REGENERATION AFTER EXARTICULATION 109 
chamber lined with moist filter paper for two days as recom- 
mended by Reed (’03). During this period the operated ex- 
tremity was moistened from time to time with a solution of 
permanganate of potash 1:1000. The animals were then 
returned to the aquaria. The above precautions almost entirely 
prevented the growth of fungus and consequent failure of the 
experiments. 
For microscopic study, the regenerating regions were removed 
and fixed for the most part in sublimate acetic or Gilson’s mer- 
curo-nitric fluid. Other fixatives, such as Zenker’s fluid, Bouin’s 
fluid and ten per cent formalin were occasionally used but on 
the whole the sublimate mixtures proved the most satisfactory. 
After hardening in alcohol for a few days, the objects were 
decalcified in a mixture of four per cent nitric acid in seventy 
per cent alcohol for three or four days. They were then imbedded 
in paraffin and sectioned. As a rule, good series were obtained, 
seven or eight micra thick, although the rather tough bone and 
cartilage from large specimens sometimes gave trouble. For 
staining Mayer’s haemalum followed by picro-acid fuchsin was 
most frequently employed. ‘This gives a brilliant differentiation 
of tissues but is not always permanent. Other stains such as 
Mallory’s connective tissue stain, borax carmine and Lyons blue, 
haemalum and congo red were also used but none proved as 
satisfactory for most purposes as the haemalum and_ picro- 
acid fuchsin combination. 
EXPERIMENTS 
Pari. I 
The fact that regeneration does occur after complete extirpa- 
tion (exarticulation) has been established by the observations 
of Kurz and the writer as stated above. In order to work out 
the detail of this process, two sets of operations were made, the 
hind limbs being used in both cases. In the first set the limb 
was amputated at the hip-joint, in the second at the knee-joint. 
Great care was exercised in making these amputations. The 
skin and muscles were first carefully divided with a small sharp 
