Giemsa Stain- Principle, Procedure, Results, Interpretation

Giemsa stain is a type of Romanowsky stain, which is a group of stains that are used to differentiate blood cells and other structures in histology and cytology. Romanowsky stains are composed of a mixture of oxidized methylene blue, azure, and eosin dyes that bind to different components of the cells based on their acidity or basicity.

Giemsa stain is named after Gustav Giemsa, a German chemist and bacteriologist who developed the stain in 1904. He initially used it to demonstrate the presence of malaria parasites in blood smears, but later it was found to be useful for other applications as well.

Giemsa stain is specific for the phosphate groups of DNA and attaches itself to regions of DNA where there are high amounts of adenine-thymine bonding. This makes it suitable for staining chromosomes and identifying chromosomal abnormalities such as translocations and rearrangements.

Giemsa stain is also a differential stain that can be used to study the adherence of pathogenic bacteria to human cells, differentiate various types of blood cells and their granules, and diagnose infections caused by parasites, fungi, and viruses. Some of the organisms that can be detected by Giemsa stain include Plasmodium spp., Trypanosoma spp., Chlamydia trachomatis, Borrelia spp., Yersinia pestis, Histoplasma spp., Pneumocystis jiroveci, and Cytomegalovirus.

In this article, we will discuss the principle, procedure, results, interpretation, and applications of Giemsa stain in detail. We will also highlight the advantages and limitations of this technique.

Romanowsky stains are a group of staining techniques used in hematology, pathology and histology to visualize and differentiate blood cells, tissue structures, and microorganisms. The most common Romanowsky stains include:

• Wright-Giemsa stain: This is one of the most widely used Romanowsky stains. It combines Wright stain and Giemsa stain to produce a purple-blue color for nuclei and granules, and a pink-red color for cytoplasm and erythrocytes. It is used for routine blood smear examination and bone marrow aspirate smears.
• Giemsa stain: This is a special stain used for examination of blood films for parasitic infections and majorly for the diagnosis of malaria. It is also used as a differential stain for various blood cells (erythrocytes, platelets, leucocytes) and cellular components such as the nuclear and the cytoplasm.
• Wright stain: This is a modification of the original Romanowsky stain that uses methylene blue and eosin as the main dyes. It is used for staining blood smears and detecting blood parasites such as trypanosomes and filariae.
• Leishman stain: This is another modification of the original Romanowsky stain that uses methylene blue and eosin as the main dyes. It is used for staining blood smears and detecting blood parasites such as Leishmania, Plasmodium, Babesia, and microfilariae.
• May-Grünwald stain: This is a two-step staining procedure whereby the first staining is done with May-Grünwald stain and a second stain of Giemsa stain which produces the Romanowsky effect (wide range of hue/color)

The principle behind all these stains is the same: they are based on the interaction of acidic and basic dyes with the acidic and basic components of the cells. The acidic dye, eosin, binds to the alkaline cytoplasm and erythrocytes, producing a red-orange coloration. The basic dye, methylene blue or its derivatives (azure A, azure B, azure C), binds to the acidic nuclei and granules, producing a blue-purple coloration. This phenomenon is known as the Romanowsky effect or metachromasia.

The Romanowsky effect was discovered by Dmitri Leonidovich Romanowsky, a Russian physician who first recognized its potential for use as a blood stain in 1891. He used a mixture of eosin and aged solutions of methylene blue that formed hues unattributable to the staining components alone: distinctive shades of purple in the chromatin of the cell nucleus and within granules in the cytoplasm of some white blood cells.

The Romanowsky stains are widely used in clinical and research settings because they provide a simple, rapid, and inexpensive method to identify and differentiate various types of cells and microorganisms. They also allow for the detection of morphological abnormalities, infections, malignancies, and genetic disorders that affect the blood cells.

