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each in a fermentation tube containing not less than 30 cc. of lactose peptone broth. These shall be incubated forty-eight hours at 37°C. and observed to note gas formation.
b. From each tube showing gas, more than 5 per cent of the closed arm of fermentation tube, plates shall be made after forty-eight hours' incubation, upon lactose litmus agar or Endo's medium.
c. When plate colonies resembling B. coli develop upon either of these plate media within twenty-four hours, a well-isolated characteristic colony shall be fished and transplanted into a lactose-broth fermentation tube, which shall be incubated at 37°C. for fortyeight hours.
For the purposes of enforcing any regulations which may be based upon these recommendations the following may be considered sufficient evidence of the presence of organisms of the B. coli group.
Formation of gas in fermentation tube containing original sample of water (a).
Development of acid-forming colonies on lactose litmus agar plates or bright red colonies on Endo's medium plates, when plates are prepared as directed above under (6).
The formation of gas, occupying 10 per cent or more of closed arm of fermentation tube, in lactose peptone broth fermentation tube inoculated with colony fished from twenty-four hour lactose litmus agar or Endo's medium plate.
These steps are selected with reference to demonstrating the presence in the samples examined of aerobic lactose-fermenting organisms.
3. It is recommended, as a routine procedure, that in addition to five 10 cc. portions, one 1 cc. portion, and one 0.1 cc. portion of each sample examined be planted in a lactose peptone broth fermentation tube, in order to demonstrate more fully the extent of pollution in grossly polluted samples.
4. It is recommended that in the above-designated tests the culture media and methods used shall be in accordance with the specifications of the committee on standard methods of water analysis of the American Public Health Association, as set forth in "Standard Methods of Water Analysis” (A. P. H. A., 1912).
The standard as recommended by the commission was submitted with the following report discussing the question of standards of purity for water in general:
First Progress Report of Commission Appointed to Recommend Standards of Purity for Drinking Water Supplied to the Public by
Common Carriers Engaged in Interstate Traffic
Limits of permissible bacteriological contamination
More than a year has been devoted to consideration and discussion of the problems raised in this connection; but, since the discussion has of necessity been conducted solely through correspondence progress has inevitably been slow, so that, even after so long a time, there remains a number of questions upon which satisfactory agreement has not yet been reached. Your commission, not wishing, on the one hand, to curtail free discussion of points upon which satisfactory agreement has not been reached, nor, on the other hand, to further delay submitting such recommendations as have been agreed upon, respectfully request that this report, though incomplete, be accepted and utilized pending the rendering of a more complete report dealing with questions not included herein.
As a preface to the recommendations which follow it is desired that a clear distinction be made between "standards of purity” and "limits of permissible impurity.” Since purity is an absolute, not a relative, quality, it is obvious that there can be no "standard of purity" other than absolute purity; that this must be the point of departure in estimating deviations from purity or degree of impurity. The recommendations here presented are limits of permissible impurity; they are in no sense "standards of purity.” They are recommended not as the nearest approximation to purity which it is desirable to attain; but, on the contrary, as the furthest deviations from purity considered permissible on the water supplies with which this report deals. In this connection it is desired also to emphasize the statement that these limits of impurity are recommended only for application to the special case in question; that is, the control of the sanitary quality of the water supplies of common carriers.
The problem before the commission has been to recommend limits of permissible impurities such as to meet the following requirements:
1. That water supplies conforming to the prescribed requirements shall be free from injurious effects upon the human body and free from offensiveness to the sense of sight, taste, or smell.
2. That supplies of the quality required shall be obtainable by common carriers without prohibitive expense.
3. That the examinations necessary to determine whether a given water supply meets the requirements shall be as few and as simple as consistent with the end in view.
In the attempt to establish limits of this kind it has been inevitable that manifold difficulties should have been encountered. The first of these is the difficulty inherent in any attempt to establish an exact line of demarcation between two such extremes as undoubtedly safe water supplies and those which should assuredly be condemned. This difficulty is enhanced by the necessity of defining uniform limits for waters from such diverse sources and subject to such varied conditions of storage as are the supplies of common carriers, and is still further enhanced by the necessity of limiting to a practicable minimum the number and kind of examinations upon which judgment must be formed. It is a fact so well established as to need no further discussion that the results of bacteriological and chemical examination of a sample of water ought always to be correlated with a knowledge of the source, treatment, and storage of the supply in order to enable a just estimate of the sanitary quality of such supply. With a full appreciation of this fact it is, nevertheless, necessary for our purpose to define limits based solely on the results of laboratory examinations, since it is often in practice impracticable to obtain first-hand authoritative information regarding the source and handling of the supplies of common carriers as actually distributed to the public. The effect of eliminating a portion of the desirable information is to necessitate somewhat more liberal limits for permissible amounts of impurities which are not actually and definitely injurious and which are removable only at great cost, and narrower limits for permissible impurities of definitely dangerous character.
