This is one of the common Romanwsky stains used in cytology. It is useful for studying cell morphology in air-dried smears. It is superior to Papanicolaou to study the cytoplasm, granules, vacuoles, basement membrane material etc. For nuclear staining Papanicolaou is superior.

Contents of the staining reagents:-

May-Grünwald solution                                               0.2%  
Methanol                                                                       99 %
May-Grünwald´s eosin-methylene blue                    0.2 %

Contains:- Eosin G, Methylene blue

Giemsa solution:-

Methanol                                                                       73 % 
Glycerol                                                                         26 %
Giemsa´s Azur-Eosin-Methylene blue                      0.6 %

Contains: Azur I, Eosin G, Methylene blue

Phosphate buffer:-

Potassium dihydrogen phosphate/ disodium hydrogen phosphate x 2H2O  (67.0 mmol/l)

Storage:-

Giemsa solution, May-Grünwald solution: protected from light at 2-25°C.
Unopened reagents may be used until the expiry date on the label.
Phosphate buffer: at 2-8°C. Unopened reagents may be used until the expiry date
on the label.

Preparation of working solutions:-

1. Buffered water: Dilute phosphate buffer with deionised or distilled water
1:20, e.g. 30 ml phosphate buffer + 570 ml deionised or distilled water.
2. Giemsa working solution : Mix 84 ml of Giemsa solution into 516 ml of
buffered water.
3. May-Grünwald working solution: Mix 360 ml of May-Grünwald solution
into 240 ml of buffered water.

Staining method:-

1. Fix the air-dried smear specimen in methanol for 10 -20 minutes
2. Stain with May-Grünwald working solution for 5 minutes
3. Stain with Giemsa working solution for 12 minutes
4. Wash with clean buffered water for 2, 5 and 2 minutes
5. Dry the slides in upright position at room temperature
6. Mount the slides with a coverslip using DPX
Any modifications to the staining procedure/working solutions may affect the
staining result, and are subject to precise method validation

Sources of errors:-

Irregular distribution of the blood smear on a glass slide may result in an erroneous cell counts. Alcohols used for wiping the skin may cause hemolysis and artifacts. Do not let the specimens dry at any stage of the staining procedure. Wash properly to avoid dye artifacts. Buffered water is strongly recommended for washing. Staining result is dependent on pH. Alkaline pH increases blue and acidic pH pink or reddish tinge in the stained specimen.

Ziehl-Neelsen stain:-