134 TWENTY-FIRST REPORT. 



Bacterial counts were made by the quantitative plating method and by 

 the direct miscroscopic method. The results per cubic centimeter were as 

 follows : 



Direct 

 SET I. Plate Method. Microscopic Method. 



Can 1 361,883 3,033.856,000 



Can 2 700 568,848,000 



Can 3 300,000 568,585,000 



Can 4 776.400 209,612,000 



Can 5 1,386,000 2,439,772,800 



SET II. 



Can 1 1,500 14,221,200 



Can 2 2,000 569,084,000 



Can 3 3,435,333 43,923.000 



Can 4 6.224.000 1,611,736 



Can 5 295,666 474,040 



This variation between the miscroscopic and plate count from the same can 

 may be explained by the fact that bacterial action had not developed far 

 enough to make up for those killed in processing, or organisms may have grown 

 and succumbed later to the byproducts produced, or the media used may not 

 have been favorable for the organisms present. However, a special effort was 

 made to see if the organisms found were microscopically the same as those 

 on the original stain from which counts were made. 



Gelatin agar shakes of pure cultures from colonies on the plates were made. 

 Duplicates were found of organisms on the aerobic and anaerobic plates, all 

 but one organi.sm, which was a strict anaerobe, bein,g facultative anaerobes. 



Besides running cultures through ordinary media aerobic and anaerobic 

 cultures were made in test tubes of sterile peas in distilled water. The anae- 

 robic culture was covered with about one inch of paraffin oil. Starch agar 

 plate streaks and starch broth cultures were also made and the thermal death 

 points determined. 



The strict anaerobe found had the cultural and morphological character- 

 istics of B. hotiiUnus; this organism lived after heating in test tubes of neutral 

 broth for 10 minutes at 15 pounds pressure in the autoclave. It was killed 

 when heated 20 minutes. This grew symbiotically with a resistant spreading 

 facultative anaerobe, but was separated by means of dilution shakes in gelatin 

 agar made in glass tubes with a rubber stopper in the bottom. As the anae- 

 robic colonies developed first, transfers made from the bottom were finally 

 successful in obtaining a pure culture. 



Of the other organism found, one appeared in five different cans, in three 

 cans from the first set and in two cans from the second set. After making 

 these cultural tests, some strikingly similar characteristics were found : 



All were spore forming rods. 



All gave the indol test. 



All caused peptonization in litmus milk, which became alkaline. 



All liquefied gelatin. 



None produced gas in any of the three sugar fermentation tubes or in 

 protein free asparagin solution, but most of them produced gas in peas. 



