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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 bad 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 baving 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.

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.

to determine with certainty whether any of the bacteria found in the effluent came down through the filtering material with the applied water. The sanitary importance of determining this point is obvious, since one of the principal objects of water filtration is the removal of pathogenic bacteria. Elaborate preparations were therefore made to settle this difficult question. The degree of bacterial purification accomplished by Filter Tank No. 8, indicated by the number of bacteria in the water before and after passing through it, is shown by the following table :

Table of Monthly Averages of the Number of Bacteria found in a Cubic Centimeter

of City Water and in the corresponding Efluent from Filter Tank No. 8.

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In January, 1890 (see Special Report on Purification of Sewage and Water, 1890, page 615), it was shown that there was a growth of bacteria in the underdrains and outlet pipe of this filter. proved by examination of the water expressed from a sterilized sponge after wiping out the pipe. This operation was repeated several times in 1891. The species present in the air and dust were obtained by exposing a sterile gelatine plate in the vicinity of the outlet pipe. These species of bacteria, as well as those found in the outlet pipe and underdrains, were quantitatively determined.

In the following table a comparison is shown of the results of the quantitative determination of the species of bacteria in the applied city water, in the effluent, the air, the outlet pipe and the underdrains. In the case of air the figures indicate the percentage which each species forms of the total number of bacteria on the plates. It is to be noted that it was during this period that the quantitative method of species determinations was developed, and, although the work was done as carefully as possible, the results are somewhat less accurate than those obtained after more experience with the method.

May

Average Results of Quantitative Determinations of the Species of Bacteria.

to August, inclusive, 1891.

SPECIES OF BACTERIA. (AVERAGE NUMBER PER CUBIC CENTIMETER.)

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It will be seen that the species are designated by numbers, and not by names. For reasons stated above, this work has been one of comparative and quantitative species determination, rather than a detailed study of the species themselves. As nearly as can be learned, however, from the comparisons made up to this time, the numbers used in these tables signify, and doubtless in most cases are identical with, previously described species, as indicated below :

No. 26 = B. cereus, Frankland
No. 24 = B. subtilis, Cohn.
No.5= B. cloaca, Jordan.
No. 35= B. fluorescens liquefaciens, Flügge.
No. 20=B. candicans, Frankland.
No. 11.2 = B. coli communis, Escherich.
No. 57 = B. ubiquitus, Jordan.
No. 55 = B. aurantiacus, Frankland.

No. 150 = red yeast. In studying the first portion of the table given above, it will be seen that species Nos. 20 and 11.0, which form more than eighty per cent.

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