SPECIAL BIOLOGICAL WORK.

The following account of the special biological work of the station has been prepared by Mr. George W. Fuller, biologist in charge.

The regular biological work has consisted in part, as in former years, in the systematic examination, both microscopical and bacterial, of the applied sewage, city water and canal water, and of the effluents of the different filters. The microscopical examinations have been made after the Sedgwick-Rafter method (see p. 397). In the bacterial analyses unusual care has been taken to make the determinations under conditions as nearly as possible parallel, in order that the results might be directly comparable. Since it has been observed, both in this laboratory and elsewhere, that nutrient gelatine, varying even to a moderate extent in its degree of alkalinity, may yield widely differing numbers of bacteria from duplicate samples of water, —a circumstance probably due to the fact that different species of bacteria require different degrees of alkalinity for their rapid development, numerous experiments have been made to determine the optimum amount of alkali necessary for the development of those species of bacteria prevailing in the waters and sewage of Lawrence. A special study has also been made of the methods available for securing the same degree of alkalinity in all samples of nutrient gelatine, but it has been found difficult to obtain an indicator sufficiently sensitive to all the compounds present in the gelatine; and it has occasionally been observed that lots of nutrient gelatine, which had been made alkaline to the same degree, so far as could be determined by the ordinary indicators, have also given widely varying numbers of bacteria in duplicate samples of the same

water.

As a step toward greater precision in manipulation, all gelatine plates have been grown not at the " room temperature," as is usual, but in a low-temperature thermostat. This thermostat, which eliminates irregular temperature differentiations, is kept at 20° C. during the summer months by passing a stream of cold water through the

water jacket, and, during the rest of the year, by a small gas flame. Devices have also been introduced for protecting the effluents from contamination by dust and air; a careful study has been made of the conditions and methods of taking samples; and these things, together with the use of a nutrient gelatine of a composition as nearly as possible uniform, and under parallel conditions, have served to increase the accuracy of the biological methods of determining the degree of purification of sewage and water. The results of the regular biological analyses have already been tabulated and discussed along with the chemical results on the foregoing pages. (See tables above, showing the chemical and biological purification effected by the different filters.)

INVESTIGATIONS UPON THE BACILLUS OF TYPHOID FEVER.

The importance of determining accurately the life-history of the typhoid fever bacillus, its deportment in sewage and in water, and the possibility of its removal from water by means of filtration, has long been recognized as an important part of the biological work. When the problem was first undertaken, it was found that there was no reliable method for its differentiation from several species of bacteria regularly present in the water of the Merrimack River at Lawrence. The potato test, upon which much dependence was generally placed, has proved to be totally inadequate, and for two reasons: first, because the typhoid fever bacillus does not under all conditions give its characteristic invisible growth on sterilized potato; and, second, because no fewer than five of the species in the Lawrence waters under some circumstances present the same characteristic.

After it had been discovered that the potato test was not diagnostic, and that it was even contradictory, it became necessary to devise other methods of differentiation. To this end a thorough study was made of the typhoid bacillus, side by side with the species of bacteria present in the water of the Merrimack River at Lawrence. The numerous special methods and media proposed for its differentiation were examined and their diagnostic value determined. This preparatory investigation was continued for several months, and resulted in a method of procedure whereby the typhoid fever bacillus can be differentiated beyond all reasonable doubt from every species of bacteria hitherto met with in the Lawrence waters. The results of this investigation are embodied in a paper which appears beyond (p. 635).

Up to this point all study and comparisons of the typhoid bacillus were made with cultures which had been obtained from prominent laboratories in New York, Paris and Berlin. These cultures had not been freshly isolated; in fact, it was believed that with one possible exception they were several years old and probably of diminished vitality, as was indicated by their inability to grow at the extreme limits of temperature which have been determined for this organism. As it was very desirable to continue these investigations with a culture of undoubted identity and of undiminished vitality, a fresh culture was necessary. Accordingly, after some difficulty and by means of the methods which have just been referred to, there was isolated from the fæces of a patient having a typical case of typhoid fever at the Lawrence General Hospital, a bacillus which, after long and careful study, appeared to be in every way identical on all culture media with typhoid cultures obtained from Prudden of New York and from Chantemesse of Paris. It also agreed very closely with the most reliable descriptions. It is of interest to note that the fæces were passed on the fifth day of the patient's illness. In the course of these investigations, eleven complete examinations of typhoid fæces and three of typhoid urine were made, in which the specific organism could not be found. It may be observed in passing that the rejection of the potato test for typhoid fever bacilli has made necessary a re-examination of many of the accepted data in this disease. A thorough study of typhoid fæces is especially required.

