8VLS 8LVvO LOZLE | Digitized by the Internet Archive in 2008 with funding from Microsoft Corporation http://www.archive.org/details/elementsofwaterb0Opresuoft a ELEMENTS OF WATER BACTERIOLOGY WITH SPECIAL REFERENCE TO ¥ SANITARY WATER ANALYSIS BY SAMUEL CATE PRESCOTT Professor of Industrial Microbiology in the Massachusetts Institute of Technology AND CHARLES-EDWARD AMORY WINSLOW Director, Division of Publicity and Education, New York State Department of Health, and Curator of Public Health, American Museum of Natural fistory, New York THIRD EDITION. REWRITTEN SECOND THOUSAND, REVISED NEW YORK JOHN WILEY & SONS, Inc. Lonpon: CHAPMAN & HALL, Lmitep TQX5 BY. - eon S. C. PRESCOTT anv C.-E. A. WINSLOW a . 7 . ‘ : “> : a" 4 < ¢ * ‘ . } - - 4 : J Es y 7 fe , 4 , ¥ ¥ * 7 ’ s : ’ # ee os “THE SCIENTIFIC PRESS) a} ROBERT DRUMMOND AND COMPANY ‘ pa aee ; BROOKLYN, N. Y- “2 < fgg arc * ai an vw > x ty * 1 . \ ¢ . * ; - ; ) t 2 axes - € 5 PREFACE TO FIRST EDITION THE general awakening of the community to the importance of the arts of sanitation—accelerated by the rapid growth of cities and the new problems of urban life—demands new and accurate methods for the study of the microbic world. Bacteriology has long since ceased to be a subject of interest and importance to the medical profession merely, but has become intimately connected with the work of the chemist, the biologist, and the engineer. To the sanitary engineer and the public hygienist a knowledge of bacteriology is indis- pensable. In the swift development of this science during the last ten years perhaps no branch of bacteriology has made more notable progress than that which relates to the sanitary examination of water. After a_ brief period of extravagant anticipation, and an equally unreasonable era of neglect and suspicion, the methods of the practical water bacteriologist have gradually made their way, until it is recognized that, on account of their delicacy, their directness, and their certainty, these methods now furnish the final criterion of the sanitary condition of a potable water. vi PREFACE TO FIRST EDITION A knowledge of the new science early became so indispensable for the sanitary expert that a special course in the Bacteriology of Water and Sewage has for some years been given to students of biology and sani- tary engineering in the Biological Department of the Massachusetts Institute of Technology. For workers in this course the present volume has been especially prepared, and it is fitting, we think, that such a manual should proceed from an institution whose faculty, graduates, and students have had a large share in shap- ing the science and art of which it treats. We shall be gratified, however, if its field of usefulness extends to those following similar courses in other institutions, or occupied professionally in sanitary work. The treatment of the subject in the many treatises on General Bacteriology and Medical Bacteriology is neither special enough nor full enough for modern needs. The classic work of Grace and Percy Frank- land is now ten years old; and even Horrocks’ valuable “ Bacteriological Examination of Water” requires to be supplemented by an account of the developments ° in quantitative analysis which have taken place on this side of the Atlantic. It is for us a matter of pride that Water Bacteriology owes much of its value, both in exactness of method and in common-sense interpretation, to American sanitarians. The English have contributed researches of the greatest importance on the significance of certain intestinal bacteria; but with this exception the best work on the bacteriology of water has, in our opinion, been done in this country. Smith, Sedgwick, Fuller, PREFACE TO FIRST EDITION vii Whipple, Jordan, and their pupils and associates (not to mention others) have been pioneers in the develop- ment of this new field in sanitary science. To gather the results of their work together in such form as to give a correct idea of the best American practice is the purpose of this little book; and this we have tried to do with such completeness as shall render the volume of value to the expert and at the same time with such freedom from undue technicality as to make it reada- ble for the layman. It should be distinctly understood that students using it are supposed to have had before- hand a thorough course in general bacteriology, and to be equipped for advanced work in special lines. Boston, March 10, 1904. Mot OY is Pe fe Oe .™» ' ¥ 4 PREFACE TO THIRD EDITION A SECOND edition of this work was called for in 1908 and it was rewritten in that year, with the inclusion of much new material in the chapters dealing with the isolation of the typhoid bacillus and of intestinal bacteria, and with the addition of a new chapter on the bacteriology of sewage and sewage effluents. In the same year there appeared an excellent volume on Water Bacteriology by Dr. W. G. Savage, which showed the English methods of investigation and interpre- tation to be closely in accord with those used in America. In the five years which have elapsed since our second edition was published, there has again been important progress along many lines in sanitary bacteriology; and in particular the publication in 1912 of a new edition of the Report of the Committee on Standard Methods of the Laboratory Section of the American Public Health Association has made necessary a change in many details of current practice. We have, therefore, prepared at this time a some- what far-reaching revision of our book. Newer ideas ix x PREFACE TO THIRD EDITION on the effect of temperature upon the viability of bacteria in water are included in Chapter I. The recent recommendations of the Committee on Standard Methods are discussed in Chapters II and IV; in particular, Chapter IV, dealing with the 37° count, has been expanded. We cannot bring ourselves to agree with the recommendation of the committee that the 37° count should replace the 20° count; but we are entirely in accord with the resolution adopted by the Laboratory Section of the American Public Health Association at its Washington meeting that both determinations should be made in ordinary routine water examinations. Indeed, this is the position we — have maintained in both our earlier editions. Chapter V, dealing with the isolation of specific pathogenes from water, has been extensively rewritten and extended. The use of the Jackson bile medium for the preliminary enrichment of the typhoid bacillus has become general since 1908 and a number of suc- cessful isolations have been reported by its use; so that this procedure promises to be of increasing importance in the future. In regard to the isolation and identification of bacilli of the colon group we feel that the time has come for a change from the usual American practice of the past. The five standard tests for “ typical B. coli” established by the Committee on Standard Methods in its 1905 report have come to seem more and more illogical and unscientific to most practical water bac- teriologists. The conviction has grown that they go either too far or not far enough. For waters, in the . ee PREFACE TO THIRD EDITION xi United States, at least, it seems clear that all of the lactose-fermenting group of bacilli are significant of pollution from human or animal sources when present in considerable numbers. The ig12 report of the Standard Methods Committee apparently takes this view in one place, while retaining the five tests in another section. We have felt it best to place ourselves fairly and fully in line with the view that the whole group of aérobic lactose-fermenting bacilli is significant and this broad definition is the one upon which we have based our general discussion of the colon group in Chap- ters VI and VII. In Chapter VIII we have discussed the subdivisions of the group as worked out by Mac- Conkey and others and their special significance with respect to recent and remote pollution as suggested by the researches of Houston and Clemesha. The use of the lactose-bile test as sufficient evidence of the presence of bacilli of the colon group has become very general and was endorsed by the Laboratory Sec- tion of the American Public Health Association at its 1912 meeting. The U.S. Treasury Department, acting on the advice of a special commission of experts, in for- mulating a standard for water supplied by common car- riers in interstate commerce has, however, recommended a lactose peptone broth enrichment medium, followed by plate isolation and a confirmatory test in lactose peptone broth. Further study is needed to determine the relative value of these two procedures. The growing importance of the application of bac- teriology to the sanitary study of shellfish has led us to include a new chapter dealing with this subject. xii PREFACE TO THIRD EDITION based largely upon the recent report of the Committee of the Laboratory Section of the American Public Health Association. Throughout the book we have resorted freely to the use of tables of actual data for the illustration of the various points discussed, believing that ample familiarity with practical examples furnishes the only sound basis for judgment in sanitary water exam- ination. For the benefit of the student the chapters have been sub-divided into sections with prominent headings indicating the general topics under discussion. Massachusetts Institute of Technology, Boston, Mass. American Museum of Natural History, NEw York,N. Y. Feb. 1, 1915. TABLE OF CONTENTS CHAPTER I PAGE THE BACTERIA IN NATURAL WATERS........-----++--e-+e ee eee I CHAPTER I THE QUANTITATIVE BACTERIOLOGICAL EXAMINATION OF WATER.. 29 CHAPTER III THE INTERPRETATION OF THE QUANTITATIVE BACTERIOLOGICAL CHAPTER IV DETERMINATION OF THE NUMBER OF ORGANISMS DEVELOPING AT THE -Bopy TEMPERATURE... 2.65.25 6-222 .-%- Se role a eens 61 CHAPTER V Tue IsoLaATION OF SPECIFIC PATHOGENES FROM WATER......... 74 CHAPTER VI Tue Coton Group oF BACILLI AND METHODS FOR THEIR ISOLATION 99 xi xiv TABLE OF CONTENTS CHAPTER VII PAGE SIGNIFICANCE OF THE PRESENCE OF THE COLON GROUP IN WATER. 140 CHAPTER VIII VARIETIES OF COLON BACILLI AND THEIR SPECIAL SIGNIFICANCE. 174 CHAPTER IX OTHER INTESTINAL BACTERIA.: .....: 3s. cess os couse eee 201 CHAPTER X THE SIGNIFICANCE AND APPLICABILITY OF THE BACTERIOLOGICAL EEXAMINATION «<3 .'s\s s/s) 5 a:0-s/0\s gaan ld-gler#le Whale ates ieee ann 215 CHAPTER XI BACTERIOLOGY OF SEWAGE AND SEWAGE EFFLUENTS...........- 228 CHAPTER XII BACTERIOLOGICAL EXAMINATION OF SHELLFISH...........--- oes 244 APPENDIX. 605.5! s. 50's 6.5 ooo cd 9 nla pa wielo eg a's ate Wed Sige» eer on 265 ELEMENTS OF WATER BACTERIOLOGY CHAPTER I THE BACTERIA IN NATURAL WATERS Bacteria and Their Nutritive Relations. Bacteria are the most numerous and the most widely distributed of living things. They are present not merely at the surface of the earth or in the bodies of water which partially cover it, as is the case with most other living things, but in the soil itself, and in the air above, and in the waters under the earth. Probably no organisms are more sensitive to external conditions, and none respond more quickly to slight changes in their environment. Temperature, moisture, and oxygen are of importance in controlling their distri- bution; but the most significant factor is the amount of food supply. Bacteria and decomposing organic matter are always associated, and for this reason a brief consideration of the general relation of bacteria to their sources of food supply must precede the study of their distribution in any special medium. The bacteria possess greater constructive ability than any animal organisms. They lack, however, 2 ELEMENTS OF WATER BACTERIOLOGY the power of green plants to build up their own food from compounds like carbon dioxide and nitrates which have no stored potential energy. The food require- ments of various bacterial types differ, however, widely among themselves. Fischer (1900) has divided the whole group into three great subdivisions according to the nature of their metabolism. The Prototrophic forms are characterized by minimal nutrient require- ments, including organisms like the nitrifying bacteria which require no organic compounds at all, but derive their nourishment from carbon dioxide or carbonates, nitrites and phosphates, or from inorganic ammonium salts. A second group of Metatrophic bacteria includes those forms which require organic matter, nitrogenous and carbonaceous, but are not dependent on the fluids of the living plant or anima]. Finally, the Paratrophic bacteria are the true parasites, which exist only within the living tissues of other organisms. These sub- divisions, like all groups among the lower plants, are not sharply defined, and the metatrophic bacteria in particular exhibit every gradation, from types which grow in water with a trace of free ammonia to organisms like the colon bacillus which normally occur on the surface of the plant or animal body, feeding upon the fluids or on the extraneous material collected upon its surface. The vast majority of bacteria belong to the second, — or metatrophic group, living as saprophytes on dead organic matter wherever it may occur in nature, and particularly in that diffuse layer of decomposing plant and animal material which we call the humus, or surface ip, ee eS oe oo by Ce ren ae a TY Pa a ee THE BACTERIA IN NATURAL WATERS 3 layer of the soil. Wherever there is life, waste matter is constantly being produced, and this finds its way to the earth or to some body of water. The excretions of animals, the dead tissues and broken-down cells of both animals and plants, as well as the wastes of domestic and industrial life, all eventually find their way to the soil. In a majority of cases these substances are not of such chemical composition that they can be utilized at once by green plants as food, but it is first necessary that they go through a decomposition or transforma- tion in which their chemical nature becomes changed; and it is as the agents of this transformation that bacteria assume their greatest importance in the world of life. We may take the decomposition of a comparatively simple excretory product, urea, as an example of the part which the bacteria play in the preparation of plant food. Through the activity of an enzyme pro- duced by certain bacteria this compound unites with two molecules of water and is converted into ammonium carbonate, COC = +220 = (NH.)2COs. NH This, however, is only part of the process. While green plants can derive their necessary nitrogen in part, at least, from ammonium compounds it is a well- established fact that this element is often obtained more readily from nitrates, and there are other bacteria which as a further step oxidize the ammoniacal nitro- + ELEMENTS OF WATER BACTERIOLOGY ~ gen to a more available form. This process of oxida- tion is known as nitrification, and takes place in a suc- cession of steps, the organic nitrogen being first con- verted to the form of ammonium salts, and these in turn to nitrites and nitrates, the oxygen used coming from the air. Several groups of organisms are instru- mental in bringing about this conversion. It is gen- erally assumed that one group attacks the ammonium compounds and changes them to nitrites; while another group completes the oxidation to nitrates. In the latter form nitrogen is readily taken up by green plants to be built up into more complex albuminoid sub- stances (organic nitrogen) through the constructive power of chlorophyll. This never-ending cycle is illustrated in the accom- panying figure, devised by Sedgwick (Sedgwick, 1889) to illustrate the transformations of organic nitrogen in nature, the increasing size and closeness of the spiral on the left-hand side indicating the progressive com- plexity of organic matter as built up by the chlorophyll bodies of green plants in the sunlight, and the other half of the figure the reverse process, carried out largely by the bacteria. In nature there are many short circuits, as, for instance, when dead organic matter is used as food for animals and built up into the living state again without being nitrified and acted upon by green plants; but the complete cycle of organic nitrogen is as indicated on the diagram. We have dwelt thus at length upon the general relation between. bacteria and organic decomposition because in this relation will be found the master key THE BACTERIA IN NATURAL WATERS 5 to the distribution of bacteria in water as well as in other natural habitats. It is true that certain peculiar forms may at times multiply in fairly pure waters; but, in general, large numbers of bacteria are found only in connection with the organic matter upon which they feed. Such organic matter is particularly abundant in the surface layer of the soil. Here, therefore, the bacteria are most numerous; and in other media their s\RAN i * WN \ \S : NITROGEN AS mh NY me . NY) + q Ui r ORGANIC NITROGEN rN ~s Sy potetatomnked Wp (ALSUMINOID AMMONIA) IN VY a YEN SNS ESS y

Y, WLLL, GALLE, 0 ) WYLTTMTTT TELL ILLETL ts Yih, bs. 77 i Ves v TENTIAL err SYA numbers vary according to the extent of contact with the living earth. Classification of Waters. Natural waters, then, group themselves from a_ bacteriological standpoint in four well-marked classes, according to their relation to the rich layers of bacterial growth upon the surface of the globe. There are first the atmospheric waters which have never been subject to contact with the earth; second, the surface-waters immediately exposed to such 6 ELEMENTS OF WATER BACTERIOLOGY: contamination in streams and pools; third, stored waters in lakes and large ponds where storage has reduced bacterial numbers and produced a state of comparative purity; and fourth, the ground-waters from which pre- vious contamination has been even more completely re- moved by filtration through the deeper layers of the soil. Bacterial Content of Various Waters. Even rain and snow, the sources of our potable waters, are by no means free from germs, but contain them in numbers varying according to the amount of dust present in the air at the time of the precipitation. After a long- continued storm the atmosphere is washed nearly free of bacteria, so that a considerable series of sterile plates may often be obtained by plating 1-c.c. samples. These results are in harmony with the observations of Tissandier (reported by Duclaux, 1897), who found that the dust in the air amounted to 23 mg. per cubic meter in Paris and 4 mg. in the open country. After a rainfall these figures were reduced to 6 mg. and 0.25 mg., respectively, With regard to what may be considered normal values for rain it is difficult to give satisfactory figures. Those obtained by Miquel (Miquel, 1886) during the period 1883-1886 showed on the average 4.3 bacteria per c.c. in the country (Montsouris) and 19 per c.c. in Paris. Snow shows rather higher numbers than rain. Janowski (Janowski, 1888) found in freshly fallen snow from 34 to 463 bacteria per c.c. of snow-water. As soon as the rain-drop touches the surface of the earth its real bacterial contamination begins. Rivulets from ploughed land or roadways may often contain several hundred thousand bacteria to the cubic centi- i ee ee oe Fe ee ey ee Paes, ee, a _ “oo ! ee - - _ ee ee) Se el ee THE BACTERIA IN NATURAL WATERS 7 meter; and furthermore the amounts of organic and mineral matters which serve as food materials, and thus become a factor in later multiplication of organisms, are greatly increased. In the larger streams several conditions combine to make these enormous bacterial numbers somewhat lower. Ground-water containing little microbic life enters as a diluting factor from below. The larger particles of organic matter are removed from the flow- ing water by sedimentation; many earth bacteria, for which water is an unfavorable medium, gradually perish; and in general a new condition of equilibrium tends to be established. It is difficult, however, to find a river in inhabited regions which does not con- tain several hundreds or thousands of bacteria to the cubic centimeter. Furthermore, heavy rains which introduce wash from the surrounding watershed may at any time upset whatever equilibrium exists, and surface-waters are apt to show sudden fluctuations in their bacterial content. Seasonal Variation of Bacteria in Surface Waters. Sharp variations in bacterial content are particularly apt to occur in the spring and fall as a result of the rain and melting snow at those seasons. The high numbers shown for various rivers in the table on page 8 illustrate this point. The rainfall is the main factor which causes these sea- sonal variations; but its specific effect differs with dif- ferent streams. The immediate result of a smart shower is always to increase contamination by introducing fresh wash from the surface of the ground. More prolonged 8 ELEMENTS OF WATER BACTERIOLOGY SEASONAL VARIATIONS IN BACTERIAL CONTENT OF RIVER WATERS. BACTERIA PER C.C. MONTHLY AVERAGE River. Year. Jan. Feb. Mar. | April. | May. | June. Thames 2.3 5..0 1905-6 | 2,075| 1,679] 1,161 277| 1,064] 382 PET SD aca ke gather dss 1905-6 | 5,192] 3,083] 1,308] 471| 1,350 598 ING@W ESR ike so 1905-6 | 1,455] 1,304 291 149 352) 108 Mississippi ?....| 1900-01] 972] 2,871] 1,795] 3,507] 2,152| 2,007 Potomac S22. 05.3 1906-7 | 4,400] 1,000] 11,500] 3,700] 750] 2,300 Merrimac ‘..... 1905 14,200] 14,800] 10,300] 3,600] 1,900] 9,600 Susquehanna ®. .| 1906 9,510| 21,228] 31,326] 39,905| 6,187| 2,903 River. Year. July. ug. | Sept. Oct. Nov. Dec. Ahames bes 5 ces. 1905-6 O52). Sos spe sete a lieshas nla tee 740 POE Ae. Seis eens 190576" | the experiments of Jordan, Russell and Zeit (1904), and Russell and Fuller (1906), which will shortly be referred to more fully, that the life of typhoid germs is shorter in water containing large numbers of other bacteria than in that of greater purity. Horrocks (1899), too, found freshly isolated typhoid bacilli alive in sterile sewage after 60 days; while they disappeared in 5 days when B. coli was also present. These phenomena may be due, however, to a struggle for oxygen, or for food, rather than to the assumed presence of highly toxic bacterial products, of which there is no independent evidence. Many investigations conducted since the pioneer researches of Downes and Blunt (Downes and Blunt, 1877) have confirmed the results reported by them, which showed that direct sunlight is fatal to most bacteria in the vegetative state and even to spores if THE BACTERIA IN NATURAL WATERS 17 the exposure be sufficiently long, while diffused light is harmful in a less degree. Opinions vary as to the degree to which light is active in destroying the bacteria in natural waters. Buchner (Buchner, 1893) found by experiment that the bactericidal power of light extends to a depth of about three meters before it becomes imperceptible. On the other hand, Procaccini (Procaccini, 1893) found that when sunlight was passed vertically through 60 cm. of drain-water the lower layers contained nearly as many bacteria after 3 hours’ treatment as before the exposure. The middle and upper portions showed a great falling off in numbers, however. But few studies have been made of the effect of light on bacteria in flowing water. Jordan (Jordan, 1900) has investigated several Illinois streams and arrived at the conclusion that in moderately turbid water, at least, the sun’s rays are virtually without action. On the other hand, Rapp has observed a considerable reduction of the bacteria in the Isar at Pullach after the period of diurnal insolation, as shown by the table on the following page. Clemesha (1912*) attributes very great importance to the action of light in the self- purification which takes place in Indian lakes and rivers; his opinion is apparently not based on com- parative experiments including and excluding this factor, but chiefly on the greater numbers of intestinal bacteria at the bottom as compared with the superficial layers of water. It is unnecessary to dwell in detail upon the effect which the lack of nutritive elements must exert upon 18 ELEMENTS OF WATER BACTERIOLOGY intestinal bacteria and soil bacteria in waters of ordinary purity. Comparative studies of culture media, to be quoted in the succeeding chapter, will show how del- icately the bacteria respond to comparatively slight changes in their food-supply. Wheeler (1906) found that typhoid bacilli would persist in almost undimin- ished numbers in sterilized water from a polluted well containing considerable organic matter and kept in the dark at 20 degrees, while in purer water or in the light they died out in from 2 to 6 weeks. EXAMINATIONS OF THE ISAR AT PULLACH (Rapp, 1903) (A) Carried out September 26, 1898, no rain having fallen for three weeks Temperature. , pee Bacteria per c.c. of the Water. of the Air. T3007; $..8> °C. 7.30 P.M. 146 sere: C. 9.0° ©, 9.30 P.M. 270 7o.5° C. 6.2°-C, 5.00 A.M. 370 16.2°'C. 82°C. 8.00 A.M. 320 (B) Carried out November 28, 1898, no rain having fallen for some time ite te OF 3.07 GC; 6.00 P.M. 266 Bie uey A ah OF 8.00 P.M. 402 iy ad OF 2.0 7G, 10.00 P.M. 482 Bete 2.0°'C, 3.00 A.M. 532 7 taht er, 2,5. Cz 7.30 A.M. 400 Whipple and Mayer (1906) have called attention to another important factor in the general problem. They find that the presence of oxygen is essential to the per- THE BACTERIA IN NATURAL WATERS 19 sistence of typhoid and colon bacilli in water, although in nutrient media both forms may thrive under anzrobic conditions. EFFECT OF OXYGEN ON VIABILITY OF TYPHOID BACILLI IN STERILE TAP WATER WHIPPLE AND MAYER, 1906 Tubes Kept in Air. Tubes Kept in Hydrogen. Period in Days aa Per Cent. peer ae Per Cent. ° 600,000 100.0 600,000 100.90 2 455,000 76.0 2,400 0.4 4 190,000 32.0 25 ©.004 8 120,000 20.0 ° 0.0 12 67,000 II.o ° 0.0 18 25,000 4.2 Bias >. 0.0 26 9,250 1.5 ° 0.0 33 2,150 0.6 ° 0.0 4° 132 0.02 ° 0.0 47 6 0.001 ° 0.0 54 ° ©.000 ° 0.0 Various inorganic constituents of the medium undoubt- edly exercise an important influence upon the life of bacteria in water; and the mutual interaction of the different substances present is a highly complex one. Thus Winslow and Lochridge (1906) report that five parts of dissociated hydrogen per million parts of tap water (0.005 normal HCl) is fatal to typhoid bacilli, while ten times as much acid is required for sterilization when 1 per cent of peptone is present to check the dissociation of the hydrogen. In Hazen and Whipple’s study of the Allegheny, Monongahela and Ohio rivers 20 ELEMENTS OF WATER BACTERIOLOGY at Pittsburgh the antiseptic effect of acid wastes was strikingly shown. (Engineering News, 1912.) Although it is hard to estimate the exact importance of each factor, the general phenomena of the self- purification of streams are easy to comprehend. A small brook, immediately after the entrance of polluting material from the surface of the ground, contains many bacteria from a diversity of sources. Gradually those organisms adapted to life in the earth or in the bodies of plants and animals die out, and the forms for which water furnishes ideal conditions survive and multiply. It is no single agent which brings this about, but that — complex of little-understood conditions which we call | the environment. If any one thing is of prime impor- tance it is probably the food-supply, for only certain — bacteria are able to multiply in the presence of the small amount of organic matter present in ordinary potable waters. As Jordan (Jordan, 1900) has said: “In the causes connected with the insufficiency or unsuitability of the food-supply is to be found, I believe, the main reason for the bacterial self-purification of streams.” Effect of Temperature upon Bacteria in Water. The effect of temperature upon the survival of bac- teria in water varies according to this primary con- dition of food-supply which has just been discussed. When bacteria are in a medium in which they are able to grow and multiply, warmth, within reasonable limits of course, favors their development, At times this may be true even of certain intestinal bacteria in water. Thus at Harrisburg, Pa., a series of B. coli examinations THE BACTERIA IN NATURAL WATERS 21 made in the midsummer of 1906 showed positive results in 7 per cent of the samples of water entering the storage reservoir and in 27 per cent of the samples leaving it. The storage period in this case was about two days and the temperature of the water in the reservoir was nearly at blood heat (Harrisburg, 1907). Clemesha (1912*) has recently made an exhaustive study of this multiplication of coli-like microbes in warm waters and has shown that it is confined to certain particular types within the colon group. For most intestinal bacteria the conditions necessary for growth and multiplication are not realized in water and an entirely different temperature effect is manifest. When a bacterium cannot multiply, the only vital activity which can take place is a katabolic wasting away, which soon proves destructive, and the higher the temperature the more rapidly the fatal result is reached. A frog in winter lives at the bottom of a pond breath- ing only through its skin and eating not at all, but as soon as the temperature rises it must eat and breathe through its lungs or perish. It is quite true that even in ice 40 per cent of typhoid bacilli perish in 3 hours and 98 per cent in 2 weeks (Sedgwick and Winslow, 1902). Recent work has shown, however, that they die in spite of the cold, not on account of it, and that the decrease is more rapid at higher temperatures, unless of course food-supply and other conditions admit of multiplication. | Houston (1911) has furnished a very clear demonstration of this temperature rela- tion by storing typhoid bacilli in water with the results tabulated on page 22. 22 ELEMENTS OF WATER BACTERIOLOGY | EFFECT OF TEMPERATURE ON SURVIVAL OF TYPHOID BACTERIA IN WATER (Houston, 1911) Percentage of Typhoid Period of Final Temperature C, serie i rg after Disappearance of Bacilll. Desi eraieccn ey taihe 46 9 weeks Beistte eS ees mre peace s hase 14 7 weeks DOM Scop ioe bien eh bps eve ee 0.07 5 weeks Tle Getic nersscene aks 0.04 4 weeks Ruediger (1911) has shown that colon bacilli are far more abundant in the Red Lake River during the winter when the river is covered with ice than in sum- mer, although the volume of the river and the amount of sewage pollution are about the same. Typhoid bacilli in celloidin dialyzers floated down the river showed only 2.5 and 3.5 per cent surviving in 2 days and 0.51, 0.89, 2.2 and 3.2 per cent surviving in 3 days when the river was not frozen, while dialyzers suspended through the ice in colder weather showed 6.1, 10.5, 17-7, 46.8 and 62.9 per cent surviving in five different experi- ments after 2 days, 31 per cent in 3 days, 19 per cent in 7 days, and 2.5 per cent in 14 days. Ruediger — attributes this greater persistence at low temperatures to the absence of poisonous waste products of other organisms and to protection from the light; but there can be little doubt that it is mainly a result of the general preservative effect of cold. From an epidemiological standpoint the conclusion that disease germs perish quickly in warm waters is amply confirmed. Almost without exception outbreaks of typhoid fever due to polluted water occur in cold weather and this is, in THE BACTERIA IN NATURAL WATERS 23 part at least, due to the greater persistence of typhoid bacilli at low temperatures. Relation between Time of Storage and Self-purifica- tion. It is obvious that the efficiency of all the agencies which tend to decrease the number of bacteria in sur- face waters will increase with the prolongation of the ‘period for which they act. Time is the great measure of self-purification; and in a comprehensive study of the self-purification of the Potomac River, the investigators of the U. S. Public Health Service have found that the time relation is close enough to be stated mathematically. Prof. Phelps expresses this in the formula log nts where ¢ is the time and m; and m2 the respective num- bers of bacteria; k, of course, will vary with the tem- perature and the other conditions discussed above. The absolute time required to make water safe will vary widely with this value &. Jordan, Russell and Zeit (1904), in an important series of experiments, added typhoid bacilli to the unsterilized waters of Lake Michigan, the Chicago River and Drainage Canal and the Illinois River, in collodion sacs suspended in the _ respective bodies of water. From the relatively pure Lake Michigan water the specific organisms could be isolated for at least a week, but in the polluted waters of the rivers and the Drainage Canal they were not found after 3 days except in a single instance. Russell and Fuller, (1906) confirmed these general results, finding that typhoid bacilli would live for 10 days in the unsterilized water of Lake Mendota, while they could be isolated only after 5 days when immersed in sewage. Other observers record much greater 24 ELEMENTS OF WATER BACTERIOLOGY viability for the typhoid bacillus. Savage (1905) added a heavy dose of the organism to unsterilized tidal mud and found it living after 5 weeks. Hoffmann (1905), after inoculating a large aquarium with a rich typhoid culture, was able to isolate the germ from the water after four weeks and from the mud at the bottom after two months. Konradi (1904) reports the per- sistence of typhoid bacilli in unsterilized tap water for over a year. These last experiments deal only with the maximum survival period for a few out of great numbers of germs introduced into the water or mud, and entirely ignore the quantitative aspects of the case. When one con- siders the proportion of the original bacteria surviving, the period necessary to bring about a reasonably safe condition is found to be much shorter. Houston (1908) has shown that when water is artificially infected with typhoid bacilli and stored, 99.9 per cent of the disease germs perish in one week, although some may persist for from 1 to g weeks. In later experiments (Houston, 1911) he finds that “ uncultivated’ typhoid bacilli added to the water directly from the urinary sediment of a disease carrier perish much more rapidly than the laboratory strains, usually disappearing entirely after one week and always after three. On a number of occasions Dr. Houston gave dramatic expression to his confidence in these negative laboratory findings by drinking half pint portions of water which a few weeks previously had contained millions of typhoid bacilli. We have plenty of practical epidemiological evidence, such as that THE BACTERIA IN NATURAL WATERS 25 offered in the Chicago Drainage Canal case and in the lawsuit over the condition of the water supply of Jersey City, to confirm the general conclusion that any water which has been stored for 4 weeks is practically safe.’ Bacteria in Ground-waters. In general we have seen that surface-waters tend continually to decrease in bacterial content after their first period of contact with the humus layer of the soil. In that other portion of the meteoric water which penetrates below the surface of the earth to join the reservoir of ground- water, later to reappear as the flow of springs and wells, this diminution is still more marked, since the filtering action of the earth removes not only most of the bac- teria, but much of their food material as well. The numbers of bacteria in the soil itself decrease rapidly as one passes downward. Kabrhel (1906) found several million per c.c. in surface samples of woodland soil, a few thousands or tens of thousands half a meter below, and usually only hundreds in centimeter samples collected at depths greater than a meter. Many observers formerly believed that all ground- waters were nearly free from bacteria, because often no colonies appeared on plates counted after the ordinary short periods of time. If, however, a longer period of incubation be adopted considerable numbers may be obtained. For convenience we may divide ground-waters into three groups, namely: shallow open wells, springs and “tubular”? (driven) or deep wells. This division is important because ordinary shallow wells form a group by themselves in respect to the possibility of aerial and 26 ELEMENTS OF WATER BACTERIOLOGY surface contamination, their water often being fairly rich in bacterial life. Egger (Wolffhiigel, 1886) examined 60 wells in Mainz and found that 17 of them contained over 200 bacteria to the cubic centimeter. Maschek (Maschek, 1887) found 36 wells out of 48 examined in Leitmeritz which had a bacterial content of over 500 per c,c. Fischer (Horrocks, 1901) reported 120 wells in Kiel which gave over 500 bacteria per c.c. and only 51 with less than that number. In the examination of 147 shallow farmyard wells by one of us (S. C. P.) it was found that 124 of the wells which contained no B. coli, and were therefore probably free from fecal pollution, averaged 190 bacilli per c.c. while 23 which gave positive tests for B. coli averaged 570 per c.c. The distribution of the two series of samples according to the number of bacteria present is indicated in the table below. BACTERIA IN SHALLOW FARMYARD WELLS PERCENTAGE OF SAMPLES IN EAcH Group Bactefia percc....:.. © |1-|11-|21—|51-—|101— | 501— | 100I—| 2001- 10] 20] 50/100} 500 | 1000 | 2000] 3000 Series I. B. coli absent.| 3 [16] 14] 16] 11] 31 5 Aol aout Series II. B. coli present...| ...).:| -8)..«.])20] -25.7' | eo 14 5 . Very similar results are reported for shallow wells used as farm water-supplies in Minnesota by Kellerman and Whittaker (1909), although the general quality of the wells examined was considerably below that of the series tabulated above. ae THE BACTERIA IN NATURAL WATERS 27 In the ordinary standard 48-hour period very few bacteria develop from normal spring-waters. Thus in an examination of spring-waters made by the Mas- sachusetts State Board of Health in 1900 (Massachusetts State Board of Health, 1901), of 37 springs which were practically unpolluted and had less than o.10 part per 100,000 excess of chlorine over the normal, 54 sam- ples were examined and gave an average of 41 bacteria per c.c. Only 6 samples showed figures over 50. It now remains to consider the other great division of ground-waters, namely, deep, “ driven,” or “‘ tubular ” wells, which, if carefully constructed, should ordinarily be free from all surface-water contamination, and should show low bacterial counts. The results tabulated below obtained by Houston in the examination of a series of deep wells of high quality at Tunbridge Wells are fairly typical. BACTERIAL CONTENT OF DEEP WELL WATERS (Houston, 1903) Bacteria per c.c. 36 6 9 4 I 16 17 4 3 12 2 4 Io 5 2 Fifteen driven wells in the neighborhood of Boston, examined in 1903, showed at the end of 48 hours an average of only 18 colonies per c.c.; and the results of certain examinations of other wells and springs, recently made by the authors, are given in the table on page 28. 28 ELEMENTS OF WATER BACTERIOLOGY BACTERIA IN DEEP WELL AND SPRING WATERS Bacteria Bacteria Town per c.c. Town. per c.c. Worcester, Mass.:..... Io Saranac Lake, N.Y. . II Waltham, Mass...... eS Ellenville, N. Y...... ° Newport, R.T....... 7 Hyde Park, Mass..... 12 It is plain that water absolutely free from bacteria is not ordinarily obtained from any source. In deep wells, however, their number is small; and the peculiar character of the organisms present is manifested in many cases by the slow development at room tem- perature (frequently no growth until the third day), the entire absence of liquefying colonies, and the abundance of chromogenic species. CHAPTER II THE QUANTITATIVE BACTERIOLOGICAL EXAMINATION OF WATER Relation of the Medium to the Number of Bacteria Obtained. The customary methods for determining the number of bacteria in water do not reveal the total bacterial content, but only a very small fraction of -it, as becomes apparent when we consider the large num- ber of organisms, nitrifying bacteria, strict anzrobes, etc., which refuse to grow, or grow only very slowly in ordinary culture media, and which, therefore, escape detection. On the one hand, certain obligate parasites cannot thrive in the absence of the rich fluids of the animal body; on the other hand, the prototrophic bacteria, adapted to the task of wrenching energy from nitrites and ammonium compounds are unable to develop in the presence of so much organic matter. Winslow (1905) in the examination of sewage and sewage effluents, found 20-70 times as many bacteria by microscopic enumeration as by the gelatin plate count. Certain special media enable us to obtain much larger counts than those yielded by the ordinary gelatin method. The Niahrstoff Heyden agar, for example, has been strongly advocated by Hesse (Hesse and Niedner, 1898) and other German bacteriologists upon this 29 30 ELEMENTS OF WATER BACTERIOLOGY ground. In this country Gage and Phelps (Gage and Phelps, 1902) showed that the numbers obtained by the ordinary procedure were only from 5 to 50 per cent - of those obtained by the use of Heyden’s Nihrstoff agar. For practical sanitary purposes, however, our methods are fairly satisfactory. Within limits, it is of no great importance that one method allows the growth of more bacteria than another. When we are using the quantitative analysis as a measure of sewage pollution the essential thing is that the section of the total bacterial flora which we obtain should be thor- oughly representative of that portion of it in which we are most interested—the group of the quickly growing, rich-food-loving sewage forms. In this respect meat-gelatin-peptone appears to be unrivalled; and it is in this respect that such media as Nahrstoff agar fail. Miiller (1900) showed that the larger counts obtained by plating on the Néahrstoff medium are due to the fact that it specially favors the more prototrophic forms, among the water bacteria themselves. Intestinal organisms and even the ordinary putrefactive germs, when plated in pure culture, show no higher counts on Nihrstoff agar than on gelatin. Gage and Adams (1904) found by plating pure cultures of the common laboratory bacteria, saprophytes and parasites, that Nihrstoff counts were actually lower than those obtained by the use of gelatin. When sewage and highly polluted waters are examined counts are slightly higher on Nihrstoff media, while with purer waters the Nahrstoff numbers are far in excess of those obtained with gelatin. Winslow (1905) found the ratio of N&ahrstoff agar QUANTITATIVE EXAMINATION OF WATER 31 to gelatin count to be 1.7 to 1.0 for sewage, and 4.8 to 1.0 for sand filter effluent. With waters of still _ better quality the ratio goes up higher, reaching a maximum when the bacteria which increase and multiply in water are most abundant. Miiiller (1900) found, for example, that water which normally showed six times as many bacteria on Niahrstoff agar as on gelatin might give a Niahrstofi-gelatin ratio of 20-30 after it had been standing for some time in the supply pipes. The table below, taken from the valuable paper by Gage and Phelps (1902), shows strikingly the different Ni&hrstoff-agar ratios for waters of TABLE SHOWING PERCENTAGES OF BACTERIA DEVELOP- ING ON REGULAR AGAR AND NAHRSTOFF AGAR FOR DIFFERENT CLASSES OF WATERS (GAGE AND PHELPS, 1902) Regular Agar Days’ Count. Class of Water. 2 3 4 5 6 7 8 Ground water.....| o 5 6 6 6 6 6 Filtered water... .. 6 7 7 7 7 7 7 Merrimac River... 6 7 7 8 8 9 9 Filtered sewage....| 14 17 18 19 19 19 19 NEWOBE. 2 2's 5 oss as 34 44 46 46 46 46 46 NGrhstoff Agar Ground water... .. 6 43 78 88 03 | 100 | 100 Filtered water.....| 37 69 80 92 98 100 100 Merrimac River...| 29 78 903 07 07 99 | I00 Filtered sewage....| 26 65 93 95 07 99 | I00 EAR eee 39 75 95 | 100 | I00 | I00 | I00 32 ELEMENTS OF WATER BACTERIOLOGY various grades of purity. It is obvious from all these facts that the effect of using the Niahrstoff medium is to increase disproportionately the bacterial counts obtained from purer waters and thus to diminish the difference in bacterial content between normal and contaminated sources. The ordinary agar and gelatin media, on the other hand, are adapted to the growth of intestinal and putrefactive forms and, therefore, serve best the prime object of bacteriological water examination. The first requisite in a procedure for water analysis is, then, that it should be adapted to the end in view, the differentiation of pure and contaminated waters. The second and equally important requirement is that the procedure should be a standard one, so that results obtained at different times and by different observers may be comparable. In this respect the work of G. W. Fuller, G. C. Whipple, and other members of the Committee on Standard Methods of the American Public Health Association has placed the art of quantitative water analysis in this country in a very satisfactory state by contrast with the varying practices which prevail in England and Germany. The first report on this question was made in 1897 (Committee of Bac- teriologists, 1898). A permanent Committee on Stand- ard Methods was then formed which reported in 1901 (Fuller, 1902), in 1904 (Committee on Standard Methods of Water Analysis, 1905), and again in 1911 (Committee on Standard Methods for the Examination of Water and Sewage, 1912), recommending in considerable detail a standard routine procedure for the quantitative 2 QUANTITATIVE EXAMINATION OF WATER 33 and qualitative bacteriological examination of water for sanitary purposes. These reports have had a far- reaching effect in simplifying and unifying the methods of water analysis. Similar results have followed from the work of the English Committee appointed to con- sider the Standardization of Methods for the Bac- terioscopic Examination of Water which reported in 1904, although this committee unfortunately did not consider the process of media making in great detail. The last report of the American Committee on Standard Methods (1912) will be adhered to in this and succeed- ing chapters unless otherwise specifically stated; and that portion of its report which deals with methods of making media will be found in full in the appendix. Standard Procedure for Quantitative Determination of Bacteria in Water. The procedure for the quantitative determination of bacteria in water consists, in brief, in mixing a definite amount of a suitably collected specimen of the water with a sterile, solidifiable culture medium and incubating it for a sufficiently long time to permit reproduction of the bacteria and the forma- tion of visible colonies which may be counted. The process is divided naturally into four stages—sampling, plating, incubating, and counting. Sampling. All samples of water for bacteriological examination should be collected in clean, sterile bottles with wide mouths and glass stoppers, preferably of the flat mushroom type. It is desirable that these bottles should have a capacity of at least 100 c.c. They should be cleaned thoroughly before using, by treatment with sulphuric acid and potassium bichromate 34 ELEMENTS OF WATER BACTERIOLOGY or with alkaline permanganate of potash followed by sulphuric acid, dried by draining, and sterilized by dry heat at 160° C. for at least 1 hour, or by steam at 115-120° for 15 minutes. If not to be used immediately the neck and stopper should be protected against dust or other contamination by wrapping with lead-foil. For transportation the bottle should be enclosed in a suitable case or box. The greatest care must be taken that the fingers do not touch the inside of the neck of the bottle or the cone -of the stopper, as the water thereby would become seriously contaminated and rendered unfit for examina- tion. It is well known that bacteria are found: abun- dantly upon the skin, and Winslow (Winslow, 1903) has shown that even B. coli is present upon the hands in a considerable number of cases. In order to obtain a fair sample, great precautions must be taken, and these will vary with the different classes of waters to be examined and with local condi- tions. Ifa sample is to be taken from a tap, the water should be allowed to flow at least five minutes (if from a tap in regular use) or for a longer period in case the water has been standing in the house-service system. In the small pipes, changes in bacterial content are liable to occur, certain species dying and others mul- tiplying. If a sample is to be taken from a pump similar pre- cautions are necessary. The pump should be in con- tinuous operation for 5 minutes at least, and preferably for half an hour before the sample is taken, in order to avoid excessively high numbers due to the growth of QUANTITATIVE EXAMINATION OF WATER 35 bacteria within the well and pump, the bacterial con- dition of the water as it passes through the ground being what we wish to determine. Thus Hereus (Hereus, 1886) in a well-water which had been but little used during the preceding 36 hours found 5000 organisms per c.c.; when the well was emptied by continuous pumping, a second sample, after an interval of half an hour, gave only 35. Maschek (Tiemann and Gartner, 1889) obtained similar results, shown in the following table: EFFECT OF PUMPING ON THE BACTERIAL CONTENT OF WELL-WATER Well-water after continuous pumping for fifteen minutes .. 458 sé S *¢ *< many hours...... 140 = ISGOr 350 Bee ees ac Games Sees eeu ee ts 5 68 oe after continuous pumping for fifteen minutes 578 i s “s many hours...... 179 Bh RSE 2s oe et Srnec’ a eer 73 After a proper interval of pumping the sample of a well-water may be collected from the pet-cock of the pump or from a near-by tap. With a hand-pump, such as is found in domestic shallow wells, the water is, of course, pumped directly into the sample bottle. The difficulties in securing an average sample from this latter source are often great, since if the flooring about the pump is not tight, as is usually the case, con- tinued pumping may wash in an unusual amount of surface pollution. In sampling surface-waters, the greatest precautions must be observed to prevent contamination from the fingers. In still waters the fairest sample is one taken 36 ELEMENTS OF WATER BACTERIOLOGY from several inches down, as the surface itself is likely to have dust particles floating upon it. The method most frequently recommended is to plunge the bottle mouth downward to a depth of a foot or so, then invert and allow the bottle to fill. Whenever any current exists, the mouth of the bottle should be directed against it in order to carry away any bacteria from the fingers. If there is no current, a similar effect can be produced by turning the bottle ‘under water and giving it a quick forward motion. In rapidly flowing streams it is only necessary to hold the bottle at the surface with the mouth pointed up-stream. For taking samples of water at greater depths, a number of devices’ have been employed, all of which are fairly satisfactory. ‘The essentials are, first, a weight to carry the bottle down to the desired depth, and, second, some method of removing the stopper when that depth is reached. The student will find one good form of apparatus described in Abbott’s “ Principles of Bacteriology ” (Abbott, 1899); an admirable one was devised by Hill and Ellms (Hill and Ellms, 1898); and Thresh (1904) figures an ingenious device for the same purpose. Miquel and Cambier (Miquel and Cambier, 1902) and other authors recommend the use of a sealed glass bulb with a capillary tube which can be broken off at the desired moment. Drew (1912) has devised an interesting sampling apparatus for use at great depths in the sea. Changes in Bacterial Numbers after Sampling. As soon as a sample of water is collected its: conditions QUANTITATIVE EXAMINATION OF WATER 37 of equilibrium are upset and a change in the bacterial content begins. Even in the purest spring-waters, which contain but few bacteria when collected, and in which the amount of organic matter is infinitesimal, enormous numbers may be found after storage under laboratory conditions for a few days or even a few hours. In some cases the rise in numbers is gradual, in others very rapid. The Franklands (Frankland, 1894) record the case of a deep-well water in which the bacteria increased from 7 to 495,000 in 3 days. Miquel (Miquel, 1891) from his researches, arrived at the conclusion that in surface-waters the rise is less rapid than in waters from deep wells or springs, and that in the latter case the decrease, after reaching a maximum, is likewise rapid and steady. Just how far protection from light, increase in temperature, and a destruction of higher micro-organisms is responsible for the increase, and to what extent an exhaustion of food-supply or the formation of toxic waste products causes the succeeding decrease, we are not aware; but the facts are well established. Whipple has exhaustively studied the details of this multiplication of bacteria in stored waters and has shown in the table given below that there is first a slight reduction in the number present, lasting perhaps for 6 hours; followed by the great increase noted by earlier observers. It is probable that there is a constant increase of the typical water bacilli, overbalanced at first by a reduction in other forms, for which the environment is unsuitable. 33 ELEMENTS OF WATER BACTERIOLOGY BACTERIAL CHANGES IN WATER DURING STORAGE (WHIPPLE, 1901) as Temp. Number of Bacteria per c.c. fait, | of face. : Sample] “ature. reds bg Initial. | , eae 6 ree, By ets sk tn c. C A #:6° 17.0° 260 215 230 900 | 27,000 B 7.6° £7:0° 260 245 255 720 | 10,850 C 7.6° E25" 260 270 231 600 2,790 D 76° L225 260 270 245 710 1,800 E 720" 2.4° 260 243 210 675 1,980 F 76% 2.4° 260 235 270 560 1,980 G 17.00. VF o5b: 77 55 58 IOI | 10,250 H tE.0° | 2258" 77 53 74 87 2,175 I 7.0" | .23.6° 77 5I 52 II,000 | 41,400 J 6.7° ||- 20:0? 430 375 246° S| Sees 385,000! oa. “bee? ae 430 345 AOB. Nic dene 750,000! L 23.2° 23.0° 510 340 230 8,000 | 20,000 M 23.2° OR ts 525 300 220 380 2,200 19,0005 per cent peptone added to the water. Wolffhiigel and Riedel (Wolffhiigel and Riedel, 1886) noted the dependence of this multiplication on the air-supply, vessels closed with rubber stoppers showing lower numbers than those plugged with cotton. Similarly, Whipple found that the multiplication of bacteria was much greater when bottles were only half full than when they were filled completely; and also, as shown in the very striking table on page 39, that the size of the bottle markedly influenced the growth. An important series of investigations by Kohn (1906) suggests that this phenomenon of multiplication dur- ing storage may be due in part to the solution of certain constituents of glass which favor bacterial life, since the increase is notably greater in bottles of the more soluble glasses. 