IOWA STATE COLLEGE OF AGRICULTURE AND MECHANIC ARTS OFFICIAL PUBLICATION Vol. XX December 28, 1921 No. 31 BACTERIAL FERMENTING LACTOSE and THEip SIGNIFKJANCE IN WATER ANALYSIS Vr.^ - /.^^ MAcXLEVINE BULLETIN 62 ENGINEERING EXPERIMENT STATION AMES, IOWA ALBERT R. MANN LIBRARY New York State Colleges OF Agriculture and Home Economics Cornell University Cornell University Library OR 105.L66 Bacteria fermenting lactose and their si 3 1924 003 218 306 The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924003218306 IOWA STATE COLLEGE OF AGRICULTURE AND MECHANIC ARTS OFFICIAL PUBLICATION Vol. XX December 28, 1921 No. 31 BACTERIA FERMENTING LACTOSE and THEIR SIGNIFICANCE IN WATER ANALYSIS By MAX LEVINE Bacteriologist IOWA ENGINEERING EXPERIMENT STATION and Associate Professor of Bacteriology, IOWA STATE COLLEGE, AMES, IOWA BULLETIN 62 ENGINEERING EXPERIMENT STATION AMES, IOWA Published weekly by Iowa State College of Agriculture and Mechanic Arts, Ames, Iowa. Entered as Second-class matter, and accepted for mailing at special rate of postage provided for in Section 429, P. L. & R., Act August 24, 1912, authorized April 12, 1920. r^ ■■':: 312450 STATE BOARD OF EDUCATION Members Hon. D. D. Murphy, Presidenl Elkader Hon. George T. Baker. Davenport Hon. Chas. R. Brenton Dallas Center Hon. P. K. Holbrook Onawa Hon. Edw. P. Schoentgen Council Bluffs Hon. C. H. Thomas Creston Hon. Pauline Lewelling Devitt Oskaloosa Hon. W. C. Stuckslager Lisbon Hon. Anna B. Lawther Dubuque Finance Committee Hon. W. R. Boyd, President Cedar Rapids Hon. Thomas Lambert Sabida Hon. W. H. GemmUl, Secretary Des Moines ENGINEERING EXPERIMENT STATION Station ConncO (Appointed by the State Board of Education) Raymond A. Pearson, LL. D President Anson Marston, C. E Professor Louis Bevier Spinney, B. M. E Professor Warren H. Meeker, M. E Professor Fred Alan Fish, M. E. E. E Professor AUen Holmes Kimball, M. S Professor 0. R. Sweeney, M. S., Ph. D Professor Fred R. White, B. C. E Chief Engineer, Iowa Highway Commission Station Staff Raymond A. Pearson, LL. D President, Ex-officio Anson Martson, C. E Director and Civil Engineer John S. Dodds, C. E Bulletin Editor Warren H. Meeker, M. E Mechanical Engineer Fred Alan Fish, M. E. E. E Electrical Engineer Allen Holmes Kimball, M. S Architectural Engineer O. R. Sweeney, M. S., Ph. D Chemical Engineer Charles S. Nichols, C. E Sanitary Engineer Louis Bevier Spinney, B. M. E Illuminating Engineer and Physicist William J. Schlick, C. E Drainage Egineer T. R. Agg, C. E Highway Engineer John Edwin Brindley, A. M., Ph. D Engineering Economist Max Levine, S. B Bacteriologist Lulu Soppeland, M. S Assistant Bacteriologist J. H. Griffith, M. S Structural Materials Engineer D. A. Moulton, B. S. in Cer. Eng Ceramic Engineer A. K. Friedrich, E. M Mining Engineer G. W. Burke, B. S. in Chem. Eng Chemist C. H. Giester, B. S Assistant Chemist Clyde Mason, B. S. in E. E., B. S. in C. E Engineer C. J. Myers, B. S. in M. E Mechanical and Hectrioal Engineer Geo. W. Rodgers Mechanician Table of Contents Page I. Characteristics of the Colon Group of Bacteria 5 II. Evidence of Two Subdivisions in the Colon Group and Tests for their Differen- tiation 17 III. Classifications of the Colon Group of Bacteria 32 IV. The Detection of the Colon Group in Water 43 V. The Colon Group as an Index of Pollution 65 VI. The Spore Forming Lactose Fermenters, and their Significance in Water Analysis 91 Appendix A. Routine Methods of Water Analysis and the Colon Index 99 Appendix B. Culture Media 108 References 119 I. CHARACTERISTICS OF THE COLON GROUP OF BACTERIA* The bacteriological analysis of water is an indirect and quantitative one. Specific pathogenic organisms are not sought nor are they likely to be detected even in a dangerous water. It devolves upon the analyst to interpret his findings and particular emphasis is placed on the determination of the presence of the colon group. The investigator and analyst should therefore be thoroughly acquainted with the characteristics, peculiarities, and idiosyncrasies of the organisms in the group, particularly with refer- ence to their distribution, viability, and differential reactions. Bacterium coli was first isolated by Emmerich from the feces of a cholera patient in 1884. It was soon recognized as a normal inhabitant of the intestinal tract of man and of other animals. For the past three decades the colon group of bacteria has been extensively studied by bacter- iologists and sanitarians especially those interested in water supply and purification. Probably as much work has been done on this as on any other group of bacteria but there is not as yet an absolute agreement as to the limitations of the group. Limitations of the Group. In the 1905 report of the Committee on Standard Methods for Water Analysis of the American Public Health Association a series of tests were described which were supposedly char- acteristic of the colon group. These tests included morphology, motility, fermentation of glucose, coagulation of milk, production of indol, reduction of nitrates, and gelatin liquefaction. Many of these reactions, as gelatin liquefaction, nitrate reduction, and indol formation, require a long incuba- tion period. If the recommendations of the committee were adhered to, it would take at least nine days to justify the inclusion of an organism in the colon group. This was found to be very impractical and the ten- dency has been to simplify the preliminary tests necessary to place an organism in this categorv. The 1917 and the 1920 Standard Methods of Water Analysis define the colon group as follows: "It is recommended that the Bact. coli group be considered as including all non-spore-forming bacilli which ferment lactose with gas formation and grow ferobically on standard solid media." This characterization is concurred in by Winslow who defines the colon group as including all aerobic non-spore forming bacilli which produce acid and gas in glucose and lactose media. Hauser modifies the definition somewhat by excluding gelatin lique- fiers. Other investigators are inclined to extend the colon group to mclude spore forming organisms which are capable of fermenting lactose with gas production and which grow asrobically on solid media. A few such strains have been recently encountered in the routine examination of water. Perry and Monfort include such spore forming types as members of the colon group. The statement of Lohnis and Smith, who have made studies *Iii conformity with the recommendations of the committee on nomenclative of the Society of American Bacteriologists the colon group is considered in the genus Bacterium. on the life cycle of bacteria, that a single species of Azotobacter may pass through as many as twelve to fourteen morphological forms including spores, is not particularly relevant with respect to Bad. coli, the life cycle of which has been carefully studied by Kellerman and Scales who note specifically that spores were not observed. It is conceivable that unfavor- able environment may lead to spore formation by members of the colon- serogenes group but the writer has never encountered nor heard of such a transformation, although he has observed a large number of cultures kept under various unfavorable conditions, nor does he anticipate such a fundamental and radical change in form. These spore forming, aerobic, lactose fermenters confuse the ordin- ary tests for Bad. coli and must be taken into consideration in interpreting water analyses, just as it is essential to differentiate the anaerobic spore forming lactose fermenters which confuse the presumptive test for Bact. coli, but there is no logical reason nor justification for placing them in the colon group. Clark and Lubs raise the question as to the reliability of lactose fer- mentation as a primary criterion. They say, "If a fundamental cultural requirement of the members of the colon-aerogenes family is that it shall ferment lactose, there is imposed the same sort of requirement for the characterization of a whole family as is imposed by the MacConkey scheme when groups within the family are separated on the basis of the fermentation of another sugar, sucrose." They point out that the fermentation of sucrose, which was formerly employed to subdivide the colon group into species and varieties (Mac- Conkey's scheme) , has been found less desirable than differentation on more recently devised tests, such as the gas ratio, the methyl-red reaction, and the Voges Proskauer test. They raise the question as to whether it is not possible that in the near future a test may be discovered which will supplant lactose fermentation as the salient and fundamental requirement for the whole colon-aerogenes family- We may well agree with Clark and Lubs that the future holds out to us promises of improved differential tests but we do not feel that, in consequence, we shall not utilize such means as are now available. Surely the classification of the organisms of this group to the best of our abiltiy on tests which are now known and used, would simplify a reclassification when these more fundamental, and we hope more reliable reactions of the future are brought out. The fact remains that the fermentation of lactose has been successfully emploved for the separation of the non-pathogenic colon group from the disease pro- ducing para-typhoid and typhoid groups. Until a test is developed which will adequately supplant this, the fermentation of lactose with acid and gas production is considered a convenient and reasonably reliable criterion for members of the colon group of bacteria. The colon group will therefore be considered to include non-sporing, Gram negative bacilli which ferment lactose with the production of acid and gas and which are capable of growing aerobically. The statement frequently made, that the group consists of aerobes which ferment lactose is somewhat confusing, for the characteristic fermentation of lactose (gas formation) is determined under anaerobic conditions. DIFFERENTIAL TESTS The colon group, as defined, is a large and complex one including a number of closely related but distinct species and varieties. The question arises whether all the members are of equal sanitary significance or whether some may not be more intimately associated with animal or particularly with human pollution thereby becoming of special significance in the interpreta- tion of water or other examinations. It may be of practical value to dis- tinguish and classify these different types and, if possible, to correlate them with their habitat. For these purposes a large number of tests have been employed. The more important reactions to be considered are: Coagulation of milk. Gelatin liquefaction. Production of indol. Motility. Fermentation of carbohydrates. Production of acetyl-methyl-carbinol. (Voges Proskauer reac- tion). Uric acid test. Methyl red test. Coagulation of milk. The test for the determination of the co- agulation of milk is made by inoculating litmus or brom cresol purple milk which is then incubated at the body temperature for 48 hours. Acid is formed, some gas develops, and coagulation usually takes place in this time. If the milk has not been clotted the tube is immersed in boiling water for a few minutes or brought to a boil over a flame and if this treat- ment induces coagulation the reaction is considered as positive. If the milk has been over sterilized in the autoclave, coagulation does not take place as readily as when the medium is sterilized by the intermittent method in the Arnold. There is no digestion of the curd and the whey, if present, is clear. The litmus may be reduced by some strains. The milk reaction has been found very valuable in identification of the colon group. Gelatin liquefaction. The liquefaction of gelatin is an important test for the differentiation of colon species. Studies by Gligler and by Johnson and Levine indicate that this test is well correlated with motility and fermentation of glycerol. Unfortunately the liquefaction of gelatin is difficult to determine. The period of incubation usually employed is fourteen days at 20 degrees C. Gage and Phelps, and later Johnson and Levine pointed out that the proportion of liquefiers recognized varies with the period of incubation as may be seen from Table I. Among 202 strains studied by Johnson and Levine, 106 (52%) were gelatin liquefiers after 34 days incubation but only 31 (15.3%) showed this reaction in 13 days. TABLE I. LIQUEFACTION OF GELATIN BY 202 COLON STRAINS FROM SOIL. Incubation days Cultures liquefied % of total liquefiers 2 17 16.0 7 17 16.0 13 31 29.2 20 38 35.8 27 61 57.5 34 106 100.0 The long incubation period necessary makes this test an inconvenient one for practical work and in some laboratories a modification has been introduced employing 37" C. for' 48 hours. This of course liquefies the gelatin and so to determine whether a physiological decomposition has taken place the tubes are immersed in ice water until control tubes solidify. Inoculated tubes which remain liquid are regarded as having been peptonized by the action of the organism in question. Indol. The production of indol from peptone is very extensively determined particularly in England where indol formers are regarded as "typical" Eact. coli. The test is usuallv carried out in the following man- ner: One percent peptone water cultures are incubated for four or five days at body temperature. The culture is acidified with 1 c. c. of a ten percent solution of sulphuric acid and then 1 c. c. of a 1-5,000 potassium nitrite is added so as to form a layer on the surface. After a period varying from a few minutes to an hour a red ring will develop at the junction of the nitrite and acidified peptone culture if indol is present. This is kno^NTi as the Salkowfsky test. A more delicate reaction is obtained by the Ehrlich test which is per- formed thus: To the culture add 3 c. c. para-dimethyl-amido-benzaldehvde and 3 c. c. of a saturated solution of potassium persulphate. Presence of indol is indicated by the production of a red coloration. The significance and value of the test has been much in dispute. Howe found it to be but slightly correlated with other characteristics and con- sequently regarded it to be of little diagnostic value. Castellani and Chalmers on the other hand, consider indol of fundamental importance in classification of colon-like forms, and Houston believes it to be of par- ticular significance for distinguishing the "typical" or "exrretal"" from the "atypical" Bad. coli. Levine found that among members of the colon group which were of intestinal origin, 91 percent formed indol, whereas of those obtained from soil only 37.3 percent were indol positive. The constancy of the reaction has been questioned. Sminiow reported that subjection of Bad. coli. to the action of carbolic acid induces a loss of the ability to produce indol but that this character is regained after several sub-cultures in nutrient broth. The source of indo] in culture media is the amino acid tryptophane, CH,CHNH,-CO0H which is decomposed with the liberation of indol. Od (X) NH Many of the irregularities reported are undoubtedly due to varying quan- tities of tryptophane in the media employed and these may be eliminated by use of tryptophane broth as suggested by Kligler. Motility. Motility may be determined either by the hanging drop method or by the use of a semi-solid medium such as Hesse agar. In the latter non-motile organisms grow along the line of inoculation with very little diffusion into the medium whereas the motile organisms grow rapidly away from the line of inoculation producing a distinct turbid zone of several millimeters, in 6 to 12 hours, which may easily be observed with the naked eye. There is considerable disagreement as to the value of motility as an index and differential test for members of the colon group. There are undoubtedly both motile and non-motile colon bacilli in the intestinal tract of man. The character seems to be quite variable as a number of prelim- inary cultures are sometimes required to make motility evident and McWeeney has reported that some strains were motile at 20 degrees and not at 37 degrees C. The statement that motile forms are characteristic of the human in- testine appears to be in error as Stocklin (quoted by McWeeney) observed 116 non-motile strains among 300 colon bacilli from feces. Levine found only 32 percent of 25 cultures from man to be motile and only 20 percent of 30 cultures from raw and septic sewage whereas colon strains obtained from animals were almost always motile (sheep 77.3%, cow 80.0%, pig 93.7%, and horse 100%). It should be noted that these results were ob- tained with the use of a semi-solid agar (nutrient agar containing 0.5% agar). The relation of motility as determined by the hanging drop and semi- solid media has recently been studied by Chen and Rettger with results indicated in Table II. It appears that for the true Bad. coli there is excellent correlation in the two methods of motility determination. Out of a total of 173 cul- tures examined, 119 strains were found to be motile by the hanging drop method and 121 with the semi-solid agar medium. With the Bact. aerogenes strains, however, out of 477 cultures observed, 122 were motile by the hang- ing drop and only 75 by the agar method. It would seem that for the aerogenes types observation of motility in semi-solid media is undesire- able. 10 TABLE II. RELATION OF HANGING DROP AND SEMI-SOLID AGAR FOR DETERMINATION OF MOTILITY. (After Chen and Rettger, 1920) Hanging drop Semi-solid agar (Hesse) Number Type of Organism Motile Non Motile Motile Non Motile Examined Bad. coli Bad. aerogenes 119 122 54 325 121 75 52 372 447 Levine, however, in a study of 151 strains of the aerogenes-cloacae group, found an excellent correlation between motility, as determined in semi-solid medium, gelatin liquefaction and starch fermentation. Thus of 89 motile organisms 81 (91.0%) liquefied gelatin and only 4 (4.5%) fermented starch; whereas among 62 non-motile organisms only 2 (3.2%) liquefied gelatin while 61 (98.5%) fermented starch. A test which cor- relates so well with other characters is probably of differential value, and, although it is not recommended at present for routine work, it may be of significance and should be included in investigational studies. MacConkey recommends that motility be observed in six hour cul- tures using dark field illumination. Castellani and Chalmers also employ motility as an important differential criterion. Fermentation of carbohydrates. The carbohydrates which have been most commonly employed in the study of the colon group are listed below: Monosaccharids. Glucose, levulose, and galactose. Disaccharids. Lactose, sucrose, and maltose. Trisaccharid. Raffinose. Polysaccharids. Starch, inulin, and glycogen. Alcohols. Glycerol, mannitol, dulcitol, and adonitol. Glucoside. Salicin. The media for fermentation tests generally consist of peptone water or broth containing one percent of the test substance. Incubation is at the body temperature for 48 hours and a positive reaction is indicated by gas production. If desired, litmus, brom cresol purple, neutral red, or the Andrade indicator may be added to the medium to observe acid for- mation. Kligler suggests that quantitive acid-production be substituted for gas-formation as an index of fermentation. He points out tliat in stand- ard meat-infusion sugar-freed carbohydrate broth media there is a rather sharp dividing line between acid-producers and nonacid-producers at 1.5 percent normal acid and that quantitive gas-production is variable and unreliable. Although quantitive gas-formation as ordinarly determined in the Smith or Durham tube is markedly inconstant and therefore of little 11 value, the fact that gas is produced at all may, nevertheless be of con- siderable significance. If a culture is inoculated into sugar broth and gas is formed, while no gas is produced in plain broth, the organism would most certainly be regarded as a fermenter of the test sugar irrespective of whether more or less than 1.5 percent acid is formed. The low titer might be due to a secondary alkali-production which masks the acid, as suggested by Rogers. It has been repeatedly observed by the author that Bad. aerogenes- in peptone dipotassium-phosphate so- lution, containing one percent or two percent glucose, may be acid to methyl red after 24 hours' incubation but alkaline after a period of 48 to 96 hours at 37 degrees C. Rogers, Clark, and Evans also determined titratable acid and selected one percent normal acid as the point of demarcation between fermenters and non-fermenters but they point out the possible errors in acid-deter- mination and give precedence to gas-formation, if positive. The author's observations are that with peptone water as a base and one percent of the test carbohvdrates, nonfermenters rarely produced as much as 0.2 percent normal acid. At what point on the acid scale are fermenters to be differentiated from nonfermenters? There is considerable disagreement as to the max- imal amount of acid formed by Bact. coli. Kligler, using meat infusion media, often obtained titers of four percent normal acid or more and sim- ilar results have been recorded by Rogers and others. Browne, however, using Liebig's meat-extract media, states that the limiting acidity for Bact. coli is 2.4% normal acid as determined by titration with phenolphthalein. Winslow and Walker determined acid-production in 12 substances by Bact. coli. The maximal acidity observed was 0.45 c. c. N/20 NaOH to the cubic centimeter of culture medium, or 2.25 percent normal acid. The writer's experience, with peptone water as the basic medium, is in entire accord with Winslow and Walker, and with Browne. Of more than 2500 titrations, none showed over 2.4 per cent normal acid. The difference in acid-production observed by various investigators is due to differences in the composition of the media employed. It is now well established that more acid is formed in meat-infusion broth than in beef-extract broth. In media containing much phosphates, as yeast water, even more acid is formed than in meat infusion broth. Acid-production should not be given precedence over gas-formation. They may be independent characters. If, however, after careful studies, it appears that there is a marked correlation between quantitative acid-pro- duction and qualitative gas-formation, then it may be feasible to supplement, if not substitute, the gas test by the acid test. In that event, the line of demarkation between fermenters and nonfermenters would have to be determined for the medium employed. Table III. shows the relation of gas-production to the amount of acid formed from sucrose, raffinose, ducitol, glycerol, and salicin in peptone water. Other test substances were observed but are not indicated because they were invariably fermented with production of gas. 12 TABLE III. RELATIONSHIP BETWEEN QAUNTITATIVE ACID-PRODUCTION AND GAS-FORMATION BY COLON GROUP. Test Substance r,a» Strain Percentage of normal acid 1 1 nf. 0-0.19 1 0.20-0.391 0.40-0.591 0.60-0.79i0.80ormore 1 1 1 1 ' ' Sucrose ' + 1 1 8 1 1 1 1 10.6 1 1 79 1 1 [ 1 98.8 1 1.2 1 I 1 48 1 19 1 1 64.0 1 25.4 1 1 Raffinose r + fNo. I % [No. 1 % 1 1 1 12 1 1.3 1 2.5 72 1 1 1 2 1 1 93.5 1 1.3 1 2.6 1 18 58 1 22.8 1 73.4 1 2 1 1 2.6 Dulcitol " + fNo. fNo. % 86 97.8 2 3.0 1 1.1 5 7.5 1 LI 23 1 37 1 34.3 1 55.2 1 ! i Salicin "No. "+ -1 43 79.7 1 1.8 ■ 1 10 3 5.6 19 82 18.6 80.4 "No. .% 6 1 1 11.1 1 1.8 Glycerol + "No. 4 10.5 5 13.2 16 13.6 23 60.5 61 i 41 51.7 i 34.7 No. % 5 I 1 13.2 i 2.6 i It will be noted that acid-production in sucrose, dulcitol, and raffinose is well correlated with the presence or absence of gas. With salicin tlie correlation is not so marked, while with glycerol the line of demarcation between gas-formers and non-gas-formers, as indicated by the quantity of acid produced, is very indistinct. The substitution of quantitative acid- production for gas-formation would therefore be particularly undesirable when working with glycerol. 13 These results are well in accord with those of Winslow and Walker, who observe: "Gas-formation coincided with acidity except in the case of dextrin." All investigators are agreed that members of the colon group normally ferment the monosaccharids with production of both acid and gas. Very detailed studies have been carried out on the products of the fermenattion of glucose, particularly the gas ratio and the H*ion concentration. These careful observations have yielded very fruitful results. The disaccharid lactose is of course fermented by all members of the group and maltose is also attacked. Sucrose is practically always de- composed by strains obtained from the soil, from grains, or from animal feces, but less frequently by strains isolated from human dejecta or sewage. The fermentation of sucrose has been recognized by many investigators as a convenient and important character for subdivision. It is the primary character in the MacConkey classification, it is employed by Jackst)n, and has been recognized by all the more recent investigators of the colon group as a most important and convenient differential characteristic. The trisaccharid raffinose is fermented by practically all strains which ferment sucrose. This marked correlation between sucrose and raf- finose fermentation was emphasized by Howe, who observed that dextrose, lactose, sucrose, and raffinose constitute a metabolic gradient, noting that fermentation of any of these carbohydrates was always accompanied by fermentation of the less complex sugar in the series. TABLE IV. CORRELATION OF SUCROSE AND RAFFINOSE FERMENTATION IN COLON GROUP. Acid and gas in raffinose Acid and gas in sucrose + - + 233 8 — 8 84 In a study of 333 strains obtained from soil, sewage, and feces of various animals and man, Levine found only 8 sucrose nonfermenters among 241 strains which attacked raffinose, whereas of 92 raffinose non- fermenters, 84 failed to ferment sucrose. Both sucrose and raffinose need hardly be employed simultaneously in a study of the colon group. The correlation between fermentability of these carbohydrates has also been observed by Winslow and Walker; Birk; Rogers, Clark and Davis; Kligler; Murray; Rogers, Clark and Lubs; and others. The fact that these two sugars are similar in chemical construc- tion (neither possesses a reacting aldehyde group), may explain the similar- ity in the behavior of colon bacilli towards them. The polysaccharids are fermented by relatively few of the? species or varieties in the colon group. Ford pointed out that the Bact. aerogenes was a starch fermenter and that a few strains also fermented inulin. Gly- cogen is very rarely attacked. Laybourn reports that Bact. aerogenes usually attacks starches from many different sources. 14 The alcohols have been very frequenlty utilized in investigational studies. Thus dulcitol was employed by MacConkey and Jackson in their classifications, but it is now being generally supplanted by other sugars. Mannitol is fermented by practically all members of the colon group except a small group observed by Rogers in his grain series. The al- cohol adonitol was recently suggested as a means of differentiating the fecal from the non-fecal Bad. aerongenes and this was adopted by the Committee of Standard Methods of Water Analysis in the 1917 report. Glycerol has been found by Kligler and later by Levine to correlate well with gelatin liquefaction and they both distinguish Bact. aerogenes from Bact. cloacae on the basis of fermentation of this material. The alco- hols are thus an important group of carbohydrates for studies of the colon group of bacteria. The glucoside salicin has been recently suggested to supplant dulcitol for classification purposes by Kligler and by Levine and Castellani and Chalmers employ it for primary subdivision of their sucrose negative strains. Salicin fermentation is an important differential test. Voge8 Proskauer reaction. (Acetyl methyl carbinol test). By the Voges Proskauer reaction is meant the production of an eosin-like col- oration in dextrose broth cultures by some members of the colon group, if made strongly alkaline with potassium hydroxide. It takes its name from the fact that it was first observed by Voges and Proskauer in 1898. The coloration develops slowly from the surface of the medium gradually extending throughout the culture. The test is ordinarily carried out by adding two or three c. c. of 10 percent potassium hydroxide to an equal volume of a 48 to 96 hour dextrose broth culture and after thoro shaking the mixture is allowed to stand exposed to the air. The characteristic eosin-like color will develop in a few hours but it is well to record after 24 hours exposure if negative in a shorter time. It is suggested that the term Voges Proskauer reaction be restricted to designate the formation of acetyl methyl carbinol from glucose but when referring to its production from other carbohydrates or alcohols, the term acetyl-methyl-carbinol test be applied. The nature of the sub- stance being tested for is thus indicated just as is the case with indol. The Voges Proskauer reaction has been found very valuable and many investi- gators have observed that it is characteristic of die colon-like organisms of the soil and grains while it is very rare to encounter intestinal members of the colon group which give this test. Methyl Red Test. Clark and Lubs in 1915 devised the socalled methyl red test which serves to split the colon group into a methyl-red- positive subgroup, found to be characteristic of the organisms obtained fiom cow feces and other intestinal sources, and the methyl -red-nen-ative subgroup, which is the predominating type in the soil and on grains. The test is made by adding a few drops of methyl red indicator to a 0.5 percent dextrose-peptone- (Witte)-dipotassium phosphate culture and noting the reaction; a yellow coloration indicates alkalinity or a negative test and a red coloration denotes acidity or a positive test. The reaction 15 correlates very well with the Voges Proskauer test and will be considered more in detail in the following pages. Uric Acid Test. Koser, in studying the utilization of nitrogen from various sources, observed that some m-cmbers of the colon group {Bad. aer- ogenes) can utilize nitrogen from uric acid, whereas others (Bad. coli) can not. He also showed this characteristic to be correlated with the methyl red and Voges Proskauer reaction. Resume. The colon group obviously includes many varieties and species of bacteria. The monosaccharids and the disaccharid lactose are always fermented with acid and gas formation; nitrates are reduced and milk is acidified and coagulated; but with respect to other tests there is considerable variation within the group. TABLE V. CHARACTERISTICS OP 333 STRAINS OF COLON GROUP FROM SOIL, FECES AND SEWAGE. SOURCE Soil Feces and sewage All strains Number Studied 177 156 333 Character No. % pos. pos. No. 1 % pos. 1 pos. No. . % pos. pos. Voges Proskauer Test Motility Gelatin Indol Sucrose* Raffinose* Dulcitol* Glycerol* Salicin* Dextrin* Inulin* Starch* 142 123 83 66 165 162 74 78 159 82 21 57 80.3 69.6 46.8 37.3 93.3 91.6 41.8 43.1 89.9 46.4 11.9 32.2 9 96 142 76 79 68 118 102 8 7 5.8 61.5 0.0 91.1 48.7 50.7 43.6 76.2 66.1 5.1 0.0 4.5 151 219 83 208 241 241 142 196 261 90 21 64 45.4 65.8 29.9 62.4 72.5 72.5 42.7 58.8 78.4 27.0 6.3 19.2 *Acid and gas formation observed. In general the reactions of the organisms isolated from the soil are quite different from those obtained from various animal feces (horse, sheep, pig, cow, and man) and sewage. In Table V. and figure 1 are shown the frequency of the various reactions. 