Staining

Giemsa Stain: Preparation, Procedure, Principle, Composition and Application

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Giemsa Stain: Staining Procedure, Principle, Result and Application
Giemsa Stain: Staining Procedure, Principle, Result and Application

The term Giemsa stain originated from a name of German chemist and bacteriologist Gustav Giemsa. He apply this stain with a combination of reagents to detect the presence of malaria parasites.

This stain is used for nucleic acid staining and histopathological diagnosis of malaria and other parasites. Giemsa Stain is a types of Romanowsky stains which is universally used for the staining of blood cells. 

It is also a differential stain, it can differentiate between human and bacterial cells and appeared as purple and pink colored bodies respectively. It furtehr utilized to study the adherence of pathogenic bacteria to the human cells.

Romanowsky stain is a mixture of mixture of oxidized methylene blue, azure, and Eosin Y. This stain is applied on a air-dried slide which is post-fixed with methanol. Methylene blue has a high affinity for acidic components of the cell such as Nucleus, becasue it is a basic dye. While Azure is a acidic dye thats why it has a high affinity for the basic components of the cells such as cytoplasm and Granules.

Romanowsky dyes are mostly prepared with Methyl alcohol (Methanol), therefore they act as a good fixative as well as the cellular stain.There are four types of Romanowsky stains such as;

  1. Giemsa Stain
  2. Leishman Stain
  3. Field’s Stain
  4. Wright’s Stain

Giemsa stain principle/giemsa stain mechanism

It is a standard staining method used to study the thin and thick smear of blood for malaria parasite, a good stain for routine examination of blood smear, and also used for morphological differentiation of nuclear and cytoplasm of Erythrocytes, leucocytes and Platelets and parasites.

Giemsa Stain contains both Acidic and Basic dyes, as result it has a affinity for both acidic and basic components of the cell. The basic dye Azure and methylene blue binds with the nucleus (acidic) and produces blue-purple color. The acidic dye Eosin binds with the cytoplasm and cytoplasmic granules and produce red color.

Reagents Required

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

Giemsa stain preparation

Nowadays laboratories uses commercially available giemsa stain by diluting them in different ratio for differen purpose. But you can prepared giemsa stain in laboratory by following these below steps;

For 500ml of Giemsa Stain Stock solution:

Giemsa stain compositionGm/L
Giemsa powder7.6
Glycerol500 ml
Methanol500 ml
giemsa stain composition
  1. Dissolve 3.8g of Giemsa powder within 250ml of methanol.
  2. Heat the solution at 60oC.
  3. Add 250ml of  glycerin.
  4. Filter the solution
  5. leave the solution to stand for about 1-2 months before use. Store the solution in a cool, dark place.

Note: During the preparation of stain, if it gets in contact with water the stain will spoilt. Therefore make sure the glasswares are dry.

Working solution Preparation

Prepare a mixture of 10ml of stock solution, 80ml of distilled water and 10ml of methanol.

Giemsa stain procedure

The procedure of giemsa staining may vary based on the staining purpose. Because different procedures are used for the study of blood cells or detection of parasites in blood smears (thin blood smear and thick blood smear).

Giemsa stain procedure for thin smear (Blood)

For thin smear, prepare a 1:20 ratio of giemsa stain by mixing 2 ml of stock solution of Giemsa stain to 40 ml of phosphate buffer solution. You may use Distilled water instead of buffer.

  1. Take a clean grease-free glass slide and make a thin smear of the blood sample or specimen on it and allow to air dry.
  2. Now, gently immerse the slide into a methanol solution for proper fixation of the smear.
  3. Allow to air dry.
  4. Add Giemsa stain on the smear and wait for 20-30 minutes.
  5. Now Rinse the stained slides by quickly lowering, once or twice, the slide in and out of a Coplin jar containing buffered water or Distilled water. (Excessive washing with buffer solution may results in decolorization of smear)
  6. Air dry the smear.
  7. Now observe the smear under a microscope.

Giemsa stain procedure for thick smear (Blood)

For tick smear, prepare a 1:50 ratio of Giemsa stain by mixing 1 ml of stock solution of Giemsa stain to 49 ml of phosphate buffer solution.

  1. Take a clean grease-free glass slide and make a thick smear of the blood sample or specimen on it and allow to air dry for 1 hour.
  2. Dip the smear within a diluted Giemsa stain.
  3. Rinse the sear with dipping distilled water or buffer solution for 3-5 times.
  4. Air dry the smear.
  5. Smear is ready to observe under the microscope.

Note: Avoid dryng of smear by an incubator or by heat, becasue it may fix the blood smear onto the slide and results in lysis of RBCs.

