14 MASS EXPERIMENT STATION BULLETIN 446 



BACTERIOLOGICAL EXAMINATION OF DISPOSAL FIELD SOILS 



This examination was undertaken to determine the extent to which the coli- 

 form bacteria from the effluent had penetrated into the soil, and the permanence 

 of the several species in the soil environment. Soil samples were taken at the 

 points indicated as A, B, C, D, and E in diagram I. Samples were taken on both 

 sides of each disposal line about midway of its length at the level of the tile and 

 two feet from the line. In the first sampling season samples were taken also at 

 two feet and five feet from the end of each line. In the second season samples 

 were taken from both sides of each line as before, but not at the ends. The 

 reason was that, in the cultivation of a field that surrounded the disposal area, 

 the soil at the ends of lines C, D, and E had been plowed practically up to the 

 tile ends and it was felt that the soil was so stirred up that representative samples 

 could not be obtained. Line C was one of two from tank M (12 hours retention) 

 and lines D and E were the two lines from tank N (8 hours retention). Control 

 samples were taken from areas in the same field that received no effluent from 

 the tanks. 



The first sampling season was in May, 1945, after the tanks had been in opera- 

 tion through the previous fall and early winter and again in the spring; and the 

 second season was in May and early June, 1946, after the tanks had been oper- 

 ated through the previous fall and early winter (table 1). Reasonable precau- 

 tions were taken to collect the samples aseptically. 



Samples were screened separately through ordinary window screen. Then 

 portions were weighed, air-dried in the laboratory, and re-weighed to establish 

 a basis for making dilutions of the moist soil in terms of dried soil. The samples 

 were kept in closed containers to keep them as nearly as possible in the same con- 

 dition as when they were taken from the field. Quantities of the samples suffi- 

 cient to give 10-gram portions of air-dried soil were suspended in dilution bottles 

 containing 100 ml. each of sterile distilled water, giving an initial 1 to 10 dilution. 

 Further dilutions were made from these suspensions. 



Since the main interest was in recovering coliform bacteria from the soils, the 

 Standard-Methods procedure (2), as employed for the examination of water and 

 sewage, was followed. This technique is the same as that employed for the stud>- 

 of coliform bacteria from the tank effluents as reported earlier in this paper. 



Preliminary trials had indicated that dilutions of lO^, lO"'^, 10^, and 10* were 

 best adapted for the purpose, so five fermentation tubes of lactose broth were 

 inoculated from each of these dilutions of each soil, 1 ml. of suspension to each 

 tube. The inoculated tubes were incubated at 3 . ° C. and read daily for gas 

 production. No tube was discarded as negative until after 72 hours of incuba- 

 tion. Eosin-methylene blue plates were streaked from all gas positive tubes and 

 incubated at 37° C. Cultures were isolated from the plates on which growth 

 occurred and were purified and held on nutrient agar slants for further study. 

 Some of the gas-positive cultures were false-positive tests caused by organisms 

 other than the coliform group. These cultures tailed to give growth on the eosin- 

 methylene blue plates. The agar-slant cultures were differentiated later on the- 

 basis ot the "IMViC" tests. 



Tables 7 and 8 give a summary of the results of the fermentation and diffe en- 

 tial tests. Table 9 is a statement of the results of the fermentation tests, Ironi 

 table 7, expressed as percentages of the numbers of fermentation tub;s inoculated 

 from the soil samples of a particular disposal line. Results from the several 

 samples for each line respectively were so much alike that all six sets of results 

 from the first series were combined, and all four from the second series, for each 



