Fungus of Loltuvi. 



loy- 



but if a paraffin Avitli a fairly low ineltin^' point Ije us<.'d, it was 

 found quite possible to obtain good and serial sections, after using 

 this fixing reagent. At a later stage Bouin's fixative was employed, 

 more particularly when dealing with the later stages in the develop- 

 ment of the grain. It was quite easy to obtain absolutely entire 

 sections after the specimens have been hxed in this way. The disad- 

 vantage lies in the fact that the starch in the endosperm was not 

 well preserved, and also after this fixative the staining reactions, 

 with the stains employed, do not seem to be as brilliant as they are 

 following upon Carnoy's fixative. 



The ether-freezing microtome was not satisfactory, owing to- 

 the difficulty in obtaining serial sections, and it was generally 

 necessary to do this. Again, it was impossible to obtain as thin 

 sections in this manner as with the paraffin method. 



Hand sections were practically useless. They can only confirm 

 the presence of the hyphae in the grain, but evidence as to their 

 absence cannot be drawn from them. 



The stain most generally employed, in fact, solely, as regards the 

 mature grain, was aniline gentian violet. i In using this stain care 

 must be taken to see that it is always fresh, as it does not keep well. 

 Its staining capacity diminishes rapidly after several days. This- 

 stain was washed out with Gram's iodine water, then with absolute 

 alcohol. Sections were next cleared in clove oil, and mounted in 

 balsam. 



Excellent results were obtained with this stain, the hyphae for 

 the most part being stained a brilliant bluish purple, and the endo- 

 sperm reacting to the iodine. 2 It far exceeded any other stain I 

 have tried, among them being Haidenhain's haematoxylin. aniline 

 safranin, erythrosin, aniline blue, etc. The aleurone cells for the 

 most part and the cells of the scutellum and embryo do not stain, 

 so that the hyphae present in these tissues stand out in striking 

 contrast to the colourless cells around them. 



This stain, used by itself, was only useful when dealing with the 

 mature grain. In studying the embryology of the grasses in ques- 

 tion it was necessary to counter-stain. Sections of the ovary 

 before, and at the time f)f fertilisation, wore stained with Bismarck 



1. One Soloid tabloid of gentian violet dissolved In 7 ccs. of abs. ale. and 

 63 CCS. of water containing 2.S ccs. of aniline solution. 



2. The colourisation of the endosperm by the Iodine Is. of cour.se. not per- 

 manent, although the hyphae retain the violet stain well. This is certainly 

 a drawback to the method, but It is more than compensjited for by the excel- 

 lent re.sults obtained, which, Indeed, are not approached by any other method. 