3 QUANTITATIVE EXAMINATION OF WATER 39 EFFECT OF SIZE OF VESSEL UPON THE MULTIPLICATION OF WATER BACTERIA DURING STORAGE (WHIPPLE, 1901) Number of Bacteria per c.c. Sample| Bottle. Incuba-| Ini- After | After | After After After tion. tial.t | 3 Hrs. | 6 Hrs.| 12 Hrs 24 Hrs. 48 Hrs. & A | r-gallon | 13° 77 63 65 47 42 175 B | 2-quart | 13° | 77 59 | 63 60 45 690 C | rquart | 13° | 77 63 63 47 46 | 325 D I-pint 13° 77 57 64 36 38 630 E 2-ounce | 13° 77 55 58 47 IOI | 10,250 F | r-gallon | 24° | 77 iS 97 | 275 290 | 300 G | 2-quart | 24° 77 92 59 62 180 250 H | r-quart | 24° | 77 84 77 46 340 goo I I-pint 24° 77 51 46 100 | 2,950 | 7,020 5 2-ounce | 24° 77 51 52 145 | 11,000 | 41,400 1 Average of five plates. Whipple’s table, quoted above, shows that the multi- plication during storage was greater at a higher tem- perature; and this is a well-recognized general rule. In order to obviate the abnormal results of storage increase it is therefore obvious that samples must be examined shortly after collection and that they must be kept cool during their necessary storage. If fairly pure waters are placed upon ice and kept between o degrees and 10 degrees, they will show no material increase in 12 hours. With polluted water, however, another danger is here introduced. Samples of such water when packed in ice show a marked decrease due to the large number of sensitive intestinal bacteria present. Jordan (Jordan, 1900) found that three samples of river-water packed in ice for 48 hours fell 40 ELEMENTS OF WATER BACTERIOLOGY off from 535,000 to 54,500; from 412,000 to 50,500, and from 329,000 to 73,000, respectively. It is, there- fore, important that even iced samples should not be kept too long; and it is desirable to adhere strictly to the recommendations of the Standard Methods Committee that the interval between sampling and examination should not exceed 12 hours in the case of relatively pure waters, 6 hours in the case of relatively impure waters, and 1 hour in the case of sewage. Plating. The bottle ccntaining the sample of water is first shaken at least twenty-five times in order to get an equal distribution of the bacteria. If the num- ber of bacteria present is probably not greater than 200, 1 c.c. is then withdrawn with a sterile 1 c.c. pipette and delivered into a sterile Petri dish of 10 cm. diameter. To this is added 5 c.c. of standard 10 per cent gelatin at a temperature of about 30° C., or standard agar (7 c.c.) at 40-42° C. Should the number of bacteria per c.c. probably exceed 200, dilution is necessary. This is best accomplished by adding 1 c.c. of the water in question to 9, 99 or 999, etc., c.c. of sterile tap water according to the amount of dilution required. The diluted sample is then shaken thoroughly and 1 c.c. taken for enumeration. In order to determine the number of bacteria originally present it is only neces- sary to multiply by the factor 10, 100, or 1000, etc. When a sample of water from an unknown source is to be examined it is generally desirable to make two check plates at each of the above dilutions, select- ing those which give nearest to 200 colonies on the plates after incubation as the ones on which to rely QUANTITATIVE EXAMINATION OF WATER 41 for the count. A much smaller number will not give average figures, and if more than 200 colonies are present on a plate many bacteria will be checked by the waste products of those which first develop and ‘the count obtained will be too low. After the addition of the diluted sample and the nutrient medium, their thorough mixture in an even layer on the bottom of the plate is obtained by careful tipping and rotation. It was formerly customary to mix the water with the gelatin in the tube before pouring into the plate, but this method is objectionable because there is always a residuum of medium remaining in the tube which will retain varying numbers of bacteria and thus interfere with the accuracy of the count. Before pour- ing the medium into the plate the mouth of the tube should be flamed to remove any possibility of con- tamination. The usual method of determining the number of bacteria in water for sanitary purposes in Germany, England and the United States has always been by the use of gelatin plates with a 2-day incubation period at 20 degrees. The 1905 Standard Methods Report of the American Public Health Association Committee recommended this procedure, which has been universally adopted. The 1912 Report, however, suggests the use of agar with a 1-day period at 37 degrees, as yielding quicker results and indicating the presence of bacteria more nearly related to pathogenic types. The com- parative value of the two methods has been well dis- cussed by Whipple (1913). The use of gelatin is not only more time-consuming, but requires the use of a 42 ELEMENTS OF WATER BACTERIOLOGY . special 20-degree incubator which is difficult to regulate. The 37-degree incubator must be provided in any case for the isolation of B. coli. On the other hand, the time seems hardly ripe for the abandonment of the 20-degree count, which has been used for 20 years all over the civilized world, and for the interpretation of which we have very complete data. There is at present no such sound basis for interpreting the 37-degree count, and in many cases, as in the control of water filtration plants, the 37-degree numbers are too small to be of any practical value. Furthermore the 20- degree count may furnish evidence of surface con- tamination as distinguished from fecal pollution, which is often of considerable value. The authors have always urged the use of the 37- degree count along with the 20-degree count as furnish- ing most valuable information; but this is very different from the substitution of one count for the other. The recommendation that the 20-degree count be abandoned, with no evidence to warrant such a revolu- tionary change, and no experimental results on which to base an interpretation of the 37-degree count, has aroused vigorous opposition from a large majority of practical water bacteriologists. At the Washington meeting of the American Public Health Association in September, 1912, it was resolved ‘‘ that in the opinion of the Laboratory Section of the American Public Health Association, ordinary routine examination of water for sanitary purposes, and in the control of purification plants, for the present should include the determination of the number of bacteria developing ed ee i~ QUANTITATIVE EXAMINATION OF WATER 43 at 20 degrees and at 37 degrees and a presumptive test for B. coli in lactose bile.” This action of the section responsible for the appoint- ment of the Standard Methods Committee appears to supersede the report of the committee itself and makes the combination of the 2c- and 37-degree counts the standard American procedure. The 20-degree count may be made on either gelatin or agar; but it is the 20-degree count which will be discussed in this chapter, leaving the body temperature count for consideration in Chapter IV. The exact composition of the medium is, of course, of prime importance in controlling the number of bacteria which will develop. The figures previously cited in connection with the discussion of Hesse’s Nahrstoff agar show how bacterial counts may vary with media of widely different composition. The table quoted on page 44 from Gage and Phelps (1902), shows the considerable differences which may be due to the presence or absence of meat infusion, peptone, etc., in media of generally similar character (compare the figures for plain gelatin, peptone, gelatin, and meat gelatin). Much slighter variations than this, however, are significant. The reaction of the medium was found as early as 1891 to be important, for Reinsch (Reinsch, 1891) showed in that year that the addition of one one-hundredth of a gram of sodium-carbonate to the liter increased sixfold the number of bacteria develop- ing. Fuller (Fuller, 1895) and Sedgwick and one of us (Sedgwick and Prescott, 1895), working indepen- dently, established the fact that an optimum reaction 44 ELEMENTS OF WATER BACTERIOLOGY existed for most water bacteria and that a devia- tion either way decreased the number of colonies developing. TABLE SHOWING PERCENTAGES OF BACTERIA DEVEL- OPING ON MEDIA OF DIFFERENT COMPOSITIONS (GAGE AND PHELPS, 1902) Medium. Days’ Count. 2 3 | 4 5 6 7 8 | 9 Wabreteff agar. :..36 5.4%. «1% 19 | 60 | 78 | 85 | 95 | 99 | 99 | I00 Nahrstoff peptone agar..... 10 | 22 | 26 | 28 | 30 | 30 1-40-Fu5g0 Peptone agar, i. 4 0. et ads II. |, 16 | 22. |:23- 24° |. ogMieaan ie oe MER TIAG AN. cine cate nara $8.) 13) )\ 20) |207 eee 17 Pla A oAr es oc Spee lees 8 | 10 | 13 | 14°) 14 | Ta} ea ee Regulariagar raccisccisteee.t 7) O° | 22 | tho), 1. eee It Niahrstoff glycerin agar ..... 6 | 10 | 1x | tx | rE | Bae II Niahrstoff meat agar........ 7 7 8 8 | 10 | 10 |] 10 Io Meat pelatin'. ¢.o4.st¥idisisgeu so 12 | 19 | 24 | 26 | 26} 26; 26} ‘26 Peptone gelatine ........... 7 | 12 | 18 | 20 | 20] 20 | 20] 20 Standard gelatin. .. 05.0.4: 8] 10 | tr | 22.) 239) E30 heehee Patsy Weal ess en a eis whe 8 T} 6.) 22 23: tots eta) eee Nahrstoff gelatin........... S| 6] 9 | 22. [19 rs seas Whipple (Whipple, 1902) has shown that not only the particular kind of gelatin used, but its exact physical condition as affected by sterilization and other previous treatments, will materially affect the results obtained. Gage and Adams (1904) found marked differences in counts as the result of the use of the two best-known commercial peptones. A long series of waters plated on agar made up with Merck’s and Witte’s peptones, respectively, showed the average relative results in the table on page 45. QUANTITATIVE EXAMINATION OF WATER 45 AVERAGE RELATIVE NUMBER OF BACTERIA ON PEP- TONE AGAR WITH DIFFERENT PEPTONES (GaGE AND ADAMS, 1904) Days | 2 4 | 6 | 8 | Io | 12 } j | TOS of a ae ack IE 51 67 89 08 oe ee 38 53 100 roo 100 10° The same authors showed that the composition of the water used exercised a marked selective action upon the development of bacteria. Agar made up with -sewage permitted a maximum growth of sewage bacteria and showed no colonies when inoculated with filtered city water. On the other hand agar made up with city water showed 100 per cent of the bacteria present in city water and river water, three-quarters of those present in sewage and less than half of those present in sewage effluents. Hesse (1904) found that the number of bacteria developing on Nahrstoff agar varied with the composi- tion of the glass tubes in which the media had previously been sterilized. The more soluble glasses yielded sufficient alkali to the medium to inhibit four-fifths of the bacteria present in certain cases. All these facts make it evident that only the strictest adherence to a standard method can ensure comparable results; the ordinary nutrient gelatin or agar should then in all practical sanitary work be made up from distilled water, meat infusion, peptone and gelatin or agar, in exact accordance with the directions of the Standard Methods Committee. 46 ELEMENTS OF WATER BACTERIOLOGY . Even the standard procedure fails to ensure uniformity in one important respect. The meat infusion which it calls for is in itself a highly variable quantity. Gage and Adams (1904), in the examination of fifteen lots of beef infusion, found variations of nearly 1 per cent in organic solids (calculated on the weight of the whole infusions), after the final filtration. The organic constituents of the meat infusion varied, therefore, among themselves by nearly the total amount of pep- tone added. Itistobe hoped that the standard methods may soon be so revised as to eliminate this necessarily uncertain constituent of nutrient media. Criticisms of detail must, however, give way to the importance of securing fairly comparable results; and the con- fusion which would follow the use by individual bac- teriologists of media made without meat would out- balance the errors inherent in the standard procedure. Incubation. Incubation should take place in a dark, well-ventilated chamber where the temperature is kept substantially constant at 20 degrees and where the atmosphere is practically saturated with moisture. It has been shown by Whipple (Whipple, 1899) and others that the number of bacteria developing in plate cultures is to a certain extent dependent upon the presence of abundant oxygen and moisture. Thus, reckoning the number of bacteria developing in a moist chamber at roo, the percentage counts obtained in an ordinary. incubator were as follows: 75 when the relative humid- ity of the incubator was 60 per cent of saturation; 82 when it was 75 per cent; 98 when it was 95 per cent. This source of error may be avoided by the use of ven- QUANTITATIVE EXAMINATION OF WATER 47 tilated dishes and by the presence of a pan of water in the incubating chamber. According to American and German practice, plates made for sanitary water analysis are counted at the end of 48 hours. The English Committee appointed to consider the standardization of methods for the Bacterioscopic Examination of Water (1904) fixed the time at 72 hours. French bacteriologists, and some Germans (Hesse and Niedner, 1906), still recommend - longer periods, and the following table from Miquel and Cambier (Miquel and Cambier, 1902) shows that many bacteria fail to appear in our ordinary procedure. It is, however, in the main, the characteristic water bacteria which develop slowly, sewage bacteria almost without exception being rapid growers. The longer period of incubation is, therefore, not only inconvenient, but undesirable, since it obscures the difference between good and bad waters. EFFECT OF THE LENGTH OF INCUBATION OF WATER BACTERIA IN GELATIN UPON THE NUMBER OF COLONIES DEVELOPING (MIQUEL AND CAMBIER, 1902) Length of Incubation. Beet: Length of Incubation. ena UMP she's ince a-c'% 39s a0. Fi .¢ days =~ .2 a. 821 MENU «Shard pxjs'aie v4 Cae 4365 NN tedays i Hele 8590 PEE oe one WG 5 Orso 254 MB ORYS 2 oe ce ee are 892 Re a ere S875 PIR GAS oct ci as yas 921 0 Sere BAG 1h 8S COWS e oo cnt ti ale O51 OEE ee 637 D4 GBYS. oo oS ee ok 976 Me OS i 3 ee | ae SEA ee 1000 OO eee 780 48 ELEMENTS OF WATER BACTERIOLOGY | Counting. The number of bacteria is determined by counting the colonies developed upon the plate, with the aid of a lens magnifying at least five diameters. For convenience in counting the plate may be placed upon a glass plate ruled in centimeter squares and set over a black tile; or the tile itself may be ruled. As has already been said, it is desirable that the number of colonies should not exceed 200, for when the number is very high the colonies grow only to a small size, making counting laborious and inaccurate, and many do not develop at all. The best results are obtained with numbers ranging from 50 to 200. When it is possible to do so, all the colonies on the plate should be counted. When tiiey exceed 400 or 500 it is often easier, and fully as accurate, to count a fractional part of the plate and estimate the total number therefrom. This should not be done, however, except in case of necessity. Ayers (1911) has suggested two counting devices which will be found very useful where a great many plates have to be handled. For getting the best possible ‘transmitted light, he places his plate on the ground- glass top of a wooden box, 7 inches square, with one side open to admit light, which is reflected upward by a plane mirror set in the box at an angle of 45 degrees. An ordinary graduated-glass counting plate may be placed between the ground-glass and the Petri dish, and the eyes are protected from direct light by a screen rising from the open side of the box. For picking colonies from a gelatin plate in a warm room, he places between the ground glass and the Petri dish a copper QUANTITATIVE EXAMINATION OF WATER 49 box with top and bottom of glass 7 inches square and 12 inches deep, through which cold water is allowed to circulate. Expression of Quantitative Results. It is customary in determining numbers to make plates in duplicate, thereby affording a check upon one’s own work. Owing to-the lack of precision in the method, the limit of experimental error is a wide one. It should be possible for careful manipulators to obtain results within ro per cent of each other, but a closer agreement than this is hardly to be expected. It has been suggested by the committee of the American Public Health Association that the following mode of expressing results be adopted in order to avoid the appearance of a degree of accuracy which the methods do not warrant. NUMBERS OF BACTERIA FROM 1-50 shall be recorded to the nearest unit 51-100 “ce “e ‘éc 5 IOI—250 sé a8 *s Io 251-500 “ce ce “ec 25 501-1000 sc 6c ce 50 IOOI—10,000 “t ne kf 100 I0,00I—50,000 nie = = 500 50,001—100,000 xf de .s 1,000 100,00I—500,000 * SS + 10,000 500,001—1 000,000 .e es ss 50,000 I ,000,001—5,000,000 re ss _ 100,000 The determination of numbers of bacteria in water in the field has frequently been attempted. Since the laboratory method of “ plating out” is difficult to use in field work, the Esmarch tube process has often been employed. This consists in introducing into a tube of melted gelatin or agar 1 c.c. of the water and then 50 ELEMENTS OF WATER BACTERIOLOGY _ rotating the tube until the medium has solidified in a thin layer on the inner wall. Other bacteriologists have devised ingenious field kits for adapting the plate method to this purpose, of which one very good form has recently been described by Van Buskirk (1912). The opportunity for air infection in work done outside a proper laboratory is, however, always great; and it is almost impossible to secure proper conditions for incubation in any makeshift establishment. On the whole, the authors are of the opinion that laboratory examinations are to be preferred to those made in the field, if a laboratory can be reached within 12 hours or so of the time of collection of the samples. The Kansas State Water Survey has made use of a device for shipping iced samples which has given excellent results. The outfit is described in the Amer- ican Journal of Public Health for May, 1914, and consists essentially of an insulated container in which the bottles are firmly held by a simple mechanism. With proper icing the samples are maintained at a tem- perature below to degrees C. for two or three days or even more. Very little change due to storage is found in the counts on agar when plates are incubated at 37 degrees C. and in winter the 20-degree counts are satisfactory on agar. Gelatine counts were found to be erratic and not dependable. The following tables kindly sent us by Prof. C. C. Young show the comparative numbers obtained in the laboratory after certain periods of storage, and in the laboratory as compared with the actual field analyses. QUANTITATIVE EXAMINATION OF WATER 50a EFFECT OF STORAGE ON BACTERIAL CONTENT Total Total B. coli per c.c. Count Count [_ Place Collected. Agar, 37 | Gelatine eg. C., | 20 Deg. | Lactose | Lactose | De t os2 24 hrs. |C., 48 hrs. Bile Broth | Bie Time—immediately CUNT ESO a 600 3,000 0.4 6.0 6.0 Settling basin........ ; 36 250 ) 0.2 0.2 Tap at 4323 Ky... .. II 1,500 ° ° ° Blackmar Well....... 18 5 ° ° ° Well at 1338 Ohio.... 7 35 0:2 ° ° Time—12 hours ORM R evar d, Sala-aip! see. 820 28,000 oO.1 2.0 2.0 Settling basin........ 50 2,000 o.1 0.2 0.2 Tap at 1323 Ky...... 25 500 ° ° ° Blackmar Well....... 30 250 ° ° ° Well at 1338 Ohio.... be) 80 O.1 O.I ° Time—48 hours EDV ORE oslo ig sic aie ties 600 55,000 o.1 0.4 0.4 Settling basin........ 20 1,800 ° 0.2 0.2 Tap at 1323 Ky...... 40 10,000 oO. oO. ° Blackmar Well....... 12 350 ° ° ° Well at 1338 Ohio.... 10 50 Oct 0.2 0.2 Time—immediately ERLVED rey Ce tt ation ds 750 6,200 8.0 6.0 6.0 Laboratory tap....... Io bere) ° ° ° Blackmar Well....... 8 300 ° ° ° Beamer Well......... 35 500 4.0 4.0 2.0 Time—12 hours RONG OE A! Wetec ears ae as 850 60,000 4.0 6.0 4.0 Laboratory tap....... 15 25 ° ° ° Blackmar Well....... 4 1,500 ° ° ° Beamer Well......... 40 1,000 4.0 4.0 4.0 Time—24 hours HRIVOES ise eee Se oie acl 920 2,900 6.0 40.0 8.0 Laboratory tap....... 20 1,400 ° ° ° Blackmar Well... .... 200 1,500 3 ° ° Beamer Well......... 100 500 0.8 4.0 I.0 506 ELEMENTS OF WATER BACTERIOLOGY | EFFECT OF STORAGE ON BACTERIAL CONTENT (Concluded) Total Total B. coli per c.c. Count Count Place Collected. Agar, 37 | Gelatine Deg. C. '20 Deg.C.| Lactose | Lactose | Dextrose 24 hrs. 48 hrs. Bile. | Broth. Bile. Time—48 hours URSV EL senza ae aioe 800 5,500 20.0 20.0 20.0 Laboratory tap....... 12 120 ° ° ° Blackmar Well....... 150 2,000 ° ° ° Beamer Well......... 120 800 0.8 2.0 I.0 Time—immediately Rivet i435 ieee sb as 2EOO bis Fens 100.0 | 100.0 | 100.0 ‘Lap at laboraterys.4.'\W) 3200 | 2.4.0; 0.8 0.8 0.8 Blackmar Well....... 7 S58 Hi ethawes 1.0 1.0 1.0 Public Well, Mass. st. 510 ball Kanade Ee 10.0 10.0 10.0 Time—24 hours RIVERS saree, S05 j-th) ACOSO AI we rohts oe I00.0 100.0 100.0 Tap at laboratory....| 1900 | ...... 0.4 0.6 0.2 Blackmar Well....... BOOr yen eeee 0.6 0.6 0.4 Public well on Mass. st. 220% Wawa 10.0 4.0 2.0 Time—48 hours WRIVEN siccc Sraicis fgiole s-ce¥s 2200 | oetiass I00.0 100.0 100.0 Zep at laboratory...) 4400. 5.635 0.6 0.6 0.4 Blackmar Well....... MOO Yl sates. 0.4 0.6 0.2 Public well on Mass.st.| 1200 | ...... 1.0 I.0 I.0 CHAPTER III THE INTERPRETATION OF THE QUANTITATIVE BACTERIOLOGICAL EXAMINATION Standards for Potable Water. The information fur- nished by quantitative bacteriology as to the antecedents of a water is in the nature of circumstantial evidence and requires judicial interpretation. No absolute stand- ards of purity can be established which shall rigidly separate the good from the bad. In this respect the terms “test”? and “analysis” so universally used are in a sense inappropriate. Some scientific problems are so simple that they can be definitely settled by a test. The tensile strength of a given steel bar, for example, is a property which can be determined. In sanitary water examination, however, the factors involved are so complex, and the evidence neces- sarily so indirect, that the process of reasoning much more resembles a doctor’s diagnosis than an engineering test. The older experimenters attempted to establish arbitrary standards, by which the sanitary quality of a water could be fixed automatically by the number of germs alone. Thus Miquel (Miquel, 1891) published a table according to which water with less than 10 bac- teria per c.c. was “ excessively pure,” with 10 to 100 51 52 EKLEMENTS OF WATER BACTERIOLOGY bacteria, “very pure,” with too to 1000 bacteria, “pure,” with 1000 to 10,000 bacteria, ‘‘ mediocre,” with 10,000 to 100,000 bacteria, ‘‘ impure,’ and with over 100,000 bacteria, ‘‘ very impure.” Few sanitarians would care to dispute the appropriateness of the titles applied to waters of the last two classes; but many bac- teriologists have placed the standard of “ purity ” much higher. The limits set by various German observers range, for example, from 50 to 300. Dr. Sternberg (Sternberg, 1892) in a more conservative fashion, - has stated that a water containing less than 100 bacteria is presumably from a deep source and uncontaminated by surface drainage; that one with 500 bacteria is open to suspicion; and that one with over 1000 bac- teria is presumably contaminated by sewage or surface drainage. This is probably as satisfactory an arbitrary standard as could be devised, but any such standard must be applied with great caution. The source of the sample is of vital importance in the interpretation of analyses; a bacterial count which would condemn a spring might be quite normal for a river; only figures in excess of those common to unpolluted waters of the same character give an indication of danger. Fur- thermore, the bacteriological tests are far more delicate than any others at our command, very minute addi- tions of food material causing an immense multiplica- tion of the microscopic flora. This delicacy necessarily requires, both in the process of analysis and the inter- pretation of results, a high degree of caution. As pointed out in the previous chapter, the touch of a finger or the entrance of a particle of dust may wholly QUANTITATIVE BACTERIOLOGICAL ANALYSIS 53 destroy the accuracy of an examination. Even the slight disturbance of conditions incident upon the storage of a sample after it has been taken may in a few hours wholly alter the relations of the contained microbic life. It is necessary, then, in the first place, to exercise the greatest care in allowing for possible error in the collection and handling of bacteriological samples; and in the second place, only well-marked differences in numbers should be considered significant. In the early days of the science, discussion ran high as to the interpretation of bacteriological analysis; and particularly as to the relation of bacterial numbers to the organic matter present in a water. Different observers obtained inconsistent results, and Bolton (Bolton, 1886) concluded that there was no relation whatever between the organic pollution of a water and its bacterial content. Tiemann and Gartner (Tiemann and Gartner, 1889) furnished the key to the difficulty in their statement that there are two classes of bacteria, the great majority of species normally occurring in the earth or in decomposing organic matter, which require abundance of nutriment, and certain peculiar water bacteria which can multiply in the presence of such minute traces of ammonia-as are present in ordi- nary distilled water. Even these prototrophic or semi-prototrophic forms, however, require a definite amount of food of their own kind. Kohn (1906) determined the minimal nutrient mate- rial requisite for certain of them and found that they could develop in the presence of 198 X 107° to 198 X 10718 per cent of dextrose, 66X107!3 to 66107!” per cent 54 ELEMENTS OF WATER BACTERIOLOGY ammonium sulphate and 66X107! to 66X107!® per cent ammonium phosphate. Similar minute amounts of organic matter are found in the purest of natural waters and under exceptional conditions certain species of bacteria may therefore multiply in bottled samples, or, at times, in a well or the basin of a spring. In normal surface-waters, such growths of the prototrophic forms do not apparently occur. Here it is found as a matter of practical experience that the number of bac- teria present depends upon the extent to which the water has been contaminated with decomposing organic matter, either by pollution with sewage or by contact with the surface of the ground. The bacterial content varies as the extent and character of the contamination varies. It measures not merely organic matter, but organic matter in a state of active decay, and like the ammonias and other features of the sanitary chemical analysis, indicates fresh organic pollution, with the added advantage that the presence of the stable nitrogenous compounds often present in peaty waters introduces no error in the bacteriological analysis. Bacterial Content of Surface-waters. In judging of a surface-water the student will be aided by reference to the figures given for certain normal sources in Chapter I; the Boston tap water with 50 to 200 bacteria per c.c. (Philbrick, 1905) and the water of Lake Zurich with an average of 71 in summer and 184 in winter (Cramer, 1885) may be taken as typical of good potable waters; and numbers much higher than these are open to suspicion, since all contamination whether contributed by sewage or by washings from the surface of the QUANTITATIVE BACTERIOLOGICAL ANALYSIS 55 ground is a possible source of danger. The excess of bacteria in surface-waters during the spring and winter months is by no means an exception to the general tule that high numbers are significant, since the peril from supplies of this character is clearly shown by the spring epidemics of typhoid fever which at the times of melting snow visit communities making use of unpro- tected surface-waters. Streams receiving direct con- tributions of sewage exhibit a similar excess of bacteria at all times, numbers rising to an extraordinary height near the point of pollution and falling off below as the stream suffers dilution and the sewage organisms perish. Miquel (Miquel, 1886) records 300 bacteria per c.c. in the water of the Seine at Choisy, above Paris; 1200 at Bercy in the vicinity of the city, and 200,000 at St. ‘Denis after the entrance of the drainage of Paris. Prausnitz (Prausnitz, 1890) found 531 bacteria per c.c. in the Isar above Munich, 227,369 near the entrance of the principal sewer, 9111 at a place 13 kilometers below the city, and 2378 at Freising, 20 kilometers further down. Jordan (Jordan, 1900), in his study of the fate of the sewage of Chicago, found 1,245,000 bacteria per c.c. in the drainage canal at Bridgeport, 650,000 at Lockport, 29 miles below, and numbers steadily decreasing to 3660 at Averyville, 159 miles below the point of original pollution. Below Avery- ville the sewage of Peoria enters and the numbers rise to 758,000 at Wesley City, decreasing to 4800 in 123 miles flow to Kampsville. Brezina (1906) found 1900 bacteria per c.c. in the Danube River above, and 110,000 at the north of the Danube canal. This number 56 ELEMENTS OF WATER BACTERIOLOGY fell to 85,000 one kilometer below, 62,000 four kilo- meters below, and 40,000 seven kilometers down the stream. Vincent (1905) records from 1000 to 46,000 bacteria per c.c. in the waters of more or less polluted French rivers. Mayer (1902), on the other side of the world, found 21 and 35 bacteria per c.c. in the Shaho River, near its source, in the vicinity of the great Chinese Wall and from 100,000 to 600,000 in the highly polluted Whangpo near its mouth. Bacterial Content of Ground-waters. In ground- waters we have seen that bacteria may occasionally be present in considerable numbers, but if so they are generally organisms of a peculiar character, incapable of development on the ordinary nutrient media in the standard time. Thus in 48 hours we often obtain counts measured only in units or tens such as have been recorded in Chapter I. When higher numbers are present, the general character of the colonies must be taken into account, since besides the slowly-growing forms certain other water bacteria, which require a comparatively small amount of nutriment, may multiply at times in a deep well or the basin of a spring. In such a case, however, the appearance of the plates at once reveals the peculiar conditions, for the colonies are of one kind and that distinct from any of the sewage species. Thus Dunham (Dunham, 1889) reports that the mixed water from a series of driven wells gave 2 bacteria per c.c., while another well, situated just like the others, contained 5000, all belonging to a single species common in the air. Except in such peculiar cases as this high numbers in a ground-water mean contamination. QUANTITATIVE BACTERIOLOGICAL ANALYSIS 57 Bacteria in Filtered Waters. The process of slow sand filtration for the purification of unprotected surface-water is essentially similar to the action which takes place in nature when rain soaks through the ground to appear in wells and springs; and it is in the examination of the effluent from such municipal plants that the quantitative bacteriological analysis finds, perhaps, its most important application. The chemical changes which occur in the passage of water through sand at a rate of 1,000,000 or 2,000,000 gallons per acre per day are so slight as to be negligible. The bacteria present should, however, suffer a reduction of 98 or 99 per cent, and their numbers furnish the best standard for measuring the efficiency of such filtration plants. At Lawrence, in 1905, Clark found an average of 12,700 bacteria per c.c. in the raw water of the Merrimac River, while the number present in the filtered water was only 7o (Massachusetts State Board of Health, 1906). Where the number of bacteria in the applied water is smaller it is difficult to obtain so high a per- centage efficiency. At Washington, for example, pro- longed sedimentation generally reduces the bacterial numbers to less than a thousand and it is almost impos- sible to secure a 99 per cent removal. The actual numbers of bacteria in the effluent are, however, much lower than at Lawrence. The monthly average results obtained for a year at these two plants are tabulated on page 58. Mechanical filtration gives similar results. Fuller at Cincinnati (Fuller, 1899) records 27,200 organisms per c.c. in the water of the Ohio River between 58 ELEMENTS OF WATER BACTERIOLOGY September 21, 1898, and January 25, 1899, while the average content of the effluent from the Jewell filter was 4oo. Data with regard to the: operation of mechanical filters are now abundant, since all over the _ world the operation of these plants is controlled by bacteriological methods. Recently Johnson (1907) has reported some interesting results from the far East. At Osaka, Japan, an average of 200 bacteria per c.c. in the raw water of the Yodo River was reduced, in 1905, to an average of 25 by slow sand filters; at Bethmangala, India, in 1906, mechanical filters treated the water of the Palar River, containing 4350 bacteria per c.c., and yielded an effluent with only 13 per c.c. (Johnson, 1907). The average monthly results obtained with the new mechanical filter plant at Harrisburg, Pa., are included in the table below for comparison with the figures REMOVAL OF BACTERIA BY NATURAL SAND FILTERS AND MECHANICAL FILTERS. BACTERIA PER C.C. IN APPLIED WATER AND EFFLUENT. MONTHLY AVER- AGES Washington, 1906. Lawrence, 1905. Harrisburg, 1906. moms | Applied | petuent. | Applied | petuent, | APP’ | petuent January ....| 1500 39 14,200 110 9,510 104 February ... 550 16 14,800 55 21,228 298 March. .4..:: 650 19 10,300 55 31,326 75 Yet | Peete 400 22 3,600 170 30,005 42 Rh eee rie 65 17 1,900 12 6,187 86 NUNC 524 30 220 17 9,600 9 2,903 31 JOY as eens 160 26 3,900 55 685 10 AUIGUSE: 60554 190 14 ‘| 19,500 37 1,637 5 September . . 130 14 13,500 44 836 12 October..... 275 16 39,800 IIo 7,575 63 November .. 220 12 8.700 70 26,224 236 December... . 700 45 oe oa 37,525 163 eee QUANTITATIVE BACTERIOLOGICAL ANALYSIS 59 recorded at Washington and Lawrence; and these may be taken as typical, since the Harrisburg plant is the latest of its type, as the Washington plant is the newest and most perfectly equipped of slow sand filters. In well-managed purification plants the bacteria in the effluent are determined daily, and any deviation from the normal value at once reveals disturbing factors which may impair the efficiency of the process. In Prussia official regulations demand such systematic examinations and prescribe 50 as the maximum number of bacteria allowable in the filtered water. In the same way the condition of an unpurified surface supply may be determined by daily bacteriological analyses and warnings of danger issued to the public, as has _ been done at Chicago and other cities. In general, any regular determination of variations from a normal standard furnishes ideal conditions for the bacteriological methods; and the detection by Shuttleworth (Shuttle- worth, 1895) of a break in a conduit under Lake Ontario by a rise in the bacteria of the Toronto water-supply may be cited as a classic example of its application. Often, however, the expert is called to pass upon the character of a water of which no series of analyses is available. In such cases an inspection of the location from which the water comes should be insisted on, as a sound interpretation of a water analysis can only be made with a reasonably full knowledge of the source of the sample. After a careful sanitary inspection, however, the comparison of the result of even a single examina- tion with the normal range for waters of the same class 60 ELEMENTS OF WATER BACTERIOLOGY may prove of great significance, as a few practical examples may make clear (Winslow, 1gor). In the spring of 1900 the city of Hartford, Conn., was using a double supply, from the Connecticut River and from a series of impounding reservoirs among the hills. A single series of plates showed from 4000 to 7000 bacteria per c.c. in the water of the river, while the reservoir water contained 300 to goo. The abandon- ment of the river supply followed, and at once the excessive amount of typhoid fever in the city was curtailed. In the fall of 1900, Newport, R. I., experienced an outbreak of typhoid fever, and when suspicion was thrown upon the surface water-supply, chemical analysis of the latter was not wholly reassuring; but there were only 334 bacteria per c.c. in the water from the taps, while a well in the infected district gave 6100. It was no surprise to find, on a further study of the epidemic, that the well was largely at fault and the public supply was not. In the case of ground-water the evidence is usually even more distinct. At Framingham, Mass., in 1903, high chlorin content in the public supply, drawn from a filter gallery beside a lake, had led to public anxiety. Five samples from different parts of the system showed averages of 1, 2, 2, 2, and 4 bacteria per c.c.; and taking this in conjunction with the other features of the bacteriological analysis, it was possible to report that any pollution introduced upon the gathering ground had at the time of examination been entirely removed. Cuvureyoy JVM "fyO As01INOD) "AK 'N ‘uinqny 3 JdA0,7 : ploydsy, jo orwopidgy sz9yeA OY} JO wax oY} Burancy oosemEO oYe’T Ul 7UW9}U0D YOO ‘| pur yuNOD [rIOT, ut sosueqy ‘Vuruq ofv95 JO 9804) LL _— ——" "2 Posey Ay Og} HO spend aoaoy ploqd sy, ‘sono prondsy, Jo sdevp OF Treen "hn } H rT bare > ee SS Ne | a hiethal f atk ala 2 a dl a? SC e 4 ong 2 4 ooaT i = 0008 oon 8 0008 a 0098 & A oor 8061 L061 ‘ies Fy ‘oag “AON "LOO "1d38 “onv Amn anne AVW "UdV “YVW ‘aad “nve ‘O30 “AON ‘LOO |‘Ld3S L e QUANTITATIVE BACTERIOLOGICAL ANALYSIS 60a 606 ELEMENTS OF WATER BACTERIOLOGY | The results presented in the diagram on page 60a offer an interesting example of the value of the total count of bacteria as compared with the estimation of B. coli. This chart, prepared by Mr. J. W. Acker- man, the Engineer of the Auburn, N. Y., Water Board, shows by the vertical. lines the total count and by the crosses the presence of colon bacilli in the water supply of Auburn drawn from Lake Owasco. In the year illustrated in this diagram, and in other years, before and after, the total count of bacteria rose sharply at the time of the spring thaw, while colon bacilli were on the whole more abundant during the summer. - The explanation of this phenomenon probably is that a certain proportion of colon bacilli are always contrib- uted by the small brooks which enter the reservoir from agricultural land which, not being of human origin, have but little significance. At the time of the spring thaws, which for the most part wash an open farming country, the normal contribution of B. coli from the fields is obscured by the rain and melting snow, while only the rise in total count registers the fact that contaminating material of all sorts is being washed into the lake. With this contaminating material, for the most part of a harmless nature, human excreta are washed in from certain points on the watershed, and in the spring of 1908 these excreta contained specific typhoid infection and an epidemic in Auburn, was the result. In this case then the total count of bacteria was a more accurate index of danger than the colon content. CHAPTER IV DETERMINATION OF THE NUMBER OF ORGANISMS DEVELOPING AT THE BODY TEMPERATURE _ Relation between Counts Made at 20° and 37°. The count of colonies upon the gelatin plate measures, as we have pointed out, the number of the metatrophic bacteria in general; and the distribution of these forms corresponds with the decomposition of organic matter wherever it may occur. In this great class, there are some species which will grow under a wide variety of conditions. These are present in most waters in small numbers, and in sources contaminated with wash from decaying vegetable matter they occur in abundance. Other metatrophic forms, however, through a semi- parasitic mode of life, have become specially adapted to the peculiar conditions characteristic of the animal body; and these bacteria possess the property of develop- ing most actively at the temperature of the human body, 37° C., which altogether checks the growth of the majority of normal earth and water forms. The determination of the number of organisms growing at the body temperature may throw light, then, on the presence of direct sewage pollution, since the bacteria from the alimentary canal flourish under such con- ditions, while most of those derived from other sources do not. Savage classifies the bacteria which may 61 62 ELEMENTS OF WATER BACTERIOLOGY be found in water under three headings: normal inhab- itants, like B. fluorescens; unobjectionable aliens (from soil), like B. mycoides, and objectionable aliens (from excreta), like B. coli. The first sort and many of the second sort are generally unable to grow at 37 degrees. This criterion is not an absolute one. Savage, (1906) reports an experiment in which unpolluted soil, which had not been manured or cultivated for at least 3 years, was added to tap water, with the result that a 20° count of 76 was increased to 1970, and a 37° count of 3 was raised to 1630. In this case most of the bacteria in the soil were capable of development at body temperature. Experience shows, however, that the numbers of such bacteria which actually reach natural waters from such sources are seldom large. The count at 37°, therefore, helps to distinguish con- tamination by wash of the soil of a virgin woodland from pollution by excreta, since in the former case the proportion of blood-temperature organisms is much smaller than in the latter. Furthermore, this method is free from much of the error introduced by the mul- tiplication of bacteria after the collection of a sample, as most of the forms which grow in water during storage cannot endure the higher temperature and _ conse- quently do not develop upon incubation. Recently, for example, water from a spring of good quality was shipped to the laboratory from a considerable distance. Gelatin plates showed 4200 bacteria per c.c., but agar plates at 37° were sterile. Significance of the 37° Count. A majority of the English Committee appointed to consider the standard- DETERMINATION OF ORGANISMS 63 ization of methods for water examination (1904) recommended the body-temperature count as a stand- ard procedure. The American Committee on Standard Methods in its 1905 Report did not recommend this method even for alternative use. In its last report (1912), however, it substituted the 37° for the 20° count, which was dropped out entirely. As we have pointed out in Chapter II, this course seems to us an unwise one, and it was formally condemned at the meeting of the Laboratory Section of the American Public Health Association in September, 1912, by the passage of a vote declaring that “ordinary routine examinations of water for sanitary purposes, and in the control of purification plants for the present, should include the determination of the number of bacteria developing at 20 degrees and 37 degrees.” By this action the body-temperature count is placed on a par with the 2o-degree count as an integral part of sanitary bac- teriological water examination, a course which has been strongly urged in earlier editions of this book. The body-temperature count must, of course, be made upon agar plates; but otherwise the procedure is much the same as that already described for the routine quantitative bacteriological examination in Chapter II. A 1.5 per cent agar medium has generally been used, but the Standard Methods Committee in its recent report recommends only 1 per cent of agar. Whipple (1913) points out that this 1 per cent agar often gives trouble from the running together of the colonies on the weaker medium. On the other hand, a 1 per cent agar gives higher counts than 1.5 per cent 64 ELEMENTS OF WATER BACTERIOLOGY agar. He emphasizes the recommendation of Jackson that the agar used should be dried at 105° C. for 30 minutes, as commercial agar itself contains more or less water. The period of incubation ordinarily adopted for body-. temperature counts is 24 hours. Lederer and Bach- mann (1911) find that with sewage effluents a 48-hour period at 37° may yield counts from two to six times as high as those obtained in 24 hours; it is ques- tionable, however, whether the higher counts thus given would compensate for the loss of time. The adoption of a 24-hour period by the Standard Methods Com- mittee in any case represents an almost universal practice. In using agar plates at 37° difficulty is some- times caused by the spreading of colonies of certain organisms over the surface of the plate in the water of condensation which gathers; this may be avoided by inverting the plates after the agar is once well set, or still better by the use of plates provided with earthen- ware tops, as suggested by Hill. The porous earthen- ware absorbs the water which condenses on it, the surface of the plate remains comparatively dry, and the percentage of “spread” plates is reduced from 30 per cent to 1 per cent (Hill, 1904). Special pains must be taken, however, to keep the atmosphere in the incubator nearly saturated with moisture or errors will be introduced by the excessive evaporation of the medium used. Use of Litmus Lactose Agar. Additional evidence as to the character of a water sample may be DETERMINATION OF ORGANISMS 65 obtained with little extra trouble by adding a sugar and some sterile litmus to the agar medium and observing the fermenting powers of the organisms present, as first suggested by Wurtz (Wurtz, 1892) for the separation of B. coli from B. typhi. It hap- ‘pens that the most abundant intestinal organisms, belonging to the groups of the colon bacilli and the streptococci, decompose dextrose and lactose with the formation of a large excess of acid. The decomposi- tion of the latter sugar, is, on the other hand, almost entirely wanting among the commoner saprophytic bacteria, and therefore lactose is most commonly used in making sugar agar, 1 per cent being added to the medium just before the final filtration (between steps 15 and 16 in the standard process of media-making given on p. 271). In pouring the plate a cubic centi- meter of sterile litmus solution should be added. After incubation the colonies of the acid-forming organisms will be clearly picked out by the redden- ing of the adjacent agar. Only those colonies which are sharply colored should be considered as significant, since certain bacteria of the hay-bacillus group pro- duce weak acid and faint coloring of the litmus. When polluted waters are examined in this manner the number of organisms developing on the lactose- agar plate will be very high, almost equalling in some cases the total count obtained on gelatin. Chick (Chick, 1901), using a lactose-agar medium with the addition of one-thousandth part of phenol, found, of colon bacilli alone, 6100 per c.c. in the Manchester ship canal; 55-190 in the polluted River Severn, and 66 ELEMENTS OF WATER BACTERIOLOGY numbers up to 65,000 per gram in roadside mud. In an examination of water from the Charles River above Boston, 37° counts ranging from 9800 to 16,900 have been found. The average result of 56 examinations of Boston sewage from July to December, 1903, showed 5,430,000 bacteria per c.c., at 20° and 3,760,000 per c.c. at 37°, of which 1,670,000 were acid formers. The average of 25 samples examined in July and August, 1904, showed 1,690,000 bacteria per c.c. at 20° and 1,400,000 at 37°; 429,000 per c.c. {were acid formers (Winslow, 1905). In unpolluted waters not only the absolute number of organisms developing at the body temperature is less, but its ratio to the gelatin count is very different. Rideal (Rideal, 1902) states that the proportion between the two counts in the case of a London water in a year’s examination was on the average one to twelve. Mathews (Mathews, 1893) in 1893 gave the following figures, the contrast between the ponds and streams, which were presumably exposed to pollution, on the one hand, and the wells, springs, and taps, on the other, being marked. Average Number of Colonies per c.c. Source of Water. Gelatin, 20°. Wurtz Agar, 37.5°. WV RUS SOTIN RS. dic si4ceiela ss) blew ae kee 1664 28 PRESEN VIIS !s {5 beg selon tareia sie td ou ye elas 153 43 POMC Rindge telsivhece vad cue Sealed as are 296 95 SEI ls a hare a-ie Aoecs batendhc eget ea 242 24 UPR AIIS yas space ape aeqek a aieaias Pate 273 IOI DETERMINATION OF ORGANISMS 67 According to the English Committee appointed to consider the Standardization of Methods for the Bac- terioscopic Examination of Water (1904), the ratio of the 20° count to the 37° count in good waters is generally considerably higher than 10 to 1. “ With a polluted water this ratio is approached, and frequently becomes 10 to 2, 10 to 3 or even less.” In 1903 Nibecker and one of ourselves (Winslow and Nibecker, 1903) made an examination of 259 samples of water from presumably unpolluted sources in Eastern Massachusetts, including public supplies, brooks, springs, ponds, driven wells, and pools in the fields and woods, with a view to testing the value of the body-temperature examination. In many cases the samples showed high gelatin counts, since some of the waters were exposed to surface wash from vacant land, but the average number of organisms developing’ on lactose agar at 37 degrees was less than 8 per c.c., as will be seen by reference to the table on the following page. The highest individual counts obtained were 95 in a meadow pool, 83 in a brook, and 74 in a barnyard well, the latter probably actually polluted. Only two samples in the whole series, one from the well above mentioned, gave any red colonies on the agar plates. For a series of shallow surface wells recently examined by one of us (S. C. P.) a similar relation is indicated in the table on page 69; 124 samples which showed no colon bacilli and were apparently unpolluted, gave an average of 190 bacteria per c.c. at 20° and 8 at 37° with less than one red colony per c.c.; 23 samples which did contain colon bacilli averaged 570 bacteria 68 ELEMENTS OF WATER BACTERIOLOGY RELATION OF 20° AND 37° COUNTS IN SAMPLES OF WATER FROM APPARENTLY UNPOLLUTED SOURCES (WINSLOW AND NIBECKER, 1903) #2 peeaeng F Sp. | sactose- Dextrose-Broth - | Oo Sear Tubes. 6 |O% | Plates, a= Oe ee) Jee § B te be a| 9 | wm 3 n Source of Samples. oe Bare eS we'd 215 eo Pee; v1 go|\eolso| P/F. (al lee S\ 33/38 /s| & [Ss lao Ce Elec essl clesleolse 4| 80 | 8c lee| & |2¢| Sa ea pe | ee"! /e° | Se lee < $21.05 IOO0O CC Reaction 1 per cent normal. The Hesse medium has been used with great success by Jackson and Melia (1909). Its general composition is as follows: EN aU ee he asi oS oats 5 gm Mute s pepton . 2... is ee Io gm. Liebig’s meat extract.......... 5 gm. 11 Is OR Se RR 8.5 gm Dastilled water... 22 ........:. 1000 C.C Reaction 1 per cent normal. Jackson (1909) recommends that the agar used should be dried for half an hour at 105°, and under 78d ELEMENTS OF WATER BACTERIOLOGY . these circumstances 4.5 grams may be used in the formula instead of the 5 grams recommended by Hesse. The medium must be stored in an ice chest with saturated atmosphere. Plates must be made in sufficient dilu- tion to give a few colonies on the plate; and where this is done the typhoid colonies are sharply dis- tinguished from those of B. coli by the fact that they grow to a considérable size, often several centimeters in diameter and show a broad translucent or scarcely turbid zone between the white opaque centre or nucleus and the perfectly circular narrow white seam or edge. Stokes and Hachtel (1912) have suggested a modifica- tion of the Hesse medium which consists in the increase of the agar to 5.5 per cent and in the addition of 10 gm. of lactose and 50 gm. of glycerin to the formula cited above. The agar is dried out at 105 degrees for half an hour and dissolved in half a liter of water. The meat extract is added to the other half liter and freed from muscle sugar by inoculation with the colon bacillus and incubation for 24 hours at 37 degrees. The sugar- free broth thus prepared is filtered and to it is added the peptone, lactose and salt. The two half liter por- tions of the medium are then mixed and boiled for 30 minutes. The medium is filtered and adjusted to a neutral reaction, the glycerin is added and the medium is tinted with azolitmin solution before tubing and steril- izing. Typhoid and para-typhoid organisms develop medium-sized, pinkish colonies with concentric rings, and may thus be distinguished from colonies of B. alcaligenes, B. proteus and other motile forms. Organ- ISOLATION OF SPECIFIC PATHOGENES 79 isms of the B. subtilis group must be eliminated by microscopic examination, using the Gram stain. Preliminary Enrichment. In most cases plate isola- tion is preceded by some sort of enrichment process designed to favor the typhoid bacilli at the expense of the members of the B. coli group. The original use of carbol broth has been already discussed. In Europe caffein media have been used for this purpose and in the United States bile media have been strongly recommended. The important fact that caffein has an inhibitory action on colon bacilli, announced by Roth (Roth, 1903) has given rise to much investigation, and offers one of the most promising methods for the isolation of the typhoid bacillus from water. Hoffman and Ficker (Hoffman and Ficker, 1904) developed methods for the isolation of B. typhi from feces and from infected water by its use in connection with nutrose and crystal violet. For the isolation from infected water solutions were prepared as follows: 1. Ten grams of nutrose dissolved in 80 c.c. of steril- ized distilled water. 2. Five grams caffein, in 20 c.c. sterilized distilled water. 3- One-tenth gram of crystal violet in 100 c.c. water. Solutions 1 and 2 were mixed by shaking together in a flask, and the mixture poured into a flask containing goo cubic centimeters of the water to be tested; 10 c.c. of solution 3 were gradually added, and the whole thoroughly mixed by shaking and then incubated at 80 ELEMENTS OF WATER BACTERIOLOGY . 