402 
SUMMARY OF CURRENT RESEARCHES RELATING TO 
)8. Teclinique.* 
(1) Collecting- Objects, including- Culture Processes. 
Four EUR, A. — Etude sur la culture des Microorganismes anaerobies. (Study on 
the culture of anaerobic micro-organisms.) 
Paris (Doin), 1889, 8vo, 73 pp. and 25 figs. 
Jeffries, J. A. — A new method of making Anaerobic Cultures. 
Med. News, 1889, p. 274. 
(2) Preparing- Objects. 
Study of the Embryology of the Earthworm.j — Mr. E. B. Wilson 
says: — “After testing many different hardening fluids, I have found 
none to compare with Perenyi’s fluid, which gives uniformly the best 
results, both for sections and for surface-views of all stages, and is far 
superior to picro-sulphuric acid or corrosive sublimate. Flemming’s 
mixture of osmic, chromic, and acetic acids gives very clear differen- 
tiation of the middle stratum of the germ-bands after staining with 
haematoxylin, but in most respects it is far inferior to Perenyi’s fluid. 
The embryos were left in the fluid from 15 to 60 minutes, placed in 
70 per cent, alcohol for a day, and kept permanently in 90 per cent, 
alcohol. 
For permanent staining no method has proved so satisfactory as 
borax-carmine followed by hsematoxylin. After being deeply stained in 
the carmine (12 hours), and extracted in acid alcohol in the usual manner, 
the embryos were treated with extremely dilute ammoniacal alcohol for 
a few minutes, to neutralize the free acid, and were then stained in 
very dilute Kleinenberg’s hsematoxylin (12 hours or more). In case of 
overstaining with hsematoxylin, the colour may be again extracted with 
acid alcohol, after which the specimens are again treated with ammo- 
niacal alcohol. This process, following treatment with Perenyi’s fluid, 
gives beautifully clear preparations, which are specially favourable on 
account of the clearness with which the cell-outlines are shown. It has 
been found desirable to imbed the specimens for sectioning as soon as 
possible after hardening, and to reduce the time of immersion in melted 
paraffin to a minimum (i. e. not more than 10 or 15 minutes). 
For surface-views of the germ-bands the borax-carmine stain should 
be very deep, and the hsematoxylin very slight, so as to give the specimen 
only a purplish colour, not a dark-blue. The germ-bands are dissected 
off on the slide, in strong glycerin. This method has, in ray experience, 
given far better results than that of osmic acid followed by Merkel’s 
fluid, so successfully used by Whitman in the study of Clejpsine. 
For the study of entire specimens of the young stages I have found 
Perenyi’s fluid, followed by alcohol, water, very dilute iodine solution, 
and glycerin, to give results superior beyond comparison to those attained 
by any other method. The iodine colours the protoplasm pale yellowish- 
brown, the cell-outlines are clearly marked, and the nuclei are stained 
deep brown. In time, most of the iodine is precipitated in the form of 
deep-brown spheres, which mar the clearness of the preparations, but 
* TMs subdivision contains (1) Collecting Objects, including Culture Pro- 
cesses; (2) Preparing Objects; (3) Cutting, including Imbedding and Microtomes ; 
(4) Staining and Injecting ; (5) Mounting, including slides, preservative fluids, &c. ; 
(6) Miscellaneous. t Journal of Morphology, iii. (1889) pp. 445-6. 
