NOTES ON THE CYTOLOGICAL TECHNIQUE 



Since osmic acid in solution is unstable a stock solution without this component was 

 generally made up. When required for use, i c.c. of 2% osmic added to 7-3 c.c. of stock 

 solution makes the complete fixative. 



STAINING 



For most sections of meiosis Heidenhain's haematoxylin used as described below was 

 successful, and with good fixation a counterstain with Bismark brown to show up cyto- 

 plasm and cell walls is a great improvement. For roots a more transparent stain is 

 needed wherever chromosome numbers are high, and for this purpose gentian violet was 

 satisfactory. With both these stains a difficulty very frequently met with in roots is the 

 presence of large amounts of some substance in the cytoplasm (perhaps tannin) which 

 holds the stain tenaciously and is liable to disfigure preparations, although, if this can 

 be tolerated, clear staining of the chromosomes even in heavily affected cells can be 

 obtained (see, for example, Fig. 187). This difficulty is less troublesome after osmic 

 fixation, but the chromosomes are as a rule easier to count after chromic fixation (in 

 roots) so that the disfigurement has usually to be accepted. The innermost layers of 

 roots are free from this difficulty but are generally less suitable for chromosome counts. 

 Feulgen staining was used very rarely with sectioned material (see Fig. ^id), though it 

 appeared to present no difficulty. With squash material this stain was sometimes bril- 

 liantly successful after acetic-alcohol fixation, notably in Equisetum and Ophioglossum. In 

 some other cases, notably meiosis in the Osmundaceae and Polypodiaceae, it failed 

 completely after this fixative and was not further investigated, since in most of these 

 species acetocarmine was found to be adequate. The schedule for acetocarmine and 

 Feulgen staining used will be described below under Squashes. 



STAINING PROCEDURE WITH HAEMATOXYLIN 



Mordant half an hour. Wash in running water for three-quarters of an hour. Stain 

 overnight. Rinse in running water (half-hour). Differentiate in alum. Wash for 4 hr. 

 Dehydrate, clear and mount. For details of the solutions and for the insertion of a counter 

 stain see next section. 



Note. In places with hard water, preparations should probably be dipped in distilled 

 water before entering the stain. This, however, was not necessary in either Manchester 

 or Leeds, where the domestic tap water is exceedingly soft. 



FORMULAE OF STAINS 



Haematoxylin was prepared and used as advised by Dame Helen Gwynne-Vaughan. The 

 dry stain, supplied by Messrs Gurr of London, was dissolved in absolute alcohol in a 10% 

 concentration and left for at least 3 months. When required for use, 5 c.c. of this solution 

 is made up to 100 c.c. with distilled water; it is then stirred with a glass rod previously 

 dipped in mordanting alum and then left for 4 days. After this, staining is at first 

 somewhat faint, though preparations become brighter with keeping. The pot improves 

 considerably after a month of constant use, and it will then remain in good condition for 

 at least a year. The stock solution will remain in good condition for several years but 



294 