37 degrees for not over 12-13 hours. At the end of the incubation period loopfuls of the solution were smeared over Drigalski-Conradi plates. By this method the B. typhi was isolated from mixtures in river water containing one typhoid bacillus to 51,867 water bacteria and colon bacilli. A number of investigations have shown that the action of the caffein is not as markedly selective as at first claimed. Kloumann (Kloumann, 1904) obtained no better results by this method than by the Drigalski- Conradi medium alone, and Willson (Willson, 1905) found that certain strains of B. typhi were inhibited, while strains of B. coli developed feebly in the presence of 0.5 per cent of caffein. In this country marked success in the isolation of the typhoid bacillus has been attained by the use of lactose bile as an enrichment medium. Jackson (Jack- son and Melia, 1909), the principal exponent of this procedure, recommends that sterilized undiluted fresh ox gall (or an 11 per cent solution of dry fresh ox gall) containing 1 per cent of peptone and 1 per cent of lactose be made up in 4o c.c. amounts in fermen- tation tubes to which varying amounts of water, up to 10 c.c. may be added. After incubation for 48 or 72 hours, he plates on Hesse agar and he finds that in the bile medium B. typhi tends to over- grow B. coli while most other organisms are entirely suppressed. ae Dr. Krumwiede has found brilliant green broth useful as an enrichment medium for the isolation of ISOLATION OF SPECIFIC PATHOGENES 81 typhoid bacilli from stools. In a personal letter he says: “If stools are inoculated into brilliant green broth (extract broth neutral to phenolphthalein with 1 per cent of glucose), there is a suppression or inhibition of many of the lactose fermenters. The dye, even in strong solutions, has little effect on the growth of pyocyaneus, proteus, paratyphoid-like (intermediates), and aérogenes (mucosus types, non dulcit fermenters) bacilli. Typhoid bacilli will multiply in dilutions of the dye that inhibit the colon types. As the feces itself reduces the activity of the dye, adjustment is necessary and can be obtained by inoculation of o.1 c.c. of the fecal suspension into broth containing I-300,000, I-400,000 and 1-500,000 of the dye. These tubes, after 15 to 18 hours’ incubation, are plated on Endo.” Concentration by Agglutination or Precipitation. A physical concentration of the typhoid bacilli precedes enrichment or isolation in the procedure recommended by many authors. Klein, as noted above, accomplished this by passing the water through a Berkefeld filter. Other workers have made use of agglutination or chem- ical precipitation for the same purpose. The phenomenon of agglutination was made the basis of a method of isolating B. typhi from water by Adami and Chopin (Adami and Chopin, 1904). Two- liter samples of the water were collected in sterilized bottles (Winchester quarts), and to each was added 20 c.c. of 1 per cent glucose broth. The sample was incubated for 18 to 24 hours at 37° C., after which 82 ELEMENTS OF WATER BACTERIOLOGY 10 c.c. portions were withdrawn and placed in long, narrow test tubes. To each of these tubes enough typhoid serum of known potency was added to make a regularly graded series, 1-50, 1-100, 1-150, and 1-200. The probable presence of the typhoid bacillus was manifest by the formation of flocculi within a quarter of an hour, and agglutination was complete in from 2 to 5 hours. The tube having the greatest dilution in which agglutination was apparent was then examined by breaking off the lower end, containing the precipitate, washing the sediment two or three times with sterile water after removing the clear supernatant liquid, and allowing the bacteria to settle again. The organ- isms remaining were plated upon various media, and examined biochemically to determine the true character of the suspected colonies. It was found that a dilution of 1 to 60 was the highest which could be used with the organisms examined, and it is therefore probable that high dilutions (greater than 1-60) cannot be success- fully used. Investigation of an organism isolated by this method was made by Klotz (1904), who’ found the culture to be not a typical B. typhi, but a form showing certain points of similarity to both B. typhi and to B. coli, and probably intermediate between them. Frost (1910) isolated a bacillus of the B. proteus group from filtered Potomac water which agglutinated with typhoid serum in high dilutions. As Klotz points out, therefore, it is evident that even when a positive result is obtained with a relatively high dilution of ISOLATION OF SPECIFIC PATHOGENES 83 typhoid serum, the action may by no means be abso- lutely specific. Schepilewski (Schepilewski, 1903) and Altschuler (Altschuler, 1903) have also used agglutination as a means of precipitating the bacteria after enrichment cultivation in broth. The former incubated the cul- ture at 37° for 24 fhours, then added a serum of high potency, allowed the mixture to stand for 2 to 3 hours, and then centrifuged. The supernatant liquid was removed, and the mass of agglutinated cells broken up by shaking with glass beads and salt solu- tion. Upon plating upon litmus lactose agar the organ- isms could be detected. In this way positive isolation was made from water containing 1 loopful of a broth culture in 50 liters of water. Altschuler’s method of enrichment was essentially like that of Schepilewski. From the surface of the culture developed at 37°, IO c.c. were removed to a tapering tube provided with a rubber tube at the bottom. Serum was added in the proportion of one part in 50, the culture agitated to release entangled non-agglutinated bacilli and the sediment run into a tube containing 1 per cent peptone and o.5 percent salt. The agglutinated mass was broken up by shaking with sand, and the culture incubated at 37° for 24 hours, then plated on Drigalski-Conradi plates. The organism was isolated from dilute suspen- sions in water (150 in 1 liter) and also from the feces of a typhoid patient from which other methods gave negative results. Precipitation Methods. A number of methods for concentrating typhoid bacilli in water by chemical 84 ELEMENTS OF WATER BACTERIOLOGY | precipitation have been tested experimentally, with some degree of promise. Vallet (Vallet, 1901) was the first to employ this principle, and made use of sodium hyposulphite and lead acetate. The mixture was then centrifuged and the precipitate dissolved in more hyposulphite. The clear solution was then plated. Schiider (Schiider, 1903) observed that the lead salt reacted harmfully upon the bacteria, and that the hyposulphite should be in excess. In his experi- ments water was allowed to stand in tall jars for 24 hours. To 2 liters of infected water, 20 c.c. of a 7.75 per cent solution of sodium hyposulphite was added, and after thorough mixing 20 c.c. of a 10 per cent solution of lead nitrate. The precipitate, after 20 to 24 hours, was treated with 14 c.c. of saturated sodium hyposulphite solution and shaken. From the clear solution 0.2 to 0.5 c.c. portions were streaked upon Drigalski-Conradi plates which were then incubated at 37° for 24 hours. Ficker (Ficker, 1904) modi- fied the process still more by using ferric sulphate, and dissolved the precipitate with neutral potassium tartrate. The final solution was then plated on Drigalski-Conradi medium. Ficker claimed that this method gives excellent results, 97-98 per cent of the typhoid bacteria being carried down with the precipitate. Miiller (Miiller, 1905), in comparing different pre- cipitation methods, adopted ferric oxychloride as the most suitable precipitant, because of its quicker and less destructive action. Willson (Willson, 1905) sug- gested the use of alum as a precipitant. He added 0.5 gr. alum per liter of water examined. The mixture fet Seo asl a la a Od a ISOLATION OF SPECIFIC PATHOGENES 85 was then centrifuged, and the precipitate suspended in a small amount of water and plated on Drigalski-Conradi medium. Nieter (Nieter, 1906) made 20 parallel experi- ments, using very pure water infected with typhoid bacilli in varying numbers. By precipitating with ferric sulphate and sodium hydrate, centrifuging, and then filtering through a sterile filter he obtained small numbers of bacteria. Using iron oxychloride as the precipitant, he confirmed the results of Miiller. By plating on malachite green agar he was often able to get positive results when the Drigalski-Conradi medium failed. By use of a combination of enrichment and chemical precipitation, Ditthorn and Gildemeister (Ditthorn and Gildemeister, 1906) isolated the typhoid bacillus from enormous artificial dilutions in water. In the typhoid fever epidemic in Posen, in 1906, it was found that the bile of those dying from the disease contained nearly pure cultures of typhoid bacilli. This led the authors mentioned to use bile and bile agar as enrich- ment media. After precipitating by Miiller’s method, the whole of the precipitate was added to too c.c. sterile ox bile and grown at 37° for 24 hours, after which time 1 c.c. portions were plated. With extreme dilutions it was found desirable to incubate for 48 to 72 hours. The results were unsatisfactory in the presence of large numbers of water bacteria. It is also pointed out that the iron oxychloride is bactericidal in 48 hours. Separation on the Basis of Motility. Drigalski (Dri- galski, 1906) has suggested the separation of B. typhi 86 ELEMENTS OF WATER BACTERIOLOGY from other bacteria in water through its greater motility. He succeeded in isolating typhoid bacilli from two springs by the following method: 5 to ro liters of water were allowed to stand one to two days in tall milk cans at room temperature. Samples were taken from the surface and plated on litmus-lactose agar (Drigalski- Conradi medium), the amount of water to be used varying with the contamination. Starkey (1906) has suggested the use of an apparatus consisting of a piece of glass tubing bent so as to give four successive connected loops. This is filled with phenol broth, inoculated at one end, and incubated anzrobically. The more actively motile bacilli find their way to the fourth loop from which they may be isolated by plating. Review of Suggested Procedures. The methods of examining water for B. typhi may be conveniently summarized as follows: i ( a. By filtration b. By agglutination Schiider’s process 1. Physical c. By chemical } Fischer’s process concentration (precipitation | Willson’s process Miiller’s process a. Parietti’s carbol broth b. Jackson’s lactose bile 2. Enrichment 4 c. Hoffmann and Ficker’s caffein Examination Process of water for d. Brilliant green broth typhoid ( a. Elsner’s gelatin medium bacilli b. Endo’s medium c. Loeffler’s malachite green me- dium . Brilliant green agar 2, Drigalski-Conradi agar 3. Isolation d e f. Hiss’s medium g a . Hesse’s medium . Morphological and _ cultural characters b. Agglutination 4. Identifica- tion ISOLATION OF SPECIFIC PATHOGENES 87 Of the comparative advantages of these methods it is still too early to speak with finality. Up to the present time the use of caffein and lactose bile has apparently been followed by the best results, and it seems likely that of the precipitation methods that employing the oxychloride of iron is the best. The suc- cessful use of malachite green and brilliant green in the isolation of typhoid and paratyphoid bacilli from feces suggests that the more general adoption of these media in water examinations would possibly prove fruitful. Identification of the Typhoid Bacillus. At the end of the process the identification of the pure cultures isolated is again- subject to considerable uncertainty. The typhoid bacillus belongs to a large group which contains numerous varieties differing from each other by minute degrees. The inability to reproduce the disease by inoculation in available test animals owing to their natural immunity is a serious drawback; and the specific biochemical characters of the organism are, as it happens, mostly negative ones, as shown by comparison with B. coli, to which it is supposed to be allied. COMPARISON OF THE CHARACTERS OF B. COLI AND B. TYPHI (HorRROCKS, Igor) B. COLI B. TYPHI (1) Surface Colonies, Gelatin (z) Much thinner than those Plates.—Thicker, and grow more _ of B. coli, and grow more slowly. rapidly than those of B. typhi. After forty-eight hours’ incuba- After forty-eight hours’ incubation tion at 22° C. they are hardly at 22° C. they are usually large _ visible to the naked eye. and characteristic. 88 ELEMENTS OF WATER BACTERIOLOGY ~ (2) Gelatin-stab.—Quick growth on the surface and along the line of inoculation. (3) Gelatin-slope.—Thick, broad grayish-white growth with a cre- nated margin. (4) Witte’s Peptone and Salt Solution.—Indol produced. (5) Milk.—Coagulated. (6) Litmus-whey, one week at 37° C. Acid produced usually requiring from 20 to 40 per cent of N/r10 alkali to neutralize it. (7) Neutral-red Glucose-agar.— Marked green fluorescence. (8) Glucose-gelatin and Lac- tose-gelatin Shake Cultures, and Glucose-agar-stab.—Marked gas formation. (9) Gelatin, 25 ver cent, incu- bated at 37° C.— Thick film appears on the surface. (10) Potato.—As a rule, a thick yellowish-brown growth. (11) Proskauer and Capaldi’s Media. No.I, after twenty hours’ growth, medium acid. No. II, Growth, medium neutral or faintly alkaline. (12) Nitrate-broth.—Nitrate re- duced to nitrite. (13) Microscopical ances.—A small bacillus often like a coccus, not motile as a rule. (14) Flagella—Usually 1 to 3, short and brittle; sometimes 8 to 12, long and wavy. (15) Agglutination.—As a rule, no agglutination with a dilute anti- typhoid serum. Appear- _ (2) Slow growth on the surface like the colonies; along the line of inoculation, the growth is much thinner, and often ends below in a few white points consisting of dis- crete colonies. (3) Thin narrow grayish-white growth, crenated margin not marked as in B. coli. (4) No formation of indol. (5) Unchanged after a month. (6) Very small amount of acid produced, requiring not more than 6 per cent of N/1o alkali to neu- tralize it. (7) No change. (8) No gas formation. (9) No film appears on the sur- face, but a general growth takes place throughout the tube. (10) Thin transparent growth hardly visible to the naked eye. (11) No. I, no growth or change in the reaction of the medium. No. II, Growth, medium acid. (12) Reduction of nitrate not so marked. (13) Usually longer than B. coli; highly motile, with a quick serpent-like movement. (14) Usually 8 to r2, long and wavy. (15) Marked agglutination with dilute anti-typhoid serum. In addition to the biochemical characters noted above the typhoid bacillus is characterized by its failure to produce gas and by its feeble acid production in lactose a ISOLATION OF SPECIFIC PATHOGENES 89 media and by its characteristic colonies on Drigalski- Conradi agar, Endo medium, Hiss medium, and Hesse ' agar. In studying its immunity reactions agglutina- tion should in all important cases be supplemented by the Pfeiffer reaction and the absorption test which will be found described in standard text-books on bacteriology. Of the many observers who have reported the isola- tion of the typhoid bacillus from water, all but the most recent are quite discredited, on account of the insuf- ficiency of the confirmatory tests, and even the latest results should be received with caution. Since the introduction of the Widal (Widal, 1896) reaction, founded on the fact that typhoid bacilli examined under the microscope in the diluted blood-serum of a typhoid patient lose their motility and “ agglutinate ” or clump together, an important aid has been fur- nished in the diagnosis. Yet serum tests are notably erratic, and insufficient to identify an organism with- out an exhaustive study of biochemical reactions. Many organisms are agglutinated by typhoid serum in a more or less dilute solution, and agglutinations are not significant unless obtained in dilutions as great as I-500 or 1-1000. The discovery of the Bacillus dysen- teriz of Shiga,! which closely resembles the typhoid bacillus, has made the identification of the latter more dubious than ever. Hiss (1904) has shown that the fermentation and agglutination reactions of the two organisms are in many respects alike, and Park and his *For an account of biology of B. dysenterie the student is referred to an article by Dombrowsky, 1903. 90 ELEMENTS OF WATER BACTERIOLOGY associates (1904) have shown that there are not less than three distinct types of dysentery bacilli Fpl 6): that group. Isolation of Typhoid Bacilli from Water. The methods we have been discussing have many of them been used chiefly for obtaining the typhoid bacillus from feces, which is much easier than its isolation from polluted water. There are, however, a number of cases in which the organism has undoubtedly been isolated from | polluted. water, as by Kiibler and Neufeld (Kiibler and Neufeld, 1899), who examined a farmhouse well at Neumark in 1899, and Fischer and Flatau (Fischer and Flatau, 1901), who discovered an organism responding to a most exhaustive series of tests for the typhoid bacillus in a well at Rellingen in 1901. In these cases the water was directly plated upon Elsner’s medium or phenolated gelatin with no preliminary process of enrichment. Willson (Willson, 1905) summarized the instances in which the typhoid bacillus had been isolated from infected drinking water, up to 1905, and included, in addition to the above-mentioned cases, the following: 1. By Losener, in 1895, from the Berlin water supply. 2. By Conradi, in 1902, from a well at Pecs in Hun- gary, by use of carbol gelatin plates. ' 3. By Jaksch and Rau, in 1904, from the water supply of Prague, and also from the river Moldau, by caffein-nutrose crystal violet agar. 4. By Strészner, in 1904, from a well near Budapest, by the same method. Several other instances in which the isolated organ- ISOLATION OF SPECIFIC PATHOGENES 91 jsms gave positive agglutination tests, as well as the usual cultural reactions, are also cited by Willson. During the last 5 years a number of successful isola- tions of the typhoid bacillus have been reported in America. An organism obtained from the water-supply of Scranton, Pa., in 1907, by simple enrichment in Parietti bouillon, was identified as the typhoid bacillus by Prof. Fox after a very careful series of tests with immune sera (Pennsylvania, 1908). The most impor- tant results have been achieved, however, by Jackson with lactose bile enrichment and subsequent plating on Hesse agar. He reports the isolation of B. typhi from 10 c.c. samples of the Grass River at Canton, N. Y., and of a pond and stream .at Hastings, N. Y., (both used as sources of water-supply) and from two 1 c.c. samples of the Hudson River near Hastings at the time of the typhoid epidemic there (Jackson and Melia, 1909). Stokes and Hachtel (1910) by the same method found organisms corresponding to typhoid in their general cultural reactions in four samples of surface-waters (two of them from an impounding reservoir of the Baltimore supply), in the sediment of a school well supposed to have caused typhoid fever, in a sewage- polluted stream and in two samples of market oysters. These organisms agglutinated with the blood of typhoid patients in 1/50 and 1/100 dilutions, but with an immune serum producing agglutination with a standard laboratory typhoid culture in dilution of 1/25,000 these water organisms would only agglutinate in dilutions of 1/250 or 1/500. Their identity must therefore be regarded as somewhat doubtful. The 92 ELEMENTS OF WATER BACTERIOLOGY same authors (Stokes and Hachtel, 1912) have more recently reported the isolation of the typhoid bacillus from the water in the neighborhood of a_ polluted oyster bed. . The search for the typhoid bacillus is usually sug- gested when an outbreak of the disease has cast strong suspicion upon some definite source of water-supply. _ By the time an epidemic manifests itself, however, the period of the original infection is long past, and the chances.are good that any of the specific bacilli once present will have disappeared. While elaborate exper- iments have shown that B. typhi may persist in sterilized water for upwards of 2 months and in unster- ilized water from 3 days to several weeks, the number of the organisms present is always very rapidly reduced. Even in highly polluted water their number is propor- tionately small; as is well shown by the experiments — of Laws and Andrewes (Laws and Andrewes, 1894) who entirely failed to isolate the typhoid bacillus from the sewage of London and found only two colonies of the organism on a long series of plates made from the sewage of a hospital containing forty typhoid patients. So Wathelet (Wathelet,1895) found that of 600 colonies isolated from typhoid stools ‘and having the appearance characteristic of B. coli and B. typhi only 10 belonged to the latter species. Epidemiological evidence confirms these results and indicates that the number of typhoid bacilli even in polluted water probably is never very great, while the fate of Lowell and Lawrence in 1890-91 and the more recent epidemics at Butler, Pa., and Ithaca, N. Y., ISOLATION OF SPECIFIC PATHOGENES 93 demonstrate that even a small number of virulent organisms can bring about an almost wholesale infection. Indeed, if the virulent organism were as abundant as some results would indicate (Remlinger and Schneider, 1897), the human ‘race would long since have been exterminated. A negative result in testing for typhoid bacilli has no significance and there is danger that it may be misinterpreted if the fact that it has been made comes to public knowledge. In spite of this danger, however, and in spite of the laborious and time-con- suming nature of the process, the increasingly large number of positive isolations in recent years indicate that it is well worth trying in cases of special importance. The search for the typhoid bacillus should of course never supersede the examination for colon bacilli, since the latter are so much more numerous in water and so much more easily identified. Because of these facts, colon bacilli will continue to be our best index of pollution, while the positive isolations of the typhoid bacillus will supply additional proof of the deadly character of a water containing it. Other Bacteria of the Typhoid Group Related to Intestinal Disease. The typhoid bacillus and the colon bacillus (which will be fully discussed in succeeding chap- ters) stand at the opposite ends of a series of many dif- ferent varieties of organisms which are intermediate in their properties between B. typhi and B. coli, all being non-spore-forming, non-liquefying rods, which produce a more or less characteristic growth on solid media. Durham (1898) divided these forms into three main divisions, grouped, respectively, about B. typhi, B. 94 ELEMENTS OF WATER BACTERIOLOGY enteritidis and B. coli. Organisms of the first division ferment neither dextrose, lactose nor saccharose; those of the second ferment dextrose but not lactose; and those of the B. coli division form gas in both these sugars. The relationship of the commonest species is indicated in tabular form below: BACTERIA OF THE COLON-TYPHOID GROUP Dextrose. Lactose. _ Species. Gas For- | Acid Pro- |] Gas For- | Acid Pro- mation. duction. mation. duction. iB, alcaligenesio3. 2s ccc None None None None Betypt. a exces eee ey None Slight None None B. dysemtenie.. sa. es. ees. None Distinct | None None Benterttidis 6 44.05.c0%e5 er Active | Strong None Slight Paratyphoid bacilli........ Active | Strong None Slight Hog cholera bacillus....... Active | Strong None Slight BNL aie Wim cieee Sa bat Active | Strong Active | Strong In the typhoid division, B. alcaligenes and B. dysen- teri are the best-known forms, besides B. typhi itself. B. alcaligenes stands at the lower end of the whole series in fermentative power. B. typhi forms a slight initial acidity in milk and a slight acidity in dextrose broth, while the reaction of B. alcaligenes in sugar media is always alkaline. B. dysenteria, on the other hand, differs from B. typhi in the direction of the B. enteritidis group, producing a well-marked acid reac- tion, but no gas in dextrose media. B. typhi and B. dysenteriz are, of course, also distinguished by their specific serum reactions. ISOLATION OF SPECIFIC PATHOGENES 95 The second great division of the colon-typhoid bacteria is the hog cholera group, or the Gartner group, as. Durham (1808) called it. As defined by him, it differed from the typhoid group by gas formation in dextrose, and from the colon group by the production of a final alkaline reaction in milk. It includes the Gartner bacillus (B. enteritidis), the hog cholera bacillus (B. cholere suis), and the paratyphoid bacilli. Some of these forms, the paratyphoid bacilli, for example, and B. enteritidis (isolated in cases of meat poisoning), produce intestinal disease in man. There is no doubt that water is sometimes the means of distributing the germs of dysentery and diarrhoea, as shown by the decrease of these diseases in Burlington, Vt., (Sedgwick, 1902), and other com- munities where pure water-supplies have been sub- stituted for polluted ones. Thresh (Thresh, 1903) described an epidemic of over 1000 cases of diarrhoea with 14 deaths, which occurred in England at Chelmsford and Widford, and was undoubtedly spread by the public water-supply. A somewhat similar epidemic of dysentery occurred in Warren and Kittanning, in Pennsylvania, in 1906, which was unquestionably due ° to contamination of the water, in this case a river- supply. It is possible that the examination of water for the B. dysenterie may in the future help to throw important light on the sanitary condition of a water. ; Starkey (1909 and 1911) believes that all organisms giving the general reactions of the Gartner and para- typhoid groups are significant and warrant the con- 96 ELEMENTS OF WATER BACTERIOLOGY demnation of a water-supply. The difficulty, however, is that while non-acid-forming bacteria of this general type are sometimes found in feces, they are also found in other habitats, and they are less abundant proportionately, in polluted than in stored and safer waters. If true dysentery and paratyphoid bacilli can be isolated and identified by serum reactions it is, of course, highly important. Houston'(1911), however, has recently tested the method suggested by Starkey (1906) for isolating these forms and found that it gave negative results even with a water artificially infected with about 14 typhoid bacilli and 21 Gartner bacilli per c.c. In his own studies Houston reports that in the examination of 13,442 microbes from polluted river water he found only one member of the Gartner group; and in another study of 20,771 colonies he found only 2 typhoid-like forms. Isolation of the Cholera spirillum. The isolation of the cholera spirillum from water can probably be accom- plished with somewhat less difficulty than is encoun- tered in the case of B. typhi. Schottelius (Schottelius, 1885) was the first to point out the necessity for grow- ing this organism in an alkaline medium, and Loeffler (Loeffler, 1893) found that its isolation from water could be successfully accomplished by adding to c.c. of alkaline pepton broth to 200 c.c. of the infected water and incubating for 24 hours at 37 degrees, when the organism could be found at the surface of the medium. Somewhat earlier than this Dunham (Dunham, 1887) had made a special study of the chemical reactions of ISOLATION OF SPECIFIC PATHOGENES 97 the cholera spirillum and found that the organism would grow abundantly in a solution containing 1 per cent peptone and o.5 per cent salt (Dunham’s solution), producing the “ cholera-red or nitroso-indol reaction.” This medium was brought into practical use by Dunbar (Dunbar, 1892), who succeeded in isolating the organisms from the water of the Elbe in 1892, during the cholera _ epidemic at Hamburg. Koch (Koch, 1893) prescribed the following method for the isolation of the organism from water: To 100 c.c. of the water to be examined is added 1 per cent pepton and 1 per cent salt. The mixture is then incubated at 37 degrees. After intervals of 10, 15, and 20 hours the solution is examined microscopically for comma-shaped organisms, and agar plate cultures are made which are likewise incubated at 37 degrees. If any colonies showing the characteristic appearance of the cholera spirillum are found, these are examined microscopically, and if comma-shaped organisms are present, inoculations are made into fresh tubes to be further tested by means of the indol reaction and by inoculation into animals. Stokes and Hachtel (1912) have suggested the use of a modified Hesse agar containing starch for the isolation of the cholera spirilla, which produce acid on such a medium, while the colon-typhoid organisms do not. The glycerin and lactose are omitted from the medium described on p. 78 and to gms. of soluble starch are added. The intestinal spirilla as a class form round, spreading, pinkish colonies on the starch medium, while colonies of other intestinal bacilli remain 98 ELEMENTS OF WATER BACTERIOLOGY _ blue. The medium is best used after the Koch enrich- ment method described above. Other pathogenic organisms have been isolated from waters, according to the accounts of numerous investi- gators, but from the sanitary point of view the typhoid and cholera bacilli are of most importance, since these are manifestly the germs of disease most likely to be disseminated through this medium. For the detection of B. anthracis and other spore-forming pathogenic bacteria which may at times gain access to water from stockyards, slaughter-houses, etc., the method suggested by Frankland (Frankland, 1894) may be adopted. The water to be.examined is heated to go degrees for 2 minutes and then plated, the characteristic colonies of the anthrax organism being much more easily dis- cerned after the destruction of tne numerous non- — sporing water bacteria. ee Es eae a ro Ng ee ne ees - : re lil Se CHAPTER VI THE COLON GROUP OF BACILLI AND METHODS FOR THEIR ISOLATION The Colon Group of Bacilli. The Bacillus coli was first isolated by Escherich (Escherich, 1884) from the feces of a cholera patient. It was subsequently found to be a normal inhabitant of the intestinal tract of man and many other animals, and to occur regularly in their excreta, and on this account it became of the _ highest interest and importance to sanitarians, since its presence in water-supplies was regarded as direct evidence of sewage pollution. Specific disease germs dre difficult to isolate even when they are present; and water may of course be grossly polluted with sewage without any specific disease germs being there at all. All sewage-polluted water, however, is potentially dangerous, since where fecal matter exists, disease germs are at any time likely to appear. A test for fecal material as distinguished from infected material is, therefore, essential; and for such a test the colon group of bacilli are specially well suited. They are not dangerous in themselves, but they are significant as indices of the probable presence of disease germs. The so-called Bacillus coli may be described as a short, 99 100 ELEMENTS OF WATER BACTERIOLOGY ~ usually motile rod, with diameter generally less than one micron and exhibiting no spore formation. It often ap- pears in pairs of rods so short as to suggest a diplococcus. It decolorizes by the Gram stain. It forms thin, irregu- lar translucent films upon the surface of gelatin, called “‘ prape-leaf colonies” by the Germans, produces no liquefaction, and gives a wire-nail-like growth in stick cultures. It forms a white translucent layer of characteristic appearance upon agar, produces a more or less abundant, moist, yellowish growth on potato, and turbidity and some sediment in broth; it ferments dextrose and lactose with the formation of gas of which ies 1s esc ge the ratio is approximately, co. 7 38 ordinarily deter- 2 mined; a strong acid reaction and gas are produced in many other sugar-containing media. The organism generally gives a characteristic reaction in esculin ' media and typically reduces neutral red, changing its color to canary yellow with a greenish fluorescence. It grows in the Capaldi-Proskauer media, forming acid in the albumin-free medium, No. 1, and giving a neutral or alkaline reaction in the peptone-mannite medium No. 2. It coagulates casein in litmus milk, and reduces the litmus with subsequent slow return of the color (red), and generally forms indol in peptone solution. Many cultures of this organism are fatal to guinea pigs when the latter are inoculated sub- cutaneously with one-half c.c. of a 24-hour bouillon culture, and most cultures produce death when this amount is inoculated intraperitoneally. Although not a spore-forming bacillus, and in general not possessing ‘i ees ae. ae THE COLON GROUP OF BACILLI 101 great resistance against antiseptic substances, B. coli is less susceptible to phenol than are many other forms, especially certain water-bacteria. We have spoken as if Bacillus coli were a single defi- nite organism. Asa matter of fact it is a name applied to a considerable group of distinct forms which may be split up almost as far as one wishes by the applica- tion of various biochemical tests. The “ colon bacillus,” as we have pointed out, usually does not liquefy gelatin and reduces neutral red and coagulates milk, and produces indol; but there are closely allied forms which differ from the type in one or more of these respects. The colon group, as Smith (1893) long ago pointed out, may first be divided into two distinct subtypes according to the action of the organisms upon saccharose. One subtype forms gas and acid in saccharose media and the other does not. Winslow and Walker (1907) have found that those strains which ferment saccharose attack raffinose also, and point out that these two sugars which behave alike are those which lack the aldehyde grouping characteristic of dextrose and lactose. The application of tests in other carbohydrate media, such as dulcite, adonite, inulin, etc., make it possible to recognize perhaps a hundred distinct types each characterized by a particular combination of reactions. The results obtained by the “colon test” will of course depend largely upon the definition of what a colon bacillus is; and there is marked disagreement upon this point among different observers. Konrich (1910) tabulates the tests used by 34 different workers, All of them defined the colon bacillus as a Gram- 102 ELEMENTS OF WATER BACTERIOLOGY — negative non-spore-forming rod, but there was unanimity in no other respect; 26 of the 34 included the formation of acid and gas in dextrose media, 22 the coagulation of milk, 21 the formation of indol, 18 the formation of acid and gas in lactose media, and 18 the failure to liquefy gelatin. No other test was used by more than 13 out of the 34 observers. Konrich (1910) himself found that of over 600 colon-like organisms from faces, all produced gas in dextrose broth, 79 per cent formed acid and 77 per cent gas in lactose broth, 65 per cent coagulated milk, 59 per cent fermented dextrose at 46°, 54 per cent reduced neutral red, 38 per cent formed indol. : Ferreira, Horta and Paredes (1908*) studied 117 strains of lactose-fermenting bacilli from human feces. All proved to be motile and Gram negative, all coag- ulated milk and produced fluorescence in neutral red media, none liquefied gelatin’ in 15 days, all but one formed indol. Dextrose, lactose, maltose, galactose, and mannit were fermented by almost all strains, while gas was formed in saccharose by 38 per cent, in dulcite by 69 per cent and in inulin by 12 per cent of the strains studied. The “ Proskauer reaction ” (apparently the Voges-Proskauer reaction, though it is not quite clearly stated) was positive only 8 times out of 117 strains in dextrose, only 7 times (out of 48 strains tested) in galactose, and not at all in dulcite or inulin; lactose and maltose, on the other hand, showed it in almost every case. Copeland and Hoover (1911) report that out of 3000 colon-like organisms fermenting lactose bile 65 per cent gave positive tests for B. coli ie THE COLON GROUP OF BACILLI 103 in milk, nitrate solution, pepton solution and gelatin; 28 per cent failed to produce indol and 5 per cent did not reduce nitrates. Numerous other results indicat- ing similar variations are cited in Chapters VII and VIil. Where shall the line be drawn? The English bac- teriologists usually require in addition to the morpholog- ical characters mentioned above, motility, non-liquefac- tion of gelatin, fermentation of dextrose and lactose media, coagulation of milk; production of indol, and reduction of neutral red. The usual American pro- cedure has included reactions in dextrose broth, milk, peptone solution (for indol), gelatin (absence of liquefac- tion), and reduction of nitrates. Of late years, how- ever, there has been a growing feeling that such arbitrary definitions went either too far or not far enough. The whole group of lactose-fermenting bacilli is characteristically of intestinal origin. That we be- lieve to have been clearly established by results to be cited later in this chapter and in the succeeding one. A differentiation between various sub-types of this group can only be properly justified by the fact that some of them are less resistant in water than others, and hence are indicative of fresh and recent pollution. There is some evidence that such is the case which will be discussed in Chapter VIII, but whatever may be concluded from the somewhat conflicting opinions on this point it appears certain that, in temperate climates at least, the whole class of lactose fermenters should be absent from safe water supplies. As the Committee on Standard Methods of Water Analysis 104 ELEMENTS OF WATER BACTERIOLOGY (1912) wisely concludes, ‘ The entire group is typical of the presence of fecal matter when water or sewage examinations are to be considered.” The Committee defines the group as a whole by the following character- istics: ‘‘ Fermentation of dextrose and lactose with gas production, short bacillus with rounded ends, non-spore-forming, facultative anaerobe, gives positive test with esculin, grows at 20° on gelatin and at 37° on agar, non-liquefying in 14 days on gelatin. Gram- staining negative.” This definition again, however, includes debatable elements. It seems to us very doubtful whether there is sufficient evidence to warrant making the esculin reaction a general criterion of the colon group; and bacteria which liquefy gelatin more or less slowly grade into otherwise identical non- liquefying forms by almost imperceptible degrees. We hesitate to add another to the long list of arbitrary definitions of the much-defined colon bacillus; but it does seem to us important to get down to rock bottom. From this standpoint we believe that the colon group may be defined as including all aerobic non-spore- forming bacilli which produce acid and gas in dextrose and lactose media. For practical purposes the test may be further reduced to positive reactions in a lac- tose fermentation medium, growth on an aerobic agar streak, and microscopic examination, since almost all forms which ferment lactose ferment dextrose as well. The relative importance of the various subdivisions of this general group will be discussed in Chapter VIII. Isolation of Colon Bacilli by Direct Plating. The Wurtz . litmus-lactose-agar plate (Wurtz, 1892), as noted in THE COLON GROUP OF BACILLI 105 Chapter IV, furnishes one ready method for the isola- tion of B. coli from water, and it was used by Sedgwick and Mathews for the purpose as early as 1892 (Mathews, 1893). The process is based upon the fact already alluded to, that B. coli readily ferments lactose with the formation of acid. If, therefore, plates are made with agar containing both lactose-and litmus, the colon colonies develop as red spots in a blue field. Since organisms other than B. coli (notably the streptococci) may also develop red colonies, it is necessary to examine them further. This is done by fishing from isolated colonies, replating and inoculating into other media for identification. The plate method of isolation is recommended by the Committee on Standard Methods of Water Analysis (1912) for sewages and polluted waters, in which colon bacilli are present in 1 c.c. or less. They recommend that Petri dishes with porous covers be used and that incubation be carried out at 40° instead of 37°. For success in the use of this method it is necessary to get a sufficient dilution so that colonies may be well isolated, and to this end it is advisable that a number of different dilutions be employed, a series of plates being prepared from each. Under any conditions the detection of the colon bacillus is seriously hampered by the development of other forms. Certain observers have therefore added phenol to the agar medium, com- bining the effect of high temperature and an antiseptic to check the growth of water-bacteria. Copeland for this purpose added to his tubes .2 c.c. of a 2 per cent solution of phenol (Copeland, 1901). Chick (Chick, 1u6 ELEMENTS OF WATER BACTERIOLOGY . 1900) found that 1.33 parts of phenol in 1000 materially decreased the number of colon bacilli which would develop, while 1 part gave very satisfactory results, the plates showing pure cultures of B. coli. The addi- tion of antiseptics in this way is always open to the objection that weaker strains may be killed and lost. In Germany the Endo medium and the Conradi- Drigalski medium have been extensively used for the direct isolation of colon bacilli with excellent results. The composition and use of these media have been discussed in Chapter V. It does not appear that they have sufficient advantages to compensate for the dif- ficulty in preparing them. The Use of Preliminary Enrichment Media in the Isolation of Colon Bacilli. The test for the colon bacillus may be made more delicate by a preliminary cultivation of the sample in a liquid medium for 24 hours at 37°, thus greatly increasing the propor- tion of these organisms present before plating. As suggested in the classic researches of Theobald Smith (Smith, 1892), this method may be made approx- imately quantitative by the inoculation of a series of tubes with measured portions of the water. If, for example, of ten tubes inoculated each with tiv of a cubic centimeter, four show B. coli, we may assume that some 4o of these organisms were present in the cubic centimeter. Irons (Irons, 1901), in a comparative study of various methods for the isola- tion of B. coli, showed that the preliminary enrichment frequently gave positive results when the results of the direct use of the agar plate were negative, and THE COLON GROUP OF BACILLI 107 concluded that “ where the amount of B. coli is small and the colony count large, the lactose plate for plating water direct is inferior to the dextrose fermentation- tube.” Gage came to a similar conclusion (Gage, 1902). The medium most commonly used in the United States: prior to 1906 for preliminary enrichment was ordinary broth to which 1.0 per cent of dextrose had been added, and the reaction brought to the neutral point. Into each of a number of fermentation-tubes of this medium a measured quantity of the water to be examined is inoculated, and the culture is incubated for 24 hours at 37.5° C. It used to be customary to incu- bate for 48 hours. Recent experience has, however, shown that a 24-hour period gives approximately the same results if the production of gas rather than any specified amount of gas is the criterion of a positive test. Longley and Baton (1907) found that of rog1 enrichment tubes giving positive tests after 48 hours only 173 showed no gas in 24 hours; of these latter only two contained B. coli. The advantage of saving a day is so great as to warrant the adoption of the shorter period. At the end of 24 hours at least, the tubes are examined for gas formation. If gas is found, a small amount of the culture should be added, after suitable dilution, to litmus lactose agar and plated. With polluted waters it will be found advantageous to plate out on the first appearance of gas (4-8 hours). _It has been shown by one of us (Prescott, 1902”) that a very rapid development of B. coli takes place in the first few hours after dextrose solutions are inoculated with intestinal material, and a nearly pure growth of 108 ELEMENTS OF WATER BACTERIOLOGY colon bacilli often results, while other bacteria multiply more slowly. With highly polluted waters gas forma- tion will probably begin within 12 hours, but with fewer colon bacilli present the duration must be increased. If the period of incubation be too long continued, trouble in the subsequent steps of the isolation may be encountered because of overgrowths by the sewage streptococci, or other forms which check the growth of the colon bacilli in the later stages of fermentation and finally kill them out. Even with pure cultures of colon bacilli Clemesha (1912) has shown that sugar- broth tubes may be almost sterile after 4 days. When it is desired to examine samples larger than 10 c.c. it becomes necessary to modify the enrichment process by adding the nutrient material to the water instead of the reverse. For this purpose dextrose broth or phenol-dextrose broth (consisting of broth with 10 per cent dextrose, 5 per cent peptone, and .25 per cent phenol) may be added to the sample of water to be enriched as suggested by Gage (Gage, 1901). Generally 10 c.c. of the broth is added to 100 c.c. of the water. The sample is thenincubated at 37° for 24 hours, and if at the end of that time growth has taken place, a cubic centimeter is inoculated into a dextrose tube. Advantages and Disadvantages of the Dextrose Broth Fermentation Tube. Experience with the dextrose broth fermentation tube as a first step in the isola- tion of colon bacilli soon led to the conclusion that a fair idea of the sanitary quality of water could be obtained from the results of this test taken by them- selves and without the further process of isolating THE COLON GROUP OF BACILLI 109 specific cultures. It appeared that a rather definite proportion of tubes showing a characteristic fermenta- tion proved on further examination to contain bacilli of the colon group; and it was therefore suggested that the dextrose broth test alone might be used as a rapid “ presumptive ” test. The underlying principle of this method is that B. coli develops rapidly in dex- trose broth with gas formation of from 25 to 7o per cent of the capacity of the closed arm of the fermenta- tion tube. Of this gas approximately one-third is carbon dioxide and two-thirds hydrogen, that is, as the gas formula is generally expressed, a i : COz 1 In testing a water by this method a series of samples, in suitable dilution, .oo1, .or, .1, 1.0, or 10 c.c., are added directly to the dextrose-broth tubes and incubated for 24 hours at 37°. On measurement of the gas, if the results above given are obtained, the reaction is considered typical. If the amount of gas is between ro and 25 percent or more than 70 per cent, or the percentage of carbon dioxide is greater than 40, the reaction is considered atypical. If no gas forms, or less than ro per cent, the test is called negative. In recent years, Irons (Irons, 1901) was perhaps the first to call attention to the value of this method, stating that “ when the dextrose tube yields approx- imately 33 per cent of COs, Bacillus coli communis is almost invariably present.” In the next year the reliability of the fermentation test as an indication of B. coli was worked out by Gage (Gage, 1902) as given in the table on p. 110: 110 ELEMENTS OF WATER BACTERIOLOGY © I c.c. 100 c.c, Number of samples tested. ..............2.0e000> 5172 1375 Number giving preliminary fermentation.......... 1036 474 Per cent of latter proved to contain coli........... 70 71 Whipple (Whipple, 1903) examined a large number of surface-water supplies by this “ presumptive test ”’ and obtained striking results, shown in the following table. The waters are arranged in six groups according to the results of sanitary inspection, group I including waters collected from almost uninhabited watersheds, and group VI waters too much polluted to be safely used for domestic purposes. PERCENTAGE OF SAMPLES OF WATERS OF VARIOUS SANITARY GRADES GIVING POSITIVE TESTS FOR B. COLI WHEN DIFFERENT AMOUNTS WERE EXAMINED (WHIPPLE, 1903) oe Group. 0.1 c.c. 1.0 c.c. 10 c.c. 100 c.c. 500 c.c. i Waa tte’. PRs 0.0 ake 20.8 50.0 50.0 ED si. cise 5.0 v fe I5.0 60.0 60.0 ALE LES GS acne ete 0.0 FIO TeN PeROre 50.0 60.0 Ve cneseipesiet atte 4.0 6.8 41.7 67.0 75.0 MASS eet alow te 5.0 13.0 75.0 100.0 100.0 Wie .y Patynceaen 5.0 20.2 75.0 80.0 100.0 In view of these results Whipple suggested the fol- lowing provisional scheme of interpretation: Presumptive Test for Bacillus Coli. Sanitary Quality. 0.01 c.c. 0.1 c.c. 1.0 C.c. 10.0 c.c, TOO c.c. ALG 09.” fect eral aeets fo) fo) ° ° -+ Reasonably safe...... ° fe) ° + + Questionable........ ° ° -- + + Probably unsafe...... ° + + + + MINSAIG. 603.5 Oteeas The Eijkman Test. Another enrichment test, which has been extensively used in Germany for checking the development of overgrowing forms and limiting the bacteria in the fermentation tube to the colon group, is the Eijkman test, which depends on the use of a high temperature (46°) (Eijkman, 1904). There is no doubt that such a procedure cuts out the water bacteria, and Christian (1905), Neumann (1906), and Thomann (1907) have reported good results from its use. Hilgermann (1909), too, urges the value of the Eijkman test and concludes that the colon-like bacilli, which fail to grow at 46°, are characteristic of compara- tively unpolluted waters. Other observers maintain, eh a PT THE COLON GROUP OF BACILLI 121 and apparently with good reason, that the conditions of this test are too severe and eliminate many intestinal bacilli of undoubted significance. Nowack (1907) found that laboratory cultures of B. coli often fail to produce gas in Eijkman’s medium at 46°, unless large numbers are introduced. With some strains an inoculation of over a million bacteria was necessary to cause gas formation. Konrich (1910) compared the Eijkman enrichment method (dextrose peptone water at 46°) and that of MacConkey (dextrose-bile-salt peptone water at 42°) with 57 water samples and obtained only about 70 per cent as many positive results with the former as with the latter method. With artificial emulsions of pure cultures and of feces even greater differences were manifest. These studies showed conclusively that incubation at 46° prevents the development of great numbers of colon bacilli and is unsuitable for an enrichment process. In comparing the two media (dextrose peptone water with and without bile salts) at the same temperature, 37°, he obtained essentially similar results. Fromme (1910) has shown that during the first 5 hours in various enrichment media colon bacilli multiply more rapidly at 46° than at 37°. After that time, however, their development is checked. At 12 hours the numbers at the two temperatures are about equal and between 12 and 24 hours the numbers increase much more rapidly at 37°. The Neutral Red Reaction. Other special media have been suggested for rapid routine water analysis, 122 ELEMENTS OF WATER BACTERIOLOGY | of which those containing “neutral red,’ one of the safranine dyes, have been somewhat fully studied. Rothberger (Rothberger, 1898) first pointed out that B. coli reduces solutions of this substance, the color chang- ing to canary-yellow accompanied by green fluorescence. Makgill (Makgill, 1901), Savage (Savage, rgor), and other English observers, as well as Braun (1906), in France, report favorable results from the use of this test; but according to American standards, Irons (Irons, 1902) and Gage and Phelps (Gage and Phelps, 1903) conclude that the group of organisms giving a positive neutral red reaction is too large a one to give very valuable sanitary information. Stokes (1904) urged the use of lactose broth with the addition of neutral red, and believed that the pro- duction in this medium of 30-50 per cent of gas with a Z gas formula and the change of neutral red to canary yellow in the closed arm of the fermentation tube was characteristic for B. coli. The Lactose Bile Test for the Colon Group. On the whole, by far the most satisfactory results in making a rapid test for the colon group have been obtained by the use of media containing bile salts, a procedure the development of which in this country we owe principally to Jackson (1906). MacConkey (1900) long ago suggested the use of media containing bile salts (sodium taurocholate) for the differentiation of B. coli and B. typhi, and bile- salts media have been used by various English observers (MacConkey, 1901; MacConkey and Hill, rgo1) for THE COLON GROUP OF BACILLI 123 the isolation of sewage bacteria. Jackson studied the action of various bile media and showed their selective inhibitory action in the striking table quoted below. His important contribution to the subject, however, was the discovery that ox bile itself could be used as a culture medium, and that it was easier to prepare, cheaper and more effective than combina- tions of meat infusion with the purified bile salts. SELECTIVE ACTION OF BILE SALTS : (JACKSON, 1906) Bacteria per c.c. Uncon- ° Suspension | Suspension * Contami- t ted of of o Weil. nated Pond.| Races, Feces. Gelatin; 20°.......... 920 2700 350,000 900,000 Sy oe 25 170 450,000 900,000 Bile agar,* 37°.......: 14 43 300,000 900,000 Lactose bile agar,* 37° . ° 25 250,000 675,000 Lactose bile agar,* 37° . ° 17 250,000 600,000 Bile agar, 37°......... ° 16 60,000 Q00,000 * Bile diluted, 1 : 1. Jackson suggested the use of fresh ox bile containing I per cent of lactose as a presumptive test instead of dextrose broth. In particular he hoped that this medium would be free to a great degree from the negative results due to overgrowths in polluted waters. He reported 275 examinations of badly contaminated waters, in which 65 per cent of the samples failed to give the dextrose-presumptive test, and only 1o per cent failed to show gas in lactose bile. In amore recent communica- tion, Jackson (1907) reports that in the examination of 124 ELEMENTS OF WATER BACTERIOLOGY 5000 samples of water at the Mt. Prospect Laboratory, the bile medium has proved uniformly satisfactory: He recommends incubation for 72 hours, results being commonly obtained, however, after 48 hours; and he considers any tube showing 25 per cent gas as positive. In a series of examinations carried out at the Institute of Technology, 16 per cent of the positive tubes showed: COMPARATIVE PRESUMPTIVE TESTS WITH DEXTROSE BROTH AND LACTOSE BILE (SAWIN, 1907) Percentage of Samples Giving Positive Tests for B. Coli. Bcdres. Dextrose Broth. Lactose Bile. 0.I c.c. | 1.0 C.c. |I10.0C.c.| 0.1 C.c. | I.0C.C. |10.0C.c. r Deep wells. :...0.... ° ° ° ° ° ° 2 Shallow wells........ ° L05)! 26 Oh) ae ° 6.0 SAAC 0! 2) ane wigs Eis T§/O.1 10,0} 25.69)" hon 8 15.0 4 Lakes os iV eee Bran T5571 Sh. Och mse $62) 235 BUR TBR et ine sw 08 ao dala 1O.0°| 8 Oi) AO Oul tO Onte see I5.0 Ox SAR a xe canene eens 10.0 | 10.5 | 26.0 | 10.0 | 10.5 | 50.0 DEAK nc detent homes ° 10.0 5.0 ° ° I5.0 Bx Lake as & eer TOO! |) 156.1 3910n) <6 5.0 | 30.0 Average, Nos. 3, 4, 5, Ope Bios Ass exe cisco 8.3 | 12.0] 23.6-]) §:0 |. 