16 Q. > o T. O Q VCT u O CO 35 o 2 c c (U «J ^ ^ "n DC 3 VJ (0 c u ■f- Q £ (D n SOi? ■•>& ••M *.» Pig 1. Percent of Positive Reactions of Coli-lilfp Bacteria from (A) Foces and Sewage and (B) Soil ~ " 17 II. EVIDENCE OF TWO SUBDIVISIONS IN THE COLON GROUP AND TESTS FOR THEIR DIFFERENTIATION. The mass of recent work clearly demonstrates that the colon group includes two quite distinct subgroups which differ culturally but partic- ularly with respect to carbohydrate and nitrogen metabolism. These sub- divisions, which will be referred to hereafter as the coli and aerogenes sections, are also quite strikingly correlated with habitat, the former pre- dominating in feces and sewage, the latter in the soil and on grains. The evidence for this subdivision together with a detailed consideration of the differential reactions employed is presented here. General Differential Characteristics. EJscherich, in 1885, distin- guished Bad. coli commune from Bact. (lactis) aerogenes by the greater plumpness of the latter, its lack of motility, and its more rapid coagulation of milk. Later Smith, in 1893 and 1895, indicated that the Bact. aerogenes produced a heavier growth and showed a tendency toward capsule forma- tion. He also remarked that gas production was more rapid from glucose and that the proportion of carbon dioxide to hydrogen was greater. Chen and Rettger observe that in glucose broth the volume of gas is seldom greater than 40 percent with the coli section, whereas the aerogense sub- group frequently produced much more gas. TABLE VI. GAS PRODUCTION IN 1% GLUCOSE BROTH f After Chen and Rettger, 1920) Number of strains Less than 40% 40% or more No. 1 % No. 1 % Coli Subgroup Aerogenes Subgroup 173 447 165 95.4 51 11.4 8 396 4.6 88.6 Burton and Rettger report also that in a modified Uschinsky medium (glucose substituted for glycerol) the coli subgroup grows very poorly, if at all, whereas the aerogenes strains show vigorous growth with almost complete utilization of the sugar. Temperature Relationships. In some very careful studies on the rate of multiplication of Bact. coli, Barber found that the rate increased with increase in temperature showing a maximum at 44 degrees to 45 de- grees C. A temperature of 40 degrees C. has often been recommended for the isolation of the colon group from water and in the Eijkman test 46 degrees C. is employed. With reference to the aerogenes section, we find that Rogers and his associates often mention the necessity for using a relatively low temperature (30 degrees C.) for growth of some strains isolated from grains. This observation is concurred in by Winslow and Cohen, arid more recently Chen and Rettger report that in studies of soil organism a large number 18 refused to grow at 37 degrees C, at least for a while. In some unpub- lished work the writer has observed that in peptone lactose media at 43 degrees C. (in a water bath) all coli culture studied (16) grew luxuriantly, as evidenced by strong turbidity in 24 hours, but 69 percent of these strains showed no gas or only a bubble in this time. Of 20 aerogenes cultures only 2 showed luxuriant growth, 2 a slight growth, while 16 did not grow at all. That these two subgroups should show this temperature relationship might be anticipated from a consideration of their respective habitats. The coli section, being most frequently encountered in the animal intestinal tract would naturally have a higher optimum growth temperature than the aerogenes section, which is more characteristic of non-fecal origin. It would be interesting to know whether the members of the aerogenes section, isolated from the intestinal contents of man can be differentiated from the soil and grain strains on the basis of temperature relationship. EVIDENCE FROM CARBOHYDRATE METABOLISM The metabolism of carbohydrates, particularly glucose, has been care- fully studied by a number of investigators. The work of Harden and Wal- pole on the products of fermentation of glucose; that of Keys and Gillespie, and of Rogers and his associates on the gas ratio; the observations on the Voges Proskauer test and on acid production particularly the recent studies on the limiting H*ion concentration have served to clarify the entire group. The Products of Fermentation of Glucose. Harden has prob- ably done the most important and extensive work on this problem. Among the products of glucose fermentation. Harden and Walpole list alcohol, acetic acid, lactic acid, succinic acid, formic acid, carbon dioxide, and hydrogen as indicated in the following table: TABLE VII. PRODUCTS OF DECOMPOSITION OF GLUCOSE (After Harden and Walpole 1905-06) Products of fermentation Per cent by weight of sugar fermented by Bact. aerogenes Bact. coli Alcohol 17.1 12.85 Acetic acid 5.1 18.84 Lactic acid 5.5 31.90 Succinic acid 2.4 5.20 Formic acid 1.0 0. Carbon dioxide 38.0 18.1 Carbon dioxide c. u. per gram glucose 198.3 91.8 Hydrogen c. c. per gram glucose 82.4 110.0 CO,/H, 2.4 .83 H,/CO, .42 1.19 19 A perusal of the table will show that Dact. coli produces a large amount of acid whereas the acidity produced by Bad. aerogenes is very much less. This is particularly ijoticeable with respect to acetic and lactic acid. It will also be noted that the Eact. aerogenes produces about twice as much carbon dioxide as Bact. coli and that the volume of hydro- gen gas formed is more nearly the same. They observed further that Bact. coli utilized only a part of the available carbohydrate whereas the Bact. aerogenes strains completely exhausted the sugar. They conclude from these observations that the two organisms act upon glucose in a totally different manner and must therefore be regarded as separate and distinct. The Voges Proskauer Reaction. If the products of glucose de- composition enumerated above are summed up, it will be found that only 69 percent of the carbon is accounted for in case of Bact. aerogenes and 87 percent with Bact. coli. This led Harden to search for the discrep- ancy which he accounted for by the presence of a crude glycol. This con- sists for the most part of 2:3 butyl eneglycol (CH3CHOH-CHOH-CH3), On oxidation it yields acelyl-methyl-carbinol (CH3, CHOH, CO. CH3), a volatile reducing substance, which, when mixed with potassium hydroxide in the presence of peptone, imparts an eosine-like coloration to the mixture on standing. Butyleneglycol is oxidized to acetyl-methyl-carbinol by Bact. aerogenes but not by Bact. coli. Neither acetyl-methyl-carbinol nor butyleneglycol give the eosin-like coloration when mixed with potassium hydroxide. In the presence of peptone, however, the coloration develops on standing in the case of the carbinol but not with the glycol. According to Harden the reaction is due to further oxidation of the carbinol (CH3CO.CHOH.CH3) to diacetyl (CH3CO.CO.CH3) which reacts with some constituent of the peptone. In a later study Harden and N orris report that in the presence of strong potas- sium hydroxide solution diacetyl reacts with proteins to give a pink color- ation together with a green fluorescence. With arginine, creatine, dicyan- amide and guanidine acetic acid, the pink coloration is also obtained but the fluorescence is absent. The reaction depends on the presence of the group NH:C (NHg) N:HR. The exact significance of R. has not been de- termined. Harden ascribed the Voges-Proskauer reaction to the production of acetyl methyl carbinol. The reaction takes its name from the fact that it was first observed by Voges and Proskauer in 1898, in their studies on the "Bacteria of Haemor- rhagic Septicaemia." They describe this observation as follows: "On addition of caustic potash, we observed a new interesting color reaction. If the tube be allowed to stand 24 hours and longer at room temperature, after the addition of the potash, a beautiful fluorescent color somewhat similar to that of a dilute alcoholic solution of eosin forms in the culture fluid particularly at the open end of the tube exposed to the air. We have investigated a few of the properties of this coloring substance, which is not produced by the action of the alkali on the sugar, and have found that it is fairly resistant to the action of the external air. After a 20 time however, it becomes paler, and finally gives place to a dirty greenish brown." Considerable work has been carried out on the Voges Proskauer test in the last five years particularly with reference to its constancy, reliability, and methods of determination. It was customary to allow the potassium hydroxide-culture-mixture to stand 24 hours, and some investigators did not record until after 48 hours. This is extremely unfortunate as it results in unnecessary loss of time. Levine, Weldin, and Johnson found that of 140 strains which gave the Voges Proskauer reaction from glucose, 130 (92.9%) were posi- tive after 5 hours. A similar result was observed with sucrose, where of 134 positive carbinol tests, 127 (94%) were obtained in 5 hours. The same was true of other substances from which acetyl-methyl-carbinol was produced as is shown in the accompanying table. They conclude from this that a period of 5 hours after the addition of the alkali is suf- ficient as a presumptive test for the Voges Proskauer reaction. TABLE Vni. COMPARISON OF FIVE HOUR AND TWENTY-FOUR HOUR RECORDS OF TESTS FOR ACETYL-METHYL-CARBINOL. Test Substzinces Total positive reactions Positive in 5 Hr. Positive Negative in 24 Hr. in 5 Hr. No. % No. % 140 134 114 131 104 14 12 18 130 127 114 116 86 11 12 17 92.9 94.8 100.0 88.5 82.7 78.6 100.0 94..! 10 7 15 i 18 3 1 7.1 5.2 Raffinose 00 Mannitol 11.5 12.3 Dulcitol 21.4 0.0 Starch 5.6 West suggested that the reaction could be hastened by heating the mixture and blowing air through it. Levine, Weldin and Johnson employed various oxidizing agents (potassium dichromate, potassium perchlorate, bleaching powder, barium peroxide, and hydrogen peroxide) all of which were capable of accelerating the reaction but best results were obtained with hydrogen peroxide. To 3 c. c. of a 48 hour culture was added an equal volume of 10 percent potassium hydroxide; the mixture was heated in a boiling water bath for 3 minutes and then 2 or 3 drops of hydrogen peroxide were added. The pink coloration appeared in one or two min- utes and persisted for several hours. An excess of hydrogen peroxide or any other oxidizing agent is to be avoided as the coloration will disappear in a very few minutes, or even instantly, if the excess is verv areat. Chen and Rettger suggest the following technique for the ^ oaes Pros- kauer test: Five or six c. c. of the culture are added to an equal volume of 10 percent potassium hydroxide in a test tube, well shaken, and incu- bated at 30 degrees C. for one to three hours, after which tlie tube is a^ain vigorously shaken until the liquid becomes foamy. A decided eosin^like coloration will develop in an hour or two. 21 The test for acetyl-methyl-carbinol is but little affected by the period or temperature of incubation of the culture. Positive reactions have been obtained after one, three, or five days at 30 to 37 degrees C. and Chen and Rettger obtained positive results in 10 to 14 hours at 30 degrees C. The kind of peptone does not influence the test, but brighter reactions are ob- tained in peptone than in synthetic media. As neither the character of the medium nor the period of incubation of culture interferes seriously with the test, the Voges Proskauer reaction should serve as a convenient and valuable index for the differentiation of aerogenes from the more objectionable coli section. Gas Production and Gas Ratio. When the presumptive test for the colon group was first suggested, it was pointed out that a volume of 25 to 75 percent gas was particularly likely to be due to colon bacilli. Escherich in 1885 determined gas ratios for Bad. aerogenes, but Theobold Smith in 1895 first called attention to the significance of the ratio of the gases evolved in the decomposition of glucose, pointing out that, whereas Bad. coli produced twice as much hydrogen as carbon dioxide, the Bad. aerogenes differed in that it produced equal volumes of these two gases. These ratios were obtained with the Smith fermentation tube and may be referred to as the crude gas ratio. The determination of the composition of the gases in the Smith tube is very inaccurate and unreliable due in part to the ab- sorption and solution of carbon dioxide and to neutralization by amphoteric substances in the culture medium which would tend to reduce the amount of carbon dioxide observed. Referring to Table VII., it will be noted that in the careful quantitative studies of glucose fermentation by Harden and Walpole, Bad. coli evolved carbon dioxide and hydrogen in ap- proximately equal volumes and not in the ratio of one to two as had been observed by Smith. On the other hand, Bad. aerogenes forms twice as much carbon dioxide as hydrogen instead of equal volumes observed with the Smith tube. These differences are easily accounted for, as has been stated above, by the loss of carbon dioxide in the Smith tube. Keyes and Gillespie carried out a series of very careful experiments on the gas ratio and their work has since been confirmed and amplified by Rogers and his associates. They conclude that the accurately determined gas ratio, obtained by growing the test organism in glucose medium in a vacuum and measuring all gas formed, is of fundamental significance and importance in studies on the colon group. The real significance of this accurately determined gas ratio was not fully appreciated until 1914 when Rogers called attention to the striking correlation between this ratio and the source of the organisms. In three papers by Rogers, Clark, and Davis (1914) and Rogers, Clark, and Evans (1914 and 1915), it is demonstrated very conclusively that the colon strains obtained from bovine feces decompose glucose with the liberation of carbon dioxide and hydrogen in about equal volume, while strains isolated from grains formed two or more times as much carbon dioxide as hydrogen. The former group {Q.02/')^.,=\:\) seemed in their other characteristics to resemble Bad. coli; the latter (C02/'H2=2:1) 22 appeared to be the Bad. oerogenes. This work of Rogers and his asso- ciates aroused considerable interest in the possible different sanitary sig- nificance of these bacteria and has stimulated considerable investigational studies. Acid Production. Studies on acid production have been carried out from two points of view. The earlier observations were restricted to qualitative tests or to the determination of total titratable acid with phen- olphthalein as an indicator but the more recent studies have concerned themselves with actual or effective acidity (i. e. the H*ion concentration) ; Evidence as to the differentiations of Bad. coli and Bad. aerogenes by both of these methods will be considered briefly. Total or Titratable Acid. In considering total acidity, we are struck with the fact that the titer is affected by the composition of the medium as was indicated in the preceding chapter. Observations of different investigators are therefore extremely difficult to compare as com- parable data can be obtained only with the same medium. Furthermore in order to avoid fallacies, it is necessary to observe considerable numbers of strains and to treat the results statistically. A few extremely high or low results will influence considerablv the average acid production of a collection of organisms. The use of unqualified averages mav therefore lead to a misconception of the acid producing properties of a group. To supplement the arithmetic mean or numerical average some statement should be made as to the distribution of the individual strains (variates) about the average. This may be indicated by the probable error or by the standard deviation. The coefficient of variability (the ratio of the stand- ard deviation to the mean) is an excellent abstract measure of variability. The modal acid production (the amount of acid most frequently formed) is usually of greater significance than the average amount of acid formed. The standard deviation is the measure of variability most commonlv employed, particularly by mathematicians. It may be expressed mathe- matically as = F? where ''n" is the number of variates or observations, "d" the deviation of the individual variates from the mean, and "f" the frequency of a devia- tion "d".The standard deviation serves to indicate whether the departures from the mean are small or great. The closer the individual organisms group themselves about the mean, or average, the smaller the standard deviation. An example may make clear the meaning and significance of the standard deviation. Suppose that the amounts of acid formed bv a group (A) of 4 organisms in glucose broth are 2.1, 2.2, 2.2, and 2.3 percent normal acid, and that those formed by another group (B) of 4 organisms are 1.9, 2, 2.4, and 2.5 percent normal acid. The average for each group is 2.2, but mere inspection shows that the organisms in Group A and those in Group B are quite differently distributed with respect to this average. In large collection of data inspection is impracticable but the standard devi- ation serves well in its place. The standard deviation in Group A is ±0.07 while for Group B it is ±0.25. The larger deviation in B denotes that the individuals in the group wander farther away from the average than do those in Group A. Acid Production from Glucose. Considering 156 strains, isolated from various sources (including sheep, horse, cow, pig, man, and sewage), the V. P. negative (coli section) gave an average of 1.82 percent normal acid with an empirical mode at 1.9 percent while the V. P. positive (aero- genes section) produced an average of 1.46 percent with a mode at 1.5 percent normal acid. Although the difference (0.36±0.04) is not very large, it appears significant for rather striking differences in acid formation between the V. P. positive and V. P. negative strains are observed with many other test substances, as maltose, sucrose, glycerol, and dulcitol. This observation is also in line with the work of Harden and Walpole, already referred to that Bad. aerogenes produces less acid than Bact. coli from glucose. TABLE IX. ACID PRODUCTION BY COLON GROUP FROM GLUCOSE AND SUCROSE. Percentage Glucose Sucrose* of All Voges- All Voges- normal strains Proskauer strains Proskauer acid Negative Positive Negative Positive 0.00-0.19 79 79 0.20-0.39 1 1 0.40-0.59 8 8 0.60-0.79 1 1 48 48 0.80-0.99 3 3 6 6 1.00-1.19 2 1 1 1.20-1.39 11 9 2 3 1 2 1.40-1.59 19 15 4 7 3 4 1.60-1.79 22 20 2 2 2 1.80-1.99 54 54 2.00-2.19 41 41 1 1 2.20-2.39 3 3 Total acid- formers 156 147 9 75 66 9 Mode 1.90 1.90 1.50 0.70 0.70 0.74 1.50 Mean 1.80 1.82 1.46 0.84 1.57 Probable error.-.. -4- .20 -H.20 ±.12 ±.23 ±.14 ±.16 Standard devi- ation ±.30 ±.30 ±.18 ±.34 ±.20 ±.23 *Only fermenters included in calculations. Acid Production from Sucrose. Sucrose is not attacked by all members of the colon group. It is practically always fermented, how- ever, with acid and gas production by the aerogenes section and many of the strains of the coli section. In the foregoing table are given the fre- 24 quency of acid production from sucrose by the V. P. negative (coli) and V. P. positive (aerogenes) strains isolated from feces and sewage. It will be observed that those V. P. negative forms which are capable of TABLE X. ACID-PRODUCTION IN FERMENTABLE SUBSTANCES BY VOCES- PROSKAUER-POSITIVE AND NEGATIVE BACILLI OF COLON GROUP Pe American rcent of -Museum Vormal Acid Lev Excess (in percent ine's of Normal) Test Substance strains strains by the V.P.+ strains American- V.P.- V.P.+ V.P.- V.P.+ Museum Levine's strains strains Glucose 1.82 1.52 1.82 1.46 -.30 -.36 Galactose 1.31 1.28 1.36 1.21 -.03 -.15 Lactose 0.96 0.85 1.31 1.26 -.11 -.05 Mannitol 1.37 1.41 1.31 1.48 + .04 +.17 Maltose 0.66 1.01 0.75 1.12 + .35 +.37 Sucrose 0.71 1.52 0.74 1.57 +.81 + .83 Raffinose 0.79 1.22 0.96 1.32 + .43 +.36 Glycerol 0.58 1.27 0.70 1.28 +.69 +.58 Dulcitol 0.83 1.15 0.81 1.30 + .32 +.49 Salicin 1.00 1.38 0.94 1.28 + .38 + .34 '.Z flc/'d- r'roducthn by Voyes- Pras/fauer Positive and /^eoati^^e Co/i-/i'ke. Bct.cte.ria. 25 attacking sucrose produce less acid than the V. P. positives. The means for the two groups are .74 and 1.57 percent respectively and the empirical modes 0.7 and 1.5 percent normal acid. Acid Production from Various Other Carbohydrates. Obser- vations on the average quantities of acid formed from various substances by members of the coli (V. P. — ) and aerogenes (V. P.-|-) subgroups, obtained from the American Museum collection and those isolated by the author from feces and sewage gave the results shown in Table X. Inspection of Table X. and figure 2 indicates that considering all of the 167 strains studied, the aerogenes strains form less acid from glucose than does the coli section and about equal quantities form galactose, manni- tol, and lactose. In all other test substances, maltose, salicin, raffinose, dulcitol, glycerol, and sucrose, the V. P. positive strains (aerogenes) give rise to considerably more acid, the excess increasing in the order named. Although the differences obtained in salicin, raffinose, and possibly glucose, may not be so significant on account of the variation observed among in- dividual strains, it is nevertheless quite striking that a small number of strains taken at random, from the American Museum collection and a few freshly isolated cultures should show such a marked parallelism as is in- dicated in Table X. and figure 2 with respect to the amount of acid formed' when they decompose such a variety of carbohydrates. The H*ion Concentration. In 1915 Clark and Lubs first pointed out that in a medium consisting of 0.5 percent anhydrous glucose, dipotas- sium phosphate, and Witte's peptone, the low ratio cultures (coli section) produced a high acidity (H^ion) which remained permament; the high ratio strains (aerogenes section) were much less acid and became progress- ively more alkaline. This difference in acidity was easily recognized by the use of the indicator methyl red which gave an acid reaction with the low ratio and an alkaline reaction with the high ratio group. This test has become known as the methyl red reaction. Principle of the Methyl Red Reaction. Michaelis and Morcora* observed that cultures of Bad. coli fermented lactose until a hydrogen ion concentration of 1x10^ was reached and then ceased their activity. They considered this point a physiological constant for the organism in question. Clark, in 1915, obtained similar results with a culture of Bact. coli in glu- cose peptone water. He found that, irrespective of the initial acidity, the final hydrogen ion concentration varied but slightly (PH 4.37 to 4.55). If we again consider the products of decomposition of glucose by Bact. coli and Bact. aerogenes (see Table VII.), it will be noted that whereas Bact. aerogenes decomposes 14 percent of glucose into acids (acetic lactic, suc- cinic, and formic) Bact. coli produces acids from 56 percent of the glu- cose. Thus, from a given quantity of sugar, the amount of acid evolved and consequently the H+ion reached will be greater for Eact. coli than for Bact. aerogenes. If now the amount of glucose in a medium is restricted to that quantity necessary to yield the limiting (inhibiting) H*ion con- centration for Bact. coli, the resulting reaction with Bact. aerogenes will *Cited by Clark and Lubs 26 necessarily be less acid. On further incubation the Bact. coli would die off, whereas the more alkaline Bact. aerogenes culture would continue to grow exhausting the available sugar, after which the reaction would become progressively more alkaline. This reversion is due partly to the decompo- sition of the peptones with the liberation of alkali but particularly to the conversion of the organic acids originally produced from the glucose into carbonates and bicarbonates, as shown by Ayers and Roup. As incuba- tion is prolonged, the difference in reaction between a Bact. aerogenes cul- ture and one of Bact. coli may be considerably increased. Clark and Lubs, in some very careful work, found that a concentra- tion of 0.5 percent anhydrous glucose, dipotassium phosphate, and Witte's peptone affords the proper combination of glucose and buffer substances for the differentiation of the coli and aerogenes sections. They recommend an incubation temperature of 30 degrees C. for 5 days, after which period, Bact. coli is acid and Bact. aerogenes will be found to be alkaline to the indicator methyl red. TABLE XI. EFFECT OF THE TEMPERATURE AND THE PERIOD OF INCUBA- TION ON THE REACTION WITH METHYL RED. Incubation at 2nd 3rd 37 C. in 4th days 6th Incubation at 30 C. in days Reaction 2nd 3rd 5th 7th Acid 143 146 12 9 12 12 146 9 12 146 9 12 139 18 10 149 6 12 148 7 12 148 Neutral 6 Alkaline 13 As to the effect of temperature and period of incubation on the re- action with methyl red, the indications are that the final reaction is reached more quickly at body temperature than at 30 degrees C. (Table XI.). With an incubation period of 5 days there is little choice between 30 de- grees C. and 37 degrees C, but if the time of incubation is reduced to two or three days, which would be very desirable for routine water work, the body temperature seems preferable. It is of course recognized that some strains of the aerogenes section will not grow well at body temperature, but from the point of view of the bacterial analysis of water it is felt that there is little value in detecting such strains. The extra expense of main- taining a 30 degree incubator for their isolation is consequentlv not war- ranted. Too much emphasis can not be placed on the necessitv of employing Witte's peptone in preparation of the medium mentioned above. The in- discriminate substitution of other peptones is bound to lead to confusion and error. Owing to the great diificulty in obtaining Witte's peptone, Clark and Lubs were led to devise a synthetic medium which consists of the following: anhydrous Na,HPO,,. acid potassium phthalate. aspartic acid. 0.7% 0.1 % 0.4% anhvdrous dextrose. 27 Incubation at 30 degree? C. for three to five days is recommended. This synthetic medium may also be used for the Voges Proskauer test by mixing with it (at the time of addition of the alkali) 0.1 percent pure casein. Evidence from Nitrogen Metabolism. In the foregoing paragraphs it was pointed out that there were two main divisions in the colon group which could be easily distinguished by differences in carbohydrate meta- bolism. It is becoming apparent that the differentiation may also be made upon nitrogen metabolism. In 1903, Rettger noted that the products of protein metabolism (mer- captan, scatol, phenols aromatic axyacids, etc.) are formed much more slowly by Bad. aerogenes than by Bad. coli. Koser (1918) in a study of 124 colon cultures found that in a medium containing uric acid as the sole source of nitrogen the V. P.-|- strains (aerogenes section) grew luxuriantly whereas the V. P. — strains (coli section) failed to grow. TABLE XII. DIFFERENTIATION OF COLI AND AEROGENES SECTIONS (After Koser, 1918) Growth in Uric Acid medium Voges Proskauer reaction Methyl Red Test Cultuies employed Pos. 1 Neg. Pos. 1 Neg. Pos. 1 Neg. Coli subgroup 74 1 74 1 74 72 1 2 Aerogenes subgroup 50 50 1 50 1 1 50 The medium employed by Koser consisted of the following: 1000 c. c. 5 grams. 0.2 grams. 0.1 grams. 1.0 grams. 30.0 grams. 