giemsa staining requires materials such as Giemsa stain, methanol, and distilled water. The technique involves fixing cells to glass slides, followed by the application of Giemsa stain diluted in methanol. After staining, the cells are washed in distilled water and observed under a microscope.
The main uses of Giemsa staining include the identification of pathogens such as Plasmodium (which causes malaria), Trypanosoma (which causes Chagas disease), and bacteria such as Chlamydia and Rickettsia. Furthermore, Giemsa staining is widely used in chromosome analysis to diagnose genetic diseases and identify chromosomal abnormalities.
Step by step guide to effectively perform Giemsa staining.
Giemsa staining is an essential technique in clinical analysis and scientific research laboratories. It allows the visualization of various cellular structures, such as chromosomes, parasites, and bacteria, through the specific staining of their components. In this article, we'll explain step-by-step how to perform Giemsa staining effectively.
Step 1: Prepare a Giemsa solution, which can be purchased ready-made or prepared from powder. The solution should be diluted in distilled water at the manufacturer's recommended ratio.
Step 2: Fix the sample to a glass slide using heat or fixatives, such as ethyl alcohol. Ensure the sample is securely fixed to avoid distortion during staining.
Step 3: Cover the fixed specimen with Giemsa solution, ensuring that the entire surface is evenly covered. Leave the slide in contact with the solution for a specific period of time, usually between 10 and 30 minutes.
Step 4: Rinse the slide under running water to remove excess stain. Gently dry the slide with absorbent paper or compressed air.
Step 5: Observe the stained sample under a light microscope using appropriate magnifying objectives. Giemsa staining will allow visualization of cellular structures with greater clarity and contrast.
Giemsa staining is widely used in various fields of biology and medicine, such as diagnosing parasitic diseases, analyzing blood cells, and identifying infectious agents. By following the steps described above correctly, accurate and reliable Giemsa staining results can be obtained.
Understand the Giemsa staining process and how it works in detail.
Giemsa staining is a method widely used in microbiology and hematology laboratories for visualizing cellular structures and detecting pathogens. Developed by Gustav Giemsa in 1902, this method is based on the affinity of basic and acidic dyes for different cellular components.
The materials needed for Giemsa staining include Giemsa stain, fixation and destaining solutions, slides, coverslips, and a microscope. Giemsa stain is a mixture of methylene blue, eosin, and glycerin and is used to stain the nuclear and cytoplasmic structures of cells.
The Giemsa staining technique involves fixing cells to a slide, followed by application of Giemsa stain for a specified period of time. After staining, the cells are washed with distilled water and decolorized to remove excess stain. Finally, the slides are dried and observed under a microscope.
The main uses of Giemsa staining include the identification of blood-borne parasites, such as Plasmodium spp., and the differentiation of blood cells in peripheral blood smears. Furthermore, this method is widely used in the identification of bacteria, viruses, and other pathogens in clinical samples.
Its operation is based on the affinity of basic and acidic dyes with different cellular components, providing clear and detailed images under the microscope.
Understand the pathological anatomy procedure with Giemsa staining for clinical analysis.
Giemsa staining is a technique used in anatomical pathology for the clinical analysis of tissue samples. Developed by German scientist Gustav Giemsa, this stain is widely used due to its ability to highlight various cellular components, such as nuclei, chromosomes, and parasites.
Giemsa staining requires a number of materials, including Giemsa stain, methylated spirit, saline solution, and glass slides. The procedure involves fixing the sample in methylated spirit, applying Giemsa stain, and washing with saline solution. After staining, the samples are observed under a microscope for analysis.
Giemsa staining is widely used in anatomic pathology laboratories to identify various pathologies, such as parasitic infections, leukemia, and autoimmune diseases. The technique allows for detailed analysis of cellular structures, facilitating clinical diagnosis and treatment monitoring.
Its use is essential for the diagnosis and treatment of various diseases, making it an indispensable technique for healthcare professionals.
Identification of the dye used to stain a blood smear.
Giemsa staining is a common method used to observe different types of blood cells in a blood smear. The main dye used in this process is Giemsa stain, which is a mixture of methylene blue, eosin, and azure B. This dye is especially effective in staining structures such as cell nuclei, chromosomes, and cytoplasmic inclusions.
Giemsa stain: foundation, materials, technique and uses
O Giemsa stain is a type of staining for clinical samples, based on a mixture of acidic and basic dyes. Its creation was inspired by the work of Romanowsky, where Gustav Giemsa, a German chemist and bacteriologist, perfected it by adding glycerol to stabilize the compounds.
The changes made to Romanowsky's original technique allowed us to considerably improve microscopic observations; therefore, the technique was named Giemsa staining.
Because it is a simple, highly functional and economical technique, 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 cellular structures. This technique stains the cytoplasms, nuclei, nucleoli, vacuoles and granules of cells, being able to distinguish fine traces of chromatin.
