so 



SCIENCE. 



[Vol. XVIII. No. 442 



lated from the separate colonies appearing on a gelatine plate 

 ■culture of this water, in every instance there has been ob- 

 tained only a negative result. To this matter of inoculation 

 with' pure cultures of bacteria we shall recur presently. 



In many of our early experiments upon nitrification we 

 used a mixture of one cubic centimetre of fresh urine with 

 two litres of tap water. This mixture was found to yield, 

 when freshly made, about .5000 free ammonia, .2000 albu- 

 minoid ammonia, .0002 nitrites, and .0250 nitrates, in 100,000. 

 This nitrogenous solution was allowed to stand at the tem- 

 perature of the room (21°-23'' C), and was tested from time 

 to time for nitrites and nitrates. The method used for the 

 determination of nitrites has been Griess's naphthalamine 

 method. This method is sufficiently delicate to detect the 

 presence of one part of nitrogen as nitrite in one thousand 

 millions. The method for determining nitrites is a modi- 

 fied form of the phenolsulphonic method of Grandval and 

 Lejoux. 



If the nitrogenous solution be first sterilized and then inoc- 

 ulated with fresh tap water, the same course is followed, 

 with the exception that the period of incubation is consider- 

 ably lengthened. If seeded with sand from a sewage filter 

 tank, or with garden soil, the whole process is materially 

 quickened, and may even be wholly completed in thirty days. 



Not only is the nitrifying organism present in Boston tap 

 water, as the above experiments clearly demonstrate, but it 

 appears to be equally common in water from all parts of the 

 State of Massachusetts. So far as our experience has gone, 

 any natural water, containing the ordinary amount of free 

 or albuminoid ammonia, contains also the nitrifying organ- 

 ism, as is shown by our long series of tests. In these natural 

 waters the nitrifying organism seems to be under wholly 

 normal conditions, and to be abundantly able to effect the 

 oxidation of the small quantities of nitrogen usually present 

 in these waters. Waters that contain high albuminoid am- 

 monia, in cases where this ammonia comes from the nitrogen 

 in infusoria, algse, etc., go through the same changes as 

 those which contain free ammonia, but more slowly. The 

 organisms in time die, the bacteria set free the nitrogen of 

 their bodies, forming free ammonia, and then in turn nitrites 

 and nitrates. 



It might, perhaps, be reasonably expected that, since the 

 nitrifying organism is undoubtedly present in all these wa- 

 ters, an examination of gelatine plate cultures of these 

 waters would reveal some particular kind or kinds of colo- 

 nies common to all, and in that way aid in sifting out the 

 nitrifying organisms. Our experience has shown, however, 

 that such a hope is unfounded. So far as the inspection of 

 gelatine plate cultures enables us to judge, no one kind of 

 colony is common to all these waters. This fact, on the 

 surface, seemed to favor the view that the power of nitrifica- 

 tion was not the property of any particular organism, but 

 was very likely possessed in common by a number of kin- 

 dred species 



The other line of bacteriological work — the inoculation of 

 nitrogenous solutions with pure cultures of isolated bacteria 

 — has been followed up from the outset, and was begun with 

 full confidence in ultimate success. It is unnecessary to 

 give a detailed account of our experiments in this direction. 

 It is sufficient to say that the nitrogenous solutions have, 

 from beginning to end, failed to nitrify. Nitrogenous solu- 

 tions of various sorts have been used, pepsin solutions, pep- 

 tone solutions, ammonium chloride solutions, Frankland's 

 solution (Zeitschr. fUr Hygiene, Bd. VI., 376), etc., all with 

 the same unfailingly negative result. A large number of 



species of bacteria have been used for inoculation, not only 

 well-known species like B. prodigiosus, B. megaterium, 

 Proteus, etc., but many species freshly isolated from water, 

 sewage, the sand of nitrifying filter tanks, and similar fa- 

 vorable situations for the nitrifying organism. The experi- 

 ments have been always prolonged for several months, and 

 in some cases for more than a year. Conditions of tempera- 

 ture, amount of surface exposed to the air, etc., have been 

 varied in many directions. Nitrogenous solutions contain- 

 ing a single species of bacterium have been poured upon 

 sterilized sand, and allowed to settle in such a way as to 

 imitate closely the conditions obtaining in filter tanks. In 

 all, more than one hundred and fifty experiments have been 

 made, covering a period of two years. In every case, with- 

 out a single exception, there was not the slightest evidence 

 of nitrification by any single species. 



There still remains a plausible explanation of this striking 

 succession of negative results. It might be that, although 

 any one species working alone was not able to effect nitrifi- 

 cation, a number of different species working together might 

 be able to produce the desired result. This was certainly 

 not an unreasonable supposition, judging from analogous 

 fermentative processes ; co-operation and combination might 

 perhaps effect more than individual and independent action. 

 Several experiments were accordingly made with a view of 

 determining this point. Here again the results were inva- 

 riably negative. Ammoniacal solutions, inoculated with 

 mixtures of several species under pure cultivation, always 

 failed to nitrify. In one experiment, for example, a nitro- 

 genous solution, found by experience to nitrify rapidly and 

 completely when seeded with garden soil, was inoculated 

 with a mixture of six different species of bacteria. These 

 six species were all isolated from soils and waters known to 

 contain the nitrifying organism. An examination of the 

 solution from time to time, by the method of gelatine plate 

 culture, showed a vigorous growth on the part of all the 

 species, but there was at no time the slightest evidence of 

 nitrification, although the experiment continued for upwards 

 of five months. 



In the course of our experiments we have found it neces- 

 sary to guard against two possible sources of error. We 

 noticed at the outset a tendency in all our solutions, whether 

 inoculated with pure cultures, or entirely free from bacteria, 

 to show an increasing quantity of nitrogen as nitrite. This 

 increase of nitrite in standing solutions is shown in the fol- 

 lowing instance. A nitrogenous solution, placed in a flask 

 stopped with cotton wool, was sterilized in the usual way, 

 and allowed to stand in the laboratory. At first no nitrogen 

 in the form of nitrite was present, but after one month .003 

 parts per 100,000 had appeared, and at the end of three 

 months .008 parts of nitrite were present. In some cases a 

 much larger amount than this appeared, although no bacte- 

 ria were in the flasks. In all these instances nitrite was un- 

 doubtedly absorbed from the air of the laboratory. Steril- 

 ized distilled water was found to absorb nitrite with the same 

 rapidity as did our nitrogenous solutions, in one case ab- 

 sorbing .0015 in a few days. If the solutiiyus were protected 

 from the free access of air, no increase of nitrite was noted, 

 and there was also no increase if they were removed to a 

 room in which little or no gas was burned. In rooms in 

 which much gas is burned it is obvious that, with the pres- 

 ent refined methods for detecting nitrites, this absorption 

 from the air, unless guarded against, may lead to erroneous 

 conclusions. This fact of nitrite absorption from the air has 

 been already noticed by Warington and other observers. 