Giemsa stain is a versatile staining technique that has several objectives and applications in cytology, histology, and parasitology. Some of the main objectives of using Giemsa stain are:

• To accurately prepare the Giemsa stain stock solution and working solution
• To stain and identify blood cells and differentiate their nuclei from the cytoplasm
• To stain and identify blood parasites such as malaria, spirochetes, and other protozoa
• To stain and identify bacteria such as Helicobacter pylori, Chlamydia trachomatis, Borrelia spp., and Yersinia pestis
• To stain and identify fungi such as Histoplasma capsulatum and Cryptococcus neoformans
• To stain and identify viral inclusions such as cytomegalovirus
• To stain and identify mast cells and their granules
• To stain and visualize chromosomes and detect chromosomal aberrations such as translocations and rearrangements

Giemsa stain is a valuable tool for diagnosing various infectious diseases, hematological disorders, genetic abnormalities, and neoplasms. It is also used for research purposes in cytogenetics, immunology, microbiology, and pathology.

Giemsa stain is a type of Romanowsky stain, which is a group of stains that are composed of a mixture of oxidized methylene blue, azure, and eosin. These stains are used to differentiate various cellular components based on their affinity for acidic or basic dyes.

Giemsa stain is specific for the phosphate groups of DNA and attaches itself to regions of DNA where there are high amounts of adenine-thymine bonding. These regions are called AT-rich regions and they tend to be located near the centromeres and telomeres of chromosomes. Giemsa stain can therefore be used to create a karyogram (chromosome map) by producing characteristic bands or patterns on the chromosomes. This technique is called Giemsa banding or G-banding and it can help identify chromosomal abnormalities such as translocations and rearrangements.

Giemsa stain is also a differential stain that can distinguish between different types of blood cells and parasites based on their cytoplasmic and nuclear characteristics. The stain contains both acidic and basic dyes that react with the opposite charges of the cellular components. Azure and methylene blue are basic dyes that bind to the acidic nucleus and other nucleic acid-rich structures, producing a blue-purple color. Eosin is an acidic dye that binds to the basic cytoplasm and cytoplasmic granules, producing a red-orange color. Methanol acts as a fixative as well as a cellular stain that enhances the staining properties of the other dyes.

The staining intensity and coloration of different cells and parasites depend on several factors, such as the pH of the staining solution, the duration of staining, the concentration of the dyes, and the composition of the cells or parasites. For example, erythrocytes stain pink, platelets stain light pale pink, lymphocytes have a dark blue nucleus and a light blue cytoplasm, monocytes have a purple nucleus and a pink cytoplasm, neutrophils have a purple-red nucleus and a pink cytoplasm, eosinophils have a blue-purple nucleus, a pale pink cytoplasm, and orange-red granules, basophils have a purple nucleus and bluish granules, and mast cells have purple granules.

Some parasites that can be detected by Giemsa stain include Plasmodium spp., which cause malaria; Trypanosoma spp., which cause sleeping sickness; Leishmania spp., which cause leishmaniasis; Borrelia spp., which cause Lyme disease; Yersinia pestis, which causes plague; Histoplasma spp., which cause histoplasmosis; Pneumocystis jiroveci, which causes pneumocystis pneumonia; and Chlamydia trachomatis, which causes chlamydia.

The reagents used in the Giemsa stain procedure are:

• Methanol: This is a solvent that is used to fix the specimen on the slide and to dilute the stock solution of Giemsa stain. Methanol also helps to precipitate the dyes on the specimen by lowering the pH of the solution.
• Giemsa powder: This is a mixture of methylene blue, azure, and eosin dyes that are derived from coal tar. These dyes are basic and acidic respectively and bind to different components of the cells and parasites. Methylene blue and azure stain the nuclei and other acidic structures blue-purple, while eosin stains the cytoplasm and other basic structures red-orange.
• Glycerin: This is a viscous liquid that is added to the stock solution of Giemsa stain to prevent evaporation and crystallization of the dyes. Glycerin also enhances the stability and solubility of the dyes in methanol.
• Water (Buffer): This is used to prepare the working solution of Giemsa stain by diluting the stock solution. The water should be buffered to a pH of 6.8 or 7.2 to optimize the staining reaction. The buffer can be phosphate buffer or distilled water with sodium bicarbonate. The buffer also helps to wash off excess stain from the slide after staining.