Furthermore, significant changes in the size, shape or color of the nucleus can be detected, where it is possible to visualize the loss of the nucleus-cytoplasm relationship.
On the other hand, it allows the identification of immature cells in the bone marrow and peripheral blood, which is important for the diagnosis of serious diseases such as leukemia. It also allows the detection of hemoparasites, extracellular and intracellular bacteria, fungi, and other pathogens.
In cytogenetics, it is widely used, as it is possible to study cell mitosis.
Giemsa stain foundation
Romanowsky-type stains use a contrast between acidic and basic dyes to stain basic and acidic structures, respectively. As can be seen, acidic dyes have an affinity for staining basic structures and vice versa.
The basic dye used is methylene blue and its oxidized derivatives (Azure A and Azure B), while the acidic dye is eosin.
The acidic structures of the cells are nucleic acids, segmented basophil granules, among others, so they are stained with methylene blue.
In the 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, since methylene blue and sky blue are characterized as metachromatic dyes, they can provide a variable tone to different structures according to the polyanion charge they possess.
This is how the strategic combination of basic and acidic dyes can develop a wide spectrum of colors, according to the biochemical characteristics of each structure, ranging from shades of pale blue, dark blue, lilac and purple in the case of acidic structures.
While the color provided by eosin is more stable, it generates colors between reddish orange and salmon.
Materials
Materials for preparing the stock solution
Preparation of the stock solution requires weighing 600 mg of Giemsa powder stain, measuring 500 ml of acetone-free methyl alcohol, and 50 ml of neutral glycerin.
Method of preparing the stock solution
Place the weighed Giemsa powder in a mortar. If there are any lumps, they should be pulverized. Then, add a sizable amount of the measured glycerin and mix thoroughly. Pour the resulting mixture into a very clean amber bottle.
The remaining glycerin is added to the mortar. Mix again to remove any remaining dye that has stuck to the mortar walls and then pour it into the same bottle.
The bottle is covered and transported for 2 hours in a water bath at 55 ° C. While in the water bath, stir the mixture lightly every half hour.
The mixture is then allowed to cool before adding the alcohol. A portion of the measured alcohol is first added to the mortar to finish washing away any remaining dye, then added to the mixture along with the remaining alcohol.
This preparation should be left to mature for at least 2 weeks. The portion used in the mother liquor 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 each time the reagent is depleted.
Materials for preparing the buffer solution
On the other hand, a buffer solution at pH 7,2 is prepared as follows:
6,77 g sodium phosphate (anhydrous) (weighed NaHPO 4 ), 2,59 g of potassium dihydrogen phosphate (KH 2 PO 4 ), and distilled water up to 1000 ml.
Final dye preparation
To prepare the final staining solution, measure 2 ml of the filtered stock solution and mix it with 6 ml of the buffer solution. The mixture is shaken.
One relevant fact that must be taken into consideration is that dye preparation techniques may change depending on the commercial installation.
Additional materials needed to make the coloring
In addition to the materials described, there must be colored bridges, t-shirts with water or a tampon for washing clothes, sheets with objects or covers, a timer to control the coloring time and blotting the paper or some material that serves to dry (gauze or cotton).
Cooperation
Coloring process
1) Before staining, the sample must be spread on a clean slide.
Samples can be blood, bone marrow, histological tissue sections, or cervical-vaginal samples. It is recommended that the pastes be thin and allowed to dry for 1 to 2 hours before staining.
2) On a coloring bridge, all the colored leaves are placed. It always works in the same order, and each leaf is clearly labeled.
3) Place a few drops of 100% methyl alcohol (methanol) on the smear and leave for 3 to 5 minutes to fix and dehydrate the sample.
4) Discard the methanol present on the leaf and let it air dry.
5) Once dry, add the final staining solution with a dropper until the entire leaf is covered. Leave it on for 15 minutes. Some authors recommend up to 25 minutes. It depends on the store.
6) Drain the stain and wash the smear with distilled water or a buffer solution in 7.2.
7) On absorbent paper, let the leaves air dry, arranged vertically with the help of a support.
8) Wipe the back of the slide with a gauze pad or cotton swab dipped in alcohol to remove any dye.
Utilities
The Giemsa staining technique is used in several areas, including: hematology, mycology, bacteriology, parasitology, cytology and cytogenetics.
Hematology
This is the most common use for this stain. With it, each and every cell present in the bone marrow or peripheral blood samples can be identified. In addition to estimating the number of cells in each series, it can detect leukocytosis or leukopenia, thrombocytopenia, etc.
Because it's sensitive to identifying immature cells, it's useful in diagnosing acute or chronic leukemia. It can also diagnose anemias such as sickle cell disease, among others.
Mycology
In this area, it is common to use Histoplasma capsulatum (intracellular dimorphic fungus) in tissue samples.
Bacteriology
In hematologic smears stained with Giemsa, it is possible to detect Borrelias sp in patients with a disease called relapsing fever. Spirochetes are abundantly observed among erythrocytes in samples collected at the peak of the fever.