4:3°) 2676 Oe River. aw cr devo 47.0 | 72.2 | 55.5 | 50.0 | 75.0 | 84.2 MO RIVED oi: ass hay steron wate 26.3 | 37 73-7 | 30.0 | 90.0 | 85.0 TEU RAVER. os pte cvs ee 36.8 | 55.1 | 68.9 | 40.0 | 73.5 | 78.1 Average, Nos. 9, 10, 11| 36.7 | 55.1 | 66.0 | 40.0 | 79.4 | 82.4 EG BSPOOK os o's the gc hioatebees 47.9. |63.2 | 72.21/60. 90.0 | 84.2 na Drainage: xs cassettes 50.0 | 73.7 | 78.9 | 84.2 | 90.0 | 90.0 Th Sewages..55).tsscene 25.0 | 25.0] 8.2 | 87. 03.7 | 81.2 Average, Nos. 12, 13,14] 40.9 | 53-9 | 53-1 | 77.2 | 91.2 |.85.1 THE COLON GROUP OF BACILLI 125 gas in 24 hours, 73 per cent after 48 hours, and the remaining 27 per cent only after 72 hours; but the Committee on Standard Methods (1912) believes that the forms which fail to develop in 48 hours are attenuated forms of little practical significance. Sawin (1907) reports comparative results with dextrose broth and bile on different classes of waters, the most striking of which are tabulated on p. 124. Like other enrichment methods which eliminate com_ peting forms it is no doubt true that the lactose bile test cuts out some weak colon bacilli. As a presumptive method, however, it is far superior to dextrose broth, giving a higher proportion of positive tests with polluted waters and a lower proportion of erroneous positive tests with waters of good quality. In an examination of 176 surface waters in eastern Massachusetts, carried out under our direction, B. coli was isolated 70 times. The dextrose-broth test was positive 120 times, an error of 70 per cent; while the bile test, alone, was positive 78 times, an error of only 11 per cent. The tabulated results of these experiments indicates fairly the merits of the bile medium for preliminary enrichment and as a presumptive test. PRELIMINARY AND COMPLETE RESULTS OF DEXTROSE BROTH AND BILE TESTS. 176 SURFACE-WATERS Preliminary Positive Final Positive Results. Results. vl (Gas Bicinatson:) (B. Coli). Dextrose broth........ 120 7O Lactose bile....... ase 78 64 126 ELEMENTS OF WATER BACTERIOLOGY © Hale and Melia (1910) have also made a valuable comparison of a number of presumptive tests as applied during a period of 2 years to 85 samples of Manhattan water (surface) and 160 samples of Brooklyn water (largely ground-water). Their principal results are tabulated below. RESULTS OF VARIOUS PRESUMPTIVE TESTS (HALE AND MELIA, 1910) 245 Samples of New York Water Percentage of Positive Resultsin Medium. 0.1 c.c. 1.0 c.c, 10.0 c.c. Dextrose broth (standard gas formula) ...| 0.4 7.0 30.7 Lactose bile. 5 ic.tcutie pales eae re oes 0.8 8.2 38.4 Lactose-peptone bile................0.. 0.8 Is78 51.2 Dextrose broth, all tubes showing 5% gas transplanted to bilesss<. ia .nuie ex 0.8 12.0 57.6 Dextrose broth, 5% gas and over, called DOSIEIVE cise se Fae Se IC 6.3 31.4 73.5 This table indicates very clearly the fallacies of the dextrose broth tube. Counting all gas formers in this medium as positive indicates much too high a value and including only the tubes showing the standard gas formula gives much too low a value. This conclusion is based on the assumption, warranted by the results of many workers, that incubation in dextrose broth followed by reinoculation into lactose bile (fourth line of the table) gives with reasonable accuracy the real number of colon bacilli present. By this standard the use of plain lactose bile with these waters is seen to give results which are also much too low; but lac- tose peptone bile approximates closely to the truth. THE COLON GROUP OF BACILLI 127 Of course it must be remembered that the advantages of lactose-bile over dextrose broth are partly due to the inhibiting effect of the bile salts and partly to the use of lactose instead of dextrose which cuts out the dextrose-positive lactose-negative group to which allusion has been made earlier in the chapter. The relative importance of these two factors, lactose and bile, is well brought out in a study by Stokes and Stoner (1909). These authors have compared a considerable series of preliminary enrichment tests followed by final isolation in dextrose broth, lactose broth and lactose bile. Of 567 colonies from positive dextrose broth tubes only 52 per cent were colon bacilli; of 3752 colonies from positive lactose broth and lactose bile tubes, 88 per cent of the lactose broth colonies and 95 per cent of the lactose bile colonies were B. coli. With sewages and heavily polluted waters in par- ticular the lactose-bile medium has proved of the greatest value. When a large proportion of sewage is present the colon bacilli are fresh from the intestine and apparently able to resist the antiseptic salts. On the other hand, the high numbers of other bacteria present make the danger of overgrowths particularly great. With waters of fair quality, such as those with which we ordinarily deal in sanitary water analysis, jactose bile is open to the same objection as phenol broth and the Eijkman test though in less degree. It inhibits not only the overgrowing forms but the weaker representatives of the B. coli group itself, and the net effect is to diminish positive results. Hale and Melia (1910) inoculated unsterilized water 128 ELEMENTS OF WATER BACTERIOLOGY (shown to contain no gas formers) with a pure culture of B. coli and stored it for different periods and under different conditions, testing at intervals by various presumptive tests. The colon bacilli lived for 8-10 days at 37°, for 38-75 days at 20°, and 77-84 days at 8° C. Comparison of presumptive tests with plate counts on litmus-lactose agar showed that a gas test in dextrose broth corresponded to an average of 4 bacteria and a positive test in lactose bile to 39 bacteria. In general the dextrose broth showed gas in one dilu- tion higher than the lactose bile; and the difference increased with the attenuation due to prolonged sojourn in water. These results of Hale and Melia make it clear that the selective action of bile salts upon the various mem- bers of the colon group may perhaps be an advantage rather than a disadvantage for practical sanitary purposes. The Committee on Standard Methods (1912) recognizes that lactose bile does inhibit certain weaker members of the colon group, but believes that these attenuated organisms indicate only remote pollution and are of little significance. They say, “In the interpretation of the sanitary quality of a water, it is best to discount the presence of attenuated B. coli and to be sure to obtain all vigorous types. The lactose bile medium accomplishes both of these objects.” The advantages of the dextrose broth enrichment of weak colon bacilli and of the elimination of gas-forming organisms other than B. coli by bile may both be obtained as pointed out above by inoculating lactose bile from THE COLON GROUP OF BACILLI - 129 the dextrose broth. Hale and Melia (1910) find that this gives results in as high a dilution as by the use of dextrose broth and with the clear-cut results of lactose bile. The Aesculin Test. A test for the colon group which has attracted much interest during the last few years is the fermentation of the glucoside aesculin. B. coli effects a hydrolytic splitting of this substance, producing sugar and a substance called aesculetin, which reacts with iron citrate to produce a dark brown salt. Harrison and van der Leck (1909) have used broth or agar made up with 1 or 2 per cent Witte’s peptone, .5 per cent sodium taurocholate, .1 per cent aesculin and .o5 per cent citrate, and find that the black colonies with a black halo produced by the colon group of organisms are highly characteristic. Of 60 samples of water which showed blackening of aesculin broth, all proved to contain B. coli. Hale and Melia (1911) have shown that two species of streptococci, the aurococcus, and the bacillus of pneumonia, also give the aesculin reaction and that in the absence of the bile salt which the aesculin medium contains a number of other forms may ferment this glucoside. On the whole we do not think it has been shown that aesculin has sufficient differential value to war- rant its inclusion in enrichment media to be used in the colon test. It appears that the anaerobic B. welchii is practically the only form outside the colon group which produces a characteristic reaction in lactose bile and not in aesculin-bile salt media. It may prove 130 ELEMENTS OF WATER BACTERIOLOGY worth while to use aesculin to exclude this form, but the more general tendency at present seems to be to eliminate the anaérobic spore formers by subsequent subcultures. The Use of Synthetic Media for the Isolation of the Colon Group. Dolt (1908), working in Prof. Gorham’s laboratory at Brown University, has attempted with success to substitute synthetic media of simple and known composition for the usual meat-infusion-peptone media used in the isolation of B. coli. He first found that colon bacilli will grow readily on a medium con- taining ‘asparagin and sodium or ammonium phosphate. He then attempted to substitute for asparagin various simple organic substances similar in their structure to the cholic acid of the bile which exerts a selective action in favor of the colon group. He finally succeeded in preparing two media which promise to be of con- siderable value in permitting the growth of B. coli while checking other forms. The first of these media is made up as follows: 500 c.c. of a 3 per cent solution of purified agar is mixed with an equal portion of a solution of 1 per cent glycerin and o.2 per cent (NH4)2HPO,. It is neutralized with sodium hydroxide and 1 per cent of lactose is added before sterilization. In the second medium 5 gm. of ammonium lactate is substituted for the glycerin and 1 gm. of NazHPO, for the ammonium phosphate. These media proved to have a considerable selective value, cutting out most of the water bacteria; but like all such selective media they cut out a good many of the colon bacilli too. The results of a single test are shown in the table below. The procedure well merits further study, however. =e THE COLON GROUP OF BACILLI 131 _COMPARATIVE RESULTS OF ORDINARY AND SYNTHETIC AGAR MEDIA (Dott, 1908) Total Colonies.| Red Colonies. B. Coli. Standard agar...... 67 38 18 Glycerin agar....... 27 9 9 Ammonium lactate agar 17 9 re) The Use of Liver Broth for the Isolation of a Maximum Proportion of Gas-forming Bacteria. The media we have been discussing, phenol broth, dextrose broth incubated at 46°, and bile, are designed to cut down the gas producers which appear in ordinary dextrose broth so that only vigorous typical members of the colon group are able to develop. For special research purposes when it is desired to get the largest possible proportion of gas formers of all kinds, there are other media which give even more positive fermentation results than dextrose broth itself. The most important of these is the liver broth of Jackson and Muer (1911) made up with beef liver, peptone, dextrose and potas- sium-acid-phosphate. The Committee on Standard Methods of Water Analysis (1912) recommends that if “a study of all gas-forming bacteria, including attenuated forms, is desirable, then liver broth should be employed in preference to the usual dextrose broth, as it gives a larger number of attenuated forms, has better rejuvenating power, and gives fewer anomalies and greater and more rapid gas production.” In order to avoid attenuation or inhibition transplants 132 ELEMENTS OF WATER BACTERIOLOGY should be made from this enrichment medium after 6-12 hours at 37°. Isolation of Pure Cultures from the Enrichment Tube. In case one does not rely upon a “ presumptive ”’ test alone but desires to study the organisms present in detail the isolation upon a solid medium, usually litmus-lactose-agar in this country, must follow the enrichment process. Since the enrichment tube was inoculated with a known amount of water all further work is purely qualitative, and it is only necessary to obtain such a number of colonies upon the lactose plate that the isolation of a pure culture shall be easy. In practice the following procedure has been found gen- erally successful. After the enrichment tubes have been incubated for 12 to 24 hours at 37°, from those which show gas, one loopful is carried over to a tube containing 10 c.c. of sterile water, and of this water one loopful is taken for the inoculation of the plate. Ordinarily this will give colonies which are sufficiently well separated, but a second plate, inoculated from the dilution water with a straight needle instead of a loop, furnishes a desirable safeguard. With practice it is possible to effect a proper seeding more rapidly by barely touching the tip of a straight needle to the broth in the fermentation tube and transferring this directly to the agar. The touch must be a very light one, how- ever, or the colonies on the plate will be too thick for proper isolation. The litmus-lactose-agar plates made in this manner should be incubated for from 12 to 24 hours at the body temperature (37°), at the end of which time, if B. coli is ae [Te i ia P | eat ye = —- i 2 7 THE COLON GROUP OF BACILLI 133 present, red colonies upon a blue field will be visible. The litmus-lactose-agar plate may become blue again after 48 hours, owing, presumably, to the formation of amines and ammonia by the action of the bacteria ‘upon the nitrogenous matter present. If the dilution is too low, the resulting colonies will be small and imperfectly developed, making it difficult to be sure of pure cultures for the subsequent tests. A great number of colonies will also prevent the change of reaction from acid back to alkaline. In the selection of those red colonies which are to be fished from the litmus-lactose-agar plate the appearance of the growths must be closely noted. A colony of irregular contour, surrounded by a very faint area of reddening, will probably belong to some member of the B. mycoides group (Winslow and Nibecker, 1903); small, compact, bright-red colonies are characteristic of the streptococci, and Gage and Phelps (Gage and Phelps, 1903) have pointed out that of these there are two types, one of a brick-red color, and of such con- sistency as to be readily picked up by the needle-point, and the other smaller and of an intense vermilion color. The colonies of the colon bacillus are usually well formed, pulvinate on the surface and fusiform when growing deeper down. If no red colonies appear on the litmus-lactose-agar plate after a positive result in dextrose broth one of four things has occurred: There may be an organism present which forms gas in dextrose but no acid in lactose; there may be present forms which individually fail to attack lactose but growing together, symbiotically, 134 ELEMENTS OF WATER BACTERIOLOGY produce gas in dextrose; B. welchii or some other form which will not grow on aerobic plates may have produced the gas; or an organism orginally present and capable of fermenting both sugars may have been overgrown and lost in the enrichment tube. If plates are made on the first appearance of gas the likelihood of the latter possibility will be reduced to a minimum. Neither of the first two contingencies has any sanitary significance; as we have seen, bacteria which ferment dextrose and not lactose are not specially characteristic of pollution. In general, therefore, the absence of red colonies on the agar plate may be considered a negative result. If red colonies are present they must be sub- cultured and examined further. The agar streak made from the litmus-lactose-agar plate shows after 24 hours certain marked character- istics. ‘The most distinct types are two, the abundant, first translucent, later whitish and cheesy growth, covering nearly the whole surface of the agar, character- istic of B. coli and its allies, and a very faint growth, either confined strictly to the streak or made up of faint isolated colonies, dotted here and there over the surface. The latter cultures are typical of the sewage streptococci, and a microscopic examination will generally settle their status at once. Of the more luxuriant growths, some of which are stringy to the needle, many will generally prove to be atypical, and if any of the weakly fermenting forms (B. mycoides) are present a dull wrinkled growth will be produced. .. The various tests which may be applied to the cul- tures after they have been isolated, the subgroups THE COLON GROUP OF BACILLI 135 into which the colon group may be divided by their use, and the significance of the results obtained will be discussed in Chapter VIII. Practical Routine Test for the Colon Group. As has been pointed out above the aggregation of lactose- fermenting bacilli which we call the colon group may be almost indefinitely subdivided by the application of a more or less elaborate series of diagnostic tests. Each observer in the past drew up a scheme of what he believed to be essential tests and called all the bacteria which failed to conform to them “ atypical.” The more of such “atypical” forms a particular worker includes the greater will be the number of positive isolations. The definition of this or any other bacterial species is more or less arbitrary; we consider as true colon bacilli those which fulfil a particular set of tests, and class as pseudo-colon organisms those which do not. If we find, having established such an arbitrary standard, that the colon bacillus, as determined by it, is found in waters known to be polluted, and. not, as a rule, in those known to be free from pollution, the sanitarian can afford to ignore the theoretical question of specific values and make confident use of the practical test. In order that results may rest on a sound basis of comparable data for various waters, it is of course essential, however, that a standard set of reactions should be agreed upon by sanitary bacteriologists. After a considerable period of uncertainty, in which each observer used the procedure which happened to appeal to him, the attainment of comparative results was made possible by the establishment of standard 136 ELEMENTS OF WATER BACTERIOLOGY - methods of procedure by bodies of authoritative posi- tion, both in England and America. In 1904 an © English Committee, appointed to consider the Stand- ardization of Methods for the Bacterioscopic Examina- tion of Water, presented a series of obligatory tests and optional tests; and in 1905 the Committee on Standard Methods of Water Analysis of the American Public Health Association drew up a set of diagnostic characters for B. coli. The latter corresponded in general with the plan developed by the Massachusetts State Board of Health (Massachusetts State Board of Health, 1899) and long in use at the Massachusetts Institute of Technology, and involved the determina- tion of morphology, motility, fermentation of dex- trose broth, coagulation of milk, production of indol and reduction of nitrates. The English standard pro- cedure corresponded quite closely to this (Committee appointed to consider the Standardization of Methods for the Bacterioscopic Examination of Water, 1904), although it differed from the American method in cer- tain respects. Since these standards were formulated 8 years ago their artificial nature has been made more and more manifest to those who have used them. A still more fundamental question, however, has pressed itself upon the practical analyst. Each lactose bile presump- tive test involves the use of a single tube of medium and the work is complete in 48 hours. The “ com- plete’ test as used in America required the use of seven different media and took 9 days to complete, since the gelatin subculture must be incubated for sill ria di io aaa THE COLON GROUP OF BACILLI 137 at least a week. Granting that the lactose bile test gave us the whole colon group and that the “ typical B. coli” giving characteristic reactions in milk, peptone, gelatin and the nitrate solution, only the more sensitive members of the group, indicative of recent pollution, was the extra information gained worth the additional trouble? Under the conditions which generally obtain, in the United States at least, it appears not. The colon test at best is an approximate one, and its results are usually only expressed in decimal fractions, positive in 10 c.c., I c.c., or .1 c.c. for example. From 7o to go per cent of the bacteria which give the lactose bile test prove to be “ typical” B. coli on any of the defini- tions ordinarily used. This makes a difference so slight as to be almost negligible. We cannot condemn a water because it contains 1o rather than 7 colon bacilli in a given proportion. It may be that under tropical conditions such as those described by Clemesha (to be discussed in a following chapter) certain forms of the colon group persist for a long time in stored waters from which disease germs have disappeared. These resistant forms must, however, be studied by a much more elaborate procedure than the 7 tests in the old American Standard method; and it seems clear that they do not occur in large numbers in temperate cli- mates. For our conditions the whole group of forms which produce gas in lactose bile should be absent from safe waters. The Committee on Standard Methods of Water Analysis in its last report (1912) apparently takes this ground, although its discussion of the problem 138 ELEMENTS OF WATER BACTERIOLOGY — is somewhat ambiguous. In order to clear up the subject, the Laboratory Section of the American Public Health Association, at its Washington meeting, in September, 1912, adopted a resolution recommending determinations of counts at 20 degrees and 37 degrees, and the lactose bile presumptive test as the standard routine procedure in water examinations. Recent Criticisms of Lactose Bile Test. During the last two years a number of workers have found that the presence of anaérobic bacilli of the B. sporogenes type, giving a misleading positive result in the lactose bile tube, may be a seriously confusing factor in inter- preting the results of examinations of certain waters. Mr. George W. Fuller has recently pointed out that at certain points on the Ohio and Mississippi Rivers the presumptive test yields results apparently far too high to be explained by ordinary pollution, and much higher than the results of a complete isolation test as shown at the same localities. Phelps and his associates in the study of the Potomac have observed that in waters showing recent pollution in large amount, the presumptive tests in both lactose bile and lactose broth were efficient and reliable. As the pollution becomes less evident, that is, as purification proceeds, however, these media give increasingly unreliable results. A study of a large number of samples in which confirmatory tests were made showed that as the purification increased, the organisms appearing as fermentation exciters in the presumptive tests were more and more frequently not of the colon type, but instead proved to be organisms THE COLON GROUP OF BACILLI 139 of anaérobic character giving large amounts of gas, even up to roo per cent, a milky appearance and a vile odor in lactose broth instead of the rather clean acid smell characteristic of B. coli. Endo plates from these tubes were either atypical or showed no growth. These organisms are probabty of the spore-forming B. sporogenes type, a group which we know from the work of Houston and others to be widely distributed. Wells found this organism in the water at Grand Forks, N. Dak., after treatment with calcium hypochlorite. They appear to grow in decom- posing organic matter, deposited sludge from sewage, etc. The occurrence of this organism was noted by A. J. McLaughlin in the report of the Commission to investigate the waters of the International Boundary (Rivers and Lakes Commission). _ The special significance of these facts is that when some waters originally highly polluted reach the final stages of purification these gas-forming anaérobes may appear and make the presumptive test less delicate and accurate. This seems to be especially the case in the examination of waters in connection with the shell- fish industry. This state of affairs is noted in the Potomac River below Washington, as will be seen by the following table kindly furnished by Mr. W. F. Wells. (See page 1394.) It is very evident from columns 2 and 5 that the greater the number of B. coli the higher is the per- centage efficiency of the presumptive test. A number of observers have also recently emphasized 1394 ELEMENTS OF WATER BACTERIOLOGY | the fact (pointed out on page 127) that lactose bile is less sensitive than lactose broth as a presumptive test, as shown by the number of positive isolations of B. coli recorded. Thus Young, of the Kansas State Water Survey, notes that out of 140 samples showing fermen- tation in the presumptive test, 116 were found to give Percent- ‘menting | Percent | te tie ‘ age no sta s9 Samples. | B. colt. | ;O789%, |B. coli tol, Organ B. coli 4 : firmed B. coli. Giesboro Point....... 770 | 205 24.1 8.1 92.5 Fort: Hoote: fo .jca. Sse .. 789 254 9.7 3.8 96.4 Fort Washington..... 778 | 123 6.2 4.8 95.2 Mt. Vernon to White- : stone Point........ 851 102 8.5 8.3 92.2 Indian Head. «3.0.53... 241 123 15.7 12.8 88.6 Possum Poimt.......: 212 66.2 | 13.8 20.8 82.4 Maryland Point...... 681 1:44 Ts 52 68.9 Pope’s: Creek, 33.063 740 .I9 34 174.0 36.6 Lower Cedar Point... 476 © .14 .16 II4.0 46272 All below Lower Cedar ; PONG, fe setich macerea 2261 .052 .057 | I10.0 47.7 positive confirmatory results for B. coli. Of these, 95 were positive in the lactose bile presumptive test, but 20 samples showed B. coli with lactose broth which were negative with lactose bile. In order to over- come these irregularities, a method of cross-inoculation was followed, using three media—lactose bile, lactose broth and dextrose bile. The procedure was as follows: Each sample of water was inoculated into the three media, using one large fermentation tube with 10 c.c. of the sample and five ordinary tubes with 1 c.c. each. Incubation for 24 hours was maintained. If the fer- THE COLON GROUP OF BACILLI 139b mentation with gas-formation had taken place in only one or two of the three varieties of media, cross-inocu- lation was made into the three media again and into dextrose broth. Streaks were also made on litmus lactose agar. Incubation was again maintained for 24 hours. It was found that organisms which would ferment all four media after cross-inoculation always proved to be B. coli, and only rarely was there any failure to confirm B. coli when all the original three media gave positive presumptive tests. These failures were ascribed to anaérobic organisms, but no isolations were made. They were so rare, however, as to be negligible, and Young recommends the use of but three media, discarding the dextrose broth. The method of cross-inoculation as above outlined appears to have several advantages with western waters at least, and probably with all waters, in that positive presumptive tests in all three media either directly or on cross-inoculation make it unnecessary to apply confirmatory tests. Furthermore, .the results are obtained in 48 hours in case all media respond positively, while if only a portion respond, the con- firmatory test is already started by the streak culture on the lactose plate. Experience seems to show that this method gives more reliable results than the use of a single presumptive medium and that the inhibitive effect of the bile and the overgrowths in lactose broth are counterbalanced. The table on page 139¢ shows the comparative results of 140 analyses of samples that showed fermentation in less than 50 per cent of the first fermentation tubes. 139c ELEMENTS OF WATER BACTERIOLOGY COMPARISON OF LACTOSE BILE, LACTOSE BROTH, AND DEXTROSE BILE FOR ISOLATING ORGANISMS OF THE B. COLI GROUP FROM WATER SAMPLES Total number of samples showing fermentation................... 140 Total number of samples confirming B. coli.....................- r16 Positive Results for Bee Per cent. B. coli with Jactogs Bile os cc, « > oe een 95 82 B. coli with lactose broth and not in lactose bile 20 17.2 B. coli with lactose broth and not in dextrose bile 6 ar B. coli with dextrose bile and not in lactose bile I4 racy B. coli with lactose bile and not in lactose broth or dextrosé ‘bile. 5 i... oe SS a aka weet 2 I.7 B. coli with Water Survey method missed by lactose bile method=. 2.66 vwuicwaewe ote 2I 18.0 SAMPLES THAT DID NOT CONFIRM Lactose Bile. Lactose Broth. Dextrose Bile. Number of samples. . : 17 6 Per: CONGE 35. Sloss esa ek ae 12.4 4:35 *POSITIVE FERMENTATION TUBES Lactose | Lactose Lactose | Lactose ||Dextrose | Dextrose Bile Bile Broth Broth Bile Bile Plus Minus Plus Minus Plus Minus No. of samples} 302 SAT 404 269 307 276 Percents coo: 44.5 55.5 60 40 59 41 The U. S. Treasury Department, on the recom- mendation of a special commission of experts appointed for the purpose by the Secretary of the Treasury, adopted on October 21, 1914, the following standard for drinking water to be supplied to the public by THE COLON GROUP OF BACILLI 139d common carriers in interstate commerce. (Public Health Reports, XXIX, 2959, Nov. 6, 1914.) “The following are the maximum limits of permis- sible bacteriological impurity: “t. The total number of bacteria developing on standard agar plates, incubated 24 hours at 37 degrees C., shall not exceed roo per cubic centimeter, provided that the estimate shall be made from not less than two plates, showing such numbers and distribution of colonies as to indicate that the estimate is reliable and accurate. “2. Not more than one out of five to-c.c. portions of any sample examined shall show the presence of organisms of the bacillus coli group when tested as follows: ““(@) Five 1o-c.c. portions of each sample tested shal] be planted, each in a fermentation tube containing not less than 30 c.c. of lactose peptone broth. These shall be incubated 48 hours at 36 degrees C. and observed to note gas formation. “(b6) From each tube showing gas, more than 5 per cent of the closed arm of fermentation tube, plates shall be made after 48 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 24 hours, a well-isolated characteristic colony shall be fished and transplanted into a lactose-broth fermentation tube, which shall be incubated at 37 degrees C. for 49 hours. “For the purposes of enforcing any regulations which 139e¢ ELEMENTS OF WATER BACTERIOLOGY - may be based upon these recommendations, the follow- ing may be considered sufficient evidence of the presence of organisms of the bacillus 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 (0). “The formation of gas, occupying ro per cent or more of closed arm of fermentation tube, in lactose peptone broth fermentation tube inoculated with colony fished from 24-hour lactose litmus agar or Endo medium. plate. “These steps are selected with reference to demon- strating the presence in the samples examined of aérobic lactose-fermenting organisms. - “3. It is recommended as a routine procedure, that in addition to five 1o-c.c. portions, one 1-c.c. portion, and one o.1-c.c. portion of each sample examined be planted in a lactose peptone broth fermentation tube, in order to demonstrate more fully the extent of pollu- tion 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.)” At present the choice of a practical method seems THE COLON GROUP OF BACILLI 139f to lie between the lactose bile test and this Treasury Department procedure. It is to be hoped that the Committee on Standard Methods of the Laboratory Section will pronounce definitely in favor of one or the other at the earliest possible moment. CHAPTER VII SIGNIFICANCE OF THE PRESENCE OF THE COLON GROUP IN WATER Colon Bacilli in the Intestines of the Lower Animals. The Bacillus coli is by no means confined to the human intestine. Dyar and Keith (Dyar and Keith, 1893) found it to be the prevailing intestinal form in the cat, dog, hog, and cow. About the same time, Fremlin (Fremlin, 1893) found colon bacilli in the feeces of dogs, mice, and rab- bits, but not in those of rats, guinea pigs, and pigeons. Smith (Smith, 1895) recorded the presence of the organism, in almost pure cultures, in the intestines of dogs, cats, swine, and cattle; and he also found it in the organs of fowls and turkeys after death. Brotzu (Brotzu, 1895) reported B. coli and allied forms as very abundant in the intestine of the dog; and Belitzer (Belitzer, 1899) isolated typical colon bacilli from the intestinal contents of horses, cattle, swine, and goats. Moore and Wright (Moore and Wright, 1900) recorded the finding of the colon bacillus in the horse, cow, dog, sheep, and hen; and in a later report (Moore and Wright, 1902) they noted its occurrence in swine and in some, but not all, the specimens of rabbits examined. In frogs it was not found. Eyre (1904) has more recently isolated typical B. coli from the intestines 140 SIGNIFICANCE OF COLON GROUP IN WATER 141 of mice, rats, guinea pigs, rabbits, cats, dogs, Sheep, goats, horses, cows, hens, ducks, pigeons, sparrows, divers, gulls, and fish of various sorts. Houston (1904) found B. coli abundant in the feces of gulls, as might be expected from their feeding habits. Houston (1905) and other recent observers have found it impossible, even by the use of elaborate series of fermentation tests, to distinguish human B. coli from those found in animals. Savage (1906) compared colon-like organisms isolated from the intestines of swine, cattle, horses, and sheep with those of human origin in respect to their action upon lactose, dulcite, mannite, raffinose, glycerine, maltose, galactose, levulose, saccharose, starch and cellulose; but he failed to find any general correlations between habitat and biochemical powers. Ferreira, Horta and Paredes (1908°) have made perhaps the most elaborate study of the distribution of colon bacilli in the lower animals. They isolated 8x lactose-fermenting bacilli from 38 species of mammals and 8 species of birds, including monkeys, bears, wolves, foxes, hyenas, lions, panthers, tapirs, a camel, deer, and ostriches from the Zoological Gardens. These cultures were studied by an elaborate series of tests and 93 per cent of them proved to be typical B. coli. Bettencourt and Borges (1908°) working in the same laboratory showed that there were no specific differences in agglutination with immune sera and in complement fixation between the colon bacilli of human and of animal origin. Konrich (1910) reports the examina- tion of 170 samples of feces from men, horses, swine, sheep, cows, goats, dogs, cats, guinea pigs, mice, rabbits, 142 ELEMENTS OF WATER BACTERIOLOGY - rats, earthworms, moles, fowls, swallows, sparrows, ducks, pigeons, geese, a jackdaw, a redstart, a blackbird, an adder, and a trout. Three out of 5 guinea pig samples, 4 out of 20 horse samples, 2 out of 3 mouse samples, 3 out of 8 rabbit samples, and 2 out of 8 earthworm samples, 14 in all, were negative; while all the rest showed B. coli. In cold-blooded animals the occurrence of B. coli is | less constant. Negative results in the frog and positive results in certain fishes, an adder and earthworms have just been quoted. Amyot (1902) failed to find the organism in the intestines of 23 fish representing 14 species. Johnson, on the other hand (Johnson, 1904), in the examination of the stomach and intestines of 67 fish caught in the polluted Illinois and Mississippi Rivers, isolated B. coli 47 times. He concluded from these results that the migration of fish from a con- taminated stream or lake to an unpolluted one may explain the occasional finding of B. coli in small samples, or the more regular detection of it in large volumes of the water. Bettencourt and Borges (1908") isolated 29 cultures of colon-like microbes from the intestines of 17 types of fishes, reptiles and amphibia. Only 8 of the 29 formed gas in lactose broth and only 2 (from an eel and an adder) proved to be typical B. coli. It should be noted, however, that the samples of fecal material were plated directly on Endo medium instead of being subjected to the more sensitive process of preliminary enrichment. Fromme (1910) reviews the work of many observers : = SIGNIFICANCE OF COLON GROUP IN WATER 143 in regard to the presence of colon bacilli in the intes- tines of cold-blooded animals (particularly fish of various sorts and oysters) and concludes that while they are regularly found in warm-blooded animals they are found often, but not regularly, in cold-blooded animals. The lower the zoological type the rarer are the colon bacilli. Alleged Ubiquity of the Colon Bacillus. Many bacte- riologists have gone further and affirmed that the colon bacillus was not a form characteristic of the intestine at all, but a saprophyte having a wide dis- tribution in nature. The first of this school, perhaps, was Kruse (Kruse, 1894), who in 1894 protested against the arbitrary conclusions drawn from the colon test as then applied. He pointed out that the characters usually observed marked, not a single species, but a large group of organisms. As ordinarily defined, he ‘added, “ the Bacterium coli is in no way characteristic of the faces of men or animals. Such bacteria occur everywhere, in air, in earth, and in the water, from the most different sources.”’ Even if the relations to milk and sugar media be considered, “ micro-organisms with these characteristics are also widespread.” Dr. Kruse gave no experimental data on which his opinion was based. In the same year Beckmann (Beckmann, 1894) isolated a bacillus which he identified by pretty thorough tests as B. coli from the city water of Strass- burg, a ground-water which he believed could by no possibility be subject to fecal contamination. Large quantities of water were used for the isolation. Refik (Refik, 1896) recorded the constant presence 144 ELEMENTS OF WATER BACTERIOLOGY of colon bacilli in water of all sorts, public supplies, wells, cisterns, and springs in the neighborhood of Constantinople, and Poujol in the succeeding year reported (Poujol, 1897) the isolation of B. coli from 22 out of 34 waters studied by him in relation to their use as public supplies. The waters were from various sources—springs, wells, and rivers—but all were of fair quality and many quite free from any possibility of contamination. Samples of 100 c.c. were used for analysis. Certain Italian observers appear to have come to even less conservative conclusions. Abba (Abba, 1895) found colon bacilli constantly present in unpolluted waters near Turin. Moroni (Moroni, 1898; Moroni, 1899) reported the examination of numerous deep and shallow wells and unpolluted springs about Parma, as well as of the public water-supply of the city, and concluded that the colon bacillus was a water form and ~ had no sanitary significance. The characters used for the identification of the species in this case were fairly exhaustive, but both Abba and Moroni used liter samples for analysis. Levy and Bruns (Levy and Bruns, 1899) gave a new turn to the discussion by emphasizing the importance of animal inoculation, already suggested by Blachstein (Blachstein, 1893) and others. They claimed that the existence of numerous para-colon and para-typhoid organisms in air, in dust, and in unpolluted water made it impossible to decide by ordinary bacteriological methods whether true colon bacilli were present in water or not. In no case, however, did representatives 'n SIGNIFICANCE OF COLON GROUP IN WATER 145 of the colon group isolated by them from water kill a guinea pig, even when 1 or 2 c.c: were injected intra- peritonally. The authors, therefore, considered patho- genicity as an attribute belonging only to the true B. coli of the intestine. This paper aroused Professor Kruse’s pupil, Weissenfeld, to a publication, in which the position of the Bonn school was carried to an extreme. Weissenfeld reported (Weissenfeld, 1900) the analysis of 30 samples of water supposedly pure, and of 26 samples considered to be contaminated. In each case a single centimeter sample was first incubated in Parietti broth, and if no growth occurred, larger samples of half a liter or a liter were examined. Colon bacilli were found in all the samples; and the patho- genicity varied independently of the source of the water. The author concluded that “ the so-called Bacterium coli may be found in waters from any source, good or bad, if only a sufficiently large quantity of the water be taken for analysis.” With regard to the question of pathogenicity as a diagnostic test for intestinal B. coli, there is little doubt of the correctness of Weissenfeld’s conclusions. This property is so variable as to have no important value. Colon bacilli freshly isolated from the intestine are frequently non-virulent, and Savage (1903) and others have shown that there is in general no correlation between pathogenic power and direct or indirect intes- tinal origin. On the other hand Weissenfeld’s work entirely fails to show that the colon bacillus, pathogenic or non-pathogenic, is a normal inhabitant of unpolluted waters. Even his own results, if the quantitative rela- 146 ELEMENTS OF WATER BACTERIOLOGY — tions be considered, furnish evidence to the contrary. In 24 of the 26 samples from bad sources; he isolated his imperfectly defined colon bacilli from 1 c.c. of the water, while in only 8 of the 30 samples of good waters could he find such organisms in that quantity. Colon Bacilli on Plants and Plant Products. The work of certain recent observers has suggested the possibility that the colon bacillus may live in a semi- parasitic fashion on plants as well as on animals. Of a series of 47 cultures of lactic-acid bacteria, recently examined by one of ourselves (Prescott, 1902°; Prescott, 1903, Prescott, 1906), 25 were found to give the reactions of B. coli. These organisms were isolated chiefly from cereals and products of milling, such as flour, bran, cornmeal, oats, barley, etc., while others were in technical use for producing the lactic fermentation. There is no evidence that any of these organisms were of intesti- nal origin, and yet they possess all the characters of typi- cal colon bacilli, even to the pathogenic action when inoculated into guinea pigs. In Germany, Papasotiriu (Papasotiriu, 1901) was meanwhile carrying on almost exactly similar investigations to Prescott’s, with identi- cal results. Other testimony is somewhat conflicting with regard to the occurrence of B. coli on plants. Klein and Houston (1900) reported the finding of typical colon bacilli in only 3 out of 24 samples of wheat and oats obtained from a wholesale house; rice, flour, and oat- meal bought at two different retail shops gave B. coli in all three cereals in one case and on none in the other. Clark and Gage (1903) were unable to isolate B. coli SIGNIFICANCE OF COLON GROUP IN WATER 147 from standing grains. Gordan (1904) could not find B. coli in .t and .or mg. samples of clean bran, but isolated it easily from that of poor quality. Winslow and Walker (1907) have recently reported the examina- tion of 178 samples of grain and 40 samples of grasses for B. coli without success. On the other hand, Diiggeli (1904) found B. coli among the bacteria occurring on the leaves of growing plants, although it was not one of the most abundant species. Barthel, too (Barthel, 1906), found B. coli widely distributed on plants from both cul- tivated and uncultivated regions. Bettencourtand Borges (1908*) examined 35 samples of vegetables and cereals purchased in open market and found 12 lactose ferment- ers, of which 6 proved to be B. coli. It should be noted, however, that the method of isolation used was direct plating on Endo-medium, which is of course less sensitive than the enrichment processes used by other workers. Neumann (1910) has recently studied the distribution of colon bacilli on and in various food substances such as bread, milk, butter and fruit. From fresh fruits immedi- ately after picking he never isolated them, but they were present in a certain proportion of all the foods which had been exposed to human contamination and the author concludes that wherever human hands have been, there will B. coli be found. Konrich (1910) in a similar series of investigations obtained positive results from 46 out of 100 .I to .5 gm. samples of cultivated plants while leaves. of trees and grasses and herbs on waste places gave about 6 per cent positive results. Hay showed colon bacilli in gi per cent of the 135 samples examined and grains in 55 per cent of 300 samples. Dr. L. A. Rogers of the U.S. Bureau of Animal Industry has recently obtained 148 ELEMENTS OF WATER BACTERIOLOGY | results which indicate that the colon bacilli found on grains are of a distinct type, which can be distinguished from fecal B. colijby their fermentative powers. Colon Bacilli in Dust and Soil. Winslow and Kligler (1912) have shown that colon bacilli may be very abundant in the dust of city streets and houses, as might naturally be expected from the fact that such dust is largely made up of horse droppings. They examined 24 samples of street dust and 72 samples of house dust (all in New York City). All of the street dusts and 63 of the 72 house dusts contained colon bacilli in at least one of three duplicate roo gram portions. In two street samples the numbers rose to 330,000 and 660,000 per gram respectively, while the largest indoor result was 60,000. The average for the indoor dusts was between 1000 and 2000 per gram and for the street dusts over 50,000 per gram. ‘This dust was dust deposited on surfaces and would only be carried up into the air by currents of some force. It is well known that colon bacilli are, as a matter of fact, rarely present in street or house air. Konrich (1910) exposed open Petri dishes of dextrose broth to the air of Jena streets for 24-hour periods, daily, for 3 months and found colon bacilli only 11 times. The colon bacilli in street dust may, however, perhaps account for the anomalous positive results sometimes obtained in reservoirs bordered by roadways. Konrich (1910) has also made important contribu- tions to the study of colon bacilli in the earth. Out of 547 samples of soil, 65% showed B. coli in por- tions of between .1 and .5 gm. The farther removed from cultivation a sample was} the less were the chances SIGNIFICANCE OF COLON GROUP IN WATER 149 of positive results. He concludes that B. coli is widely distributed in the outer world. It is almost always found in soil from cultivated fields or from traveled places. The farther a source is removed from travel and from cultivation the more rarely is the colon bacillus found; but it is never altogether absent. On plants or parts of plants it is frequently found when they come from cultivated land; on plants from waste places it is rarely found. It seems probable that colon bacilli may be even more widely distributed in a thickly settled and intensively cultivated country like Ger- many than in the United States. The Number of Colon Bacilli, not Their Mere Pres- ence, as an Index of Water Pollution. The more important practical conclusions to be drawn from these various investigations seem to be as follows: 1. Bacteria corresponding in every way to B. coli are by no means confined to animal intestines, but are widely distributed elsewhere in nature. 2. The finding of a few colon bacilli in large samples of water, or its occasional discovery in small samples, does not necessarily have any special significance. 3. The detection of B. coli in a large proportion of small samples (x c.c. or less) examined is imperatively required as an indication of recent sewage pollution. 4. The number of colon bacilli in water rather than their presence should be used as a criterion of recent sewage pollution. With these qualifications the value of the colon test was never more firmly established than it is to-day. _ Whether or not originally a domesticated form, it is 150 ELEMENTS OF WATER BACTERIOLOGY clear that the colon bacillus finds in the intestine of the higher vertebrates an environment better suited to its growth and multiplication than any other which occurs in nature. Houston (1903*) records the number of B. coli per gram of, normal human feces as between 100,000,000 and 1,000,000,000. It is almost certain that the only way in which large numbers of these organisms gain access to natural waters is by pollution with the domestic, industrial, and agricultural wastes of human life. If pollution has been recent, colon bacilli will be found in comparative abundance. If pollution has been remote the number of colon bacilli will be small, since there is good evidence that the majority of intestinal bacteria die. out in water. If derived from cereals or the intestines of wild animals, the number will be insignificant except perhaps in the vicinity of great grain-fields or where the water receives refuse from grist-mills, tanneries, dairies, or lactic-acid factories. The first recognition of the necessity for a quantita- tive estimation of colon bacilli in water we owe to Dr. Smith, who in 1892 (Smith, 1893°) outlined a plan for a study to be made by the New York Board of Health on the Mohawk and Hudson Rivers. Burri (Burri, 1895) pointed out that the use of so large a sample as a liter for examination would lead to the condemnation of many good waters. Freudenreich (Freudenreich, 1895) at the same time indicated the necessity for taking into account the number of colon bacilli present. He recorded the isolation of the organisms from unpolluted wells, when as large a quantity of water as 100 c.c. was used, ard concluded that it was entirely absent only from SIGNIFICANCE OF COLON GROUP IN WATER ‘151 waters of great purity and present in large numbers only in cases of high pollution. This author also quoted Miquel as having found colon bacilli in almost every sample of drinking-water if only a sufficient por- tion were taken for analysis. The practical results of the application of the colon test from this standpoint have proved of the highest value. As originally outlined by Dr. Smith, it con- sisted in the inoculation of a series of dextrose tubes with small portions .of water, tenths or hundredths of the cubic centimeter. It was first used by Brown (Brown, 1893) in 1892 for the New York State Board of Health, and it showed from 22 to g2 fecal bacteria per c.c. in the water of the Hudson River at the Albany intake, and from 3 to 49 at various points in the Mohawk River between Amsterdam and Schenectady. In some previous work at St. Louis, the colon bacilli in the Mississippi River were found to vary from 3 to 7 per c.c. Hammerl (Hammerl, 1897) used the presence of Bacillus coli as a criterion of self-purification in the river Mur. He considered, in spite of the position taken by Kruse, that when a water contained large numbers of colon bacilli, as well as an excess of bacteria in general, it might be considered to be contaminated by human or animal excrement. As, however, the organism would naturally be present in large quantities of such a water as that of the Mur, he used no enrichment process, but made plate cultures direct; he defined the B. coli as a small bacillus, non-motile or but feebly motile, growing rapidly at 37° C., coagulating milk and forming gas in sugar media. In general, Hammerl 152 ELEMENTS OF WATER BACTERIOLOGY failed to find colon bacilli in the river by this method, except immediately below the various towns situated upon it; at these points of pollution he discovered a few colon colonies upon his plates, not more than 4 to 6 per c.c. of the water. He concluded that “ the Bacterium coli, even when it is added to a stream in great numbers, under certain circumstances disappears very rapidly, so that it can no longer be detected in the examination of small portions of the water.” The most important work upon the distribution of B. coli has been that carried out in England by the bacteri- ologists of the local government board, by Dr. Houston in particular. This investigator (Houston, 1898; Hous- | ton, 1899*; Houston, 1900*) made an elaborate series of examinations of soils from various sources to see whether the microbes considered to be characteristic of sewage could gain access to water from surface wash- ings free from human contamination. In the three papers published on this subject the examination of 46 soils was recorded. In only 10 of the samples was B. coli found, and of these 10, 9 were obviously polluted, being derived from .sewage fields, freshly manured land, or the mud-banks of sewage-polluted rivers. The author finally concluded that ‘as a matter of actual observa- tion the relative abundance of B. coli in pure and impure substances is so amazingly different as to lead us to suspect that not only does B. coli not flourish in nature under ordinary conditions, but that it tends to even lose its vitality and die.” ‘In brief, I am strongly of opinion that the presence of B. coli in any number, whether in soil or in water, implies recent pollution of SIGNIFICANCE OF COLON GROUP IN WATER 153 animal sort.’ Pakes (Pakes, 1900) stated on the strength of an examination of “about 300 different samples of water,”’ no particulars being published, that water from a deep well should not contain B. coli at all, but that water from other sources need not be con- demned unless the organism was found in 20 c.c. or less. When colon bacilli were found only in greater quantities than 100 c.c. the water might be considered as probably safe. Horrocks (Horrocks, 1910), after a general review of English practice, concluded that ‘“‘ when a water-supply has been recently polluted with sewage, even in a dilution of one in one hundred thousand, it is quite easy to isolate the B. coli from 1 c.c. of the water.” ‘I would say that a water which contained B. coli so sparingly that 200 c.c. required to be tested in order to find it had probably been polluted with sewage, but the contamination was not of recent date.” Chick (Chick, 1900) found 6100 colon bacilli per c.c. in the Manchester ship canal, 55-190 in the polluted River Severn, and numbers up to 65,000 per gram in roadside mud. On the other hand, of 38 unpolluted streams and rivulets, 31 gave no Bacillus coli and the other 7 gave 1 perc.c. or less. The Liverpool tap water, snow, rain, and hail showed no colon bacilli. One of the first elaborate applications of the colon test was made by Jordan in the examination of the fate of the Chicago sewage in the Desplaines and Illinois Rivers. In these studies of self-purification (Jordan, 1901) the analyses were made quantitative by the examination of numerous measured samples, fractions of the cubic centimeter; and the method employed 154 ELEMENTS OF WATER BACTERIOLOGY was enrichment, either in dextrose-broth fermentation tubes or in phenol broth, with subsequent plating on litmus lactose agar. The cultures isolated were tested as to their behavior in dextrose broth, pep- tone solution, milk, and gelatin; of the dextrose tubes made directly from the water all were con- sidered positive which gave more than 20 per cent gas in the closed arm, with an appreciable excess of hydrogen. The results were very significant. In fresh sewage a positive result was obtained about one-third of the time in one one-hundred-thousandth of a cubic cen- timeter and almost constantly in one-ten-thousandth of a cubic centimeter. The Illinois and Michigan canal proved almost as bad, giving positive results on 7 days out of 28 in dilutions of one in a hundred thousand and on 28 days out of 32 in a dilution of one in ten thousand. At Morris, 27 miles below Lockport, where the canal enters the bed of the Desplaines River, and 9g miles below the entrance of the Kankakee, the principal diluting factor, the numbers were so reduced that positive results were obtained only on 11 days out of twenty in one-thousandth of a cubic centimeter, on: 20 days out of thirty in one-hundredth of a cubic centi- meter, and on 20 days out of 23 in one-tenth of a cubic centimeter. At Averyville, 159 miles below Chicago, colon bacilli were isolated on only 4 days out of 27 in one-tenth of a cubic centimeter, and on 13 days out of 31 in one cubic centimeter. A comparison with certain neighboring rivers showed this to be about the normal value for waters of similar character, as the following table extracted from Professor Jordan’s paper will show: SIGNIFICANCE OF COLON GROUP IN WATER 155 NUMBER OF B. COLI PRESENT IN CERTAIN RIVER WATERS (JORDAN, 1901) 0.1 C.c. I c.c. Source of Sample. No. Days | No. Days | No. Days | No. Days Water B. Coli Water B. Coli Examined.