In regard to the physical and chemical properties which render water disagreeable to sight, taste, or smell without producing any concrete harmful effect a difficulty is encountered in that the degree of offensiveness is not accurately measurable, being largely dependent upon individual taste and habits. Again, regarding many of the constituents found in natural waters, as, for example, various mineral salts, it is impossible in the present state of our knowledge to definitely specify the ill effects, if any, which given amounts of these substances may produce. Limits upon these impurities must, accordingly, be so placed as to allow the public an ample margin of safety; but to do this raises the question as to how far it is justifiable to tax the carriers to eliminate impurities whose deleterious effects are so doubtful. This question is the more difficult since it is generally impracticable to remove the mineral salts present in waters by measures of practicable application, and consequently narrow limits to the permissible amounts of such substances will have the effect of eliminating many sources of supply-sources which will frequently be the only ones readily available and may yield waters conforming with entire satisfaction to the other more important sanitary requirements. Finally, it is in regard to permissible amounts of mineral constituents that the greatest difficulty is encountered in framing such requirements as shall require only simple methods of examination for their enforcement.
It is because of these difficulties and because of the minor sanitary importance of regulating the chemical impurities of water supplies that these questions have been left for further discussion and this report drafted to deal only with the more important question of regulating bacteriological impurities.
The definition of limits for the permissible bacteriological pollution of the water supplies in question is at once more important and simpler than defining limits for permissible chemical impurities. It is more important because the most dangerous of all the impurities which may be present in drinking water are diseaseproducing bacteria and other parasitic disease germs. It is simpler than the other problems mentioned, because as the result of a vast deal of careful study which has been devoted to the bacteriological quality of water supplies and their effects upon the public health it is now possible to define with reasonable precision what constitutes a bacteriologically safe water supply. Finally, strict regulations, requiring common carriers to provide water of undoubtedly safe bacteriological quality, are thoroughly justified, because where water of the requisite degree of purity is not obtainable from a convenient natural source, it is entirely practicable to obtain a pure supply from a moderately polluted source by comparatively simple and inexpensive processes of purification. The art of water purification has, in fact, progressed to the point where there is no longer any excuse whatsoever for using bacteriologically polluted water.
The vast majority of the bacteria found in drinking water supplies are entirely harmless, and regulations designed to insure freedom from disease-producing bacteria must, accordingly, be concerned more with the character than the numbers of the bacteria present. The isolation of the disease-producing bacteria which may be present even in highly polluted waters is so nearly impossible, because of the difficulty of separating them from the much more numerous harmless bacteria with which they are associated, that in actual practice the attempt to directly demonstrate the presence of disease-producing bacteria is seldom made. The bacteriological examination of water supplies has therefore been developed, rather, along the line of determining the number and proportion of certain broad classes of bacteria present in a given volume of water, namely:
1. Bacteria developing distinct colonies within forty-eight hours at 20°C. on standard gelatin culture medium.
2. Bacteria which developed distinct colonies within twentyfour hours at 37°C. on standard agar culture medium.
3. Bacteria belonging to the B. coli group.
It is beyond the scope of this report to enter into a detailed discussion of the interpretation of results of bacteriological examination of water supplies. In general, reliable interpretations can be made only by those having a sufficiently broad knowledge of bacteriology to enable them to apply the established general principles to each case as it is presented.
The bacteria developing on standard gelatin at 20°C. include a relatively large proportion of harmless bacteria which are normally inhabitants of soils and natural waters free from dangerous pollution. The number of bacteria, as estimated by the standard gelatin count, serves in a general way as an index of the cleanliness of the sample; but to properly interpret the results of such a count it is necessary to have knowledge of the source of the sample examined, the nature of the pollution to which it has been exposed, and the opportunities afforded for multiplication of the harmless varieties of bacteria present. On account of the rapid multiplication of harmless varieties of bacteria, which may take place when water is stored in small containers at moderate temperatures, and the impossibility of making approximately correct allowance for such multiplication, it is believed that the attempt to establish a limit for bacteria developing on gelatin is not practicable for the purposes of the control of supplies of common carriers.