Microscopy Results with phosphate buffer acc. to Weise pH 6.

Cell type Giemsa’s staining Pappenheim’s

1.09204 Giemsa’s Azure Eosin Methylene Blue Solution staining
1.09203 Giemsa’s Azure Eosin Methylene Blue Nuclei red to violet purple to violet
Lymphocytes plasma blue plasma blue
Monocytes plasma dove-blue plasma dove-blue
Neutrophilic granulocytes granules light violet granules light violet
Eosinophilic granulocytes granules red to grey- granules brick-red to
blue dark violet
Basophilic granulocytes granules dark-violet granules dark violet
In Vitro Diagnostic Medical Device to black
Thrombocytes violet violet
Erythrocytes reddish reddish
Giemsa’s azure eosin methylene blue for Blood parasites nuclei bright red

microscopy Technical note

Staining of blood and bone marrow smears and clinical-cytological
specimens
Principle
The typical color of cell nuclei, namely purple, is due to molecular interaction
between eosin Y and an azure B-DNA complex.
Both dyes build up the complex later. The intensity of the staining depends on
the azure B content and on the ratio azure B/eosin Y. The staining result can
be influenced by several factors such as the pH of the solutions and buffer
solution, buffer substances, fixation, staining time.
Sample material
Air-dried blood and bone marrow smears
Clinical specimens in cytology, e.g. urine sediment, sputum, FNAB, imprints,
lavages.
Reagents
Merck Cat. No. 1.09204 Giemsa’s azure eosin methylene blue solution
100 ml, 500 ml, 1 l, 2.5 l
Merck Cat. No. 1.06009 Methanol GR 1 l, 2.5 l
Merck Cat. No. 1.04091 Glycerol GR 1 l, 2.5 l
Merck Cat. No. 1.09468 Buffer tablets acc. to Weise pH 7.2 1 pack (100 tabs)
Merck Cat. No. 1.11374 Buffer tablets acc. to Weise pH 6.8 1 pack (100 tabs)
Merck Cat. No. 1.09203 Giemsa’s azure eosin methylene blue 25 g, 100 g
Preparation
1. Buffer solution
Dissolve 1 buffer tablet* in 1 l distilled water.
*1.11374 or 1.09468 depending on the required reaction color
The microscope used should meet the requirements of a medical diagnostic
laboratory.
Use a full-flow filter for filtering the dilute staining solution prior to use.
Sample preparation
All samples must be treated using state-of-the-art technology.
All samples must be clearly labeled.
Suitable instruments must be used for taking samples and for their
preparation. Follow the manufacturer’s instructions for application/use.
Diagnostics
Diagnoses must only made by authorized and trained persons.
Valid nomenclatures must be used.
Further tests must be selected and performed according to recognized
methods.
Storage
Store the staining solution at +15°C to +25°C and the dye at +5°C to
+30°C.
The solution and the dye must be used by the expiry date stated.
Shelf-life
After first opening the bottle, the contents can be used up to the expiry date
when stored at +15°C to +25°C (solution) resp. +5°C to +30°C (dye).
The bottles must be kept tightly closed at all times.
Instructions for use
For professional use only.
In order to avoid errors, the staining process must be carried out by
qualified personnel.
National guidelines for work safety and quality assurance must be followed.
Microscopes equipped according to the standard must be used.

2. Diluted Giemsa’s solution for manual staining Protection against infection

Dilute 10 ml Giemsa’s azure eosin methylene blue solution with 190 ml buffer Effective measures must be taken to protect against infection in line with
solution, mix well, leave to stand for 10 min, and filter if necessary. laboratory guidelines.

3. Diluted Giemsa’s solution for staining with MIRASTAINER Instructions for disposal
Slowly add 25 ml Giemsa’s solution to 275 ml buffer solution, mix and leave to Used solutions and solutions that are past their shelf-life must be disposed
stand for 10 min, and filter if necessary. of as special waste according to local disposal guidelines.
In addition to taking back used or expired products via its Retrologistik
4. Giemsa’s azure eosin methylene blue solution service, Merck also provides technical support for local disposal solutions.
Dissolve 0.76 g Giemsa’s azure eosin methylene blue in 50 ml glycerol and
heat for 3 h at 60°C on a water bath, add 50 ml methanol, leave to stand for Auxiliary reagents
5 days and filter. Merck Cat. No. 1.04699 Immersion oil 100 ml, 500 ml
Merck Cat. No. 1.15577 Immersion oil 100 ml
Procedure acc. to DIN ISO 8036-1
Air-dried smears Merck Cat. No. 1.07961 Entellan® new 100 ml, 500 ml
Staining rack / Staining cuvette Hazard classification
Methanol 3-5 min Merck Cat. No. 1.09204, Merck Cat. No. 1.09203
Diluted Giemsa’s solution 15-20 min Please observe the hazard classification on the label and the information
Rinse with buffer solution 2 x 1 min given in the safety data sheet.
Dry The Merck safety data sheet is available on the Internet.
Staining with MIRASTAINER®
Reagents Time Station Dip
Main product components
Merck Cat. No. 1.09204
Methanol 3 min 2 on
C.I. No. 52015 + Azure 4.1 g/l
Diluted Giemsa’s solution 20 min 3 on
C.I. No. 45380 2.5 g/l
Buffer solution 1 min 4 on
contains CH3OH
Running water (rinse) 2 min 5 on
Dry 3 min 6 -
Merck Cat. No. 1.09203
Pappenheim’s staining C.I. No. 52015 + Azure 57 %
C.I. No. 45380 42 %
Staining with May-Grünwald’s solution and Giemsa’s solution

Cover the smear with 1 ml May-Grünwald’s solution 3 min March 2003

Add 1 ml buffer solution, mix and stain 3-5 min
Cover with diluted Giemsa’s solution, stain 15-20 min Merck KGaA, 64271 Darmstadt, Germany, phone +49 6151 72 0
Rinse with buffer solution
Dry