| Found. | Examined.} Found. Tllinois River, Averyville. . . 27 4 31 13 Mississippi River, Grafton. . 34 be) 35 23 NE GEV a %c0e-) 8 arcs fos 00 22 2 23 6 Sangamon River.......... 25 14 27 21 Missouri River..........--. 32 13 31 21 These results harmonize rather closely with those previously recorded by Brown and Fuller and indicate that in the larger rivers where the proportionate pollu- tion is not extreme, colon bacilli may be isolated in about half the 1-c.c. samples examined. Such rivers are of course inadmissible as sources of water-supply, accord- ing to modern sanitary standards, unless subjected to purification of some sort. Hunnewell and one of us (Winslow and Hunnewell, 1902”) examined a considerable series of normal waters for B. coli, testing 1 c.c. from each by the dextrose- broth method and a larger portion of 100 c.c. by incuba- tion with phenol broth as described in Chapter VI. The samples were obtained from the public supplies of Taunton, Boston, Cambridge, Braintree, Brookline, Needham, and Lynn in Massachusetts, and Newport, R. I., from the Sudbury River, from the ocean, from the waters of springs bottled for the market, from ponds, pools of rain and melted snow, springs, brooks, shallow wells, and driven wells in various towns near 156 ELEMENTS OF WATER BACTERIOLOGY the city of Boston. For comparison 50 samples of polluted waters from the Charles, Mystic, Neponset, and North Rivers were examined. The colon bacillus was defined by gas production in dextrose broth, coagulation of milk, reduction of nitrates, formation of indol and failure to liquefy gelatin; and organisms which lacked the power to reduce nitrates or to form indol were classed in the “ Paracolon group.” The results are summarized in the following table: PRESENCE OF B. COLI IN POLLUTED AND UNPOLLUTED WATERS (WINSLow AND HUNNEWELL, 1902) Unpolluted Waters I ¢c.c I00 c.c Samples Examined... 6c. gckiein hota «ees 157 153 Dextrose broth positive. i... 6.66 cscawees 40 76 Lactose plate positive... 2.645 J. cscaws' 13 31 SOLON SOU ii ae. scree Suk teeters oars a 5 It IPATACOLON-BTOUD 2 ascents cues naisesteiet mor 5 5 Bi Coace Brow ss sie <5'ch oacae aes esis aes Be 5 DITEPLOCOCCUS' STOUDs.. s.ocitc ranch votes 3 nae) Polluted Waters I c.c 100 c.c. Samples examined ...32.0% akc eicd Shaie ve ore 50 48 Dextrose broth positive................. 50 37 Lactose plate positives ac ss ss40sestenaicen 50 26 SOLON ETOUD 5. Gz cesrecwiern oo; Seletete me ie 18 4 Paracolon Stroup... ceca t vtunecee ene 6 ieee Be ClOaCey Sai tauean se cas tiiainsnsahrieete I Si Ale Streptococcus. @roup soos; sss tease sees 25 22 As the authors pointed out, these tables indicate that bacteria capable of growth at the body temperature and fermenting dextrose and lactose are infrequently found SIGNIFICANCE OF COLON GROUP IN WATER 157 in unpolluted waters, and colon bacilli are very rarely present. In 157 samples, typical colon bacilli were found only 5 times out of 157, in 1 c.c. Lactose ferment- ing organisms appeared in only 8 per cent of the nor- mal samples and in too per cent of the polluted ones, in 1c.c. Incidentally it may be pointed out that these tables well illustrated the dangers of overgrowths, particularly in large samples. It is clear that the strep- tococci had killed out colon bacilli, originally present, in a large proportion of the 100-c.c. samples of polluted waters and in some of the 1-c.c. samples, since, in so many cases, gas formation was followed by the isola- tion of the streptococcus alone. Colon Bacilli in Surface Waters. Clark and Gage (1903) have published the results of certain studies of DISTRIBUTION OF TOTAL BACTERIA AND B. COLI IN SURFACE-WATERS (CLARK AND GAGE, 1903) B. Coli Populati f - Per Cent Positive Tests. Lake. Gietceahed oor pinay aba Guba Square Mile. Pe I ce. 100 C.c. 1* 1400 612 13.3 33-0 2 356 319 3-5 17-2 3 116 103 0.0 0.0 y fore) 170 0.0 14.0 5 62 87 0.0 9.0 6* 60 48 254 4-5 4* 50 66 4.6 21. 8 47 133 0.0 9.0 9 42 131 0.0 6.7 10* 40 31 0.0 6.2 II 8 28 0.0 j 7.7 I2 42 107 0.0 | 9-3 * Shores used for pleasure resorts. 158 ELEMENTS OF WATER BACTERIOLOGY Massachusetts ponds which indicate clearly the coin- cidence of the distribution of B. coli in single centimeters of surface waters, with actual sanitary conditions. They show also the slight significance of the test for this organism in larger volumes of water. Almost every source gave positive tests in 100 c.c., while with 1-c.c. samples only those lakes appear suspicious which are, in fact, exposed to dangerous pollution. Houston (1905) gives the following table, which may be taken as another fair example of the distribution of B. coli in small streams and lakes. Of the two lakes studied, Loch Ericht is free from the pollution of human or domesticated animals, while Loch Laggan receives some drainage from farm lands; both are of large size. The brook and river samples were collected from adjacent streams. DISTRIBUTION OF B. COLI IN SURFACE-WATERS (Houston, 1905) Percentage of Samples showing B. Coli in each Dilution. Ditution, =| to.res.| T52Ce| Shoes |. Trews | epee Brooks and river. AEG 53.8 34.6 3.8 he Loch bage@an: jas: ssa 1,2 33.0 49-4 16.4 BOCHSTiChts see! siccats coeok Waters aie 1.0 19.0 80.0 As an example of a heavily polluted stream, on the other hand, the table on page 159 may be cited. It shows in a striking way the increase of B. coli in the Thames on its passage through London and its progressive purification below. i SIGNIFICANCE OF COLON GROUP IN WATER 159 The river at the lower stations in this table was con- siderably diluted with sea-water, yet it showed clearly its large proportion of sewage. Normal sea-water, even in the neighborhood of the shore, shows B. coli only in large samples. Houston (1904), in another communication, reports the examination of 168 samples of sea-water near the English coast. None of the samples showed B. coli in 1 c.c.; 97 samples gave negative results in 10 c.c.; 45 in 100 c.c., and 4 had no B. coli even in 1000 C.c. B. COLI IN THE RIVER THAMES AT VARIOUS POINTS (Houston, 1904°) Percentage of Positive Results Place. Sek bok ceo aor [cenail ceaees to aeoee c.c. c.c. c.c. c.c. c.c. Sunbury...... ice prescel vsen fh 70-01 38.8 BON Cire nas Hampton...... Sth oh Seclh Sic Ose [ERC A wt BR Barking....... weve 4.2 | 45.8 | 45.8 4.2 Crossness...... Surrey Cee ae Mae We 2 Pe a es Cy cd ee II.I Purfleet....... ta Ml i ries 3-0] 9.1 | 33-3 | 30-2 I MERGES oe Sas ss Dest patlsts 2.8 | 22.2 | 41.7 | 33-3 Mucking...... give hae SOs) S97 1 Bese cook Chapman..... E.Ort 45601 50.0-|) . 8 3 Barrow Deep ..| 12.0 | 36.0 | 40.0 | 12.0 : Gartner (1910) has collected some interesting data in regard to the ratio between the number of colon bacilli and of total bacteria in waters of different quality. The results from four different sets of experi- ments by Konrich at Jena, Houston at London, Noble in New York, and Hill at Giessen, may be combined as on the following page : 160 ELEMENTS OF WATER BACTERIOLOGY RATIO OF TOTAL BACTERIA TO COLON BACILLI IN WATERS OF DIFFERENT CLASSES Ratio of Plate Count to B. Coli. Coli titer— Smallest Por- | tion of Water Jena. London. New York. Giessen. Showing | B. Coli. 20° 20°. 37°. 24°. 37°. TOO GIG: cs eA og aie 60,500 | 11,800 TONG. 6905 15,100 1,810 1950 280 TiCiGin pe 183 1,288 255 213 47 D0 Cine wis 20.7 352 34 20 az. BOL CO. a 4.1 46 BAL) is oad 8 Ween 87 O.00T CC) SRG eed Rene bal een aes 20 O-000L C.C.. Fe} Saisie eas tha te ol eevee soa ace ee 4.9 ee. o oc) eX Ca | Me rem ier in: ACEO | Linnie Gag eT AS 0.4 The rather regular decrease in the ratio of the total count to the B. coli count with an increase in the actual number of colon bacilli is very interesting. Prof. Gartner apparently holds that this fall in the ratio of the plate count to the “ coli titer” indicates a fallacy in the method of the latter and in particular he emphasizes the absurdity of the lowest figures in the table which indicates that there were twice as many colon bacilli as bacteria of all sorts. It seems to us that the last phenomenon is quite as likely to be due to an error in the plate count as to a failure in the enrichment pro- cedure. Unless dilutions are very carefully made plates inoculated with waters containing tens and hundreds of thousands of bacteria per c.c. are pretty likely to be so crowded that only a portion of the bac- teria with which they are sown are able to develop. As to the diminishing ratio with increasing coli-content, it is exactly what might reasonably be expected. One- tenth to one-quarter of the bacteria in sewage may be a a ee! SIGNIFICANCE OF COLON GROUP IN WATER 161 colon bacilli, and the greater the amount of sewage in water, the nearer this ratio will be approached. Colon Bacilli in Ground-waters. With ground-waters the story is the same. Even in sources of excellent quality we should expect to find, and we do sometimes find, colon bacilli in large volumes of water. Abba, Orlandi, and Rondelli (1899) showed by experiments with B. prodigiosus at Turin that when bacteria are present in great numbers on the surface of the ground, a few may penetrate for a considerable distance and ultimately reach the sources of ground-waters. The chance that disease germs could survive this process in a soil so impervious as to allow colon bacilli to appear only in large samples of water, is infinitesimal. An interesting contribution to the bacteriology of ground-waters was made by the Massachusetts State Board of Health (Massachusetts State Board of Health, 1g0r) in connection with the examination of the spring- waters bottled for the sale in the State. Ninety-nine springs were included in this study, and in almost every instance 4 samples were examined, 2 taken directly from the spring by the engineers of the board and 2 from the bottles as delivered for sale to the public. In the water of one spring B. coli was found twice, once in a sample from the spring and once in the bottled sample. This spring was situated in woodland, but was unprotected from surface drainage, and the method of filling bottles subjected it to possible contamination. In 5 other cases B. coli was found once in the sample from the spring; all were subject to pollution from dwellings or cultivated fields, and 4 of the 5 were shown 162 ELEMENTS OF WATER BACTERIOLOGY — to be highly contaminated, chemically. In 7 other cases B. coli was found in the bottled samples alone; 3 of these sources were of high purity, but the bottling process furnished opportunity for contamination. Clark and Gage (1903), in the examination of 170 samples of water from tubular and curb wells of good quality used as sources of water-supply, found B. coli only 5 times, once in 1 c.c. and 4 timesin too c.c. Horton (1903), from a study of ground-waters in Ohio, concluded that the presence of B. coli in wells and springs was indicative of serious pollution. Houston (1903”) makes an instructive comparison of some more or less polluted shallow wells at Chichester with deep ground-waters of high quality at Tunbridge Wells. The following table shows the value of the 1 cubic-centimeter sample in discriminating between good and bad waters. DISTRIBUTION OF B. COLI IN GOOD AND BAD WELL WATERS ‘ (Houston, 1903”) Percentage of Positive Tests . Chichester Shallow Tunbridge Wells, Quantity of Water. Wells. Deep Wells. 100 C.C. go 25 TO'C\C) 80 6 I t.c. 45 ° 6. £.C.C; 20°," ° In a subsequent investigation, Houston (1905) exam- ined still larger samples of water from the Tunbridge Wells for B. coli: 49 samples of 100 c.c. each showed no B. coli, and 27 liter samples showed B. coli only once. SIGNIFICANCE OF COLON GROUP IN WATER 163 Kaiser (1905) reports an interesting correlation between total numbers and B. coli in a series of 38 well waters. Of 11 wells containing over 200 bacteria per c.c. go per cent showed colon-like organismsin liter samples. Of 12 wells containing from 50 to 200 bacteria per c.c. 67 per cent gave colon-like organisms; of 26 wells with less than 50 bacteria per c.c., only 27 per cent showed positive results. Fromme (1910) brings out the relation between B. coli and total numbers in 120 samples of well waters near Hamburg in the table below. RELATION BETWEEN TOTAL NUMBERS OF BACTERIA AND B. COLI (FROMME, 1910) Colony Count. Number of Samples. B. ca eg} = Over 200 35 40.0 50-200 19 15.8 Under 50 66 3.0 Similar data obtained by one of us for some American sources have been cited in Chapter I. Even Konrich (1910), who is exceedingly sceptical as to the value of the colon test, has shown that an increase in the colon content of the Jena water supply (a ground-water) always followed a heavy rain which washed through some of the colon bacilli in the soil. Colon Bacilli in Filtered Waters. One of the most important applications of the colon test is in the control of the operation of municipal water filters. It has been used for this purpose for 10 years or more at Lawrence, and Fuller laid stress upon its results in his classic experiments on water purification in the Ohio valley. At Cincinnati he records the presence of 164 ELEMENTS OF WATER BACTERIOLOGY colon bacilli in 60 per cent of the 1-c.c. samples from the Ohio River, while the effluent from either slow sand or mechanical filters gave positive results only half the time in samples of 50 c.c. The results of the examina- tions carried out at Lawrence for 6 years are brought together in the table below from the Annual Reports of the Massachusetts State Board of Health. B. COLI IN MERRIMAC RIVER AND LAWRENCE FILTER EFFLUENT Mein a Per Merrimac River, Piece Vee Per pec Bienes iP Number B. coli per CORE Or eae Samples containing ea Sample containing B. coli. ae B. coli. 1900 99.7 87 18.1 1901 * * * 1902 99.0 73 4.0 1903 99.0 78 4.2 1904 I00.0 72 8.0 1905 100.0 118 4-7 * Not given. At Harrisburg, Pa., mechanical filtration combined” with chlorin disinfection has yielded the results tabulated below: B. COLI IN RAW AND TREATED WATER AT HARRISBURG, PA. (HARRISBURG, 1913) Per Cent Positive Tests in 1 c.c. Year. : Raw Water. Treated Water. £906 71.9 Te 1907 64.0 1.0 1908 65.7 ar 1909 63.1 1.0 1910 55.4 0.2 IQII HRS 0.6 I9I2 46.9 0.8 . SIGNIFICANCE OF COLON GROUP IN WATER 165 At Washington the most complete slow sand- filtration plant yet constructed has yielded the results tabulated below, for which we are indebted to the courtesy of Mr. F. F. Longley: B. COLI IN POTOMAC RIVER AND WASHINGTON FILTER EFFLUENT Dalecarlia Reservoir Inlet. Sey ee ee 1906. Samples. Samples. Number Number Tested. - ue | Tested. + IO c.c. I c.c. 10 c.c. I. c.c. February. . I5 5 3 24 ° ° March. ..... 24 12 3 27 ° ° ‘See 18 9 6 25 I ° i.) 0), 25 3 I 27 ° ° jJume: ....... 26 9 8 26 ° ° EY ee asc : 20 8 9 21 I ° August.....- 26 21 14 27 I I September.. . 10 4 I 25 2 ° October. .... Io 3 2 27 I ° November. 8 3 ° 25 2 ° December. . 9 4 4 24 2 2 1907 January.. - 9 5 3 26 3 3 February. . 8 2 2 23 ° ° March...... 8 7 4 26 ° ° 1303 1 Pea 9 4 I 26 I ° i age ee 23 21 15 26 ° ° Jane. =. Se.) - 25 20 17 25 ° ° MUEY; 101 Bf 26 II 8 26 ° ° August...... 27 13 8 27 ° ° September. 24 15 13 25 I ° It must be remembered that in the Washington plant filtration is supplemented by thorough sedimentation, preliminary and subsequent. The entire credit for the good effluent obtained is not therefore due to the filters. At Lawrence it has been shown that removal of colon 166 ELEMENTS OF WATER BACTERIOLOGY bacilli in storage reservoirs and pipe systems may be considerable. The figures obtained in 1900 at various points in the distribution system may be cited as an example. PERCENTAGE OF SAMPLES OF WATER CONTAINING B. COLI. LAWRENCE, MASS. (MASSACHUSETTS STATE BOARD OF HEALTH, 1901) Effluent of Outlet of Sey Tap Experi- Filter. Reservoir. City Hali. ment Station. In 1 c.c....... 18 9 4 2 Tn 160 CiCi 5 3% 38 23 16 16 Fromme (1910) in the examination of a filtered water averaging 35 bacteria per c.c. obtained the following results in various quantities of water. PERCENTAGE OF SAMPLES OF WATER CONTAINING B. COLI. (FROMME, 1910) Number of Samples. Volume Examined. Per Cent Positive. IOI 200 C.C. 30.9 412 TO C.Cc. 2.9 800 EC.C, 0.25 In regard to the proportion of positive colon tests permissible in a filter effluent, Clark and Gage (Clark and Gage, 1900) reported some specially instructive observations made when certain of the underdrains of the Lawrence filter were relaid in the autumn of 1808. In doing this work the sand on some of the beds was seriously disturbed; and in December, after the work was completed, B. coli was found in 1 c.c. of the filtered SIGNIFICANCE OF COLON GROUP IN WATER 167 effluent in 72 per cent of the samples examined. In January and February the organisms were found in 54 per cent and 62 per cent of the samples, respectively, while in March the number fell to a normal value of 8 per cent. Corresponding to this excess of B. coli in the city water, there were 12 cases of typhoid fever in December, 59 cases in January, 12 in February, and 9 in March, all during the early part of the month. The authors conclude that “ when filtering a river- water as polluted as that of the Merrimac, it is safe to assume that when B. coli is found only infrequently in 1 c.c. of the effluent, the typhoid germs, necessarily fewer in number and more easily removed by the - filter, have been eliminated from the water.” The results of the daily tests carried out at municipal filter plants are frequently expressed in monthly or yearly averages, as in some of the cases quoted above. It must be remembered, however, that averages of this sort are accepted only by courtesy and with the implied assumption that conditions are approximately constant during the period averaged. When it is said that an acceptable effluent may show B. coli in 3 or 4 per cent of the samples tested the statement is true only for a series of samples collected and examined at the same time. If in a given month 3 per cent of the 1 c.c. samples tested show B. coli, the effluent may or may not be safe. If on each of 20 days 3 B. coli or thereabouts were present in too c.c. of the water it is probably a safe one. If on 19 days no B. coli were present, and on the twentieth day too c.c. showed 60 B. coli, the average result would be the same, but 168 ELEMENTS OF WATER BACTERIOLOGY — the water on one day was of a dangerous character. With properly managed filter plants marked varia- tions do not occur from day to day and average results are generally reliable. It is wholly misleading, how- ever, to compare such results with the average exami- nations of an unfiltered surface water. With surface waters daily variations are the rule and a low monthly average of colon tests may include and cover up dangerous and significant high numbers at particular periods. Summary of American and Foreign Opinion as to the Value of the Colon Test. The general results of the studies of the colon tests which have now been carried out in great numbers all over the world may be sum- marized by a few further citations. In America the fact that the number of colon bacilli in a water measures the degree of its pollution is now universally accepted. The same conclusion has been established in England by the elaborate investigations of Houston and his pupils. Savage, for example, concluded (Savage, 1902) from a study of a large num- ber of water supplies in Wales that even in surface © waters, exposed to animal contamination from adjacent grazing grounds, B. coli is not present in 2 c.c. unless other pollution is present. In a more recent review of the whole subject, the same author (Savage, 1906) concludes that ‘“ there is no evidence or observations which have ever shown that B. coli, reasonably defined, is present in any numbers in sources which have not been exposed to some form of fecal contamination.”’ In Germany, Petruschky and Pusch (Petruschky _ SIGNIFICANCE OF COLON GROUP IN WATER 169 and Pusch, 1903) examined a considerable series of waters from different sources by incubating measured samples with equal amounts of nutrient broth and iso- lating upon agar. In 45 samples of well-waters they found B. coli 7 times in .or c.c., 9 times in .1 c.c., and 7 times in 1 c.c. In the other 22 cases it could not be found in 1 c.c. and in 4 cases not in too c.c. One sample showed it only in 600 c.c. and 1 not in 750 c.c. Of 29 river-waters, only 2 failed to give positive results in .r c.c. and 14 showed B. coli in .oor of a’ c.c. or less. In sewage the number varied from 1 to 1,000,000 per c.c. The authors conclude that a quantitative estima- tion of the B. coli content furnishes a good measure of the fecal pollution of water. There is still a school of bacteriologists in Germany, however, who are inclined to place little value on the colon-test. We have pointed out how Kruse and his pupils at Bonn led in the attack on it in 1894. Fourteen years later Kruse (1908) concluded after a full summary of the literature that the colon test was on the whole less valuable than the gelatin count, although he admitted that when the test is made quantitative it is valuable as a supple- ment to the plate count. Konrich (1910), working in Gartner’s laboratory at Jena, after perhaps the most exhaustive study ever made of the whole subject, concludes, that to include the colon test in forming judgment on the sanitary quality of a water is to complicate the procedure without improving it and that one would do well to omit the test except in certain special cases. The German criticisms of the colon test are based 170 ELEMENTS OF WATER BACTERIOLOGY mainly on two considerations, the inaccuracy of the method itself and the difficulties in its interpretation. They hold on the one hand that the errors in the enrich- ment process and the consequent lack of correspondence between duplicate determinations are so great that the whole process is worthless. It is of course true that chance errors of distribution and the overgrowth which often occurs in the lower tubes of a dilution series do occasionally lead to individual erroneous results. If several dilutions are made with duplicates in each dilution, and particularly if reliance is placed, as it should be placed, not on single determinations, but on the average of several tests, results are, however, obtained which are in accord with each other and with the results of practical epidemiological experience. The other objection brought forward by many Ger- man sanitarians is that the wide distribution of the colon bacillus leads to its presence even in considerable num- bers in waters which are really of good sanitary quality. It is undoubtedly true that colon bacilli are often found in surface waters which receive no sewage but which are polluted only with the wash from roadways or cultivated land. Even dust blowing in from a road- way may perhaps contribute an appreciable pollution, as we have pointed out above. Girtner (1910) cal- culates that in the soil of a cultivated field 100 meters square there are 15,000,000,000 colon bacilli and points out that it is no wonder that a rain should wash a few of them through into neighboring wells. Konrich (1910) goes so far as to say that to reject on principle all water containing B. coli in 1 c.c. portions SIGNIFICANCE OF COLON GROUP IN WATER 171 is impossible, since many regions have no other water available. On the other hand, it may be urged that it is better to be on the safe side if possible. There is no doubt that in the United States there is no difficulty in secur- ing water supplies, either from the ground or by storage or filtration, which only rarely contain colon bacilli in 1c.c.samples. It may, perhaps, be that in the thickly settled and intensively cultivated parts of Germany this is not the case. Where it is possible to obtain such waters we believe it to be wise to do so. Ground waters to which the colon bacilli from cultivated soil penetrate and surface waters which still contain many colon bacilli from fields and roadways may be generally inocuous but there is always the chance that infectious material may find its way to the soil and may enter, along with the intrinsically harmless colon bacilli of manure. The two most recent German contributions to the subject are on the whole favorable to the colon test if wisely and intelligently applied. Fromme (1910), from Dunbar’s laboratory at Hamburg, concludes from a survey of the literature and from his own studies that “‘ the finding of colon bacilli in water is a valuable indicator of the quality of the water ” and recommends it particularly for ground-water spring waters and filter effluents; and Prof. Gartner, the head of the Hygiene laboratory at Jena, after a thorough discussion of previous work, comes to a much more conservative conclusion than his pupil, Konrich (Gartner, 1910). While emphasizing the shortcomings of the process and deploring the tendency “ to set B. coli on a high 172 ELEMENTS OF WATER BACTERIOLOGY — throne and dance before it’ he recognizes that the isolation of this organism has its place. “ My aim will have been realized,” he says, ‘‘if I have been able to show that the colon test may be useful under certain circumstances, but that it must be viewed with great caution and that, moreover, not the mere numbers of colon bacilli, but a careful consideration of the local situation and all the circumstances bearing on the special case are absolutely essential to the formation of an opinion.” With Prof. Gértner’s emphasis on the importance of a sanitary inspection all experienced bacteriologists will certainly find themselves in agree- ment. Some of the best French bacteriologists are strong supporters of the value of B. coli as an indicator of pollution. Gautié (1905) holds that the quantitative determination of B. coli is of the highest importance in water analysis; and Vincent (1905), in an excellent review of the subject, gives strong reasons for main- taining the same position. He finds B. coli absent from spring and well-waters of good quality and present in polluted water in proportion to its pollution. He concludes finally that water containing B. coli in .1 to 1.0 c.c. is unfit to drink, while if the organism is found in 1.0 to 10.0 c.c. it is of doubtful quality. In Portugal too the trend of opinion is strongly in favor of the colon test—the Anglo-American procedure, as it is called in the publications of Dr. Bettencourt and his associates (Ferreira, Horta and Paredes, 1908°). Altogether the evidence is quite conclusive that the absence of B. coli demonstrates the harmlessness of a SIGNIFICANCE OF COLON GROUP IN WATER 173 water as far as bacteriology can prove it. That when present, its numbers form a reasonably close index of the amount of pollution, the authors above quoted have proved beyond reasonable cavil. It may safely be said that when the colon bacillus is found in such abun- dance as to be isolated in a large proportion of cases from 1 c.c. of water, it is generally proof of the presencz of serious pollution. The standard here suggested is much more lenient than the one set forth by the U. S. Treasury Depart- ment for drinking water supplied to the public by com- mon carriers (see p. 139d). Instead of permitting some 20 or 40 colon bacilli for each 100 c.c. of water as the ordinary test of five 1-c.c. samples does, the Treasury Department condemns a water containing over 2 colon bacilli in too c.c. It must be remembered, however, that this government standard was “ recommended only for application to the special case in question”; and the Commission pointed out that, with water served on trains, it was necessary to set “ narrower limits for permissible impurities of definitely dangerous character ” than would be the case with waters where a sanitary inspection of the source could be made. | Mr. George W. Fuller and his colleagues, in testify- ing before the International Joint Commission on the Pollution of Boundary Waters, recommended a stand- ard for raw waters before filtration of 500 B. coli per roo c.c. of water expressed as an annual average and determined from a considerable number of confirmatory tests made throughout the year. CHAPTER VIII VARIETIES OF COLON BACILLI AND THEIR SPECIAL SIGNIFICANCE Tests Used for Subdividing the Colon Group. The group of colon bacilli as defined in Chapter VI includes all aerobic Gram-negative bacilli which produce acid and gas in dextrose and lactose media. By the applica- tion of various tests, mainly bio-chemical, it may be further split up, almost at will, into a great number of varieties. The principal tests which have been used for this purpose are as follows: I. Nun wn Motility. . Coagulation of milk. Production of indol. Liquefaction of gelatin. . Reduction of nitrates. . Reduction of neutral red. 7: Fermentation of various carbohydrates other than dextrose and lactose. 8. Voges and Proskauer reaction. 9. Character of colonies on various solid media. ro. Aesculin reaction. 11. Growth and fermentation at 46°. Motility is seldom determined in actual routine work in this country from the general belief that its demon- 174 VARIETIES OF COLON BACILLI 175 stration is both burdensome and needless. Motility is a fluctuating and uncertain property and one which frequently requires repeated preliminary cultivations to make manifest. Furthermore, non-motile colon bacilli are common in the intestine and are probably as charac- teristic of pollution as the motile forms. McWeeney (1904) found non-motile B. coli abundant in feces and observed cases where the organisms were motile at 20° and not at 37°. He quotes Stécklin as hav- ing found 116 non-motile strains among 300 otherwise normal B. coli from feces. Evidence that non-motile bacteria, otherwise resembling B. coli, occur in unpol- luted water would furnish the only basis for requiring this test as a routine procedure. No such evidence exists. The great body of data which connects the presence of B. coli with pollution includes all B. coli whether motile or not, since scarcely any bacteriologists observe this property in actual practice. Howe (1912) has recently come to the conclusion that motility has no diagnostic value. MacConkey, however (MacConkey, 1909), after carefully reviewing the various characters suggested for use in studying colon bacilli, retains this one as important. He recommends that it be made in a drop of a 6-hour broth culture on an ordinary slide with a 4-inch objective and dark ground illumina- tion. Failure to show motility indicates in particular B. lactis-aerogenes and B. pneumoniz of his classifica- tion (see p. 191). Coagulation of milk is one of the most generally ac- cepted tests for the colon group; and as a rule most lactose fermenting forms give a positive reaction. The 176 ELEMENTS OF WATER BACTERIOLOGY common practice in this country is to incubate litmus milk tubes for 48 hours at 37° and then heat to boiling. Tubes which have not coagulated spontaneously fre- quently do so on heating. Biffi (1906) has pointed out that milk to be used for this purpose should not be — sterilized in the autoclave. Temperatures above 100° so alter the milk as to make its coagulation much slower. Konrich (1910) concluded from his experiments that the coagulation of milk by the colon bacillus is often not due to acid production, but to the secretion of a specific coagulating ferment, since he found that the addition of an amount of acid similar to that produced by the organism failed to coagulate. The production of indol in a peptone solution is an- other test very generally used in this country and in England as diagnostic of ‘‘ typical” B. coli. The usual procedure has been to incubate a tube of an aqueous solution containing 1 per cent peptone and .OI per cent sodium nitrite for four days at 37° and to test for indol by adding 1 c.c. of a .o2 per cent solution of sodium or potassium nitrite and 1 c.c. of a 1 to 1 solution of sulphuric acid. Both the tube and the reagents should be cooled on ice before mixing, and the tube should be left in a cool place for an hour afterward to allow time for the characteristic rose-red color of nitroso-indol to develop. Marshall (1907) and other German and English work- ers have shown that this nitrite and sulphuric acid test for indol often gives incorrect results and that the test modified by Béhme from Ehrlich is both more sensitive and more accurate. Two solutions are used; No. 1 is VARIETIES OF COLON BACILLI. 177 made up of 4 parts of para-dimethyl-amido-benzalde- hyde, 380 parts of absolute alcohol, and 80 parts of con- centrated HCl; No. 2 is a saturated aqueous solution of potassium persulphate; 5 c.c. of 1 is added to Io c.c. of a broth culture and then 5 c.c. of 2 is added and the whole shaken. A red color indicates indol. Mac- Conkey (1909) who was at first inclined to discard indol as a routine test, believes that when made in this way it is of much value. Howe (1912), from a statistical study of 630 strains of intestinal colon bacilli, concluded that indol, ammonia, and nitrite tests were but slightly correlated with general vigor and had but slight classificatory significance. It does not necessarily follow, however, that this is true of the forms which occur in stored waters. The so- called ‘“‘ atypical B. coli” are of course rare in feces but they may occur in sufficient numbers to be important in waters which have been remotely polluted. The liquefaction of gelatin is another test generally applied in any detailed study of the colon group. The longer the tubes are kept the higher will be the propor- tion of positive results, for there are many slowly lique- fying forms grading by almost imperceptible degrees into the commoner non-liquefying type. The table cited from Gage and Phelps (1903) on p. 186 shows that of a series of 1908 cultures from various waters, sewages, and shellfish 8 per cent liquefied gelatin in 4 days, 10 per cent in 7 days, 13 per cent in 10 days, and 17 per cent in 14 days. The reduction of nitrates to nitrites has been used in the United States as one of the five standard tests for 178 ELEMENTS OF WATER BACTERIOLOGY ~ B. coli, but it has never gained wide acceptance in Eng- land or Germany. The usual practice has been to incubate for 4 days at 37° and to test for nitrites by adding a drop of each of the following solutions in suc- cession: A; Suiphanilicgcid 5.) ciaceskae ede .5 gram Acetic acid (28% aol.) :.4..-: Svan vance 150.0 C.c. B. Naphthylamine chloride............... o.I gram Distilled watets.. ox .k:scens sda weaeemes 20.0 C.C. Acetic:acid (25% sol.) eee ee ee 150.0 C.c. A red or violet coloration indicates the presence of nitrites. In making the nitrite test it is important to remember the possibility that appreciable amounts of nitrite may be present in the media—either derived from the air or from the use of impure peptone (Wherry, 1905). In the case of the nitrite reaction control tubes should always be tested from the same batch of media and only a distinct red color should be considered positive. The nitrite test is particularly subject to variations of un- explained origin. Of two duplicate tubes inoculated in the same way, one may show a strong reaction and the other none. The reduction of neutral red has been extensively used in England and less in this country. It has been re- ferred to in Chapter VI. as one of the tests suggested for use as a presumptive indicator of the colon group as a whole. MacConkey (1909) concludes that both the ni- trate test and the neutral red test should be dropped from the procedure used in identifying colon bacilli, since so many organisms give these reactions that they have little significance. Houston, however, in the important investigations which will shortly be discussed, used the VARIETIES OF COLON BACILLI 179 production of a greenish fluorescence in neutral red broth as one of his tests of “ typical” B. coli and ap- parently found it valuable. Fermentation of various carbohydrate media has be- come the most common method of subdividing the bacilli of the colon group during the last few years, largely as a result of the work of MacConkey. Sugar broths for this test are generally put up in fermentation tubes of some sort so that the gas formation may be observed and perhaps measured, while acid production may be indicated by the addition of litmus or accurately deter- mined by titration. The old-fashioned fermentation tube with a bulb and a stand has given way in most water laboratories to a plain bent tube of even bore and, more recently, to a still simpler device, a small vial inverted in an ordinary test-tube of sugar broth. The air in the top of the vial is driven out on sterilization and the presence or absence of gas can be easily deter- mined, although it is not possible to measure its quan- tity with accuracy. If an ordinary bent tube is used the amount of gas in the closed arm may be conven- iently measured by the Frost gasometer (Frost, 1901). If a measurement of the gas ratio is desired a few centi- meters of strong sodium or potassium hydrate are added and mixed with the broth by cautiously tipping the - tube; a second measurement determines the amount of gas absorbed (assumed to be COz). It has been pointed out in Chapter VI that the gas ratio appears to be a reaction of slight importance as thus determined. The list of fermentable substances used by various 180 ELEMENTS OF WATER BACTERIOLOGY observers in classifying colon bacilli is a long one. It was pointed out by Smith (1893) long ago that.saccharose divides these organisms into two groups, and Winslow and Walker (1907) have found that strains which attack saccharose generally ferment raffinose also. MacConkey (1909) has made an exhaustive study of the fermentative reactions of the group and suggests an elaborate classification based on the use of a long series of carbohydrate media. ‘Howe (1912), from his recent statistical study of 630 strains of intestinal bacilli, concluded that fermenta- tion tests in mannite, dulcite, and starch media are of little value in the classification of colon bacilli, since they are not closely correlated with other characters. It should be noted, however, that he worked only with fresh intestinal strains and it is possible that types characterized by definite reactions in these media may be rare in faeces but may develop so as to be important in stored waters. Two noteworthy studies of the fermentative reactions of the colon group have recently been published by Kligler and by Rogers, Clark, and Davis, which will be discussed in detail on pages, 200-200). The Voges and Proskauer reaction has been extensively used by MacConkey and his followers in England and by Bergey and Deehan (1908) in this country. After the carbon dioxide in the fermentation tube has been absorbed by caustic soda, if the tube be allowed to stand, an eosin-like color gradually develops in the open arm, due to the presence of acetyl-methyl-carbinol. West (1909) points out that the test used by Rivas, VARIETIES OF COLON BACILLI 181 the boiling of 1-4 c.c. of a 48-hour dextrose broth culture with 5 c.c. of a 10 per cent caustic soda solu- tion, is a quick method of obtaining the Voges and Proskauer reaction. A yellow color is produced under these conditions by the sugar alone, and a pinkish eosin-like color when the acetyl-methyl-carbinol is present. The reaction is hastened by shaking or blowing into the tube to promote oxidation. West confirms the conclusion of MacConkey and Bergey that this reaction is characteristic of the B. lactis- aerogenes and B. cloace types (saccharose positive, dulcite negative organisms). The characters of colonies on various solid media, such as Endo agar or Conradi-Drigalski agar appear to be of minor importance, usually depending on one of the simple fermentative reactions for their differential value. The aesculin test and the Eijkman test (fer- ‘mentation at 46°) have been discussed in connection with their use as enrichment procedures in Chapter VI. Biological Significance of Variations in the Colon Group. The general view among water bacteriologists has been that forms differing from the “ typical” B. coli in one or more respects represented original intes- tinal types weakened by a prolonged sojourn in an unfavorable environment. As Whipple says (Whipple, — 1903), “ The type form of Bacillus coli is one which can be defined within reasonably narrow limits, but when the organism has been away from its natural habitat for varying periods of time, and has existed under abnormal conditions, its ability to react nor- mally to the usual tests appears to be greatly im- 182 ELEMENTS OF WATER BACTERIOLOGY paired. Its power to reduce nitrates may be lost, or on the other hand, may be increased; its power to produce indol may be lost, or on the other hand, it may be increased; its power to coagulate milk, even, is sometimes reduced, although seldom entirely lost; its power to ferment carbohydrates may be altered so that the amount of gas obtained in a fermentation- tube, as well as its ratio of H to COz, is quite abnormal. But in spite of all these facts, the bacillus tested may have been originally a true Bacillus coli,” The results obtained by Peckham (1897) suggest that the indol reaction in particular is highly variable. By successive daily transfers in peptone broth she was able to increase the amount of indol produced by nor- _ mal B. coli, and by a longer continuance of the same process to again weaken and abolish the power of form- ing it. Gas formation too was slackened in the cul- tures grown for too many transfers in the same medium. © Horrocks (1903) found that B. coli kept in unsterilized well-waters and tap waters and in sterilized sewage and Thames water for 2 to 3 months showed only a feeble indol production and a delayed action on milk and neutral red. These modified forms are sometimes called “ atypical B. coli,” or “ para-colon bacilli,” and Vincent gives them the picturesque name, “‘ microbic satellites of B. coli.” ' Such anomalies are most frequent with cultures freshly isolated from water, and they may often be avoided, as Fuller and Johnson (1899) have shown, by subjecting the organism to a process of preliminary cultivation. For this purpose the American Public VARIETIES OF COLON BACILLI 183 Health Association Committee recommends three suc- cessive cultivations in broth at 20 degrees, each of 24 hours’ duration, inoculation from the last broth tube of a gelatin plate which is incubated for 48 hours at 20 degrees, inoculation of an agar streak from one colony on the plate and incubation of this streak for 48 hours at 20 degrees. Often, however, the differences between types of the colon group indicate something much more funda- - mental than temporary weakening due to unfavorable environment. In particular sudden more or less perma- nent mutations may suddenly appear. Twort (1907) reports that by continued cultivation in sugar media he was able to develop fermentative power in certain mem- bers of the Gartner group which lacked such powers before. The work of Massini, Miiller, Sauerbeck, Konrich and others (well summarized by Konrich, 1910) has also shown that mutations capable of fermenting sugars may suddenly arise from a parent strain lacking this power. Burri (1910) has contributed to the same problem and has found that the latent power to ferment a given sugar is released by growing the organism on that particular sugar, but that as Konrich and the others show only a certain proportion of the cells develop this power. The most important recent studies of bacterial mutation have been made by Penfold. In his latest communication (Penfold, 1912) he shows that many bacteria of the colon-typhoid group pro- duce a mutant capable of fermenting lactose, that all strains of the typhoid bacillus produce dulcite and 184 ELEMENTS OF WATER BACTERIOLOGY iso-dulcite mutants, that many paratyphoid and Girtner group bacilli produce raffinose mutants, and that other mutations also occur. The general phenomena are the same in each case. A strain which normally fails: to ferment a given carbohydrate is grown upon a solid medium containing that carbohydrate. As the colonies develop there appear upon them raised papille of a different consistency from the rest of the colony and colored red if litmus be present. Transplants from the papille give pure cultures of a strain fermenting the carbohydrate in question and forming no papille. Transplants from the other portions of the colony give the original strain, ‘non-fermenting, but capable of producing fermenting mutants as before. The identity of derivative strain in all other respects has been made clear by exhaustive cultural tests and serum reactions; and Penfold has shown that the whole proc- ess may be repeated, starting from an isolated single cell. The work of MacConkey and Clemesha, which will be discussed shortly, is based on the assumption that a great number of minute subdivisions of the colon group, whether they have arisen by the gradual modify- ing effect of an unfavorable environment, or by muta- tions, or in some other way, are for practical purposes fairly permanent entities which they describe and name as definite species. Distribution of Types of the Colon Group in Waters of Various Kinds. The sanitary importance of a study of these minor types within the colon group depends on the assumption that a certain set of characters is VARIETIES OF COLON BACILLI 185 generally associated with forms fresh from the intes- tine and may therefore be called typical. Such “typ- ical” B. coli are understood as a rule to be motile, to clot milk, produce indol, reduce nitrate and neutral red and to fail to liquefy gelatin. It seems clear that forms having these characters predominate in the intestine itself while differing or “atypical” forms bear to them somewhat the relation implied in Vincent’s term, satellites. Houston (1903*) examined in detail ror cultures of coli-like microbes isolated from feces and found that 72 per cent of the cultures were typical in all respects, while 11 per cent more differed only in being non-motile. The remaining 17 per cent were atypical, reacting abnormally to milk, indol, neutral red, litmus whey or Capaldi and Proskauer’s medium. In a later investigation, Houston (1904) made a careful study of the distribution of the atypical forms in faces, sewage, polluted water, and the filtered water-supplies of London. According to his ingenious system of nomenclature, “fl” indicates an organism which pro- duces green fluorescence in neutral red broth; “ag,” one which forms acid and gas in lactose media; “ in,” one which produces indol; and “ ac ” one which acidifies and clots litmus milk. The combination of all these properties gives “ Flaginac,” or typical B. coli; “ aginac’”’ is a form which fails to reduce neutral red; “ flagac,” one which fails to form indol, etc. ‘“ Flaginac”’ B. coli form the great majority of coli-like microbes in feces, but Houston found that in filtered water they are outnumbered by atypical forms, of which he recognized thirty-five distinct types. ELEMENTS OF WATER BACTERIOLOGY 186 *Y}MOIS O1jslIajOeIeYO-UOU ‘AjULOS AIOA & BUIATB ‘soIny[No sn00000}dal}s pure rude uO MOIB 0} pales YOIYM saingjno Burpnpouy, gS lo |S Io |Zr l€x jor |g |¢ log |xZ |rZ gg |v og jgo61/¢ |S [xx |1g |zofz| SZ |eserer rete ee s[eq0y, gb liz |v. |S Jo€ |oz |Sz |Sz |Z £9 |gg |zv jeg |S [Lo |ZSS lo |g ox |zZ [62 BL a eee: <7 2 Meg sss aceloree vale 2] Iv ig |o oz gz |gz |gx [Sx jo |zZ [6S |Z9q |Log Jo |oS [6€ |€z |b |o Irs |es CAI [ess Sof aia aa gigs eloasas fv ig jz |9 jor [6 |6 [6 |o |tg |SZ [Zo |16 |€ gq fore |x lz |€E lho Jere | zl |r-eereeeee "*°** USaTTEUS OS |Z |e jo |Sr Jor 6 |6 |r |hg |SZ JoL |gg jo {SZ jovr |x Ig |b |£Q logr | vres |rvt street eee d39 ‘aseVMag gs jf |S |S |€r |6 |Z Ih |b |€g |xZ lod j06 |J€ |¥g lofzilp |v [6 [€g |\Sghr} of |recceee: "++ *JoyeM PoldILy 6S jor |v jor jo€ |gz |Sz |zz |r |69 |€L |zg log |€z |1g |ofz |x |€z lo jog [Elz | vr+° [eerste JOAN IPWILID]T (e) fe) fe) (e) fe) fe) {e) fe} oh + a. oh O° + “* fe) ro) fe) — re 13 (o) gq mW Oliol/s (rial saz]s Cay (psoas (at sel aL a ae | co) ey = Lo WA ak ae) PeISlelSlololslslelal ele leslie lseizlelol P| | wes Oe (BISISISIS [El S/EIEIS lee Flsele |e! ele] 2 | azo 8] & PIsis ie | #(F1/8/8 1/8/5188] 3 |or|e 181318] g | 88 eo! sal G2 Beye] 2] s ojge| |Elalelea| Se ee & ala] ez @ | fh : Ba pi Ey ‘WIOT OU, o | 2 Es = ere: = 3 e - Mal ay B pay “1° | gag |8e ge | ae ‘amyfng jo sosmog 2 *peyenbryT en hs a § & g se ot + 5 Ks a | & g os 2 8 *soinyng Py gS g "qeIg UTeIED Pao | GE) joqejieg | & | ps Pe) ee | RG : *sainy[nD jo quad Jog a *yeoljg 1e3y 8 (€061 ‘SqTaHd GNV ADNVD) HLIVAH AO auvod ALVIS SLLASQHOVSSVIN AHL JO NOILVIS 7NAWINAIXA AONANMVI AHL LV IIOO “A YOL NOLLVNINVXA ANILNOW AHL NI SISAL SNOIAVA DNISSVd SHYNALINO AO ADVINAOUAAd VARIETIES OF COLON BACILLI 187 On the whole, much of the English evidence tends to the assumption that the atypical forms, or “ paracolon bacilli,” generally represent weakened strains from the intestinal B. coli stock. As Savage says, “we know that nearly all the coli-like organisms in feces are quite typical B. coli, that in sewage a good many atypical varieties are present, and that in contaminated water and soil the proportion present is still larger.” The data tabulated on p. 186 from Gage and Phelps (1903) lead to a similar conclusion. About 60 per cent of the cultures isolated from polluted river water, filtered water, and sewage proved to be typical B. coli, while 41 and 43 per cent of those isolated from spring water and shellfish, respectively, and 48 per cent of these from ice belonged in this class. Contradictory results, indicating a higher proportion of typical forms outside the body than within it, have been obtained by Konrich (1910) in the examination of 2387 different strains isolated in about equal propor- tions from feces, earth, and water. Of the 2387 coli- like microbes studied, 308 were excluded by micro- scopic examination (showing abnormal morphology or positive Gram reaction) or by their liquefaction of gelatin. The other 2079 strains were tested in sugar | media and peptone water with the results tabulated on p. 188. We are inclined to attribute these results of Konrich’s largely to the technique which he employed. It seems to be clearly stated in his paper that he obtained his fecal cultures by diréct plating on solid media, while his earth and water samples were treated to preliminary 188 ELEMENTS OF WATER BACTERIOLOGY BIOCHEMICAL REACTIONS OF 2079 ORGANISMS OF THE COLON GROUP (KonRICH, 1910) Percentage of Positive Results. Medium. Reaction. Fecal | Earth | Water Strains. | Strains. | Strains. Dextrose broth........| Gas production.....| 100- 100 100 Dextrose broth, 46°....| Gas production... .. 59 82 82 Lactose broth. ....... Gas production... .. 77 87 92 Lactose broth: 063s). Acid production. ... 07 I0O 100 Mane iecte te oe aortic Coagulation........ 65 84 80 Neutral red dextrose SRANG Teor ieeres tyne Fluorescence....... 54 68 65 Peptone solution...... Indol production... . 38 57 57 ENdO SRF: 5. 05 ies xas Deep red _ colonies with greenish luster| 60 77 76 enrichment in sugar broths. Such an enrichment would undoubtedly tend to increase the proportion of typical B. coli. Konrich’s own experiments on the storage of pure cultures of colon bacilli in water showed a gen- eral, though not invariable, relative increase of atypical forms as the sojourn in water became long continued. Houston (1911) has recently pointed out the danger that the selective action of enrichment media may confuse the normal relations of the bacterial flora; and in order to obtain a more accurate idea of the relations involved he made an elaborate study of raw water, stored water, and stored and filtered water by direct plating, preceded by the use of various physical concentration methods. The results indicated in the table on p. 189 are of interest. VARIETIES OF COLON BACILLI 189 CHARACTERISTICS OF DEXTROSE FERMENTING = BAC- TERIA FROM RAW, STORED, AND FILTERED WATERS (Houston, 1911) s Per Cent Positive Results. g a Gas Production. Sa 2 ag Source. a| gis 2 ae S| £\8glas| Slag] §| #| 4) s | 83 2) 3/2*/82| g]2*| 2| 8] 8) 8 \S3 Raw river on eee 320] 76 | 49 | 35 | 57 | 44115] 7 | 0-3} 63 | 15 Stored water. .| 232) 57 | 40 | 31 | 35 | 25 | 6 | 0.4) .. | 66] 6 Stored and fil- tered water.