It is also possible to visualize intracellular bacteria as Rickettsia sp e Chlamydia trachomatis in infected cells.
parasitology
In the field of parasitology, Giemsa staining has allowed the diagnosis of parasitic diseases such as malaria, Chagas disease and leishmaniasis.
In the first two parasites, Plasmodium sp e trypanosoma cruzi, respectively, can be seen in the peripheral blood of infected patients and can be found in various stages depending on the stage of the disease.
To improve the search for parasites in the blood, it is recommended to use Giemsa stain mixed with May-Grünwald stain.
Similarly, 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 widely used technique for this purpose.
However, in cases of resource scarcity, it can be used, offering similar functionality to the Pap smear and at a lower cost. However, it requires expertise from the examiner.
cytogenetics
A key feature of Giemsa staining is its ability to bind strongly to regions of DNA rich in adenine and thymine. 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 different regions of the chromosomes.
Research demonstrating the effectiveness of Giemsa staining
Cannova et al (2016) compared three staining techniques for the diagnosis of cutaneous leishmaniasis.
For this purpose, samples obtained from an experimental animal were used ( Mesocrisetus auratus) experimentally inoculated with Leishmanias.
The authors demonstrated that Giemsa staining was superior to Pap-mart® and Gaffney staining. Therefore, they considered Giemsa staining 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 has the necessary conditions to create a favorable contrast, making it possible to clearly distinguish the structures of the amastigotes, both intra- and extracellularly.
The other techniques (Pap-mart® and Gaffney) also did this, but in a weaker way and therefore more difficult to visualize. This is why Giemsa staining is recommended for the parasitological diagnosis of leishmaniasis.
Similarly, a study by Ramírez et al (1994) evaluated the validity of Giemsa and Lendrum stains in conjunctival smears for the identification of Chlamydiatrachomatis.
The authors determined that Giemsa and Ledrum stains have the same specificity, but Giemsa proved to be more sensitive.
This explains why Giemsa staining is currently the most frequently used for the diagnosis of chlamydial infections, especially in resource-poor settings.
Recommendations for good coloring
The leaves should not be dried too quickly. You should wait for the appropriate amount of time to air dry them. Approximately 2 hours.
Color immediately after 2 hours for best results.
For the stains to set and blend better, the sample should be spread across the sheet in a thin, even layer.
The preferred blood sample is capillary, as the smear is taken 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 the latter usually deforms the cells.
Common mistakes in Giemsa staining
When using this coloring technique, mistakes can be made. These are evidenced by sudden changes in the structure's hue.
Extremely blue coloration
It could be due to:
- Very thick stains
- Exceeding the coloring time
- Wash insufficiently.
- Use of reagents well above neutral (alkaline) pH.
Under these conditions, the colors of the following structures are distorted, so that erythrocytes, instead of dying salmon-pink, will turn green, eosinophil granules, which should be dyed brick-red, will turn bluish or gray, and so on. deviation from the usual shades.
Excessively pink coloration
It could be due to:
- Insufficient staining time.
- Prolonged or excessive washing.
- Not very dry
- Use of very acidic reagents.
In this specific case, structures that are normally tinted blue will be barely visible, while structures that are tinted pink will have very exaggerated hues.
Example: Red blood cells will appear bright red or deep orange, nuclear chromatin will appear pale pink, and eosinophil granules will stain bright red.
Presence of precipitates in the smear
The causes may be:
- Use dirty or poorly washed sheets.
- Do not let the stain dry well.
- Leave the fixing solution for a long time.
- Improper washing at the end of coloring.
- Inadequate filtration or no filtration of the dye being used.
Presence of morphological artifacts
Morphological artifacts may appear in the stains, making it difficult to visualize and interpret the structures. This is because:
- Type of anticoagulant used, such as heparin.
- Use of dirty, damaged or oily leaves.
Storage mode
After preparation, the dye should be kept at room temperature (15-25°C) to prevent dye precipitation. It should be stored in a tightly closed amber container.
References
- Cannova D, Brito E and Simons M. Evaluation of staining techniques for the diagnosis of cutaneous leishmaniasis. Salus . 2016; 20 (2): 24-29.
- PanReac Applichem Reagents ITW. Giemsa staining. Version 2: JMBJUL17 CEIVD10ES. Castellar del Vallés, Spain.
- Clark G. Staining procedures (1981), 4th. Williams & Willkins.
- Applied Clinical Chemistry. Giemsa Stain for Diagnostics vitro . Distributor: cromakit.es
- 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 Chlamydiatrachomatis. Sanit Panam Bowl. 1994; 116(3): 212-216.
- Casas-Rincón G. General Mycology. 1994. 2nd Ed. Central University of Venezuela, Library Editions. Venezuela, Caracas
- «Giemsa staining.» Wikipedia, The Free Encyclopedia . September 1, 2017, at 01:02 UTC. December 6, 2018, en.wikipedia.org.