0.5 grams. Distilled ammonia free water NaCl Magnesium sulphate CaClj Dipotassium phosphate Glycerol Uric acid Sterilization is in the autoclave at 13 to 15 pounds for 15 minutes. A slight turbidity, presumably due to the finely divided precipitate of cal- cium sulphate may be evident immediately after autoclaving but disap- pears on cooling. A solid uric acid medium may be prepared by the addition of 1.5 percent washed agar. He suggests the differentiation is due to the inability of the strains in the Coli Sedion to break down the purin ring and thus obtain nitrogen for growth from the uric acid. NH — C --0 I I O'C C —NH\ I II ^=o NH- -NH^ 28 Evidently scrupulous care must be employed in the preparation of the medium to avoid introduction of any nitrogenous compounds which might be present in imperfectly cleaned glass-v»rare or ordinary tap v^ater or even distilled water, for the nitrogen thus introduced might serve to pro- mote growth of organisms of the coli subgroup and thus obscure the differential reaction. The reliability of this uric acid differential test has been confirmed by Chen and Rettger in a study of 640 strains isolated from soil and from intestinal tracts of man and various animals. They note, however, that half the coli strains isolated from the soil grew in the uric acid medium. TABLE XIII. GROWTH OF THE COLON GROUP IN URIC ACID MEDIUM (After Chen and Rettger, 1920) Growth Pos. Neg. Total Aerogenes strains from soil (V. P. +) 447 447 Coll strains from feces (V. P. — ) 173 173 Coli strains from soil (V. P. — ) 10 10 20 They showed further that xanthine may be substituted for uric acid and that all the coli strains from the soil failed to grow in the xanthine medium. It is probable that xanthine will be found preferable to uric acid for dif- ferentiation of the coli from the aerogenes types but there may be an intermediate group resembling the former with respect to the Y. P. and methyl red reactions but which differs from it in its ability to attack uric acid. The temperature of incubation employed bv Koser was 37 degrees C. for four days. Chen and Rettger employed a temperature of 30 degrees C. for three to five davs. Differentiation on Solid Media. The coli and aerogenes sections also present several cultural differences particularly with respect to the appearance of colonies on some solid media. The aerogenes colonies are generally larger more opaque and more convex than those of the coli sub- group. On litmus lactose agar the former sometimes revert to an alkaline reaction. These differences, however, are very difficult to detect on litmus lactose agar but may be readily observed in the Endo and particularly on eosin-methylene-blue agar. Ferreira, Horta and Paredes noted that Bad. aerogenes and Bact. clo- acae produced a rose color on Endo agar but that the metallic luster, so characteristic of Bad. coli, was absent. Levine employing a simplified fuchsin sulphite (Endo) agar notes striking differences between the col- onies of the coli and aerogenes subgroups. The coli strains formed deep red button-like colonies with a greenish metallic sheen (by reflected light) and were usually three or four m. m. in diameter when well isolated. The aerogenes colonies are lighter colored, markedly convex, and do not show the metallic luster. 29 Wood records that on neutral-red-bile salt lactose agar, the V. P.-|-M. R. — strains may develop mucoid colonies which are generally paler than the V. P.— M. R.4- strains. Another distinct differentiation may be obtained with the modification of the (Holt-Harris) eosin-methylene-blue agar (see appendix). On this medium the well isolated coli colonies are about three m. m. in diameter appearing very dark, almost black by transmitted light and by reflected light they seem to be button-like, often concentrically ringed with a dis- tinct greenish metallic sheen. Those of aerogenes, on the other hand, are larger, tend to run together, are markedly convex, very much lighter in color, (when viewed by transmitted light), and a metallic sheen is rarely observed. That these cultural differences are quite reliable, at least for routine work, is indicated by a report of Levine, who found that 96.9 per- cent of 122 colonies picked as of the coli section from their appearance on eosin methylene blue agar and 82.4 percent of 102 colonies supposedly of the aerogenes section were confirmed by subsequent tests. Correlation of Reactions. The studies of carbohydrate and nitrogen metabolism and cultural characters, all indicate that the colon group em- braces two distinct sections which may be readily distinguished by a num- ber of tests including (1) the gas ratio, (2) acidity to methyl red, (3) the Voges Proskauer reaction, (4) growth in uric acid medium, and (5) ap- pearance of colonies on agar (Endo and eosin-methylene-blue). Although no investigator has employed all these reactions simultan- eously, they are known to be very strikingly correlated. Thus Clark and Lubs in 1915 observed a perfect correlation between the gas ratio and the methyl red test. Levine pointed out that the strains which were positive for methyl red were negative for the V. P. test and vice versa. The cor- relation between these two reactions had been extensively confirmed. Johnson; Burton and Rettger; Chen and Rettger; Hulton; Greenfield; and many others have observed an almost perfect correlaction. Clark and Lubs (1917) correlated the gas ratio with the V. P. and M. R. reactions, while Koser and Chen and Rettger have shown that the uric acid test, the V. P. and methyl red reaction were strikingly confirmatory of each other. Choice of a Routine Differential Test. The question naturally arises as to which test shall be employed or at least given preference in future studies of this group. A choice of a test must of course be de- pendent upon the nature of the work at hand. For investigational studies it should be emphasized that the carefully determined gas ratio, as is urged by Rogers, Clark, etc., is of fundamental importance and that in order to throw further light upon the reliability of the other reactions men- tioned it is desirable that they should all be considered and observed. The problem, however, is quite different when applied to routine water an- alysis. There the time available is limited, the apparatus and other lab- oratory facilities are at a minimum, and the skill of the analyst, we must regrettably admit, is too frequently not comparable with that of the chem- ist. 30 For routine water work the selection of a differential test must be governed to a great extent by the following considerations: 1. The medium should be simple, easy to prepare, and permit of a considerable degree of variation. 2. The reaction should be distinct and constant. 3. The test must be one which can be completed in a short time, preferably not more than 24 hours. In the light of these considerations it becomes evident that the gas ratio is out of the question. That it is permanent and constant has been well demonstrated by the investigations of Rogers and his associates, but the skill and apparatus necessary together with the time required removes it from the possibility of a routine water test. The uric acid reaction is a convenient and reliable test but the scrup- ulous care required in preparation of the medium can not be obtained at the present time at least, in laboratories which are concerned with routine water analysis. The period of incubation, three to five days, is also a disadvantage. The choice thus becomes limited to the \oges-Proskauer and methyl red tests. Opinions are quite at variance as to which is preferable. The methyl red reaction has been urged by Clark and Lubs and by Winslow. The necessity for employing Witte's peptone together with the fact that the reaction is based on a delicate adjustment of the source of carbon and buffer substance and the insatiable desire among bacterialogists to deviate from the media recommended and to make individual substitutions has lead to numerous difficulties in the application of this test in practice. The synthetic medium of Clark would of course eliminate some of these difficulties. The methyl red reaction is simple, reliable, and when care- fully performed, constant, but the period of incubation for accurate dif- ferentiation is quite long (3 to 5 days), too long, in fact, to be conven- iently employed as a routine test. The tendency recently has been toward the V. P. reaction. Thus Chen and Rettger conclude from a study of 640 strains that the V. P. reaction is even more satisfactory than the methyl red test in that it is simple in operation, and when correctly carried out, is thoroughly constant in its results. Clark and Lubs, on the other hand, point out that the production of acetyl methyl carbonol, being the result of secondary reaction, and pos- sibly synthetic to some extent, may not be intimatelv connected with the main course of the fermentation and the quantity produced may be verv slight, thus giving a faint V. P. test even though the fermentation may be very vigorous. Nevertheless, the reaction has been found to be very con- stant and it has proven very satisfactory in the hands of practicallv all who have tried it. The advantages of this reaction are: 1. Any peptone medium in which the organisms will grow and which contains glucose (in a wide range of concentration) is suitable. It is preferable, however, to have the medium as free from color as pos- sible. ^ 31 2. The reaction may be obtained after 14 to 24 hours incubation at 30 degrees or 37 degrees C. 3. The brand of peptone employed does not affect the intensity of the reaction. It is interesting to note that in case of a mixture of coli and aerogenes types, the methyl red test will be acid and the V. P. reaction will be positive. Undoubtedly many instances are recorded as examples of inter- mediate strains when they were really due to impure cultures. This was the experience of Johnson and Levine who found that by repeated puri- fication, the proportion of strains which would not show perfect correlation was considerably reduced. Chen and Rettger record that of 18 strains which persisted in giving methyl red positive and V. P. positive reactions in all media, which they employed, only four continued to give non-corre- lating reactions after purification, although contaminating organisms could not be demonstrated. Resume. The gas ratio, Voges Proskauer, methyl red, and uric acid tests are strikingly correlated. The inembers of the colon group which produce acetyl methyl carbinol, are capable of using the' nitrogen from the purin ring of uric acid, give an alkaline reaction with the methyl red test, and in the decomposition of glucose, yield a relatively small quantity of acid and two or more times as much COj as Hj. On the other hand, the organisms, which do not produce acetyl methyl carbinol, can not utilize the nitrogen from the purin ring, give an acid reaction with methyl red, break down glucose with the production of a relatively large amount of acid and liberate CO, and Hj in approximately equal volumes. The colon group therefore includes two distinct subdivisions which are characteristically of different sources. These have been designated the coli and aerogenes sections. Their characteristics are tabulated below: TABLE XIV. DIFFERENTIATION OF THE MAIN SUBDIVISIONS OF THE COLON GROUP. Section Gas Ratio M. R. Test V. P. Test Growth Uric Acid medium Habitat Coli 1.0 (low ratio) acid neg. Negative (no growth) Predominates in feces and sewage Aerogenes 1.5 or more ( high ratio ) alk. pos. Positive (good growth) Predominates in soil and on grains 32 III. CLASSIFICATIONS OF THE COLON GROUP OF BACTERIA. Several attempts have been made to classify the numerous organisms of the colon group for the most part on the basis of acid and gas produc- tion from various carbohydrates. The reliability of such studies is some- times questioned on the ground that fermentation reactions are inconstant and may be easily acquired or lost. Smirnow grew colon strains in media containing various chemicals (3.0% glucose, 4.0 '7f sodium chloride, O.-T^'r sodium sulphate, and 0.25 to 0.75% phenol) and found that after successive transfers, for periods of one to three months, indol formation and later fermentation of carbohydrates were suppressed. Reversion to the original characteristics took place rapidly, however, when grown on ordinary media. Bronfenbrenner and Davis found colon organisms in food which fer- mented lactose slowly when first isolated but after cultivation on lactose media the rate of decomposition of this substance became normal. Twort, Penfold, and others, observed spontaneous mutations such as the loss or acquistion of the ability to ferment various carbohydrates. After a careful survev of the data on biological variations and muta- tions, Winslow concludes that "Taking the great mass of colon typhoid strains, as they are isolated from the bodies or intestines of man and animals, and cultivated under standard conditions, fermentative character- istics exhibit a high degree of constancy and what is even more important a higher degree of correlation with other biochemical and serological and pathogenic properties." From the practical water analysis view point, we are especially in- terested in the effect of a long sojourn in water or soil on the biological activities of Bad. coli. Houston repeatedly found that the proportion of strains not forming indol was much greater among those isolated from purified than from raw waters. He thought this was due to a loss of indol producing power as a result of unfavorable conditions encountered in water. He speaks of such indol negative forms or strains, which differ from the supposedly original Bad. coli commune, as "atypical" Bad. coli. Horrocks in 1903 exposed Bad. coli in various types of soils and waters for two to three months, and Savage in 1905 observed the effect of tidal mud. They came to the conclusion that alterations in character- istics were not induced and that there was no evidence that Bad. coli ever becomes "atypical." MacConkey placed a broth culture of Bart, coli in a sterile Pasteur candle which was then suspended in tap water. The water was changed occasionally. Fact, coli was isolated from the candle at irreaular inter- vals up to 358 days and examined biologically and biochemically. In no instance was a loss or gain of a character detected. It seems that the remarkable facts are not that an organism may oc- casionlly show a biological variation, but that, considering the simplicity of the bacterial cell, such variations are so infrequent. 33 Theobald Smith, Kligler, Winslow and others have reported that with cultures of Bad. cloacae motility and fermentation of carbohydrates per- sisted after the power to liquefy gelatin had disappeared. Digestion of gelatin is generally considered a reliable differential test. We therefore need have no compunction about utilizing the apparently more persistent fermentation reactions in studies on classification of the colon group. Theobald Smith, 1893, suggested that sucrose fermentation may be employed to subdivide the colon group into two subgroups and in 1901 Durham suggested the name Bad. coli communior for the sucrose positive variety and noted that the fermentation of starch was distinctive of Bact. ladis aerogenes. MacConkey's Classification. In 1905 MacConkey divided lactose fermenting (acid and gas) bacilli into four groups on the basis of fer- mentation of sucrose and dulcitol. TABLE XV. MacConkey Acid and gas Type species group Sucrose Dulcitol I 1 - - Bact. acidi lactici II - + Bact. coli (commune) III + + Bact. neapolitanum IV + — Bact. (lactis) aerogenes In 1909 he recognized in each group a number of varieties which were distinguished on the basis of their reactions to the Voges Proskauer test, motility, indol production, gelatin liquefaction, fermentation of inulin adonitol, and inosite. The probable existence of 128 different strains or 32 varieties in each of his four subdivisions was suggested. Many of these have been isolated, described, and given specific names. Others have been merely indicated by a number in his classification as shown in Table XVI. Bergey and Deehan's Varieties. Very similar to MacConkey's classification is that of Bergey and Deehan (1908). They employed eight characters — fermentation of sucrose, dulcitol, adonitol, and inulin; gelatin liquefaction, indol production, motility, and the Vogese Proskauer reaction — and from a consideration of all possible combinations recognized the pos- sible existence of 256 varieties. The Jackson Classification. In 1911, Jackson proposed a classi- fication resembling that of MacConkey but preference is given to dulcitol over sucrose for the primary division. Each of the four groups thus formed, which are regarded as species, are then further divided on raf- finose and mannitol into four varieties designated (A, B. C, and D), and further differentiation may then be made on motility, indol, gelatin lique- faction, fermentation of other carbohydrates, etc., giving subvarieties, in- dicated by numerical suffixes (A^, Ag, Bg, etc.). This scheme which was included in the Standard Method for a Water Analysis for 1912 is detailed in Table XVII. 34 TABLE XVI. MACCONKEY'S CLASSIFICATION OF LACTOSE FERMENTING BACILLI. d a MacConkey 2 o >-» Group '5 Variety ( Name) •3 s: o o B O T3 a 3 O a,' > O-J s 1— 1 1 — + + T — — — 2 B. acidi lacti (Huppe) — — + + — — _ Group I. 3 B. levans + + — — + - 1 + (Nos. 1-32) 4 B. Grunthal, B. sidcatus gasoformans. - + + — — — 1 — Sucrose — B. castellus Dulcilol— 5 B. vesiculosus - — + — — — 6 — + — — + 7 8 — + — — — — ~- 33 - '+ + ! + — Group II. 34 B. coli communis + + (Nos. 33-64) B. cavicida Sucrose — 35 B. Schafferi* + Dulcitol+ 36 + + — — — — 65 B. oxytocus perniciosus + '+ + + 1 + + 66 — + + — 67 + + + Group III. 68 B. rhinoscleroma, B. Friedlander + + Nos. 65-96) 69 + + — + + Sucrose + 70 + + — — + — — Dulcitol+ 71 + + — — 1 — 72 B. neapolitanus 4- 1 _ 73 + + — — -! + 74 + — — — 1- 75 — — — — + 1 + 97 + — T +~ + -1 + . 98 — — - + + + 1 + 99 — — - + + + — 100 — + + + — 101 — + + — + — Group IV. 102 + + - + - + (Nos. 97-128) 103 B. lactis aerogenes, B. dysenteriae vitulorum, B. capsulatus (Pfeiffer) - + 1 + + Sucrose+ 104 B. gasoformans non-liquefaciens* — i + 1 -1 + ± Dulcitol— 105 + + _ -1- + 106 + + — 107 B. coscoroba 4- 108 B. cloacae + + -(- + 1 109 1 - + - — -1- *Usually produce only a small amount of gas. 35 w cq + +I + I eas « - - w ►J 3 Q + c;? 05 + + I + I rt 5 :: :: = + + + I I ni3 3 3 3 5mMUQ ■E..2 > .E ^ ^ ts i 36 Castellan! and Chalmers' Grouping. These authors have recently suggested a rather unique classification of bacteria in which the organisms generally considered as members of the colon group are distributed among three Tribes and three Genra as follows: Tribe Encapsulateae. (Castellani and Chalmers, 1918. Bacillaceae growing well on ordinary media, without endospores; neither fluorescent nor chromogenic; aerobes; facultative anaerobes; not liquefying gelatin; possessing capsules in animal tissue. The genus Encapsulatus includes two lactose fermenting (acid and gas) species differentiated on fermentation of inosite. Inosite not fermented. 1. Encapsulatus acidi lactici Inosite fermented with acid and gas. 2. Encapsculatus lactis aerogenes Tribe Proteae. (Castellani and Chalmers, 1918) Bacillaceae grow- ing well on ordinary media ; without endospores; aerobes; neither fluorescent nor chromogenic; aerobes; facultative anaerobes; not 1 iquefying gelatin. They recognize two lactose fermenting species in the genus Cloaca which are different on sucrose fermentation. Sucrose fermented with acid and gas. 1. Cloaca cloacae. Sucrose not fermented 2. Cloaca levens. All other members of the colon group are placed in their Genus Escher- ichia of the Tribe Ebertheae. Tribe Ebertheae. (Castellani and Chalmers, 1918). Bacillaceae growing well on ordinary laboratory media; not forming endospores; aer- obes, and often facultative anaerobes; without fluorescence pigment forma- tion or gelatin liquefaction; without polar staining; Gram negative; with- out a capsule. Genus Escherichia. (Castellani and Chalmers, 1918.) Ebertheae which ferment glucose and lactose completely with acid and gas; milk clotted. I. Indol positive division. (Smith) A. Sucrose fermented — Communior section of Durham. 1. Dulcitol fermented with acid and gas. a. Adonitol fermented with acid and gas. (1) Motile 1. Escherichia oxYtocus (2) Non motile. 2. Escherichia metacoli b. Adonitol no change. (1) Motile. (a) Agglutinated by pseudocoli serum 3. Escherichia pseudo-coli (b) Not agglutinated (late fermentation of sucrose) 4. Eschericltia coliforrnis (2) Non motile. 5. Escherichia nrapolitanus 2. Dulcitol not fermented, a. Motile (1) Inosite fermented with acid and gas. 6. Escherichia pseudocoloidella (2) Inosite no change 7. Escherichia pseudocoloides 37 b. Non motile. 8. Escherichia pseudocoscoroba B. Sucrose not fermented — Communis section of Durham. 1. Dulcitol fermented with acid and gas. a. Salicin fermented with acid production. 9. Escherichia cavicida b. Salicin fermented with acid and gas. (1) Motile. 10. Escherichia coli (2) Non motile. (a) Inosite not fermented. 11. Escherichia coloidella (b) Inosite fermented with acid and gas. 12. Escherichia coloides c. Salicin no change. 13. Escherichia metacoloides 2. Dulcitol — no change, a. Motile. (1) Maltose — acid and gas. 14. Escherichia paragrilnthali (2) Maltose — no change. 15. Escherichia griinthali h. Non motile. (1) Maltose — acid and gas. 16. Escherichia colitropicalis (2) Maltose — no change. 17. Escherichia vesiculosus II. Indol negative division (Smith). 18. Escherichia coli mutabilis The prominence given to the indol reaction and the fermentation of dulcitol, adonitol, and maltose by Castellani and Chalmers is quite at var- iance with the practices of American investigators. From the extensive recent studies on carbohydrate and nitrogen metabolism it appears that the acidi-lactici types are more closely affiliated with the strains in their Genus Escherichia, than with the lactis-aerogenes forms and that the Genus Cloaca of Castellani and Chalmers should be associated with the lactis- aerogenes forms rather than with the proteus group. A very serious objection to such classifications as those of MacConkey, Bergey and Deehan, and Jackson is their extreme flexibility and complexity; for, as the number of fermentable substances or other characters observed increases, the number of "varieties" increases geometrically (approaching infinity) and soon produces a most unwieldy scheme. The number of "varieties" is given by the formula 2° where "n" is the number of char- acters studied. Thus with eight characters there are 256 possible combi- nations or "varieties." This number rises to 1,024 with 10 characters and to 65,536 when 16 tests are considered. It is aparent therefore that to regard each character as of similar and equal differential value will quick- ly result in an unwieldy grouping. Another objection to these classifications is the arbitrary manner of selecting the order in which reactions are to be employed for division. Organisms which are very closely related may be far separated in two 38 schemes of classification if the authors happen to select different characters for the initial subdivision. Statistical Classification of the Colon Group. To offset these objections, Winslow suggested the utilization of the statistical method first employed by Andrews and Border and Winslow and Winslow in studies on Coccaceae. Individual characters are not considered paramount and inde- pendently but only in relation to each other. Howe first attempted the statistical method in 1912. This investigator made a detailed study of acid and gas production and various other tests on 630 strains freshly isolated from human intestinal contents. He con- cluded that indol, nitrate reduction, motility, fermentation of dulcitol and mannitol, and starch were not correlated with other characters and were consequently not of classificatory value. He recognized only two groups the sucrose positive. Bad. communior, and the sucrose negative. Bad. com- munis. Rogers and his associates in 1914 and 1916 studied a large number of colon strains from milk, grains and bovine feces and on the basis of ac- curately determined gas ratio from dextrose concluded that two distinct groups may be distinguished. One, referred to as the low ratio group, produced carbon dioxide and hydrogen in equal volumes and includes about 52 percent of the strains from milk, 99.6 percent of the strains frojn bovine feces, and only 4.8 percent of their grain strains; whereas the high ratio group, which was characterized by the production of two or more times as much carbon dioxide as hydrogen, included 47.5 percent of the milk strains, only 0.5 percent of the bovine fecal strains, and about 95 percent of the strains obtained from grains. There is thus a very strong correlation between these subdivisions and the source. Kligler (1915) suggested that salicin be substituted for dulcitol, in subdividing coli-like bacteria, pointing out that salicin fermentation cor- relates better with the Voges-Proskauer reaction than does dulcitol de- composition. He recognizes a sucrose negative-salicin negative group (B. acidi-ladici) ; sucrose negative-salicin positive group [B. communis) ; su- crose positive-salicin negative group [B. communior) and sucrose positive- salicin positive (B. aerogenes) . B. cloacae is differentiated from B. aero- genes by its inability to ferment glycerol. The characterization of B. communior as salicin negative is probably untenable. The term B. coli-communior was first employed by Durham to describe members of the colon group which fermented sucrose and which were motile. Later Ford recognized it as a species B. communior. Of 77 motile sucrose fermenting bacilli of the coli section, 56 (73%) were found by the writer to be salicin fermenters. It is felt therefore that Bad. communior should not be described as a salicin non-fermenter. Where the principle of correlation has been employed the best cor- related character has apparently been picked out by inspection of the data. Inspection is a tedious and difficult procedure, entirely inapplicable where the number of characters considered is large, and it does not permit of a concise statement of the degree of correlation which exists between differ- 39 ent reactions. The author feels that considerable information in an ab- stract, concise, and workable form may be obtained from a study of the coeificients of correlation. The Coefficient of Correlation. Where we are concerned merely with the presence or absence of characters the coefficient of correlation be- tween any two characters may easily be determined. Suppose that it is desired to know if the characters X and Y are correlated and that a study of a number of organisms showed that 'a' cultures are positive for both X and Y; 'b' organisms positive for X but negative for Y; 'c' cultures are negative for X and positive for Y; and 'd' strains are negative for both X and Y. The distribution of the organisms is first tabulated thus: . a J, '-T'd The degree of association, or the coefficient of correlation, is then expressed, according to Yule, by the formula ad-bc (1) ad+bc If 'ad' is equal to 'be' the coefficient becomes or 0; which ad _|- be indicates that there is no correlation whatever. ad the formula becomes 7"-:=l; indicating a perfect positive correlation. If — be 'a' or 'd' is zero then we have ' = — 1; showing a perfect negative cor- relation. It should be observed that an absolute positive correlation exists in reality only if both 'b' and 'c' are zero and an absolute negative cor- relation when both 'a' and 'd' are zero. In order to avoid coefficients of 1 or — 1 where only one group — 'a', 'b', 'c', or 'd' — is zero. Yule gives the formula a (a + b + c + d) - (a + c) (a + b) (2) \/ (a + c) (b + d) (a + b) (c + d) In practice, however, a few strains are almost always found in each of the four groups and Yule suggests the use of the simpler formula (1). Some caution should therefore be employed in interpreting coefficients of 1 or — 1. It was assumed that if the coefficient between two characters is numeric- ally greater than 0.5 they may be regarded as correlated, but if less than Sccrose •5a//c//y •A — / 7 43 - /32 2 lodo/ ■/- _ ■f- 89 7 - -9 82 y- _ ■¥- 7S SZ - 39 '^6 ■^■ _ /■ e? /O - 76 9 7x3-^:^^/32 -/.OP 7xS i-^3 x/3S •^.99 73xa6-SSx39 _ f^rt/'a/ Corre/'fJ 87':9-i~76x./o'^ 40 0.3 there is probably no association. A few examples of correlation co- efficients actually obtained in the course of this study are given to illus- trate the method of calculation. The principle of correlation should not be applied indiscriminately to collections of data for systematic purposes. Certain characters and properties have been universally accepted as reliable and appropriate for bacterial differentiation; thus, staining reactions such as the Gram and acid fast stains, spore formation, and aerobiosis and anaerobiosis, hardly need to be bolstered up by correlation with other characters to justify their taxonomic value. On the other hand the significance of such characters as motility, indol production, and fermentation of certain substances, is still debatable. Motility is regarded by many as a highly variable property. Perhaps it is in reality a reliable morphological difference. Certainly if it could be shown that this character goes hand in hand with several others, more reliance and attention should and would be given to motility. The same is true of the indol test. In dealing with gas formation from carbohydrates, alcohols, or polysaccharids, the question naturally arises as to which substance should be given preference for subdivision, or whether all are to be considered of equal taxonomic value. The lack of a criterion for determining the most significant fermentable substances has led to con- siderable confusion. It has already been pointed out how subdivision on every character studied results in an infinite number of varieties. Where we are dealing with a number of characters each of which is assumed to be of equal taxonomic significance, it would certainly be desircible and advantageous to subdivide on that character which gives the greatest amount of information as to the manner in which the resulting subgroups react with respect to other characters. It is under such circumstances that the principle of correlation of characters may be legitimately, conveniently, and advantageously employed. The Method of Selecting the Best Correlating Character. The following example will illustrate the method of selecting the best correlated character for the purpose of subdivision of a group of organisms. Let us take for instance a group of 89 strains of the coli section, which were found to be non-fermenters of sucrose, and which it is required to further divide on one of the following characters: — motility, indol production, dulcitol, glycerol, or salicin fermentation. Tabulation is first made, as indicated below, so as to show the relation of each character to every other character and also to facilitate the calculation of correlation coefficient which are then determined for each pair of characters and recorded as in- dicated in Table XVIII. For subdivision that character is selected which gives the highest co- efficient of correlation with the greatest number of other characters. Thus, in the group under consideration, motility is not well correlated with any other character. Dulcitol and glycerol each have a high correlation co- efficient with salicin but not with any other character. Salicin fermenta- tion, on the other hand, is well correlated with three characters — glycerol, 41 TABLE XVIII. (a) SHOWING CORRELATION OF CHARACTERS AMONG 89 SUCROSE NEGATIVE STRAINS OF THE COLI SECTION. /io/iMy Indol Dulatol Glycerol Sal/do \ -h — + — i- — Y- — •h — 1 ^ S3 45 8 22 31 43 /O 33 20 - 36 34 2 II 25 26 10 18 18 1 / 45 34 79 29 50 6/ 18 5/ 28 - 8 2 10 4 6 3 2 10 1 7^ 22 // 29 4 33 27 6 28 5 - 31 25 50 6 56 42 /4 23 33 1 i- 43 26 61 8 27 42 69 48 21 - /O 10 18 2 6 14 20 3 17 1 / 33 /6 J/ 28 2'5 48 3 51 - 20 /8 26 /O 5 35 21 /7 38 TABLE XVIII. (b) COEFFICIENTS OF CORRELATION FOR EACH PAIR OF CHARACTERS IN TABLE XVIH. (a). 