clxii 
THE VOYAGE OF H.M.S. CHALLENGER. 
a collencliymatous mesoderm, but since it is always necessary to prepare thin slices for the 
investigation of the finer histological details, but little is gained by this. 
Dissociation and teasing must be resorted to for the study of separate histological 
elements, and the spicules must be isolated from the soft parts for comparison, measure- 
ment, and illustration. Under the head of dissociation and teasing I have nothing to 
add to what is already well known, but with regard to the separation of spicules and the 
preparation of thin slices, it may prove useful if I describe here in detail those methods 
which I have found most successful. 
Preparation of Thin Slices . — The simplest and most generally useful method is that 
known as the paraffin process ; but sometimes in special cases and for special purposes 
freezing may be resorted to. Although the hard parts of the Tetractinellida are always 
siliceous, spongin being the only other substance present in the skeleton in addition to 
opal, yet it sometimes happens that the sponge contains a considerable quantity of 
calcium carbonate which has been introduced as foreign matter from without, either 
during the life of the Sponge or subsequently on being scraped up by the dredge ; this 
calcareous matter is usually in the form of fine mud or consists of isolated tests of 
Foraminifera and other organisms ; frequently also calcareous organisms, more especially 
Foraminifera, grow attached to the outer surface of the Sponge. As a preliminary to 
staining all traces of calcium carbonate must be removed. This is best accomplished by 
an alcoholic solution of nitric acid; a 1 per cent, solution of the acid is made with 60 
per cent, alcohol, and this is added to the object placed in 60 per cent, alcohol, drop 
by drop, till an occasional bubble of gas is set free ; the preparation is then left to stand 
for some hours and a further quantity of acid alcohol added till no more carbon dioxide 
is liberated, the preparation is then transferred to pure 70 per cent, alcohol, which is 
changed till every trace of calcium nitrate is extracted. The advantage of this method in 
avoiding any unnecessary osmosis is sufficiently obvious. Fortunately but few of the 
Challenger specimens needed to be treated in this manner, but “ en revanche ” in some 
few cases other mineral bodies, such as fragments of pumice and grains of quartz sand, 
were present and there is no process by which these can be removed. My experience of 
hydrofiuoric acid, as might a priori have been expected, is altogether unsatisfactory. 
When siliceous fragments occur lying loosely in the canals of the Sponge the razor will 
generally tear them out, and fairly satisfactory slices may be obtained, as in the case of 
Tetilla sandalina ; but when, as in Psammastra murra/yi, numerous quartz grains occur 
firmly embedded by fibrous tissue in a dense cortex, one has to abandon all hopes of a 
thin slice, and to put up with a very bad example of a thick one. 
For staining I have found hsematoxylin most generally useful ; picrocarmine and 
other carmine dyes have also been used, and with picrocarmine especially very elegant 
results may be obtained ; this stain is particularly well adapted for use with the freezing 
process. 