In addition to these necessary investigations, which covered a period of more than six months, experiments were made which indicated that typhoid fever germs are able to survive in the water of the Merrimack River, but in greatly diminished numbers, for a period of at least three weeks, - sufficiently long to enable these germs to pass from the Lowell sewers to the service pipes of the Lawrence water supply. The conditions were also studied for the application of these germs, in large numbers, to the several water filters. The composition of the nutrient media, in which the germs could be best grown and applied to the filters, was investigated, both with respect to the typhoid fever bacillus itself and to the ordinary species present in the river water. These and other details were worked out and have since been applied in the numerous experiments upon water filtration which are in progress during the present year, some of which are summarized in the preceding section.

DETERMINATIONS OF SPECIES OF BACTERIA.

In the course of the investigations upon typhoid fever bacilli much was learned concerning the species of ordinary water bacteria. It became necessary to compare the species of bacteria found in the effluents with those in the water or sewage applied to the several filters. Filter Tank No. 8, filtering at first city water and afterwards river water, was selected for exhaustive study, and the results are given in detail below. It soon became evident that the degree of bacterial purification in this case must be learned, not so much by study of the morphological and physiological characteristics of a few species of bacteria in the applied water and in the effluent, as by a long-continued and systematic comparison of all of the species, and the number of each present in the water before and after its passage through the filter. To this end, it became necessary to devise means by which the species could be readily recognized, and also for estimating the number of each species of bacteria present in the sample of water.

A method was devised which consisted in examining all of the colonies on the gelatine plate, and estimating the number of each kind presenting marked characteristics. This placed together in groups, kinds superficially resembling one another. One or more colonies of each group were "fished," and ten or more colonies possessing no marked characteristics on the gelatine plate (including many non-liquefying colonies lying beneath the surface) were taken at random; and, in case of less than ten such colonies, all were taken. Fortunately plates were seldom seen in this work which contained more than twenty-five colonies. To facilitate the separations, dilutions of one to one hundred were made of the river water. After the species of bacteria were recognized the number present of those possessing marked characteristics on the gelatine plate was easily calculated, and of the remainder each species was given its aliquot portion. During the earlier part of the work the following tests were used in the species determinations:

1. Examination in the hanging drop, of bouillon culture two days old. 2. Examination of stained preparations from a seven-days-old agar culture, with particular reference to spore formation.

3. Growth on the gelatine plate.

4. Growth in the gelatine tube.

5. Growth on the agar plate incubated at 37° C.

6. Growth on the inclined agar tube (both at 20° C. and at 37° C).

7. Growth in bouillon.

8. Growth on potato.

9. Reduction of nitrates in peptone solution.

10. Formation of turbidity or of gas, or of both, in closed arm in the Smith test.* 11. Coagulation of milk (observations made both before and after boiling). 12. Fermentation of milk (quantitative determination of acid or alkali).

With a thorough knowledge of the species in a particular case, the amount of labor in identifying them may be much reduced. Thus it was learned that in many cases the growth on potato, under the mica plate on gelatine, in bouillon, and the examination in the hanging drop, gave no differentiations which were not as well shown by the other tests. They were accordingly omitted in some cases, although they were made from time to time, to ascertain that no new species of bacteria had become prominent without detection. The conditions of determination were made as nearly as possible parallel, both with regard to time and temperature. On account of the large number of determinations necessary, it was not possible in this particular inquiry to follow each culture carefully, day by day. In fact, at the outset it was apparent that this method of procedure might result in giving groups of closely related species, rather than the rigid separation of individual species. But in this investigation it was far more important to obtain an accurate quantitative determination of all the groups of species in both applied water and effluent than a more detailed study of a limited number. A careful study and classification of the data obtained has readily indicated the appearance from time to time of new species, and as a matter of fact this method of species determination, after several months' trial, has proved to be trustworthy.

A close examination of the species included in the several groups has revealed the important fact that the microscopic flora of the Merrimack River includes not an infinite number of kinds of bacteria but only about thirty prevailing species.

TOTAL REMOVAL OF BACTERIA FROM THE APPLIED WATER BY FILTER TANK No. 8.

This filter, which is described on pp. 607-617, has filtered city water intermittently, at the average rate of 183,000 gallons per acre daily, during 1891. Beginning in May, 1891, the attempt was made

Centralblatt für Bakteriologie, Bd. VII., p. 502.