Description: Papanicolaou stain (also Papanicolaou's stain and Pap stain) is a multichromatic staining histological technique developed by George Papanikolaou, the father of cytopathology. Pap staining is used to differentiate cells in smear preparations of various bodily secretions; the specimens can be gynecological smears (Pap smears), sputum, brushings, washings, urine, cerebrospinal fluid, abdominal fluid, pleural fluid, synovial fluid, seminal fluid, fine needle aspiration material, tumor touch samples, or other materials containing cells. Pap staining is a very reliable technique. As such, it is used for cervical cancer screening in gynecology. The entire procedure is known as Pap smear.
The classic form of Pap stain involves five dyes in three solutions:
When performed properly, the stained specimen should display hues from the entire spectrum: red, orange, yellow, green, blue, and violet. The chromatin patterns are well visible, the cells from borderline lesions are easier to interpret, the photomicrographs are better, and the stained cells are pretty. The staining results in very transparent cells, so even thicker specimens with overlapping cells can be interpreted.
On a well prepared specimen, the cell nuclei are crisp blue to black. Cells with high content of keratin are yellow, glycogen stains yellow as well. Superficial cells are orange to pink, and intermediate and parabasal cells are turquoise green to blue. Metaplastic cells often stain both green and pink at once.
Fixation: Fix smear according to standard procedure. For example, 95% alcohol or 100% methanol.
Procedure 1 (Standard Method):
95% Ethanol 15 minutes (fixation)
Rinse in tap water
Harris or Gill Hematoxylin 1-3 minutes (Time vary with selection of hematoxylin solution)
Rinse in tap water or Scott's tap water
95% Ethanol 10 dips
OG-6 stain for 1.5 minutes.
95% Ethanol 10 dips
EA-50, or Modified EA-50, or EA-65 stain for 2.5 minutes.
95% Ethanol 10 dips, 2 changes
100% Ethanol 1 minute
Clear in 2 changes of xylene, 2 minutes each
Mount with permanent mounting medium
Procedure 2 (Modified Pap Procedure):
95% Ethanol 15 minutes (fixation)
Distilled water 10 dips, 2 changes
Gill Hematoxylin 2 minutes
Distilled water 10 dips
Scott's tap water 1 minute
Distilled water 10 dips, 2 changes
95% Ethanol 10 dips, 2 changes
OG-6 stain for 1-2 minutes
95% Ethanol 10 dips, 3 changes
EA-50 or EA-65 stain for 6-10 minutes
95% Ethanol 20-30 dips, 3 changes
Absolute ethanol 10 dips
Clear in xylene
Mount with permanent mounting medium
Procedure 3 (Rapid Economic, Acetic Acid, Papanicolaou Stain Method):
Blotting was done after each step. Mount by D.P.X
References:
Rapid Economic, Acetic Acid, Papanicolaou Stain Method