Can any one help me get a protocol for giemsa staining for bone marrow of wistar albino rats?

Manual Staining of Bone Marrow Preparations: Wright's and Wright-Giemsa Stain

Wright's or Wright-Giemsa stains are usually the preferred staining method for bone marrow aspirate smears. These are methanol-based staining solutions with similar dye composition to the diff-quick stain but require longer stain contact time for adequate staining. The Wright's and Wright-Giemsa stains have a buffer step as well. Since Wright's stains are methanol based they do not require a fixation step prior to staining, although you might prefer to do so first to reduce water artifact that can occur on humid days or with aged stain.

In the dip method of staining, the smears are first dipped in methanol to fix the specimens and then placed in Wright's or Wright-Geimsa stain for 10-15 minutes to stain. The smears are next moved to a mixture of stain and 6.8 pH phosphate buffer (usually one part stain to 2-3 parts buffer) and allowed to stain for at 20-30 minutes. After staining, they are given a quick rinse in distilled water and allowed to air dry before mounting or cover-slipping.

When using a staining rack, the marrow slides or coverslips are first flooded with enough stain to cover the slide and stained for 10-15 minutes. Then, a 6.8 pH buffer is carefully added without overflowing and gently mixed by blowing until a green metallic sheen forms. This is allowed to stand for 20-30 minutes and then rinsed off with distilled water. The slides or coverslips are then air dried and mounted.

Staining times can be extended for extremely cellular marrows; however, care must be taken when using the rack staining method. Extended times can lead to evaporation of the stain and cause excessive precipitation. Both the stain and buffer can be topped up if necessary to prevent this from occuring, while additional rinse time may be needed.

Wright's and Wright-Giemsa stains, when performed properly, give sharp and clear nuclear, cytoplasmic, and granule detail. There can be variation in the quality of the stain from batch to batch, dependent on the manufacturer's quality control, storage, and shipping conditions. Many manufacturers age their stains for a minimal amount of time before shipping and assume that there will be additional standing time at the distributor before it reaches your lab. This may work for peripheral blood staining, but it is not ideal for bone marrow staining. It is advisable, if possible, to keep a separate stock of Wright's stain for bone marrow staining which is kept at least 6 months before use. Like a fine wine, the older Wright's stain gets, the better the quality and clarity of the final stain.

May Grunwald/Giemsa for Stain Bone Marrow

Fixation:

1. Smears: Draw a curetted fragment lightly across a sterile slide in a serpentine path. Don’t allow the streak to dry completely. Start with step #4.

2. Sections: Zenkers’s or other well-fixed tissue. Start with step #1

Sections:

Paraffin, 6 microns

Staining Procedures:

1. Deparaffinize and hydrate to distilled water.

2. If use Zerker’s contains mercury, remove mercuric chloride crystals with Lugol’s or Gram’s Iodine solution, clear with Sodium Thiosulfate, 5% for 3 minutes. If Zenker’s use is contained no mercury, go to step #4

3. Wash in tap water, 10 minutes and rinse in distilled water for two changes

4. Place promptly in Methyl Alcohol, two changes, three minutes each.

5. Stain with May Grunwald Solution or Jenner Solution 1A3 minutes. Add an equal volume of distilled water and allow to stand one minutes. Drai, no rinsing. Alternatively, stain in Working Jenner’s (dilute 1:1 with distilled water) 6 minutes.

a. Sections: Stain in Working Giemsa (freshly dilute 2.5ml Giemsa Stock Solution in 50ml distilled water, do not reuse), 45 minutes

b. Smears: Cover for 12 minutes with Working Giemsa (15 drops of Giemsa Stock Solution to 10 drops of distilled water)

a. Sections: Differentiate each slide individually in Acetic Acid, 1% with gentle agitation, checking often under the microscope until nuclei are well defined. Rinse quickly in distilled water.

b. Smears: Differentiate in distilled water, agitating for approximately 5 seconds and checking under the microscope.

8. Section only: dehydrate quickly in 95% alcohol and then absolute alcohol, and clear in two changes of xylene.

a. Sections: Mount with Permount or DPX

b. Smears: Blot with fine grain filter paper and mount.

Stain Results:

Nuclei, Bacteria Blue

Cystoplasm Pink-Rose

Maximow's Method for Bone Marrow

Fixation:

Zenker’s / Formalin

Sections:

Paraffin at 6 microns

Staining Procedures:

1. Deparaffinize and hydrate to distilled water.

2. Mayer’s Hematoxylin for 15 minutes. Wash in tepid tap water for 15 minutes.

3. Stain in working solution overnight. Prepare working solution fresh each time as follows:

a. Eosin Y, 0.1%, Aqueous - 10 ml

Triple distilled water or Phosphate Buffer - 100 ml

Azur II, 0.1%, Aqueous - 10 ml

Glacial Acetic Acid - 2 drops

4. Differentiate in 95% alcohol until blue ceases to come out into alcohol and erythrocytes and collagen are pink.

5. Dehydrate in absolute alcohol, clear in xylene, two changes each.

6. Mount with Permount.

Stain Results:

Nuclei Blue

Basophile leucocyte, mast cell Purple to Violet

Cartilage Purple

Erythrocytes, eosinophile granules Pink

Cytoplasm Blue to Pink