100 



JO) was transferred by means of a sterile pipette to 10 c. c. of fluid agar (cooled to 

 41° C. ), and after thorough shaking was poured into a sterile Petri dish. 



(2) The remainder of the culture was then plunged for 10 minutes into water at 

 46.05° C. ; it was then cooled at room temperatures for a few minutes and 1 c. c. taken 

 out by means of another sterile pipette, put into another tube of melted agar (10 c. c. 

 at 41° C), and when thoroughly shaken poured into a second sterile Petri dish. 



These two dishes were then kept at living-room temperatures and compared from 

 time to time by turning them bottom up under the microscope. Result: May 18 

 (1) agar uniformly milky cloudy. Under the microscope innumerable small colonies 

 are to be seen, dumber of colonies estimated at 8,000 to 10,000 per field (Zeiss 16 

 mm. and 12 comp. oc.) (2) This jDlate was also milky cloudy, but the colonies 

 were larger and not nearly so numerous, about 95 per cent having been destroyed by 

 the heat. 



These two plates were kept under observation for a week or two, but with no con- 

 flicting results. 



IX. June 3, 1897, six tubes of stock 245, a beef broth made feebly alkaline to 

 litmus by means of sodium carbonate, were selected for this experiment. Each was 

 inoculated with a loop from tube 4, June 2^ a 26-hour culture in stock 245, which was 

 not yet distinctly clouded, but became so after a few hours. Four tubes were heated, 

 but not until over an hour after inoculation (room temperature 28° C. ) The tem- 

 perature of the bath was unusually variable, ranging from 46.70° to 47. 10° C, it being 

 most of the time below 47° C. 



Results: (1) Two of the tubes Avere cooled slowly at room temperature. These 

 tubes were examined at intervals of a few days until July 29, but both remained 

 clear. 



(2) Two of the tubes were cooled quickly under running water. One of these 

 tubes remained clear for 56 days, after which the experiment Mas discontinued. 

 The other remained clear until the sixth day. It then became feebly clouded, and 

 contained numerous small zoogloeae, most of the germs seeming inclined to pass at 

 once into this state, i. e., growth was retarded but not all of the germs were killed. 

 June 10, feebly clouded, zoogloeae larger, numerous, ragged. June 14, moderately 

 clouded; slight rim on tube at level of liquid; the larger zoogloeae are distinctly yel- 

 low. June 16, well clouded with rolling clouds on shaking. Considerable distinctly 

 yellow precipitate. A thin pellicle in shape of a delicate membrane thickly dotted 

 wath small zoogloe^e is present. This membrane sinks on gentle shaking, breaking 

 up into ribbons which are fine granular under X6 Zeiss aplanat. June 28, copious 

 yellow precipitate. The pseudo-pellicles have all settled. July 6, abundant yellow 

 precipitate. Fluid nearly clear. On this date the other three (sterile) tubes were 

 reinoculated from this tube, but they remained clear. 



(3) Two of the tubes were kept as checks. One of them became contaminated 

 with a white organism growing best on the bottom of the tube ( Oospora?) . The other 

 remained clear until after the third day. On the fifth day it was distinctly but 

 f^^ebly clouded, and the surface layers contained small zoogloese which streamed 

 down cloudily on gentle shaking. June 9, clouded more than yesterday, but not 

 heavily so. June 10, well clouded with considerable yellow precipitate. June 14, 

 a pellicle consisting of yellowish more or less united zoogloeie. June 16, well clouded 

 with rolling clouds on shaking. No new pellicle. The broken one (shaken down 

 on the 14th) has not gone to pieces, but lies on the bottom with hundreds of tiny 

 zoogloeee embedded in it very regularly. June 28, a copious yellow precipitate. 

 July 6, fluid nearly clear, i. e., becoming exhausted of nourishment; otherwise as 

 before. July 29, washed out; precipitate yellow. 



X. On June 3 three tubes of stock 244b (-^20 gelatin) were converted into 3 

 poured plates as follows: 



(1) One cubic centimeter of the cloudy (luid from tube 4, June 2 (see IX), was 



