296 CYTOLOGICAL METHODS. 



Differentiate in- 

 Absolute alcohol . . . . .80 parts. 



Methyl alcohol 40 



Aq. dest 100 



till no more red comes out (three to five minutes). 



Wash in 80 per cent, alcohol, absolute and clove oil, xylol and 

 xylol balsam ; nuclei and cytoplasm, blue, glycogen red. 



It is a good plan when working on glycogen to prepare triplicate 

 slides, one for iodine stain, one for Best ; the other slide is brought 

 down to water and spat upon and set aside : the glycogen is dissolved 

 by the diastase of the saliva, the latter is washed off in water and the 

 slide stained as usual for Best's carmine. Comparison between the 

 first slide and this one will assist in properly identifying glycogen ; 

 (procedure of Dr. B. R. G. RUSSELL, Imperial Cancer Research Bureau). 



One generally succeeds at first trial with such material as the liver of 

 a rabbit, but with invertebrate materials, especially from paraffin 

 sections, even though soaked in 1 per cent, celloidin, the results are often 

 disappointing. This can be overcome by practice and by slight modi- 

 fication in the time used for differentiation. For delicate material it 

 seems best to work with celloidin sections. 



649. ZIEGLWALLNER'S Alcoholic Flemming for Glycogen and Fat. 



Neither the iodine nor Best's carmine method preserves fat as well as 

 glycogen. Zieglwallner has worked out the following method for 

 preserving both fat and glycogen. Fix small pieces of tissue in this 

 mixture for twenty-four to forty-eight hours : 



1 per cent, chromic acid in 80 per cent, alcohol 15-0 



2 per cent. Os0 4 in water . . . .4-0 

 Acetic acid . . . . . . 1 -0 



In 100 c.c. of this mixture there would be 50 per cent, alcohol. 

 If a corrosive sublimate fixation is necessary use this mixture in the 

 same way : 



Concentrated aq. sol. corrosive sublimate . 20-0 

 2 per cent. Os0 4 in water . . . .20-0 



Acetic acid . . . . . .10-0 



Alcohol absolute . . . . . .50-0 



In washing out, a little iodine will be necessary. Transfer the pieces 

 of tissue to 70 per cent., then upgrade and imbed in celloidin. 



In order to preserve the brownish black colour of the osmic stain of 

 fat, which soon disappears when the sections are brought to balsam, 

 one may convert the reduced osmic into its sulphide by adding a small 

 quantity of Na^S to the 70 per cent, alcohol which replaces the fixative. 

 Imbed in celloidin or wax : stain as by the iodine, or better in Best's 

 carmine method, from celloidin. Dr. J. A. Murray informs me that it 

 is generally necessary to stain sections first in warm iron alum, then 

 warm hsematoxylin, and then to differentiate in the cold with acid 

 alcohol. Afterwards proceed to Best's carmine. 

 PAUL BUCHNER (Praktikum der Zelknlehre /., Berlin, 1915) fixes 



