40'4 University of California Publications in Agricultural Sciences [Vol. 2 



The buds are put in the Carnoy fixative and, as for Crepis, the 

 tips of the involucral scales are removed in order to facilitate pene- 

 tration of the fixative between the florets. The buds remain in the 

 first fixative from three to ten minutes, when they are changed into 

 Xavashin's formalin-chromic-acetic acid. Carnoy 's fluid seems to 

 prepare the way through the tissue for the second fixative, but in 

 the short time of application it does not dehydrate or collapse the 

 tissue. Kihara (1924) describes a combination Carnoy-Flemming's 

 fluid which is based on the same principle as the combination here 

 described. 



As for the stains, Heidenhain's is too little transparent for Crepis 

 chromosomes which usually must be counted in side ^dew. Both 

 iodine-gentian violet and iron-acetocarmine (in smears) give good 

 transparent stains, but of these tw^o the gentian violet gives the most 

 contrast and the clearest, most unquestionable counts. Iron-aceto- 

 carmine applied on Carnoy-fixed material does not seem to be inferior 

 to the fresh fixed and stained smears. The Carnoy-Navashin fixative 

 is not so adequate for iron-acetocarmine smears as the Carnoy fluid, 

 because the material is not brittle enough to be pulled out in very 

 small, thin pieces. Perhaps the application of absolute alcohol for 

 some time would give the Carnoy-Navashin fixed material the con- 

 sistency that is necessary for making good smears. 



The best way of applying the gentian violet for Crepis w^as to omit 

 the iodine treatment before the staining, going directly from 70 per 

 cent alcohol to 1 per cent gentian violet in water, applied for 5 

 minutes. After rinsing in water the slides were put in a solution of 

 1 gram iodine and 1 gram iodide of postassium in 100 cc. of 70 per 

 cent alcohol. Here they stayed for 30 to 45 seconds and thereafter 

 they were rushed through 70 per cent, 95 per cent, and absolute 

 alcohol to pure clove oil with some orange G. Here the differentiation 

 took place and after washing in xylol with a few drops of absolute 

 alcohol in it they were put in pure xylol and afterwards mounted in 

 balsam. The differentiation is often so perfect that the plasm is 

 totally unstained if no orange G has been applied. In sectioned 

 material it is necessary that one be able to .see the limits up and 

 down of the section in order to be sure that the pollen mother cell 

 has not been cut. The orange G stains the plasm sufficiently to make 

 its structure visible and still it gives so good a contrast to the gentian- 

 violet stained and transparent chromosomes that these are very clearly 

 differentiated from the plasm. 



