GIEMSA STAINING TECHNIQUE – PRINCIPLE, PREPARATION, PROCEDURE & OBSERVATIONS

As you know, that there are various types of Blood cells are present in our body, having different shapes and sizes, which take up the stains as per their structures. Some of the Components of the blood cells are Basophilic i.e. they have a great affinity for acidic dyes whereas some of the components are Acidophilic i.e. they have a high affinity for Basic Dyes and then there are some components of the cells which are neutral and has the high affinity for neutral stains.

Therefore, the stains used in Hematology laboratory are the combination of these three types of stains. Besides the dyes, a buffer is added to the stains which act as the mordant and helps in the binding of the stain with the cells.

Romanowsky stains are such types of stains that are universally employed for the staining of blood cells. Almost all the Romanowsky group of stains has two essential components i.e. Methylene blue & Eosin or Azure dye. Methylene blue is a basic dye which has the high affinity for the acidic components of the cell i.e. Nucleus and Eosin/ Azure is the Acidic dye which has the high affinity for the basic components of the cells i.e. the cytoplasm and Granules in some cells.

Most of the Romanowsky stains are prepared with Methyl alcohol (Methanol) so that they act as a fixative as well as the cellular stain. There are 4 different types of Romanowsky stains commonly used in Hematology laboratory for staining the blood cells –

• Giemsa Stain
• Leishman Stain
• Field’s Stain
• Wright’s Stain

In this Article, I’ll discuss Giemsa Staining Technique used in Hematology Laboratory.

A brief Description of Giemsa Stain….

Giemsa stain is a mixture of Azure, Methylene blue, and Eosin dye. Giemsa stain is a differential stain that is used to variably stain the various components of the cells and it can be used to study the adherence of pathogenic bacteria to the human cells. It differentially stains the human and bacterial cells and appeared as purple and pink colored bodies respectively.

The Giemsa stain is one of the best stains for malaria and other blood parasites and also satisfactory as a routine bloodstain to stain the Peripheral blood smear for the examinations of blood film under the microscope.

PRINCIPLE OF GIEMSA STAINING

Giemsa stain is commonly used when there is a need to examine the Blood smear for the Parasites but is a good stain for routine examination of blood smear and used to differentiate nuclear and cytoplasmic morphology of the various cells of the blood like Platelets, RBCs, WBCs as well as the parasites. This stain is the most dependable stain for blood parasites, particularly in thick blood smears.

The working principle of the Giemsa stains is the same as described above. As it is a type of Romanowsky stains, it contains both the Acidic and Basic dyes which have the affinity for Basic and Acidic components of the Blood cells respectively. The acidic dye, Eosin & Azure variably stains the Basic components of the cells i.e. the cytoplasm, Granules etc. and the Basic dye, Methylene blue stains the Acidic components, especially the Nucleus of the cell. The stain must be diluted for use with water buffered to pH 6.8 or 7.2, depending on the specific technique used.

The Standard Operating Procedures (SOPs) are different for almost every laboratory as per the quality of the staining solution & buffer they are using and the purpose of staining. Do not consider this article as the only correct way of staining the Blood smear with Giemsa stain.

MATERIALS REQUIRED FOR THE GIEMSA STAIN

• Giemsa Stain (Stock Solution)
• Microscopic Glass Slide
• Phosphate buffer (pH 7.2)
• Graduated pipettes
• Measuring cylinder
• Distilled Water
• Pasteur pipette
• Coplin Jar
• Blood Specimen – The specimen used usually consists of fresh whole blood collected by finger puncture (capillary puncture) or the EDTA anticoagulated whole blood, collected by venipuncture and it should be less than 1-hour old for better results. The blood specimen can also be collected in Heparin or Sodium Citrate for the identification of parasites.

Nowadays, commercially prepared Giemsa stain solution is used in most of the laboratories which are then diluted in various ratios for different purposes. However, you can easily prepare it in the laboratory. Follow the link to learn more….

Check out how to prepare Giemsa Stain (Stock Solution) in laboratory

PREPARATION OF GIEMSA STAIN IN LABORATORY

Similarly, the commercially prepared phosphate buffer solution is used in laboratories which can be adjusted as per the pH required using HCl or NaOH solution. However, you can easily prepare it in the laboratory. Follow the link to learn more…

Check out the Preparation of Phosphate Buffer Solution in Laboratory

PREPARATION OF PHOSPHATE BUFFER SOLUTION IN LABORATORY

PROCEDURE OF GIEMSA STAINING

The Procedure of Giemsa staining varies as per the purpose of staining that means whether the staining is done for the examination of Blood cells or to find the Parasites in the blood smear and accordingly the Blood smears are prepared as Thin Blood films or Thick blood films. Here, I’m explaining the procedure of staining 3 different types of smears as the thin smear, Thick smear and the Combination of Thick and Thick smear on the same slide…. So let’s start with staining the thin blood smear.

Staining the Thin blood smear with Giemsa stain

⇒ Prepare a thin blood smear on a clean and dry microscopic glass slide and air dry it.

If you don’t know how to prepare it, check it out here, the preparation of thin blood Smear

PREPARATION OF PERIPHERAL BLOOD SMEAR

⇒ Firstly fix out the air-dried thin blood smear in the absolute methanol by dipping the blood smear quickly (two dips) in a Coplin jar containing absolute methanol.

