Stainsfor blood and bone marrowOverview 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

Advantagesof the hematological staining methodsfrom 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-Grünwald 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 10 ml Giemsa's solution to 190 ml buffer solution (pH 6.8 or 7.2), mix well, allow to stand 10 minutes and filter if necessary.

For staining automat Add 15 ml Giemsa's solution to 220 ml distilled water and 65 ml buffer solution (pH 6.8 or 7.2) mix well, allow to stand for 10 minutes and filter if necessary.

Procedure Staining rack May-Grünwald's solution 3 min Buffer solution (1ml) add, mix, stain 3–5 min Dilute Giemsa's solution 15–20 min Rinse with buffer solution Drying

Coplin jar Blood smears Air dried May-Grünwald's solution 3–5 min Dilute Giemsa's solution 15–20 min Wash in buffer solution 2 x 1 min Drying

Staining automat May-Grünwald's solution 4 min Dilute Giemsa solution 20 min Wash in buffer solution 1 min Tap water 2 min Drying 3 min

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 1. Buffer solution pH 6.8 (7.2)

Dissolve 1 tablet in 1l distilled water.2. Dilute May-Grünwald's solution:

Dilute 30 ml May-Grünwald's solution with 150 ml distilled water and 20 ml buffer solution.

For staining automat Add 30ml buffer solution and 220 ml distilled water to 50 ml May-Grünwald's solution, mix and allow to stand for 10 minutes before using.

Procedure Staining rack May-Grünwald's solution 3 min Buffer solution (1ml) add, mix, stain 6 min Rinse with buffer solution Drying

Coplin jar Blood smears Air dried May-Grünwald's solution 3 min Dilute May-Grünwald's solution 6 min Wash in buffer solution 2 x 1 min Drying

Staining automat May-Grünwald's solution 3min Dilute May-Grünwald's solution 6 min Wash in buffer solution 1 min Tap water 2 min Drying 3 min

Result Nuclei: red-purple to blue-violet Lymphocytes: plasma blue Monocytes: plasma grey-blue Granulocytes: - neutrophilic: granules light violet - eosinophilic: granules brick-red to dark violet - basophilic: granules dark violet

to black

Thrombocytes: violet Erythrocytes: reddish grey

May-Grünwald

Solution 1. Buffer solution pH 6.8 (7.2) Dissolve 1 tablet in 1l distilled water. 2. Dilute Giemsa's solution (1:20): Add 10 ml Giemsa's solution to 190 ml buffer solution (pH 6.8 or 7.2), mix well, allow to stand 10 minutes and filter if necessary.

For staining automat Slowly add 25 ml Giemsa solution to 275 ml buffer (pH 6.8 or 7.2) mix well, allow to stand for 10 minutes and filter if necessary.

Procedure Staining rack Methanol 3 min Dilute Giemsa's solution 15–20 min Rinse with buffer solution Drying

Coplin jar Blood smears Air dried Methanol 3–5 min Dilute Giemsa solution 15–30 min Wash in buffer solution 2 x 1 min Drying

Staining automat Methanol 3 min Dilute Giemsa solution 15–30 min Wash in buffer solution 1 min Tap water 2 min Drying 3 min

Result Nuclei: purple to violet Lymphocytes: plasma light blue Monocytes: plasma dove blue Granulocytes: - neutrophilic: granules delicate violet - eosinophilic: granules red to grey-blue - basophilic: granules strong violet Thrombocytes: violet Erythrocytes: weak red Blood parasites: nulei bright red

Giemsa

Solution1. Buffer solution pH 6.8 (7.2) Dissolve 1 tablet in 1l distilled water.2. Dilute Wright's solution: Dilute 30 ml Wright's solution with 150 ml distilled water and 20 ml buffer solution.For staining automatAdd 30 ml buffer solution and 220 ml distilled water to 50 ml Wright's solution.

