NET DETERIORATION IN LAKE ERIE 153 



laboratory. A drop of slime was put into a test tube containing 

 some twine and a nutrient solution which was known to favor the 

 growth of cellulose-attacking bacteria more than the growth of organ- 

 isms which utilize other material for food. In a short time the nutrient 

 solution clouded, there was formation of membranes on the surface 

 and of gas bubbles, all of which indicated vigorous growth of the 

 "planted" organisms. 



The next step in isolating these organisms was to transfer some of 

 the culture to an agar culture medium containing finely divided 

 cellulose and special nutrient material, the purpose of which was to 

 favor the growth of the bacteria in which we were interested. 



The culture medium used was that recommended by Dubos (1928) 

 and consisted of: Sodium nitrate (Na NO3), 0.50 grams; di-potassium 

 phosphate K2HPO4, 1 gram; magnesium sulphate (Mg SO4 7H2O), 

 0.50; potassium chloride (KCl), 0.50; ferrous sulphate (Fe SO4 7H2O), 

 0.01; and distilled water, 1,000 cubic centimeters. The reaction was 

 pH.7.3. 



Later experiments have shown that ammonium sulphate serves 

 better than sodium nitrate. Apparently some or all of the organisms 

 are facultative anerobes and resist higher oxygen tensions when, 

 ammonium salts are supplied as nutrients. 



The use of cellulose agar was suggested by the work of McBeth and 

 Scales (1913). The preparation of cellulose with the copper treatment 

 was not satisfactory, and a much better preparation was made by 

 dissolving 75 grams of very pure absorbent cotton in 16 cubic centi- 

 meters of 72 per cent sulphuric acid, precipitating by diluting, and 

 then washing thoroughly. The agar medium was made by adding 1 

 per cent by weight of the suspended cellulose and 1.5 per cent agar to 

 Dubos medium. 



The mixture was poured into Petri dishes and allowed to incubate 

 at 25° C. for several days. At the end of 10 days the surface of the 

 agar was examined. A number of colonies of bacteria were found, ^ 

 but not all of them were the same in appearance. Some had clear 

 zones around them in the otherwise cloudy agar, showing that they 

 had removed the suspended cellulose for use as a foodstuff. The 

 colonies grew until the fourteenth day but remained small, though 

 they were active enough to remove the cellulose from large areas 

 surrounding them. Two typical plates 18 days old are shown in 

 Figure 3. The cellulose-digesting bacteria from these colonies were 

 examined under the microscope, and some of them were transferred 

 to test tubes to prepare cultures for later experiments upon preserva- 

 tive treatments. 



Morphology of the bacteria. — The organisms most commonly 

 observed in the decomposed cotton fibers and in cultures prepared for 

 study, were round-ended rods 1.3^ to 2.6/i by 0.4)u occurring singly. 

 The cultures give a microscopic picture which suggests that there are 

 other organisms present. This is indicated by the size of the rods 

 and their reaction to the Gram stain, which may be either positive or 

 negative. In hanging drop preparations some of the organisms 

 appear to be motile but most of them give no indication of vital 

 movement. Flagella could not be demonstrated with the Caesares- 

 Gil stain. No spores have been observed. There is often present 



' The occurrence of halo-forming bacterial colonies in the agar was not completed at 10 days, for other 

 colonies put in their appearance at 11, 12, 13, and 14 days. 