The Giemsa stain stock solution is a concentrated solution of Giemsa powder, methanol and glycerol that can be stored for a long time and used to prepare the working solution for staining. The stock solution should be prepared with high-quality reagents and filtered before use to ensure optimal staining results. The preparation of the stock solution requires the following steps:

• Place about 50-100 methanol-cleaned glass beads into a dark or amber glass bottle with a screw cap . The glass beads help to dissolve the Giemsa powder and prevent caking.
• Weigh 3.8 g of Giemsa powder (preferably Biological Stain Commission grade) on an analytical balance, and pour it into the bottle containing the beads through a funnel .
• Gently pour about 250 mL of pure, high-grade, acetone-free methanol into the bottle, ensuring that all dry stain is washed into the bottle . Tighten the screw cap on the bottle, and shake it in a circular motion for 2-3 minutes.
• Add 250 mL of high-grade, pure glycerol to the mixture, slowly . Tighten the screw cap on the bottle again, and shake it for 3-5 minutes.
• Heat the solution up to ~60°C for about 1.5-2 hours . Let it cool to room temperature.
• Filter the solution through Whatman filter paper #1 or a similar filter into another clean, dry, dark or amber glass bottle . Label the bottle with the date of preparation and store it in a cool, dark place away from light and heat .
• Let the bottle stand tightly closed and protected from light for one week before use. Alternatively, some sources suggest leaving it to stand for about 1-2 months before use.

The stock solution can be stored for up to one year if kept under proper conditions. However, it is advisable to check its quality periodically by staining a control slide and observing the staining intensity and quality.

The working solution of Giemsa stain is prepared by diluting the stock solution with buffered water (pH 7.2) just before staining the blood films. The working solution should be used within 15 minutes of preparation and discarded after use. The concentration of the working solution depends on the method of staining: rapid (10%) or slow (3%).

To prepare 10% Giemsa working solution, follow these steps:

• Place 90 mL of buffered water into a clean beaker or tube.
• Filter the Giemsa stock solution through Whatman #1 filter paper and transfer it to a 25 to 50 mL container.
• Add 10 mL of the filtered stock solution to the buffered water using a dry pipette and mix well.

To prepare 3% Giemsa working solution, follow these steps:

• Place 97 mL of buffered water into a clean measuring cylinder.
• Filter the Giemsa stock solution through Whatman #1 filter paper and transfer it to a 25 to 50 mL container.
• Add 3 mL of the filtered stock solution to the buffered water using a dry pipette and mix well.

About 3 mL of stain is required for each slide with a blood film.

Thin blood smears are used to identify the species and stage of the malaria parasites, as well as to estimate the parasite density. The procedure for thin film staining is as follows:

• On a clean dry microscopic glass slide, make a thin film of the specimen (blood) by spreading a drop of blood with the edge of another slide or a spreader. The film should be uniform and cover about two-thirds of the slide.
• Leave the slide to air dry completely. Do not heat-fix the slide, as this may distort the morphology of the parasites and cells.
• Dip the slide briefly (2-3 dips) into pure methanol in a Coplin jar or a staining tray. This will fix the smear and prevent it from washing off during staining. Alternatively, you can apply methanol with a cotton swab or a spray bottle. Let the slide air dry for 30 seconds.
• Flood the slide with 5% Giemsa stain solution (prepared by adding 10 ml of stock solution to 80 ml of distilled water and 10 ml of methanol) in a Coplin jar or a staining tray. Make sure that the entire film is covered with the stain and that there are no air bubbles on the surface.
• Leave the stain on for 20-30 minutes. The optimal staining time may vary depending on the pH and temperature of the staining solution, as well as the age and thickness of the smear. A longer staining time may result in darker and more contrasted staining, while a shorter staining time may result in lighter and less distinct staining. You may need to adjust the staining time according to your preference and experience.
• Flush the slide gently with tap water or distilled water to remove excess stain. Do not use a strong stream of water, as this may wash off the smear or cause streaking. You can also dip the slide in and out of water to wash it.
• Keep the slide in a vertical position and let it air dry. Do not blot or wipe the slide, as this may damage the smear or cause artifacts.
• Examine the slide under a microscope using oil immersion objective (100x). Scan the entire film systematically and look for malaria parasites inside or outside the red blood cells. Identify the species and stage of the parasites based on their morphology and appearance.