77o/r7//y lodo/ Ot//cM O/ycem/ 5a/ia'o 77oWifY -.50 -t.2Z ^.25 t.Z5 Inc/ol -.^0 -.07 -.08 ^/.OO ffi/7afol i-.22 *.07 i:20 t.78 5/ycero/ r.e5 -.08 *.20 t.86 5alicin t.25 t/.OO ^.78 -f.86 dulcitol, and indol, showing coefficients of +0.86, +0.78, and +1.0 re- spectively. Subdivision is therefore made upon salicin. For each of the resulting subgroups new correlation tables are made and further subdivision again carried out on the best correlated char- acter. A point is very quickly reached where further subdivision, upon correlated characters, is no longer feasible. These groups are regarded as species and to each was assigned, as far as possible, the name of the MacConkey variety which it most resembled. Levine's Classification. The following classification is suggested by the author, based upon a study of 333 strains obtained from soil, sewage, and the feces of man, horse, sheep, pig, and cow. The characters employed are the methyl-red and Voges-Proskauer re- actions, indol production, motility, gelatin liquefaction and gas formation from sucrose, raffinose, dulcitol, glycerol, salicin, dextrin, inulin and corn 42 starch. Other fermentable substances — lactose, maltose, galactose and mannitol — were also observed but as these substances were all attacked with gas formation they need not be considered. As was discussed in detail in Chapter II, the investigations of Harden, Smith, Rogers and others on carbohydrate and nitrogen metabolism have demonstrated adequately and conclusively that the colon group includes two distinct subgroups, the methyl-red positive- Voges Proskauer negative- uric acid negative group designated herein as the coli section, and methyl red negative Voges Proskauer positive-uric acid positive group or aerogenes section. These two main sections are recognized and each is subdivided into species on the basis of correlated characters as described above. KEY TO THE MORE IMPORTANT SPECIES OF THE COLON GROUP. The colon group includes all non sporing Gram negative short rods, fermenting glucose and lactose with acid and gas production and which grow aerobically. I. Not producing acetyl methyl carbinol (Voges Proskauer reaction negative) ; acid to methyl red, and carbon dioxide and hydrogen produced in approximately equal volumes from glucose. Can- not utilize uric acid as a source of nitrogen. COLI SECTION A. Sucrose not attacked. 1. Salicin fermented with acid and gas. 1. Bact. coli 2. Salicin not attacked. 2. Bact. acidi-lactici B. Sucrose fermented with acid and gas. 1. Motile. 3. Bact. communior 2. Non-motile. a. Salicin fermented with acid and gas. 4. Bact. neapolitanum b. Salicin not fermented. 5. Bact. coscoroba II. Producing acetyl methyl carbinol (Voges Proskauer reaction posi- tive) ; alkaline to methyl red, and forms two or more times as much carbon dioxide as hydrogen from glucose. Capable of utilizing uric acid as a source of nitrogen. AEROGENES SECTION A. Glycerol and starch fermented with acid and gas formation; non motile, gelatin not liquefied. 6. Bact. aerogenes B. Glycerol and starch not fermented; motile, gelatin liquefied. 7. Bact. cloacae In the 1917 and 1920 Standard Methods of Water Analysis the coli section is not differentiated into species. The aerogenes section is sub- divided on the fermentation of adonitol into a fermentin" variety sup- posedly of fecal origin and a non-fermenting variety whicli is re"-arded as of non-fecal origin. The value of adonitol for this distinction has not as yet been adequately investigated and is not generally accepted. 43 IV. THE DETECTION OF THE COLON GROUP IN WATER. Various methods have been suggested for the isolation of members of the colon group. They are all based on the principle that all members of this group of organisms are capable of decomposing lactose with acid and gas production. Media are employed which contain lactose and some indicator to show whether fermentation has taken place. Isolation may be accomplished (1) directly by plating on solid dif- ferential media or (2) indirectly on solid differential media, after pre- liminary enrichment in some fluid medium. The mediums most commonlly employed for direct isolation are: Litmus lactose agar. Phenolated litmus lactose agar. Endo agar. Conradi Dragalski agar. MacConkey agar. Litmus lactose agar consists of nutriment agar, neutral to phenolph- thalein, which contains sufficient litmus or azolitmin to give a distinct blue color. Fermentation of lactose with the production of acid is indicated by the formation of red colonies. The technique for direct isolation is merely to place definite quan- tities of the test sample (1.0 c. c, 0.1 c. c, 0.01 c. c, etc) into petri dishes to which are added about 10 c. c. of litmus lactose agar. (In some labor- atories the litmus is added to the petri dish and then lactose agar poured in.) The plates are incubated at 37 degrees to 40 degrees C. for 24 hours after which members of the colon group will appear as red colonies on a blue background. As there are other organisms, particularly the strep- tococci, which are capable of fermenting lactose with acid production, the mere appearance of acid colonies is not absolute proof of the presence of the colon group. In the process of sterilization, lactose is sometimes broken down so that organisms other than the colon group may then produce slightly acid reactions. They must therefore be examined fur- ther. Suspicious colonies are fished to agar and a Gram stain is made to insure that the organism is a Gram negative non-spore-forming rod. From the agar slant various other tests (fermentation of carbohydrates, gelatin liquefaction, milk coagulation, the V. P., methyl red, etc.) may be carried out. Acid produced by the growth of fermenting forms diifuses through the medium, often coloring considerable portions of the plate, making it extremely difficult to detect the acid producing colony. The medium exerts very little inhibitory action and overgrowths of non-colon forms are not infrequent. These disadvantages may be overcome to some ex- tent by (1) the use of porous tops to prevent spreaders, (2) by increasing the concentration of agar to 3 percent thereby diminishing considerably the diffusion of acid, and (3) by the addition of selective antiseptics to inhibit the growth of forms other than the colon group. The use of the higher temperature (37 degrees C.) and preferably 40 degrees C, exerts 44 some inhibitory action on the ordinary water forms and many soil forms. This may be further increased by the addition of small quantities of phenol to the agar medium. Chick found that 0.1 percent (1 part per thousand) gave very excellent results. In using phenolated media, it must always be borne in mind that there is danger of inhibiting some members of the colon group as well. Endo agar consists of nutrient lactose agar containing 3 percent agar with basic fuchsin decolorized by sodium sulphite as an indicator. The lactose fermenting organisms produce red colonies often with a distinct metallic luster, the medium itself being colorless or slightly pink. The reaction is presumably due to the production of acid and aldehyde by the lactose fermenting organisms. These products react with the fuchsin sulphite combination liberating the fuchsin. The high concentration of agar serves to check diffusion of acid and also to eliminate many of the water forms. It was thought at one time that the indicator itself exerted inhibitory action but this does not seem to have been adequately proven. Conradi-Drigalski agar consists of litmus lactose agar to which has been added some nutrose and crystal violet. The crystal violet checks the growth of many forms particularly the streptococci but is supposedly non-inhibitory for the colon group in the concentration employed. Both the Endo and the Conradi Drigalski have been found very valuable for the isolation of the para typhoid and typhoid bacilli from stools but they have never found favor in the United States although they are the media of preference in Germany for direct isolation of the colon group from water. MacConkey agar (also known under the name Rebipelagar) consists of peptone lactose agar with 0.5 percent bile salts (sodium glycocholate and taurocholate) , the function of the latter being to check saprophytic forms. The inhibitory action of bile is well illustrated in Table XX. TABLE XX. SELECTIVE ACTION OF BILE SALTS. (After Jackson 1906) *Bile diluted, 1:1. Bacteria per. c. c. Uncontaminated well Contaminated ponH Suspension of feces Suspension of feces Gelatin 20° 920 2700 350.000 900.000 Agar 370 25 170 450.000 900.000 Bile Agar *370 14 43 300,000 900,000 Bile Agar 370 16 25 60.000 900.000 Lactose Bile Agar *370 250,000 675,000 Lactose Bile Agar *370 17 250,000 600,000 where it will be noticed that the bile salts exerted a much greater re- straining action on forms generally found in water than on the organisms (almost exclusively Bact. coli) present in a suspension of feces. The 45 indicator in the Rebipelagar is neutral red. Acid producing colonies are distinctly red. This medium is very extensively used in England but has not found favor in the United States for, as will be shown later in the discussion of the presumptive test for Bad. coli, there is considerable evi- dence that the bile salts may inhibit some strains of Bad. coli. Reference to the foregoing table shows that in one of the fecal suspensions a count of 450,000 on agar was reduced to 60,000 by bile. The isolation of the colon group by direct plating is recommended for highly polluted waters and sewage where the number of coli strains are quite large, being present in one c. c. or less. If, however, it is necessary to employ large samples (5 c. c. or 10 c. c.) as is essential in routine water work, it becomes extremely inconvenient to plate out. Furthermore, if the incidence of the colon group is small compared to other forms, they may be missed. Although theoretically the colon colonies are clearly dif- ferentiated from others, it is the practical experience of all investigators that when growing on a plate with a large number of alkali formers, the acid of Bad. coli and related forms may be neutralized, and the characteris- tic reaction thereby masked, so that a colon colony may thus escape de- tection. On the other hand, in the process of sterilization of medium the lactose may be decomposed in part and it will then be found that some soil forms will produce an acid reaction on solid media. Acid production on any of the solid media described above can therefore not be relied upon to always indicate the colon group, and so further tests must be resorted to as has been mentioned in discussing litmus lactose agar. For the isolation from relatively large quantities of water, or for an admixture with numerous other forms, the best results are obtained by the indirect or so-called preliminary enrichment method. The procedure con- sists of three steps: 1. Growth in a favorable liquid medium containing some constituent which will indicate the probable presence of the colon group. This is spoken of as a preliminary enrichment tube and is often employed as a pre- sumptive test. 2. Isolation from this preliminary enrichment tube on some one of the solid media described above. If typical colonies are formed the pres- ence of members of the colon group is considered partially confirmed. 3. Suspicious colonies on (2) are examined, by fishing to agar and then testing for coagulation of litmus milk, gas in lactose broth, V. P., etc. This would constitute a completely confirmed test: The media most commonly employed for preliminary enrichment are: Glucose broth Phenol broth Liver broth Lactose broth Lactose bile salts broth •Eijkman test (glucose peptone water at 46° C.) Lactose peptone bile 46 Glucose broth consists of ordinary broth, neutral to phenolphthalein, with 1 percent glucose. This medium was the one most commonly em- ployed in the United States prior to 1906. The probable presence of the colon group is indicated by the production of gas after 24 to 48 hours incubation at body temperature. Phenol broth consists of ordinary broth containing 0.1 percent car- bolic acid which exerts a selective antiseptic action on the normal water bacteria thereby supposedly favoring the growth of the colon types. This medium is employed by the French, but it has been observed that the phenol exerts an antiseptic action on some members of the colon group and that its employment is particularly objectionable with water of fairly good quality. Liver broth has been found particularly efficacious for isolation of colon types and in 1912 the Standard Methods for Water Analysis of the A. P. H. A. recommended that, "If a further study of all gas forming bacteria, including attentuated forms, is desirable, then liver broth should be employed in preference to the usual dextrose broth, as it gives a larger number of attentuated forms, has better rejuvenating powers, and gives fewer anomalies, and greater and more rapid gas production." For routine water work, however, liver broth has not found favor. Since 1906, there has been a tendency to substitute lactose for dextrose in the preliminary enrichment mediums. It is quite surprising that this was not done sooner considering that the essential characteristic of the colon group is lactose fermentation. Lactose broth, which is nutrient broth containing 0.5 to 1.0 percent lactose, is most commonly employed in the United States at the present time and is the medium recommended in the Standard Methods of Water Analysis of the A. P. H. A. of 1917 and 1920. It is supplanting the lac- tose peptone bile medium of Jackson, which was extensively used between 1912 and 1917. In both of these mediums the probable presence of the colon group is indicated by gas production from the lactose. Lactose bile salt broth of MacConkey consists of 0.5 percent lactose, 2% peptone, 0.5 percent sodium taurocholate with neutral red as an in- dicator. The colon group will produce gas and acid indicated by red coloration of the liquid after 24 to 48 hours at 37° C. Technique for Isolation. The steps in tlie isolation of the colon group from water may be summarized as follows: (1) Place various quantities of the water under examination (10 c. c, 1 c. c, 0.1 c. c, etc.) into Smith or Durham fermentation tubes containing the preliminary enrichment medium. (2) Incubate at the body temperature (37^ C.) for 24 hours. If gas is formed, it is taken as presumptive evidence that Bact. coli or its close allies are present. (3) The next step is then to isolate these organisms on some one of the solid media enumerated above. This may be done in one of the fol- lowing ways: 47 Method 1. Barely touch the liquid in the preliminary enrichment tube, showing gas, with the point of a platinum needle, wash off this needle in a tube of melted litmus lactose or Endo agar and pour into a petri dish. Incubate at the body temperature for 24 hours. Method 2. Pour L. L. A., Endo or other differential agar into petri dishes and allow them to solidify and dry in the incubator. Dip a small loop into a positive preliminary enrichment tube and wash it off in a tube of sterile salt solution. Place a loop of this wash water in the center of a plate containing the differential medium and spread it over the surface with the aid of a sterile glass rod bent at right angles. Incubate 20 to 24 hours at body temperature. Method 3. Dip a platinum needle, whose end is bent at an angle of about 120°, into the positive preliminary enrichment tube, stab into a plate containing the differential medium (to remove the excess organisms) and then make a Series of streaks on this plate about a quarter of an inch apart, taking care to always streak in the same direction and to lift the needle at the end of each stroke. With a little practice, very excellent isolations may be obtained by this simple method. This was first demon- strated to the author by Mr. Greenfield of the Illinois Water Survey and it has proved very satisfactory. Incubation is, as before, at body tem- perature for 24 hours. TABLE XXI. RELATIVE GROWTH OF BACT. COLl AND SEWAGE STREPTO- COCCI FROM POLLUTED WATER IN GLUCOSE BROTH. (After Prescott and Baker 1904) Sample No. Initial number of red colonies per c. c. on L. L. A. Type Number found in million per c. c. after various periods of growth First gas noticed after 11 hrs. 16 hrs.|23 hrs. 39 hrs. 63 hrs. 1 2 10 Bad. coli 20 76 150 11 Strep. 25 85 420 hrs. 7 35 Bad. coli 130 140 200 20 8 10 Strep. 1 1 20 1 110 400 1 45 hrs. 8 460 Bad. coli 332 420 405 24 6 Strep. 210 350 105 150 hrs. If characteristic colonies are present on these agar plates the test is regarded as partially confirmed for the colon group. For further con- firmation, it would be necessary merely to fish a well isolated colony and 48 to determine its Gram characteristics and other reactions, particularly fer- mentation of lactose broth, as previously explained. The objections to the preliminary enrichment method are: (1) It is difficult to estimate the incidence of the colon forms for by employing Ic. c, 0.1 c. c, etc., there is considerable error introduced in calculating the number of organisms present in a unit volume of water. (2) There is a danger of loss of the colon forms which may be killed off before isolation is attempted either by (a) products of their own meta- bolism or (b) by over-growths of other organisms. In glucose broth inoculated with a mixture of Pact, coli and Strepto- cocci, the former usually predominated for the first 24 hours, but thereafter the number of Streptococci was considerably greater, in some instances having completely choked out or killed the colon types in 39 hours. The danger of losing colon strains due to overgrowth and death may be minimized considerably, if not completely eliminated, by (1) reducing the concentration of fermentable substances to 0.5 percent (as recommended by Burling and Levine) and (2) attempting isolation at the earliest pos- sible time (after 12 to 24 hours or at the first appearance of gas). It will be noticed from Table XXI. that at the first appearance of gas the colon forms were far in excess of the Streptococci, in fact practically in pure culture. With these precautions the preliminary enrichment method will be found very reliable and convenient. THE PRESUMPTIVE TESTS The method for isolation of members of the colon group described above, comprising as it does the reaction of the isolated organism in milk, production of indol, reduction of nitrates, gelatin liquefaction, and mo- tility was in general use between 1905 and 1910. The method, however, was soon found to be tedious and inconvenient for routine work. The time required for such a complete analysis was at least nine days, whereas for practical purposes it is desirable to have a report available within 24 to 48 hours. Furthermore, there is no evidence that the determination of the characters enumerated shed any light upon the sanitary significance of the bacilli isolated. For routine water work, it is essential to have some test which is simple and rapid and which can be relied upon as a reason- able index of the probable presence of the colon group in a large pro- portion of instances. Such a test is known as a presumptive test for the colon group. From his wide experience with the glucose broth pre- liminary enrichment method, Whipple in 1903 observed that a consider- able portion of tubes showing characteristic fermentation were found, on further examination, to contain colon-like organisms. He therefore sug- gested that dextrose broth alone might be employed as a rapid presumptive test of the probable presence of Bact. coli. The media which have been and are most commonly employed for the presumptive test are (1) glucose broth, (2) lactose peptone Ijile, and (3) lactose broth. Other media have also been suggested, as for example the Eijkman test (glucose peptone 49 water at 46° C.) and neutral red broth, but these are not employed to any extent in the United States and will not be considered further. The Glucose Broth Presumptive Test. The test consists of the determination of the amount of gas and the ratio of hydrogen to carbon dioxide in glucose broth after 24 hours incubation at the body temperature. Production of 25 to 70 percent gas, one-third of which is carbon dioxide and the remainder hydrogen (that is H/C02=2 to 1) is con- sidered a "typical" test and is regarded as indicating the presence of Bad. coli or closely related bacilli. Thus Irons stated that when the pro- portion of carbon dioxide is approximately 33 percent Bact. coli commune is almost invariably present. If the amount of gas is (a) between 10 and 25 percent, (b) more than 70 per cent or (c) if the proportion of carbon dioxide is more than 40 percent of the total volume of gas formed, the reaction is considered "atypical" and the probable presence of the colon group is doubtful. If there is less than 10 percent gas, or if no gas is formed at all, the probability of isolation of the colon group is very slight and the test is considered negative. The early studies indicated that the colon group was successfully iso- lated from about 70 percent of positive presumptive tests. More recent and detailed studies have shown, however, that the proportion of presump- tive tests confirmed by further observation is not constant but is effected by the character, treatment, etc. of the water under examination and the season of the year. Objections to the Glucose Broth Presumptive Test. The ob- jections to the glucose broth presumptive test may be summarized as fol- lows: 1. The volume of gas produced is not a significant criterion of the probable presence of the Bact. coli or its close allies. 2. The gas ratio, as determined in the Smith tube, is unreliable. 3. There are many glucose fermenting species which are incapable of attacking lactose. 4. The reliability of the glucose presumptive test varies with (a) the season of the year, and (b) differs for raw and treated waters. 5. The glucose broth medium is particularly subject to overgrowths. That 25 to 75 per cent gas is not necessarily characteristic of the colon group and that even pure cultures produce as little as 10 percent gas has been demonstrated by Fuller and Ferguson, Longley and Batton, Hale and lyielia, and others. They also have shown that the so-called 'typical' gas ratio is not at all characteristic of the colon group. Thus of 818 tubes of sterilized water, inoculated with Bact. coli but containing no other gas former, only 474 or 58 percent gave the typical gas volume and gas ratio. The errors inherent in the gas ratio as determined in the Smith tube have already been discussed in detail in Chapter II. Fermentation of glucose with gas production is not a very reliable in- dication that the organism will also ferment lactose. There are many species of bacteria capable of attacking the monosaccharids, but which 50 are inert as respects the more complex disaccarid lactose. Thus Clark and Gage in 1903 pointed out that there were 58 well described species which gave the presumptive test in dextrose broth of which 23 are widely separated from the colon group and since then many other glucose-positive- lactose-negative forms have been described. Prescott and Winslow call attention to the seasonal variation in the reliability of this presumptive test noting that Fromme (1910) isolated colon bacilli from 87 percent of positive glucose broth tubes in the fall and winter (Oct. to Mar.) but only 66 percent from similar tubes in the spring and summer (Apr. to Sept.) In stored or filtered waters the incidence of glucose fermenters which do not attack lactose is considerably greater than in raw waters. The reports of Houston for 1909-11, which are summarized below, show that only 9.5 percent of coli-like bacteria isolated from raw river water after preliminary enrichment in glucose broth failed to ferment lactose but that with filtered and ground waters 38.3 percent of the isolated bacilli did not attack this disaccharid. TABLE XXII. GLUCOSE AND LACTOSE FERMENTATION BY BACTERIA FROM RAW AND FILTERED WATERS. (After Houston 1909 to 1911) COLI-LIKE Raw River waters Filtered waters* Number of Strains 7,657 10,620 Glucose+ ; Lactose — Glucose-I- ; Lactoses- 9.5% 90.5% 38.3% 61.7% *Includes ground waters. TABLE XXIII. EFFECT OF NATURAL AND ARTIFICIAL PURIFICATION ON INCIDENCE OF GLUCOSE AND LACTOSE FERMENTING BACILLI IN PULTA WATER. (After Clemesha 1912) Intake Settled Filtered *No. of strains % Lactose negative ♦No. of strains % Lactose negative •No. of strains % Lactose negative October (Heavy rain) 40 80 40.0 40 50.0 40 50 November (River muddy but no rain locally) 31.2 80 16.2 80 48.7 December (River clearing) 60 70.0 60 85.0 60 91.6 January (River clear) 100 1 61.0 100 71.0 60 60 100.0 February (River clear) 80 93.7 80 98.7 98.2 *A1] glucose fermenters. 51 Clemesha (1912) has made numerous observations on the relative incidence of glucose and lactose fermenting bacilli in waters of India subjected to various periods of storage, exposure to sunlight, and filtration. In Table XXIII. taken from his book on the bacteriology of Surface Waters in the Tropics are shown the effects of treatment and the influence of self purification on the relative incidence of glucose fermenting bacilli which are not lactose fractors. The increased incidence of glucose positive-lactose negative organisms in waters during dry weather is particularly striking. Thus during the rainy month of October 40 to 50 percent of the strains studied were of this type, the number gradually increasing as the river cleared and the forces of natural purification (sedimentation, sunlight, etc.) manifested them- selves until in February, 93.7 to 98.2 percent of the glucose fermenting strains were non-lactose fractors. The glucose broth presumptive test is therefore particularly unreliable as an index of the colon group when dealing with treated and stored waters. Clemesha attributes the marked increase of glucose -[-, lactose — , bacilli in India stored waters to the presence of an organism which he designates Bacillus P. This bacillus is widely distributed in nature, ex- tremely resistant and capable of multiplying in water. The important characteristics of the organism are given below: Morphology, etc. Gram positive, motile bacillus. Litmus Milk. Slight acidity on top (occasionally). No coagulation. Indol. Negative. Gelatin, Not liquefied in ten days. Fermentation. Glucose acid and gas. Sucrose acid and gas. Lactose. Faint trace of acid on long cultivation in laboratory. Dulcitol Mannitol Adonitol Inulin Neither acid nor gas Voges Proskauer Reaction. Strongly positive. Bacillus P. of Clemesha resembles the B. proteus but differs from it in being Gram +, and giving a positive Voges Proskauer reaction. Not only is gas production in glucose broth a poor index of probable presence of Bad. coli but the organism may be present even in the absence of gas formation, particularly if the water has a high bacterial count or contains streptococci. It is not infrequently observed with this medium that small samples of water (1 c. c. or 0.1 c. c.) may be positive whereas larger portions (10 c. c.) may be negative. Upon considering that there are numerous organisms capable of attacking glucose with acid produc- tion, this choking off of the colon bacilli may be readily explained by the inhibiting action of the acids formed. 