2IO CALIFORNIA ACADEMY OF SCIENCES. [Proc. 30 Ser. 



culture are accompanied by bacteria which multiply only 

 too rapidly. In spite of thorough sterilization of the 

 slides, cover- glasses, vessels «and instruments used in 

 connection with the cultures, and the utmost pains to pre- 

 vent the introduction into the cultures of organisms from 

 outside, I have failed in most cases to make pure cultures, 

 because the lichen fragments themselves carried bacteria, 

 and substances nutritious for them, into the culture-media. 

 In many cases, however, the number of bacteria was con- 

 stantly small. 



For most of my cultures I used the "hanging-drop" 

 method of the bacteriologist, employing for the purpose 

 slides with two concavities, ground sufficiently deep to 

 avoid contact of the drop with the bottom of the concavity. 

 The cover-glasses were sealed upon the slide with vaseline. 

 If the concavities are reasonably large and deep (e. g. 

 13 mm. diameter, i mm. depth) a hanging-drop surrounded 

 by an adequate volume of air, and large enough to supply 

 food-material for several weeks, may be used. It must be 

 admitted, however, that hanging-drop cultures have only a 

 limited usefulness, for the food-supply presently becomes 

 exhausted, and there may accumulate in the drop undue 

 amounts of the dissolved excreta of the organisms present. 



Various methods have been employed by others in pre- 

 paring material for culture. As culture-media for the 

 spores and lichen fragments, I have experimented with 

 boiled tap-water, with the moisture carried by the spores 

 expelled upon the cover-glass, with a drop of the ordinary 

 nutrient beef-gelatine of the bacteriologist, with similar 

 nutrient agar-agar, and with the distilled water used for 

 general purposes in the laboratory. The distilled water 

 was poisonous. I did not consider it necessary to deter- 

 mine the reason for this, though one would suspect it of 

 being acid and possibly of containing traces of copper or 

 other injurious salts. The distilled water prepared in 

 quantity in the chemical laboratory, and supplied by it to 

 the other laboratories of this University, is sufficiently pure 



