Stains: For Blood and Bone Marrow

Stains
for blood and bone marrow

Overview of the classic staining methods,
fast stain and foil staining
Focussing your hematology targets.
All reagents from one source
Fast and safe processes
Easy to use
Quick and sure results in less than 30 minutes
All stainings at room temperature
First-class and reproducible staining pictures
The same careful fixation for all stainings
According to the standard literature
Advantages
of the hematological
staining methods
from Merck
Hematological
staining methods
Hematological staining
For the staining of blood and bone marrow smears are mixtures of acid (eosines) and basic (methylene blue, azure) dyes
especially suitable. With the basic dyes the acid parts of cells such as nucleic acid are stained. The acid dyes stains above all
basic proteins, such as lysosomal enzymes in the granula. The staining picture with the staining intensity and color is deter-
mined from the dye solution, the quality of the staining is determined by the chosen staining method as well as their perform-
ance. Beside that the pH of the used water and the chosen staining times play an important role.
From the great number of the hematological staining methods some have due to their excellent results become widespread.
There are the classic staining methods according to Giemsa and May-Grnwald as well as the combination of both (Pappen-
heim), the staining according to Wright, the Hemacolor fast stain as well as the foil staining without dye solutions with
Sangodiff.
The water quality at the blood smear staining
The quality of the staining pictures becomes determined in high grade through the quality of the used water. If the water reacts
acid, the eosines-effect of the dye solution becomes raised. Reddish shades dominates. If the water is alkaline, it comes to one
reinforcement of the azure-metachromasie and simultaneously to an extenuation of the eosin-effect so that a bluish-grey shade
prevails. Determining is the pH, which should be as neutral as possible, i.e. the pH of the water should be in the range of 6.8 to
7.2. Optimal reproducible staining pictures are accomplished by the application of buffer solutions according to Weise.
According to desired staining results the pH of the used buffer solution is to choose.
Storage and documentation of preparations
For the documentation of the preparations the mounting medium Entellan new is highly recommended. As soon as the slide
has dried, few drops of Entellan new are brought up on the preparation. With a coverslip glass under avoidance of air bubbles
the slide is covered. After a drying time of about 20-30 minutes the smear can examined under the microscope and stored in
an archive. The so preserved preparations remain over 10 years colorstable.
Solution
1. Buffer solution pH 6.8 (7.2)
Dissolve 1 tablet in 1l distilled water.
2. Dilute Giemsa's solution (1:20):
Add 10ml Giemsa's solution to 190ml
buffer solution (pH 6.8 or 7.2), mix
well, allow to stand 10 minutes and
filter if necessary.
For staining automat
Add 15ml Giemsa's solution to 220ml
distilled water and 65ml buffer solution
(pH 6.8 or 7.2) mix well, allow to stand
for 10 minutes and filter if necessary.
Procedure
Staining rack
May-Grnwald's solution 3min
Buffer solution (1ml)
add, mix, stain 35min
Dilute Giemsa's solution 1520min
Rinse with buffer solution
Drying
Coplin jar
Blood smears Air dried
Wash in buffer solution 2 x 1min
Drying
Staining automat
Dilute Giemsa solution 20min
Wash in buffer solution 1min
Tap water 2min
Drying 3min
Result
Nuclei: purple to violet
Lymphocytes: plasma light blue
Monocytes: plasma dove-violet
Granulocytes:
- neutrophilic: granules light violet
- eosinophilic: granules brick-red
to red-brown,
- basophilic: granules dark
violet to black
Thrombocytes: violet
Erythrocytes: reddish
Pappenheim
Solution
2. Dilute May-Grnwald's solution:
Dilute 30ml May-Grnwald's solution
with 150ml distilled water and 20ml
buffer solution.
Add 30ml buffer solution and 220ml
distilled water to 50ml May-Grnwald's
solution, mix and allow to stand for
10 minutes before using.
Procedure
Staining rack
add, mix, stain 6 min
Drying
Coplin jar
Dilute May-Grnwald's
solution 6min
Drying
Staining automat
Dilute May-Grnwald's
solution 6min
Tap water 2min
Drying 3min
Result
Nuclei: red-purple to
blue-violet
Lymphocytes: plasma blue
Monocytes: plasma grey-blue
Granulocytes:
to dark violet
- basophilic: granules dark violet
to black
Erythrocytes: reddish grey
May-Grnwald
Solution
2. Dilute Giemsa's solution (1:20):
Add 10 ml Giemsa's solution to 190ml
buffer solution (pH 6.8 or 7.2), mix
well, allow to stand 10 minutes and
Slowly add 25ml Giemsa solution to
275ml buffer (pH 6.8 or 7.2) mix
well, allow to stand for 10 minutes and
Procedure
Staining rack
Methanol 3min
Drying
Coplin jar
Methanol 35min
Drying
Staining automat
Methanol 3min
Tap water 2min
Drying 3min
Result
Monocytes: plasma dove blue
Granulocytes:
- neutrophilic: granules delicate
violet
- eosinophilic: granules red to
grey-blue
- basophilic: granules strong
violet
Erythrocytes: weak red
Blood parasites: nulei bright red
Giemsa
Solution
2. Dilute Wright's solution: Dilute 30ml
Wright's solution with 150ml distilled
water and 20ml buffer solution.
distilled water to 50ml Wright's solution.
Procedure
Staining rack
Wright's solution 1min
Drying
Coplin jar
Dilute Wright's solution 6min
Drying
Staining automat
Dilute Wright's solution 6min
Tap water 2min
Drying 3min
Result
Nuclei: red violet to
blue-violet
Monocytes: plasma dove-blue
Granulocytes:
to red-brown
to black
Erythrocytes: reddish-grey
Wright
Solution
2. Dilute Leishman's solution:
Dilute 30ml Leishman's solution with
150ml distilled water and 20ml
buffer solution.
distilled water to 50ml Leishman's
solution.
Procedure
Staining rack
Leishman's solution 1min
Buffer solution (1ml )
Drying
Coplin jar
Dilute Leishman's solution 6min
Drying
Staining automat
Dilute Leishmany's solution 6min
Tap water 2min
Drying 3min
Result
Granulocytes:
- neutrophilic: granules violet
Erythrocytes: red
Leishman
Solution
Buffer solution pH 7.2:
Procedure
Coplin jar
Hemacolor solution 1
(fixing solution) 5 x 1sec
(colour reagent red) 3 x 1sec
(colour reagent blue) 6 x 1sec
Wash in buffer solution 2x10sec
Drying
Staining automat
Hemacolor solution 1 30sec
Wash in buffer solution 10sec
Tap water 10sec
Drying 3min
Result
Granulocytes:
- neutrophilic: granules light
violet
to red-brown
Hemacolor
Solution
Buffer solution pH 6.8 (7.2):
Procedure
Staining foils
Methanol 10sec
Add 1 drops buffer solution
and cover with stain foil 10min
Remove foil
Wash with buffer solution 1-2min
Result
Granulocytes:
- neutrophilic: granules light
violet
to red-brown
Sangodiff
The kit is for automatic blood smear

