950

to 1904.

THE AMERICAN JOURNAL

OF

PUBLIC HEALTH

FIGURE III from that for Boston in the period 1900 ITS RATE OF DECLINE Both cities, however, show the BIRTH RATE AND THE CITY OF LONDON BY SEASON FOR winter peak with the falling off in the spring. When we examine the rates of decline we find that the average annual decrease and the percentage annual decrease vary with the size of the birth rate, At indicating that the decline in the birth rate is differential as to season. This form of differential decrease is evidently tending toward the elimination or the smoothing out of the seasonal curve for births. Since time intervals of 22 and 35 years are too short for us to consider that this change is indicative of a biological change in mankind, it is obvious that we must look for the explanation in the forces of environment. While many of the factors which affect birth rate, such as birth con- that these two populations are becoming trol, nutrition, etc., come to mind as pos- more and more uniform in their producsible reasons for this differential decline tion of offspring throughout the season. by months, it is unlikely that it is due to any single one of them. Whatever the exREFERENCES 1. M. B. Social Consequences of the planation may be, it is of interest to note ness Hexter,pp. 11. Houghton Mifflin Company. BusiCycle,
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T8sj~fIrl

THE SIGNIFICANCE OF BACTERIOPHAGE IN SURFACE WATER*

LLOYD ARNOLD, M.D.
Department of Bacteriology, Pathology and Preventive Medicine, Loyola University School of Medicine, Chicago

BACTERIOPHAGE is capable of causing dissolution or lysis of certain susceptible bacteria; this lytic process is transmissible in series and the active principle increases in potency as bacteria are lysed. Several investigators have
reported the presence of this bacteriophagic substance in surface water.' If surface water does contain such a potent lytic principle, then its effect upon parasitic and pathogenic bacteria found in such water would be of interest from a public health standpoint.
* Read before the Laboratory Section of the American Public Health Association at the Fifty-fourth Annual Meeting at St. Louis, Mo., October 21, 1925.

We wish to mention briefly some of the characteristics of the bacteriophage before recording our experiments. 1. A susceptible bacteria must be viable and must be in an environment in which it is growing before the bacteriophage can cause lysis of the bacteria. 2. When susceptible bacteria are placed in a fluid medium, containing bacteriophage, this substance adheres or is adsorbed by the bacteria from the medium. When such sensitized strains are put in an environment that allows reproduction to, take place, the bacteriophage causes lysis of the young bacteria with an increase in the potency of the active lytic principle.

SIGNIFICANCE

OF

BACTERIOPHAGE

IN

SURFACE WATER

951

3. Phage-sensitive bacteria are more susceptible to adverse environment than are resistant strains of the same bacteria.2 The experimental procedure is as follows:

A sample of surface water is passed through a Berkefeld candle and some of the filtrate added to nutrient broth and seeded with a susceptible strain of bacteria. Subcultures are made upon the surface of agar plates to determine the presence of irregular co'lonies or sterile areas on the agar after 24 hours' incubation. -In some instances 0.5 c.c. of the filtered water added to 10 c.c. of a young broth culture of B. typhosus is sufficient to cause a marked phagic action when immediately subcultured, and leads to clearing of the cultures in a few hours. Where there is only a trace of bacteriophage, the filtered water is added in equal parts to double strength nutrient broth, seeded with a susceptible strain of bacteria and after 24 hours' incubation this culture is passed through the Berkefeld filter and the filtrate added to nutrient broth in the same manner as the original water filtrate from a highly polluted region. This is necessary in order to cause the increased concentration of the phagic substance to make it detectable in. quantitative amounts. The faintest trace of bacteriophage can in this way be determined, and by several such passages it can be made as potent as is desired for experimental work. The strong or potent phage made in this way does not differ culturally or antigenically from a phage of high potency isolated from the same surface water close to a domestic sewerage source. As the phage increases in potency it causes lysis of strains that seemed previously to be resistant to its lytic action. If a susceptible strain of B. typhosus is added to some Chicago River water close to the State Street sewer opening and subcultures are made into broth or upon agar, the B. typhosus that develops is rapidly lysed and 18- to 24-hour old

broth cultures are cleared of visible bacterial growth (Chart I). Susceptible strains disappear from such water more rapidly than do resistant strains. Phagesensitive strains do not multiply in polluted water and consequently never develop a resistance to the phagic activity. Sensitive B. typhosus, B. coli and several different B. dysenteriae strains have been kept in contact with the filtered water for 7 days. Such strains are as sensitive to phage as the original culture before contact with the bacteriophage containing
water.

