TABLE XV.[A]—ANTISEPTIC EFFECT OF CHLORINE

Table XVI shows the effect of a concentration of 1.0 p.p.m. of chlorine; the hypochlorite at this concentration acted almost entirely as a germicide or disinfectant.

TABLE XVI.[B]—EFFECT OF CHLORINE AS A DISINFECTANT

Table XV shows a recovery of the anabolic functions after treatment with 0.1 p.p.m. of chlorine but since this was obtained by plating on such a suitable medium as nutrient gelatine, it is probable that reproduction in water having a low organic content would be still further diminished. This is indicated by the results obtained.There is no evidence of any marked difference in the resistance of ordinary water bacteria to chlorine and these are the first to be affected by the added germicide. The common intestinal organisms are also very susceptible to destruction by chlorine and there is considerable evidence that B. Coli is slightly more susceptible than many of the vegetative forms usually found in water. The specific organisms causing the water-borne diseases, typhoid fever and cholera, are, on the average, not more resistant than B. coli.

The spore-forming bacteria usually found in water are those of the subtilis group, derived largely from soil washings, and B. enteritidis sporogenes, from sewage and manure. The spores of these organisms are very resistant and survive all ordinary concentrations. Wesbrook et al.[1] found that 3 p.p.m. of available chlorine had little effect on a spore-forming bacillus isolated from the Mississippi water and the author has obtained similar results with B. subtilis.

Thomas,[2] during the chlorination of the Bethlehem, Pa., supply, found four organisms that survived a concentration of 2 p.p.m. of available chlorine: Bact. Ærophilum, B. cuticularis, and B. subtilis, all spore formers and M. agilis.In practice no attempt is made, except in special cases, to destroy the spore-bearing organisms as they have no sanitary significance and the concentration of chlorine required for their destruction would cause complaints as to tastes and odours if the excess of chlorine were not removed. Such doses are unnecessary and result in waste of material. It is found that, when the dose is sufficient to eliminate the B. coli group from 25-50 c.cms. of water, the majority of the residual bacteria are of the spore-bearing type. Smeeton[3] has investigated the bacteria surviving in the Croton supply of New York City after treatment with 0.5 p.p.m. of available chlorine as bleach. Table XVII gives the results obtained.

The organisms of the B. subtilis group outnumbered all the others, 66 (62.8 per cent) belonging to this group alone. This group contained B. subtilis—Cohn (36 strains), B. tumescens—Chester (15 strains) B. ruminatus—Chester (13 strains), and B. simplex—Chester 1904, (2 strains). Three of the four coccus forms were classified as M. luteus. No intestinal forms were found.Clark and De Gage[4] in 1910 directed attention to the fact that the bacterial counts, made at 37° C. on chlorinated samples, were often much greater than the counts obtained at room temperature. “This phenomenon of reversed ratios between counts at the two temperatures,” they stated, “has been occasionally observed with natural water, but a study of the record of many thousands of samples shows that the percentage of such samples is very small, not over 3-5 per cent.... On the other hand 20-25 per cent. of samples treated with calcium hypochlorite show higher counts at body temperature than at room temperature.” Clark and De Gage were unable to state the true significance of this phenomenon but were of the opinion that it was not due to larger percentages of spore-forming bacteria in the treated samples. Other observers, on the contrary, have invariably found the spore-formers to be more resistant to chlorine and thermophylic in type.

TABLE XVII.—ORGANISMS SURVIVING TREATMENT
NEW YORK

The removal of intestinal forms is, of course, merely a relative one and when large quantities of treated water are tested their presence can be detected.The author, in 1915, made a number of experiments to ascertain whether the B. coli found after chlorination were more resistant to chlorine than the original culture. The strains surviving treatment with comparatively large doses were fished into lactose broth and subjected to a second treatment, the process being repeated several times. The velocity of the germicidal reaction with the strains varied somewhat, but not always in the same direction, and the variations were not greater than were found in control experiments on the original culture. No evidence was obtained that the surviving strains were in any way more resistant to chlorine than the original strain; in considering the results it should be borne in mind that the surviving strains were cultivated twice on media free from chlorine before again being subjected to chlorination.