Giemsa stain procedure for Chlamydia trachomatis

Follow the above-mentioned steps but use 1:40 ratio of Giemsa stain, prepare by mixing 0.5 ml stock Giemsa solution to 19.5 ml buffered water. After that leave the stain for  90-120 minutes.

Result and Observation

Giemsa Stain: Staining Procedure, Principle, Result and Application
Giemsa Stain: Staining Procedure, Principle, Result and Application | Image Source: https://en.wikipedia.org/wiki/Giemsa_stain#:~:text=Giemsa%20stain%20is%20a%20classic,leukocyte%20nuclear%20chromatin%20stains%20magenta. and https://www.researchgate.net/figure/Peripheral-blood-smear-May-Gr-unwald-Giemsa-stain-600x-In-detail-medium-sized_fig1_324003139

A = Giemsa stained Trypanosoma parasites (Chagas disease pathogen), B= Whirling disease section stained with Giemsa stain, C = “Owl’s-eye” viral inclusions, associated with Cytomegalovirus infection, D = Peripheral blood smear, May-Gr€ unwald Giemsa stain, 600x. In detail: medium-sized myeloblasts, promonocytes, and atypical neutrophils with a characteristic “clumped” chromatin, in irregularly compacted nuclei, and hypergranular cytoplasm can be noticed.

Cytoplasm and cytoplasmic granules appears red in color while nucleus appears blue-purple in color.

  • Red blood cells: Mauve-pink
  • Neutrophils: Reddish purple nuclei with pink cytoplasm
  • Eosinophils: Purple nuclei, faintly pink cytoplasm and 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.
  • Nuclei of host cells: Dark purple
  • Nuclei of WBCs: Dark purple
  • Cytoplasm of host cells: Pale blue
  • Cytoplasm of white cells: Pale blue or grey-blue
  • Melanin granules: Black green
  • Bacteria: Pale or dark blue
  • Chlymadia trachomatis inclusion bodies: Blue-mauve to dark purple depending on the stage of development
  • Borrelia spirochetes: Mauve-purple
  • Yersinina pestis coccobacilli: Blue with dark stained ends (bipolar staining)
  • Malaria parasite: Malaria parasites have a red or pink nucleus and blue cytoplasm. If P. vivax is seen, the Schüffner dots are seen as an even carpet of pink dots in the cytoplasm of red blood cells. If P. falciparum is observed, Maurer clefts will be seen as unevenly distributed, coarse bodies in the red cell cytoplasm.

Interpretation

  • The basic dye Azure and methylene blue interacts with the acidic nucleus and produces blue-purple color.
  • The acidic dye Eosin binds with the cytoplasm and cytoplasmic granules which are alkaline-producing red-orange coloration.

Application/Imporatance of Giemsa stain

Giemsa stain used for these following pu

  1. It is used for Giemsa banding (G-banding) to stain chromosomes. It is frequently applied to generate a diagrammatic representation of chromosomes (idiogram).
  2. Used to examine the adherence of pathogenic bacteria to human cells. It differentiates human cells and bacterial cells by staining them as purple and pink.
  3. Used for diagnosis of some spirochete and protozoan blood parasites and malaria.
  4. Used in Wolbach’s tissue stain ( i.e staining hematopoietic tissue and for the identification of bacteria and rickettsia).
  5. Used to stain peripheral blood smears and bone marrow specimens.
  6. Used to observe the chromosomes, identifying chromosomal anomalies (i.e translocation and rearrangement).
  7. Used to identify Mast cells.

Advantages of Giemsa Stain

  1. Giemsa Stain is Readily available.
  2. It is easy to prepare.
  3. Easily maintain and use.

References

  1. https://en.wikipedia.org/wiki/Giemsa_stain#:~:text=Giemsa%20stain%20is%20a%20classic,leukocyte%20nuclear%20chromatin%20stains%20magenta.
  2. https://chlorine.americanchemistry.com/Science-Center/Chlorine-Compound-of-the-Month-Library/Methylene-Blue-Part-2-The-Chemists-Indicator/
  3. https://microbenotes.com/giemsa-stain-principle-procedure-results-interpretation/
  4. https://answers.yahoo.com/question/index?qid=20080712002122AAAhrqK
  5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC540181/
  6. https://clinicalgate.com/preparation-and-staining-methods-for-blood-and-bone-marrow-films/
  7. https://paramedicsworld.com/hematology-practicals/giemsa-staining-technique-principle-preparation-procedure-interpretation/medical-paramedical-studynotes
  8. https://www.researchgate.net/publication/24346194_Histopathology_for_the_diagnosis_of_infectious_diseases
  9. https://microbeonline.com/giemsa-stain-principle-procedure-and-results/
  10. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1453983/

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