ZOOLOGY AND BOTANY, MICROSCOPY, ETC. 
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over the worm and move it slowly to and fro. This movoment causes 
the worm to straighten. As soon as the Nematode assumes the desired 
position pipette in the fixing solution between the slides, continuing the 
motion of the upper slide till the worm is dead. By this method a 
specimen can be obtained which is perfectly straight and sound. 
Pressure on a delicate worm may be avoided by pasting a piece of paper 
on the upper surface of the second slide, and using that as a handle. 
As a killing liquid Mr. Stiles generally uses a solution of corrosive 
sublimate — f- 70 per cent, alcohol -f- a few drops of acetic acid heated 
to 50° ; this passes through the cuticle very rapidly. 
Methods of Studying Development of Amphiura squamata*— 
Mr. E. W. MacBride fixed his specimens with corrosive sublimate in 
distilled or in sea water ; with a mixture of three parts sublimate and 
one part acetic acid ; with chromic, picric, and glacial acetic acid ; with 
Flemming’s solution, alcohol of 30 per cent., or alcohol (hot) at 70 per 
cent., with a few drops of corrosive ; with one-fifth to 1 per cent, osmic 
acid ; or with osmic acid followed by Muller’s fluid for 18 to 20 hours. 
He found that the only liquid which gives reliable results is osmic acid, 
though there are certain disadvantages in its use, for it renders the 
animals very brittle and has little penetrating power. The shrinkage 
which follows its use is entirely prevented, and the brittleness is 
diminished if the osmic acid is followed by Muller’s fluid. All liquids 
which decalcified as well as fixed were of no use as they gave rise to 
cavities by the evolution of gas in the still soft tissues. The method 
finally adopted by the author was to kill the animals in a solution 
of about half per cent, osmic acid allowed to act for ten minutes or so ; 
after mixing with water they were transferred to Muller’s fluid for 
18 to 20 hours, then put at once into alcohol of 30 per cent, and brought 
slowly up into alcohol of 90 per cent. In this last they were hardened 
for a night ; two or three drops of nitric acid were then added to some 
fresh alcohol of 90 per cent., and the animals were immersed in this 
till decalcification was complete, a process which occupied not more than 
twenty hours. 
Double staining was used in order to be certain about the boundaries 
of sinuses, since the ordinary plasma of Echinoderms stains with great 
difficulty. Mayer’s paracarmine was used as a nuclear stain ; this has 
the great advantages that it acts rapidly, and that all superfluous stains 
can be extracted by 70 per cent, of alcohol, which can be allowed to act 
for an indefinite time. The plasma stained was applied on the slide ; 
two were found to give good results — solution of picric acid in turpentine, 
and Mayer’s oxidized haemoglobin or “ haematein.” The advantage of 
the former is that it can be used with the shellac method of mounting 
without any danger of staining the mounting agent. For embryos pre- 
served in glacial acetic acid Mayer’s hEemalaun was used ; this gives a 
blue nuclear stain and colours much of the plasma a faint yellow. 
The embryos were imbedded in paraffin and cut into series of 
sections in a plane parallel to the line joining the madreporite with 
the mouth, and at the same time perpendicular to the plane of the disk. 
The specimens were always carefully oriented before being cut, a point 
* Quart. Journ. Micr. Sci., xxxiv. (1892) pp. 131-4. 
