Pacific Salpidae — YoUNT 
277 
study. Especial thanks are due Dr. P. B. van 
Weel who informed me of the effectiveness 
of toluidin blue as a stain for salps. 
Mr. O. E. Sette, in charge of the Pacific 
Oceanic Fishery Investigations of the United 
States Fish and Wildlife Service (referred to 
in the remainder of this report as POFI), 
kindly permitted me to use the plankton 
samples of POFI. In addition, Mr. J. E. King 
of POFI was helpful in the final judgment as 
to which plankton samples would be used, as 
well as in obtaining the samples. 
The figure of Thetys vagina agg. (Fig, 2^b) 
was drawn by Mr. James Park, illustrator of the 
Department of Zoology and Entomology, 
University of Hawaii. 
MATERIALS AND METHODS 
The animals used were taken from a large 
series of plankton captures made by POFI 
(principally from cruise 5 of the "Hugh M. 
Smith”). The area of capture extended from 
27°N. to 5°S. and from 176°W. to 155°W. 
The animals were studied by staining with 
toluidin blue. Apparently the idea of staining 
the animals has not been used by anyone 
previous to this report except Berner (1954), 
who used rose bengal. 
Salps cannot be advantageously stained in 
an alcoholic stain, as alcohol dehydrates the 
test. Some water-soluble stains (such as rose 
bengal) were found to be rather effective, but 
they did not result in good differentiation 
between test and other body structures. P. B. 
van Weel suggested the use of toluidin blue, 
which he had used on salps as a vital stain 
and which, he had found, differentiated be- 
tween body tissues and test of the living 
animal. It has proved a most important con- 
tribution to this study. 
Toluidin blue can be mixed in any water 
medium, whether it contains formalin, is 
salty, or is fresh. The dry stain was sprinkled 
into the fluid until the latter became a deep 
blue or purple color. The most satisfactory 
strength is 0.15 gram stain to 500 cubic cen- 
timeters water. The animal was placed in the 
stain from 5 to 30 minutes. While it was in 
the stain, a small pipette or hypodermic needle 
was used to inject stain through the mouth 
or cloacal opening in order to stain body 
tissues; this is an essential step of the staining 
process. When body muscles and other in- 
ternal organs became rather dark, the animal 
was washed in a vessel containing clear water 
to remove excess stain. It was then ready for 
study, best done with a white background, 
but a black background or even removal from 
the water may be useful. There are, however, 
two disadvantages to the use of the stain: 
(1) it is not permanent in the preservative, 
and (2) the natural coloration of the animal 
becomes obscured. The many advantages of 
the stain, however, greatly outweigh these 
disadvantages. 
Some salps, such as the solitary forms of 
C. hakeri, C. floridana, and H. komaii, require 
a different method than the one just de- 
scribed. In the first two species, the muscles 
are so delicate that they do not stain well at 
the usual concentration; in the latter species 
the test absorbs stain heavily, thereby ob- 
scuring structures beneath it. The animals 
were placed in clear water, not in the stain. 
Then a stain of 1 gram to 500 cubic centi- 
meters of water was injected slowly into the 
body cavity until it was completely filled, 
and left until the muscles became dark. In 
this way, the test is not stained, but the body 
structures are. 
Toluidin blue differentiates by staining the 
test pink and the body tissues varying degrees 
of blue. Thus the test and various organs of 
the body become completely visible and yet 
retain their transparency to a large degree. 
Stiasny (1926) reported that the test is use- 
ful in combination with other structures, es- 
pecially muscles, for distinguishing species. 
However, extensive examination of the stained 
tests shows that in the aggregate form there 
is often no basic type of test. It is apparently 
not as useful as he thought, as I found great 
variation in test structure of both solitary and 
aggregate forms. 
