DIAGRAM VRELATION OF DOSAGE TO OXYGEN ABSORBEDRelation of dosage to oxygen absorbed
DIAGRAM VRELATION OF DOSAGE TO OXYGEN ABSORBED
Another method which has been generally adopted for military work during the war, consists in the addition of definite volumes of a standard chlorine solution to several samples of the water and, after a definite interval, testingfor the presence of free chlorine by the starch-iodide reaction. The details of the method of Gascard and Laroche, which is used by the French sanitary service, have been given by Comte.[12]One hundred c.cms. of the water to be examined are placed in each of 5 vessels and 1, 2, 3, 4, and 5 drops of dilute Eau de Javelle (1 : 100) are added and the contents stirred. After twenty minutes, 1 c.cm. of potassium iodide-starch reagent (1 gram each of starch, potassium, iodide and crystallized sodium carbonate to 100 c.cms.) is added and the samples again stirred. The lowest dilution showing a definite blue colour is regarded as the dose required, and the number of drops is identical with that required of the undiluted Eau de Javelle for 10 litres of water when the same dropping instrument is used. The actual concentration represented by these dilutions depends necessarily upon the size of the drops and the strength of the undiluted Eau de Javelle, but one drop per 100 c.cms. usually represents approximately 1 p.p.m.
In Horrocks’s method, as used in the British army, a standard bleach solution is added and is almost immediately followed by the zinc iodide-starch reagent. The two methods were compared by Massy,[13]who found that the French method gave an average result of only 0.06 m.gr. per litre (0.06 p.p.m.) higher than the English method. Water in the Gallipoli campaign required from 0.21 to 1.06 p.p.m. as determined by both methods.
Diénert, Director of the Paris Service for investigating drinking water, adds 3 p.p.m. of available chlorine and allows the mixture to stand fifteen minutes after shaking; the residual chlorine is then titrated with thiosulphate. The amount absorbed is increased by 0.5 p.p.m. and in the opinion of Diénert this dosage is correct for a contact period of three hours.
For military camps where a standpipe usually provides a reasonable contact period, it has been found good practice to add sufficient chlorine to give a rich blue colour with the starch-iodide reagent and subsequently reduce the dosage gradually until the water, after standing one hour, gives but a faint reaction to the test reagent. This method should be checked up as soon as possible by bacteriological examinations. An example of this method is given inTable XIV.
[1]Nissen. Zeit. f. Hyg., 1890,8, 62.[2]Delépine, J. Soc. Chem. Ind., 1911,29, 1350.[3]Phelps. Water Supply Paper No. 220, U. S. Geo. Survey.[4]Wesbrook, Whittaker, and Mohler, J. Amer. Public Health Assoc., 1911,1, 123.[5]Lederer and Bachmann. Eng. Rec., 1912,65, 360.[6]Harrington. J. Amer. Waterworks Assoc., 1914,1, 438.[7]Ellms. Eng. Rec., 1911,63, 472.[8]Heise. Philippine Jour. Sci., 1917,12, A, 17-34.[9]Norton and Hsu, Jour. Inf. Dis., 1916,18, 180.[10]Langer. Zeit. f. Hyg., 1916,81, 296.[11]Longley. J. Amer. Public Health Assoc., 1915,5, 920.[12]Comte. J. Pharm. Chim., 1916,14, 261.[13]Massy. J. Pharm. Chim., 1917,15, 209.
[1]Nissen. Zeit. f. Hyg., 1890,8, 62.
[2]Delépine, J. Soc. Chem. Ind., 1911,29, 1350.
[3]Phelps. Water Supply Paper No. 220, U. S. Geo. Survey.
[4]Wesbrook, Whittaker, and Mohler, J. Amer. Public Health Assoc., 1911,1, 123.
[5]Lederer and Bachmann. Eng. Rec., 1912,65, 360.
[6]Harrington. J. Amer. Waterworks Assoc., 1914,1, 438.
[7]Ellms. Eng. Rec., 1911,63, 472.
[8]Heise. Philippine Jour. Sci., 1917,12, A, 17-34.
[9]Norton and Hsu, Jour. Inf. Dis., 1916,18, 180.
[10]Langer. Zeit. f. Hyg., 1916,81, 296.
[11]Longley. J. Amer. Public Health Assoc., 1915,5, 920.
[12]Comte. J. Pharm. Chim., 1916,14, 261.
[13]Massy. J. Pharm. Chim., 1917,15, 209.
A disinfectant is usually described as a substance capable of destroying bacteria and other micro-organisms, and an antiseptic as one that restrains or retards their growth or reproduction. This distinction is entirely arbitrary as the ability of a substance to kill organisms or merely inhibit their growth depends upon the concentration employed.
Chlorine and hypochlorites, even in minute doses, exert a toxic effect that is sufficient to produce death in organisms but when still smaller concentrations are employed the toxic effect is transient and the reproductive faculty may be entirely regained.
The enumeration of bacteria by means of solid media depends upon the ability of the organism to reproduce at such a rate as to produce a visible colony within the period of incubation and any substance that prevents the growth of a visible colony is classified as a disinfectant; if on further incubation the bacterial count approximates that of the untreated sample the added substance has acted mainly as an antiseptic. In practice no substance acts entirely as an antiseptic as the organisms present have varying degrees of resistance and the less viable ones are killed by doses that are only antiseptic to the more resistant ones. An example of an antiseptic effect followed by a mild disinfectant action, caused by small doses of bleach is shown inTable XV. In this experiment the water designated as control was from the same source as the treated water. In order to make the bacterial count in this water approximately the same as in the treated water, the original count was reduced by diluting the sample with water from the same source, sterilised by boiling, and afterwards reaërated with sterile air.
Table XVIshows 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 XVshows 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 thatB. Coliis slightly more susceptible than many of the vegetative forms usually found in water. The specificorganisms causing the water-borne diseases, typhoid fever and cholera, are, on the average, not more resistant thanB. coli.
The spore-forming bacteria usually found in water are those of the subtilis group, derived largely from soil washings, andB. 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 withB. 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, andB. subtilis, all spore formers andM. 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 theB. coligroup 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 XVIIgives the results obtained.
The organisms of theB. subtilisgroup outnumbered all the others, 66 (62.8 per cent) belonging to this group alone. This group containedB. subtilis—Cohn (36 strains),B. tumescens—Chester (15 strains)B. ruminatus—Chester (13 strains), andB. simplex—Chester 1904, (2 strains). Three of the four coccus forms were classified asM. luteus. No intestinal forms were found.
Clark and De Gage[4]in 1910 directed attention to thefact 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.
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 theB. colifound 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 ofB. colifound 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 inTable 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.InTables XIII(p. 44) andXV(p. 51), it will be noticed that, after the preliminary germicidal action has subsided, a second phase occurs in which there isa 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.
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 adequateexplanation. 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 ofB. coli, the usual index of pollution, there is but little information. H. E. Jordon[6]reported that, of 201 samples, 21 gave a positiveB. colireaction 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 thefollowing 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 theB. colicontent is very apparent.
During 1915 and 1916 the author endeavoured to duplicatethese 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 inTables VI,VIIIandIXonpages 33and37.
In every case there was persistent diminution in the number ofB. coliwith 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 developmenton nutrient agar. TheB. coligroup behaved differently and persistently diminished in every case. IfB. typhosusacts in a similar manner toB. 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 assub judiceuntil 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 ofB. colithan 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 containingB. coliin 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 2B. coliper 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 ofB. coliand 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 inTables XXandXXI.
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.