A number of the strains that survived several treatments were cultivated in lactose broth and the acidity determined quantitatively. All the cultures produced less acid than the original culture, and the average was materially less than the original. These results point to a diminution of the bio-chemical activity by action of the chlorine.A point of perhaps more scientific interest than practical utility is the relative proportion of the various types of B. coli found before and after treatment with chlorine. The author, in 1914, commenced the differentiation of the types by means of dulcite and saccharose and obtained the results shown in Table XVIII. These figures are calculated from several hundreds of strains.

Although there is a slight difference in the relative proportions of the types found at Ottawa and Baltimore, both sets of results show definitely that there is no difference in the resistance of the various types to chlorination.Aftergrowths. In Tables XIII (p. 44) and XV (p. 51), it will be noticed that, after the preliminary germicidal action has subsided, a second phase occurs in which there is a rapid growth of organisms. This is usually known as aftergrowth. When the contact period between chlorination and consumption is short, the reaction does not proceed beyond the first phase, but when the treated water is stored in service reservoirs the second phase may ensue. At one purification plant, where the service reservoirs are of large capacity, the aftergrowths amounted to 20,000 bacteria per c.cm. although the water left the purification plant with a bacterial count usually lower than 50 per c.cm.

TABLE XVIII.—TYPES OF B. COLI SURVIVING CHLORINATION

Regarding the nature of this aftergrowth, there has been a considerable difference of opinion: some regard it as the result of the multiplication of a resistant minority of practically all the species of organisms present in the untreated water; others, that it is partially due to the organisms being merely “slugged” or “doped,” i.e. are in a state of suspended animation, and afterwards resume their anabolic functions; whilst others believe that with the correct dosage of chlorine, only spore-forming organisms escape destruction and that the aftergrowth is the result of these cells again becoming vegetative.

The aftergrowths obtained under the usual working conditions vary according to the dosage of chlorine employed, and none of the above hypotheses alone provides an adequate explanation. When the dosage is small, a small number of active organisms, in addition to the spore bearers, will escape destruction, and others will suffer a reduction of reproductive capacity. The flora of the aftergrowth in this case will only differ from the original flora by the elimination of a majority of the organisms that are most susceptible to the action of chlorine and the weaker members of other species of greater average resistance. As the dose is increased these factors become relatively less important until a stage is reached when only the most resistant cells, the spores, remain. The resultant aftergrowth must necessarily be almost entirely composed of spore-bearing organisms. A small number of the most resistant members of non-sporulating organisms may also be present but they will, in the majority of instances, form a very small minority. This is the condition that usually obtains in practice and it is necessary to consider whether the aftergrowth may have any sanitary significance.

Concerning the secondary development of B. coli, the usual index of pollution, there is but little information. H. E. Jordon[6] reported that, of 201 samples, 21 gave a positive B. coli reaction immediately after treatment, 39 after standing for twenty-four hours, and 42 after forty-eight hours. These increases were confined to the warm months, the cold months actually showing a decrease. The following figures, taken from the author’s routine tests for 1913 and 1914, show a similar tendency, but an analysis of the results by months did not show that this was confined to the warm season. The sequence of the results from left to right, in the following Table, is in the same order as the contact period. Approximately 290 samples were taken at each sampling point.

At station No. 2 the germicidal action was still proceeding but at No. 5, representing an outlying section of the city, the increase in the B. coli content is very apparent.

During 1915 and 1916 the author endeavoured to duplicate these results under laboratory conditions and entirely failed. These experiments, which were made with the same materials as were in use at the city chlorination plant, but in glass containers, were usually only carried to a forty-eight hours contact, as this was the extreme limit for the city mains; one, however, was prolonged to five days. Many experiments were made under varying conditions, with similar results. Typical examples are given in Tables VI, VIII and IX on pages 33 and 37.

