282 
Journal of Agricultural Research 
Vol. XI, No. 6 
cut them with the freezing microtome if the operator is careful not to 
cut them too thin. The advantage in having sections of such large size 
lies in the fact that the observer can explore the whole of the suscep¬ 
tible regions for evidence of the mycelium of the parasite under condi¬ 
tions really favorable for its discovery. 
No killing solution is used, but, so far as can be observed, no shrinkage 
at all occurs in the hyphae, and but little in the host cells of fresh speci¬ 
mens. The blocks of bark and wood should be cut into pieces approxi¬ 
mately }4 to % inch on a side. The prepared blocks are then soaked 
for about io minutes in a io per cent aqueous solution of gum arabic 
containing 0.5 per cent of phenol. Blocks cut from fresh specimens can 
be put to soak at once. If the specimen is old and dry, the blocks should 
be boiled before being put into the gum arabic. The same strength 
gum-arabic solution is used as a freezing medium. If the material be 
embedded in paraffin, the usual process of sectioning and mounting can 
be followed. Excellent sections of young pine bark extending from 
cambium to epidermis can be easily cut 5 fx thick from paraffin material. 
However, for ordinary diagnosis or morphological study of the fungus 
hyphae sections 15 to 20 ix thick, cut on the freezing microtome, are very 
satisfactory. Such sections should be removed from the knife with a moist 
camers-hair brush and rinsed in several changes of clean water. They 
can be handled very conveniently in bevel-ground Syracuse watch glasses. 
(b) Stains. —The stains, safranin and lichtgruen, should be kept in 
dropper bottles of 25-to-50-c. c. capacity and used from the dropper or 
pipette. Similar bottles should be used for absolute alcohol, clove oil, 
and xylol. The writer uses the alcoholic solution of safranin almost 
exclusively. This method is described by Chamberlain 1 as follows: 
The alcoholic solution is made by dissolving 1 g. of the alcoholic safranin in 100 
c. c. of 95 per cent or absolute alcohol, and, after the safranin is completely dissolved, 
adding 50 c. c. of distilled water. 
This stain may be used over and over again, but it should preferably 
be filtered back into the drop bottle after each staining. A convenient 
stock solution of lichtgruen is a 1 per cent in 95 per cent alcohol. A con¬ 
venient strength for use is one-half stock, made by diluting the stock 
with an equal volume of 95 per cent alcohol. For thin sections, how¬ 
ever, from paraffin material, better results can be obtained by the rapid 
use of the stain at full strength. In addition to the regular drop bottle 
pipettes, there should be two or three others of good capacity and small 
tube opening for use in draining off the stains or excess reagents during 
the steps of the process. 
(c) Staining process. —The sections should be rinsed several times 
and then drained of all excess water and a generous quantity of safranin 
added. This stain should remain for from two to four hours, after 
1 Chamberlain, C. J. methods in plant histology, ed. 3, P- 51. Chicago, 1915. 