Thick blood films are used to detect and quantify malaria parasites in blood samples. They are more sensitive than thin films, but they do not allow the identification of the species or the morphology of the parasites. Therefore, thick films should always be accompanied by thin films for confirmation and speciation.

The procedure for thick film staining is as follows:

• On a clean dry microscopic glass slide, make a thick smear of blood (about 2-3 drops) and spread it evenly over an area of about 1 cm in diameter. Do not make the smear too thick or too thin, as this will affect the quality of staining and examination.
• Let the smear air dry completely for several hours or overnight. Do not use heat or an incubator to dry the smear, as this will damage the parasites and cause artefacts.
• Do not fix the smear with methanol, as this will dissolve the red blood cells and make the parasites invisible.
• Stain the smear with diluted Giemsa stain (prepared by taking 1 ml of the stock solution and adding to 49 ml of phosphate buffer or distilled water). The optimal pH of the stain should be between 6.8 and 7.2. If the pH is too high or too low, the staining will be poor or uneven.
• Flood the slide with the stain and leave it for 10-15 minutes. Alternatively, you can dip the slide into a staining jar containing the stain for the same duration.
• Wash the slide gently with buffered water or distilled water for 3-5 minutes. Do not use tap water, as it may contain chlorine or other chemicals that can interfere with the staining. Do not rub or wipe the slide, as this may remove the parasites.
• Let the slide air dry completely before examining it under a microscope using an oil immersion lens (100x magnification).

The Giemsa stain results in different coloration of the various components of the blood cells and parasites, depending on their affinity for the acidic or basic dyes. The general color scheme is as follows :

• The nucleus of the blood cells appears blue-purple due to the binding of azure and methylene blue, which are basic dyes.
• The cytoplasm and cytoplasmic granules of the blood cells appear red-orange due to the binding of eosin, which is an acidic dye.
• The erythrocytes appear pink due to the presence of hemoglobin, which has a weak affinity for eosin.
• The platelets appear purple due to the presence of granules that bind azure and methylene blue.
• The lymphocytes have a dark blue nucleus and a light blue cytoplasm.
• The monocytes have a purple nucleus and a pale blue cytoplasm.
• The neutrophils have a purple-red nucleus with lobes and a pink cytoplasm with fine granules.
• The eosinophils have a blue-purple nucleus with lobes and a pale pink cytoplasm with coarse orange-red granules.
• The basophils have a purple nucleus with lobes and a bluish cytoplasm with dark purple granules.

The Giemsa stain can also be used to identify various blood parasites, such as malaria, spirochetes, trypanosomes, microfilariae, etc. The parasites usually appear blue-purple due to their DNA-rich nuclei and cytoplasmic structures. However, some parasites may have distinctive features that can help in their identification :

• The malaria parasites (Plasmodium spp.) appear as ring-shaped or oval structures within the erythrocytes. They may have pigment granules that appear black or brown. Depending on the stage of development, they may also have multiple nuclei or vacuoles. The species of Plasmodium can be differentiated by the size, shape, number, and distribution of the parasites within the erythrocytes.
• The spirochetes (Borrelia spp., Treponema spp., etc.) appear as thin, spiral-shaped bacteria that may be seen outside or inside the blood cells. They may be difficult to visualize due to their small size and low contrast. They may require special stains or dark-field microscopy for better detection.
• The trypanosomes (Trypanosoma spp., Leishmania spp., etc.) appear as elongated, flagellated protozoa that may be seen outside or inside the blood cells. They may have a kinetoplast, which is a large mass of mitochondrial DNA that appears as a dark dot at one end of the cell. They may also have a nucleus and an undulating membrane along their body.
• The microfilariae (Wuchereria spp., Brugia spp., Loa loa, etc.) appear as slender, thread-like worms that may be seen outside or inside the blood cells. They may have a sheath, which is a transparent covering that extends beyond their body. They may also have a tail tip, which is a blunt or pointed projection at one end of the worm.