ProcedureStaining rackWright's solution 1 minBuffer solution (1ml) add, mix, stain 4 minRinse with buffer solutionDryingCoplin jarBlood smears Air driedWright's solution 3 minDilute Wright's solution 6 minWash in buffer solution 2 x 1minDryingStaining automatWright's solution 3 minDilute Wright's solution 6 minWash in buffer solution 1 minTap water 2 minDrying 3 min

ResultNuclei: red violet to blue-violetLymphocytes: plasma light blueMonocytes: plasma dove-blueGranulocytes:- neutrophilic: granules light violet- eosinophilic: granules brick-red to red-brown- basophilic: granules dark violet to blackThrombocytes: violetErythrocytes: reddish-grey

Wright

Solution1. Buffer solution pH 6.8 (7.2) Dissolve 1 tablet in 1l distilled water.2. Dilute Leishman's solution: Dilute 30 ml Leishman's solution with 150 ml distilled water and 20 ml buffer solution.For staining automatAdd 30 ml buffer solution and 220 ml distilled water to 50 ml Leishman's solution.

ProcedureStaining rackLeishman's solution 1minBuffer solution (1ml) add, mix, stain 5 minRinse with buffer solutionDryingCoplin jarBlood smears Air driedLeishman's solution 3 minDilute Leishman's solution 6 minWash in buffer solution 2 x 1minDryingStaining automatLeishman's solution 3 minDilute Leishmany's solution 6 minWash in buffer solution 1minTap water 2 minDrying 3 min

ResultNuclei: purple to violetLymphocytes: plasma blueMonocytes: plasma grey-blueGranulocytes:- neutrophilic: granules violet- eosinophilic: granules brick-red- basophilic: granules dark violetThrombocytes: violetErythrocytes: red

Leishman

SolutionBuffer solution pH 7.2:Dissolve 1 tablet in 1l distilled water.

ProcedureCoplin jarBlood smears Air driedHemacolor® solution 1 (fixing solution) 5 x 1 secHemacolor® solution 2 (colour reagent red) 3 x 1 secHemacolor® solution 3 (colour reagent blue) 6 x 1 secWash in buffer solution 2x10 secDryingStaining automatHemacolor® solution 1 30 secHemacolor® solution 2 3 secHemacolor® solution 3 6 secWash in buffer solution 10 secTap water 10 secDrying 3 min

ResultNuclei: purple to violetLymphocytes: plasma light blueMonocytes: plasma dove blue Granulocytes:- neutrophilic: granules light violet- eosinophilic: granules brick-red to red-brown- basophilic: granules dark violetThrombocytes: violetErythrocytes: reddish

Hemacolor®

SolutionBuffer solution pH 6.8 (7.2):Dissolve 1 tablet in 1l distilled water.

ProcedureStaining foilsBlood smears Air driedMethanol 10 secAdd 1 drops buffer solution and cover with stain foil 10 minRemove foilWash with buffer solution 1-2 min

ResultNuclei: purple to violetLymphocytes: plasma light blueMonocytes: plasma dove blueGranulocytes:- neutrophilic: granules light violet- eosinophilic: granules brick-red to red-brown- basophilic: granules dark violetThrombocytes: violetErythrocytes: reddish

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 Nuclei: purple to violetLymphocytes: plasma blueMonocytes: plasma grey-blueGranulocytes:- neutrophilic: granules violet- eosinophilic: granules brick-red- basophilic: granules dark violetThrombocytes: violetErythrocytes: red

Auto-Hemacolor®

Ordering informationMethod Product Merck Cat. No. Package size

Pappenheim May-Grünwald'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-Grünwald May-Grünwald's 1.01424. 100 ml, 500 ml, 1 l, 2.5 l eosin methylene blue solution

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 eosin methylene blue solution

Leishman Leishman's 1.05387. 500 ml eosin methylene blue solution

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 for preparing buffer solution acc. to Weise

Weise buffer tablets pH 6.4 1.11373 100 tablets for preparing buffer solution acc. to Weise

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 KGaA64271 Darmstadt, GermanyPhone +49 (0) 6151/72-0Fax +49 (0) 6151/72-20 00E-mail: microscopy@merck.dewww.merck-chemicals.com

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