Prepared by
ROY ELLIS
IMVS Division of Pathology
The Queen Elizabeth Hospital
Woodville Road, Woodville, South Australia 5011
Principle
Giemsa's stain is a member of the Romanowski group of stains, which are defined as being the black precipitate formed from the addition of aqueous solutions of methylene blue and eosin, dissolved in methanol. The variants of the Romanowski group differ in the degree of oxidation (polychroming) of the methylene blue stain prior to the precipitation.
The stain class was originally designed to incorporate cytoplasmic (pink) staining with nuclear (blue) staining and fixation as a single step for smears and thin films of tissue (spread preparations of omentum). Minor modifications of working stain concentration and staining time have been made over the years for fixed tissue sections.
The Romanowski stains are extremely tedious to prepare, and are best purchased as the commercially available pre-made stock stain.
Technical Points
1. (step 2) - Usually the staining is performed at room temperature overnight, however, increasing the stain temperature shortens staining time. Sections stained at 37°C for several hours, (staining time assessed by microscopical examination), produce better results than sections stained at 60°C for a shorter period. The higher the staining temperature, the greater the intensity of blue staining, but without the equivalently increased red staining - see technical point 2 below.
2. Differentiation with acetic acid will vary according to the staining time and temperature, but it is generally achieved within 30 secs. The differentiating agent removes only the blue dye component, thus increasing the apparent intensity of the red component.
Method
1. Bring sections to distilled water
2. Stain with diluted Giemsa's stain made up fresh (see technical point 1)
3. Rinse in distilled water
4. Differentiate with 0.5% aqueous acetic acid (see technical point 2)
5. Dehydrate rapidly
6. Clear and mount
Results
bile pigments.........................................................green
collagen, muscle, bone..............................................pale pink
micro-organisms, fungi, parasites.................................purplish-blue
starch granules, cellulose............................................sky blue
pigments (native colour is yellow/brown, or if fixed in dichromate containing fixative)...green
nuclei..................................................................dark blue to violet
erythrocytes..........................................................salmon pink
cytoplasm…………………...............................................varying light blue shades
Reagent Formulae
1. Giemsa's stain, stock solution – obtain from commercial sources.
Giemsa reagent improves with age, expiry is unimportant.
Giemsa stain, working solution
Giemsa stock solution ------ 40 drops
Distilled water --------------- 40 ml
The diluted stain keeps well, but is best made up fresh each time.
2. Acetic acid 0.5%
References
Faraco 1938
Giemsa G,(1902),Zentbl
Lillie, R.D. 1965, Histopathologic Technique and Practical Histochemistry, 3rd edition, McGraw-Hill
Lillie, R.D. 1977, H.J. Conn's Biological Stains, 9th edition, Williams and Wilkins