staining on Hema-Tek* Slide Stainer.
Use the solutions contained in the set to
fix, stain and wash blood smears.
* Trademark of Ames Company, Division Miles
Laboratories Inc., Elkat, Indiana, USA
Result
Granulocytes:
- neutrophilic: granules violet
Erythrocytes: red
Auto-Hemacolor
Ordering information
Method Product Merck Cat. No. Package size
Pappenheim May-Grnwald's 1.01424. 100 ml, 500 ml, 1 l, 2.5 l
eosin methylene blue solution
Giemsa's 1.09204. 100 ml, 500 ml, 1 l, 2.5 l
azure eosin methylene blue solution
May-Grnwald May-Grnwald's 1.01424. 100 ml, 500 ml, 1 l, 2.5 l
Giemsa Giemsa's 1.09204. 100 ml, 500 ml, 1 l, 2.5 l
azure eosin methylene blue solution
Wright Wright's 1.01383. 100 ml, 500 ml, 2.5 l
Leishman Leishman's 1.05387. 500 ml
Hemacolor Hemacolor staining set 1.11674. 100 ml of each solution 1, 2 and 3
for blood smears 3 buffer tablets pH 7.2
Hemacolor staining set 1.11661. 500 ml of each solution 1, 2 and 3
for blood smears 6 buffer tablets pH 7.2
Hemacolor-solution 1 1.11955. 2.5 l
fixing solution
Hemacolor-solution 2 1.11956. 2.5 l
colour reagent red
Hemacolor -solution 3 1.11957. 2.5 l
colour reagent blue
Sangodiff Sangodiff staining foils 1.15332. 100 staining foils,
for manual blood smear staining buffer solution pH 6.0
Auto-Hemacolor Auto-Hemacolor staining set 1.15213. 200 ml stains,
for automatic blood smear staining 500 ml buffer solution and
1 l wash solution
Additional reagents
Application Product Merck Cat. No. Package size
Waterquality Weise buffer tablets pH 7.2 1.09468 100 tablets
for preparing buffer solution acc. to Weise
Weise buffer tablets pH 6.8 1.11374 100 tablets
Weise buffer tablets pH 6.4 1.11373 100 tablets
Fixation Methanol GR f.a. ACS, ISO, Reag. Ph Eur 1.06009. 1 l, 2.5 l
Mounting media Entellan new 1.07961. 100 ml, 500 ml
Entellan 1.07960. 500 ml
Neo-Mount 1.09016. 500 ml
Immersion oil Immersion oil 1.04699. 100 ml, 500 ml
Immersion oil acc. to ISO 8036 mod. 1.15577. 100 ml
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Further information:
Merck KGaA
64271 Darmstadt, Germany
Phone +49 (0) 6151/72-0
Fax +49 (0) 6151/72-20 00
E-mail: microscopy@merck.de
www.merck-chemicals.com
We provide information and advice to our customers on application technologies and regulatory matters to the best of our
knowledge and ability, but without obligation or liability. Existing laws and regulations are to be observed in all cases by
our customers. This also applies in respect to any rights of third parties. Our information and advice do not relieve our
customers of their own responsibility for checking the suitability of our products for the envisaged purpose.

Stains: For Blood and Bone Marrow

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The kit is for automatic blood smear

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