296 
BULLETIN OP THE BUREAU OF FISHERIES 
species, especially shape and pigment marldng, which make them easily distinguished 
subsequently, but the first attempt at identification of field collections is possible 
only by counting the vertebrae. This information, together with knowledge of the 
adult fish fauna of the locality, permits us to narrow down the possibilities and very 
often to detect the species immediately. Unfortunately, the importance of the 
vertebral count has only recently been recognized, and thus the older descriptions 
of fish do not contain it. Wherever possible during the work, we have made these 
counts of the adults as well as the young. In some small specimens strong light is 
sufficient to reveal the spinal column, but usually it is necessary to stain and clear, 
and in larger fishes to bisect. The limited time of the survey prevented extensive 
staining, and when the spinal column was not readily shown, myomere counts were 
made which, although not identical, correspond to the vertebral count sufficiently and 
are constant enough for the usual requirements. 
No detailed rules can be laid down for the technique of staining all young fishes, 
for the variation in size, permeability, and general reaction seems to make each 
species and often each specimen a problem in itself. Most processes are long and 
require considerable watching, but painstaking care and patience will surely produce 
results worth the effort. (See fig. 121 and other photographs of stained and cleared 
specimens in the following text.) The several methods used with success by the 
author are briefly outlined below. 
VERY SMALL SPECIMENS WITH CARTILAGINOUS SKELETONS 
Approved stain. — “New Methylene Blue” (Chromatine Blue Violet), National 
Aniline & Chemical Co., Buffalo, N. Y. 
Preparation of staining solution. — One gram of dry methylene blue dissolved in 
400 cubic centimeters of 70 per cent alcohol, acidulated with a few drops of 10 per cent 
hydrochloric acid. (Do not keep stock solution acidulated but add acid imme- 
diately before use.) 
Wash formalin-preserved specimens in distilled water and rim gradually up to 
70 per cent alcohol, then place in staining solution and examine frequently until 
stained a deep midnight blue. The time varies from a few hours to more than a 
week. Usually at least 3 or 4 days are necessary. Wash in successive changes of 
acidulated 70 per cent alcohol until the color ceases to wash out of the tissues. Place 
in 95 per cent alcohol for 1 to 2 hours, and finally into oil of cloves. Peppermint 
oil, xylol, and pyridin can be used successfully, but oil of cloves is preferred inasmuch 
as its clearing powers are very effective, and it can be used directly from 95 per 
cent alcohol. 
LARGE SPECIMENS WITH BONY SKELETONS 
Approved stain. — Alizarine sodium sulphonate. 
Preparation of staining solution. — Aqueous solution for Method I: Saturated 
• solution of alizarine in distilled water. Alcoholic solution for Method II : Saturated 
solution of alizarine in 70 per cent alcohol. 
Method I (most rapid but apt to be less effective): Soak formalin preserved 
specimens in distilled water for at least 1 hour. Stain slightly with aqueous alizarine 
solution (depth of stain must be determined by experiment). Avoid overstaining. 
Dehydrate, and clear in xylol from absolute alcohol. 
