NOTES ON THE CYTOLOGICAL TECHNIQ.UE 



or spoiled when the shde and cover-shp were first separated, and the benefit was then 

 reaped of the prehminary photographs. 



IV. Modification of the acetocarmine technique. Orcine and Lacmoid were both tried as a 

 substitute for carmine (La Cour and Darhngton), though no benefit was felt. For the 

 Pteridophyta, carmine when properly used is very satisfactory, and the reagent is also 

 stable enough to remain in good condition for a year with only occasional filtering. 

 Lacmoid, under certain circumstances, had the disadvantage of rendering the chromo- 

 some brittle and was discarded for this reason. 



Experiments were also made with alternative mountants such as 'Euparal', and 

 though satisfactory these showed no advantage over balsam and were therefore not 

 extensively employed. Euparal, indeed, has a disadvantage over balsam in that it is 

 soluble in the immersion fluid that was used (Leitz 'Objektol'), which precludes the 

 making of high-power observations near the edge of a slide. For this reason alone it 

 would have been discarded. 



V. Feulgen squashes. These were only used sparingly as a check on certain troublesome 

 genera such as Equisetum, which had proved difficult to complete by other means. Both 

 in Equisetum and Ophioglossum very beautiful preparations were obtained by this means, 

 though the much smaller size of the individual chromosomes after this treatment than 

 when swollen by acetocarmine meant that little if any additional facts were learnt. The 

 chromosomes also became somewhat brittle and liable to separate at secondary con- 

 strictions. 



The procedure adopted was as follows : 



(i) Fix in acetic-alcohol for lo min. to half an hour. 



(ii) Transfer to water. 



(iii) Hydrolyse in normal HCl at 60° C. for 10-20 min. according to the material. 



(iv) Transfer to Schiff 's reagent (see p. 295) in the dark for half to 2 hr. 



(v) Transfer to 45% acetic acid on a slide, tease out, cover, warm gently and squash 

 gently under a piece of blotting paper. 



(vi) Examine at once, and if successful transfer to balsam by McChntock's method 

 (p. 296 above). 



Examples of this technique are reproduced in Figs. 218a and 220. 



OBSERVING 



There was nothing worthy of special note about the methods of observing exceot 

 perhaps the very close use of photography as an integral part of observation at 

 every stage (for further details see Appendix 2), and the liquid used as an immersion 

 fluid. The frequent necessity of making detailed observations on unringed liquid 

 mounts made the change from cedar oil as the immersion fluid to ' Objektol ' very advan- 

 tageous. Objektol (which is obtainable commercially from Messrs. Leitz of London) 

 does not stiffen on exposure to air and can be washed oflT with water. This means that 

 its presence is not a difficulty when slides have to be processed further, since it merely 

 disperses in the acetic acid, and being very liquid throughout the examination reduces 

 considerably the risk of damage to the slide by accidental movement of the cover-slip 

 which can occur when cedar oil has become stiff. 



297 



