Giemsa Stain- Principle, Procedure, Results, Interpretation

Table of Contents

• Introduction
• Objectives of Giemsa stain
• Principle
• Reagents Used
• Procedure
• Staining procedure 1: Thin Film staining
• Staining Procedure 2: Thick Film Staining
• Results
• Interpretation
• Applications Giemsa stain
• Advantages
• Limitations

Introduction

Giemsa stain was given that name by a German chemist scientist who used a number of different chemicals to show that malaria parasites exist.

It is a member of the Romanowsky stain subclass. These neutral stains were applied to an air-dried slide that had been post-fixed with methanol. They are composed of oxidised methylene blue, azure, and Eosin Y. Romanowsky stains are used in the pathological inspection of materials like blood and bone marrow films as well as the display of parasites like malaria. They are also used in the differentiation of cells. The Romanoswsky stain comes in four varieties:

• Giemsa stain
• Jenner Stain
• Wright stain
• May-Grunwald Stain
• Leishman stain

Objectives of Giemsa stain

• To precisely produce the stock solution for the Giemsa stain
• to colour and distinguish blood cells
• to distinguish the nuclei from the cytoplasm of blood cells

Principle

The gold standard staining method, giemsa stain, is used for both thin and thick smears to evaluate blood for malaria parasites, to do a regular examination for other blood parasites, and to morphologically distinguish parasites from erythrocytes, leucocytes, and platelets.

According to the affinities of acidity and basicity for blood cells, it contains both acidic and basic dyes, like all Romanowsky stains do. A basic dye, such as azure and methylene blue, binds to the acid nucleus to produce a blue-purple hue. The cytoplasm and cytoplasmic granules, which are alkaline and produce red colouring, are drawn to eosin, an acidic dye. To precipitate the dyes and bind simple components, the stain has to be buffered with water to a pH of 6.8 or 7.2.

Giemsa stain, a type of differential stain that has been around for a while and is commonly used in cytogenetics and histopathology to diagnose the following conditions:

1. Spirochetes, Chlamydia trachomatis inclusion bodies, and other blood parasites include malaria.
2. Borrelia species
3. Pestis Yersinia
4. Hetoplasma species
5. jiroveci Pneumocystis cysts

Reagents Used

• Methanol
• Giemsa powder
• Glycerin
• Water (Buffer)

Procedure

Preparation of the Giemsa Stain Stock solution (500ml)

1. Add 3.8g of Giemsa powder to 250ml of methanol and stir to dissolve.
2. Heat the mixture to 60 oC.
3. After that, slowly add 250ml of glycerin to the mixture.
4. Before using, filter the solution and let it stand for one to two months.

Preparation of Working solution

1. 80 ml of distilled water, 10 ml of methanol, and 10 ml of stock solution are added.

Staining procedure 1: Thin Film staining

1. Make a thin film of the specimen (blood) on a spotless, dry microscope slide, and allow it to air dry.
2. Put the smear through two to three dips in pure methanol to cure it, then let it dry naturally for 30 seconds.
3. For 20 to 30 minutes, completely cover the slide with 5% Giemsa stain solution.
4. Wash with tap water, then wait till it dries.

NOTE: In an emergency, wait 5–10 minutes before using the Giemsa stain solution.

Staining Procedure 2: Thick Film Staining

1. Add a generous layer of blood, and let it air dry on a staining rack for one hour.
2. Put a thin layer of Giemsa stain on the heavy blood stain (prepared by taking 1ml of the stock solution and adding to 49ml of phosphate buffer or distilled water, but the results may vary differently).
3. Wash the smear by submerging it in distilled water with a buffer for three to five minutes.
4. Leave it to dry.

Results

Blood cells' nuclei are blue-purple in color, while their cytoplasm and cytoplasmic granules are red.

The colour of the erythrocytes will be pink.

• Orange-red granules, a pale pink cytoplasm, and a blue-purple nucleus are characteristics of eosinophils.
• Neutrophils will have a pink cytoplasm and a purple-red nucleus.
• Basophils will have bluish granules and a purple nucleus.
• The cytoplasm and nucleus of lymphocytes are both light blue in colour.
• Monocytes will have a pink cytoplasm and a purple nucleus.
• Purple granules will be seen in platelets.

Interpretation

A basic dye, such as azure and methylene blue, binds to the acid nucleus to produce a blue-purple hue. Eosin is an acidic dye that is drawn to the reddish-orange colour of the cytoplasm and cytoplasmic granules, which are alkaline-producing.

Applications Giemsa stain

1. The DNA phosphate groups are only visible with the Giemsa dye. It binds to areas of DNA that have a lot of adenine-thymine bonding.
2. Giemsa stain is used to stain chromosomes in Giemsa banding (G-banding), and it is frequently used to display chromosomes diagrammatically (idiogram).
3. Giemsa stain, which distinguishes between human and bacterial cells by colouring the former purple and the latter pink, can be used to analyse the adhesion of harmful bacteria to human cells.
4. It can be used to make a histopathological diagnosis of malaria as well as some blood parasites caused by spirochetes and protozoa.
5. Additionally, it is utilised in Wolbach's tissue stain, which is used to stain hematopoietic tissue and to identify bacteria and rickettsia.
6. For bone marrow samples and peripheral blood smears, the traditional blood film stain Giemsa stain is used. Red blood cells stain pink, platelets stain a light pale pink, leukocyte nuclear chromatin stain magenta, lymphocyte cytoplasm stain sky blue, monocyte cytoplasm stain pastel blue, and so on.
7. The fungus Histoplasma, Chlamydia bacteria, and Mast cells can all be stained using the Giemsa stain in addition to identifying chromosomal abnormalities like translocation and rearrangement.

Advantages

easily accessible, prepareable, maintainable, and usable

Limitations

Giemsa stain that is working has to be prepared just before usage.