The purpose of a differential stain is to colour the cells concerned in such a way that two populations can be distinguished from each other and from the background. This is certainly true of staining mixtures used to differentiate `live' and `dead' mammalian spermatozoa. In practice, the distinction is between eosinophilic and non-eosinophilic cells with a secondary stain (e.g. nigrosin) which appears only to provide a background.
The degree of background staining can be varied from none to intense by varying the concentration of the secondary stain. At very low concentration, the non-eosinophilic spermatozoa tend to merge with the background, their outline is difficult to distinguish clearly and, consequently, they may be overlooked in a count. As the concentration of the background stain is increased,