Giemsa’s Solution

The Universal Stains


Giemsa’s azure eosin methylene blue solution is one of the most popular microscopy stains, commonly used in hematology, as well as in histology, cytology and bacteriology for the medical diagnosis of human samples. This highly versatile solution is suitable for staining a diverse range of specimens.





Quality Means Clarity

Giemsa’s solution is one of the core products of MilliporeSigma’s microscopy portfolio. As it is intended for the investigation of human material, our Giemsa’s solution is registered as an “in vitro diagnostic medical device” (IVD), and bears the CE mark. It is produced to the highest quality standards to enhance the visibility of cell structures, and ensure clear results. Other advantages include excellent batch consistency, long shelf life, and detailed analysis certificates.

Applications

  • Blood
  • Bone marrow 
  • Sputum
  • Urine
  • Body effusions
  • Lavages
  • FNAB (fine needle aspiration biopsy)
  • Imprints
  • Paraffin sections (e.g. stomach biopsy for detection of Helicobacter pylori) 
  • Lymph nodes
  • Spleen samples 
  • Tonsils

Features and Benefits

  • Proven protocols for application
  • Batch-to-batch consistency
  • Three-year shelf life
  • IVD, CE marked
  • Different package sizes for more flexibility
  • Certificate of Analysis (CoA) available for each batch

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Panoptic Methods

In Giemsa staining, it is important to carefully follow the instructions for the specific type of material being investigated in order to obtain reliable results with highly differentiated cell structures. The following procedures describe staining of blood and bone marrow smears, paraffin sections and clinical-cytological specimens.

Preparation
  1. Buffer solution: 
    Dissolve 1 buffer tablet in 1 L distilled water
  2. Giemsa’s solution for manual staining:
    Dilute 10 mL Giemsa’s solution with 190 mL buffer solution, mix well, leave to stand for 10 min, and filter if necessary.
  3. Giemsa’s solution for staining with staining automat:
    Slowly add 25 mL Giemsa’s solution to 275 mL buffer solution, mix and leave to stand for 10 min, and filter if necessary. 
  4. Giemsa’s solution from dry dye: 
    Dissolve 0.76 g Giemsa’s azure eosin methylene blue (dry dye) in 50 mL glycerol and heat for 3 h at 60°C on a water bath, add 50 mL methanol, leave to stand for 5 days and filter.
Procedure of Air-Dried Smears
Staining with Staining Automat
Reagents
Time
Methanol 3 min
Dilute Giemsa’s solution 15 – 20 min
Buffer solution 1 min
Running water (rinse) 2 min
Dry 3 min
Staining Rack/Staining Jar
Reagents
Time
Methanol 3-5 min
Dilute Giemsa’s solution 15 – 20 min
Rinse and Buffer solution 2 x 1 min
Dry 3 min

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Giemsa Staining of Blood Samples

For hematology, carefully dilute Giemsa’s solution in the ratio 1:20 with phosphate buffer solution acc. to Weise pH 6.8 or 7.2. The table below shows the results for phosphate buffer solution pH 6.8.

Results
Neutrophilic Granulocytes
Color
Nuclei Red to violet
Lymphocytes Plasma blue
Monocytes Plasma dove-blue
Eutrophilic granulocytes Granules light violet
Eosinophilic granulocytes Granules red to gray-blue
Basophilic granulocytes Granules dark-violet
Thrombocytes Violet
Erythrocytes Reddish
Blood parasites Nuclei bright red
Blood smear, Giemsa's stain Autohemacolor
Blood smear, Giemsa's stain  Blood smear, Giemsa's stain
Blood smear, Giemsa’s stain Blood Smear, Giemsa’s Stain
Blood smear, Giemsa’s stain Blood smear, Giemsa’s stain

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Pappenheim Staining of Blood Samples

Alternatively, blood samples can be stained via the Pappenheim method using a combination of May-Grünwald’s solution and Giemsa’s solution.

 Pappenheim stain blood smear  Pappenheim Pappenheim stain, blood smear  Pappenheim Stain, blood smear 
Pappenheim stain blood smear Pappenheim Pappenheim stain, blood smear Pappenheim stain, blood smear
Procedure Staining with Staining Automat
Reagents
Time
May-Grünwald’s solution 4 min
Dilute Giemsa’s solution 20 min
Rinse with buffer solution
acc. to Weise pH 6.8
1 min
Running water (rinse) 2 min
Dry 3 min 
Results
Cell Type
Color
Nuclei Purple to violet
Lymphocytes Plasma blue
Monocytes Plasma dove-blue
Neutrophilic granulocytes Granules light violet
Eosinophilic granulocytes Granules brick-red
to dark violet
Basophilic granulocytes Granules dark violet
to black
Thrombocytes Violet
Erythrocytes Reddish

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Giemsa Staining of Paraffin Sections of Bone Marrow Samples

For bone marrow samples, the specimen should be decalcified in a gentle decalcifying solution, such as OSTEOSOFT®, so that immunohistochemistry can be successfully processed. It is also critical to use separate xylene or Neo-Clear® rinse baths, as any ethanol traces in the solutions may result in the preparations being discolored.

Pretreatment of Bone Marrow and Iliac Crest Biopsy Material

Place the fixed biopsy materials in OSTEOSOFT® decalcifying solution for 6 hours to gently remove any calcification, then transfer to histoprocessing. Carefully cut smaller blocks and, if required, treat again with OSTEOSOFT® for an additional 20 minutes.

Staining Procedure
Step
Time
Deparaffinate and rehydrate the sections using standard methods
Distilled water 10 sec.
Undiluted, filtered Giemsa's solution 15 min
0.1% acetic acid 10 sec.
Distilled water 10 sec.
2-propanol 10 sec.
2-propanol 10 sec.
2-propanol 10 sec.
Xylene or Neo-Clear® 5 min
Use Entellan® New to cover the preparations moistened with xylene, or use Neo-Mount® to cover those moistened with Neo-Clear®
Results
Cell Type
Color
Cell nuclei, cells Blue, dark blue
Collagen, osteoid Pale blue
Eosinophilic grains Red
Acidophilic mucopolysaccharides, mastocytes, cartilage matrix Reddish violet
Acidophilic materials Orange red
 Bone marrow section   Lymph node section
Bone marrow section Lymph node section

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Giemsa Staining of Spirochaetes

For spirochaetes (also known as spirochetes), diluted Giemsa’s solution can be used for 12- to 24-hour staining. Alternatively, staining time can be reduced to 6-8 hours by adding a small amount of potassium carbonate solution. Staining results are the same for either modification.

Results
Cell Type
Cell Type
Spirochaeta pallida Pink
Other spirochaetes Reddish to bluish-violet

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Ordering Information

Product
Package Size
Order Number
Giemsa’s azure eosin methylene blue solution 100 mL
500 mL
1 L
2.5 L
1.09204.0100
1.09204.0500
1.09204.1000
1.09204.2500
Giemsa’s azure eosin methylene blue (dry dye) 25 g
100 g
1.09203.0025
1.09203.0100
May-Grünwald’s eosin methylene blue solution 100 mL
500 mL
1 L
2.5 L
1.01424.0100
1.01424.0500
1.01424.1000
1.01424.2500
Methanol for analysis EMSURE® ACS, ISO, Reag. Ph Eur 1 L
2.5 L
1.06009.1000
1.06009.2500
Glycerol 85% suitable for use as excipient EMPROVE® exp Ph Eur, BP 1 L
2.5 L
1.04091.1000
1.04091.2500