Use of the Feulgen Reaction in Cytology I. Effect of Fixatives on the Reaction

1. In vitro experiments with nucleic acid impregnated agar blocks, using four different types of fixatives, show that the Feulgen nuclear stain gives two types of staining curves, depending on the presence or absence of chromic acid in the fixative. 2. With fixatives not containing chromic acid the maximum stain is produced by hydrolysis at 60⚬ C. extending from 4 to 8 minutes; after that time a gradual falling off of the stain takes place, until at 30 minutes no further stain is visible. With fixatives containing chromic acid the maximum stain is produced by hydrolysis at 60⚬ C. for 5-30.minutes. 3. That retention of the stain with increased hydrolysis is due to chromic acid has been demonstrated by testing the individual ingredients of the fixatives separately. In this case only chromic fixation produces the type of staining curve with the prolonged maximum. 4. None of the ingredients of the fixatives, if present at the concentration normally used, interfere with the stain. Chromic acid at high concentrations (above 5 per cent) and acting for a considerable length of time will prevent the stain, owing to destruction of the nuclear material. 5. Tannins, which have been considered by Milovidov to inhibit the stain, have the same effect as chromic acid in retaining it, except when a fixative is used that contains formalin. In that case there is considerable weakening of the stain. 6. Various plants from the different divisions of the plant kingdom, when treated with the four fixatives in the same way as the agar blocks, give the same results. A stain, usually a strong one, is obtained in all cases. In some cases the stain is not pronounced owing to dispersion of the chromatin in widely separated chromocenters or to the presence of a diffuse reticulum in a large nucleus. In one case, Marchantia, all types of nuclei show only a weak stain, suggesting a chemical difference, at least as regards part of the nucleic acid present. These are considered to be possible explanations of the frequently reported negative results. 7. It can be concluded in general that for the production of a satisfactory stain 5 minutes' hydrolysis with any of the regularly used cytological fixatives is sufficient.