| 225] 69 | 24 | 34/51 | 25/ 5] 3] -- | 40] 6 The strains having the combination of positive reac- tions in lactose and peptone solution made up 53 per cent of those isolated from the raw waters, 46 per cent of those from the stored waters, and 34 per cent of those from the stored and filtered waters. MacConkey’s Classification of the Colon Group. MacConkey is not satisfied with this general classifica- tion of colon bacilli into typical and atypical forms, but wishes to go much further, believing that even the so-called “typical B. coli” should rather be con- sidered a complex including a considerable number of ° definite individual types. After a detailed study of the reactions of the lactose-fermenting bacteria of feces he outlined a new classification based on fermentative reactions in the rarer sugars (MacConkey, 1905). Using saccharose and -dulcite he first divided the lactose- fermenting organisms into four groups as indicated in the table on p. Igo. 1909 ELEMENTS OF WATER BACTERIOLOGY PRIMARY SUBDIVISIONS OF THE COLON GROUP Saccharose. Dulcite. Type. _ - B. acidi-lactici = + B. coli (or B. communis) + oa B. neapolitanus (or B. communior) - —_ B. aerogenes The fourth group, fermenting saccharose, but not dulcite, was further subdivided by MacConkey into the B. coscoroba type, which does not liquefy gela- tin or give the Voges and Proskauer reaction, the B. lactis-aerogenes type, which does not liquefy gelatin, but does give the Voges and Proskauer reaction, and the B. cloace type, which liquefies gelatin and gives the Voges and Proskauer reaction. Records of the preva- lence of the four principal groups in human and animal feces and in milk are given in MacConkey’s two papers (1905 and 1909) as well as their relative numbers in a suspension of feces in water after various intervals of time. The results do not, however, appear to us to justify any important practical conclusions. In his later paper MacConkey (1909) carried the sub-division of the colon group much further. He isolated 497 lactose-fermenting bacilli from the feces of man and animals, from sewage, water, grains, etc. All were Gram-negative, fermented lactose, coagulated milk and reduced nitrate. They were subdivided by their action on gelatin, pepton and various fer- mentable substances and by their motility into over 100 types of which the more important have received Po VARIETIES OF COLON BACILLI 191 names. The principal types of this classification are indicated in the table below: MacCONKEY’S CLASSIFICATION OF THE COLON GROUP (MacConkEy, 1909) an Fermentation of |, 4 Bs £= 8) ils os Group. | No. Name. EL PC) be ee ia se) 2/%218 Sis 2 > aes 53/2 138|/3|3| 2/8/2183 3 \|Si2 |4/a|2|a\a eo : BB ois Waclws ke a EE —)4+)+)—]-]4+]-]-I- 2 | B. acidi-lactici....... —|—|+/-|-—|[|+]-|-[- ari levels... 2s. e: +/+)-—]-—/]-]-|4+/-/]+ 4 | B. Griinthal......... =f ey Hf a SE 5 B. vesiculosus... .... —{—/+]—]-—]—|-|]=-]— If. | 34 | B. coli communis.....) —| +] +/—{|+]—]—|]—|]— 35 | B. Schafferi......... —|—/|+/-—14+]-l|-|-|- Ill. | 65 | B. oxytocus pernicio- | Se ey rae +)—}+)+})4+/4+/}/4+/4+]+4+ 68 | B. pneumoniz....... —}|—j}-/|+/+/+]-/|4+]- BE Nasiaie a safcatsare saver ks Khe —|+)+i+i4+i-|-j-|- 72 | B. neapolitanus...... —|—|+14+/4+]/-l|-|-|]- I Gee Saas Bete drop eras +)+i—}[+i4+]-—-|-!}- I+ IV. |103 | B. lactis aerogenes...| —| —| —|+|—|+]—|+]4+ 104 | B. gasoformans...... —}—}—-j}+}]-—-|4+)]-l[4]/+ 107 | B. coscoroba........ —|—|+/+]-l|-|-|-|- roS | B. cloace. .......... Fit +i — 1H et In this country the MacConkey classification was first adopted by Bergey and Deehan (1908). These workers used 8 diagnostic characters, motility, indol production, liquefaction of gelatin, the Voges-Pros- kauer reaction, and the fermentation of saccharose, dulcite, adonite and inulin. They tabulated 256 different combinations of these 8 characters and in the examina- 192 ELEMENTS OF WATER BACTERIOLOGY tion of 92 colon-like bacilli from 50 samples of milk, 8 of sewage and 1 of kefir they found 43 of the possible combinations. Copeland and Hoover (1911) have recently urged the importance of these fermentative reactions in the rarer carbohydrates in the study of the colon group. They confirm the positive Voges and Proskauer reaction reported by other observers for B. lactis-aerogenes and B. cloace and point out that B. lactis-aerogenes is the only form in a considerable series studied which gives a brown coloration in aesculin media in one day. On the other hand they record a positive dulcite reac- tion for B. lactis-aerogenes and B. cloace which is highly confusing and makes it difficult to interpret their results. Both these names according to the usage of “MacConkey, which has been accepted for the past five years, are applied to dulcite-negative saccharose. positive organisms. Still another classification of the colon group is Jack- son’s modification of MacConkey’s scheme in which MacConkey’s four primary groups are symmetrically subdivided according to reactions in mannite and raffi- nose with motility, indol production, nitrate reduc- tion, liquefaction of gelatin and coagulation of milk as secondary differential characters (Jackson, 1911). Under each of the four groups, B. communior (MacConkey’s B. neapolitanus), B. communis (MacConkey’s B. coli), B. aerogenes (MacConkey’s Group IV), and B. acidi- lactici, he distinguishes four types, A (fermenting both mannite and raffinose, B (mannite+, raffinose—), C (mannite—, raffinose +), and D (fermenting neither VARIETIES OF COLON BACILLI 193 mannite nor raffinose); and he indicates reactions in other media by subscript letters. These types with their subtypes are fully discussed in the last report of the Committee on Standard Methods of Water Analysis (1912). Clemesha’s Investigation of Stored Waters in India. The most suggestive contribution to this subject which has been made in recent years is a book by Major W. W. Clemesha of the Indian Medical Service on The Bacteriology of Surface Waters in the Tropics (Clemesha, 1912*), in which a vigorous argument is made for the MacConkey classification in practical water work. Major Clemesha’s researches show the prevalence of considerable numbers of all of MacConkey’s primary types in human and bovine feces as indicated in the table below, although the relative proportions found in England and in India do not correspond very closely. Clemesha’s percentages are of special importance because they are based in each case on over 1000 colonies. RELATIVE PROPORTION OF MacCONKEY’S GROUPS IN HUMAN F#CES AND IN COW DUNG Human Feces. Cow Dung. Group. MacConkey. Clemesha. MacConkey. Clemesha._ I 34 53 17 poe] 2 38 17 25 9 3 15 7 48 16 4 I2 | 22 12 35 Both in human feces and in cow dung Clemesha finds the prevailing types to be B. coli, B. Griinthal, 194 ELEMENTS OF WATER BACTERIOLOGY and B. coscoroba, the three together usually making up 75 per cent all the lactose-fermenting organisms present. A very interesting point brought out in these investigations was the occurrence of “ epidemics,” of particular types which at certain periods become suddenly frequent, usually prevailing in human feces, cow feces and water supplies at the same time. (It should be noted for the benefit of anyone studying Clemesha’s book that the tabular classification of the colon group at the end contains a serious misprint. B. lactis-aerogenes, B. gasoformans, B. coscoroba and B. cloace are there given as_ saccharose negative, whereas they should be saccharose positive.) The discussion in the text, however, appears to refer to the orthodox MacConkey types. Clemesha (1912*) made a number of experiments on the relative resistance of the various lactose-fermenting types by placing feecal emulsions, with or without sand, in shallow dishes in the sunlight and at various intervals isolating 10 colonies of the predominant types and working out their fermentative reactions. In general the experiments showed B. coli to be the dominant form at the beginning. It quickly disappeared, however, and after a few hours B. lactis aerogenes, B. acidi-lactici, B. cloace and others appeared. At the end of the experiments, often on the second day, B. Griinthal or B. cloacze were generally the only forms surviving. In a long series of examinations of Red Hills Lake Clemesha obtained 138 colonies of lactose-fermenting organisms during rainy periods and of these 59 belonged to MacConkey’s Group I, 1o to Group II, 14 to Group III and 55 to VARIETIES OF COLON BACILLI 195 Group IV. Of 280 colonies isolated during dry periods, 37 belonged to Group I, 22 to Group III and 221 to Group IV. When the forces of self-purification had been at work, Group II (B. coli) entirely disappeared and Group IV (B. cloace and B. coscoroba) was pre- dominant. B. Griinthal was the commonest of the Group I forms. B. cloace was especially prevalent in bottom samples. A study of a number of rivers in Bengal gave the results tabulated below. RELATIVE PREVALENCE OF CERTAIN LACTOSE- FERMENTING TYPES IN BENGAL RIVERS MacConkey Dry Weather, | Wet Weather, Group. Types. Dec.-June. | July-Nov. I B. Griinthal and B. vesiculosus 41 23 II B. coii communis. .......... 3 13 IV. B. lactis aerogenes ........-. 7 19 Do RSD COACH. Stee cin cis ein ae oe II 4 There are many irregularities in Dr. Clemesha’s results. For example, B. aerogenes, as well as the other _ representatives of Group IV, was more abundant in Red Hills Lake during dry periods than at times of rain. On the whole, however, it does seem clear that his results justify a general classification of the lactose- fermenting organisms into three main groups accord- ing to resistance. B. coli communis and B. oxytocus perniciosus (representing MacConkey’s Groups II and III, both fermenting dulcite) are sensitive organisms found in numbers only where pollution is fresh. B. lactis- aerogenes, representing the subgroup of MacConkey’s 196 ELEMENTS OF WATER BACTERIOLOGY Group IV which ferments adonit, but does not form indol or liquefy gelatin, occupies a somewhat inter- mediate position, appearing in waters which have been fairly recently polluted and later disappearing again. Finally B. Griinthal and B. vesiculosus (MacConkey’s Group I, negative in both saccharose and dulcite) and B. cloace and B. coscoroba (of MacConkey’s Group IV, dulcite negative and saccharose positive), are highly resistant organisms which occur in relatively high proportions in stored waters. B. cloace is most abundant in bottom sediments and B. Griinthal and B. vesiculosus in sunned surface-waters. The moral drawn by Major Clemesha is that for Indian conditions with waters stored in warm sunned lakes and large rivers, where sensitive fecal bacteria have ample opportunity to die out and resistant fecal bacteria have an ample opportunity to multiply, it is not proper to condemn water containing any members of the colon group without distinguishing between the more and the less resistant forms. For example, he quotes 239 examinations of which only 74 showed no B. coli according to the English standard, which closely - corresponds to our own, while 165 showed what we should call positive results. Of the 165, however, 69 contained only the highly resistant B. Griinthal and 59 contained mixtures of other forms not belonging to MacConkey’s Group II (saccharose negative, dulcite positive). Thus of the 239 samples 31 per cent would have been passed by Houston’s standard, 53 per cent would have been condemned by Houston’s standard, although containing only resistant forms which Clemesha — eee —— VARIETIES OF COLON BACILLI 197 believes to be unimportant, and 16 per cent would be condemned by Clemesha as containing true B. communis. Major Clemesha does not claim that these results necessarily indicate any change of procedure in dealing with the waters of temperate climates. Indeed, the experience of English and American bacteriologists offers pretty conclusive evidence that waters so stored as to be safe do not contain large numbers of lactose- fermenting organisms of any type. In other tropical countries and perhaps in warm summer weather, the Indian conditions may possibly be duplicated (as we know they are in the case of the forms fermenting dextrose but not lactose); and the experiments reported in this book deserve the careful consideration of water bacteriologists and sanitarians. The results obtained by Houston (1911) in London unfortunately do not correspond at all with these Indian data. Houston studied in detail the reactions of about 800 strains of dextrose-fermenting bacteria from raw river-water, stored water, and stored and filtered water. Comparison of the relative prevalence of types from these three sources ought to furnish some confirmation of Major Clemesha’s conclusions, even although the extreme conditions of warmth and sun- light are lacking. We find, however, on careful study of the figures that they do not. The Houston types corresponding to B. communis, B. Schafferi and B. neapolitanus (sensitive forms) are on the whole but little more prevalent in the raw than in the stored and filtered waters. On the other hand the types corresponding to B. Griinthal, B. vesiculosus, B. cos- 198 ELEMENTS OF WATER BACTERIOLOGY coroba and B. cloace (Clemesha’s resistant types) are less abundant in the filtered and stored than in the raw water. Houston’s lactose-fermenting forms clas- sified in MacConkey’s four great groups show the rela- tions indicated in the table below, which are almost the reverse of what should be expected if the dulcite- fermenting forms (Groups II and III) were indicative of recent pollution. DISTRIBUTION OF MacCONKEY’S GROUPS IN RAW, STORED, AND FILTERED WATER AT LONDON Percentage in Group. "Reactions. Type. Raw | Stored |Filtered Water. |Water. | Water. I |Saccharose—Dulcite—|B. acidi-lactici...} 34 39 37 II |Saccharose— Dulcite+|B. coli.......... 23 25 38 III |Saccharose+Dulcite+|B. neapolitanus. .| 15 26 9 IV |Saccharose+ Dulcite —|B. lactis-aerogenes| 28 Io 16 Statistical Classification of the Colon Group. From a biological standpoint, there is a twofold difficulty with such a classification as that of MacConkey and Jackson. In the first place it is enormously complex, or soon becomes so, as new investigators add new diagnostic tests. In the second place, it is entirely arbitrary in its choice of the order in which particular tests are to be used in splitting up the group. Closely related forms may be widely separated if they chance to differ in the one respect first chosen for dichotomic division. The best basis for a classification following natural bio- logical lines seems to us to be the statistical method first VARIETIES OF COLON BACILLI 199 suggested by Andrewes and Horder (1906) and Winslow and Winslow (1908) in the study of the cocci. The essential point about this method is that the characters of the organisms studied are not considered indepen- dently, but in relation to each other. The individual reactions are first studied quantitatively in a considerable series of allied strains, so that those types of reaction which are manifested by a large number of strains may be distinguished from the rarer intermediate variations. In the second place, the correlations be- tween different characters are used as a basis for group- ing the types on the assumption that a coincidence in several characters indicates a closer relationship than any single character alone. The statistical method has been applied to the colon group in three recent studies which promise at last to lay a foundation for a sound knowledge of the relation- ships of the colon group. Howe (1912) in his investigation dealt with 630 strains of fresh intestinal colon bacilli. He concluded from his exhaustive study that in bacilli of this type isolated directly from stools, the characters of motility, indol formation, ammonia production, nitrate reduction, fermentation of mannite, dulcite, and starch were not sufficiently correlated with each other or with other characters to be of classificatory value. Dextrese, lactose, saccharose and raffinose he found to-constitute a natural metabolic gradient, in the order named, fermentation of any member ofthe series implying fermentation of those preceding it. Fifty-three per cent of his strains fermented all four sugars, 5 per cent all 200 ELEMENTS OF WATER BACTERIOLOGY but raffinose, 41 per cent attacked dextrose and lactose only, and 1 per cent dextrose alone. Rogers, Clark and Davis (Journal of Infectious Diseases, 1914, XIV, 411) have made a most impor- tant contribution to our knowledge of the colon group. The most striking point is perhaps the fact that when ‘the gas produced by these organisms in dextrose broth is collected under a vacuum and analyzed with proper precautions, two groups may be clearly distinguished by the ratio of hydrogen to carbon dioxide produced. One well-defined group including half the B. com- munior strains, all the B. communis strains, one-fifth of the B. aérogenes strains and four-fifths of the B. acidi-lactici strains studied gave a constant ratio of about one part of carbon dioxide to one part of hydro- gen, while the rest gave a ratio of over 1.4 to 1.0 and usually 2 to 1 or more. The high-ratio group was usually saccharose-positive, dulcite-negative. The low-ratio group included strains of more widely differing reactions. In a subsequent study, Rogers, Clark and Evans (Journal of Infectious Diseases, 1914, XV, too) present a most significant comparison of the types of colon bacilli present in milk and in bovine feces, respectively. Of 125 strains isolated from milk, about half belonged to the high-ratio group defined above (presumably corresponding to B. aérogenes), while in 150 cultures from bovine feces this high-ratio group was found only once. The organisms of the low-ratio group in both milk and bovine feces were divided rather sharply into a saccharose-positive dulcite-positive type (presum- ably B. communior) and a saccharose-negative generally dulcite-negative type (presumably B. acidi-lactici). — ee VARIETIES OF COLON BACILLI 200a Kligler (Journal of Infectious Diseases, 1914, XV, 187) has analyzed the reactions of 80 strains of bacteria of the colon group in the collection of the American Museum of Natural History. His most important conclusion is, that after the primary division of the | lactose-fermenting bacteria has been made on the basis of saccharose fermentation (separating B. com- munis and B. acidi-lactici on the one hand, from B. communior and B. aérogenes on the other), salicin rather than dulcite is the carbohydrate that is of next importance. The tables quoted below indicate that the salicin fermentation is much more closely correlated with the Voges and Proskauer reaction, the liquefaction of gelatin and the fermentation of glycerine than is that of dulcite. CORRELATION OF DULCITE AND SALICIN FERMENTA- TION, RESPECTIVELY, WITH OTHER CHARACTERS OF BACILLI OF THE COLON GROUP. (Kticter, 1914.) Or- | Indol Vv and P Glycerin Gelatin Salicin gan-| Posi- | Posi- Fer- | Lique- Fer- isms.| tive. tive |menters.| fiers. | menters. Saccharose-positive Dul- % % % % % cite-positive........... 20] 50 20 85 5 50 Saccharose-positive Dul- cite-negative........... 20 25 70 7° 15 go Saccharose-negative Dul- cite-positive. .......... 9 | 100 ° 100 ° 89 Saccharose-negative Dul- cite-negative........... 8 | 63 ° 88 ° 38 Dulcite Fer- Saccharose-positive Sali- te rk cin-positive............ 28 21 68 68 14 35 Saccharose-positive Sali- cin-negative........... eH) key ° 100 ° 83 Saccharose-negative Sali- cin-positive............ II | 100 ° 100 ° 73 Saccharose-negative Sali- cin-negative........... 6! so ° 83 ° 17 200b ELEMENTS OF WATER BACTERIOLOGY Kligler, therefore, defines B. aérogenes as saccharose- positive salicin-positive (generally dulcite-negative); B. communior as saccharose-positive salicin-negative (generally dulcite-positive); B. communis as saccharose- negative salicin-positive (generally dulcite-positive) ; and B. acidi-lactici as saccharose-negative salicin- negative (generally dulcite-negative). B. cloace may be separated from B. aérogenes not only by the lique- faction of gelatin, but by the failure to ferment glycerine. Kligler’s general summary of the relationships of the dextrose fermenting bacteria is reproduced below: ce ond + Salicin+ Salicin— Salicin+ Salicin— Salicin+ Salicin— Gelatin Gelatin— Indol+ Indol+ Indolt Indol + Indol- B.proteus B.sulpestifer B.communior B.coli communis B,acidi-lactici Paravolon b, B.entiritidis (cholerse-suis) Glycerint Glycerin— Gelatin— Gelatin+ Indol # Indol— + B.acrogenes _B.cloacae Since Rogers and his colleagues did not use salicin in their tests, while Kligler did not determine the gas ratio, it is uncertain how far their results correspond, and it is to be hoped that their classifications may soon be compared in detail by the simultaneous appli- cation of both procedures. The fact that Rogers and his associates find the high-ratio group characteristic of milk but not of feces, and their recent suggestion (at the Jacksonville meeting of the A. P. H. A.) that this high-ratio group is derived from grains and grasses may prove of the greatest significance in sanitary water analysis. CHAPTER IX OTHER INTESTINAL BACTERIA Ir would be an obvious advantage if the evidence of sewage contamination, furnished by the presence of the colon group, could be reinforced and confirmed by the discovery in water of other forms equally char- acteristic of the intestinal canal. The attention of a few bacteriologists in England and America has been turned in this direction during the past few years; and two groups of organisms, the sewage streptococci and the anaerobic spore-bearing bacilli, have been described as probably significant. . Significance of the Sewage Streptococci. The term “sewage streptococci,’ as generally used, covers an ill-defined group, including many cocci which do not occur in well-marked chains. Those most commonly found grow feebly on the surface of ordinary nutrient agar, producing faint transparent, rounded colonies, but under semi-anaerobic conditions flourish better, giving a well-marked growth along the gelatin stab and only a small circumscribed film on the surface. They are favored by the presence of the sugars and ferment dextrose and lactose, with the formation of abundant acid but no gas. They are seen under the microscope as cocci, occurring as a rule in pairs, 201 202 ELEMENTS OF WATER BACTERIOLOGY | short chains, or irregular groups. They do not show visible growth and do not form indol and nitrite in the standard peptone and nitrate solutions; most of them do not liquefy gelatin, though occasionally forms are found which possess this power. Until recently no systematic study of the varicus species found in the intestine had been made and all cocci giving the char- acteristic growth on agar and strongly fermenting lactose are commonly included as “ sewage streptococci.” Although the significance of the streptococci as sewage organisms is not established with the same definiteness which marks our knowledge of the colon group, these forms have been isolated so frequently from polluted sources and so rarely from normal ones that it now seems reasonable to regard their presence as indicative of pollution. Although originally reported by Laws and Andrewes (Laws and Andrewes, 1894), their importance was not emphasized until 1899 and 1900, when Hous- | ton (Houston, 1899, 1900”) laid special stress upon the fact that streptococci and staphylococci seem to be characteristic of sewage and animal waste, the former being, in his opinion, the more truly indicative of dangerous pollution, since they are “ readily demon- strable in waters recently polluted and seemingly altogether absent from waters above suspicion of con- tamination.” In six rivers recently extensively sewage- polluted, he found streptococci in from one-tenth to one ten-thousandth of a c.c. of the water examined, although in some cases the chemical analysis would not — have indicated dangerous pollution. On the other hand, eight rivers, not extensively polluted, showed OTHER INTESTINAL BACTERIA 203 no streptococci in one-tenth of a c.c., although the chemical and the ordinary bacteriological tests gave results which would condemn the waters. Horrocks (Horrocks, 1901) found these organisms in great abun- dance in sewage and in waters which were known to be sewage-polluted, but which contained no traces of Bacillus coli. He found by experiment that B. coli gradually disappeared from specimens of sewage kept in the dark at the temperature of an outside veranda, while the commonest forms which persisted were varieties of streptococci and staphylococci. In America attention was first called to these organisms by Hunnewell and one of us (Winslow and Hunnewell, 1902*), and the same authors later (Winslow and Hunne- well, 1902”) recorded the isolation of streptococci from 25 out of 50 samples of polluted waters. Gage (Gage, 1902), from the Lawrence Experiment Station, has reported the organisms present in the sewage of that city, while Prescott (1902”) has shown that they are abundant in fecal matter and often overgrow B. coli in a few hours when inoculations are made from such material into dextrose broth. In the monograph of Le Gros (Le Gros, 1902) of the many streptococci - described, all without exception were isolated, either from the body or from sewage. Baker and one of us (Prescott and Baker, 1904), found these organisms present in each of 50 samples of polluted waters. On the other hand, in the study of 259 samples of presuma- bly unpolluted waters, by the method of direct plating, Nibecker and of the authors (Winslow and Nibecker, 1903) found streptococci in only one sample. Clemesha 204 ELEMENTS OF WATER BACTERIOLOGY > (1912°) finds that streptococci in India are present in .coo1 or .cooor gm. of feces, but are rare in waters unless very grossly polluted. In a series of bottle experiments in the laboratory and in the study of an artificially polluted tank outdoors he showed that they disappear very rapidly in water, within 2 or 3 days at the most. Gordon (1904) showed that certain strep- tococci are abundant in normal saliva and are found in air which has been exposed to human pollution but not in normal air. On the whole there can be no doubt of the fact that streptococci occur on the surfaces of the human and animal body more commonly than anywhere else in nature. Isolation of Sewage Streptococci. The isolation of these organisms either from plates or liquid cultures is easy. On the lactose-agar plate, made directly from a polluted water, the colonies of the streptococci may generally be distinguished from those of other acid- formers by their small size, compact structure, and deep-red color, which is permanent, never changing to blue at a later period of incubation. Developing somewhat slowly, however, they may be overlooked if present only in small numbers. In the dextrose- broth tube, streptococci will generally appear in abun- dance after a suitable period of incubation. Prescott and Baker, in the work above mentioned, found that with mixtures of B. coli and streptococci in which the initial ratios of the latter to the former varied from 1:94 to 208: 1, the colon bacilli developed rapidly during the early part of the experiment, reaching a maximum after about 14 hours, and then diminishing OTHER INTESTINAL BACTERIA 205 rapidly. The streptococci first became apparent after to to 15 hours and reached their maximum after 20 to 60 hours, according to the number originally present. Applying the same method to polluted waters, similar periodic changes were observed; nearly pure cultures of B. coli were first obtained, then the gradual displace- ment of one form by the other took place, and at length RELATIVE GROWTH OF B. COLI AND SEWAGE STREPTO- COCCI FROM POLLUTED WATERS IN DEXTROSE BROTH (PRESCOTT AND BAKER, 1904) | Sample Number........... | 2 ee i Red _ colonies inal sam | from I c.c. of original sam- ple on litmus lactose agar a{silelz|s || x | | 4) Io 9 : 8 55 a 460/1250) 105 | 68. 200} 185| 400 130) 332| 420] 410 B. coli] 0} 20) Strept o| 0o| 16 B. coli 200} 76) 130) 270) 220) 210| 140) 420| 285} 410 Number found, | |hrs. in millions per cubic centime- ter, after; | 23 growth in dex- } |hrs Strept 40] 25) 20) 10) 45) 390 20 210) 75| 145 B. coli 280 150| 385) 370, 300) 570, 200) 405) 320) 300 ° } | | } recs trose broth for Strept 140 85 280 170} 3900/1700) II0) 350) 370) 350 Various peri- Z Sol Bee iar ee pa ee esc DORIS cece xs 32 B.coli 0 O} 25} IXO} © 210)" ZO) 24) 10S “\Strept 474/420] 480) 300] 390] 170 400) 105) 250 63 B.coli 0} o ° ° oOo} 12 8 o o o ol "|\Strept 2) o o| 45 | 2} 45) r50| 86! 170 First gas noted after Chee): | ro} 10 9| 9 r9| 8} xo} 6} 6 8 the streptococci were present either in pure culture or in great predominance as shown by the accompany- ing tables. The samples of water were plated directly upon litmus lactose agar and the plates were incubated at 37° for 24 hours, when the red colonies were counted. At the time of plating, 1 c.c. from each sample was also inoculated into dextrose broth in fermentation tubes, 206 ELEMENTS OF WATER BACTERIOLOGY > which were likewise incubated at 37°. After various periods, as indicated by the tables below, the tubes were shaken thoroughly and 1 c.c. of the contents withdrawn. This was diluted (generally 1-—1,000,000,) with sterile water, plated on litmus lactose agar in the usual way, and incubated for 24 hours. The colonies of B. coli and streptococci were distinguished micro- ’ RELATIVE GROWTH OF B. COLI AND SEWAGE STREPTO- COCCI FROM POLLUTED WATERS IN DEXTROSE BROTH (PRESCOTT AND BAKER, 1904) Sample Numbers. <= stains wae 18 | .19.| 20 | 21°] 22) 23.4084 lege ey of original sample on litmus lac- Red colonies developing from I c.c. | BORG) AAT oss ieee se ele SisiPibieee osetia ale *| Strept. Olsael sess fe) oO fe) oO fe) B. coli} 266] roo] 88] 350] 510} 380] 330] 160 ~ "| Strept.| 150 0} 40] 140] 240] 128] 80} 220 Number found, in mil- lions per cubic centi- B. coli} 520] 610| 72} 700/1000} 740} 100] 300 meter, after growth{ i in dextrose broth for 28 various periods.... ‘| Strept.| 800] 860) 670|1080/2500/4380]... .|3900 B. coli fs) Oo} r0| 22| 36 », 7 40 hrs. | Strept.| 252] 330] 260| 22! 66] 60| 52 52 B. coli Io} 16) 38) 20] 70} 35} Io} 27 hrs. ‘| Strept.| 40} 16) 3.8] 31] 412| 25] 10} 30 scopically, and by difference in color and general characters. . The successive growth of these two intestinal groups in the same dextrose-broth tube suggests the following method for the detection of both B. coli and sewage streptococci. Inoculate the desired quantity of water, preferably —— OTHER INTESTINAL BACTERIA 207 I c.c., into dextrose broth, in a fermentation tube, and incubate at 37°. After a few hours’ incubation examine the cultures for gas. Within 2 or 3 hours’ after gas formation is first evident, plate from the broth in litmus lactose agar, incubating for 12 to 18 hours at 37°. If at the end of this time no acid-produc- ing colonies are found, it is probably safe to assume that there were no colon bacilli present. On the other hand, if red colonies are developed, these must be fur- ther examined by the regular diagnostic tests for B. coli. After the first plating from the dextrose broth, replace the fermentation tube in the incubator and allow it to remain for 24 to 36 hours, then plate again on litmus lactose agar. This plating should give a nearly pure culture of streptococci if these organisms were originally present in the water. Streptococci as Indicators of Recent Pollution. The comparative relation of the streptococci and the colon bacilli to sewage pollution is still somewhat uncertain. Houston (Houston, 1900) held that the former microbes imply “ animal pollution of extremely recent and there- fore specially dangerous kind,” and Clemesha’s experi- ments led to the same conclusion. Horrocks (Horrocks, 1901), on the other hand, maintains, largely on the ‘strength of certain experiments with stored sewage, that the streptococci persist after colon bacilli have disappeared and indicate contamination with old sewage which is not necessarily dangerous. These discordant results are probably to be explained by the difierent media in which the viability ‘of the bacteria was com- pared. It seems likely that in sewage where there is a 208 ELEMENTS OF WATER BACTERIOLOGY. large amount of organic food material present the streptococci may kill out the colon bacilli as they: do in the fermentation tube, and as we know they fre- quently do in shellfish. This would explain Horrocks’ results. On the other hand, there is good evidence that the streptococci are less resistant than B. coli to the unfavorable conditions which exist in water of ordinary organic purity. In waters of potable char- acter B. coli is frequently present without the strep- tococcus; and a negative test for streptococci has little significance. A positive test, on the other hand, furnishes valuable confirmatory evidence of pollution. This evidence is of course of special importance when through the activity of the streptococci themselves, or from any other cause the colon isolation has yielded an erroneous negative result. The English Committee appointed to consider the standardization of methods for the bacterioscopic examination of water (1904) by a majority vote rec- ommended the enumeration of streptococci, as a routine procedure in sanitary water analysis, but ‘ in this country the Committee on Standard Methods of Water Analysis (1912) has concluded that ‘‘the information afforded by the occurrence of these organisms seems to be of less value than in the case of B. coli and it is believed that for the present at least, the streptococcus test is of subordinate importance.” Use of the Streptococci to Distinguish between Human and Animal Pollution. There seems some reason to hope that the streptococci may prove of assistance in the important task of differentiating human and animal OTHER INTESTINAL BACTERIA 209 pollution, a task in which all other tests have so far failed. Unlike the colon bacilli, streptococci from the intestines of cattle and men appear to belong to dis- tinct types. The recognition of this fact we owe primarily to Gordon (1905), who made an elaborate study of the fermentative power of the streptococci in a long series of carbohydrate media. His work and that of Houston (Houston, 1904; Houston, 1905", Houston, 1905”) have made it clear that the streptococci of the herbivora differ from those found in the human body in their low fermentative power. In their review of the genus, Andrewes and Horder (1906) describe the type characteristic of the herbivora under the name, Str. equinus, and define it by its failure to ferment lactose, raffinose, inulin or mannite, or to reduce neutral red. Five other types are described from the human mouth and intestine; all of them ferment lactose, and most reduce neutral red and fer- ment raffinose. The commonest intestinal form clots milk, reduces neutral red and ferments saccharose, salicin, coniferin and mannite. The specific types of the genus Streptococcus, grade into each other by almost imperceptible degrees, and streptococci ferment- - ing lactose and raffinose and reducing neutral red are sometimes found in bovine feces; but the studies made in this country by Winslow and Palmer (1910) confirm the conclusions of the English observers that there are specific differences between the streptococci of the human, bovine, and equine intestines. The most im- portant of these results are indicated in the table below: 210 ELEMENTS OF WATER BACTERIOLOGY COMPARATIVE FERMENTATIVE ‘POWER OF STREPTO- COCCI FROM THE HORSE, THE COW, AND MAN (WINSLOW AND PALMER, I9gI0) Percentage of Positive Results (300 Strains). Streptococci. Lactose. Raffinose. Mannite. EV URNA 95 oss acne Mose cece 62 Re 28 EQUINE. Santee ese 8 4 Hu BOVINE. o's cis 5s.cs Saoirse 52 28 6 ‘The rarity of lactose-fermenting streptococci in the horse makes it probable that this group can be used for distinguishing pollution by street washings from that due to domestic sewage; and the fact that a considera- bly larger proportion of human strains attack mannite and a considerably larger proportion of bovine strains ferment raffinose should make it possible to use the ratio between results in these two media to distinguish between the wash from pastures and cultivated land and sewage. Clemesha (1912*) in India found that ' both human and bovine fecal streptococci fermented raffinose, saccharose, and salicin, but not mannite. Fuller and Armstrong, however (Journal of Infectious Diseases, 1913, XIII, 442), confirm the results obtained by Winslow and Palmer, finding that lactose-fermenting streptococci of any kind are rare in horse dung, that the streptococci of bovine feces characteristically fer- ment lactose and raffinose, while the fermentation of lactose and mannite is usual among human fecal strains. The results of these workers were more con- sistent than those tabulated above, 65 per cent of the human strains being of the S. fecalis type (man- —* OTHER INTESTINAL BACTERIA 211 nite-positive), while 64 per cent of the bovine strains were of the S. salivarius (raffinose-positive) type. Rogers and Dahlberg (Journal of Agricultural Re- search, 1914, I, 491) also find that streptococci from bovine feces typically ferment raffinose and not man- nite, while those from the mouth of the cow are fre- quently of the S. fecalis type. The Anaerobic Spore-forming Bacilli. The English bacteriologists have ascribed much importance as indicators of sewage pollution to another group of organ- isms, the anaerobic spore-forming bacilli, of which the form described as B. aerogenes capsulatus (Welch and Nuttall, 1892) and now called B. welchii, and the form isolated by Klein (Klein, 1898; Klein, 1899) in 1895 in the course of an epidemic of diarrhoea at St. Bartholomew’s Hospital, described under the name of B. enteritidis sporogenes (now called B. sporogenes) are types. The procedure originally described by Klein for isolating B. sporogenes is as follows: a portion of the sample to ke examined is added to a tube of sterile milk, which is then heated to 80° C. for 10 minutes to destroy vegetative cells. The milk is next cooled © and incubated under anaerobic conditions, which may be accomplished most conveniently by Wright’s method. A tight plug of cotton is forced a quarter way down the test-tube, the space above is loosely filled with pyrogallic acid, a few drops of a strong solution of caustic potash are added, and the tube is tightly closed with a rubber stopper. After 18 to 36 hours at 37° the appearance of the tube will be characteristic if the B. sporogenes 212 ELEMENTS OF WATER BACTERIOLOGY is present. ‘ The cream is torn or altogether dissociated by the development of gas, so that the surface of the medium is covered with stringy, pinkish-white masses of coagulated casein, enclosing a number of gas-bubbles. The main portion of the tube formerly occupied by the milk now contains a colorless, thin, watery whey, with a few casein lumps adhering here and there to the sides. of the tube. When the tube is opened, the whey has a smell of butyric acid and’ is acid in reaction. Under the microscope the whey is found to contain numerous rods, some motile, others motionless.” Since this organism is not present in very large num- bers, even in sewage, the test of a water-supply must be made with large samples, and the concentration of at least 2000 c.c. of water by filtration through a Pasteur filter is recommended by Horrocks as a necessary prelude (Horrocks, 1901). The Committee on Standard Methods of Water Analysis (1912) recommends the following enrichment procedure for the isolation of B. sporogenes which avoids physical concentration. Vari- ous dilutions of the water to be tested are incubated in fermentation tubes containing liver broth for 24 hours at 37°. If B. sporogenes is present gas will be evolved and a characteristic ‘‘ vile odor” will be produced. If this reaction is obtained the contents of each posi- tive tube is transferred to an Erlenmeyer flask or large test-tube and heated at 80° C. for 10 minutes to destroy vegetative cells. One c.c. of broth containing sediment is withdrawn from the bottom of each flask and enriched once more in a fresh liver broth tube. B. sporogenes will now usually be present in pure culture showing OTHER INTESTINAL BACTERIA 213 large sluggishly motile bacilli containing spores. A gelatin stab culture made from these 24-hour liver broth tubes will show after 48 hours incubation at 20° a dis- tinct liquefying anaerobic growth beginning about 2 cm. below the surface with gas bubbles at the top of the liquefied area. In order to obtain absolutely pure - cultures it is necessary to fish from liver broth tubes only 3-5 hours old as only young vegetative cells will grow on plates. Transplants from the closed arm of such tubes will grow on dextrose liver agar plates incu- bated under anaérobic conditions. Another method for isolating B. sporogenes suggested by Wells is simpler than the foregoing. He recommends the use of freshly-boiled lactose broth tubes (heated in Arnold Sterilizer) brought to a temperature of 70 degrees C., at which temperature the inoculation is made. The inoculated tubes are then kept at 7o degrees C. for 10 minutes and subsequently incubated at 37 degrees C. By this means the spore forms of B. sporogenes develop into vegetative cells in the course of 24 hours and give rise to a rapid evolution of gas. Aérobic plates made from these tubes will generally give no growth. On the other hand, if B. coli were present, gas would ordinarily be produced in 18 hours and aérobic plates, either on litmus agar or Endo medium would show an abundance of colonies. The organisms of the B. sporogenes group are large stout bacilli often occurring in chains. They liquefy gelatin vigorously and on agar produce fine discrete gray colonies. They vigorously ferment dextrose, lac- tose and saccharose, producing acid and gas, and in sugar 214 ELEMENTS OF WATER BACTERIOLOGY agar each colony will be marked by one or more gas bubbles surrounded by a delicate whitish fringe. The organism is strongly pathogenic for guinea pigs, by which character it is distinguished from the B. butyricus of Botkin. B. welchii differs from B. sporogenes chiefly in lacking motility and in forming spores with less read- iness (Klotz and Holman, 1911). The researches of Klein and Houston (Klein and Houston, 1898, 1899) have shown that the B. sporogenes occurs in English sewage in numbers varying from 30 to 2200 per c.c. and that it is often absent in considerable volumes of pure water. In Boston sewage it may usually be isolated from .or or .oor of a c.c. (Winslow and Belcher, 1904). Since the spores of an anaerobic bacillus may persist for an indefinite period in polluted waters, their presence need not necessarily indicate recent or dangerous pollution. Vincent (1907) and other French observers consider the determination of the total number of anaerobic bacteria as significant, since the decomposition of organic matter is accompanied by anaerobic growth. It is not claimed, however, that bacteria of this type are characteristic of animal more than of vegetable decompositions, and the total anaerobic count apparently adds nothing of impor- tance to the information gained by the ordinary gelatin plate method. The property of liquefaction was for- merly believed to be of significance, inasmuch as the liquefying bacteria were regarded as indicative of pollu- tion. This position is, however, no longer tenable, since many bacteria, typical of the purest waters, may cause liquefaction. CHAPTER X THE SIGNIFICANCE AND APPLICABILITY OF THE BACTERIOLOGICAL EXAMINATION Sanitary Inspection and Sanitary Analysis. The first attempt of the expert called in to pronounce upon the character of a potable water should be to make a thorough sanitary inspection of the pond, stream, well or spring from which it is derived. Study of the pos- sible sources of pollution on a watershed, of the direc- tion and velocity of currents above and below ground, of the character of soil and the liability to contamina- tion by surface-wash are of supreme importance in interpreting the analyses to be made. In many cases, however, the results of the sanitary inspection will be found to be by no means conclusive. If house or barnyard drainage or sewage is actually seen to enter a water used for drinking purposes it is obviously unnecessary to carry out delicate chemical or bacteri- ~ ological tests to detect pollution. On the other hand, no reconnoissance can show certainly whether unpurified drainage from a cesspool does or does not reach a given well; whether sewage discharged into a lake does or does not find its way to a neighboring intake; whether pollution of a stream has or has not been removed by a certain period of flow. Evidence upon 215 216 ELEMENTS OF WATER BACTERIOLOGY these points must be obtained from a careful study of the characteristics of the water in question, and this study can be carried out along two lines, chemical and bacteriological. Sanitary Chemical Analysis. A chemical examina- tion of water for sanitary purposes is mainly useful in throwing light upon one point—the amount of decom- posing organic matter present. It also gives an his- torical picture which may be of much value. Humus- like substances may be abundant in surface-waters quite free from harmful pollution, but these are stable compounds. Easily decomposable bodies, on the other hand, must obviously have been recently introduced into the water and mark a transitional state. ‘“ The state of change is the state of danger,” as Dr. T. M. Drown once phrased it. Sometimes the organic mat- ter has been washed in by rain from the surface of the ground, sometimes it has been introduced in the more concentrated form of sewage. In any case, it is a warn- ing of possible pollution, and the determination of free ammonia, nitrites, carbonaceous matter, as shown by ‘oxygen consumed,” and dissolved oxygen yield important evidence as to the sanitary quality of a water. Furthermore, nitrates, the final products of the oxida- tion of organic matter, and the chlorine introduced as common salt into all water which has been in contact with the wastes of human life, furnish additional infor- mation as to the antecendents of a sample. The results of the chlorine determination are indeed perhaps more clear than those of any other part of the analysis, for chlorine and sewage pollution vary together, due allow- BACTERIOLOGICAL EXAMINATION 217 ‘ance being made for the proximity of the sea and other geological and meteorological factors. Unfortunately, it is only past history and not present conditions which these latter tests reveal, for ina ground-water completely purified from a sanitary standpoint such soluble con- stituents remain, of course, unchanged. Thus, in the last resort, it is upon the presence and amount of decom- posing organic matter in the water that the opinion of the chemist must be based. Information Furnished by Bacteriological Examina- tions. The decomposition of organic matter may be measured either by the material decomposed or by the number of organisms engaged in carrying out the proc- ess of decomposition. The latter method has the advan- tage of far greater delicacy, since the bacteria respond by enormous multiplication to very slight increases in their food-supply, and thus it comes about that the standard gelatin-plate count at 20° roughly corresponds, in not too heavily polluted waters, to the free ammonia and “‘ oxygen consumed,” as revealed by chemical analysis. If low numbers of bacteria are found, the evidence is highly reassuring, for it is seldom that water cou'd be contaminated under natural conditions without the direct addition of foreign bacteria or of organic matter which would condition a rapid multiplication of those already present. The bacteriologist in such cases can declare the innocence of the water with justifiable certainty. When high numbers are found the interpreta- tion is less simple, since they may exceptionally be due to the multiplication of certain peculiar water forms. Large counts, however, under ordinary conditions, 218 ELEMENTS OF WATER BACTERIOLOGY — when including a normal variety of forms indicate the presence of an excess of organic matter, derived in all probability either from sewage or from the fresh wash- ings of the surface of the ground. In either case danger is indicated. A still closer measure of polluting material may be obtained from the numbers of colonies which develop on litmus-lactose-agar at 37°, since organisms which thrive at the body temperature, and particularly thos: which ferment lactose, are characteristic of the intestinal tract and occur but rarely in normal waters. Gage (Gage, 1907) has shown that by counts at 20, 30, 40, and 50° C., information may be quickly obtained which is of great assistance in judging the character of the water. ‘““Modern methods of bacterial examination of water, consisting usually of determinations of the numbers of bacteria by means of plates incubated at room tempera- ture, and of tests for the presence or absence of one or two specific types, occasionally lead to an erroneous interpretation of the quality of a water, owing to the fact that they do not yield adequate data by which abnormal and inaccurate results may be separated from those which are truly indicative of purity or pollution. Furthermore, as several days must elapse before the bacterial tests can be completed, the results when obtained may have passed their usefulness. If, however, we can so modify our procedure that the varied char- acter of the bacteria in waters of different classes may be quickly and accurately recognized, the value of bacterial water analysis will be enormously increased. BACTERIOLOGICAL EXAMINATION 219 Much of this information may be obtained by the use of selective media, selective temperatures, or by a proper combination of the two. ‘By the use of litmus-lactose-agar in place of agar or gelatin we obtain similar counts of total bacteria, and in addition are able to separate those bacteria into two groups, which do and do not produce acid fermenta- tion of lactose, and the numbers of the two classes of bacteria so obtained indicate more completely the character of the water than would the numbers of either class alone. By incubating our plates at temperatures of 30 or 40° C. we are able to obtain counts in 12 to 18 hours, which counts, while smaller than those on plates incubated for a longer period at a lower temperature, appear to be fully as significant. If we increase our number of determinations by incubating duplicate plates at two or more temperatures, the various results and the ratios between them furnish a check upon one another in addition to increasing the available data upon which to base an interpretation.” (Gage, 1907.) Finally, the search for the Bacillus coli furnishes the most satisfactory of all single tests for faecal contamina- tion. This organism is preeminently a denizen of the alimentary canal and may be isolated with ease from waters to which even a small proportion of sewage has been added. On the other hand, it is never found in abundance in waters of good sanitary quality, and its numbers form an excellent index of the value of waters of an intermediate grade. The streptococci appear to be forms of a similar significance useful as yielding a certain amount of confirmatory evidence. 220 ELEMENTS OF WATER BACTERIOLOGY The full bacteriological analysis should then consist of three parts, the gelatin-plate count, as an estimate of the amount of organic decomposition in process; the total count, and the count of red colonies, on litmus-lactose-agar, as a measure of the organisms which form acids and thrive at the body temperature; and the study of a series of lactose bile tubes for the isolation of colon bacilli. Special Advantage of the Bacteriological Examination. The results of the bacteriological examination have, in several respects, a peculiar and unique significance. First, this examination is the most direct method of sanitary water analysis. The occurrence of nitrites or free ammonia in a small fraction of one part per million, or of chlorine in several parts per million, do not in themselves render a water objectionable or dangerous. They merely serve as indicators to show that germ-containing and germ-sustaining organic mat- ter is present. By a determination of the chlorine and study of the relations of carbon and _ nitrogen, it is possible to determine with some degree of accuracy whether this organic matter is of plant or animal origin, and hence to rate its objectionable or dangerous char- acter. By the bacteriological examination, on the other hand, we are able to determine directly whether particular kinds of organisms characteristic of sewage are, or are not, actually present in the water. What we dread in drinking-water is the presence of pathogenic bacteria, mainly from the intestinal tract of man, and it is quite certain that the related non-pathogenic bacteria from the same source will behave more nearly BACTERIOLOGICAL EXAMINATION 221 as these disease germs do than will any chemical com- pounds. In the second place, the bacteriological methods are superior in delicacy to any others. Klein and Houston (1898) showed by experiment with dilu- tions of sewage that the colon test was from ten to one hundred times as sensitive as the methods of chemical analysis; and studies of the self-purification of streams have confirmed their results on a practical scale. Thus in the Sudbury River it was found that while chem- ical evidences of pollution persisted for 6 miles beyond the point of entrance, the bacteria introduced could be detected for 4 miles further (Woodman, Winslow, and Hansen, 1902). The statement is sometimes made that while bac- teriological methods may be more delicate for the detection of pollution in surface-waters, contamination in ground-waters may best be discovered by the chemical analysis. That such is not the case has been well shown by Whipple (Whipple, 1903) who cites the fol- lowing two instances in which the presumptive test revealed contamination not shown by the chemical analysis: “ A certain driven-well station was located in swampy land along the shores of a stream, and the tops of the wells were so placed that they were occasionally flooded at times of high water. The water in the stream was objectionable from the sanitary standpoint. The wells themselves were more than 100 feet deep; they pene- trated a clay bed and yielded what may be termed arte- sian water. Tests for the presence of Bacillus coli had invariably given negative results, as might be naturally 222 ELEMENTS OF WATER BACTERIOLOGY expected. Suddenly, however, the tests became positive and so continued for several days. On investigation it was found that some of the wells had been taken up to be cleaned, and that the workmen in resinking them had used the water of the brook for washing them down. This allowed some of the brook-water to enter the system. It was also found that at the same time the water in the brook had been high, and because of the lack of packing in certain joints at the top of the wells the brook-water leaked into the suction main. The remedy was obvious and was immediately applied, after which the tests for Bacillus coli once more became negative. During all this time the chemical analysis of the water was not sufficiently abnormal to attract attention. On another occasion a water-supply taken from a small pond fed by springs, and which was practically a large open well, began to give positive tests for Bacillus coli, and on examination it was found that a gate which kept out the water of a brook which had been formerly connected with the pond was open at the bottom, although it was supposed to have been shut, thus admitting a contam- inated surface-water to the supply.” Whipple also calls attention to the report on the Chemical and Bacteriological Examination of Chichester Well-waters by Houston (Houston, 190i), in which the results of chemical and bacteriological examinations of thirty wells were compared. It was found that the bacteri- ological results were in general concordant and satis- factory. The wells which were highest in the number of bacteria showed also the greatest amount of pollu- tion, as indicated by the numbers of B. coli, B. sporo- te cn a elie Taba BACTERIOLOGICAL EXAMINATION 223 genes, and streptococci. On the other hand, the chlorine and the albuminoid ammonia showed no correspondence with the bacteriological results. Vincent (Vincent, 1905) cites an interesting case of the detection of progressive pollution of a ground- water by bacteriological methods. The well of a military camp in Algeria showed 200 bacteria per c.c. before the arrival of a regiment of troops. Its sub- sequent history is indicated in the table below: PROGRESSIVE POLLUTION OF A WELL (VINCENT, 1905) | Bacteria per c.c. |Bacillus coli per c.c. Before arrival of troops......... 