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CARNEGIE INSTITUTION OF WASHINGTON 
The method of antigen preparation includes the following steps: (1) 
Collecting and starving the animals; (2) obtaining the body fluids (where 
present) and grinding the tissues with the addition of sterile salt solution; 
(3) vigorously shaking the ground tissues in a shaking machine; (4) filter¬ 
ing the extracts, ending with a final sterilizing filtration through Seitz 
filters; (5) storing in sterile condition in 5 ml. serum vials; and (6) testing 
for sterility and protein concentrations. 
In some cases the extracts were concentrated by blowing with electric 
fans before the final filtration. 
What appear to be satisfactory antigens for use in the production of 
antisera, were obtained for 28 genera distributed as follows: Porifera, 2; 
Ccelenterata, 1; Sipunculoidea, 1; Annelida, 3; Crustacea, 5; lVIollusca, 7; 
Echinodermata, 6; Prochordata, 3. 
In addition, other antigens representing seven more genera were obtained 
which will be suitable for titration, even though not concentrated enough 
for injection. They are distributed as follows: Ccelenterata, 4; Mollusca, 
1; Echinodermata, 1; Prochordata, 1. 
Finally, samples of the blood sera of three of the lower Vertebrata were 
obtained, to be used in the study of Prochordate affinities. 
The next step in the investigation will be the production of the precipi¬ 
tating antisera and this will be followed by the making of the actual titra¬ 
tions by means of which further light may be thrown on the systematic 
relationships of the species studied. The results should help to make more 
clear the proper location of those species of “uncertain systematic position/’ 
and to make more quantitative the expression of the interrelationships of 
all the forms studied. 
Report on Tissue Culture, by L. R. Cary 
My work during this season was confined to a study of the behavior in 
vitro of tissues of Ptychodera bahamensis. The routine procedure for 
making the cultures, described in my report of last year, gave uniform 
success. 
Sterilization of tissue fragments by irradiation with ultra-violet from a 
mercury vapor-bulb was equally efficient. This procedure resulted in a 
marked saving of time. There was no apparent deleterious effect on the 
tissues from an exposure of 2% minutes at 15 cm. All bacteria were 
destroyed or rendered incapable of reproduction lor several days. After 
a week, bacterial activity sometimes became very marked in cultures pre¬ 
pared by this procedure. A peptic digest of entire Ptychoderas was used 
as a nutrient medium in the preparation of all cultures. 
Last season almost complete failure resulted in attempts to obtain satis¬ 
factorily stained preparations from the cultures, as a permanent record of 
their activities. Consequently an extensive series of photomicrographs 
of cultures throughout the period of their development was made during 
the first two weeks of this season. Later a series of micro-cinematographs 
of the growth of explants from the digestive caeca were taken. 
The structure of Ptychodera makes it possible to choose as an explant 
a small fragment of tissue from the dorsal side of the mid-section of the 
body which will contain: (1) The ectodermal epithelium; (2) nerve cell; 
(3) cells from the digestive (hepatic) caeca; (4) muscle cells; and (5) con¬ 
nective tissue cells. In a culture from such an explant the first visible 
growth is that of nerve fibers. Then endoderm cells from the caecum begin 
to migrate as flask-shaped bodies. Their stalks become very long and 
TORTUGAS LABORATORY 
283 
slender. Before these primary migrants have separated from the explant, 
other cells, to which the former are attached, move out from the mass of 
tissue. This process continues until often a chain of ten or more endoderm 
cells extends out from the explant. In the meantime many muscle cells 
become separated from the tissue mass. Their behavior varies greatly. 
Some become greatly elongated and show marked amoeboid activity. Others 
shorten to masses of rectilinear shape. These soon swell until a clear vacuole 
surrounds the central mass of protoplasm. Later these may elongate and 
become extremely active. 
By the time the greatest activity of the above-mentioned cells has been 
passed, a mass migration of the ectodermal epithelial cells takes place. 
This has the appearance simply of a flowing of the cell mass. Soon the 
other types of cells are entirely overgrown and hidden by the migrating 
ectoderm cells. The culture now appears as a smooth mass of rounded 
cells and all evidence of earlier activity of other elements is lost. 
When an explant is selected which consists of csecal endoderm alone, 
migration continues until no central mass is distinguishable. The com¬ 
ponent cells of the culture move about actively over the cover-slip. The 
protoplasmic strands between cells become highly attenuated. Relatively 
large areas are seen where only these connecting strands are to be found. 
At the time of the division of the cells, their characteristic pigment 
granules may be passed on entirely to one daughter cell. The other now 
appears as a clear mass of protoplasm. When freed in this manner from 
the large granules, the cells exhibit unusual amoeboid activity. They lose 
their characteristic shape and can not easily be distinguished as descend¬ 
ants of their parent cells. 
Subcultures were easily obtained from many slides. When a fragment 
of the original explant was used, activity was always very marked. Indeed, 
subcultures from slides on which there had been no growth were usually 
very active as though the explant had been released from some inhibiting 
influence operating in the original culture. 
Subcultures containing pure csecal endoderm cells were easily obtained 
and kept alive for a period of two weeks without transfer. When transfers 
were made every 48 hours, these cultures could be carried on indefinitely. 
Studies Upon Tortugas Sponges, by M. W. de Laubenfels 
In continuation of my work on sponges at Tortugas, particularly with 
reference to methods of regeneration, cylindrical species were cut trans¬ 
versely and kept under observation while the injury was repaired. Some¬ 
times disks were cut from such sponges, kept between glass, and watched 
while the new surface was formed, in this case not across the wound but 
out to the glass. Active moving about of cells in and from the sponge 
was involved, and some metamorphosis of one cell sort into another. 
The commercial sponge Spongia officinalis (Linne) was more abundant 
this year than earlier, but was unsuited for experimental work with dis¬ 
sociated cells, because its cells refused to come out of suspension even 
when centrifuged. 
A few observations were made upon sponges eaten by fishes under normal 
conditions, and upon the effect of feeding suspensions of sponge cells 
to others. 
The specific descriptions of sponges by early students of the West Indian 
fauna are often so brief as to be worthless. There are excellent recent 
papers, but the number of species of which they treat is not large, and 