52 This lack of constant relation between the dextrose broth fermentation and the actual presence of the colon group has resulted in a gradual abandon- ment of the glucose broth presumptive test in favor of media contain- ing lactose. The Lactose Peptone Bile Presumptive Test. The use of bile containing lactose was first suggested in this country by Jackson in 1906. He recommended fresh ox bile containing 1 percent lactose but the in- clusion of 1 percent peptone was soon found to be desirable. The medium is prepared by adding 1 percent peptone and 1 percent lactose to fresh undiluted ox gall (or a 10% solution of dried ox gall) which is placed in fermentation tubes and sterilized in the usual manner. He found, how- ever, that a longer period of incubation than is employed in dextrose broth presumptive test is necessary and recommended 72 hours. For or- dinary work a period of 48 hours is ample. In this medium, the pro- duction of 10 percent or more gas is considered a positive presumptive test. The proportion of positive presumptive tests found on further study to contain the colon group is very high, Stokes and Stoner reporting 95 and Gumming 87 percent. Its superiority to dextrose broth is quite marked. Thus Prescott and Winslow, from a study of 176 surface waters in eastern Massachusetts, obtained 120 positive fermentations with dextrose broth and 78 with lactose bile; but Bad. coli was isolated from only 70 (58%) of the dextrose as compared with 64 (82%) of the positive bile tubes. Reliance on the dextrose broth presumptive test would have intro- duced an error of 70 percent as compared to an error of 11 percent for the lactose bile medium. The superiority of lactose bile, as a presumptive test, is evident. This is due partly to the selective antiseptic action of the bile salts but also to a considerable extent to the use of lactose which automatically elimin- ates the dextrose positive-lactose negative organisms. It should also be noted that the presence of lactose may indirectly inhibit non-lactose fer- menters for by stimulating the growth of the colon group, the resultant acid production through fermentation of the disaccharid may in turn in- hibit markedly the growth of water forms. In lactose media the probabil' ity of the colon group being overgrown is considerably reduced as com- pared with glucose media where acid may be produced by many other bacilli resulting in the retardation of the colon group itself. Consideration of the example cited above indicates, however, that although lactose bile is superior as a presumptive test, the actual number of positive isolations with this medium (64) was about 8.5 percent less than was obtained with dextrose broth (70). Apparently therefore the inhibitory action of the bile is not restricted to water forms, but is exerted to some extent, on the colon group as well. The Committee on Standard Methods (1912) suggested that this in- hibitory action is exerted on the weaker members of the colon group, strains which have become attenuated, so to speak, and which are indicative only of remote pollution and therefore of apparently little significance. Thus they say, "In the interpretation of the sanitary quality of a water, it 53 is best to discount the presence of attenuated Fact, coli and to be sure to obtain all vigorous types. The lactose bile medium accomplishes both these objects." This contention, that bile affects the weaker strains, must be regarded merely as an assumption, for it is not substantiated by actual observations. Jordan, in a careful study of the subject, concludes that there is no re- lation between attenuation and the antiseptic action of the bile. He found that freshly isolated strains were inhibited to as great or even greater degree than old strains. Gumming, in a comparison of lactose bile and lactose broth, found that with sewage preliminary enrichment in lactose bile yielded only 25 percent as many colon forms as were obtained with lactose broth. In a similar study with river water, the bile method gave 50 to 70 percent as many colon organisms as the broth. These results would indicate quite the converse of the contention of the 1912 report of the Committee on Standard Methods that "Attenuated B. coli does not re- present recent contamination, and all B. coli not attenuated grow readily in lactose bile." The 1917 and 1920 reports of the Committee on Standard Methods of Water Analysis recommend the use of lactose broth for preliminary en- richment and the presumptive test on the ground tha it gives a higher total number of positive isolations. The concensus of opinion at present seems to be in favor of the substitution of broth for bile. Lactose Broth vs. Lactose Bile. Jordan in 1913 called attention to the greater proportion of successful isolations of fact, coli with lactose broth. Of forty — 5 c. c. portions of Lake Michigan water inoculated into lactose broth and lactose bile media, colon bacilli were isolated from 42 percent of the former and only 30 percent of the latter. Similarly one hundred and fifty — 1 c. c. samples yielded 31 percent positive isolations with lactose broth compared to 22 percent when lactose bile was em- ployed for preliminary enrichment. Creel in a study of drinking waters on railroad trains reports the following frequencies of the colon group in comparative tests: Water samples containing colon group 91 Positive from lactose bile only 18 Positive from lactose broth only 45 Positive from both media 27 The inhibitory action of the bile is very marked. About 50 percent of the colon bacilli isolation would have been lost if reliance had been placed on lactose peptone bile alone. Similarly Dr. Gumming in the Potomac River studies calculates the number of colon bacilli to be 84 per c. c. by lactose broth enrichment followed by confirmatory tests as compared to 47 when lactose bile was employed and followed by confirmatory tests, again indicating that about half of the colon bacilli were lost when preliminary enrichment was car- ried out in the bile medium. Obst comes to the same conclusion. Houser of the Cincinnati Water Supply prefers the broth medium. Ritter in a detailed comparison of 54 both media concludes that if both lactose broth and lactose bile are positive in 24 hours, the probability of the presence of the colon group is very great (about 98% confirmed by subsequent tests) and that, in general, if there is fermentation in both bile and broth media the presumptive test is reliable in about 75 percent, but if either medium is negative, then the proportion of positively confirmed tubes is very much less, even as low as 25 to 45%. This high proportion of positively confirmed presumptive tests, when both broth and bile are positive, may find a ready explanation in the observations of Creel. Most of the spurious presumptive tests are due to the presence of spore forming anaerobic lactose fermenters. Creel found that some of these anaerobes are capable of growing in broth but not in bile. He calls this "Group A" whereas another "Group B" will grow readily in bile but not in broth. Thus a positive test in both lactose broth and lactose bile automatically eliminates both of these anaer- obic spore formers and thereby accounts for the high proportion of pos- itively confirmed tests. Opposed to the views expressed above is that of Hale who recently (1917) stated that from a careful detailed, and extensive comparative study on the two media at the Mount Prospect Laboratory, he considers the results were all in favor of bile, that gas formation was more rapid, produced in larger amounts, and that the B. Clostridium welchii types (an aerobic spore formers) were less frequent. Probably his experiences may be explained by the fact that, in these experiments, he employed a 5 per- cent dried bile, whereas the observations of other investigators were con- cerned with the original medium, containing either undiluted ox gall or 10 percent of the dried bile. The work of Salter and the author in the Laboratory of the Iowa En- gineering Experiment Station indicated quite clearly that bile may inhibit or stimulate multiplication of Bact. coli, depending upon the concentration of bile salts. With less than 0.5 percent bile salts a stimulating action on the rate of multiplication was observed, whereas higher concentrations were markedly inhibitory. The effect was studied by observing changes in the generation time (See Table XXIV.). Since bacteria divide by simple fission, the number of organisms pres- ent at any time in an actively growing culture may be expressed thus: b = B X 2° Where "B" is the initial number of bacteria "n" the number of generations "b" the number of bacteria after "n" generations If the time elapsed is "t" then "g" the generation time — equals t t • — or "n" equals — . J Thus b = B X 2" = B X 2g' t log2 ° "~ log b — logB 55 TABLE XXIV. EFFECT OF CONCENTRATION OF COMMERCIAL BILE SALTS ON GROWTH OF BACT. COLI IN 0.5 PERCENT PEPTONE WATER. (Temp, of incubation 37o C.) Concentration Bacteria per c. c. After Average generation of bile salts hrs. 2 krs. 4 hrs. 8 hrs. time in minutes Control 0.0% 124 410 12,800 14.600.000 28.7 0.1% 124 595 17,900 23.700.000 36,000,000 27.5 0.2% 124 515 17,900 26.6 0.3% 124 450 17,000 24,700,000 27.7 0.5% 124 520 20,200 21,500,000 27.8 0.7% 124 425 13,400 10,800.000 29.5 1.0% 124 435 7,150 1,650,000 35.4 The experience of Hale, that CI. Welchii forms were less frequently encountered in bile than in broth, may merely mean that in the waters he worked with Creel's anaerobe of "Group A" were more frequent than "Group B". In other laboratories, the reverse might be true. The relative merits of lactose broth and lactose bile may be summed up as follows: 1. Lactose bile is a more reliable presumptive test but a greater proportion of the colon group may be detected by preliminary enrichment in lactose broth. 2. If the proper concentration of bile salts could be determined the bile medium would probably be preferable. For the present, considering the difficulty of obtaining bile of constant composition or the chemically pure salts, and in view of our insufficient knowledge as to the optimum concentration of bile salts, it seems best to employ lactose broth as a more uniform medium may thus be obtained in different laboratories. It is very probable that if a standardized evaporated bile were available a con- concentration of 1 to 2 percent in lactose peptone water would be superior to lactose broth. THE LACTOSE BROTH PRESUMPTIVE TEST. The use of lactose broth for preliminary enrichment and as a pre- sumptive test has received considerable impetus through the investigations of the Public Health Service and has been accepted by the Committee on Standard Methods of Water Analysis of the A. P. H. A. (1917). Factors Afifecting the Preparation of Lactose Broth. In 1917, the Standard lactose broth consisted of 0.3 percent beef extract and 0.5 percent peptone with 1.0 percent lactose, the reaction being neutral to phenolphthalein. The medium was tubed and sterilized in the autoclave at 15 pounds (120" C.) for 15 minutes. Hasseltine pointed out that sterilization in the autoclave was objection- able as it brought about a breaking down of the lactose resulting in a con- siderable increase in the number of unconfirmable presumptive tests as com- 56 pared with the lactose broth medium employed in the Public Health Ser- vice. The Public Health Service lactose broth is made as follows: Nutrient broth neutral to phenophthalein is prepared in bulk and sterilized in the autoclave. A sufficient quantity of a 20 percent solution of lactose in distilled water, previously sterilized in the Arnold for an hour and a half, is then added to make a concentration of 1 percent. The medium is then distributed aseptically into sterile Smith fermentation tubes, which are then heated in the Arnold for 30 minutes. Hasseltine found that the "Standard" lactose broth gave 73 percent more positive fermentations and yielded 10 percent fewer confirmations than the Public Health Service medium. With reference to these observations, it should be pointed out,- however, that in the preparation of the Standard broth, the period of sterilization was quite prolonged. He states that the broth was in the autoclave for about an hour (25 minutes to raise the pressure to 15 pounds, 15 minutes at that pressure, and about 20 minutes to allow the pressure to fall sufficiently to permit opening the autoclave without blowing out or wetting the stoppers). In the autoclave in which steam must be generated this prolonged period is of course probably neces- sary but in autoclaves provided with pressure steam, the entire period of sterilization may be reduced to 25 or at most 30 minutes; with such an instrument, the very significant objections raised by Hasseltine do not apply. In fact, Mudge, in a careful study of the relative effects of tem- perature and pressure as compared with time of exposure, concludes that the time factor is the more vital one in the decomposition of lactose. He maintains that heating in streaming steam for three successive days, as is ordinarily done in the Arnold, will cause a greater hydrolysis of lactose and maltose than sterilization in the autoclave at 15 pounds for 15 minutes. These conclusions are based upon the relative increase in monosaccharids as indicated in Table XXV. By use of bacterial cultures confirmatory re- sults were obtained. TABLE XXV. EFFECT OF METHOD OF STERIIJZATION ON DECOMPOSITION OF CARBOHYDRATES. (After Mudge 1917) Method of sterilization Lactose Maltose Sucrose Raffinose Autoclave 15 minutes Trace* 1 Autoclave 30 minutes 1.5* 3 Autoclave 60 minutes 2.0 Very great Slight Arnold 1st day 4 Arnold 2nd day 1 Very great Arnold 3rd day 2 Very great ♦Figures indicate relative degree of reduction as determined by Barfoed's method for monosaccharids. Mudge also makes an interesting observation as to the cause of in- creased acidity in sterilized culture media. As is well known, a neutral 57 solution of amino acids become acid on the addition of neutral formalde- hyde. Sugars may be considered as polymers of formaldehyde, and, as some sugars have aldehyde groups capable of reacting, it is suggested that the increased acidity in sterilized sugar media may be due to the interaction of the amino acids in the medium with the aldehyde group of the sugar. This reaction takes place very slowly and does not become evident before 30 minutes autoclaving. In Table XXVL this contention is very clearly demonstrated. Neither asparagine, maltose, lactose, nor rafinose become acid on autoclaving, even for an hour, but if maltose or lactose are heated in the presence of asparagine a distinct acidity develops in 30 min- utes, and a much more marked acidity after one hour. With raffinose, on the other hand, there is no increase in acidity. The disaccharids (maltose and lactose) contain reactable aldehyde groups whereas the trisaccrid, raffinose, does not. The contention of Mudge that it is the presence of an unstable sugar molecule together with an amino acid which gives rise to the acidity on sterilization appears very plausible. TABLE XXVL EFFECT OF AN AMINO ACID (ASPARAGINE) ON PRODUCTION OF ACID FROM SUGARS, BY AUTOCLAVING. (After Mudge 1917) Acidity ( % normal) after autoclaving for Medium 15 min. 30 min. 60 min. Asparagine 0.0 0.0 0.0 Maltose 0.0 0.0 0.0 Lactose 0.0 0.0 0.0 Raffinose 0.0 0.0 0.0 Asparagine + Maltose 0.0 0.44 0.58 Asparagine -|- Lactose 0.0 0.34 0.64 Asparagine + Raffinose 0.0 0.0 0.0 In the revised Standard Methods for 1920, two important modifications in the preparation of lactose broth were recognized: (1) The method of sterilization recommended for autoclaving is 15 pounds for 15 minutes provided the total time of exposure to heat is not more than a half-hour. "Otherwise a 10 percent solution of the required carbohydrate shall be made in distilled water and sterilized at 100° (for one and a half hours), and this solution shall be added to sterile nutri- ment broth in amounts sufficient to make a 0.5 percent solution of the carbohydrates and the mixture shall then be tubed and sterilized at 100^ C. for 30 minutes, or it is permissable to add by means of a sterile pipette directly to a tube of sterile neutral broth enough of the carbohydrates to make the required 0.5 percent. The tubes so made shall be incubated at 37° C. for 24 hours, as a test for sterility." (2) The concentration of lactose is reduced to 0.5 percent. This seems to be desirable as it tends to eliminate the danger of loss of the colon group through destruction by acid formed in fermentation. 58 Clemesha, in 1912, found that in peptone bile salt, neutral red broth containing 1 percent lactose, pure cultures of Bad. coli and Bact. aerogenes begin to fall off in numbers after 24 hours. Burling and Levine observed that the concentration of carbohydrates influences markedly the viability of the colon group in culture media. With 0.3 percent glucose or lactose, the number of viable organisms reached a maximum of 100 to 1,000 mil- lion in about 10 hours and remained constant for about 7 days. Increasing the amount of carbohydrates to about 0.5 percent resulted in death of over 99 percent of the organisms in a week, whereas with a concentration of 1 percent of the carbohydrates a count of 100 million, after 24 hours, was reduced to less than 10,000 (a reduction of 99.9 percent in 72 hours.) In preparing carbohydrate media the author has used the following method with excellent results: The broth containing 0.5 percent of the carbohydrate is distributed in sterile Durham fermentation tubes, using ordinary precautions but not necessarily strict asepsis, and autoclaved at 10 pounds for 10 to 15 minutes in a pressure steam sterilizer. Immediately after removing from the auto- clave, the tubes are cooled by placing in running tap water after which they are incubated at 37" C. for 24 to 48 hours to eliminate unsterile tubes. Several tubes are inoculated with Bact. enteritidis or Bact. paratyphosum and incubated for 24 hours to detect hydrolysis as shown by gas formation. Wagner and Monfort have recently reported that if gentian violet (1-20,000 to 1-100,000) be added to lactose broth, the medium may be prepared by a single heating in the Arnold or simply pasteurization. If further experience proves gentian violet non-inhibitory to the growth of Bact. coli, such a medium would be practically ideal, for not only is the danger of inversion of the lactose eliminated, but growth of the spore forming lactose fermenters is also reported to be checked. In recording the presumptive test with lactose broth (this applies to lactose bile also), the production of 10 percent or more gas is regarded as a positive presumptive test. If the amount of gas is less than 10 percent the test is regarded as questionable. If there is no gas it is of course negative. Experience indicates that where the quantity of gas is more than 70 percent the fermentation is very likely due to anaerobic spore formers rather than to the colon group. Reliability and Value of the Lactose Broth and Lactose Bile Presumptive Tests. In studies on the Ohio and Mississippi Rivers, Fuller noticed that the results with these presumptive tests were far be- yond what could be explained by ordinary pollution and much higher than results obtained by a complete isolation test. Similarly in studies on the Potomac River, Cummings found these tests were reliable when pol- lution was excessive, but as purification progresses, the correlation be- tween the presumptive and confirmatory tests become less marked. Using Endo agar for confirmation 92 percent of positive presumptive tests were confirmed in regions close to the source of pollution but in the vicinity of oyster beds, which were further removed from pollution, only 47.5 per- cent of the positive presumptive tests were found to contain members of 59 the colon group. The reliability of these presumptive tests therefore varies directly with the degree of contamination or inversely with the remoteness in time and distance from the source of pollution. The non-confirmed presumptive tests are due, for the most part, to lactose fermenting spore producing anaerobes which persist for a long time in water and which are probably of very little sanitary significance. Hall and Ellefson have suggested the addition of gentian violet to lactose broth to restrict the growth of these anaerobes, and recently Meur and Harris indicate that brilliant green may be employed in lactose bile to eliminate these spurious presumptive tests. This will be considered more in detail in a subsequent chapter. The reliability of the presumptive test also varies with the treatment which the water has received. Hauser, using lactose broth, obtained con- firmations in 97.4 percent of raw water samples as compared with 86.1 percent of samples taken at the outlet from the clear water reservoir. The effect of chlorination on the reliability of the presumptive test is particularly marked and is shown in the following able: TABLE XXVI. SHOWING CORRELATION OF RATE OF GAS PRODUCTION WITH CONFIRMATION OF THE PRESUMPTIVE TEST FOR COLON GROUP IN LACTOSE BROTH. Untreated supplies Chlorinated supplies Rate of Gas Production Number of tubes showing gas Percentage of gas tubes con- firmed Number of tubes showing gas Percentage of gas tubes con- firmed Rapid (10 percent or more in twenty-^ jur hours) 684 97.7 57 44.0 Moderate (less than 10 per- cent in twenty-four hours) 193 91.2 15 20.0 Slow (no gas in 24 hours; 10 percent or more in forty-eight hours) 276 73.2 156 6.4 Very slow (no gas in twenty-four hours ; less than 10 percent in forty- eight hours) 33 45.5 26 0.0 The results indicated are based on a study of 1559 water analyses in the Advanced Sector of the American Expeditionary Forces at Dijon, France, comprising waters from both treated and raw supplies and from various sources. An interesting correlation is noted between the rate of gas production and the probability of confirmation. It appears evident that 1. The positive presumptive test (10 percent or more gas in 24 hours) is a very reliable index of the probable presence of Bact. coli 60 when dealing with untreated waters but it is not to depended upon when testing chlorinated waters; 97.7 per cent of the former and only 44 per cent of the chlorinated samples showing gas, were confirmed for Bad. coli or its close allies. 2. The doubtful presumptive test (less than 10 percent gas in 24 hours, or more than this quantity in 48 hours where 24 hours was nega- tive) is only a fair index of the probable presence of Bact. coli in un- treated waters, while for chlorinated specimens it is practically negligible, as 75.9 percent of gas tubes from untreated, and only 7.6 percent of those from chlorinated samples were successfully confirmed. 3. A small amount of gas in 24 hours is a more reliable index for Bact. coli than 10 percent or more gas in 48 hours. The rate of gas pro- duction seeems more significant than the total volume of gas formed as a presumptive test. Similar results were obtained by Graff and Mote. Of 966 instances of gas production in lactose peptone bile from settling basins (unchlorin- ated) 41 percent were positive. Of 844 instances from reservoirs (after chlorination) 12 percent were confirmed, and 1063 gas tubes from taps only 5 percent were confirmed. They also noted that vigorous gas pro- duction in 24 hours yielded positive tests for Bact. coli in a greater percent of cases, from all sources, than was obtained if gas was not noted previous- ly to the 48 hour period. It is thus apparent that gas production in either lactose broth or lactose bile, particularly with treated, partially purified, or chlorinated waters, cannot be relied upon as an index of the presence of the colon group. Confirmation must be restored to, except possibly for water known to be polluted. CONFIRMATORY TESTS it is merely necessary to streak out or smear on the surface of a differential medium (litmus lactose agar, Endo agar, or Eosin-Methylene-Blue agar) which is then included at 37° C. for 24 hours. If typical colonies develop, the presence of the colon group in the presumptive test is considered con- firmed. This is known as the partially confirmed test and for routine work is sufficient, although it must be borne in mind that there are a few spore forming lactose fermenters which are capable of growing aerobically. If the colonies developing on this differential medium are not typical it becomes necessary to further study these colonies to determine that they are Gram negative non-spore formers and that they are capable of pro- ducing gas from lactose. The latter may be determined by merelv fish- ing a colony into lactose broth which is incubated for 24 hours. If gas is formed and if the Gram stain showed the colony to be a non-spore form- ing Gram negative short rod, the test constitutes what is spoken of in the Standard Methods as the completed test for the colon group. If no growth at all develops on the confirmatory differential medium the assumption is that the gas in the presumptive test tube was due to strict anaerobes and the colon group is recorded as absent. It is con- 61 ceivable, of course, that such a test may be due to death of the colon types. A transfer from this unconfirmed presumptive test to another tube of lactose broth, which on incubation shows gas production, would be excellent evidence that the gas forming organism in question was an an- aerobe and that the negative confirmatory test was not due to death or over- growth of the gas producer. The media for confirmatory tests which have found most favor among American bacteriologists are litmus lactose agar, and Endo agar; and more recently the simplified Eosin-Methylene-Blue agar was suggested by the author. The litmus lactose agar has been discussed in detail in con- nection with the isolation of the colon group. It will merely be added here that Meyer suggests that by the addition of 3 percent agar, giving a stiifer and dryer medium, more favorable results are obtained than with the ordinary medium. The Endo Agar. This is perhaps the most extensively employed confirmatory medium at the present time. It is the medium of choice of the Public Health Service and is recommended in the Standard Methods (1917 and 1920). The colon colonies produce a distinct red coloration, often with a metallic sheen, and are quite easy to detect. Unfortunately there has been, in the past, considerable lack of uniformity in the prepara- tion of Endo. The method of adjustment of the reaction is very crude and there was lack of agreement as to the quantity of indicator to be em- ployed. Thus Kendall, and Prescott and Winslow recommended 0.1 c. c. of a saturated basic fuchsin and 1 c. c. of a 10 percent sodium sulphite per 100 c. c. of agar. The Army Laboratory Manual, employed exten- sively during the war, recommends 0.18 c. c. of fuchsin and 2.5 c. c. of a 10 percent sodium sulphite. In the Hygienic Laboratory Endo medium the indicator consists of 0.5 c. c. of basic fuchsin and the equivalent of 2.5 c. c. of a 10 percent sodium sulphite solution. Some investigators, Rob- inson and Rettger, have recommended decolorization with sodium acid sulphite in place of sodium sulphite. The appearance of the colon colonies will depend to a considerable extent on the concentration of the indicator, being very intense red and showing the characteristic metallic sheen only with the higher concentra- tions. The description of a typical colony of Bad. coli on one of these mediums may therefore not tally at all with its appearance on supposedly the same medium prepared in some other laboratory. It thus becomes essential to detail, in each instance, the method of preparation of the medium employed. We may well exclaim with Morse and Wolman, "What shall the standard test for Bad. coli include if a mere difference in the proportion of fuchsin and sulphite in two Endo media results in the one showing typical colonies in 75 percent of the tubes, in which Bad. coli is present, while the other shows the same colonies in only 14 percent?" The apparent difference is due to a confusion of the term "typical" Bad. coli colony. What is considered typical for one medium with one con- centration of dye will of course not be typical for another. 62 One of the great difficulties in the preparation of Endo medium is the adjustment of the reaction. In most of the methods the reaction is adjusted on the phenolphthalein scale to some definite point and then a quantity of sodium carbonate is added to make it more alkaline. This is a rather inaccurate method and results in some batches being very ser- viceable while others are almost worthless. Levine suggested a modified and simplified Endo medium which re- quires no adjustment of reaction and which need not be filtered. The medium consists of 1 percent Difco peptone, 0.3 percent dipotassium phos- phate, 0.5 percent agar, and I percent lactose. One-half c. c. of a satur- ated basic fuchsin, decolorized by 2.5 c. c. of a 10 percent sodium sulphite, as recommended by the Hygienic Laboratory, is employed as an indicator for each 100 c. c. of the medium. Aside from the simplicity of prepar- ation, an advantage claimed is that Bad. coli may be differentiated from Bad. aerogenes. The former possess a distinct metallic sheen, the colonies are flat and button-like, and about two or three m. m. in diameter; whereas the latter usually produces considerably larger colonies which are convex and a metallic sheen is rarely observed. The disadvantage of the medium is that dififusion of color, due to acid production, is very rapid. This may be reduced by increasing the content of agar but when that is done the differentiation between Bad. coli and Bad. aerogenes becomes less dis- tinct. All of the Endo mediums above have the disadvantage of instability. Exposure to light or air induces a deep red coloration which interferes seriously with the detection of acid formers thus making it necessary to prepare the medium fresh and at frequent intervals. Kahn suggests the use of Endo medium in tubes instead of plates inoculating from the presumtive test onto the slant and also into the butt in a manner analagous to the Russel double sugar medium. He states that Endo agar prepared and sterilized in tubes may be kept from three to four weeks without deterioration and that anaerobes will not develop in the butt of the Endo agar tube. Thus the test for acid production under aero- bic and gas formation under anaerobic conditions by members of the colon group is determined simultaneously thereby performing both partially confirmed and a complete test, described above in a single tube. This use of the Endo medium is very interesting and suggestive and worth trial and consideration. The Eosin Methylene Blue Agar Confirmatory Medium. Le- vine, in 1917, suggested a modification of the eosin-methylene blue agar (first described by Holt, Harris and Teague) for the confirmation of the presumptive test. The medium is prepared in the following manner: Distilled water 1000 c. c. Peptone (Difco) 10 gm. Dipotassium phosphate 2 gm. Agar -15 gm. 63 Boil ingredients until dissolved and make up any loss due to evapora- tion. Place measured quantities in flasks and sterilize at 15 pounds for 15 minutes. Just prior to use, add to each 100 c. c. of the melted agar, prepared as above, the following constituents: Sterile (20%) lactose solution 1 gm. or 5 c. c. Aqueous (2.0%) eosin (yellowish) solution.