200 ° 6 days-after arrival............. 770 ° 14 days after arrival............ 4,240 I 41 days after arrival............ 6,960 2 60 days after arrival............ 14,900 10 Thirdly, negative tests for Bacillus coli and low bac- terial counts may be interpreted as proofs of the good quality of water, with a certainty not attainable by any other method of analysis. Many a surface-water with reasonably low chlorine and ammonias has caused epi- demics of typhoid fever; but it is impossible, under any natural conditions (except perhaps in a well polluted with urine) that a water could contain the typhoid bacillus without giving clear evidence of pollution in the bile tube or on the lactose-agar plate. In the examination of springs, especially those used for domestic supplies at country houses, the authors have found that the bacteriological examination offers a 224. ELEMENTS OF WATER BACTERIOLOGY more delicate and more certain index of the quality than may be obtained by chemical analysis. In a number of instances, springs located in pastures have become slightly polluted by animals, but to so small an extent that the chemical examination gave no indi- cation of trouble. The bacteria, however, increased greatly in number, and colon bacilli could be readily isolated: from 75 per cent of the 1-c.c. samples of a long series used in making the presumptive test. A single case may suffice as an illustration. This was a spring located on a hill in Hopkinton, Mass. The chemical analysis was as follows: Color Se oisea ds ahiiaiek Ganesan eee ee None BB big 5 1c\ ln See Ween Pelpee aara Oae Pear es ABR UTS None Sedimentaie ecco et arctan cite ie eee Sea None Odorithob) was etic cwaseaio Athen een eee None Odori(cold) ins e ese nin seeseo sites eee ee None Parts per Million. Ota SONGS icc igre ates acces sini haleshee ea ane per 33 .0000 TOSS ON IEHILION ais. Wt, ocis sonteteenste tats ee als 7.0000 Pixed Pestle ns -ctin ie wes ies elon tiate sores eae 26.0000 TOP ATESS oie siare Wins GeO Ae leew ae orcas tee ee II .0000 ChIGHNE Aaah ein Rapa srt ceo ae 10.0000 Nitrogen as— Albuminoidtamimonias s:. sh ..22 ease als otlalene tun 0.0000 Free aMmonlasecs ce ccg sic sis aan wears 0.0000 INitsites: Socre ooo certs sac ones TA ELT 0.0000 INGErStES ic eiclecs amaler vate eaten aa eeane 0.0000 The bacteriological examination showed a total count of 375 bacteria per c.c. and a 37° count of 350 per c.c. The presumptive tests for Bacillus coli showed that gas-producing organisms were present in a majority of 1-c.c. samples, and typical colon bacilli were isolated. In this case the contamination was brought about by cattle gaining access to the area immediately surround- BACTERIOLOGICAL EXAMINATION 225 ing the spring; but the same conditions might easily have led to infection from human beings. Fromme (1910) cites several interesting examples of temporary pollution detectable only by _bacteri- ological tests. The most striking case was that of an artesian well. Its average bacterial content had been 38 per c.c. and colon bacilli were absent from 200 c.c. In May, 1908, this well became polluted from a broken stable drain 10 meters away. The number of bacteria rose to 4370 and colon bacilli were found in 10 c.c. sam- ples. The source of pollution was removed, but the well water in July still contained 7100 bacteria and B. coli in 1 c.c. In September the number had fallen to to5 and colon bacilli were present in 200 c.c. In Novem- ber the bacteria numbered 120 and colon bacilli were absent from 200 c.c. At no time did chemical tests give any indication of danger, while the bacteriological data obviously measured very delicately a comparatively slight but real pollution and its gradual disappearance. Similar results have been reported by Savage and Bulstrode (Savage, 1906) in the examination of the water-supply of Bridgend. It seems to the writers that the real application of chemistry begins where that of bacteriology ends. When pollution is so gross that its existence is obvious and only its amount needs to be determined, the bacteri- ological tests will not serve, on account of their exces- sive delicacy. In studying the heavy pollution of small streams, the treatment of trades wastes, and the purification of sewage, the relations of nitrogenous compounds and of oxygen compounds are of prime 226 ELEMENTS OF WATER BACTERIOLOGY importance. In other words, when pollution is to be avoided, because the decomposition of chemical sub- stances causes a nuisance, it must be studied by chem- ical methods. When the danger is sanitary and comes only from the presence of bacteria, bacteriological methods furnish the best index of pollution. In the study of certain special problems the para-_ mount importance of bacteriology is generally recognized. The distribution of sewage in large bodies of water into which it has been discharged may thus best be traced on account of the ready response of the bacterial counts to slight proportions of sewage, particularly since the ease and rapidity with which the technique of plating can be carried out make it possible to examine a large series of samples with a minimum of time and trouble. The course of the sewage carried out by the tide from the outlet of the South Metropolitan Dis- trict of Boston was studied in this way by E. P. Osgood in 1897, and mapped out by its high bacterial content with greater accuracy than could be attained by any other method. Some very remarkable facts have been developed by similar studies as to the persistence of separate streams of water in immediate contact with each other. Heider showed that the sewage of Vienna, after its discharge into the Danube River, flowed along the right bank of the stream, preserving its own bacterial characteristics and not mixing per- fectly with the water of the river for a distance of more than 24 miles (Heider, 1893). Jordan (Jordan, 1g00), in studying the self-purification of the sewage discharged from the great Chicago drainage canal, BACTERIOLOGICAL EXAMINATION —=§ 227 _ found by bacteriological analyses that the Des Plaines and the Kankakee Rivers could both be distinguished flowing along in the bed of the Illinois, the two streams being in contact, yet each maintaining its own indi- viduality. Finally, the quickness with which slight changes in the character of a water are marked by fluctuations in bacterial numbers renders the bacteri- ological methods invaluable for the daily supervision of surface supplies or of the effluents from municipal filtration plants. In the commoner case, when normal values obtained by such routine analyses are not at hand, the problem of the interpretation of any sanitary analysis is a more difficult one. The conditions which surround a source of water supply may be constantly changing. No en- gineer can measure the flow of a stream in July and deduce the amount of water which will pass in February; yet the July gauging has its own value and significance, so a single analysis of any sort is not sufficient for all past and future time. If it gives a correct picture of the hygienic condition of the water at the moment of examination it has fulfilled its task, and this the bacteriological analysis can do. The evidence fur- nished by inspection and by chemical analysis should be sought for and welcomed whenever it can be obtained, yet we are of the opinion that, on account of their directness, their delicacy, and their certainty, the bacteriological methods should least of all be omitted. CHAPTER XI BACTERIOLOGY OF SEWAGE AND SEWAGE EFFLUENTS Bacteriological and Chemical Examination of Sewage. The first object of modern sewage disposal is the oxida- tion of putrescible organic matter. Chemical, rather than bacterial, purification is usually the prime requisite; and chemical tests therefore serve best as criteria of the results obtained. Bacteria are the agents in the process of sewage purification; but the most generally useful measure of the work accomplished is the chemical oxidation attained. ‘‘ To employ a simile, it is a case of the saw and the 2-foot rule—the saw will do the cutting, but the rule will measure the work cut.” (W. J. Didbin.) In certain cases, however, bacterial as well as chemical purity must be effected, in view of special local require- ments. The sewage from a contagious disease hospital, for example, should be freed from infectious material as a factor of safety. Sewage discharged into a body of water adapted for bathing may well be so treated as to protect those using the water. In the case of seaboard cities where sewage effluents are likely to contaminate oyster beds and other layings of edible shellfish the problem assumes great importance. Where bacterially impure effluents are discharged into streams used for 228 BACTERIOLOGY OF SEWAGE 229 sources of water-supply the town taking water may protect itself by filtration. It should so protect itself, at any rate, from the pollution necessarily incident to surface waters; and, unless the bacterial condition of a stream or lake is made very materially worse by the discharge of sewage effluents, it is fair that the respon- sibility of purification should rest on the water works, rather than on the sewage purification plant. Shell- fish, on the other hand, cannot be purified. Either pollution must be prevented, or the industry abandoned. Under such circumstances sanitary authorities may rightly demand, as they have demanded at Baltimore, that bacteria, as well as putrescible organic matter, shall be removed in sewage treatment. Under such circumstances the bacterial control of purification plants is as essential as in the case of water filters. Methods of Bacteriological Examination of Sewage and Effuents. In England, considerable attention has been devoted to this subject, and numerous methods have been recommended as furnishing valuable criteria of the bacterial quality of sewage effluents. Houston (z902°), for example, suggests various tests involving the use of litmus milk, peptone solution, gelatin tubes, and neutral-red broth, as well as the inoculation of animals. He considers the determination of the num- bers of B. coli and B. sporogenes as of greatest moment, while the identification of streptococci is of value in certain cases and the enumeration of liquefying bacteria, spore-forming aerobes, thermophilic bacteria, and hydro- gen sulphide producing bacteria is of subsidiary impor- tance. Rideal (1906) has recently recommended a some- 230 ELEMENTS OF WATER BACTERIOLOGY what less extensive series of tests, including aerobic and anaerobic counts, both at 20 and 37°, with the determination of the number of liquefiers and the num- ber of spore-formers. The results attained do not seem to warrant any such elaborate procedure. As far as the authors are aware, the determination of liquefying bacteria, anaerobic bacteria and thermophilic bacteria does not add any information of material importance to that obtained from the total count. Some test for specific sewage organisms is of course desirable. Here again, however, the determination of B. sporogenes and sewage streptococci tells the observer little more than can be learned from the routine use of the colon test. In the United States the practise of sewage bacteriologists is crystallizing around the total count and the estimation of B. coli. In the absence of evidence as to the specific value of other data, the routine control of filter plants may well be limited to these two determinations. The total count of bacteria should be made, as in the case of waters, at 20°. Determinations carried out in duplicate at 37° give additional information of considerable value. The ratio of the 37° count to the 20° count varies with different sewages. At Boston the body temperature count is 70 to 80 per cent of the total count; at Lawrence it appears to be propor- tionately much lower (Gage, 1906). In using either medium, it is well to add lactose and litmus and note the number of red colonies, as a check on the enumera- tion of B. coli. It should be borne in mind, as Lederer and Bach- BACTERIOLOGY OF SEWAGE - 931 mann (1911) have recently pointed out, that the sampling error is a very serious one with sewage. Duplicate tests made at 1-minute intervals for a period of 10 _ minutes in their experiments gave extreme values of 190,000 and 550,000 per c.c. The determination of the number of colon bacilli in ‘sewage and effluents should furnish an integral part of bacteriological sewage analysis, since it is important to know whether the decrease of intestinal bacteria in the process of purification is proportional to the reduc- tion of total bacteria. The State Sewerage Commis- sion of New Jersey has adopted this procedure in its supervision of the disposal plants in that State; and the results seem amply commensurate with the labor involved. Asin the case of polluted waters the enumera- tion of B. coli may be carried out, either by the study of the red colonies which appear on litmus-lactose-agar plates inoculated with the sample directly, or by the use of a preliminary enrichment process. The com- plete identification of B. coli seems unnecessarily tedious, however, where the organisms are present in such abundance. Some approximate presumptive method is indicated here, if anywhere; and the experience with polluted water, reviewed in Chapter VI, points to the Jackson bile medium as the most promising one. Experience at the Sewage Experiment Station of the Massachusetts Institute of Technology has shown that this presumptive test in general yields good results. As pointed out above, a 48-hour incu- bation period at 37° is required. All tubes showing 20 per cent gas at the end of this time may be con- 932 ELEMENTS OF WATER BACTERIOLOGY sidered positive tests for the colon group, without serious error. | Numbers of Bacteria in Sewage. The total number of bacteria and the number of colon bacilli naturally vary widely in the sewages of different cities and towns. European sewages, being more concentrated, show as a rule higher numbers than are found in America. Results compiled from various sources show from 1,000,000 to 5,000,000 bacteria in the sewages of Essen, Berlin, Charlottenburg, Leeds, Exeter, Chorley, and Oxford, 2,000,000 to 10,000,000 in the sewages of Lon- don, Walton, and W. Derby and over 10,000,000 in the sewages of Paris, Ballater and Belfast (Winslow, 1905). The number of colon bacilli in English sewages varies from 50,000 to 750,000. In American sewages, on the other hand, bacteria are somewhat less numerous. At Lawrence the determinations made from 1894 to 1gor showed on the average 2,800,000 bacteria per c.c. At Worcester, Eddy reported 3,712,000 in 1901 (Eddy, 1902); at Ames, Iowa, Walker (1901) found 1,248,256 in the same year. At Columbus, Johnson (1905) reports an average of 3,600,000 bacteria per c.c.; the individual numbers varied from 320,000 to 27,000,000. The number of colon bacilli varied from 50,000 to 1,000,000 and averaged 500,000. Day samples of Boston sewage collected three times a week, from October, 1906, to April, 1907, showed an average of 1,200,000 bacteria per c.c. In the summer months numbers are notably higher than at other seasons in many sewages. Thus in 1903, Boston sewage con- tained 2,995,000 bacteria in July, 4,263,600 in August, BACTERIOLOGY OF SEWAGE 233 11,487,500 in September, 3,693,000 in October, 587,100 in November, and 712,000 in December (Winslow, 1905). There is also a marked diurnal variation in the bacterial content of sewage, since the flow con- tains a smaller proportion of intestinal matter at night than at other times. For example, a series of hourly samples at the Sewage Experiment Station of the Massachusetts Institute of Technology showed the - following results: BACTERIA IN BOSTON SEWAGE—AVERAGES FOR EACH FOUR-HOUR PERIOD. AUGUST 13-14, 1903 (WINSLOW AND PHELPs, 1905) Period. Bacteria per c.c. POOREST IO AMO oicnis cactus cas SG nis Bae wet eae 1,800,000 RAEN MES SOO DME 5 wg eb onlin och dba s, be Sin cin © se 3,200,000 RESO BE tn ia sNccity Wk Dacis ee < ores 4,600,000 PEARED RO DM cco oc eisai le ote aie cle siele a sioiaexd 3,500,000 PIERO A590 AM, Sage act se vessels esis cesses ¢ T 000,000 ARES he BOAR hao Sra atnid else = casie, a aielaisty saibie Se 400,000 It is evident that many published results of bacterial examinations of sewage are in excess of the average values, since they refer in most cases to day samples only. | Bacterial Content of Sewage Effiluents. The bacterial content of sewage effluents varies widely according to the process of purification adopted and the efficiency of the particular plant. The only process which yields a notably purified effluent from the bacteri- ological standpoint is that of filtration through sand. Processes of this type when operated with care may give a bacterial purification well over 99 per cent as 234 ELEMENTS OF WATER BACTERIOLOGY shown by bacteriological examinations at the Brockton (Mass.) filters, reported by Kinnicutt, Winslow and Pratt (1910) as follows: BACTERIA IN SEWAGE AND EFFLUENTS AT BROCKTON, AVERAGE OF FOUR EXAMINATIONS, AUTUMN OF 1908 Bacteria per c.c. Colon Bacilli per Gelatin 20°. c.c. Lactose Bile. PIO WANE rscnc cin ates sletionie semctas 3,150,000 150,000 BAMUENE Aes Se x5is ares kate wo 4 nie’ s 1,900 400 - JT rah: ETS Ra ger ats at Coy ted erent Pa 6,300 I5 Apes IVa eels Sioa chee ees 6 125 ° M3 BE io ai ahe ahora anctele e's viele I,400 5 BD pNP Eee ten teats. 2,000 I Such high efficiencies as this table indicates are often not realized under the actual working condi- tions of a municipal plant. At Vineland, N. J., for example, the intermittent filters show a reduction of go to 95 per cent in total bacteria and a somewhat higher reduction of B. coli. The results of three examinations made in 1906 are given below. BACTERIA IN SEWAGE AND SAND FILTER EFFLUENT AT VINELAND, N. J. (N. J. State SEWERAGE COMMISSION, 1907) Bacteria per c.c. B. Coli in Date Sewage. Effivent. Sewage. Effluent. March 23): 4:3 480,000 20,000 ©.0001 C.c. | O.OT C.c. TU 265 si :500 496,000 61,000 ©.0001 C.c. | 0.001 C.C. TUL, 26 0.0055 s.0 511,000 38,000 ©.0000I ¢.Cc. | 0.001 C.C. BACTERIOLOGY OF SEWAGE 235 The newer bacterial processes, contact beds, and trickling filters naturally show a much less satisfactory bacterial removal than sand filtration beds. In the Columbus experiments, Johnson (1905) found from 1,000,000 to 2,000,000 bacteria in the effluents of con- tact beds and from 750,000 to 1,900,000 in the effluent from trickling filters. At the experiment station of La Madeleine, in Lille, Calmette (1907), reports 5,000,000 bacteria per c.c. in the crude sewage, 2,900,000 in the second contact effluent and 800,000 in the effluent from the trickling bed. Of 20,000 B. coli per c.c. applied to the filters, the contact system delivered 4000 and the trickling bed 2000 per c.c. The average results of examinations made three times a week at the Sewage Experiment Station of the Massachusetts Institute of Technology, during two different periods, were as follows: BACTERIA IN SEWAGE, SEPTIC EFFLUENT AND TRICKLING EFFLUENT AT BOSTON (WINSLOW AND PHELPS, 1907) B. Coli. Bacteria per c.c. — ppt mag c.c.* : Jaly-Sept., r006. Oct., ere sg Fn, : Per Cent Per Cent No. Reduc- No. Reduc- Per Cent. tion. tion. Sewage... 2.2.5. £:400;000}) 20/25 oa 1,200,0001) "2 =. 65 Septic effluent... .| 1,650,000! Inc. 750,000! 38 66 Effluent from trickling bed..| 750,000} 42 200,000} 83 35 Septic tank and trickling bed..| 750,000) 42 180,000} 85 35 * Jackson bile test. 236 ELEMENTS OF WATER BACTERIOLOGY The following average data for two of the largest trickling filter plants in the United States are cited by Kinnicutt, Winslow and Pratt (1910). BACTERIAL CONTENT OF SEWAGE AND EFFLUENTS FROM TRICKLING FILTERS Bacteria per c.c. Place. ‘Period. Screened Septic Filter Sewage. Effluent. Effluent. Reading, Pa.-...<. 1908-1909 | 3,100,000 | 1,800,000 600,000 Columbus, Ohio. .. 1909 2,370,000 | 1,050,000 560,000 It is obvious that effluents of this character cannot be considered satisfactory from the standpoint of bacterial purification. As Houston concluded, after a careful review of the subject, ‘‘ The different kinds of bacteria and their relative abundance appear to be very much the same in the effluents as in the crude sewage. Thus, as regards undesirable bacteria, the effluents frequently contain nearly as many B. coli, proteus-like germs, spores of B. enteritidis sporogenes and streptococci, as crude sewage. In no case, seemingly, has the reduc- tion of these objectionable bacteria been so marked as to be very material from the point of view of the epidemiologist ” (Houston, 1902°*). Experimental studies with specific bacteria have confirmed these conclusions. Houston (1904”) found that B. pyocyaneus appeared in the effluent of a trickling bed 10 minutes after application to the top and con- tinued to be discharged for 10 days. In septic tanks and contact beds, the same germ persisted for 10 days. ‘BACTERIOLOGY OF SEWAGE 237 Rideal (1906) quotes experiments by Pickard at Exeter, which show that typhoid bacilli may persist for 2 weeks in a septic tank and that contact bed treatment only effects a go per cent removal of these organisms. Disinfection of Sewage Effluents. Where bacterial purity is required, some special process of disinfection must be combined with the contact bed or the trickling filter. For this purpose treatment with chloride of lime or other chemicals is rapidly gaining ground as an important adjunct to bacterial disposal plants; -and in connection with this process bacteriological control is an essential. Rideal (1906) first showed at Guildford that 30 parts of available chlorine per million would reduce the number of bacteria in crude sewage from several mil- lions to 50,000, while 50 parts would reduce their number to 20 per c.c. Colon bacilli were reduced from one million per c.c. to less than one per c.c. by 30 parts of chlorine. In septic effluent 25 to 44 parts of chlorine per million reduced B. coli from two and a half to four and a half million per c.c. to less than one per c.c. With contact effluents smaller amounts of chlorine proved efficient, The primary effluent required 20 parts per million, the secondary effluent 10.6 parts per million and the tertiary effluent 2.5 parts per mil- lion to reduce the number of B. coli so that this organism could not be isolated in 5 c.c. In this country Phelps and Carpenter (1906) demon- strated the practical usefulness of bleaching powder disinfection, at the Sewage Experiment Station of the Massachusetts Institute of Technology. As indicated 238 ELEMENTS OF WATER BACTERIOLOGY in the table below smaller amounts of chlorine than were used by Rideal will give good results with more dilute American sewages. ae BACTERIA IN TRICKLING FILTER EFFLUENT BEFORE AND AFTER TREATMENT WITH CHLORIDE OF LIME (56 PARTS PER MILLION AVAILABLE CHLORINE) (PHELPS AND CARPENTER, 1906) Bacteria per c.c. B. Coli, Jackson Bile Test. Date. Before. After. senee ee Cc. eee. 1906 August 11....] 270,000 69 + 0 +:0 coin ATG 2 gl 020;000 41 Oo. oO" + o SON FAA | ES5IO0O 406 + + + 0 fe ee 230,000 21 0) 4.0 °. 0 wet TOs jase 250,000 37 + 0 ° Oo “ 38...,| 110,000 40 6/9 5 a | ota « ar 90,000 54 + 0 oO re (258. sl). (220,000 22 oO oO Been A ORS Ae | Weaver aa tataee Tie Me niente nie + 0 oO Average...... 240,000 86 33%+ 22%+ Average removal.... 99.96% 99.993 % The success of chemical disinfection varies with the character of the sewage or effluent treated, since the organic matter present consumes a certain amount of the disinfectant and renders it inoperative. Dis- cordant results are therefore reported from different sources. An important series of experiments carried out in Ohio by Kellerman, Pratt, and Kimberly (1907) showed good results with sand filter effluents and BACTERIOLOGY OF SEWAGE 239 contact effluents. Septic sewage, on the other hand, required large amounts of chlorine to produce a rea- sonable bacterial reduction. The table on page 240 shows the results obtained at Marion, Ohio. In Germany, on the other hand, Schumacher (1905), Kranepuhl (1907), and Kurpjuweit (1907) found larger amounts of chlorine necessary, in the neighborhood of 60 parts per million parts of sewage. Their tests were somewhat severe, however, the criterion of success being the absence of B. coli in a large proportion of liter samples. Standards for Sewage Effiluents. The science of sew- age bacteriology is in its infancy; and it is difficult to give any general rules for the interpretation of bac- teriological examinations designed to indicate whether disposal plants are successful or not. Houston stated provisionally that the 20° count should be under 100,000 and the 37° count under 10,000, while B. coli should be absent from .oor c.c. and B. sporogenes from .1 c.c. (Houston, 1902”). This standard seems to us far too lenient. Either organic purity alone is necessary, as at many sewage disposal plants, or a higher grade of purity than this should be attained. It seems wisest’ at the present time to avoid fixing any general standards of purity for sewage effluents. Each case should be judged intelligently on its own merits. In general, however, where bacterial purification is indicated at all, it seems fair to demand that the effluent should be of such a quality as not to increase materially the bacterial content of the body of water into which it is discharged. 240 ELEMENTS OF WATER BACTERIOLOGY BACTERIA IN SEPTIC EFFLUENT, CONTACT EFFLUENT, AND SAND EFFLUENT AT MARION,O., BEFORE AND AFTER TREATMENT WITH CALCIUM HYPOCHLORITE (KELLERMAN, PRATT, AND KIMBERLY, 1907) Bacteria per c.c. Average Availatle Date. | Effluent. | Chlorine. 20° C. 37° C. Total Count. Parts per Million. Untreated.| Treated. | Untreated.| Treated. 1907 : Apr. 11 | Septic 4.3 850,000] 1,100,000} 1,200,000] 240,000 Apr. 12 | Septic 6.2 4,400,000} 550,000} 850,000! 260,000 Apr. 15 | Septic 7.6 600,000] 400,000} 450,000] 190,000 Apr. 28 | Contact 2.9 110,000 2;500) .s «sag Meee em eee Apr. 29 | Contact 5.0 65,000 1,600 73,000 370 Apr. 30} Contact 4.4 500,000 800} 160,000 400 Mar. 21} Sand 3.8 49,000 570 9,800 150 Mar. 22| Sand 3.0 56,000 140 7,000 60 Mar. 26] Sand i Red 70,000 4,000 20,000 160 Bacteria per c.c, Average Date. | Effluent. Chogne. 37° C. Red Colonies. B. Coli. Parts per illion. Untreated.| Treated. | Untreated.} Treated. 1907 Apr. 11 | Septic 4.3 55,000 7,400 Apr. 12 | Septic GHZ 60,000 15,000 Apr. 15 | Septic 7.6 100,000 51,000 Apr. 28 | Contact Bie? WN deen oat | asks aes 20,000/Not ino. 5 Apr. 29 | Contact 5.0 10,000 ° 15,000!‘ 0.5 Apr. 30} Contact 4-4 21,000 3 20,000] ‘* 1.0 Mar. 21] Sand 3.8 1,300 ° r,000| “©5760 Mar. 22] Sand 250 800 ° 2,000] ‘* 1.0 Mar. 26] Sand Tas 4,000 I 2,000|In I.0 BACTERIOLOGY OF SEWAGE 241 Bacteriology of the Sewage Filters Themselves. Before leaving the subject of sewage bacteriology, brief reference must be made to the importance of bacteri- ological studies in relation to the processes of sewage purification which bring about the removal of the organic matter itself. Nothing is more necessary to the development of the present art of sewage disposal than knowledge of the micro-organisms concerned and of the conditions which favor their activity; but such knowledge is woefully deficient. Something is known of the nitrifying organisms long ago discovered by Winogradsky. More recent work, like that of Schultz- Schultzenstein (1903), Boullanger and Massol (1903) and Calmette (1905), has cleared up many points concerning these forms; but much remains to be done. In regard to the reducing action of bacteria in the septic tank and contact bed we are almost wholly in the dark. Septic tanks work well with some sewages and badly with others; and the presence or absence of the right bacteria is probably largely responsible for the different results. In some cases, as at Plainfield, N. J., the seeding of a tank with cesspool contents has produced a material improvement in septic action. Knowledge of the kinds of bacteria involved would make it possible to substitute scientific control for such empiricism and might well lead to improved methods of a more intensive character than are yet available. The work already done upon a laboratory scale furnishes promise of such results. The student 242 ELEMENTS OF WATER BACTERIOLOGY who wishes to follow out this line of investigation will find a good summary of what is already known of the hydrolysis and denitrification of nitrogenous bodies and the decomposition of cellulose and other carbohydrates in Rideal’s ‘‘ Sewage and the Bacterial Purification of Sewage ”’ (1906). Gage (1905) has made a suggestive study of the bacteria which carry on the reducing changes in sewage which deserves the attention of all who are interested in the more theoretical aspects of sewage treatment. His method consisted in plating sewages and effluents, isolating typical cultures and determining their power to decompose peptone and nitrates with the produc- tion of ammonia and free nitrogen. The rate of gelatin liquefaction, the amount of nitrate reduced, the amount of free ammonia formed, and the amount of nitrogen liberated were quantitatively determined for each culture thus isolated. The numerical values obtained, multiplied by the number of bacteria, apparently of the same type, observed in the plates, gave coefficients of the liquefying, denitrifying, ammonifying, and nitrogen-liberating power of the effluent; and these coefficients may be considered as measures for a given sample of the tendency of the bacterial flora to set up certain changes. The results of further studies made by Clark and Gage (1905), on sewages and on sand, contact, and trickling effluents, show that there may be important differences between various sewages in this respect which must render their purification more or less easy. They indicate that the effluents obtained from intermittent sand BACTERIOLOGY OF SEWAGE 243 filters in cold weather contain larger numbers of ammo- nifying and denitrifying bacteria than appear at other seasons, which may help to explain the poorly nitrified effluents obtained in the winter season. Along these lines research work in sewage bacteriology promises to be fruitful of results. CHAPTER XII BACTERIOLOGICAL EXAMINATION OF SHELLFISH Shellfish and Disease. The pollution of areas devoted to the growing of shellfish and the consequent pollution of the shellfish themselves is a matter of much sanitary importance. Oysters, clams and mussels are the shellfish commonly used as food, and since they are likely to be eaten in an uncooked or partially cooked condition, it is important to be assured as to their character from the bacteriological standpoint. In their normal habitats, in clean sea-water, or in river estuaries free from pollution, shellfish are unquestionably free from dangerous bacteria, although their feeding habits make it probable that the types of bacteria indigenous to the waters in which they are found might be present in considerable numbers. With the pollu- tion of streams by unpurified sewage the areas in which oysters and clams develop may easily become infected by organisms of intestinal types, and there is, therefore, offered an easy means for the typhoid bacillus and other pathogenes to pass from the sewage directly into the intestinal tract of the consumer of the raw oysters or clams. The history of this subject is well summarized by Newlands and Ham (1910), from whose excellent report the following paragraphs are adapted: 244 : EXAMINATION OF SHELLFISH 245 Attention was first drawn to the danger from shell- fish by the remarkable outbreak of typhoid fever which occurred in Middletown, Conn., in 1894, as a result of the serving of raw oysters at college fraternity banquets. The oysters used in this case were all derived from a certain portion of Long Island Sound, where they had been put down, or planted, in order to fatten. Investigation showed that the stream entering the Sound at this point was highly polluted, and furthermore, that at a nearby house there were two severe cases of typhoid fever from which the intes- tinal discharges were turned into the drain and thence into the stream without disinfection. The course of the passage of the bacteria from the patient suffer- ‘ing with the disease to the oyster and so on to the young men at the banquets was, therefore, traced out in a most complete and thorough way. This investigation, which was conducted by Prof. H. W. Conn, of Wesleyan University, caused immediate invest- igations to be set on foot in England and in this coun- try. Two.years later there followed a report by the Local Government Board of Great Britain dealing with pollution of shellfish along the English coast, and the matter has also received much attention in this country. A study of the literature reveals only a few references to oysters as carriers of disease germs previous to 1880. In that year Cameron, in a paper entitled “ Oysters and Typhoid Fever,” read before the British Medical Association, suggested that outbreaks of typhoid fever and cholera might be caused by eating oysters. 246 ELEMENTS OF WATER BACTERIOLOGY In 1893 Thorne-Thorne, in a report to the Local Gov- ernment Board, wrote that, in his opinion, certain cases of cholera which had occurred that year at various inland towns in England were due to eating contam- inated oysters from beds at Grimsby, where there had been a small cholera epidemic. Following the suggestions embodied in this report the English Govern- ment began a series of investigations which have made many important additions to our present knowledge of the subject. In 1902 the famous oyster epidemics at Winchester and Southampton, England, were proven beyond reasonable doubt to have been caused by contami- nated oysters taken from grounds at Emsworth. Here again we have to deal with banquets given in different cities where the only common source of infection appears to have been contaminated oysters. Of the 267 guests at these banquets 118 were attacked with intestinal disorders and 21 cases of typhoid fever developed, 5 of which were fatal. Although a great many sensational attacks have been made against oysters as carriers of disease germs which have been based on little or no evidence, the above-mentioned investigations and others, among which might be mentioned those of Thresh, Marvel, and Soper, have brought out sufficient trustworthy evidence to show that contaminated oysters must be considered as a real factor in the dissemination of typhoid fever and-other water-borne diseases. An esti- mate of the extent to which such illness is due to oysters would be impossible at the present time. The EXAMINATION OF SHELLFISH 247 Royal Sewage Commission after an extensive investi- gation on this subject came to the following conclusion: “‘ After carefully considering the whole of the evidence on this point, we are satisfied that a considerable num- ber of cases of enteric fever and other illness are caused by the consumption of shellfish which have been exposed to sewage contamination; but in the present state of knowledge, we do not think it possible to make an accurate numerical statement. “Moreover an examination of the figures which have been placed before us as regards those towns in which the subject has been most carefully studied shows that there may be occasional errors. Indeed the witnesses themselves recognized that absolutely accurate figures were not obtainable. “We are far from denying that isolated cases may have been due to contaminated shellfish, but we must remember that the possibility of some of them being due to other causes cannot be altogether excluded.” In the above-mentioned cases, where oysters have been proven or reasonably suspected of being the cause of disease, it was found that the oysters in ques- tion had been floated or grown in heavily polluted water where direct contact with specific infection could be proven or readily assumed. The Wesleyan epidemic is a case in point. Oysters had undoubtedly been floated in the contaminated waters at Fair Haven for a number of years previous to 1894 without any noticeable effect on the health of persons eating them, but specific infection of the water from two patients in a house near by was followed by a serious epidemic. 2948 ELEMENTS OF WATER BACTERIOLOGY | Valuable studies of the relation between shellfish and disease have recently been published by Bulstrode (x911) and Wilhelmi (1911) and Stiles (1912). Effect of Cookery upon Polluted Shellfish. It should be noted that it is unfortunately not only raw shellfish which are responsible for the spread of disease. Most of the processes of cookery to which these foods are subjected are insufficient to destroy pathogenic germs. Clark (1906) found that clams and oysters in stews and fried and scalloped in the usual manner were generally free from colon bacilli and streptococci. With steamed clams, however, the bacteria present could not be destroyed except by a temperature high enough and prolonged enough to ruin the clams for eating. Rickards (1907) confirmed these results as to the danger from steamed clams, while he found fried clams and clams in chowder and scalloped oysters to be practically sterilized. Oyster stew, however, is not exposed to long continued heat as is clam chowder, and fried oysters are less thoroughly heated than fried clams in the ordinary processes in use. Oysters in both of these forms and fancy roast oysters still contained colon bacilli and streptococci. Buchan (1910) finds that the ordinary methods of cooking mussels do not remove the risk of typhoid infection. Bacteriological Examination of Shellfish. Without further discussing the general sanitary aspects of the subject it is important to consider just how one may determine whether the oysters from a given region are polluted or not. The methods which have been developed for this work are essentially modifications EXAMINATION OF SHELLFISH 249 of the methods used in water examination, involving sometimes total counts of bacteria at different tem- peratures, but especially the application of the various tests for the determination of the colon bacillus, since here, as in water examination, this organism may be taken as an index of pollution and its occurrence in considerable numbers must be looked upon not merely with suspicion, but as a practical proof that the supernatant waters are polluted and that the shell- fish themselves may contain organisms of pathogenic importance, such as B. typhi, B. dysenteriz, B. sporo- genes and others. Determinations of the pollution of the water above the beds are sometimes made as bearing indirectly and inferentially on the possi- bility of the pollution of the shellfish contained therein. Results of the two determinations are not always in close agreement, however, owing to the rapidly changing local conditions due to tide, etc. The gen- eral relations and the individual variations between water and shellfish determinations are well _ illus- trated in the table on page 250 from the report by Newlands and Ham (1910) on conditions in New Haven Harbor. Study of the methods of examination of shellfish has been conducted with great care at the Lawrence Experiment Station by Gage, at the Sanitary Research Laboratory at the Institute of Technology by Phelps, at Brown University by Gorham, and in New York by Pease. Other officials of the Shellfish Commissions of different States have also carried out investigations upon this subject. The Lawrence Experiment Station 250 ELEMENTS OF WATER BACTERIOLOGY BACTERIA IN WATER AND SHELLFISH, NEW HAVEN HARBOR Water. © Oysters. Station. Av. Number Average | Average Samples Bacteria per c.c. Number | Number ves Character. Taken. |, | B. Coli *| B. Coli *| go ¥8°0".| of Bottom. 37° C. | 20°C, | Ber c-c: | Per c.c. Pie Ferry St : : Bridge I2 210 1260 AS Fi alee eee Soft Tomlin- ; son Br I5 910 2650 SHS TG oe Jai ws aiaterahe oe No. 1 15 510 1680 SI I Savane ot ee ab aa rR No. 2 15 375 910 WUE Peres ta pen eee ve No. 3 16 255 835 9 72 16 re Buoy 10 I5 155 450 EO) He Deena seme Min 0. 4 15 160 1720 Day AN onretes Ae) A ad 8 No. 5 17 615 1340 74 308 13 7 Buoy 5 23 315 715 oD by CAPS ee Wier res ye aS Buoy 8 15 205 410 Bie dy sg scalen hie metre ts No. 6 16 145 485 8 37 6 Seaweed No. 7 ai 215 740 29 425 II Hard No. oB. . II 220 260 7 6. 10 r No. 9A 13 100 185 9 4 Io re No. 9 12 195 200 17 37 10 me No. 7A II 120 240 10 255 Ir BM No 16 180 270 7 370 6 No. 10 23 300 615 9 100 I Soft No. 11 II 405 510 8 Io 4 “¢ Buoy 6 21 815 1690 ODP it Ske: srt ha teeta eae Buoy 3 17 175 590 6 201 8 Hard No. 12 I4 620 I1I90 4 6 5 “ No. 13 7 240 120 Bae) 10 8 xs No. 14 I2 285 Ir00 I- 7 8 7 Buoy 4 4 375 | 1400 4 0. 15 7 455 1680 I 45 15 Hard No. 16.. 14 280 1025 I- I- 3 Soft No. 17.. 14 300 1260 ble) PI ead, barra Seve Hard No. 19.. I 800 300 No. 20.. Io 135 860 I— 10 8 Hard Buoy 2. II 375 905 2 , No. 22. 8 305 560 I— 7.3 3 Hard Buoy tr. 6 IIs 9095 I- 4 12 es o. 18. 10 255 675 I- No. 24. 4 710 1340 4 9 10 Hard No. 23 6 450 240 I— te No. 25 2 130 1000 I- No. 26 5 130 405 I— No. 27 Io 630 605 Tier cepa. Bees Soft No. 28 4 415 1400 I- 4 3 Mudandsand No. 29 5 370 1700 Times) Sor Fe sgul) warene te Soft No. 30 5 185 440 I— I- 15 Hard No. 31 7 320 130 I— I— 15 SF No. 32 5 70 1050 I- 3 15 vi No. 33 4 485 405 I- I- ae) rly No. 34 4 535 495 I— I 10 ae No. 35 4 120 270 I- I 15 Sticky * Jackson's lactose bile presumptive test used. Minus sign after figure 1 indicates that the average was less than 1. EXAMINATION OF SHELLFISH 251 method was published in the Massachusetts State Board of Health Report for 1905 (Clark, 1906). This method consisted in the total counts of bacteria de- veloping at 20° and 37° and the fermentation reaction in dextrose broth. Experience indicated that it was. not merely necessary to examine the stomach contents of the oysters but the “ shell water ” as well was sub- jected to examination. With the advent of lactose bile as a better medium for the development of B. coli without interference with other typés of bacteria, the substitution of this medium for dextrose broth was commonly made, and this is now one of the standard media employed for the determination. It has been noted that the superiority of lactose bile to dextrose broth is greatest in water examinations when the water is most polluted. In the study of shellfish the danger of overgrowths is even greater than in polluted waters, since the organic matter in the oyster and its surrounding shell water furnishes a culture medium for many bacteria. Streptococci are particularly abundant. As pointed out in Chapter TX, streptococci die out more rapidly than colon bacilli in potable waters, but where organic matter is present in abundance the former may survive the latter. We have compiled the table on page 252 from the results of Clark (1906). In all cases except in that of the shell water there is a considerable difference between the dextrose fermentation tests and the colon isolations, indicating an overgrowth by strepto- cocci and other forms, of colon bacilli originally present. The B. sporogenes is also very frequently 252 ELEMENTS OF WATER BACTERIOLOGY responsible for such anomalous results in shellfish examinations. COLON BACILLI AND STREPTOCOCCI IN DIFFERENT PORTIONS OF THIRTY CLAMS Per Cent of Samples Showing Fermenta- : Seer B. Coli and tion in : Strepto- Dextrose B. Coli. coach: eee ie Shell qwaters, vii sot gee 90 83 47 40 O51 | CUR aie See ti iAtee tae es 77 53 25 t5 Stomach (intestine)... . 55 35 22 12 Rectum (intestine)..... 82 45 43 13 TAVER Ss cdi, EVE ee 39 18 15 3 Visceral tissue........ 18 8 7 2 It will be noted from Clark’s table above that the shell liquor is not only freer from overgrowths than the portions of the body of the clam, but that the propor- tion of positive reactions is in each case higher. Since the shell water is of course easier to examine than the macerated animal, this is now generally adopted as the standard material for examination. Self-purification of Shellfish. In connection with the bacteriological examination of shellfish for colon bacilli certain investigations have been carried out which are of great importance from the commercial as well as from the sanitary standpoint. Phelps (1911) has shown that oysters which develop in waters sub- ject to sewage pollution may be purified or entirely freed from colon bacilli by the removal of the oysters themselves to waters of purer character, when, after EXAMINATION OF SHELLFISH 253 sufficient time has elapsed, the oysters will have cleansed themselves through their metabolic processes and become entirely safe even for consumption in the raw state. It is of considerable importance to determine the length of time necessary for this self-purification to take place. Obviously, from the commercial stand- point it is desirable to make it as short as possible, while from the sanitary standpoint it must be long enough to insure a thorough and satisfactory removal of all traces of polluted matter. Oyster beds which are free from pollution or which are sufficiently good for the re-laying for polluted oysters are difficult to find and limited in areas because of their nearness to sources of pollution. The investigations in question were conducted by Phelps in the Providence River and the upper part of Narragansett Bay. The oysters were removed from heavily polluted regions and car- ried to waters which were practically free from pollu- tion, where they were planted. Examinations were made from day to day in order to determine the length of time that these particular oysters showed pollution and it was found that within 4 days the organisms of the colon type were practically all eliminated. It must be borne in mind that, if shellfish are care- lessly opened and handled, they may suffer a considerable additional pollution in the process, and may therefore be much worse instead of better than when they were taken. This is well brought out by the table on page 254, taken from a report by Stiles (1911) in which shucked market oysters show much worse pollution than market oysters in the shell. 254 ELEMENTS OF WATER BACTERIOLOGY COMPARATIVE BACTERIOLOGICAL CONDITION OF MAR- KET OYSTERS, SHUCKED AND IN THE SHELL (STILES, Ig11). Average per c.c. Liquor. Bacteria. Number : 0) Samples. Bile Plain Agar. Salt | B. Coli. | Strepto- Agar. coccl, 25° 377 37° Shell oysters. .... 36 6,000} 1,000 200 7 ce Shucked oysters. .} 33 |867,000|268,000] 45,000} 74,000 | 8000 Seasonal Variation of Bacteria in Oysters. It has been observed by Gorham (1912) and others that the examination of oysters from certain regions made in the summer failed to agree with the similar analyses from the same beds made in the winter. With the advent of cold weather there seems to be a great improvement in the sanitary quality, so that oysters taken from beds in close proximity to the outfalls of large sewers show in the colder months entire absence of any evidence of contamination, judged solely by the bacteriological data. Thus Gorham found in the sum- mer of 1910 that all oysters on the beds in the Prov- idence and Warren Rivers and the upper part of Narra- gansett Bay were so badly polluted by sewage as to be unfit for food. Colon bacilli were found in the “shell water” of every oyster in amounts as small as or of a cubic centimeter or less. Chemical and bacteriological examination of the waters over these EXAMINATION OF SHELLFISH 255 beds showed them to be heavily sewage polluted. In December of the same year the analyses of the oysters were strikingly different, although the condition of the water was apparently unchanged. In the examina- tion five oysters were selected in each case and the average total number of bacteria per cubic centimeter was determined and the presence of colon bacilli was tested by the bile tube and subsequent isolation and identification of the organisms. The table on page 256 shows the numbers of bacteria found, and the propor- tion of the five oyster samples in which colon bacilli were present in cubic centimeter amounts and also in o.1 and o.o1 of a cubic centimeter. The conclusions arrived at by Gorham are that during the cold weather the oysters assume a condition of rest or hibernation, during which time ciliary move- ment ceases and the process of feeding is suspended. No organisms are therefore taken in from the outside water and those inside the oyster are gradually elim- inated, so that the total number of organisms is reduced very considerably and the oyster becomes — practically free from colon bacilli. Standard Methods for the Examination of Shellfish. The examination of shellfish for pollution is regarded as of such importance by the American Public Health Association (1912) that a committee was established to report upon methods of examination and estima- tion of the numbers of colon bacilli found. The following abstract of the second report of this committee gives the recommendations for standard methods for bacteriological examination of shellfish ELEMENTS OF WATER BACTERIOLOGY 256 SEASONAL VARIATION IN THE BACTERIAL CONTENT OF OYSTERS (GoRHAM, 1912) Ea 3232] Proportion of Five eos a Oysters Showing B. Coli Tempera- Date. 2 NaS Rawat Score.| Present in ores goo > Water in hea eae ASS] Ic.c. | 0.1 c.c.|0.0rc c. Bep No. 8. PROVIDENCE RIVER Dec. 20, 1910} 1000 3 I ° 4 |oo1rcec, {—r1° Jan. 14,1911 | 750 5 3 I 41 Pkt aa 80 4 iB ° 23 | 0.01 c.c r= Jans 27. Sec: 23 5 3 ° 32 Feb. 10 i654 130 2 2 ° 4 | 220.¢.¢ 0.1° Feb. 28..... 140 ° ° ° © | 0.0001 c.c.} 1° | i ET me Aiepte Me 200 5 4 ° 4I | 0.01 C.c L7s~ ADTH TAs... 275 5 2 ° 23 | 0.01 C.c 8.5° April 28..... 700 5 5 4 410 | 0.0001 ¢.c.| 12.5° May t2: 2.3: 1700 5 5 5 500 | 0.0001 C.c.| 15° Bep No. 44. PROVIDENCE RIVER Jan. 7, 1911..| 425 5 5 I 140. Wit. ase 0.25° Heb 27... 250 4 ° fo) 4 lia °° Feb. 28..... 240 5 I fo) TA eeu tae Or5* March 11 100 5 2 ° 23:1 Soca niee 2° April 14..... 210 2 ° ° Dit" sien o. April 28..... 1000 5 5 4 410.) Naieate Irs" May 12..... 1100 5 5 4 AIO 7} ct bara 14.75° Bep No. 204. WARREN RIVER Jan. 25,1911 | 600 5 4 I 50’ Kuk s eae °° Peb::10; <5 5 140 ° ° ° o | t.0c.c °° Feb. 28,-....: 400 fe) fe) ro) o | 0.01 c.c 0.75° March 4 750 cis a o* ret | ee ST °.75° March 11 60 I ° ° t |‘Ovon cs x2 March 14....] 3400 ° ° ° © | 0.01 <.c 8.75" April 28..... 1050 5 5 4 410 | 0.01 C.c 134 Bep No. 205. WARREN RIVER Dec. 22, 1910| 250| 3 re) ° $0 [ i aeads —1° Feb. 10, 1911 | 325 fe) ° 0 o | £-0.c.c. °° Peb:-28;...3.. 450 4 2 ° 14 | 0.01 cc te March 4..... 600 2 2 I Baleaeas 0.78" Marchir....| 85 2 I ° 3 | 0.01 cc 2° April 14..... 325 I I ° 2 |0.01 C.c 8.25° April 28..... 4000 a 5 5 500 | 0.01 c.c r¢° * Only three oysters used. EXAMINATION OF SHELLFISH 257 which were adopted by the Association at its meet- ing in 1912: RECOMMENDATIONS FOR STANDARD METHODS FOR THE BACTERIOLOGICAL EXAMINATION OF SHELLFISH Oysters in the Shell Selection of Sample. Twelve (12) oysters of the average size of the lot under examination, with deep bowls, short lips, and shells tightly closed, shall be picked out by hand and prepared for transportation to the laboratory. As complete a record of such data as is possible to obtain shall be made covering the following points: The exact location of the bed from which the sample has been selected. . The depth of the water over the bed at time of collection. The state of the tide. The direction and velocity of the wind. Other weather conditions. The day and hour of the removal of the stock from the water. The conditions under which the stock has been kept since removal from the water and prior to the taking of the sample. The day and hour of the taking of the sample. Transportation of the Sample. The oysters so selected shall be packed in suitable metal or pasteboard containers of such size and shape that a number of them can be enclosed in a shipping case capable of satisfactory refriger- ation by means of ice. The important points in this con- nection are: A. The prevention of the mixing of the oyster liquor of different samples, and of the mixing of the ice-water with the oysters. 