IMMUNOLOGY

Experiment 4

Aim of the Experiment

Geimsa staining of Blood smear for Leukocytes identification

Introduction:

Giemsa stain is a type of Romanowsky stain, named after Gustav Giemsa, a German chemist who created a dye solution. It was primarily designed for the demonstration of malarial parasites in blood smears, but it is also employed in histology for routine examination of blood smear.

Uses of Giemsa Stain

Apart from staining malarial parasites, Giemsa stain has a variety of applications in Microbiology and Pathology:

• Giemsa stain is used to obtain differential white blood cell counts.

• It is also used to differentiate nuclear and cytoplasmic morphology of the various blood cells like platelets, RBCs, WBCs.

• In Microbiology, Giemsa stain is used for staining inclusion bodies in Chlamydia trachomatis, Borrelia species, and if Wayson’s stain is not available, to stain Yersinia pestis. Giemsa stain also is used to stain Histoplasma capsulatum, Pneumocystis jiroveci, Klebsiella granulomatis, Penicillium marneffei and occasionally bacterial capsules.

• This stain is also used in cytogenetics to stain the chromosomes and identify chromosomal aberrations. It is commonly used for G-banding (Giemsa-Banding)

Principle

Giemsa stain is a differential stain and contains a mixture of Azure, Methylene blue, and Eosin dye. It is specific for the phosphate groups of DNA and attaches itself to where there are high amounts of adenine-thymine bonding. Azure and eosin are acidic dye which variably stains the basic components of the cells like the cytoplasm, granules etc. Methylene blue acts as the basic dye, which stains the acidic components, especially the nucleus of the cell.

Methanol act as a fixative as well as the cellular stain. The fixative does not allow any further change in the cells and makes them adhere to the glass slide.

In house preparation from Geimsa Powder

1. Weigh the required amount of powder stain, and transfer to a clean, dry 1litre capacity bottle. Add methanol and mix well.

2. Measure and add glycerol and mix well.

3. Place the bottle of stain in water bath at 50-60°C or at 37°C for up to 2 hours with frequent mixing.

4. Label the bottle and store in a cool, dark place with a firm stopper.

NOTE: If water gets in contact during any steps of preparation of stain, the stain gets spoilt, therefore use, dry glassware and store in conditions where there would be no water contact.

• Filter the stain using Whatman filter paper no.1 and dilute with water buffered to pH 7.2 to make working solutions

Procedure

1. Wear gloves when performing this procedure.

2. Fix air-dried film in absolute methanol by dipping the film briefly (two dips) in a Coplin jar containing absolute methanol.

3. Remove and let air dry.

4. Stain with diluted Giemsa stain (1:20, vol/vol) for 20 min. For a 1:20 dilution, add 2 ml of stock Giemsa to 40 ml of buffered water in a Coplin jar.

Observation and Result

• Macroscopically, blood films appear purplish. If blue, the buffered water was too alkaline; if pink to red, the buffered water was too acid.

• Microscopically, RBCs appear pinkish gray, platelets appear deep pink, and WBCs have purple-blue nuclei and lighter cytoplasm. Eosinophilic granules are bright purple-red, and neutrophilic granules are purple. Basophilic stippling within uninfected RBCs is blue.

• Slight variation may appear in the colors described above depending on the batch of stain used and the character of the blood itself, but if the various morphological structures are distinct, the stain is satisfactory.

Reference Material

1. Protocol

2. Blood smear and Giemsa stain

3. What blood looks like under microscope

4. Blood smear evaluation

Questions

1. What diseases require preparation of blood smear?

2. What are the criteria of a good blood smear?

3. What is the principle of Giemsa staining?

4. Describe different leukocytes which can be observed under the microscope after Geimsa staining.