— 2 c. c. Aqueous (0.5%) methylene blue solution 2 c. c. Pour medium into petri dishes, allow it to harden in incubator and inoculate in the ordinary way. Smearing the surface with a glass rod seems preferable to the streaking method sometimes employed. There is no adjustment of reaction and filtration of medium is not necessary. Test tubes may be substituted for petri dishes if desired. The value of such a change is (1) the reduction of expense, as only 3 or 4 c. c. of medium is needed for every test tube while about 15 c. c. is usually em- ployed with petri dish, and (2) test tubes may be stored for long periods whereas the medium in petri dishes would have to be prepared at intervals of a week or less. TABLE XXVn. DIFFERENTIATION OF BACT. COLI AND BACT. AEROGENES ON EOSIN-METHYLENE BLUE AGAR. Size Conflu Bad. coli ( 1 ) Well isolated colonies are 2 — 3 m.in. in diameter, Neighboring colonies show little tendency to run together. Bad. aerogenes (2) Well isolated colonies aie larger than coli; usually 4-6 m.m. in diameter or more. Neighboring colonies (2) run to- gether quickly. Elevation Colonies slightly raised; surface flat or slightly concave, rarely convex. Colonies considerably raised and markedly convex; occasionally the center drops precipitately. Appearance by trans mitted light Dark almost black centers which extend more than % across the diameter of colony; inter nal structure of central dark portion difficult to discern. Centers deep brown; not as dark as Bact. coli and smaller in proportion to the rest of the colony. Striated internal structure often observed in young colonies. Appearance by re- flected light Colonies dark, button-like, often concentrically ringed with greenish metallic sheen. Much lighter than Bad. coli. Metallic sheen not observed except occasionally in de- pressed center when such is pre sent. (1) Two other types have heen occasionally encountered: One resembles the type de- scribed, except that there is no metallic sheen, the colonies being wine colored. The other type of colony is somewhat larger (4 m. m.) grows effusly. and has a marked crenated or irregular edge, the central portion showing a very distinct metallic sheen. These two varieties constitute about 2 or 3 percent of the colonies observed, (2) A small type of aerogenes colony about the size of the colon colonies, which shows no tendency to coalesce, has been occasionally encountered. 64 The advantages claimed for the eosine-methylene-blue medium are (1) ease of preparation, as neither fihration nor adjustment of the reaction is required, and (2) relative permanency. (Petri dishes pouredi have been used after a week or longer, if stored in the ice box) and (3) the medium affords an excellent differentiation between Bact. coli and Eact. aerogenes, as described in Table XXVII above. The method of preparation of the test tubes is briefly as follows : The medium prepared as above is poured aseptically into sterile test tubes and allowed to solidify so as to give a long slant without a butt. To confirm the presumptive test, inoculation is made on the surface of these slants with a straight platinum needle. If desired the medium may be placed in test tubes and sterilized in the Arnold or in the auoctlave in the ordinary manner. In that case it will be found that the agar, when first removed from the sterilizer, is fluorescent like eosin but as it cools the typical wine color returns. The reliability of the differentiation of Bact. coli and Bact. aerogenes is indicated in the following table: TABLE XXVIII. RELIABILITY OF PRESUMPTVE DIFFERENTIATION OF BACT. COLI AND BACT. AEROGENES ON EOSIN-METHYLENE BLUE AGAR. Designation 1918-1919, France 1917, Iowa from appearance on agar Nnmber of colonies tested Percentage correct Number of colonies tested Percentage correct Bact. coli 87 94.2 102 96.9 Bact. aerogenes 55 85.5 122 82.4 Thus in 1917, of 122 colonies fished, as probably Bact. coli, 96.9 per- cent were proved to be such and in 1918 and 1919, 94.2 percent of 87 colonies fished were correctly designated from their appearance on eosin- methylene blue agar. The proportion of Bact. aerogenes, correctlv indenti- fied was 82.4 percent and 85.5 percent in the series in Iowa and France respectively. The medium therefore appears to be quite reliable for the routine presumptive differentiation of these two groups of colon bacteria and may therefore be of considerable value if in the future it is consid- ered desirable to distinguish these forms because of their probable differ- ent sanitary significance. 65 V. THE COLON GROUP AS AN INDEX OF POLLUTION Safe Water. A safe water for human consumption may be defined as one which is free from harmful constituents important among which are disease producing microorganisms. The logical and most direct pro- cedure to determine the potability and safety of a water would be to determine the presence or absence of pathogenic bacteria but unfortun- ately this task is an impossible one for routine and recourse must there- fore be taken to an indirect index of the probable presence of harmful germs. Since the diseases transmissable through water are primarily of intestinal origin the detection of the presence of intestinal material natur- ally leads to the presumption that a potential danger exists, for if such material is present it is very probable 'and certainly possible that intes- tinal disease germs are also present. A number of tests both chemical and bacteriological have been sug- gested as indicators of intestinal pollution. The bacterial examination, by reason of the large number of bacteria present in feces and sewage and the ease with which they may be detected in water, is a particularly delicate test. Three groups of bacteria have been regarded as indicators of pol- lution : The colon group. Sewage streptococci. Spore forming anaerobes. An organism to be considered an ideal index of fecal pollution should have the following characteristics: 1. It should be distinctively and ch'aracteristicall]^ of human or animal intestinal origin. 2. It should be absent or extremely rare in nature outside of the intestinal tract. 3. It must be capable of easy and rapid detection. 4. Its incidence in water should bear some constant relation to the sanitary survey or our knowledge as to the probability of pollution, par- ticularly with sewage. 5. It should be distinctly more viable and more resistant in water and to treatment than are the intestinal pathogens [Bact. typhi, Bad. dysen- teriae, etc.), but not excessively so. Such an ideal index is not available but the general concensus of opin- ion among English and American bacteriologists is favorable to the em- ployment of the Colon group for this purpose. Although bacteria of this group are not restricted in habitat to the intestinal tract of man being characteristic also of the intestinal tract of the lower animals, it is never- theless true that there is a correlation between the quantitative incidence of a least the coli section and known pollution. The whole group is easy of detection as will be seen from the following considerations. It is more viable than Bact. typhi but yet dies off relatively quickly; colon bacilli are present in relatively large numbers in water known to be polluted but only infrequently in natural supplies. A correlation has been established 66 between the incidence of the colon group in drinking water and the ty- phoid fever rate in the community. Viability of Bad, coli and Bad. typhi in Water. These organ- isms do not find favorable conditions for growth in natural waters. The food supply is apparently insufficient or unavailable, the temperature is unfavorable, other organisms exert a deleterious effect, and in consequence a diminution in number rapidly ensues. It is conceivable that under cer- tain conditions of extremely high temperature, as may occur in summer, there may be an initial increase but death soon results. A convenient method for appraising the relative viability of two organisms is to com- pare the rates at which they die off under a given set of conditions. The rate of death of bacteria may be expressed by 1 N K = Ti-To -log — where, "N" is the number of organisms at the beginning, i. e. time "To". "n" is the number of organisms at the end of the time, "T^". "K" is a measure of the rate of death. If the logarithms of the number of surviving bacteria at different periods are plotted against the elapsed time it will be observed that the points fall along a straight line intersecting the "x" axis at an angle whose tangent is numerically equivalent to "K". The greater the rate of death the greater will be "K". In the following tables are given the number of surviving Back coli and Bad. typhi when stored in water and exposed to the air. It will be observed that the rate of death of Bact. typhi is considerably higher than Bact. coli, though the latter organism also dies off rather rapidly. TABLE XXIX. VIABILITY OF BACT. COLI IN WATER AT 20O C. (After Hinds 1916) Elapsed time in hours Count Rate of death "K" 690,000 2 600,000 .069 6 420,000 .083 13 244,000 .079 24 123,000 .071 36 10,000 .1181 48 3,400 .111 72 120 .120 ..0.094 TABLE XXX. VIABILITY OF BACi TYPHI IN WATER AT 20O C. (After Hinds 1916) Elapsed time in hours Count Rate of death "K" 118,000 1.38 2 7,500 1.38 4 500 1.37 6 280 1.01 8 230 .78 10 150 .67 l:i 30 .69 14 30 .59 24 2 .46 Average "K" Average "K" 0.87 The writer made some observations at the Massachusetts Institute of Technology in 1913 on the effect of temperature on the viability of Bact. coli in distilled and conductivity water containing C. P. salt. The results are shown in figures 3 and 4 where it is apparent that the higher the tem- perature the greater the rate of death, and that the presence of gases in solution also affects the death rate. 67 Houston artificially infected Thames River water with pure cultures of Bact. typhi and noted the proportion of survivors are infleuced by the temperature of storage. The results, based on ten experiments at each temperature, are summarized below: TABLE XXXI. EFFECT OF TEMPERATURE ON VIABILITY OF BACT. TYPHI IN THAMES RIVER. (After Houston 1913) Temperature of storage Surviving Bact. typhi per c. c. after 1 week* Bact. typhi absent from 1 c. c. after Oo C. 47,766 9 weeks 50 C. 14,894 7 weeks 100 c. 69 5 weeks 180 C. 39 4 weeks 270 C. 19 3 weeks 370 C. 5 2 weeks *Initial number 103,328 per c. c. r ^^-C ^^^^ • -^- • ~~r 6~ o a "^ \ 1 'lability in Dis. ^illed M- t co/i 7ter coni dining IBSTi Aj <^CI cc P) /c '0 i 00 36 o , 30 I I/. \ \ Ti phoK 1 De an 1th f d fate; Inci lenc : of Color Gra 'P \ in riltei Eln ird 1 Ue r c I.Y ■f \ / \ Y N \ a Typh( lion Ir .id f? le J_ \ \ \ \ \ \ 1 t 1 1 \ \ \ \ \ ^ \ \ \ \ 1 1 1 1 \ \ '"" — \ r;.'" I I I 20 0$ Si 1903 'en 1505 Ofc '07 08 'OS /?I0 II '12 '13 'It I9i5 K. '17 The English and American bacteriologists uphold it. The Germans have objected strenuously to its inclusion in water work. The contention of Kruse, Konrich and other German investigators that colon bacilli may be found in any water irrespective of its source, provided a sufficiently large sample is taken, and that of Chick, Houston, and other English author- ities, that these organisms are not present in water unless it has received 71 sewage pollution, led Prescott and Winslow to conclude that it was the number rather that the presence of colon bacilli that should be used as a criterion for recent sewage pollution. Thus they state that the find- ing of a few colon bacilli in large samples of water or their occasional isolation from small samples is not of any special significance but if these organisms are present in a large proporotion of small samples (I c. c. or less) then the evidence of recent sewage pollution is significant. Gartner (1910) estimates the number of colon forms in cultivated soil at 1,500,000 per square meter and naively remarks that it is no wonder the rain should wash a few of them into neighboring wells. The recent work of Johnson and Levine, Burton and Rettger, Chen and Rettger and others have shown quite conclusively that the organisms in the soil are markedly different from those habitually present in the human and animal intestine. Furthermore the "B. coli" of Houston and other English in- vestigators does not include many of the soil forms for they regard only lactose positive-indol positive varieties as "typical" B. coli, whereas most of the Bad. aerogenes and Bad. cloacae are indol negative. German opinion is gradually becoming more favorable toward acceptance of the colon test, provided that preliminary enrichment is carried out at 46*^ C. (Eijkman test). This procedure, as has been pointed out before, serves to eliminate the Bact. aerogenes and Bad. cloacae or characteristic soil types. It appears that the contentions of the German school were really against these soil forms as indices of pollution. It thus becomes possible to reconcile their opinions with those of English and American bacteriol- ogists if we recognize that not only is the numerical incidence of the colon group significant as an index of pollution, but that the type of organism must also be considered. THE COLI AND AEROGENES SECTIONS AS INDICES OF POLLUTION The question arises as to whether the aerogenes and coli sections are to be accorded the same sanitary significance. Considerable work is still needed along these lines, but the following is presented as to (1) their distribu- tion in nature, (2) correlation of their relative incidence with the sanitary qualities of water, (3) relative viability in water, and (4) relative resis- tance to treatment and chlorination. Incidence of the Coli and Aerogenes Sections in Feces. Rogers, Clark, and Evans called attention to the scarcity of the high ratio group (aerogenes section) in cow dung. Only one colony of 150 fished was of this type. Levine observed that among 117 cultures isolated from cow, horse, sheep, pig, and man, not a single organism proved to be Ba€t. aero- genes. A study of the literature employing the Voges-Proskauer test as a means of differentiation of the two colon sections disclosed that these ob- servations, as to the scarcity of the aerogenes types in human and animal feces has long been recognized although not adequately appreciated. Thus MacConkey (1905) remarks on the scarcity of Bact. (ladis) aerogenes in human feces. In the examination of 241 strains obtained from 72 23 human stools, only 4 gave the Voges-Proskauer reaction and of these 3 were obtained from a single sample. At the same time, a study of 51 strains from 5 samples of horses and 48 from 6 samples of cow manure did not yield a single aerogenes culture. In 1909 he made further ob- servations with similar results. Ferriera, Horta and Paredes reported 8 out of 117 lactose fermenting strains from human feces, and 2 of 81 strains obtained from 46 different species of animals to belong to the aerogenes section as indicated by a positive Proskauer test. The work of Clemesha is particularly significant because of the large number of cultures examined. Of 1207 human strains, about 6 percent were found to be of the aerogenes type but a large number of these came from a single sample. Among 1029 cultures from cow dung. Bad. [lactis) aerogenes was encountered in about 10 percent. He records that in cow dung, the Bad. aerogenes was found in small numbers, and Bad. cloacae was sometimes very common, but that in human stools these types were very rare and that a sudden increase in their prevalence was never ob- served. Clemesha's observations were confirmed to a considerable extent by R. G. Archibald of the Wellcome Tropical Research Laboratory in an investigation of the water of Khartoum. TABLE XXXVI. INCIDENCE OF AEROGENES TYPES AMONG COLON BACILU IN HUMAN DEJECTA. Investigators No. of strains studied % (V Aerogenes Section . P._1_:M. R ) MacConkey (1905^ 241 1.7 Ferriera, Horta and Paredes (1908) 178 5.6 MacConkey (1909) 117 6.8 Archibald (1911)* 100 0.0 Clemesha (1912) 1207 4.6 Levine (1916) 25 0.0 Hulton (1916) 10 0.0 Rogers. Clark and Lubs (1916) 113 5.8 Rogers, Clark and Lubs (1918) 177 t23.0 Darling (1919) 20 0.0 Wood (1919) 33 0.0 Stokes (1919) 141 §16.3 Chen and Rettger (1920) t 173 0.0 Total 2534 5.9 ^Includes strains obtained from latrines, directly from feces, and fecal suspensions exposed to action of sunlight. +These comprised 46 strains isolated for the most part by special methods and 31 of these strains were obtained from a single specimen. The true incidence of aerogenes types is therefore considerably less than shown in the table. §A11 Bact. cloacae. Jlncludes animal strains. 73 Recent observations by Rogers and his associates, Darling, Chen and Rettger, Stokes, and others are entireh' in accord with the opinions recorded that the aerogenes section is rare in feces of man and animals. The relative presence of the coli and the aerogenes sections in human and various animal feces is shown in Tables XXXVI. and XXXVII. TABLE XXXVII. INCIDENCE OF AEROGENES TYPES AMONG COLON BACILLI IN ANIMAL DEJECTA. Investigators Animal source No. of strains studied % Aerogenes Section (V.P._|_ ; M.R_) MacConkey (1905) Horse & cow 99 1.0 Ferriera, Horta & Paredes (1908) Misc. 81 2.5 MacConkey (1909) Horse 67 4.5 MacConkey (1909) Misc. 87 0.0 Archibald (1911) Cow & goat* 20 40.0 Clemesha (1912) Cow 1029 10.7 Rogers, Clark & Evans (1914) Cow 150 0.7 Levine (1916) Misc. 92 0.0 Darling (1919) Misc. 93 0.0 Wood (1919) Misc. 99 7.0 Stokes (1919) 15 13.0 Total 1832 7.4 *Smgle samples from each species. Incidence of the Coli and Aerogenes Sections on Grains and in Soil. Many investigators have reported the presence of the colon group on grains and plants. Prescott found lactose fermenting bacteria in flour, bran, corn meal, oats, and barley. Similar observations were made by Papasotiriu in Germany. Cline and Houston found, what they regarded typical colon baccili, in 3 out of 24 samples of wheat, oats, rice, etc. Bettencourt and Borges (1908) succeeded in isolating such organ- isms from vegetables and cereals. Konrich (1910) frequently observed the colon group on cultivated plants and that even about six percent of plants obtained from waste places showed colon bacilli. Fifty-five percent of 300 samples of grain showed members of the colon group. These investigators, however, did not differentiate between the BacL coli and Bact. aerogenes. Rogers, Clark, and Evans were the first to point out that the colon-like forms, occurring on grains, were distinctly dif- ferent from those observed in feces. They isolated 166 cultures from 33 samples of dried grains and 2 samples of green oats. Of these only 8 (4.8%) were the low ratio (coli section); (91.0%) were of the 74 high ratio type (aerogenes section), and 7 (4.2%) produced only hydrogen. They noted further that none of the types found on grains were identical with those obtained by them from bovine feces, the low ratio types from grains being pigmented. Stokes isolated 77 strains from corn meal, grape nuts, post toasties, corn flakes, puffed wheat, quaker oats, rye, corn, wheat, barley, and oats, of which 57 (74.0%) were of the aerogenes cloacae type. It is appar- ent therefore that on grains and plant products, the high ratio V. P. positive, methyl-red negative group predominates. Houston, in 1897 and 1898, examined a number of samples of orchard, garden pasture, and virgin soil, including polluted and unpolluted areas, concludes that the true colon bacilli are very rarely or never found in virgin soils, while they are present in large numbers in other areas, especially those which have been contaminated from animal sources. Konrich, in a study of 547 specimens of soils in Germany, detected the colon group in 65 percent of 0.1 to 0.5 gram samples. He notes that TABI^E XXXVIII. SOURCE, TREATMENT, ETC., OF 42 SAMPLES OF SOIL EXAMINED IN 1915. A B C D E H I II III IV VI VII VIII IX X XI XII XIII XIV Corn field Corn field Corn field Corn field Corn field Corn field Expt. plot 114 Expt. plot 113 Expt. plot 112 Expt. plot 111 Expt. plot 110 Expt. plot 109 Expt. plot 108 Expt. plot 107 Expt. plot 106 Expt. plot 105 Expt. plot 104 Expt. plot 103 Expt. plot 102 Expt. plot 101 Manured, corn grown for three years Manured, corn grown for three years Manured, corn grown for three years Manured, corn grown for three years Manured, corn grown lor three years Manured, corn grown for three years, 4 tons manure annually 2 tons manure annually. , 1 ton manure annually 4 tons of clover chopped and ploughed under annually 2 tons of clover chopped and ploughed under annually 1 ton of clover as in IV 2 tons of oat straw chopped and ploughed under annually No treatment, check plot 2 tons of timothy chopped and ploughed under annually 1 ton timothy as in IX 8 tons of clover once, in four years. 8 tons of manure once in four years. 28 tons peat annually Timothy grown each year and stubble plowed under 1913 I Corn 1913 I Corn 1913 ICom 1913 1913 1913 1914 1914 1914 1914 1914 1914 1914 1914 1914 1913 1913 1914 1914 Corn Com Corn None None None None None None None None None None None None None 14 4 3 2 9 18 i I 1 I I 1 1 I 26 I 24 I 14 Timothy | 18 75 TABLE XXXVm. (continued) Sample Source Soil treatment or other remarks 5J-a >M 9 f^o 9 / II 1 9 O 31 % SO.0 5.? 35.6 3£ Z9.0 /l/o 1 8 5 3 Z 1 12 Z 7 39 % Z.6 £0.5 7.7 77 5.1 Z.6 30.8 5.1 17.9 ^an lyo O Z 1 5 5 1 II 25 7. 8.0 4.0 20.0 £0.0 4.9 44.0 Total 89 62 77 7 9 33 18 ZO 18 333 The Bad. neopolitanum was present only in bovine feces and sewage, comprising 20 percent of the bovine and 7.7 percent of the sewage. Bad. coscoToba occurred as follows: sheep 22.7 percent, pig 3.2 per- cent, sewage 5.1 percent and human 4.0 percent. The relation of sucrose fermentation and the source is especially em- phasized. The sucrose fermenting strains are relatively uncommon in human feces, whereas they constitute the predominating type in animal feces and in the soil. This low incidence in human feces is confirmatory of the observations of numerous other investigators. In this connection, it may be well to recall that when Durham sug- gested the name B. coli communior for the sucrose fermenting variety be- cause of its greater prevalence, his observations were based on the intestinal contents of animals for which this fact holds true, but, as has been pointed out above, the sucrose positive strains are relatively scarce in msm. Bad. coli, like Bad. communior was isolated from all of the sources tested, but a rather distinct correlation with the source is observed with the varieties Bad. coli-communis and Bad. coli-immobilis. The former comprise 1.1 percent of soil; 21 percent of horse; 4.5 percent of sheep; 45 percent of cow; 35.6 percent of pig; 2.6 percent of sewage; and 20 percent of human strains. Bad. coli-immobilis was not obtained from the soil, horse, sheep or cow, but it made up 3.2 percent of the pig, 30.8 percent of the sewage, and 20 percent of the human strains. TABLE LI. FERMENTATION OF SUCROSE BY BACT.-COLI-LIKE BACTERIA FROM HUMAN FECES. Investigators Number of organisms studied Number of sucrose fermenters Percentage of sucrose fermenters Houston, 1902-3 100 30 30 MacConkey, 1905 and 1909.... 419 ~ 142 33.9 Ferreira, Horta, Paredes, 1908 117 44 37.6 Winslow and Walker 1907 25 8 32 Howe, 1912 540 324 60 Clemesha, 191Z 1200 348 29 Browne, 1915 175 20 11.3 Levine, 1916 25 3 12 Total .- 2601 919 35.3 Bact. acidi-lactici was not obtained from the horse nor sheep, and only rarely from the cow (5.0%) or soil (4.0%). The motile variety Bact. acidi-lactici var. Grunthali was particularly abundant among the pig cul- tures (29%) and rare in sewage (5.1%) and man (4%). The non-motile Bact. acidi-lactici var. immohili was restricted to man and sewage entirely, comprising 44 percent of the human and 17.9 percent of the sewage strains. If the sucrose negative forms are more indicative of human pollution it would be anticipated that they would be more prevalent in the more in- tensely polluted waters. Observations by the writer on 78 samples col- lected in France were as follows: Among 34 samples in which the coli section was present in 1 c. c. or smaller quantities, sucrose negative strains were detected in 23 or (68%), whereas of 44 samples containing the coli section only in 10 c. c. or larger quantities but 13 (29%) showed sucrose negative coli strains. That is, the more polluted supplies apparently did contain a greater proportion of sucrose non-fermenters. More extensive work on the correlation of species of colon bacilli with the source, character of pollution, and history of waters is certainly desirable. Resume. From considerations of the requirements for an index of pollution, the colon group appears to be a convenient and desirable one. It is not however, an ideal indicator for the species which it comprises are not all of equal sanitary significance. The evidence seems to be clear and definite that the colon group com- prises two subgroups or sections which are characteristically of different habitat; one, typified by Bact. coli, is present in large numbers in feces and sewage, whereas the other, exemplified by Bact. aerogenes, is rare in 90 such objectionable matter but predominates on the presumably harmless soil and grains. The aerogenes group, as indicated by laboratory experi- ments and observations in the field, is much more viable in water, where it will persist for long periods, and seems capable of growing, to some extent, in stored treated supplies. It is not possible from our present limited knowledge of these two groups to put forth any definite rules for interpreting the significance of their presence in water, but it is felt that the presence of Bad. aerogenes alone should not be regarded as objectionable as is the presence of the Boot, coli in equal numbers. If the sanitary survey is favorable and there is no evidence of the true Bact. coli types in a water supply under different weather conditions, then a considerably greater number of Bact. aerogenes may be tolerated. The presence of Bact. aerogenes alone (i. e. not associated with Bact. coli) in a supply may indicate merely remote pollution or soil contamina- tion which is not as objectionable and certainly not as dangerous as sewage pollution. Differentiation of the coli and aerogenes types in routine water an- alysis is obviously desirable as it may assist in the detection of the probable source and nature of the contamination. 91 VI. THE SPORE FORMING LACTOSE FERMENTERS AND THEIR SIGNIFICANCE IN WATER ANALYSIS. Spore forming lactose fermenters are not infrequently encountered in water. They are for the most part anaerobes resembling the CI. welchii (B. aerogenes capsulatus of Welch) or the CI. enteritidis sporogenes group of Kline, but recently spore formers capable of growing on aerobic plates have been reported and isolated by Meyers, Ewing and Ellms and Hinman and Levine. They interfere seriously with the presumptive test for the colon group. Sanitary Significance. \ery little is known as to the source, dis- tribution, or pathogenicity of the aerobic sporing types. Ellms reports their presence in feces while Ewing emphasizes that they were present in water only after heavy rains, so that they may represent soil forms. No reliable conclusion can be drawn at present as to their sanitary significance. The anaerobes may be frequently encountered, often in large numbers, in the intestinal tract of man. Kline and Houston report 30 to 2,200 CI. (enteritidis) sporogenes per c. c. in sewage, whereas in waters of good quality such forms are often absent even from large volumes. These obser- vations have sometimes led to the contention that the presence of CI. (enter- itidis) sporogenes or closely allied bacteria in water is indicative of fecal pollution. The employment of these organisms as indices of dangerous pollution appears to the author unwarranted, undesirable and impractical for, 1. They are not characteristic of the human intestine. 2. There is a very little correlation between the incidence of Sporogenes and Welchii types in water and the sanitary survey. 3. They are extremely resistant. The anaerobic lactose fermenters are verv widely distributed in nature. They are encountered in large numbers in manures from various animals, in decomposing organic materials, and in the soil. They cannot be considered distinctive or characteristic of the human intestinal tract. In surface waters, the number of spores is remarkably constant, quite independent of the degree of sewage pollution as indicated by sanitary in- spection. Thus Gumming reports "Unlike B. coli which varies many thousand percent, from several hundred per c. c. to less than 1 in 10 c. c, according to the intensity of pollution, these spores were found often in the best river water in 10 c. c. and seldom showed an average much above 4 or 5 per c. c " "Their number furnishes no clue to the degree of pollution and puri- fication as does the number of B. coli " "The generally uniform distribution of organisms of this group in surface water, even in those not highly contaminated with sewage, and with no considerable increase in polluted waters, indicates that this group is not, as has sometimes been supposed, an organism characteristic of the intestine." 92 ■3 Pathogenic for guinea pigs, pigeons and mice; less so 'or rabbits. Many strains of ow pathogenic- ity. Specific sol- uble toxin. Pathogenic foi guinea pigs, jigeons, rab- Dits, and mice. Produces speci- ic soluble tox- Pathogenic for mice and gui- lea pigs; rab- ttits relatively Unsusceptible. J*roduces specif- ic affKlutinins and soluble tox- in. 'S e 0. ^0 'e 0, 1 ■g 0. 1 a ^ ri a >,G '0 f b'c = w. . P c ■s:§ <§£ 3 c .