258 ELEMENTS OF WATER BACTERIOLOGY B. The icing of the samples, if they are not to arrive at the point of laboratory examination inside of 36 hours, or if the outside temperature is above 50° F. It is not necessary to enclose the oysters in an absolutely tight container, providing the above conditions are main- tained. Condition of Samples. Record shall be made of the general condition of the oysters when received, especially whether the shells are open or closed; of the presence of abnormal odors; and of the temperature of the stock. Technical Procedure. The bacteriological examination shall be started as soon as possible after the receipt of the sample. The oysters shall be thoroughly cleaned with a stiff brush and clean running water and then dried. The edges of the shell shall be passed through the flame or burned with alcohol. The opening of the shell shall be accomplished by either of the following methods: A. By the use of a sterile oyster knife in the usual manner. B. By drilling through a flamed portion of the shell near the hinge with a sterile drill. The drill shall be sterilized, and the site of the operation on the shell shall be flamed at least once during the drilling process. Bacterial Counts. Bacterial counts shall be made of a composite sample of each lot obtained by mixing the shell liquor of five oysters. Agar shall be used for the culture medium and in general the procedure shall be in accordance with the method recommended for the exam- ination of water by the Committee on Standard Methods of Water Analysis of the American Public Health Association. The water used for dilution purposes shall contain 1 per cent of sodium chloride, in order to approximate the natural salinity of oyster liquor. The agar plates shall be incubated at 20° C. for three days and the colonies then counted. EXAMINATION OF SHELLFISH 259 Determination of Bacteria of the Bacillus Coli Group. The quantitative determination of the presence of B. coli shall be in accordance with the following procedure: Measured quantities (1.0, 0.1, 0.01 c.c., etc., or their equivalents in dilutions) of the shell water of each of 5 oysters selected from the dozen, shall be placed in fer- mentation tubes containing lactose peptone bile, prepared according to the method recommended by the Committee on Standard Methods of Water Analysis. These shall be ‘incubated for three days at 37° C., and the presence or absence of gas noted daily. For all ordinary purposes of routine work a development of from tro to 85 per cent of gas during this time period shall constitute a positive test indicating a presumption of the presence of at least one bacterium of the Bacillus coli group in the quantity of shell water tested. But no final B. coli rating based on these results shall be used for official approval or condemnation unless positive confirmatory tests for the presence of organ- isms of the B. coli group shall have been obtained from the tube of highest or next highest dilution from each oyster, showing the presence of gas. These confirmatory tests shall be begun immediately upon noting the formation of gas, and carried out in accordance with the procedure recommended by the Committee on Standard Methods of Water Analysis. Statement of Resulis. The results of the bacterial counts shall be expressed as Number of Bacteria per c.c. The results of the tests for B. coli shall be expressed either in the form of the following arbitrary numerical system to be known as “ The American Public Health Association Method of Rating Oysters for B. coli; or in Estimated Number of Bacteria of the B. coli Cio per c.c. of the Sample.” 260 ELEMENTS OF WATER BACTERIOLOGY The American Public Health Association Method of Rating Oysters for B. Coli.* ; The following values shall be assigned to the presence of bacteria of the B. coli group in each of the 5 oysters examined, these figures being the reciprocals of the greatest dilutions in which the test for B. coli was positive: If present in 1.0 c.c. but not in o.1 c.c., a value of 1. If present in 0.1 c.c. but not in o.o1 c.c., a value of 10. If present in o.o1 c.c, but not in o.cor c.c., a value of 100, etc. The sum of these values for the 5 oysters gives the total value for the sample and this figure shall be taken as the ‘rating for B. coli.” The results shall be expressed in the following tabular form: RESULTS OF TESTS FOR B. COLI IN DILUTIONS INDICATED Oysters. 1.0 C.c. 0.1 c.c. 0.01 c.c. Nears I 0 + ° 10 2 + i ° Io 3 a ° O° 7 4 + ° ° I 5 + ° O° I Total, or rat\ing for B. coli =23 + = presence of bacteria of the B. coli group in fermentation tube test with lactose bile. o = failure to demonstrate presence of bacteria of the B. coli group. Estimated Number of B. Coli per c.c. If the standard B. coli rating above described is divided by 5, or, in general, if the rating is divided by the number of oysters tested, the result will be approximately the number of B. coli * Where the term B. coli is used, it refers in all cases to bacteria of the B. coli group and not to the specific prototype. EXAMINATION OF SHELLFISH 261 per c.c. of shell water. Partly because it does not do this exactly, but also for simplicity and the avoidance of fractions, the method of stating results as an arbitrary rating is preferred by the committee. Practical experience with the method also has appeared to justify this preference. Illustrations of the Application of the Method of Rating Oysters for B. Coli. Sometimes results similar to the following are obtained, that is, one or more oysters may show positive results in small quantities of shell water, while an equal number may show negative results in larger quantities. In this case the next lower numerical value shall be given to the positive results in the high dilutions, and such positive results shall be considered as being trans- ferred to a lower dilution giving negative results in another oyster. This is done on the theory that inconsistent results, mathematically considered, may follow naturally from an unequal distribution of the bacteria in the shell water. This recession of the assigned values, however, shall not be carried beyond the point where the number of such recessions is greater than the number of instances where other oysters in the series failed to give positive B. coli results. As examples of the method of obtaining the rating for B. coli, the following illustrations are given. They repre- sent results that may be met with in practice: CASE A.—RESULTS OF B. COLI TESTS IN DILUTIONS INDICATED Oysters. 1.0 c.c. 0.1 c.c. 0.01 C.c. ep rae: > + tr ° 10 as 4: a ° 10 3 + 15 ° 10 4 + ° ° 10 (not 1) 5 “i i i 10 (not 100) 50=rating 262 ELEMENTS OF WATER BACTERIOLOGY CASE B.—RESULTS OF B. COLI TESTS IN DILUTIONS ok INDICATED Oysters. ho ce. 0.1 c.c. 0.01 c.c. Samana I a a of 10 (not 100) 2 + +} + Io (not 100) 3 + ° ° I 4 ° ° ° I (not o) 5 ° ° ° I (not o) 23 =rating CASE C.—RESULTS OF B. COLI TESTS IN DILUTIONS INDICATED Oysters. 1.0 c.c. 0.1 c.c. 0.01 c.c. wee I + + fo) Io 2 ++ + ° Io 3 - =; ni 100 4 = a + 10 (not 100) 5 oS ° ° 10 (not 1) 140= rating Oysters removed from the Shell (Opened or Shucked Stock). Except as hereinafter stated, all the procedures and requirements for the examination of opened oysters, i.e., shucked stock, shall be those specified for the examination of oysters in the shell. Selection and Preparation of Sample. The stock in the container from which the sample is to be taken shall be thoroughly mixed, and one or more wide-mouthed sterile jars of a total capacity of one quart shall be each half filled with the sample by means of a clean ladle or other instru- ment sterilized by flaming alcohol. The jar or jars shall be so sealed as to exclude all possibility of contamination from without. a Ag rm EXAMINATION OF SHELLFISH 263 Transportation of Samples. When the time between the collection of the sample and its examination exceeds 3 hours, or if the outside temperature is above 50° F., the sample shall be thoroughly refrigerated by means of ice placed around, but not in, the sample jars. Technical Procedure. The bacteriological examination shall be begun as soon as possible after taking the sample. The sample shall be thoroughly shaken at least 25 times immediately before opening. Bacterial Counts. The procedure specified for oysters in the shell shall be followed: Determination of Bacteria of the Bacillus Coli Group. The procedure specified for oysters on the shell shall be followed, but attention is called to the fact that higher dilutions than 7}, c.c. are usually required. Triplicate fermentation tubes shall be inoculated from each dilution of the sample. Statement of Results. The results of the bacteriological examination of the opened oysters, or shucked stock, shall be expressed in the same way as that specified for oysters in the shell, except that in the calculation of B. coli rating the values for the results of the positive fermentation tests after confirmation shall be recorded for each of the inocula- tions of each dilution. In order that the rating from these triplicate tests may be compared with that obtained from testing 5 oysters in the shell, the sum of the values for the triplicate tests shall be multiplied by 3. If, instead, the’ sum is divided by 3, the result will give approximately the number of B. coli per c.c. Clams and Other Shellfish The methods for examining clams and shellfish other than oysters shall be those given above. Certain modi- fications are necessary in the method of handling the samples - and the opening of the shells, etc. 264 ELEMENTS OF WATER BACTERIOLOGY Clams are more likely to lose water during transportation than oysters. It is therefore necessary to take greater precautions to separate different samples of clams from each other than in the case of oysters. In opening soft clams it has been found that if two incisions are made through the mantle the shell water may be poured out without opening the shell. Hard clams are more difficult to open, but if the shell — be struck over the dorsal muscle with a small hammer an opening will be formed permitting the insertion of the knife to cut the muscle. Sometimes clams and other shellfish contain too little liquor to make all of the tests above described. This is always the case when the shells are very small. Under these conditions the water from two or more shellfish shall be taken together and tested and considered as one. _ Standards of Interpretation. As in the case of water it is neither practicable nor desirable to attempt to formulate any hard and fast standard for passing or condemning shellfish. It is very clear from the work carried out by the English Commission, and at Lawrence, Boston, Providence and New Haven in this country that shell- fish from entirely unpolluted regions are free from colon bacilli and that the proportion of positive tests for these organisms increases with the increase in pollution. Just where to draw the line, however, it is not easy to say. According to Newlands and Ham (1910), the stand- ards of permissible pollution adopted by various English and American workers vary from a positive test in 1 c.c. to a positive test in o.1 c.c. of shell liquor. The Bureau of Chemistry of the United States Department of Agriculture condemns oysters sold in interstate com- merce which show three positive tests out of five in 0.1 c.c, portions, and the same standard has been adopted by the Rhode Island Shell Fish Commission. ew ate - - APPENDIX! PREPARATION OF CULTURE MEDIA In view of the marked influence upon bacteriological reactions of variations in culture media caused by differ- ences both in ingredients and in technique of preparation, it is necessary that uniform methods be used in order to obtain comparable data. In specifying the various ingre- dients used in culture media it is the intention that they shall be uniform in quality, but it is not the intention that the recommendations as to ingredients and technical manipulations shall stand in the way of true progress as to improvements. When, however, improved or modified methods are used, the variations from the standard methods shall be plainly set forth together with the reasons for the modifications. INGREDIENTS Distilled water shall be used in the preparation of stand- ard culture media. Infusions of fresh lean meat, and not meat extract, shall be used as the basis of various media. Peptone shall be that of Witte (dry from meat). Gelatin shall be the best French brand, so called. It shall be as free as possible from acids and other impurities, and shall be of such a character that a 10 per cent solution 1 From the Report of the Committee on Standard Methods of the American Public Health Association. 265 266 APPENDIX prepared in the usual way shall not soften when kept at a temperature of 25° C. Commercial agar in threads shall be of as high a grade as can be obtained. Agar may be purified by washing. The various sugars, such as dextr se, lactose, and sac- charose, shall be as nearly as possible the chemically pure compounds designated. Unusual effort to obtain such sugars is considered to be necessary. Glycerine shall be double distilled. In place of litmus, a 1 per cent aqueous solution of . Kahlbaum’s azolitmin may be used. | Of the various other ingredients used, nearly all of which are of a mineral nature; special effort shall be made to see that they are chemically pure products within the full meaning of this expression. STERILIZATION Sterilization in the autoclave seems to be preferable to that in flowing steam. Both in the lowering of the melting point of gelatin and in the breaking down of sugar media the time of sterilization has a greater effect than does the temperature within the standard limits. It is, therefore, suggested that small containers be used and that media be sterilized in the autoclave at 120° C. (15 pounds pressure) for 15 minutes. A shorter period than this, in practice, sometimes results in incomplete sterilization, and a longer. time results in the inversion of sugars or the lowering of the melting point of gelatin. Agar media should be melted before placing in the autoclave. An important point in the sterilization of gelatin and sugar media is to have the sterilizer hot when the media are introduced, so that heating to the point of sterilization will be accomplished as quickly as possible. Also when sterilization is complete the media should be cooled rapidly. This not only reduces the time of heating, thus preserving APPENDIX 267 the gelatin and sugar, but also assists in the actual sterili- zation. It is also advisable in the use of the autoclave to keep the small pet cock at the bottom partially open so that steam is escaping during sterilization. This insures the removal of all air. If practicable, store media at room temperature for two days to see that sterilization is complete. In intermittent sterilization, media shall be placed on each of three successive days in streaming steam for 30 minutes after the steam fills the sterilizer. REACTION Phenolphthalein shall be the standard indicator used in obtaining the reaction of all media. Turmeric paper possesses similar properties, and its use is advised where phenolphthalein is not available. Titrations and adjustment of reactions shall be made as follows: : Put 5 c.c. of the medium to be tested into 45 c.c. dis- tilled water. Boil briskly one minute. Add 1 cc. of phenolphthalein solution (5 gm. of commercial salt in 1 liter of 50 per cent alcohol). Titrate while hot (preferably while boiling) with N/2o0 caustic soda. A faint but distinct pink color marks the true end-point. This distinct pink color may be more precisely described as a combination of 25 per cent of red (wave length approximately 658). with 75 per cent of white as shown by the disks of the color top (described under Record of Tints and Shades of Apparent Color, p. 10 of Standard Methods Report). In practice, titration is continued until the pink color of alkaline phenolphthalein matches that of the fused disks. All reactions shall be expressed with reference to the phenolphthalein neutral point and shall be stated in per- centages of normal acid or alkaline solutions required to . heutralize them. Alkaline media shall be recorded with the minus (—) sign before the percentage of normal acid 268 APPENDIX needed for their neutralization, and acid media with the plus (+) sign before the percentage of normal alkaline solution necessary for their neutralization. The standard reaction of culture media shall be ++1.0 per cent. If it differs from 1 per cent by more than o.2 per cent it should be readjusted. Wherever reactions other than the standard above given are used it shall be clearly stated in all results of bacterial work, and the reasons therefor also stated. STORAGE OF MEDIA It is recognized by the committee that it is desirable to prepare media in large quantities in order to guard against discrepancies in composition; but, all things con- sidered, the complications resulting from the varying amounts of heating incident to withdrawing portions from time to time and tubing it, are believed to more than offset this advantage. Consequently, when possible, media shall be put at once into tubes and placed in cold storage. To guard against changes due to evaporation all media not used promptly shall be stored in a moist atmosphere, preferably in an ice-box, or else the flask shall be sealed by dipping the cotton plug in paraffin. NUTRIENT BROTH Nutrient broth shall be prepared as follows: Infuse soo gm. chopped lean meat 24 hours with 1000 c.c. dis- tilled water in refrigerator. Restore loss by evaporation. Strain infusion through cotton flannel. Add 1 per cent peptone. Warm on water bath, stirring until the pep- tone is dissolved. Heat over boiling water (or steam) bath 30 minutes. Restore loss by evaporation. Titrate. Adjust reaction to +1 per cent by adding normal hydro- chloric acid or normal sodium hydrate, as required. Boil APPENDIX 269 2 minutes over free flame, constantly stirring. Restore loss by evaporation. Filter through absorbent cotton and cotton flannel, passing the liquid through until clear. Titrate and record final reaction. Tube, using 10 c.c. in each tube. Sterilize. SUGAR BROTHS Sugar broths shall be prepared in the same general manner as the standard nutrient broth, with the addition of 1 per cent of dextrose, lactose, saccharose or other sugar, just before sterilizing. The removal of muscle-sugar by inoculation with B. coli is unnecessary if small amounts of gas are disregarded. Ii, however, test-tubes of small dimensions are used and the presence of any gas, however small, is taken to indicate gas formation the removal of muscle sugar is necessary. The reaction of sugar broths shall be neutral to phenol- phthalein. Sterilization may be done in streaming steam, as usual, or in the autoclave at 120° C. (15 pounds pressure) for I5 minutes. Sterilization in the autoclave seems to be preferable to that in flowing steam. For the routine work of testing samples in water for B. coli, especially large volumes of water are to be mixed with broths of such strength that the resulting mixture . will be one of normal strength. Dextrose broth made with Liebig’s Beef Extract is not equal in effectiveness to that made of fresh beef extract and should not be sub- stituted for the latter. LACTOSE BILE The lactose bile medium consists of sterilized undiluted - fresh ox gall (or a ro per cent solution of dry fresh ox gall) _ to which has been added 1 per cent of peptone and 1 per cent of lactose. The addition of peptone is important. 270 APPENDIX GELATIN No gelatin media should be employed having a melting point below 25° C. The percentage of gelatin added may be increased to bring the melting point up to the desired figure. With most gelatin on the market 11 per cent “seems to be preferable to the standard of 10 per cent, provided the gelatin is weighed out without correcting for contained moisture, as appears to have been the custom. Ten per cent, or even 20 per cent of moisture commonly occurs in laboratory gelatin, and unless this is taken into account in weighing, the stiffness of the media is materially affected and the bacterial results obtained considerably modified. All gelatin should be tested for moisture before using by drying a sample for half an hour at 105° C. The stock should be kept under uniform conditions in tight containers, so that the percentage of water present may then be properly accounted for and the weight on a dry basis be used in making up the medium. AGAR For bacterial counts 10 gm. of agar per liter should be used. The smaller amount seems to be sufficient to carry the added water and the medium is less stiff. This appears to give higher and more consistent counts. Fifteen grams may be employed for keeping cultures. North medium is especially valuable for keeping cultures, particularly cocci. This is composed as follows: 500 c.c. Extract of 500 gms. of chopped beef. 500 c.c. Distilled Water. 1o gm. Agar. 20 gm. Gelatin. 20 gm. Peptone. 5 gm. Sodium Chloride. Reaction neutral. It is well to determine the reaction of the media after sterilization, as during this process the reaction often changes APPENDIX 271 and the final results should correspond to the acidity recommended by the standard methods. What has been said regarding the necessity of correcting for moisture in gelatin applies with equal force to agar and for the same reasons. NUTRIENT GELATIN AND AGAR Nutrient gelatin and agar shall be prepared as follows: Gelatin. Agar. I. Boil 15 gm. thread agar in 500 c.c. water for half an hour and make up weight to 500 gm. or digest for 15 minutes in the autoclave. Let this cool to about 60° C. 2. Infuse 500 gm. lean meat 24 Infuse 500 gm. lean meat 24 hours hours with 1000 c.c. of dis- with 500 c.c. of distilled water in tilled water in refrigerator. refrigerator. 3. Make up any loss by evaporation. 4. Strain infusion through cotton flannel. — Weigh filtered infusion. 6. Add 1 per cent Witte’s pep- Add 2 per cent of Witte’s pep- tone and 1o per cent Gold tone. Label sheet gelatin on dry basis. ‘ Pi Warm on water bath, stirring till peptone and gelatin are dissolved and not allowing the temperature to rise above 60° C. s: To 500 gm. of the meat infusion add 500 c.c. of the 3 per cent: agar, keeping the temperature below 60° C. 9. Titrate, after boiling one minute to expel carbonic acid. Io. Adjust reaction to +1.0 per cent by adding normal hydrochloric acid or sodium hydrate as required. Il. Heat over boiling water (or steam) bath for 40 minutes. 12. Restore loss by evaporation. - 313. Readjust to +1.0 per cent if necessary and boil 5 minutes over free flame, constantly stirring. 14. Make up loss by evaporation. 272 APPENDIX 15. Filter through absorbent cotton and cotton flannel, passing the filtrate through the filter until clear. 16. Titrate and record the final reaction. ry. Tube, using 10 c.c. of medium in each tube. 18. Sterilize 15 minutes in the autoclave at 120 degrees, or for 30 minutes in streaming steam on three successive days. Put the gelatin at once into ice-water till solidified. 19. Store in the ice-chest in a moist atmosphere, to prevent evaporation. LACTOSE LITMUS AGAR Lactose litmus agar shall be prepared in the same manner as nutrient agar, with the addition of 1 per cent of lactose to the medium just before sterilization. The reaction shall be made neutral to phenolphthalein (see p. 267). If the medium is to be used in tubes the sterilized azo- litmin solution shall not be added until just before the final sterilization. If the medium is to be eed in Petri dishes the steril- ized azolitmin solution shall not be added to the medium until it is ready to be poured into the dishes. More colonies and better general results are obtained on the lactose litmus agar plates, when the litmus and lactose are each sterilized separately and added to the plate with the neutral agar at the time of planting. Good results can, however, be obtained, if the agar and lactose are mixed and sterilized in an autoclave at 120° C. for 15 minutes only. The azolitmin on the market varies considerably, much of that sold being entirely unreliable for the purpose. A 1 per cent solution of Kahlbaum’s azolitmin, if boiled for 5 minutes, readily dissolves and needs no correction for acidity if added to standard agar. Many bacteriologists prefer pure litmus to azolitmin, and it is therefore sug- gested that its use be made optional. Both total and red colonies may be counted after from 18 to 24 hours APPENDIX 273 when incubated at 37° C. Such tests are sometimes used in the control of filtration plants. LIVER BROTH This medium is made from a hot infusion of beef liver instead of fresh meat, and is, in other respects, with the exception that phosphate is added the same as dextrose broth, but it is a richer food medium for bacteria. It gives gas formation with all species which ferment dextrose and develops attenuated bacteria, whether gas-forming or not, to a better degree than does beef broth. It -is also especially suited to the rejuvenation of species in pure culture. FORMULA SS AON oe aia og p55 ak vaca uabaws uae 500 gm pane CWC Soe oe sick Spee a ec cec eee eee Io gm PRCRINOHE distro ene Sh Sa pees Io gm Di-Potassium Phosphate (K2HPO,)........... I gm Water 1. Chop 500 gm. of beef liver into small pieces and add 1000 c.c. of distilled water. Weigh the infusion and container. 2. Boil slowly for 2 hours in a double boiler, starting cold and stirring occasionally. 3. Make up any loss in weight by evaporation and pass through a wire strainer. 4. To the filtrate add 10 gm. of peptone, 10 gm. of ° dextrose and 1 gm. of potassium phosphate. 5. After warming this mixture in a double boiler and stirring it for a few minutes to dissolve the ingredients, titrate with N/20 sodium hydrate, using phenolphthalein as an indicator, and neutralize with normal sodium hydrate. 6. Boil vigorously for 30 minutes in a double boiler, and 5 minutes over a free flame with constant stirring to prevent the caramelization of the dextrose. 7- Make up the loss in weight by evaporation and filter through cotton flannel and filter paper. 274 APPENDIX 8. Tube and sterilize in an autoclave for 15 minutes at 120° C. (15 pounds). Other valuable liver media (for use in the identification of B. sporogenes and other species) are prepared as given below: LIVER GELATIN 1. Proceed as in steps 1, 2, 3, in preparing liver broth. 2. Cool the filtrate to 50° C. Add ro per cent of sheet gelatin and stir a few minutes until dissolved. 3. Add 1 per cent of peptone, 1 per cent of dextrose and o.1 per cent of potassium phosphate. 4. Stir until the ingredients are dissolved, keeping the temperature below 50° C., and then proceed as in steps 5, 6, 7, 8. LIVER AGAR 1. Chop 500 gm. of beef liver into small pieces, add 500 c.c. of distilled water, and boil slowly for 2 hours, stirring occasionally. 2. Add 5 gm. of agar (dried at 105° C. for 30 minutes) to 500 c.c. of distilled water and digest for 30 minutes in an autoclave at 120° C. (15 pounds pressure). 3. After making up the loss by evaporation, pass the liver infusion through a wire strainer, add 500 c.c. of the filtrate to the agar solution and proceed as in steps 4, 5, 6, 7, 8, in preparing liver broth. It is very important to note that liver broth should not be exposed to the high temperature attained in the auto- clave any longer than 15 minutes, as prolonged heating above the boiling-point causes caramelization of the carbo- hydrates, rendering the medium less delicate for bacterial development. For the rejuvenation of attenuated cultures, especially B. sporogenes, the addition of very small pieces of liver tissue, which have been sterilized in Petri dishes APPENDIX 275 in the autoclave for 15 minutes improves the rejuvenating properties of the medium. They should be added to the tubes after sterilization. Bacterial growth being very rapid in this medium, pre- liminary POSTS at 37° C. should be concluded between 6 and 12 hours. HESSE AGAR Agar (absolutely dry): 2 06500 02625 6o. eee 4.5 gm enbime,) WHE oo oss oka eas Sanee vet xdaas Io gm. Baemer's extract of Beek... oo. es ye. ce exes 5 gm SaMMR pee ea. iy. irae goa Bat 5 lata aes al oo aS 8.5 gm MPIRIIINOML WALES lk i vrs els oleae eh Sask wi Sees I00o C.c Dissolve 4.5 gm. of dry agar in 500 c.c. distilled water by heating over a free flame, making up loss in weight by evaporation. Into another vessel 500 c.c. of distilled water is poured and to this is added 10 gm. of peptone, 5 gm. of Liebig’s Beef Extract, and 8.5 gm. of salt. This is heated until all is dissolved and the loss in weight by evaporation is made up by adding distilled water. Add the two solutions together; boil 30 minutes; make up loss in weight with distilled water, filter through absorb- ent cotton held in the funnel by cotton flannel, passing the filtrate through several times until perfectly clear. Test the reaction; adjust, if necessary, to +1.0 per cent normal acid, and tube, using 10 c.c. of medium in each tube. Sterilize for 15. minutes at 120° C. (15 pounds’ pressure) in an autoclave. Cool with running water and store in an ice-chest the air of which is saturated with moisture. CONRADI-DRIGALSKI MEDIUM These authors have modified lactose litmus agar by adding to it nutrose and crystal violet and by using 3 per cent of agar instead of 1 per cent. The crystal violet strongly inhibits the growth of many other bacteria, especially cocci, which would also color the medium red; the 3 per 276 APPENDIX cent agar makes the diffusion of the acid which is formed more difficult. Three pounds of chopped beef are allowed to stand 24 hours with 2 liters of water. The meat infusion is boiled one hour and filtered. Twenty gm. of Witte’s peptone, 20 gm. of nutrose, and 10 gm. of NaCl are then added, and the mixture boiled another hour. After filtration and the addition of 60 gm. of agar the mixture is boiled for 3 hours, made alkaline and filtered. In the meantime 300 c.c. of litmus solution (Kahlbaum) are boiled for 15 minutes with 30 gm. of lactose. Both solutions are then mixed and the mixture, which is now red, made faintly alkaline with ro per cent soda solution. To this feebly alkaline mixture 4 c.c. of hot sterile 10 per cent soda solution are added and 20 c.c. of a sterile solution (1 to 1000) of crystal violet (Hiéchst B.). ENDO MEDIUM Make 3 per cent agar as follows: 1. To 1 liter of cold water add 30 gm. of powdered agar by sifting successive small portions upon the surface and allowing them to settle. 2. Add 10 gm. of Witte’s peptone and 5 gm. of Liebig’s meat extract. 3. Heat in double boiler until the ingredients are thoroughly dissolved. 4. Neutralize the sodium carbonate, using litmus as an indicator, and then add 10 c.c. of a 10 per cent solution of sodium carbonate. ; 5. Store the medium in flasks in lots of 100 ¢.c. or in larger known quantities, the flasks being large enough for the other ingredients to be added later. 6. Sterilize 2 hours in streaming steam. It is essential to use powdered agar and cold water, since the agar settles more readily in cold water. APPENDIX 277 To use Endo medium: (a) make a 1o per cent solution of sodium sulphite in water; (b) make a ro per cent solution of basic fuchsin in 96 per cent alcohol; (c) add 2 c.c. of the fuchsin solution to 10 c.c. of the sulphite solution, pre- pared as above, and steam a few minutes in an Arnold sterilizer. 7. Add 1 gm. of chemically pure lactose to each 100 C.c. of Endo medium. 8. Melt the Endo medium in streaming steam and add 3 c.c. of the fuchsin-sulphite solution described above. 9. Pour plates, and allow them to -harden thoroughly in the incubator. The lactose used must be chemically pure, and the sul- phite solution must be made up fresh every day. HISS’ MEDIA Two media are used: one for the isolation of the typhoid bacillus by plate culture, and one for the differentiation of the typhoid bacillus from all other forms in pure culture in tubes. Plate Medium. The plate medium is composed of 10 gm. of agar, 25 gm. of gelatin, 5 gm. of sodium chloride, 5 gm. of Liebig’s extract, 10 gm. of dextrose, and 1000 c.c. of water. When the agar is thoroughly melted the gelatin is added and completely dissolved by a few minutes’ boiling. The medium is then titrated, to determine its reaction, phenolphthalein being used as an indicator. The requisite amount of normal hydrochloric acid or of sodium hydrate solution is added to bring it to the desired reaction; Le., +2. To clear the medium add the whites of one or two eggs, well beaten in 25 c.c. of water, boil for 45 minutes, and filter through a thin filter of absorbent cotton. Add the dextrose after clearing. The reaction of the medium is most important; it should contain never less than 2 per cent of normal acid. 278 APPENDIX Tube Medium. The tube medium contains agar 5 gm., gelatin 80 gm., sodium chloride 5 gm., meat extract 5 gm., and dextrose ro gm. to the liter of water; reaction +1.5. The mode of preparation is the same as for the plate medium, care being taken always to add the gelatin alter the agar is thoroughly melted, so as not to alter this ingredient by prolonged exposure to high temperature. The dextrose is: added after clearing. MILK The milk to be used as a culture medium shall be as fresh as possible, “ Certified Milk” being ordinarily the best obtainable in city laboratories. It shall be placed in a refrigerator over night to allow the cream to rise and the suspended matter to settle. The skimmed milk shall be siphoned off into a flask for use. It will be found more convenient, however, to allow the milk to stand in a separa- tory funnel. Should the milk be too acid the reaction shall be corrected to +1 by the addition of normal sodium hydrate. It is then ready ‘to be tubed and sterilized. Litmus milk shall be prepared as above, with the addition of sterile r per cent azolitmin. As it is impossible to make each lot of litmus milk with the same shade of color, it is recom- mended that a control tube be always exposed with the inoculated tubes for purposes of comparison. © NITRATE BROTH Dissolve 1 gm. peptone in 1 liter of tap water, and add 0.2 gm. of nitrite-free potassium nitrate. PEPTONE SOLUTION FOR INDOL TEST Broth which has been inoculated with B. coli to remove the muscle sugar contains toxins which interfere with the proper growth of many species, hence peptone solution, APPENDIX 279 (x per cent peptone in water) is recommended for use in the indol test. ZESCULIN MEDIA Broth. Dissolve 10 gm. peptone, 5 gm. commercial sodium taurocholate (or glycocholate), o.1 gm. esculin and o.5 gm. soluble iron citrate in 1 liter distilled water. Tube in 5 c.c. quantities in test-tubes and sterilize. It is preferable to keep the iron citrate solution separate and to add it to the rest of the media.as used, since the iron causes a precipitate. Sometimes the iron citrate will not readily dissolve. In such case a few drops of ammonia will cause immediate solution, especially on warming. The excess of ammonia should then be boiled off. Agar. This medium is made in the same manner as the broth with the addition of the usual amount of agar. APPARATUS . Few definite requirements need be made respecting apparatus. The quality of the glass shall be such as not to be easily acted upon by the reagents used, and all glass- ware shall be scrupulously clean when used. When nec- essary it shall be sterilized by dry heat for one hour at about 150° C. A slight browning of the cotton stoppers. is a good index of proper exposure. In some operations, as for example, the determination of the thermal death point, it is necessary to use test-tubes of a definite size and thickness. For this purpose the standard size culture tube shall be 15 cm. long, 1.6 cm. in diameter, and of medium weight. Tubes to be filled with gelatin for quantitative work may be those described as 6 in. X? in. “ heavy.” ; The standard loop for making transfers shall be pre- pared as follows: 280 APPENDIX Bend the end of a piece of No. 27 platinum wire about ro cm. long over a bit of No. ro wire, and fasten the loop thus formed into a glass rod to serve as a handle. A loopful of culture shall be interpreted as meaning all the fluid that the loop can hold. That is, the fluid shall form a bi-convex body and shall not be simply a film covering the space in the loop. : The standard fermentation tube shall be a tube 1.5 cm. in diameter, bent at an acute angle, closed at one end and provided with a bulb at the other which is large enough to receive all the liquid contained in the closed portion. The length of the closed end of the tube shall be about 14cm. The bulb shall have a diameter of about 3.8 cm. INCUBATION There shall be two standard temperatures of incubation— 20° C. and 37° C., the first corresponding to ordinary room temperature, the second to blood heat. The temperature of the incubators shall not be allowed to vary from these two standards by more than 1° C. in either direction. The atmosphere of the incubator shall be kept moist, preferably near the point of saturation. The incubator shall be ventilated so as to insure a reasonably good circulation of air in order to prevent the accumulation in the incubator of gases which might be prejudicial to the development of the bacteria. No definite period of incubation can be prescribed which will be suitable for all the work of species determination, but in reporting results the period used shall always be stated and form a part of the report. General statements as to the necessary periods will be found in connection with the principal tests. REFERENCES ApBa, F. 1895. 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Baker, 203, 204. Barthel, 147. Baton, 107, 111, 117. Bachmann, 64, 231. Beckmann, 143. Belcher, 213. Belitzer, 140. Bergey, 180, 191. Bettencourt, 141, 142, 147, 172. Biff, 176. Blachstein, 144. Blunt, 16. Bolton, 53. Borges, 141, 142, 147. Boullanger, 241. Braun, 122. Brezina, 55. Brotzu, 140. Brown, 151. Bruns, 144. Buchan, 248. Buchner, 17. Bulstrode, 225, 248. Burri, 150, 183. AUTHORS Calmette, 235, 241. Cambier, 36, 47. Cameron, 245. Carpenter, 237. Chick, 65, 105, 153- Chopin, 8r. Christian, 120. Clark, H. W., 57, 72, 111, 146, 157, 162, 166, 242, 248, 251. Clark, W. M., 118, 199. Clemesha, 17, 21, 108, 114, II5, 184, 193, 210. Cramer, 54. Conn, 245. Conradi, 76. Copeland, 102, 105, 192. Davis, 118, 190. Deehan, 180, 191. Dibdin, 228. Ditthorn, 85. Doebert, 77. Dolt, 130. Downes, 16. Drew, 13, 36. Drigalski, 76, 85. Duclaux, 6. Diiggeli, 147. Dunbar, 97. Dunham, 56, 96. Durham, 93, 95- Dyar, 140. 308 Eddy, 232. Egger, 26. Eijkman, 120, Ellms, 36. Elsner, 75. Endo, 76. English Committee, 136. Escherich, 99. Eyre, 140. Fehrs, 15. Ferguson, 116. Ferreira, 102, 141. Ficker, 80, 84. Fischer, 2, 26, go. Flatau, go. Fox, oI. Frankland, 12, 37, 98. Fremlin, 140. Freudenreich, 16, 150. Fromme, 113, I19, 121, 142, 163, 171, 225. Frost, 16, 179. Fuller, 16, 32, 43, 57, 116, 182. Gaehtgens, 77. Gage, 8, 9, 30, 31, 43, 44, 45, 46, 69, 70, 72, 107, 108, 109, IIT, 122)°533) 3465-7575: 202, 1106, 177, 186, 187, 203, 218, 230, 242, 249. Garré, 15. Gartner, 14, 35, 53; 159, 170, 171. Gautié, 172. Gildemeister, 85. Gordan, 147, 2009. Gorham, 249, 254, 256. Hachtel, 70, 91, 97. Hale, 116, 126, 127, 129. Ham, 244, 249, 250, 264. Hammerl, 151. Hammond, 113, 115, 116. INDEX OF AUTHORS Hankin, 75. Hansen, 221. Harrison, 120. Hazen, 10. Heider, 226. Heraeus, 35. Hesse, 29, 45, 47- Hilgermann, 120. Hill, 36, 64. Hiss, 78, 89. Hoffmann, 24, 80. Holman, 213. Hoover, 102, 192. Horder, 199, 209. Horhammer, 15. Horrocks, 15, 26, 87, 153, 182, 203, 207, 212. Horta, 102, 141. Houston, 11, 21, 22, 24, 27, 96, II5, 141, 146, 150, 152, 158, 159, 162, 178, 185, 189, 197, 202, 207, 200, 213, 222, 236, 239. Howe, 175, 177, 180, 199. Hunnewell, 115, 118, 203. Huntemiiller, 15. Irons, 106, 109, 120, 122. Jackson, 64, 78, 81, 91, 122, 123, 131, 192. Janowski, 6. Johnson, 58, 142, 182, 235, 232. Jordan, 14, 16, 17, 20. 23, 39, 55 153, 155, 226. Kabrhel, 25. Kaiser, 163. Keith, 140. Kellerman, 26, 238, 240. Kimberly, 238, 240. Kinnicutt, 234, 236. Kisskalt, 9. INDEX OF AUTHORS Klein, 75, 81, 146, 211, 213. Kligler, 148. Klotz, 82, 213. Kloumann, 80. Koch, 97. Kohn, 38, 53- Konradi, 24. Konrich, ror, 102, 121, 141, 147, 148, 163, 169, 179, 171, 176, 183, 187, 188. Korschun, 15. Kranepuhl, 239 ‘Kruse, 143, 169. Kiibler, go. Kurpjuweit, 239. Laws, 92, 202. Lederer, 64, 231. LeGros, 203. Lemke, 77. Lentz, 77. Levy, 144. Lochridge, 19. Loeffler, 77, 96. Longley, 107, 111, 117, 165. Lubenau, 87. MacConkey, 122, 175, 177, 178, 180, 184, 189, 190, IgI. Makgill, 122. Marshall, 176. Marvel, 246. Maschek, 26, 35. Mass. State Board of Health, 136, 161. Massol, 241. Massini, 183. Mathews, 66, 105. Mayer, A., 18, 19. Mayer, G., 56. McWeeney, 175. Melia, 81, 91, 116, 126, 727, 129. Miquel, 6, 37, 47, 51, 55- 309 Moore, 140. Moroni, 144. Muer, 131. Miller, 30, 31, 36, 84, 183. Neufeld, go. Neuman, 13, 120, 147. Newlands, 244, 249, 250, 264. Nibecker, 12, 67, 68, 111, 133, 204. Niedner, 45, 47- Nieter, 85. Nowack, 77; 121. Nuttall, 211. Orlandi, 161. Osgood, 226. Otto, 13. Pakes, 153. Palmer, 209. Papasotiriu, 146. Paredes, 102, 141. Parietti, 74. Park, 80. Pease, 240. Peckham, 182. Penfold, 183. Petruschky, 168. Phelps, 30, 31, 43, 44, 71, 111, 113, II5, 116, 122, 133, 177, 186, 187, 233, 237, 249, 252. Philbrick, 10, 11, 54. Poujol, 144. Pratt, 234, 236, 238, 240. Prescott, 12, 26, 43, 67, 107, 146, 203, 204. Procaccini, 17. Pusch, 168. Rapp, 17, 18. Refik, 143. Reinsch, 43. Remlinger, 93. 310 INDEX OF Reynolds, 1109. Rickards, 248. Rideal, 66, 229, 237, 242. Riedel, 38. Rivas, 180. Rogers, 118, 199. Rondelli, 161. Roth, 80. Rothberger, 122. Ruediger, 22, 23. Russell, 13, 16. Sauerbeck, 183. Savage, 24, 62, 122, 141, 145, 168, 214, 225. Sawin, 124, 125. Schepilewski, 83. Scheurlen, 14. Schneider, 93. Schottelius, 96. Schiider, 84. Schultz-Schultzenstein, 241. Schumacher, 230. Sedgwick, 4, 21, 43, 95, 105. Shell Fish Commission, A.P.H.A., 255. Shuttleworth, 59. Smith, ror, 106, 140, 150, 180. Soper, 246. Spitta, 14. Stamm, 117. Starkey, 86, 95, 96. Sternberg, 52. Stiles, 248. Stokes, 79, 91, 97, 122, 127. Stokvis, 15. Stoner, 127. Swellengrebel, 15. AUTHORS Thoman, 75. Thomson, 75. Thorne-Thorne, 246. Thresh, 36, 95, 246. Tiemann, 14, 35. Tietz, 77. Tully, 73. Twort, 183. Vallet, 84. Van Buskirk, 50. van der Leck, 129. Vincent, 56, 172, 213, 223. Walker, 101, 147, 180, 232. Wathelet, 92. Weissenfeld, 145. Welch, 211. West, 180. Wheeler, 18. Wherry, 178. Whipple, 18, 19, 38, 39, 41, 44, 46, 63, 71, 72, 110, 118, 181, 221. Whittaker, 26. Widal, 89. Wilhelmi, 248. Willson, 80, 84, 90. Winslow, 12, 19, 2I, 20, 30, 34, 60, 66, 67, 68, 71, 101, 111, 118, 133, 147, 148, 155, 180, 199, 203, 209, 213, 221, 232, 233, 234, 236. Wolfthiigel, 26, 38. Woodman, 221. Wright, 140. Wurtz, 65, 104. Zagari, 15. Zeit, 16, 23. SUBJECT INDEX Acid-forming organisms, 65. Acid wastes, antiseptic effect of, 20. Aesculin, 129. Aesculin bile medium, 1209. Aesculin medium, preparation of, 279. Aesculin test, 1209. Agar, drying of, 78. Agar, for body temperature count, 63. Agar, preparation of, 270. Agglutination of typhoid bacilli, 81, 82, 83. Anaerobic bacteria, 2or. Anaerobic spore-forming bacilli, 2rt. Anglo-American procedure, 172. Aniline dyes, use of, 77. Antagonism, 16. Anthrax, occurrence in water, 98. Apparatus, treatment of, 279. Arbitrary standards, fallacy of, 51. Atmospheric waters, 5. Atypical colon bacilli, 135. Azolitmin, 272. Bacillus, anaerobic spore-forming, 211. Bacillus acidi lactici, 189. Bacillus aerogenes, 1809. Bacillus alcaligenes, 94. Bacillus anthracis, 98. Bacillus coli, 94, 90. cultural features, 100. discovery, 90. distribution in waters, 152. effect of temperature on, 113. fermentation reactions, ror. importance of numbers, 149. index of pollution, 219. in cold-blooded animals, 142. in ground waters, 113. in sewage, 230. in soils, 152. isolation of, 104, 132. isolation by Conradi-Drigalski medium, 106. : isolation by Endo medium, 106. isolation by synthetic media, 130. mutations in, 183. occurrence, 99. occurrence in animals, 140, 141. occurrence on plants, 146. pathogenicity, roo. positive isolations, 110. preliminary enrichment of, 106. quantitative test, 138. standard tests for, 115. streak characteristics, 134. ubiquity of, 143. ~ Bacillus coli and sewage strepto- cocci, 205, 206. 311 312 B. communis, 181. B. communior, 181. a ’ B. dysenteriae, 89, 94. B. enteritidis, 94. B. mycoides, 133. B. sporogenes, 211. characteristics of, 213. ‘growth in liver broth, 212. in sewage, 213. isolation of, 211. occurrence of, 212. B. typhi, 94. identification of, 87. comparison with B. coli, 87. in oysters, 91. isolation from water, 89, go. isolation of, go. small numbers in water, 92. B. welchii, 129, 134, 138, 211. Bacteria, affected by temperature, 20. as agents of decomposition, 3. count of as index of efficiency of filters, 50. counts of on different media, 44. development at high tempera- tures, 69. ’ distribution, 1. diurnal variation of in sewage, 233. effect of composition of media on, 19. effect of light, 16, 17. effect of rainfall on, 7. effect of storage on, 10. estimation of, 48. expression of counts of, 49. factors influencing diminution, £3; field determinations of, 49. food requirements, 2 in contact effluents, 240. in disinfected effluents, 240. Bacteriological Bacteriological SUBJECT INDEX Bacteria in driven ita 27. in dust and air, 6. in earth, 6. in filtered waters, 57. in ground waters, 25. in lakes and ponds, 12. in ocean, 13. in oysters, seasonal variation of, 254, 250. in polluted streams, 7. in rain and snow, 6. in sand effluents, 234, 240. in septic effluents, 240. in sewage, 232. in sewage effluents, 233. in shallow wells, 26. in springs, 26. in surface waters, 54. in trickling filter effluents, 236. in unpolluted streams, 7. in water, 5. in water and shellfish, 249, 250. metatrophic, 2 microscopic enumeration of, 29. mineral nutrients for, 53. multiplication in stored waters, 37: nitrifying, 4 number of as index of purity, 60. numbers of in sewage, 232. occurrence, 1 paratrophic, 2 pathogenic in water, 74. prototrophic, 2, 29. relation to character of food, 29. quantitative methods of deter- mination, 20. seasonal variation of, 7. sedimentation of, 14. examination of shellfish, 244, 248. examination of sewage, methods of, 229. oP Ti ene 5 ee SUBJECT INDEX Bacteriological examinations of water, significance of, 217. Bacteriological methods for super- vision of filtration plants, 227. Bacteriological methods for super- vision of water supplies, 227. Bacteriological methods in detect- ing sewage distribution, 226. Bacteriology of sewage, 228. Bacteriology of sewage effluents, 228. Bacteriology of sewage filters, 241. Bacteriological examination, 220. advantages of, 220. certainty of, 223. delicacy of, 221. Bile, importance of, 127. Bile media, 81, 122. Bile salts, 122. selective action of, 123, 128. Body temperature count, 42, 61. relation to hot weather, 70. _ Caffein, selective action of, 80. * Calcium hypochlorite, 238. Carbon dioxide, absorption of, 117. Chemical disinfection, 238. Chlorine disinfection, relation to counts, 72. Cholera red reaction, 97. Cholera spirillum, isolation from water, 96. media for, 96. Clams, 244, 263. Cold, action of on bacteria, 21. Colon bacilli, 64, 99. as index of pollution, 168. as index of self-purification, 153- atypical, 135, 177. colonies of, 133. comparison with B. typhi, 87. 313 Colon bacilli, distribution in waters, 152. effect of temperature on, 22. * flaginac,” 185. importance of numbers, 149. increased survival in cold weather, 22. in cultivated soil, 170. in dust, 148. in filter effluents, 166. in filtered waters, 163. in foods, 347. in fruits, 147. in grains, 146. in ground waters, 161. in sewage, 231. in sewage effluents, 231. in shallow wells, 162. in shellfish, 252. in soil, 148. in surface waters, 157. in unpolluted waters, 155. isolation of, 104. isolation by bile media, 122. on plants, 146. standard tests for, 115. “‘ typical,” 176. ubiquity of, 143. varieties of, 174. viability at different tempera- tures, 128. Colon group, 99. characteristics, 104. distribution in water, 185. Jackson’s classification of, 192. McConkey’s classification of, 180. statistical classification, 198. tests for, 174. variations in, 181. Colon test, ror. Colon typhoid group, 94. reactions of, 95. 314 Comparison of sand and mechan- ical filters, 58. Composition of médium, impor- tance of, 43. Confirmatory tests, 136. Conradi-Drigalski medium, 76. for B. coli, 106. preparation of, 275. Contact beds, 235. Counting, 48. Crystal violet, 76. Culture media, ingredients for, 265. preparation of, 265. reaction of, 267. sterilization of, 266. titration of, 267. uniform methods for, 265. - Deep wells, bacteria in, 27. Dextrose broth, 107. advantages of, 108. comparison with lactose bile, 125. disadvantages of, 108. Dextrose test, failure of, 116. Diluting samples, 40. Disinfection of sewage, 237. Disinfection of sewage effluents, 237, Distribution of types of colon group in waters, 184. Division of colon group, 174. Dunham’s solution, 97. Dysentery, spread by water, 95. Eijkman test, 120. Endo medium, 76. for B. coli, 106. preparation of, 276. Examination of shellfish, standard methods for, 255, 257. Examination of shell water, 251. SUBJECT INDEX Expression of quantitative re- sults, 49. 4 Fermentation of lactose, 114. Fermentation test, 105, 109, 179. effect of temperature on, 112. exceptions to, 111. interpretation of, 110. Field kits, 49. Field methods, 50. Filter plants, routine control of, 230. Filtered waters, bacteria in, 57. Filtration in Japan, 58. * Flaginac ” B. coli, 185. Food supply, importance of, 20. Gartner bacillus, 95. Gas-forming bacteria, growth in liver broth, 131. Gas production in vacuo, 118. Gas ratio, 100, 109. effect of age on, 117. unreliability of, 118. Gelatin liquefaction, 177. Gelatin plates, use of, 41. Gelatin, preparation of, 270. Green plants, food requirements, Ground waters, 6. bacterial content of, 56. B. coli in, 113. Hesse medium, 78. drying of, 78. preparation of, 275. High temperatures, of, 60. Hiss agar. medium, 78. preparation of, 277. Hog cholera bacillus, 94. significance Incubation, 46, 280. SUBJECT INDEX Incubation period, 41, 47- for body temperature count, 64. Incubator, necessity for moisture in, 46. Indol test, 176. Infusoria, destruction of bacteria by, 15- Interpretation of results, 51. Intestinal bacilli, 103, 201. Isolation of B. coli, 104, 132. by bile media, 122. Tsolation of cholera spirillum, 97- Isolation of streptococci, 203. Lactose bile, 81, 122, 126. action of bacteria on, 102. advantage of, 127. comparison with dextrose broth, 125. decomposition of, 64. preparation of, 269. Lactose fermentation, 114. importance of, 115. Lactose fermenting bacilli, 102. effect of storage on, 115. “Lamirasacsal,” streptococci, 210- “ Larasacsal ” streptococci, 210. Light, destructive effect on bac- teria, 16, 17. Litmus lactose agar, 64, 104. counts, 65. incubation of, 132. preparation of, 272. Liver agar, preparation of, 274. Liver broth, 131. preparation of, 273. Liver gelatin, preparation of, 274. Malachite green agar, 77- McConkey’s group in filtered water, 198. 315 McConkey’s groups in raw waters, 198. McConkey’s groups in stored waters, 198. Mechanical filtration, 57. Methods of estimation, 48. Middletown outbreak, 245. Milk, preparation of, 278. Mussels, 244. Mutations in B. coli, 183. Nahrstoff-agar ratio, 31. Nahrstoff-gelatin ratio, 31. Nahrstoffi-Heyden agar, 29. ‘Neutral red, 122. Neutral red reaction, 12r. Neutral red test, 178. Nitrate broth, preparation of, 287. Nitrates, 3. Nitrite test, 177- Nitrogen cycle, 4. Nitroso-indol reaction, 97. Nutrient broth, preparation of, 268. Nutrose, 76. Obligate parasites, 29. Overgrowth, effect of, 1109. Oysters, 244. B. coli in, 259. bacterial counts of, 258. bacteriological examination of, 257- opened, examination of, 262. rating for B. coli, 260. sampling, 257- Oysters and typhoid, 246. Para-colon bacilli, 187. Para-typhoid bacilli, 94. 316 Pathogenes in water, 74. Pathogenic bacteria, 74. Peptone, importance of, 44. Petri dishes, 105. Phenol agar, 105. Phenol broth, 119. Phenol dextrose broth, 108. Plate method, 105. Plating, 40. Polluted shellfish, ery on, 248. Polluted waters, isolation of B. coli from, 107. Pollution, progressive, 223. of shellfish, 245. temporary, 225. Porous tops, 64, 105. Precipitation of typhoid bacilli, 81, 83, 84. Preliminary enrichment, 106. Presumptive test, 110. Presumptive tests, various, 126. Presumptive tests with bile media, 124. Progressive pollution, of, 223. Prototrophic bacteria, 29. Protozoa, reduction of bacteria by, 15. Pumping, effect of on bacteria, 35. effect of cook- detection Quantitative bacteriological de- termination, 29. interpretation of, 51. Quantitative results, expression of, 49. Rainfall, effect of on bacteria, 7. Reaction, importance of, 43. Reaction of culture media, 267. Reaction, optimum, 43. . SUBJECT INDEX Reducing bacteria, importance of, 242. - Relation between room tempera- ture and body temperature counts, 61, 67, 71. Room temperature counts, 61. Saccharose, fermentation of, 180. Samples, dilution of, 40. Samples, icing of, 39. Sampling, 33. Sand filtration, 57. Sanitary chemical analysis, sig- nificance of, 216. Sanitary inspection, 215. importance of, 172. Seasonal variation, 72. Sedimentation, 7. Sedimentation of bacteria, 14. Self-purification, 20. Selective media, 219. Selective temperatures, 219. Sewage, bacteria in, 214. bacteriology of, 228. colon bacilli in, 231. Sewage effluents, bacteriology of, eee). colon bacilli in, 231. standards for, 239. Sewage examination, use of bile medium in, 231. Sewage sampling, error of, 231. streptococci, 134, 201. as index of pollution, 202. isolation of, 204. Sewage streptococci’ and B. coli, growth in dextrose broth, 205, 206. detection of, 206. Sewage and sewage effluents, 228, 220. Shallow wells, bacteria in, 26. SUBJECT INDEX Shallow wells, body temperature count in, 67. _ Shellfish and disease, 244. bacteriological examination of, 248, 251. bacteria in shucked, 254. careless handling of, 253. colon bacilli in, 252. self-purification of, 252. standards of interpretation, 264. streptococci in, 252. Shell water, examination of, 251: Significance of 37° count, 62. Specific sewage organisms, tests for, 230. Springs, bacteria in, 26. Standard methods, 32, 33, 41- necessity of, 45. Standard reaction, 268. Standards for sewage effluents, 239. Staphylococci in sewage, 202. Storage, effectiveness of, 25. effect of on bacteria, 10, 21. effect of duration of, 23. } effect on lactose fermenters, 115. | Storage of samples, effect of, 37. ‘Stored waters, 6. Streptococci, 64, 133. antagonism to colon bacilli, 208. comparative fermentations by, | 210. from different animals, 200. in sewage, 202. in saliva, 204. in shellfish, 252. in polluted waters, 203. in stored sewage, 207. indicative of recent pollution, 207. index of pollution, 220. isolation of, 203. on animal bodies, 204. Streptococci, 209. Streptococci, “‘ lamiracsal,”’ 210. Streptococci, “ larasacsal,”’ 210 Streptococcus equinus, 209. Sugar broths, preparation of, 269. Sugar reactions, 179. Sugars, action of bacteria on, 102. Surface waters, 5. bacterial content of, 54. Swimming pools, bacteria in, 72. Synthetic media, 130. varieties of, 208, Temperature, effect on B. coli, 113. effect on bacteria in water, 20. effect on fermentation test, 112. Temporary pollution, detection of, 225. Titration of culture media, 267. Toxic products, effect of, 15. Trickling filters, 235. Typhoid, occurrence weather, 22. in cold Typhoid and shellfish, 245. Typhoid bacilli, agglutination of, 81, $2, 83. artificial infection of water with, 24. developing on malachite green media, 77. . effect of oxygen on, 19. enrichment in caffein media, 79. enrichment of, 75. examination of water for, 74. in polluted waters, 23. in pure culture, 16. in tap water, 24. in unsterilized waters, 23. isolation by lactose bile, 81. isolation of, 76. media for, 76, 77, 78, 79. precipitation of, 81, 83, 84. 318 Typhoid, preliminary enrichment of, 79. separation by motility, 85. small numbers in water, 92. summary of isolation methods, 86. uncultivated strains in water, 24. viability in mud, 24. viability in sewage, 16. viability in water, 15. “ Typical ” B. coli, 176. SUBJECT INDEX Unpolluted waters, body tempera- ture count in, 68. Urea, decomposition of, 3. Voges-Proskauer reaction, 180. Waters, classification of, 5. Wells, bacteria in, 26, 56. 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