We have found the bacteriophage to be present in variable amounts in the Chicago River water. The concentration is in direct proportion to the concentration of domestic sewage. The farther away from domestic sewerage source the more diluted is the bacteriophagic substance. We have always found bacteriophage in the water at the opening of the State Street sewer (drainage from theloop district) that was active B. typhosus, B. coli, B. dysenteriae group, B. paratyphosus B. and Staphylococcus. Trade wastes cause a rapid disappearance of the bacteriophage from water. The presence of bacteriophage in surface water is too variable to be used as a criterion of domestic sewage pollution; it -does not compare with the recognized bacteriological and chemical methods in determining pollution. From our results so far it would seem that the bacteriophage in water would exert an influence upon the future development of the pathogenic or parasitic strains of bacteria of the gastro-intestinal group, such as' B. coli, B. typhosus, B. dysenteriae, etc. If such water with its pathogenic ba'cteria comes in contact with food material, milk, etc., the growth is accompanied with lysis and it would require several days for a good growth of phage-resistant strain to develop. This can play a very important role from an epidemiological standpoint in gastrointestinal infections. One c.c. of a 24-hour old culture of

952

THE AMERICAN JOURNAL OF PUBLIC HEALTH

The Lake Michigan 24-hour culture gave
a massive regular growth. On the fifth day the Chicago River subculture gave a

typhoid bacteria was added to 50 c.c. of Berkefeld filter Chicago River water, the same amount of bacteria was added to 50 c.c. of Berkefeld filtered Lake Michigan water obtained from an intake 4crib some distance away from the shore.
CHART I IB. TYPHOSUS ADDED TO BERKEFELD FILTERED LAKE MICHIGAN WATER AND ALSO CHICAGO RIVER WATER AND SUBCULTURED AFTER 3 HOURS CONTACT INTO

heavy regular growth. Upon examination this culture was resistant to phagic action. This experiment was repeated several times and the viable bacteria were counted by the usual dilution method.
The results are shown in Chart I. These graphs represent the average results of ersn heaeaerslso

5RROTH.*gah

6 experiments. The importance from a

9
a 7

public health standpoint of contamination

of milk supply or other material with B. typhosus from the two sources does not need to be dwelt upon. Surface water containing a high degree of domestic sewage pollution contains bacteriophagic substances. Phage-sensitive pathogenic bacteria in such water are not as liable to be the cause of an epidemic as the same number of bacteria in a surface water that is free of bacteriophagic substances. Bacteriological examination of water for pathogenic bacteria can be influenced by the phagic content of such water. The impregnation of water filters with bacteriophage active against certain pathogenic gastrointestinal bacteria offers a new field of investigation in water purification problems. Of equal importance is the utilization of these tranmissible bacterial autolysins in sewage disposal problems

5
4

3 2

I
O

,"............... . , , . ,
2
4

.
96
lZO hours*

12

24

.8

72

* Heavy line-B. typhosus from Lake Michigan water, Water.

Dotted line-B. typhosus from Chicago River

These

are

the average results of 6 experiments.

After Ijwas transferred to 10 c.c. of nutrient

I I
I

12 hours' contact 1 c.c. from each

broth and subcultured after 6 and 12 hours and then every 24 hours for 7 days. Dne standard loop was transferred to the surface of an agar plate and smeared with i bent glass spreader. The subcultures from the Chicago River water gave the same growth at the 6-hour period as the ILake Michigan water. The 12-hour IChicago River subculture showed bacREFERENCES 1. Dumes. Sur la presence du bacteriophage dans tteriophagic action, the growth was not so sain saus la Compt. rend. and.many of the colonies were lintestinbiol.,'83:1314, terre et daus 1920. 'rood and*oodmany of the colonies were Soc. de P. C. Der Bacteriophage l'eau. die Selbstreinund Flu, moth eaten or irregular in outline. igung des Wassers. Centralbi. f. Bakteriol u. Parae Beitrage E. Kritische Fhe 24-hour Chicago River subculture SzurZ.dansky, der Wirkungs und experimentelle BacterioFrage moeglichkeit der Iwas sterile, also the 48-hour subculture. phagem, etc. Ztschr. f. Hyg. N. Infectionsskrankh.,
I

NOTE.-The author is indebted to Arthur E. Gorman, Sanitary Engineer, Chicago Department of Health, for 1: cooperation in securing the water samples and for I nany helpful suggestions during the progress of this Iwork.
I

iis

103:164, 1924. Nakashima, T. Beitrag zum Vorkommen und Verhalten des bacteriophagen Lysins in Abwassern. Centralbl. f. Bakteriol., Abt. I, Orig. 94:303, 1925. 2. Arnold, L. Twort-d'Herelle's Phenomenon. Abst. Bact., 8:7 (Jan.), 1924.