260 Hill . — The Histology of the Sieve- Tubes of Angiosperms. 
callus and protoplasm shall be possible in the same section. With the 
Safranin method it is possible, if deeper staining is required, either to 
remordant with any of the proper mordants and restain with Safranin or 
with Gentian Violet, or else to use the Acid Blue method after the first 
staining with Safranin. 
The Acid Blue method, which is sometimes a useful alternative to the 
Safranin staining, may be used on sections cut from fixed or fresh material, 
but cannot be used directly with any success on material which has been 
mordanted for Safranin ; intensification can be brought about by repetition 
of the process. By this method results are somewhat artificial ; it is not 
always easy to be certain whether the effects produced are due to precipi- 
tation between the Iodine and the dye taking place in empty spaces, or 
whether they represent the staining of actual structure. In addition to this 
it is not possible to stain the callus properly with Water Blue or other 
reagents, since the action of the acid distorts the callus, and the action of 
the Water Blue tends to upset the protoplasmic staining. The unchecked 
use of this method has led Kuhla 1 into error with regard to the structure 
of the sieve- plates, &c. of Cimirbita , and though the modification of Meyer’s 
Acid Violet (Pyoktanin) method, by which staining takes place in watch- 
glasses and not on the slide, gives fairly reliable results, it can only be 
used usefully in comparison with the Safranin method. 
It may be as well to remark here that the success of the modification 
of the Acid-Violet method is due to the employment of Benzyl Blue 2 , 
a peculiar impure dye made some twenty-five years ago by the Actien- 
Gesellschaft, &c. of Berlin. A dye is still sold under the same name, but it is 
now made by a different process, with the result that it is practically useless 
for the method. 
A third method which has recently been devised is, in principle, like 
the Safranin method, over which, in some cases, it has certain advantages. 
Sections of fixed material may be mordanted with a weak solution of Iron 
Alum, and after a brief washing staining is effected by an Aniline water 
solution of Water Blue made up after the manner of the Safranin solution. 
By this method the cell-walls are not stained at all, which is an advantage 
over the Safranin method, and the Water Blue in this case does not stain 
the callus. Intensification of the staining is possible by repetition of the 
process, and a very dark staining of protoplasm and threads is obtained 
with scarcely any coloration of the cell-wall, and without any precipitation 
effects. 
The stained sections are mounted for observation either in Glycerine 
with Iodine, or a mixture of Glycerine and Zinc Chloride with Iodine, or in 
Glycerine Jelly, and there is nothing to add to what has been already 
published. Preparations so mounted are found to be quite clear at the end of 
1 Kuhla, Bot. Zeit, 1900, pp. 38 et seq. 2 The value of this dye was discovered by Gardiner. 