⇒ Take out the slide from the Coplin jar and let it air dry.

⇒ Diluting the Giemsa Stain for Thin Blood smear: For staining the thin blood smear the Giemsa stain is used in 1:20. To make 1:20 dilution of Giemsa stain add 2 ml of stock solution of Giemsa stain to 40 ml of phosphate buffer solution in a clean Coplin jar. You can also use the Distilled water instead of buffer but the results may vary.

⇒ Now, stain the Methanol fixed Blood smear with diluted Giemsa stain (1:20, v/v) for 20 min. For this, put the slide in the Coplin jar containing the diluted Giemsa stain or you can also put it on a staining rack or on any flat surface with the smear side up and pour the stain over the smear so that it equally covers the smear.

⇒ Now Wash out the stained slides by quickly dipping, once or twice, the slide in and out of a Coplin jar containing buffered water or Distilled water.

Do not overexpose the smear to buffered water (phosphate buffer solution). Excessive washing may decolorize blood smear.

⇒ Let the smear dry well in the air.

Staining the Thick blood smear with Giemsa stain

⇒ Prepare a thick blood smear on a clean and dry microscopic glass slide and air dry it.

⇒ If you don’t know how to prepare it, check out the preparation of Thick blood Smear

PREPARATION OF PERIPHERAL BLOOD SMEAR

⇒ Do not dry the thick blood smear in an incubator or by heat, because this will fix the blood smear onto the slide and interfere with the lysis of RBCs.

Note: If a rapid diagnosis of the malaria parasite is required, thick films can be made slightly thinner than usual, allowed to air dry for 1 hour at room temperature, and then stained with Giemsa stain.

⇒ Diluting the Giemsa Stain for Thick Blood smear: For staining the thick blood smear the Giemsa stain is used in 1:20. To make a 1:50 dilution of Giemsa stain, add 1 ml of stock solution of Giemsa stain to 49 ml of phosphate buffer solution in a clean Coplin jar. You can also use the Distilled water instead of buffer but the results may vary.

⇒ Now, stain the Air-dried Blood smear with diluted Giemsa stain (1:50, v/v) for 50 min. For this, put the slide in the Coplin jar containing the diluted Giemsa stain or you can also stain it on a staining rack or on any flat surface with the smear side up and pour the stain over the smear so that it equally covers the smear.

⇒ Wash out the Blood smear by placing it in the buffered water or Distilled water for 3 to 5 min.

⇒ Let the smear dry well in the air.

Staining the Thin and thick blood smear on the same slide with Giemsa stain

⇒ Prepare a thin and thick smear of the specimen on the same slide which can be done by dividing the slides into two parts and then making the thin smear on one side and thick on the other side. Allow the smear to air dry.

⇒ Now, carefully fix the air-dried thin blood smear in absolute methanol by dipping the thin smear side quickly (2-3 dips) in a Coplin jar containing absolute methanol. Be sure that the Methanol or its fumes get to the thick film which is done by slightly tilting the slide.

Note: The Introduction of even a small quantity of Methanol into the Thick smear will interfere with the lyses of the RBCs in the thick blood smears during staining.

⇒ Take out the slide from the Coplin jar and let it air dry with the thick smear up. Be sure that the slide is thoroughly dried on both the thin & thick smear side before staining.

⇒ Diluting the Giemsa Stain for Thin and thick blood smear on the same slide: For staining the Thick & thin blood smear on the same slide the Giemsa stain is used in 1:50. To make a 1:50 dilution of Giemsa stain, add 1 ml of stock solution of Giemsa stain to 49 ml of phosphate buffer solution in a clean Coplin jar. You can also use the Distilled water instead of buffer but the results may vary.

⇒ Now, stain the Air-dried Blood smear with diluted Giemsa stain (1:50, v/v) for 50 min. For this, put the slide in the Coplin jar containing the diluted Giemsa stain or you can also stain it on a staining rack or on any flat surface with the smear side up and pour the stain over the smear so that it equally covers the smear.

⇒ Place the slide in the Coplin jar in a way that the thick smear should be down to prevent the debris of RBCs from falling down onto the thin blood smear.

⇒ Now it’s the crucial step of rinsing the slide…… Rinse the thin smear side by quickly dipping the smear in and out of a Coplin jar of phosphate buffer (one or two dips) and then Wash the thick smear side for 3 to 5 min using phosphate buffer or distilled water. Be sure that the thick smear is immersed but do not allow the water to cover any part of the thin smear. Otherwise, the thin smear will get decolorized.

⇒ Let the smear dry well in the air.

OBSERVATIONS OF GIEMSA STAINED SMEAR UNDER THE MICROSCOPE

The various blood cells will be observed under the microscope as follows:

⇒ Red Blood Cells: Pink color.

⇒ Neutrophils: Reddish Purple nuclei with pink cytoplasm.

⇒ Eosinophils: Purple nuclei, faintly pink cytoplasm & 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.

RUHI RANA

Hi, I am Ruhi Rana, the author of this Article. I am a Medical Laboratory Technologist and Medical Content Writer. I love to teach people about various Medical & Paramedical concepts.