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were intended to serve a double purpose—one to determine the biology of the IPotomac water, and the other to serve as preliminary to a proposed investigation concerning the pollution of the water supplies, where the sanitary condition of one or more States was threatened or involved. It was intended to collect the data of at least a year before publishing the final results, yet it is believed that the inquiry has been sufficiently far advanced to furnish conclusions of value. This investigation has been assigned to my colleague, Passed Assistant Surgeon Sprague, who submits the following report: HYgiENIC LABoratoRY, Washington, D.C., March 10, 1898. SIR: I have the honor to submit for your consideration the following report of the bacteriological examination of Potomac River water from July 1, 1897, to February 28, 1898, inclusive: In order that sufficient data might be obtained from which to draw reliable conclusions, it was determined to make semiweekly bacteriological analyses of our drinking water for a period of at least one year. Realizing the necessity of certain fixed factors in our work, upon which would depend the value of comparisons to be drawn, preparations were made to secure the desired uniformity. Accordingly, sufficient media was prepared in June, 1897, to last through the entire year. Using, as we have, for each examination, the same culture material for the bacteria, any sudden variation in the biological character of the water can in no instance be attributed to the employment of different media. There must also be a common source from which to obtain the water, and it should represent as nearly as possible the water which flows in our mains. In the basement of the Marine Hospital Bureau building, in which the hygienic laboratory is located, is a tap which is kept constantly running in the operation of a vacuum apparatus, and from this source all of our samples have been collected. It is evident that these samples must very accurately represent the condition of Potomac water as it flows in the city pipes. The temperature has been taken with a centigrade thermometer, graduated to tenths of one degree, and a note made of the clearness or turbidity. The water was always collected in a sterile graduate, provided with a sterile paper cover, and thus conveyed to the laboratory. By means of a sterile pipette 5 cubic centimeters of the water was introduced into each of four fermentation tubes, two of which contained 1 per cent lactose-peptone bouillon, while the other two contained 1 per cent dextose-peptone bouillon. The duplicate tubes were used for purposes of control as well as to furnish a broader basis upon which to conduct further research for the colon group of organisms and other sewage bacteria. The fermentation tubes were then placed in a special incubator kept at a constant temperature of 39.5°C., and allowed to remain undisturbed for a period of forty-eight hours. At the same time 1 cubic centimeter of the water was introduced into a flask containing 99 cubic centimeters of freshly sterilized distilled water. The flask was then agitated sufficiently to make an even mixture, and 1 cubic centimeter was placed in each of six Petri dishes. Into the Petri dishes was then poured a tube of 2 per cent glycerine agar, which was thoroughly mixed with the 1 cubic centimeter of diluted Potomac water. Three of these plates were allowed to develop for forty-eight hours in the dark at room temperature, which varied from 25°C. to 30°C. The remaining three were placed in a Novy jar, and by repeated exhaustion of the air and by the introduction of hydrogen a pure atmosphere of the latter gas was obtained. These plates were then allowed to develop under the same conditions of temperature, darkness, and time as the first three. At the expiration of forty-eight hours the colonies were counted and an average taken, which, multiplied by 100, gave the number of organisms in 1 cubic centimeter of undiluted Potomac water. To those grown in the atmospheric air the name aërobes is given, and to those grown in hydrogen anaërobes. At the same time that the colonies were counted the fermentation tubes were examined and the amount of gas, if any, in the arm of the tube was recorded in inches. A hanging drop from each of the tubes was next examined for motile bacilli. If there was no fermentation and no motile organisms present, the tube was destroyed and no further examination made; but if motility, with or without fermentation, was present that tube was considered suspicious, and the examination of all such was continued for the purpose of detecting intestinal or sewage organisms. It may here be noted that the typhoid bacillus and the majority of the colon bacilli possess the power of moving more or less rapidly through a liquid medium in which they may be suspended. Hence the necessity of further study of any bacillus possessing that character. In order to identify the sewage organisms it is necessary to separate them from the numerous harmless bacteria constantly present in water and from which they can not always be distinguished by their morphology. To secure this separation a small loop of the suspicious culture was transplanted into a specially prepared medium known as Elsner's, from the name of the bacteriologist who first made use of it. Without going into the precise method of preparation, it suffices to say that it is an iodized potato gelatine so standardized as to alkalinity that it corresponds exactly in its reaction to a chemically pure solution of calcium hydrate. This particular method of standardizing this medium is a modification of the method originally employed, suggested by Grimbert, and it has been found to give most excellent results. The peculiarity of this medium is that the growth of bacteria other than the bacillus of typhoid fever, the colon bacilli, and sewage organisms is either entirely inhibited or so limited that the intestinal and sewage bacteria, if present, are readily isolated. In passing, it may not be amiss to state that very recently Professor Roux, of the Pasteur Institute of Paris, was heard to say that, in his opinion, Elsner's method was the best yet devised for isolating fecal bacteria. Every batch of Elsner prepared was tested with a pure culture of typhoid and colon to prove that they would grow thereon if present. Inasmuch as gelatine was the substance employed to produce solidity, it became necessary to keep the Elsner plates in a cool chamber, the temperature of which was kept at or below 20°C. The low temperature interfered to some extent with the rapidity of development of the bacteria, and for this reason the plates were retained for a period varying from five to seven days. No growth occurring at the end of that time, further investigation was abandoned; but in case of a growth appearing it was examined more fully for the purpose of ascertaining its character. If the growth was found to be a bacillus, it was at once transferred to several different media in order to learn its behavior and to enable it to be classified. A bacillus obtained from a colony on an Elsner plate, after going through the stages previously described, was studied as to the following points: Did it possess motility? Did it ferment lactose and dextrose peptone bouillon? Did it produce indol after forty-eight hours' growth, at a temperature of 37° C., in Dunham's peptone solution? Did it liquefy gelatine? Did it acidify or coagulate litmus milk, and how long a time was required for any change in this medium? What was the nature of its growth on potato? How did it stain by Gram's method? According to the answers obtained to the foregoing questions was based our determination of the name of the bacillus. While, as can be seen from the above, every effort was made to find the typhoid bacillus, it must be acknowledged that up to the present time it has not been isolated from Potomac water; but the colon bacilli, which very closely resemble the typhoid, and are always found in connection with the last-named germ, were isolated on numerous occasions, as will presently be shown. In addition to the 70 examinations of water taken directly from the tap,55samples