- ^ si c 1-5 S s-i « f^ (S «■ = « i: oi D - ■3 i « g & C OJ Oj ill E.S Q^ - <-3 s 5^ I Liquefac- 1 Liquefaction tion of of Coagulated Gelatin | Serum ai — Ml IS ^ I t> £0 w — ■tj^ fl tH p. ^ 0-& 3 a i-lll -f 1 1 1 1 + + + + 1 . 1 1 Hi t-l 0. CQ j 5 2 -- — 11. 6b llll •r c =3 S E 1 Gram Stain + 1 -1- >v + 1 >-. c + 1 >> + ! + ;^ + 1 >» + 1 + a 1 + + m -h + CD i 1 II 2 «^ IT *•? p-» •" C? K C . — 05 am "i a c It Is. Eoa G c |oa G 93 Furthermore, as these anaerobes are extremely resistant they would persist in water for considerable periods beyond any possible danger of transmission of intestinal disease (as typhoid, dysentery, cholera) the or- ganisms of which (being non spore bearing) die off quite rapidly in water. Again they resist disinfection and other methods of water purification, which experience has shown efficient and sufficient for elimination of disease producing microorganisms. The presence of these spore formers in a treated water is entirely insignificant as an index of danger from ty- phoid or similar diseases. It is conceivable, however, that some members of the Welchii group may themselves be responsible for intestinal disturbances. Herter in this country, and Klein in England have considered CI. welchii as a cause of diarrhea in children. Such affections have been traced in a few instances to milk. As an index of specific infection, the determination of the Welchii group in water analysis may possibly be of value, but if employed for this purpose, it would obviously be necessary to first determine the particular species or variety which is an intestinal pathogen. The terms CI. ivelchii and CI. (enteritidis) sporogenes, as employed by water works opeartors and analysts, designate not a specific organism but the whole group of anaerobic lactose fermenters. Some of these are known to be responsible for serious wound infections but many are harmless and no specific strain has been definitely proven to be pathogenic when restricted to the intes- tinal tract. The Anaerobic Sporing Lactose Fermenting Species and Vari- eties. These are for the most part rather long rods, possessing central or subterminal spores usually slightly larger than the diameter of the cell. During the war considerable work on anaerobes was in progress and an extensive literature has developed which was carefully summarized in the Special Report No. 39 of the Medical Research Committee of England. Table LIII., taken for the most part from the foregoing report sum- marizes the characteristics of the lactose fermenting anaerobes. TABLE LIII. FERMENTATION REACTION OF LACTOSE FERMENTING SPORING ANAEROBES. Species a ea Ci 3 3 i 13 O 3 o 3 cn O 3 Q 1 3 c CI. welchii* + + + + + + — -1- - — + 1 + CI. oedematis + 4- + + + — — + — CI. chauvoei + + + + + + — — — - — CI. aerofetidum + + + + + — — — — + — — CI, butyricum + + + + — — — + — CI. multi-fermentans + + + + — + — + -f- CI. tertium + -L -f + + + + — — + + CI. sphenoides + + + + + -+- -+- •- - + -+- — *Four types have been differentiated on iniilin and glycerol fermentation. Type I. InullnJ-, glycerol_|_. Type TI. Inulin — , glycerol_|_. Type III. glycerol — . Type IV. Inulin — , glycerol — . Inulin _[_, 94 KEY TO THE MORE COMMON SPORE PRODUCING LACTOSE FERMENTING ANAEROBES. I. Gelatin liquefied (generally pathogenic). A. Coagulated serum, liquefied (non-pathogenic) 1. CI. aerofetidum B. Coagulated serum not liquefied (pathogenic) 1. Non motile 2. CI. welchii 2. Motile a. Sucrose fermented salicin not attacked 3. CI. clmuvoei h. Sucrose not attacked, salicin fermented 4. CI. oedematis II. Gelatin not liquefied (non pathogenic) A. Non motile or very faintly motile 5. CI. tertium B. Motile 1. Salicin not fermented 6. CI. hutyricum 2. Salicin fermented a. Glycerol and inulin fermented 7. CI. multifermentans b. Glycerol and inulin not fermented. 8. CI. sphenoides Creel made a very interesting and, from the standpoint of the pre- sumptive test, an important observation in his study of drinking waters on railroad trains. He found two types of anaerobes which he designates "Group A and B" respectively. "Group A" comprises very long Gram- negative bacili (6-9 microns long and about 0.5 microns in width) whose spores are very much larger than the diameter of the cell. It is particularly significant to note that this group grows very rapidly producing consider- able gas in lactose broth, under anaerobic conditions, but that no gas is evolved in the lactose bile medium. Creel's anaerobes of "Group B" were found to be non-motile, capsulated, resembling in general CI. welchii. This group will not produce gas in lactose broth unless the medium has been freshly boiled or steamed in the Arnold until all air is expelled but in or- dinary lactose bile, on the other hand, gas is formed very readily. These observations may explain the controversy as to the relative value of lactose broth and bile as a presumptive test for the colon group. In dealing with waters containing anaerobes of "Group A" lactose bile will prove more reliable whereas if "Group B" is the predominating anaerobe then lactose broth will be found to give a higher proportion of confirmed presumptive tests. Isolation of Anaerobes. Several methods have been suggested and employed for the isolation of anaerobic spore forming lactose fevmenters from water. That originally described liv Kline is as follows: Milk is inoculated with the water under examination and heated for 10 minutes at 80^ C. to destroy vegetative cells. The tube is then cooled, made anaerobic, and incubated at the body temperature. In about 24 to 36 hours, a characteristic, so-called "sporogenes" reaction will be ob- 95 served. This is described by Kline as follows: "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 color- less, 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." The method employed by Creel, which was said to be very efficient for isolation in pure culture is given herewith. Petri dishes are selected having covers considerably larger than the inner plates. Agar is poured into the inner dish, allowed to harden and then inoculated by smearing over the surface with material from a broth or lactose bile tube suspected of containing an anaerobe. The inoculated plate is then inverted into the large cover. Three grams of pyrogallic acid are placed in the cover, one c. c. saturated potassium hydroxide is inserted with a pipette, and the two dishes are immediately sealed with melted paraffin. Incubation is at the body temperature. In the Standard Methods of Water Analysis A. P. H. A. 1912, a de- tailed procedure for the detection of these anaerobes is recommended as given below: "B. spoTogenes* is indicated by a vile odor which is produced in the liver broth fermentation tubes used in the regular test for general gas form- ing bacteria. The specific tests are made as follows: 1. Inoculate various dilutions (usually 0.1, 1.0 and 10.0 c. c.) of water, or of sewage in higher dilutions, into fermentation tubes containing liver broth and incubate for 24 hours at 37° C. If B. sporogenes is pres- ent in the dilutions used, there will be vigorous gas formation, accompanied by an offensive odor, and numerous large spores will be present. 2. Transfer the entire contents of each tube showing gas plus char- acteristic odor into separate sterile Erlenmeyer flasks or large test tubes and heat to SO'^ C. for 10 minutes. 3. One (1) c. c. (not more) of broth containing sediment is with- drawn from the bottom of each of the flasks or tubes which have been heated, and is planted separately into a second set of sterile liver broth fermentation tubes and incubated for 24 hours at 37° C. after which time gas formation and characteristic odor will again be observed. Microscopic examination will reveal the presence of numerous large sluggishly motile bacilli containing spores. Usually B. sporogenes is now present in pure culture. 4. A stab culture made from this 24 hour liver broth culture into dextrose liver gelatine or nutrient gelatin will demonstrate the presence of B. sporogenes by the following characteristic growth. After 48 hours incubation at 20° C., a distinct anaerobic growth will be observed begin- *Term employed to designate the Welchli group and not a specific organism. 96 ning about two centimeters below the surface. Liquefaction will be well advanced and gas bubbles will accumulate at the top of the liqufied area. 5. In order to obtain colonies of B. sporogenes on agar plates it is necessary to transplant a few drops of broth and sediment from the second set of fermentation tubes, in step 3, into a third set of tubes and incubate for three to five hours at 37° C. After that period a distinct anaerobic growth will be observed in the closed arm, and a few bubbles of gas will be seen at the top. The B. sporogenes is now in the vegetative state and this ^ is the only condition in which it will grow on the plates. The contents of the closed arm are transferred to the open bulb by tilting forward, and plated in dilutions of 1.0 to .00001 c. c. on dextrose liver agar, and incubated for 12 to 18 hours in hydrogen at 37*^ C. Typical colonies will then be visible consisting of one or more gas bubbles sur- rounded by a delicate white fringe. The plate cultures also have a dis- agreeable cheesy odor. 6. From one of these typical colonies a deep stab culture is made into dextrose liver agar and incubated for 24 hours. A distinct anaerobic growth will be observed along the line of puncture and sometimes the agar is split into two or three layers by the gas evolved. 7. A sub-culture may also be made into litmus milk and incubated for 48 hours, anaerobically, after which time there will be a complete separation of curd and whey and a strong odor of butyric acid. Some- times the curd adheres to the sides of the tubes and has a peculiar shredded appearance." It should be emphasized that none of the foregoing methods will yield all the sporing lactose fractors. Detailed procedures for isolation of specific forms together with the special media necessary are described in the English Report on Anaerobic Bacteria and Infections, to which ref- erence has already been made. The Aerobic Sporing Lactose Fermenters, Meyers, in 1918, isolated a spore forming lactose fermenter capable of growing on the sur- face of Endo agar, from the water supplies of Newport and Covington, Kentucky and from tannery wastes. The organism is Gram negative, grows readily on Endo agar, pro- ducing a red colony in 24 hours, which shows a distinct metallic luster after 48 hours, and in Clark and Lubs medium it is alkaline to Methyl Red and positive for the Voges Proskauer Reaction. The more important characteristics as detailed by Meyers are: Agar slant. Growth quite distinctive. At 37^ C., in 24 hours, thin transparent veil-like growth over entire surface except the very top. Growth lobate along upper edge. Microscopically — in 24 hours mainly vegetative forms, in 48 hours spore-bearing forms and later only free spores. Endo's plates at 37^ C. In 24 hours, colonies pink with red center, irregular contour, one to two m. m. diameter, little or no sheen. Colonies 48 hours, deep red, much sheen in colonies and surrounding medium. Lat- ter point distinctive. 97 Gelatin stab at 20° C. In 48 hours, beginning liquefaction; in 72 hours liquefaction infundibuliform, slight precipitate. Carbohydrates. In standard extract broth to which has been added one percent of the following carbohydrates, acid and gas are formed: (1) glucose, (2) laevulose, (3) raffinose, (4) maltose, (5) sucrose, (6) lactose, (7) inulin, (8) starch, (9) glycerol, (10) mannitol. No acid or gas and little growth in dulcite broth, which remains clear and limpid. In other broths gas usually appear in 24 hours. Media uniformly clouded, slight stringy precipitate, no pellicle. Media 48 hours, slightly viscous. Clark and Lubs. Typical reaction of 'Grain' type coli, in 48 hours at 37*^ C, i. e. reaction alkaline to methyl red, Voges-Proskauer test posi- tive. Indol production in 1 percent peptone, four days at 37*^ C. No indol detected when tested for by the nitrite and by Ehrlich's para-dimethyl- amido-benzaldehyde method. Glucose-neutral-red broth. Same reaction as Bad. coli i. e. yellow fluorescence with gas formation. Litmus milk at 37'-' C. In 24 hours acid, in 48 hours partially reduced, coagulated with extrusion of whey; beginning digestion of curd. Lactose bile at 37° C. In 96 hours, no gas or growth. Chromogenesis. None. Ewing, in 1919, reported a similar bacillus in the water supply of Baltimore and noted that its presence was associated with heavy rainfall. TABLE LIV. ELIMINATON OF SPURIOUS PRESUMPTIVE TESTS. (After Hall and Ellefson 1918) Series Gentian violet concentration Positive presumptive tests Colon group present % Presumptive tests confirmed A None 1-100,000 21 20 12 15 57.1 75.0 B None 1-20,000 44 33 26 27 59.1 81.8 C None 1-20,000 85 74 58 61 68.2 82.4 D Samples heated 60O.C 30 min. None 1-20,000 81 5 3 1 3.7 20.0 Inhibition of Growth of Spore Formers. Hall has suggested the use of gentian violet in the lactose broth presumptive test tube to eliminate spurious presumptive tests. In a series of examinations with water and pure cultures, he found that 1-20,000 to 1-100,000 gentian violet in lactose broth exerted but little inhibitory influence on the growth of Bact. coli; whereas the anaerobes were almost completely checked. Table LIV. shows the marked inhibitory action of gentian violet on the anaerobes. Not only are spurious presumptive tests eliminated but the total of successful isolations of colon bacilli is increased. This 98 would indicate that the presence and growth of the Welchii group is detri- mental to the successful isolation of Bact. coli. This work of Hall and Ellefson has recently been confirmed by Wagner and Monfort. Ellms (1919) reports sporing lactose decomposing aerobes from the water supply of Milwaukee, and the feces of children. These strains differ from those of Meyers and Ewing in that they are Gram positive and are acid to methyl red. The importance of these aerobic spore formers to the bacteriologist and engineer is apparent. Being much more resistant than the non-sporing colon group, they would naturally survive the ordinary methods of water treatment, and as they are capable of growing aerobic- ally, they may be mistaken for Bact. coli, Bact. aerogenes, etc., in the or- dinary routine examination. Their presence in a water may conceivably account for the apparently poor results sometimes obtained in purification processes. Muer and Harris, of the Mount Prospect Laboratory, observed that in lactose peptone bile a concentration of 1-700 to 1-1000 brilliant green would not appreciably affect the volume of gas produced by Bact. coli in seven days, whereas CI. ivelchii would not produce gas until the brilliant green was diluted to 1-50,000. This observation is quite remarkable for, as is well known, brilliant green is frequently used to inhibit Bact. coli. The writer has observed a dilution of even 1-2,000,000 has a marked inhibitory effect on the rate of growth of Bact. coli in peptone water. Their results, however, are very distinct and significant. Probably the bile reacts in some way with brilliant green. It is well known, for example, that in eosin brilliant green agar a much higher concentration of brilliant green can be employed without affecting Bact. typhi than in the Andrade brilliant green medium of Krumwiede. The following table shows very clearly the in- hibitory action of brilliant green on the anaerobic spore formers. It ap- pears also that the growth of the anaerobes interferes with the isolation of Bact. coli. In 17 samples Bact. coli was isolated when brilliant green lactose bile was employed but was missed when plain lactose peptone bile was used. TABLE LV. COMPARISON OF 115 SAMPLES OF WATER PLANTED IN BOTH PLAIN LACTOSE PEPTONE BILE AND BRILLIANT-GREEN LACTOSE BILE. (After Muer and Harris 1920) Medium Number of dilutions from which Bact. coli was isolated Number of dilutions from which CI. welchii was isolated Plain lactose-peptone bile 34 18 Brilliant-green lactose bile 51 The chief interest of the water bacteriologist in these spore formers is that the anaerobes interfere and confuse the presumptive test rendering confirmation necessary, while the aerobic forms complicate the confirmatory test as well, making it essential to resort to more detailed identification tests where there is reason to suspect this type present. 99 APPENDIX A. ROUTINE METHODS OF WATER ANALYSIS AND THE COLON INDEX. Although the American Public Health Association has been issuing standard methods of water analysis for some fifteen years, there is still a marked lack of uniformity in the methods employed in different labora- tories. Thus Norton (1918), in a tabulation of 23 laboratories, found that 13 employed lactose broth, 8 lactose bile, 1 lactose and dextrose broth, and 1 lactose agar and dextrose broth for primary inoculation or prelim- inary enrichment. The most commonly employed routine methods are given here. I. The Treasury Department Standard for the Examination of Water on Interstate Common Carries. The following method for the examination of water on Interstate common carriers has been formu- lated by a committee of prominent sanitarians. The permissible limits of bacteriological impurity are stated as follows: 1. The total number of bacteria developing on standard agar plates, incubated 24 hours at 37° C, shall not exceed 100 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 10 c. c. portions of any sample ex- amined shall show the presence of organisms of the Bacillus coli group when tested as follows: (a) Five 10 c. c. portions of each sample tested shall 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 37^ C. and observea to note gas formation. (b) From each tube showing gas, occupying more than five per- cent of the closed arm of the 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° C. for 48 hours. For the purpose of enforcing any regulations which may be based upon these recommendations the following may be considered sufficient evidence of the presence of organisms of the 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 (b). The formation of gas, occupying 10 percent 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's medium plate. 100 These steps are selected with reference to demonstrating the presence in the samples examined of aerobic lactose-fermenting organisms. 3. It is recommended, as a routine procedure, that in addition to five 10 c. c. portions, one 1 c. c. portion, and one 0.1 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 pollution in grossly polluted samples. 4. It is recommended that in the above-designated tests the culture media and methods used shall be in accordance with the specifications of the committee on Standard Methods of Water Analysis of the American Public Health Association, as set forth in "Standard Methods of Water Analysis" (A. P. H. A., 1912). II. English Procedure (After Savage). Add 0.1 and 1.0 c. c. of water respectively to tubes of lactose bile salt broth in double tubes. Add 10 c. c. to a similar tube, but containing lactose bile salt broth of double strength. To the remainder in the bottle, after all the different amounts of water have been withdrawn for the different parts of the examination, add the contents (about 10 c. c.) of a tube of four times strength neutral red broth. Replace the glass stopper. Four times strength bile salt broth may be used, and, if the examination is for B. coli alone, is preferable, but by using neutral red broth the mixture is also avail- able for the examination for streptococci. If a 2-ounce sample is collected, the amount remaining in the bottle will be about 30 c. c. If a large sample of water is collected, then 50 c. c. should be added by sterile pipette to a tube of four times strength neutral red broth large enough to hold the added water. The tubes are labeled, incubated at 37'' C, and examined after 24 and after 48 hours. If the 0.1, 1.0, and 10 c. c. tubes show no gas after 48 hours, it can be assumed that B. coli is absent in these amoimts. Then, in every case, the larger amount (i. e. the 30 c. c. in the bottle) should be examined for this organism. The alteration of the red color to yellow, with the presence of fluorescence, is an indication of the probable presence of B. coli. If gas is present in the tubes containing smaller amounts, use the one showing gas in the tube with the least quantity of added water for in- oculating plates of solid media. In this way it can be definitely ascertained whether B. coli is present or absent in 50 c. c. or less, and if present, approximately in what numbers. To isolate the B. coli group organism, a trace of the positive tube selected is distributed over the surface of a plate containing neutral red lactose bile salt agar (L. B. A.), fuchsin agar or some other medium se- lected. L. B. A. is recommended as most suitable. Several colonies should be subcultivated and worked out. Subcultivation upon or in the following five media is recommended for routine work, i. e. : (a) Gelatine slope (for morphology, motility, cultural appearance, and liquefaction). 101 (b) Litmus-milk at 37° C. (c) Lactose-peptone litmus solution (in a double tube). (d) Peptone water (for indol production). (e) Saccharose peptone litmus solution (in a double tube). in. American Public Health Association. Standard Method. The 1920 report of the Committee on Standard Methods of Water Analysis of the American Public Health Association suggested the following: It is recommended that the B. coli (colon)* group be considered as including all non-spore-forming bacilli which ferment lactose with gas for- mation and grow aerobically on standard solid media. The formation of 10 percent or more gas in a standard lactose broth fermentation tube within 24 hours at 37^ C. is presumptive evidence of the presence of members of the B. coli group, since the majority of the bacteria which give such a reaction belong to this group. The appearance of aerobic lactose-splitting colonies on lactose-litmus- agar or Endo's medium plates made from a lactose-broth fermentation tube in which gas has formed confirms to a considerable extent the presumption that gas-formation in the fermentation tube was due to the presence of members of the B. coli group. To complete the demonstration of the presence of B. coli as above defined, it is necessary to show that one or more of these aerobic plate colonies consists of non-spore-forming bacilli which, when inoculated into a lactose-broth fermentation tube, form gas. It is recommended that the standard tests for the B. coli group be either (a) the Presumptive, (b) the Partially Confirmed, or (c) the Com- pleted test as hereafter defined, each test being applicable under the circumstances specified. A. Presumptive Test. 1. Inoculate a series of fermentation tubes with appropriate graduated quantities of the water to be tested. In every fermentation tube there must always be at least three times as much medium as the amount of water to be tested. When necessary to examine larger amounts than 10 c. c. as many tubes as necessary shall be inoculated with 10 c. c. each. 2. Incubate these tubes at 37*^ C. for 48 hours. Examine each tube at 24 and 48 hours, and record gas-formation. The records should be such as to distinguish between: (a) Absence of gas-formation. (b) Formation of gas occupying less than 10 percent of the closed arm. (c) Formation of gas occupying more than 10 percent (10%) of the closed arm. More detailed records of the amount of gas formed, though desirable for purposes of study, are not necessary for carrying out the standard tests pre- scribed. *Parenthesis author's. 102 3. The formation within 24 hours of gas occupying more than 10 per cent. ( 10 % ) of the closed arm of fermentation tube constitutes a positive presumptive test. 4. If no gas is formed in 24 hours, or if the gas formed is less than 10 percent (10%), the incubation shall be continued to 48 hours. The presence of gas in any amount in such a tube at 48 hours constitutes a doubtful test, which in all cases requires confirmation. 5. The absence of gas formation after 48 hours' incubation consti- tutes a negative test. (An arbitrary limit of 48 hours' observation doubt- less excludes from consideration occasional members of the B. coli group which form gas very slowly, but for the purposes of a standard test the exclusion of these occasional slow gas forming organisms is considered immaterial ) . B. Partially Confirmed Test. 1. Make one or more Endo's medium or lactose-litmus-agar plates from the tube which, after 48 hours' incubation, shows gas formation from the smallest amount of water tested. (For example, if the water has been tested in amounts of 10 c. c, 1 c. c, and 0.1 c. c. gas is formed in 10 c. c, and 1 c. c, not in 0.1 c. c. the test need be confirmed only in the 1 c. c. amount). 2. Incubate the plates at 37° C, 18 to 24 hours. 3. If typical colon-like red colonies have developed upon the plate within this period, the confirmed test may be considered positive. 4. If, however, no typical colonies have developed within 24 hours, the test cannot yet be considered definitely negative, since it not infre- quently happens that members of the B. coli group fail to form typical colonies on Endo's medium or lactose-litmus-agar plates, or that the colon- ies develop slowly. In such case, it is always necessary to complete the test as directed under "C" 2 and 3. C. Completed Test. 1. From the Endo's medium or lactose-litmus- agar plate made as prescribed under "B", fish at least two tvpical colon- ies, transferring each to an agar slant and a lactose broth fermentation tube. 2. If no typical colonies appear upon the plate within 24 hours, the plate should be reincubated another 24 hours, after which at least two of the colonies considered to be most likely B. coli, whether typical or not, shall be transferred to agar slants and lactose broth fermentation tubes. 3. The lactose broth fermentation tubes thus inoculated shall be in- cubated until gas formation is noted; the incubation not to exceed 48 hours. The agar slants shall be incubated at 370 q f^^ 4g hours, when a microscopic examination shall be made of at least one culture, selecting one which corresponds to one of the lactose broth fermentation tubes which has shown gas-formation. The formation of gas in lactose broth and the demonstration of non- spore-forming bacilli in the agar culture shall be considered a satisfac- tory completed test, demonstrating the presence of a member of the B. coli group. 103 The absence of gas-formation in lactose broth or failure to demon- strate non-spore-forming bacilli in a gas-forming culture constitutes a negative test. APPLICATION OF PRESUMPTIVE, PAROALLY CONFIRMED, AND COMPLETED TESTS. A. The Presumptive Test. When definitely positive, that is showing more than 10 percent (10%) of gas in 24 hours, is sufficient: (a) As applied to all except the smallest gas-forming portion of each sample in all examinations. (b) As applied to the smallest gas-forming portion in the examina- tion of sewage or of water showing relatively high pollution, such that its fitness for use as drinking water does not come into consideration. This applies to the routine examinations of raw water in connection with control of the operation of puri- fication plants. 2. When definitely negative, that is showing no gas in 48 hours, is final and therefore sufficient in all cases. 3. When doubtful, that is showing gas less than 10 percent (10%) (or none) in 24 hours, with gas either more or less than 10 per- cent in 48 hours, must always be confirmed. B. The Partially Confirmed Test. 1. When definitely positive, that is, showing typical plate colonies within 24 hours, is sufficient: (a) When applied to confirm a doubtful presumptive test in cases where the latter, if definitely positive, would have been suffi- cient. (b) In the routine examination of water-supplies where a sufficient number of prior examinations have established a satisfactory index of the accuracy and significance of this test in terms of the completed test. 2. When doubtful, that is, showing colonies of doubtful or negative appearance in 24 hours, must always be completed. C. The Completed Test. The completed test is required as applied to the smallest gas-forming portion of each sample in all cases other than those noted as exceptions under the "presumptive" and the "par- tially confirmed" tests. The completed test is required in all cases where the result of the confirmed test has been doubtful. IV. Modification of A. P. H. A. Method. The following pro- cedure, which is a modification of the A. P. H. A. method has proved very satisfactory and convenient in the hands of the author. 104 PROCEDURE FOR THE EXAMINATION OF WATER. (BACTERIOLOGICAL) Steps in procedure Further tests required A 1. Plate two 1 c. c. and one 0.1 c. c. or other ap- propriate portions of the sample on plain agar, and incubate for 24 hours at 37^ C. 2. Inoculate 10 c. c, 1 c. c. or other portions of the sample into lactose broth (or lactose peptone water). Incubate at 37" C. 3. Optional if a very rapid result is neces- sary. 8 to 10 hours after incubation perform a Preliminary Confirmatory test as follows : Divide an Eosine-methylene blue, (or Endo, or Litmus-lactose) agar plate into sectors. Streak out a drop or loop of a fermentation tube con- taining 10 c. 0. of the sample on one of the sectors, and in a similar manner streak out other fermentation tubes on the remaining sectors. Incubate at 37° C. B 1. Count agar plates made the previous day, re- cord and discard petri dishes. 2. Record presence or absence of gas after 24 hours incubation as follows: 10% or more, — Positive; less than 10%, — Doubtful; no gas, — Negative. 3. Gas formation (any amount) accompanied by a positive preliminary confirmatory test, con- stitutes what is known as a Partially Con- firmed test for the colon group. 