APPENDIX 1 

 NOTES ON THE CYTOLOGICAL TECHNIQUE 



FIXATION 



The best fixatives for sections of most members of the Pteridophyta are half-strength 

 chromacetic-formaHn for roots and 2BD for sporangia. Full-strength chromacetic- 

 formalin can also be used for sporangia and for these is better than the half strength. 

 The formulae of these fixatives are given below. Sporangia should always be momentarily 

 dipped in alcohol (70-90%) or acetic-alcohol as a preliminary to fixation, though care 

 should be taken not to carry over excess of alcohol into the aqueous fixative ; a con- 

 venient method is to dip a sorus or sporangium into spirit for just long enough to wet it, 

 then to put it momentarily in contact with a piece of fabric to absorb the surplus alcohol 

 before transferring quickly to the proper fixative. Very large sporangia such as those of 

 the Psilotales should be punctured before fixing. 



Bleaching before staining is of course necessary after the osmic fixative. This can be done 

 in the usual way by leaving wax-free sections in diluted hydrogen peroxide overnight. 



For squash preparations of every kind acetic-alcohol fixation is generally satisfactory. 

 The precise concentration is not critical, i : 3 glacial acetic acid : absolute alcohol is 

 correct for some genera, e.g. Polypodium, though for others i : 2 is sometimes better. In 

 cold weather, fixation of several days (3 days to a week) is required for successful carmine 

 mounts, though in the height of summer overnight is sufficient. For Feulgen squashes 

 10 min. to half an hour's fixation is often satisfactory. 



FORMULAE OF AQ.UEOUS FIXATIVES 

 I. Chromacetic-formalin: 



Mix in equal parts immediately before use. For the half-strength fixative dilute with 

 an equal volume of water after mixing. 



II. 2BD (La Cour, 1931): 



Chromic acid 1% 100 c.c. 



Potassium bichromate 1% 100 c.c. 



Saponin o-i g. 



Osmic acid 2% 30 c.c. 



Acetic acid 5% 30 c.c. 



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