4 MEMOIRS OF THE NATIONAL ACADEMY OF SCIENCES. 



From made soil, although many years old and paved over, numerous colonies were obtained 

 from a depth of 9 feet. No actual count was made, but the number of bacteria at this depth was 

 apparently as great as at the surface. 



Method of collecting samples. — Whenever possible, the samples were taken from the wall of a 

 freshly made excavation, the wall being dug away to a depth of several inches just before taking 

 the sample, and care was exercised that the pick should not touch the spot from which the earth 

 was taken. A short tube of thick glass, previously plugged and sterilized, was bored into the 

 fresh surface until a sufficient quantity was obtained. In all other instances the earth borer of 

 Frankel was used, but was not found satisfactory, besides being difficult to operate, the soil being 

 a very stiff' clay containing numerous pebbles. 



Method of examination of samples. — Two methods of examination have been suggested. Iu 

 one — that preferred by Frankel in making counts — a portion of the earth to be examined is put 

 into liquefied gelatin and thoroughly broken up with a sterile rod, after which it is mixed by 

 tilting the tube up and down, revolving it at the same time, and lastly rolled. The second method 

 consists in crushing the earth iu a tube of sterile water and washing it well by agitation. A 

 certain amount of the water is then introduced into the liquid medium and roll tubes made. Both 

 methods were tried, the former being found to be more satisfactory. It was used exclusively, with 

 the modification of pouring the gelatin into Petri dishes instead of making roll tubes; and iu 

 every instance dilutions were made from the original tube, iu order to better isolate the colonies. 

 To determine the temperature at which the cultures should be kept, duplicate plates on agar-agar 

 and gelatin were made from several samples of earth, following the second method described. 

 After washing the earth thoroughly in sterile water, equal quantities of the water were mixed 

 into tubes of liquid agar agar and gelatin and theu poured into Petri dishes. All of the gelatin 

 and a duplicate series of the agar-agar plates were kept at room temperature, the others being 

 placed in the incubator at 35° to 36° C. At the end of thirty-six hours a count showed that those 

 plates kept at room temperature contained the greater number of colonies. In all the subsequent 

 experiments gelatin has beeu used and the plates kept at room temperature. The wisdom of this 

 has beeu confirmed by the subsequent study of the individual organisms isolated, since quite a 

 large proportion of them grow better at room temperature than at 35° to 36° C, and some will not 

 grow at all at the latter temperature. 



Study of organisms. — The bacteria isolated have each been studied as to their morphology, 

 both in stained preparations and in the hanging drop; as to motility and cultural peculiarities; 

 all have been stained for flagella after the method of Loftier, and all have been placed in an 

 atmosphere of pure hydrogen aud the growth noted. The culture media employed have been 

 those given in the text-books, and the percentages have not been changed except in very hot 

 weather, when it was found uecessary to add gelatin in the proportion of 12 per cent instead of 

 the 10 per cent used during most of the work. 



CONCLUSIONS. 



As stated at the outset, the data gained during this study are not sufficient to base any 

 positive conclusions upon. The following propositions have been substantiated to a greater or 

 less extent: 



(1) Made soils, as commonly found, are rich in organic matter, aud excessively damp through 

 poor drainage. 



(2) They furnish conditions more suited to the multiplication of bacteria than do virgin soils, 

 unless the latter are contaminated by sewage or offal. 



(3) Made soils contain a larger number of bacteria per gram, and the number of species is 

 greater, while the deeper layers are as rich, both in numbers and varieties, as are the upper ones. 

 After some years the number in the deeper layers probably becomes proportionately less. 



(4) There is a greater probability that made soils may contain pathogenic bacteria. 



THE CULTIVATION OF ANAEROBIC BACTERIA. 



Among the organisms which find their habitat in the upper layers of the soil, several of the 

 most interesting and important, from a pathological point of view, belong to the class of strict 



