Fixation in the context of diagnostic cytopathology today traces its roots to Dr. Papanicolaou's use of equal parts of diethyl ether and 95% ethyl alcohol. He first described this fixative in a 1917 paper about the existence of a typical estrous cycle in the guinea pig – although there is no mention of that fixative in that paper. Thus, Papanicolaou published no rationale for his choice of alcohol and ether. This fixative became the standard in diagnostic cytopathology after Dr. Papanicolaou recorded its use in the 1942 monograph that he co-authored with Dr. Herbert Traut.

Papanicolaou did not publish his rationale for including ether in alcohol. Ether has no fixative properties. Since it is an effective fat solvent, ether may serve as an adjuvant, which speeds alcohol's penetration into cells. In blinded comparisons with cells fixed only in 95% alcohol, no discernible difference is evident.

Ether, of course, is highly volatile and explosive. More than a few non-explosion proof refrigerators have exploded when storing ether that was ignited by a spark. Consequently, its use was generally discontinued by the late 1950s, leaving 95% ethanol alone as the standard fixative for Pap smears (Figure 6.1). Indeed, the very first issue of Acta Cytologica published in 1957 included a write-in symposium that addressed “experiences with various methods of fixation of smears.”

In practice, a fresh cellular sample is spread onto a glass slide and immediately fixed by plunging it into alcohol (i.e. wet-fixation). Air-drying should not be allowed to occur either before, during, or after fixation.