a primitive Lichen . 581 
appeared in the hanging-drop cultures. There appeared to be no develop- 
ment of Botrydina in either. 
Cultures were made on sterilized sandstone and on a sterilized porous 
plate, but these also were unsuccessful, the Botrydina showing no develop- 
ment. 
Finally, cultures were made in Petri dishes on 3 per cent, agar dissolved 
in 0-25 per cent. Knop’s solution. In these the Botrydina lived for some 
time seemingly unchanged, though as before some cultures developed 
a fungus, and others a unicellular green alga. In one case only were there 
any signs of multiplication of the Botrydina . In this culture the globular 
structures had been broken up before infecting, and examination, after 
several weeks, showed the presence of very small, but fully-formed, globules. 
There was no indication how these had appeared, and so, in spite of all 
precautions, the repeated attempts at procuring a pure culture of Botrydina 
seemed to have failed, while no light had been thrown upon the formation 
of the envelope. 
The Envelope. 
At the same time that the culture experiments were being carried out, 
attempts were made to get some clear idea as to the structure of the 
globular bodies and the nature of the envelope, but here again results were 
at first unsatisfactory. Various stains were tried, but no clear preparation 
could be obtained. Stains penetrated with great difficulty, and when once 
they had done so, stained so deeply that the structure could not be 
observed. The extreme resistance of the envelope to chemical reagents was 
further shown by the fact that in material soaked for three days in Eau de 
Javelle, washed in water, and placed in 70 per cent, alcohol for three days, 
the green colour of the cells in the interior was still retained. Moreover, 
the envelope was not dissolved by the action of concentrated caustic 
potash for 24 hours, and was only dissolved by concentrated hydrochloric 
acid after a period of two days. 
On testing for the presence of cellulose, both with iodine and sulphuric 
acid, and with a solution of chlor-zinc-iodine, no trace of it could be 
detected. Neither is it likely that the envelope is of a gelatinous nature, 
as stated by both Rabenhorst and Kirchner ; 1 for it does not swell in water, 
neither is it dissolved by water even on boiling, and it only stained with 
great difficulty in methylene blue. 
As the envelope was thus shown to be neither cellulose nor a gelatinous 
one, the question arose of what does it consist ? The absence of true cellu- 
lose and the great resistance of the envelope to the action of acids and 
alkalies led to the suggestion that it was of the nature of fungus -cellulose. 
1 These authors both state that the pseudo-parenchymatous investment is formed by the increase 
in size of the gelatinous walls of the original green cells. 
R r 2 
