IOWA ACADEMY OP SCIENCES 
263 
nuclei of the neurilemma on these fibrils have more of a tendency to 
roundness and that the individual fibrils end as such and not in a bulb. 
He states that the endings have little of the medullary sheath. 
This investigation was begun in 1904, and was presented in somewhat 
different form to the faculty of Ohio University in June, 1905, as a 
thesis for the degree of A. M. Research has been continued since then 
as time would permit. The animal thus far used for observation was 
the common North American toad {Bufo lentiginosus) and the method 
employed essentially that of Sihler's. The microscope chiefly used has 
a Zeiss aprochromatic oil immersion two millimeter lens. 
Fresh tissue was macerated, stained, teased, and mounted in glycer- 
ine on slides. 
The macerating fluid is made as follows: 
Ordinary acetic acid 1 part 
Glycerine -.1 part 
Chloral hydrate 1 per cent, solution in distilled water 6 parts 
The muscles should be in bundles of net more than three or four 
millimeters in diameter. Those of the foot or fore limb of the toad or 
frog are already of convenient size. 
The staining material is prepared as follows: 
Well ripened Erlich’s Haematoxylin 1 part 
Glycerine 1 part 
Chloral 1 per cent, solution in distilled water 6 parts 
The muscle should be macerated from 12 to 20 hours depending upon 
the age of the animal. It would do no harm to use stronger acetic acid 
for older animals. The object is to dissolve the inter-muscular connec- 
tive tissue, which is accomplished when the muscle no longer offers much 
resistance to teasing. One should begin testing the matter as soon as 
12 hours have passed. When properly macerated the tissue should be 
left in glycerine until it becomes saturated, which requires about two 
hours. 
Before putting the tissue into the staining fluid it should be further 
teased into bundles not more than one or two millimeters in diameter. 
All teasing should be done in glycerine. Staining requires from 3 to 10 
days. When properly stained the muscle fibres have a color ranging 
from wine color to nearly black. Nervous and muscular material are 
stained almost black, while connective tissue remains quite pale. The 
darkened nuclei of the capillaries which are everywhere so abundant are 
a good index to the intensity of the staining process, since nerve struc- 
tures take the stain at about the same^rate with them. All the parts do 
not stain uniformly. Parts that are over-stained can be readily reduced 
in color by subjecting them to a weak solution of acetic acid. A con- 
venient way to accomplish this is to immerse them in glycerine, to which 
has been added a small amount of acetic acid. The stained tissue may 
be kept unchanged almost indefinitely in glycerine. 
A convenient way to find nerve structure is to tease quite a number 
of muscle bundles still smaller until each remaining part contains per- 
haps half a dozen fibres. These in a shallow glass dish may be examined 
