Autoclaved Sewage 



Again;, with an initial inoculiim of 100 - 50, Duff and associates 

 (194.0) noted definite multiplication (between the first and fourth day) 

 recovering the last colony on the twenty-second day. A second experiment 

 gave similar results,, except that viable organisms were recovered up to 

 the thirty-fifth daj-. 



Experiments were carried out by Duff and associates (194.0) with 

 varying dilutions of autoclaved sewage supernatant. An inoculum of 350 

 * 50 B. salmonicida per cc. was planted into 2-liter amounts of decimal 

 dilutions of sewage supernatant in tap water. In all cases the numbers 

 of B. salmonicida after first dropping below 100 per cc. within the first 

 10 days, quickly multiplied to over 200,000,000 per cc, remaining 

 greatly in excess of 2,000 per cc. for periods of 23 to 29 days, vihen 

 the numbers dropped below 2,000 per cc. The ejqjeriment was terminated 

 on the thirtieth day with a B. salmonicida population of over 1,000 per 

 cc. still in existence. 



Commenting on their results. Duff and associates (194.0) pointed 

 out that it is well known that simply confining a fluid containing a 

 bacterial population in laboratory glassware may lead to a multiplication 

 of the bacteria per unit volume (Winslow 1928, Waksman and Carey 1935, 

 Zobell and Anderson 1936). They felt that if one could consider that 

 the addition of a B. salmonicida population to a sterile sewage 

 suspension meant merely the addition of an inoculum to a relatively poor 

 nutrient medium, there would be no need for further explanation. But 

 their results indicated that a very similar multiplication occurs in 

 widely varying dilutions of sewage. Multiplication of B. salmonicida 

 to over 200,000,000 per cc. occurred between 9 and 16 days in dilutions 

 of 1:1,000 to 1^1,000,000 suggesting that dilution of nutrient is not a 

 major factor, although when nutrient is completely eliminated (as in 

 tap water control) no multiplication is possible. It is therefore 

 possible that some of the multiplication of the pathogen in their 

 experiments may be attributable to confinement of its menstruum in 

 laboratory glassware. Should this be the case, such findings might have 

 no direct bearing on the behavior of the pathogen under field conditions 

 in which the organism is released into practically an infinite volume of 

 fluid. 



Ether-Treated Sewage 



Duff and associates (1940) also studied the behavior of B. 

 salmonicida in ether- treated sewage. In this experiment a portion of 

 the original living sewage microflora was retained alive in sewage. 

 This result vfas effected by exposing sewage to ether vapor as follows. 

 Two liters of fluid were placed in a 4-liter flask, closed tightly with 

 a rubber stopper. From the stopper was suspended a wide vial containing 

 30 cc. of ether. The flask was shaken frequently, so as not to spill 