The interpretation of the Giemsa stain results depends on the clinical context and the differential diagnosis of the patient. The hematopathologist will examine the morphology, quantity, distribution, and proportion of the blood cells and parasites and compare them with the normal reference ranges and patterns. Any abnormal findings will be reported along with their possible causes and implications. For example:

• An increase in the number or percentage of lymphocytes (lymphocytosis) may indicate an acute viral infection, chronic lymphocytic leukemia, lymphoma, or other lymphoproliferative disorders.
• A decrease in the number or percentage of neutrophils (neutropenia) may indicate an acute bacterial infection, drug-induced bone marrow suppression, autoimmune disease, or congenital neutropenia.
• A presence of malaria parasites within the erythrocytes (malaria parasitemia) may indicate an active infection with Plasmodium spp., which can cause fever, chills, headache, anemia, splenomegaly, and other complications.
• A presence of spirochetes within or outside the blood cells (spirochetemia) may indicate an infection with Borrelia spp., which can cause Lyme disease or relapsing fever; or Treponema spp., which can cause syphilis or yaws.

Giemsa stain has a wide range of applications in cytology, histology, parasitology, and cytogenetics. Some of the main uses of Giemsa stain are:

• Giemsa stain is specific for the phosphate groups of DNA. It attaches itself to regions of DNA with high amounts of adenine-thymine bonding . Giemsa stain is used in Giemsa banding (G-banding), to stain chromosomes and it is often used to create a diagrammatic representation of chromosomes (idiogram) . It can identify chromosomal aberrations such as translocations and rearrangements .
• Giemsa stain is a differential stain that can distinguish between different types of cells and cellular components based on their affinity for the acidic or basic dyes . Giemsa stain can be used to study the adherence of pathogenic bacteria to human cells, differentiating human cells as purple and bacterial cells as pink . It can also be used to differentiate nuclear and cytoplasmic morphology of the various blood cells like RBCs, WBCs, and platelets .
• Giemsa stain is a gold standard staining technique for malaria and other blood parasites. It can be used for histopathological diagnosis of the Plasmodium species that cause malaria and some other spirochete and protozoan blood parasites such as Trypanosoma, Leishmania, Toxoplasma, Giardia, etc. . Giemsa stain can also be used to visualize the classic "safety pin" shape in Yersinia pestis .
• Giemsa stain has some other applications in microbiology and pathology. It can be used to demonstrate Helicobacter pylori (bacteria) in gastric biopsies , Chlamydia trachomatis inclusion bodies in conjunctival scrapings , Borrelia spp. in skin biopsies , Histoplasma spp. in tissue sections , Pneumocystis jiroveci cysts in bronchoalveolar lavage specimens , and Wolbachia bacteria in Drosophila melanogaster . Giemsa stain can also be used to identify mast cells and "owl`s-eye" viral inclusions associated with cytomegalovirus infection .

Giemsa stain is a widely used technique in hematology, cytogenetics, histopathology and microbiology. It has several advantages and limitations that should be considered when applying it.

Some of the advantages are:

• It is readily available, easy to prepare, maintain and use. The stock solution can be stored for a long time and the working solution can be prepared shortly before use.
• It is a differential stain that can distinguish between different types of blood cells, nuclei, cytoplasm and granules based on their affinity for acidic or basic dyes. It can also differentiate between normal and abnormal cells, such as those infected by parasites or bacteria.
• It is specific for the phosphate groups of DNA and can be used to visualize chromosomes and identify chromosomal anomalies like translocation and rearrangement. It can also be used to create a diagrammatic representation of chromosomes (idiogram).
• It can be used to stain various microorganisms, such as malaria parasites, spirochetes, chlamydia, borrelia, yersinia, histoplasma and pneumocystis. It can also be used to identify mast cells and rickettsia.

Some of the limitations are:

• The working solution must be prepared shortly before use and cannot be stored for long periods. The pH of the solution must be adjusted to 6.8 or 7.2 for optimal staining results.
• The staining procedure requires careful timing and temperature control. The duration and temperature of staining may vary depending on the type of specimen and the desired result. Overstaining or understaining may affect the quality and interpretation of the stain.
• The staining results may vary depending on the quality of the reagents, the preparation of the slides, the fixation method and the type of microscope used. The stain may fade over time or be affected by exposure to light or chemicals.
• The staining technique may not be specific enough to identify some microorganisms or cellular structures that require special stains or immunohistochemical methods. For example, Giemsa stain cannot differentiate between gram-positive and gram-negative bacteria or between different species of malaria parasites.

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