TABLE XIX.—AFTERGROWTHS OF B. COLI

In every case there was persistent diminution in the number of B. coli with increase of contact period. Determination of the bacterial count on nutrient agar showed that, in several experiments, the aftergrowth had commenced, and in some instances there was evidence that the second cycle was partially complete i.e. the number had reached a maximum and then commenced to decline. The time required for the completion of the two cycles, comprising the first reduction caused by the chlorine, the increase or aftergrowth, and the final reduction due to lack of suitable food material, is dependent upon several factors of which the dosage and temperature are the most important. With a small dosage the germicidal period is short and the second phase is quickly reached; with large doses, the second phase is not reached in forty-eight hours; the higher the temperature the quicker is the action and the development of the aftergrowth. These statements refer only to the bacteria capable of development on nutrient agar. The B. coli group behaved differently and persistently diminished in every case. If B. typhosus acts in a similar manner to B. coli, the laboratory experiments show that aftergrowths are of no sanitary significance and can safely be ignored, but as the results obtained in practice are contradictory to the laboratory ones, the matter must be regarded as sub judice until more definite evidence is available.

It is common knowledge that samples of water from “dead ends” of distribution mains show high counts and much larger quantities of B. coli than the water delivered to the mains. This is another phase of aftergrowth problem that often causes complaints and can only be eliminated by “blowing off” the mains frequently or by providing circulation by connecting up the “dead ends.” One extreme case of this description might be cited. A small service was taken off the main at the extreme edge of the city to supply a Musketry School two miles away and was only in use for a few months in the summer season. This service pipe delivered water containing B. coli in a considerable percentage of the 10 c.cm. samples and in a few instances in 1 c.cm., although the water delivered to the city mains never exceeded 2 B. coli per 100 c.cms. and averaged about one-tenth that quantity. No epidemiological records of the effect of this water are available because it was put through a Forbes steriliser before consumption.

In some instances the rate of development of the organisms after chlorination is greater than in the same water stored under similar conditions. This is especially noticeable in the presence of organic matter and has been ascribed to the action of the chlorine on the organic matter with the production of other compounds that are available as food material for the organisms.

Houston, during the treatment of prefiltered water Lincoln in 1905, found that although the removal of B. coli and other organisms growing at 37° C. was satisfactory, there was almost invariably an increase in the bacteria growing on gelatine at 20° C. This was ascribed to the action mentioned above and the chemical results supported this view, more organic matter being found in the filter effluents than in the prefiltered water. Rideal’s experiments with sewage at Guildford indicate that a similar action may occur in contact beds. The addition of bleach to the prefiltered water at Yonkers also resulted in an increased count and in these instances the aftergrowths are due to a disturbance of the equilibrium by the action of the chlorine on the zooglea and other organic matter invariably found in ripe filters. Similar results can be produced by the addition of chlorinated water to small experimental sand filters. This is shown by the results in Tables XX and XXI.

TABLE XX.—AFTERGROWTHS IN SAND

TABLE XXI.—AFTERGROWTHS IN SAND

It is observable that the effect of small doses was comparatively small and transient; large doses of bleach reduced the bacteria very materially but the reduction was not maintained and the subsequent increase was abnormally rapid.

BIBLIOGRAPHY

[1] Wesbrook, Whittaker and Mohler. J. Amer. Pub. Health Assoc., 1911, 1, 123.

[2] Thomas. Jour. Ind. and Eng. Chem., 1914, 6, 548.

[3] Smeeton. Jour. of Bact., 1917, 2, 358.

[4] Clark and De Gage. Rpt. Mass. B. of H., 1910, p. 319.

[5] Thomas and Sandman. J. Ind. and Eng. Chem., 1914, 6, 638.

[6] Jordan, H. E. Eng. Record, 1915, May 17.