TOXICITY OF HEAVY METALS 65 



a cubic centimeter) was placed in a separate glass cylinder (2 cm. high 

 and 3 cm. in diameter), to which spores from a stock culture were then 

 transferred. These inoculations were made in order, beginning with the 

 weakest solution of the lighter metal. A platinum needle was used for 

 this purpose, flamed and washed to sterilize and clean it after each 

 inoculation. 



In each case the tip of the needle was dipped a single time in the well 

 mixed mass of spores which had been prepared on the agar surface, as 

 already described, and the spores that adhered were washed off in the culture 

 solution. Thus, approximately the same number of spores were inoculated 

 into all of the glass dishes and the solutions were then ready for the 

 preparation of the hanging drops. 



The drop cultures were made after much the same method as that 

 described by Clark. 15 Van Tieghem cells were used, small cylinders of 

 glass tubing, with ground ends, 9 mm. high and 12 mm. in diameter, which 

 were cemented to ordinary microscope slides by means of beeswax. Two 

 cells were affixed to each slide. The culture solutions in the glass dishes, 

 into which spores had already been inoculated, were thoroughly mixed 

 with a glass rod. By means of this rod, a drop of the liquid was then 

 placed upon a flamed cover glass. 



A small drop of the culture solution from the corresponding flask with- 

 out spores was placed in the bottom of the Van Tieghem cell and the cover 

 bearing the drop culture was inverted over it. Duplicate drop cultures 

 were made from each concentration of solution, both cultures being placed 

 on the same slide. As has been shown by Clark, the presence at the 

 bottom of the culture cell of a small amount of the same solution as that 

 from which the hanging drop is composed prevents evaporation from the 

 drop and hence obviates marked alteration in its concentration, even if the 

 cultures remain in the thermostat for a considerable time. Without this 

 precaution the solution contained in the hanging drop is apt to become 

 markedly more concentrated during the period of an experiment, which 

 might lead to erratic results. The covers were sealed in position with 

 petrolatum. It was not found necessary to take the precaution recom- 

 mended by Clark, of first allowing the expanding air to escape through 

 a small opening in the seal, possibly because the temperature of the thermo- 

 stat here used was only a little above the temperature at which the prepara- 

 tions were made. 



For ease in handling the cultures, the slides were placed in sheet metal 

 trays, which could be piled one upon another in the thermostat so as to 

 form a rack. These trays were 15 cm. wide and 20 cm. long, with vertical 



15 Clark, J. P., On the toxic effect of deleterious agents on the germination and development of cer- 

 tain filamentous funtn. Bot. Gaz. 28: 289-327, 378-404. 1890. 



