Nuances of the Papanicolaou stain.

The impressive list of achievements of Dr. G. N. Papanicolaou and his tedious journey from normal to abnormal human cell includes the importance of wet fixation of cells and the development of the unique polychromatic Pap stain. The 5-dye Pap stain method evolved through 2 salient phases. The first being the development of wet fixation using alcohol-ether to enhance cellular transparency and the second phase saw the introduction of various cytoplasmic counterstaining methods using orange G and EA (light green, Bismarck brown, eosin) and phosphotungstic acid, facilitating the distinction of cell types. The specific characteristics of the staining method is, the cellular transparency combined with crisp nuclear staining, achieved through tailored cellular fixation and cytoplasmic staining using variable dye and pH combinations. With little modifications if any the Pap stain continues to be applied uniformly globally. However, institutional supply of dyes and chemicals from different companies make minor modifications, that remain consistent, an essential part of the staining protocol. This chapter describes the preparation and principles of various components of the stain that are being currently used in our department.