CHART 3

MAP
SHOWING THE
B AS I N

OF THE

POTOMAC RIVER.

Scale of Miles.

Stations at which colon bacilli were found.
· · · sewage bacteria were found.

" " neither colon nor sewage was found

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vere furnished the hygienic laboratory by courtesy of the Geological Survey. They were taken at Westernport, on the north branch of the Potomac; at Romney, on the south branch; at Port Republic, on the Shenandoah, and at all the princi. pal intervening towns down to Point of Rocks. It will thus be seen that the conlition of the entire river and its tributaries was thoroughly investigated. These samples were collected under strict aseptic precautions, were promptly forwarded to the laboratory, and no unnecessary delay occurred in beginning the bacteriological analysis. It has been claimed by some investigators that a bacteriological analysis of water made some hours or days after its collection was worthless, because of the death of the organisms which would occur in the time of the interval between the time of collection and the beginning of the analysis.

For the reason that some slight changes, either by death or growth of the organ

isms, probably would occur in twenty-four or forty-eight hours, no count of the d. bacteria was made from these samples; but anyone familiar with the bacteriolog

ical analysis of water, observing the behavior of many of these samples, would IS found have said that if tinie had destroyed any of the bacteria present in the fresh sam

ple he would want ages to elapse before drinking the same. There were positive indications that some of the samples were literally teeming with bacteria; and the chart of the river showing the points at which the samples were collected and the points where the colon bacilli were found will show that in nearly every instance where sewage contamination could be expected it was present. For the reason above stated, the examination of these samples was limited to the search for sewage bacteria.

Reference to the accompanying chart, No. III, will show the places at which samples were collected marked with a circle, and those places at which the colon bacilli were found are marked with red. Briefly, colon bacilli were found in thirty instances and sewage bacteria were found in six others.

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Number of bacteria found in Potomac water at each examination.

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333 1,133

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