60 
PACIFIC SCIENCE, Vol. VI, January, 1952 
Fig. 1. Map of the Hawaiian Islands showing the localities sampled. 
(6) Place in 4 per cent potassium hydrox- 
ide for 4 to 6 hours, until vertebrae can be 
seen clearly. 
(7) Place in alizarin staining solution (see 
below), stain for 10 hours, then turn the fish 
over and stain the other side for 5 to 10 hours. 
(8) Place, successively, in a series of 4 per 
cent potassium hydroxide-glycerin solutions 
(90:10; 80:20; 60:40; 20:80; 0:100), each for 
12 hours; if a solution turns dark red-blue, 
repeat that particular solution. 
(9) Preserve in 100 per cent glycerin, 
adding two or three small crystals of thymol 
to prevent the growth of molds; all parts of 
the fish, except the bones, are now trans- 
parent. 
(10) Place the fish in a container filled with 
glycerin and, using a standard Kodak en- 
larger, project the image on photographic 
paper, expose, and develop (Fig. 2); several 
fish may be photographed simultaneously. 
(11) Using the photograph, count the 
number of vertebrae. 
In the direct method, the fish were pre- 
pared in the following manner: 
(1) Wash in running water for 1 to 3 
hours. 
(2) Using a scalpel, slice the flesh from 
the caudal peduncle to the head on the left 
side of the fish, cutting to, but not into, the 
column; using scissors, snip the flap of flesh 
diagonally across the head, thus exposing the 
base of the skull and the first vertebra; using 
scalpel, scrape the remaining flesh from the 
column on the left side of the fish and remove 
viscera from body cavity. 
(3) Place the fish in staining solution for 
4 hours. 
(4) Transfer to water or to 5 per cent 
formaldehyde solution; some de-staining will 
occur. 
(5) Count the vertebrae under the low- 
power binocular microscope, using a pointer 
(eyelash hair) in the ocular as a reference 
point. 
A stock solution of stain was made by dis- 