4. If 10% or more gas is formed, and the prelim- inary confirmatory test is negative or was not made, the result is regarded as a positive pre- sumptive test for the colon group. 5. If less than 10% gas is formed, and the pre- liminary confirmatory test is negative or was not made, the result is regarded as a doubtful presumptive test, and should be confirmed. None 'D' except for sew- age or raw water 'D' C 1. If no gas was formed after 24 hours' incuba- tion, but is present after 48 hours, the test is regarded as doubtful and inconclusive. This must be confirmed. 'D' 105 PROCEDURE FOR THE EXAMINATION OF WATER. (BACTERIOLOGICAL) Steps in procedure 1. To confirm the presence of the colon group in a tube showing gas, streak out a loop of the medium onto eosine methylene blue agar (or Endo or litmus-lactose agar.) Incubate over- night at 370 C. (a) Presence of characteristic colonies on the agar constitutes a Partially Confirmed Test for the colon group. Record whether the colony resembles the coli or aerogenes sections. (b) If no growth develops on the agar plate, it is considered that the gas produced in the fermentation tube was due to anaero- bic organisms and not to coli-Iike forms. Record probably anaerobe. (c) If characteristic colonies of Bad. coli or its close allies are not present, the plates must be examined further before reporting absence of the colon group. 1. To determine whether Bad. coli or its close allies is present on a negative or questionable eosine methylene blue (Endo, or litmus-lac- tose) agar confirmatory plate. (a) Pick one or two colonies which most re- semble Bad. coli or Bact. aerogenes and plant into lactose broth (or lactose pep tone water), and incubate for 24 hours at 37° C. If 10% or more gas is formed, record colon group present. (b) A Gram stain should be made of the col- ony before inoculation into lactose broth to insure that a Gram-negative coli-like form in pure culture is being fished. Clark and Lubs medium or glucose broth may be inoculated, if desired to test for the M. R. or V. P. reaction , respectively. Further tests required None None THE COLON INDEX Estimation of the Incidence of the Colon Group. It is apparent that all agree in the use of the preliminary enrichment method for the de- termination of the presence of the colon group but a moment's thought 106 will show that it is extremely difficult to estimate the number of colon forms present from the result obtained. Perhaps the point can best be illustrated by considering a specific example. Suppose a sample of water was examined and gave the following re- sult for colon types: 1 c. c.+ ; 0.1 c. c.+ ; and 0.01 c. c— . What was the incidence of the colon group per unit volume? Should 10 colon bacilli present per c. c. be recorded? It might be said, as is commonly stated, that there were more than 10 but less than 100 colon forms, and yet it is conceivable that there might be less than 10. Assume that there were five Bact. coli per c. c. In that case, in taking out a 0.1 c. c. sample the analyst would be just as likely to catch a Bact. coli as to miss one. The mere detection of the organism in a sample is not necessarily a safe criterion for regarding the organism constantly present in that quantity of water. On the other hand, the absence of colon forms in 0.01 c. c. is no justi- fication for stating that such organisms would be absent if another 0.01 c. c. sample were taken, for if there were 50 colon bacilli present per c. c, the analyst would be just as likely to catch an organism as to miss one in a single 0.01 c. c. sample. It is apparent, therefore, that from the an- alysis presented it is extremely difficult to express by a single figure, the number of colon types present per unit volume. If, however, instead of having taken one portion of each dilution ten had been employed, a very much closer approximation to the actual number of organisms could be made. Similarly if the dilutions indicated were taken on ten different days a reasonably close estimate could then be made of the average num- ber of colon bacilli present during that period. In water works operation, and for comparison of the efficiencies of different plsmts, we are not concerned with a single analysis but with a series of analyses extending over a long period, perhaps a month or a year. A number of methods have been suggested for calculating the in- cidence of the colon group or what is known as the "Colon Index." The most commonly employed method, and the one recommended by the A. P. H. A. is that of Phelps. More complicated but probably more accurate methods are described by McCrady, Wolman and Weaver, and Stein. The Phelps method is based on the assumption that the most probable number of organisms present in any specimen is the reciprocal of the highest positive dilution; thus in the above example (1 c. c.+ ; 0.1 c. c.+ ; 0.01 c. c— ) 10 colon forms per cubic centimeter would be considered the most probable number. To obtain the colon index for a month or a year it is merely necessary to add the reciprocals of the highest positive dilutions for the individual (daily or otherwise) tests and divide by tlie total number of tests; this will give the average, but not necessarily the most probable, number for the period under consideration. An example follows: 107 Results of Daily Test Probable incidence Day 1 c. c. 0.1 c. c. 0.01 c. c. 0.001 c. c. of colon group 1 + + — 10 2 + + + — 100 3 + + — — 10 4 + + — — 10 5 + + — — 10 6 + — — 1 7 + + — — 10 Total (for estimating averages) 151 Average of 7 tests 21.6 per c. c. In 1915 McCrady proposed a method for the calculation of the most probable number of colon bacilli from a series of fermentation results but his methods have not been employed because of the cumbersome cal- culations involved. Wolman and Weaver, in 1917, simplified the McCrady formula and presented some graphs by means of which the tedious com- putations are almost entirely removed. If A=total number of tubes inoculated with 10 c. c. B=total number of tubes inoculated with 1 c. c. C=total number of tubes inoculated with 0.1 c. c. a= number of 10 c. c. tubes positive b=number of 1 c. c. tubes positive c=number of 0.1 c. c. tubes positive then X — ^the most probable number of colon bacilli per 100 c. c. — may be obtained by trial substitutions of values for x in the following equation. lOOA - lOB - 1C=- 100a 1— .9^ 10b 1— .99^ Ic 1— .999== From the charts accompanying Wolman and Weaver's paper, the values for x corresponding to any proportion of positive tests may be read off directly when dealing with a single dilution. When concerned with several dilutions the above formula must be employed, but the val- ues of (1— .9=^), (1 — .99''), and (1 — .999==) for any assumed value of X are also given on these charts so that the actual mathematics involved is reduced to merely simple arithmetic. Stein in 1918, from a consideration of the laws of probability, evolved a curve from which the colon index, together with its reliability could be read directly if (1) the number of observations (2) the proportion of positive tests, and (3) the size of the test samples, were known. For further details the reader is referred to the original papers by Stein, M. F. Journal of Bacteriology, IV, 1919, p. 243, and Wolman and Weaver, Journal of Infectious Diseases, XXI., 1917, p. 287. In the Pub- lic Health Journal (Canadian) IX., 1918, p. 201, McCrady presents a set of tables for the interpretation of fermentation-tube results, which almost completely relieve the analyst of mathematical calculations. 108 APPENDIX B.— CULTURE MEDIA Numerous mediums have been utilized in the bacteriological examina- tion of water. The preparation of the more important is described here. I. ADJUSTMENT OF REACTION OF CULTURE MEDIA (A. P. H. A. 1920) 1. Phenol Red Method for adjustment to a hydrogen- ion concentration of PH+ = 6.8-8.4. Withdraw 5 c. c. of the medium, dilute with 5 c. c. of distilled water, and add 5 drops of a solu- tion of phenol red (phenol sulphone phthalein). This solution is made by dissolving 0.04 grams of phenol red in 30 c. c. of alcohol and diluting to 100 c. c. with distilled water. Titrate with a 1:10 dilution of standard solution of NaOH (which need not be of known normality) until the phenol red shows a slight but distinct pink color. Calculate the amount of the standard NaOH solution which must be added to the medium to reach this reaction. After the addition check the reaction by adding 5 drops of phenol red to 5 c. c. of the medium and 5 c. c. of water. 2. Titration with phenolphthalein. (For the convenience of those who wish to retain the use of this method for the present it is given here, but it is recommended that as soon as possible the more accurate meth- od of determining the hydrogen-ion concentration be substituted.) In a white porcelain dish put 5 c. c. of the medium to be tested, add 45 c. c. of distilled water. Boil briskly for one minute. Add 1 c. c. of phenolphthalein solution (5 grams of commercial salt to one liter of 50 percent alcohol). Titrate immediately with a n/20 solution of sodium hydrate. A faint but distinct pink color marks the true end point. This color may be precisely described as a combination of 25 percent of red (wave length approximately 658) with 75 percent of white as shown by the disks of the standard color top made by the Milton Bradley Educational Co., Springfield, Mass. All reactions shall be expressed with reference to the phenolphthalein neutral point and shall be stated in percentages of normal acid or alkali solutions required to neutralize them. Alkaline media shall be recorded with a minus ( — ) sign before the percentage of normal acid needed for their neutralization and acid media with a plus ( + ) sign before the percentage of normal alkali solution needed for their neutralization. The standard reaction for culture media for water analysis shall be 4-1.0 percent, as determined by tests of the sterilized medium. As ordin- arily prepared, broth and agar will be found to have a reaction between +0.5 and +1.0. For such media no adjustment shall be made. The re- action of media containing sugar shall be neutral to phenolphthalein. Whenever reactions other than the standard are used, it shall be so stated. II. STANDARD STOCK MEDIA A. Nutrient Agar (A. P. H. A., 1920). 1. Add 3 grams of beef extract, 5 grams of peptone and 12 grams of agar, dried for one-half hour 109 at 1050 C. before weighing, to 1,000 c. c. of distilled water. Boil over a water bath until all agar is dissolved, and then make up the loss by evaporation. 2. Cool to 450 C. in a cold water bath, then warm to 65° C. in the same bath, without stirring. 3. Make up lost weight and adjust the reaction to a faint pink with phenol red, or if the phenolphthalein titration is used, and the reaction is not already between +0.5 and +1, adjust to +1. 4. Filter through cloth and cotton until clear. 5. Distribute in test-tubes, 10 c. c. to each tube, or in larger contain- ers, as desired. 6. Sterilize in the autoclave at 15 pounds (120° C.) for 15 minutes after the pressure reaches 15 pounds. B. Nutrient Gelatin (A. P. H. A., 1920). 1. Add 3 grams of beef extract and 5 grams of peptone to 1,000 c. c. of distilled water and add 100 grams of gelatin dried for one-half hour at 105^ C. before weighing. 2. Heat slowly on a steam bath to 65° C until all gelatin is dissolved.* 3. Make up lost weight and adjust the reaction to a faint pink with phenol red, or if the phenolphthalein titration is used, and the reaction is not already between +0.5 and +1, adjust to +1. 4. Filter through cloth and cotton until clear. 5. Distribute in test-tubes, 10 c. c. to each tube, or in large con- tainers as desired. 6. Sterilize in the autoclave at 15 pounds (120° C.) for 15 minutes after the pressure reaches 15 pounds. C. Nutrient Brotb. (A. P. H. A., 1920). 1. Add 3 grams of beef extract and 5 grams of peptone to 1,000 c. c. of distilled water. 2. Heat slowly on steam bath to at least 65*^ C. 3. Make up lost weight and adjust the reaction to a faint pink with phenol red, or if the phenolphthalein titration is used, and the reaction is not already between +0.5 and +1, adjust to +1. 4. Cool to 25*^ C. and filter through paper imtil clear. 5. Distribute in test-tubes, 10 c. c. to each tube. 6. Sterilize in the autoclave at 15 pounds (120*^ C.) for 15 minutes aftpr the pressure reaches 15 pounds. D. Media for Indol Test. (A. P. H. A., 1920). To 1,000 c. c. of distilled water add 0.3 gram tryptophane, 5 grams dipotassium hydro- gen phosphate (K,HP04), and 1 gram peptone. Heat until ingredients are thoroughly dissolved, tube (6 to 8 c. c), and sterilize in autoclave for 15 minutes after the pressure reaches 15 pounds. Some American peptones are standardized to contain a uniform amount of tryptophane. If such peptone is used the tryptophane in the above formula may be omitted and the peptone increased to 5 grams. E. Litmus Milk. (After Prescott and Winslow). 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 *The solution of the gelatin will be facilitated by allowing it to soak in the cold one-half hour before heating. 110 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 separatory 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 1 percent azolitmin. As it is impossible to make each lot of litmus milk with the same shade of color, it is recommended that a control tube be always exposed with the inoculated tubes for the purposes of comparison. III. MEDIA FOR PRELIMINARY ENRICHMENT OR THE PRE- SUMPTIVE TEST. A. Lactose Broth, (Standard Methods A. P. H. A., 1920). Sugar broths shall be prepared in the same general manner as nutrient broth with the addition of 0.5 percent of the required carbohydrate just before sterili- zation. The removal of muscle sugar is unnecessary as the beef extract and peptone are free from any fermentable carbohydrates. The reaction of sugar broths shall be a faint pink with phenol red or, if on titration with phenolphthalein the reaction is not already between neutral and +1, adjust to neutral. Sterilization shall be in the autoclave at 15 pounds (120" C.) for 15 minutes after the pressure reaches 15 pounds, provided the total time of exposure to heat is not more than one-half hour; other- wise a 10 percent solution of the required carbohydrate shall be made in distilled water and sterilized at 100° C. for IV2 hours, and this solution shall be added to sterile nutrient broth in amounts sufficient to make a 0.5 percent solution of the carbohydrate and the mixture shall then be tubed and sterilized at 100*^ C. for 30 minutes, or it is permissible to add by means of a sterile pipette directly to a tube of sterile neutral broth enough of the carbohydrate to make the required 0.5 percent. The tubes so made shall be incubated at 3T^ C. for 24 hours as a test for sterility. B. Lactose (Peptone) Bile. The lactose bile medium consists of sterilized undiluted fresh ox gall (or a 10 percent solution of dry fresh ox gall) to which has been added 1 percent of peptone and 1 percent of lactose. The addition of peptone is importcuit. C. Lactose Bile Salt Broth. (After Savage). Sodium taurocholate 5 grammes Lactose 5 grammes Peptone 20 grammes Water 1000 c. c. These constituents are heated together until the solids are dissolved. The mixture is filtered, and sufficient neutral litmus solution is added to give a distinct color. The medium is then distributed into Durham's fer- mentation tubes and sterilized by steaming for twentv minutes on three successive days. The sodium taurocholate prevents the growth of many saprophytic bacteria. Ill The presence of fermenting organisms, including B. coli is shown when the medium turns red (due to acid production) and gas is formed in the inner tube. D. Glucose Broth. Same as lactose broth, substituting glucose for the disaccharid. E. Liver Broth. (After Prescott and Winslow). 1. 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 Beef Liver 500 gm. Peptone 10 gm. Dextrose 10 gm. Di-Potassium Phosphate (KjHPOJ 1 gm. Water 1000 gm. 2. Chop 500 gm. of beef liver into small pieces and add 1000 c. c. of distilled water. Weigh the infusion and container. 3. Boil slowly for 2 hours in a double boiler, starting cold and stir- ring occasionally. 4. Make up any loss in weight by evaporation and pass through a wire strainer. 5. To the filtrate add 10 gm. of peptone, 10 gm. of dextrose and 1 gm. of potassium phosphate. 6. 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. 7. Boil vigorously for 30 minutes in a double boiler, and 5 minutes over a free flame with constant stirring to prevent the caramclization of the dextrose. 8. Make up the loss in weight by evaporation and filter through cotton flannel and filter paper. 9. Tube and sterilize in an autoclave for 15 minutes at 120*^ C. (15 pounds). The following media have been suggested for the elimination of spur- ious presumptive test: F. Gentian Violet Lactose Broth. (Hall and Ellefson). The medium consists of 1 percent lactose broth containing 1-20,000 gentian vio- let. 112 G. Brilliant Green Lacto§e Bile (Muer and Harris). The com- position of the medium used is as follows: Distilled water 1,000 grams Oxgall (dried) 50 grams Peptone 10 grams Lactose 10 grams Brilliant-green 0.1 grams Directions for Preparation 1. Heat 1 liter of distilled water in double boiler until water in outer vessel boils. 2. Add 50 grams of dried ox gall and 10 grams of peptone, stirring until all ingredients are dissolved. 3. Continue boiling for one hour. 4. Remove from flame and add 10 grams of powdered lactose. 5. Filter through cotton flannel until clear. 6. To each liter of the filtrate add 10 c. c. of a 1 percent solution of brilliant-green. 7. Tube and sterilize in autoclave for 15 minutes at 15 pounds pres- sure. IV. MEDIA FOR DIRECT ISOLATION OR CONFIRMATION OF PRESUMPTIVE TEST. A. Litmus Lactose Agar. (Wurtz Agar). (Standard Methods A. P. H. A., 1920). Litmus-lactose-agar shall be prepared in the same manner as nutrient agar with the addition of 1 percent of lactose just before sterilization. The reaction shall be a faint pink with phenol red, or, if on titration with phenolphthalein the reaction is not already between neutral and -fl, adjust to neutral. One c. c. of sterilized litmus or azolitmin solution shall be added to each 10 c. c. of the medium just before it is poured into the petri dish, or the mixture may be made in the dish itself. B. Fuchsin Sulphite (Endo) Agar. Endo agar consists of nutrient lactose agar containing basic fuchsin decolorized with sodium sulphite as an indicator. Many modifications have been described. Those more commonly employed and method of use are listed below: (a). Endo's Medium (Standard Methods A. P. H. A., 1920) 1. Add 5 grams of beef extract, 10 grams of peptone and 30 grams of agar dried for one-half hour at 105° C. before weighing, to 1,100 c. c. of dis- tilled water. Boil on a water bath until all the agar is dissolved and then make up the loss by evaporation. 2. Cool the mixture to 45° C. in a cold water bath, then warm to 65° C. in the same bath without stirring. 3. Make up lost weight, titrate, and if the reaction is not already between neutral and +1, adjust to neutral. 4. Filter through cloth and cotton until clear. 5. Distribute 100 c. c. or larger known quantities in flasks large enough to hold the other ingredients which are to be added later. 113 6. Sterilize in the autoclave at 15 pounds (120° C.) for 15 minutes after the pressure reaches 15 pounds. 7. Prepare a 10 percent solution of basic fuchsin in 95 percent alcohol, allow to stand 20 hours, decant and filter the supernatant fluid. This is a stock solution. 8. When ready to make plates melt 100 c. c. of agar in streaming steam or on a waterbath. Dissolve 1 gram of lactose in 15 c. c. of dis- tilled water, using heat if necessary. Dissolve 0.25 gram anhydrous sodium sulphite in 10 c. c. water. To the sulphite solution add 0.5 c. c. of the fuchsin stock solution. Add the fuchsin-sulphite solution to the lactose solution and then add the resulting solution to the melted agar. The lac- tose used must be chemically pure and the sulphite solution must be made up fresh. 9. Pour plates and allow to harden thoroughly in the incubator be- fore use. (b) Endo's Medium (Hygienic Laboratory Modification) . 1. The Hygienic Laboratory-Endo medium consists of a 3 percent agar which is titrated and corrected to +0.5 to phenolphthalein, to which is added 3.7 cubic centimeters of a 10 percent solution of anhydrous sodium carbonate per liter. For convenience it is flasked, sterilized, and stored in 200 cubic centimeter quantities. When ready to use, the following ingredients are added to 200 cubic centimeters of agar as follows; 2. Dissolve 2 grams C. P. lactose in 25 to 30 cubic centimeters of distilled water, with the aid of gentle heat. 3. Dissolve 0.5 gram of anhydrous sodium sulphite in 10 to 15 cubic centimeters of distilled water. 4. To the sulphite solution add 1 cubic centimeter of saturated so- lution of basic fuchsin in 95 percent alcohol. 5. Add the fuchsin-sulphite solution to the lactose solution, and then add the whole to the agar. Pour plates at once and, after hardening, dry for 15 minutes in the incubator. (c) Endo's Medium (Med. Dept. U. S. Army Modification). 1. Into a container put 1 liter of tap water, marking the level of the fluid. Add 30 grams of thread agar, 10 grams of peptone, 5 grams of NaCl, 5 grams of beef extract. Cook until dissolved — it is best to autoclave thirty minutes at 15 pounds; filter through sterile gauze or cotton, li necessary clear with egg. For this purpose, for each liter beat up the white of one egg with 10 c. c. of warm water until the egg is well mixed. Add this to agar cooled to 55° C, mix thoroughly, heat for 30 minutes or autoclave and filter through cotton. 2. This stock agar is kept on hand in quarter-liter flasks or bottles. Agar is standardized just before use and reaction adjusted to 0.2 percent acid to phenolphthalein. Before use, fuchsin and sodium sulphite are added. A filtered, saturated solution of basic fuchsin in 95 percent alcohol is kept on hand. A 10 percent solution of dry sodium sulphite crystals in sterile water is freshly made. 114 3. Teague has shown that a 10 percent solution of crystalline sodium sulphite can be heated for twenty minutes at 15 pounds pressure with prac- tically no change, and that the 10 percent sodium sulphite solution covered with a layer of liquid petroleum about one cm. thick and sterilized in the autoclave can be kept at room temperature for three weeks and probably much longer with but very slight change. 4. One and eight-tenths c. c. of fuchsin solution is added per liter to the agar. After this has been done the sodium sulphite solution is added gradually until the hot agar is almost decolorized — usually about 25 c. c. to the liter. A pale rose color should be present in the hot agar, which fades to a very faint pink on cooling; 10 grams of lactose is dis- solved in a little water, filtered and added to each liter. Various fuchsin solutions may differ and the absolute quantities given above may not be exactly the proper balance in separate lots. These are approximate, however, and the proper balance can easily be attained by a little preliminary testing in which sodium sulphite solution is added to small quantities of fuchsin solution in a test-tube. The finished product is poured into large sterile Petri dishes. The cover is left off until the agar is hard. Smears are made on these plates. It is helpful to lay a piece of filter paper into the lid of the petri plate in order to absorb liquid evaporating from the agar in the incubator. If there is not enough filter paper for this, the plate should be placed up- side down in the incubator. (d) Endo'8 Medium (Kendal's Modification). (1) Preparation of Agar. — (a) Prepare plain, sugar-free nutrient agar, using 15 grams of agar per liter. (b) Adjust the reaction to a point just alkaline to litmus. (c) Flask the agar, 100 c. c. to a flask, and sterilize in the autoclave. (2) Preparation of Indicator — (a) Prepare a 10 percent solu- tion of basic fuchsin in 96 percent alcohol. This solution is fairly stable if kept away from light. (b) Prepare a 10 percent aqueous solution of chemically pure anhy- drous sodium sulphite (1 gram in 10 c. c. water). This solution does not keep. (c) Add 1 c. c. of "2, a" to 10 c. c. of "2, b" and heat in the Arnold sterilizer for 20 minutes. The color of the fuchsin is nearly dis- charged if the solutions are of proper strength. This solution must be prepared each day — it does not keep. (3.) Preparation and Use of Endo medium — (a) Add 1 gram of C. P. lactose (free from dextrose) to 100 c. c. of agar and place in the autoclave until melted and the lactose is thoroughly dissolved. (b) Add a sufficient volume of "2, c" (about 1 c. c.) to impart a faint pink color to the medium. (c) Pour into sterile Petri dishes and allow to harden in a dark place with the covers partly removed. When cool the medium should be color- less when viewed from above and a very faint pink when viewed from the 115 edge. The medium must be kept in a dark place because the color is re- stored by the action of daylight. (e) Endo's Medium (Robinson and Rettger's Modification). Water 1,000 c. c. Agar (powdered) 25 grams Peptone (American brand) 10 grams Meat extract (Liebig's) 5 grams Sodium carbonate (10% sol.) 10 c. c. Lactose, c. p 10 grams Fuchsin (sat, alcoh. sol.) 5 c. c. Anhydrous sodium bisulphite (10% sol.) 10 c. c. Dissolve the agar, meat extract, and peptone. Make neutral to litmus paper, steam in the autoclave at 12 to 15 pounds extra pressure for 35 to 40 minutes, filter through absorbent cotton and cheesecloth, add the sodium carbonate solution, and heat for about 10 minutes in a boiling water bath. Introduce the lactose and the fuchsin into the hot liquid. The medium will now be brilliant red. Finally add the bisulphite solution. The hot medium is light red in color, is filled into large test-tubes, 20 cubic cen- timeters in each tube, and sterilized for five to seven minutes at 10 pounds extra pressure. When the medium has cooled completely it should be of a light pink or flesh color in the tubes, but transparent and practically colorless in the large Petri dishes. The tubed agar may be kept for sev- eral weeks in the refrigerator. (f) A Simplified Endo's Medium (Levine) The medium is pre- pared as follows: Distilled Water 100 c. c. Peptone (Difco) 10 grams Dipotassium phosphate (K2HPO4)- 2 to 5 grams Agar... 15 to 30 grams The ingredients are boiled until dissolved and any loss due to evapora- tion is made up with distilled w^ater. No adjustment of reaction is made and filtration is not necessary if the medium is to be used for streak plate cultures. Measured quantities are placed in flasks or bottles and sterilized for 15 minutes at 15 pounds. For use, the agar prepared as above is melted and the following ma- terials added to each 100 c. c. of medium. 20 percent lactose solution. 1 gram or 5.0 c. c. 10 percent (saturated) alcoholic solution of basic fuchsin 0.5 c. c. Freshly prepared 10 percent sodium sulphite solution 2.5 c. c. Plates are poured, allowed to harden in the incubator, and inoculated in the ordinary way. (g) Fuchsin (Endo) Agar (Savage's Modification). 1. Peptone, 10 grams; Liebig's extract of beef, 10 grams; sodium chlorid, 5 grams, are boiled up in an enamelled dish with 1 liter of distilled water. The mix- ture is then poured into a flask, 30 grams of powdered agar added, and the whole heated in the autoclave at 115° C. for one hour. The flask is 116 removed, and, after cooling to about 60^ C, the white of one egg mixed with a little distilled water is added. The contents are coagulated by heating in current steam in the usual way, filtered, and the filtrate made up to 1 liter. The mixture is made neutral, litmus paper being used as the indicator. Then 19 c. c. of normal sodium carbonate solution and 10 grams of chemically pure lactose are added. The flask is replaced for 30 minutes in the steam sterilizer. Almost invariably there is a consider- able precipitate, and the mixture has to be again filtered. 2. Seven c. c. of the fuchsin solution (see below) are added, fol- lowed by 25 c. c. of a quite freshly prepared 10 percent sodium sulphite solution. The mixture becomes much less red, but is not immediately decolorized. It is then tubed, conveniently into small flasks, each con- taining 50 to 60 c. c. of media, and sterilized in current steam for two days, 30 minutes each day. 3. The fuchsin solution is made as follows: Three grams of pow- dered crystalline fuchsin are placed in a dry flask, and 60 c. c. of abso- lute alcohol are added. The contents are thoroughly mixed, and the flask, tightly stoppered, allowed to stand for exactly 24 hours at 20° to 22° C. The alcoholic extract is then decanted and preserved in a clean glass-stop- pered bottle. Made in this way a uniform fuchsin extract is obtained which keeps well, and the same quantity of fuchsin is added each time a fresh batch of medium is prepared; a matter of much importance. The medium must be stored in the dark, since light gradually turns it red. When solidified it is almost free from color. (h.) Conradi-Drigalski Agar (After Prescott and Winslow) These authors have modified lactose litmus agar by adding to it nutrose and crystal violet and by using three percent of agar instead of one percent. The crystal violet strongly inhibits the growth of many other bacteria, es- pecially cocci, which would also color the medium red; the 3 percent agar makes the diff^usion of the acid which is formed more difficult. Three pounds of chopped beef are allowed to stand 24 hours with two 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 three 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 10 percent soda solution. To this feebly alkaline mixture 4 c. c. of hot sterile 10 percent soda solution are added and 20 c. c. of a sterile solution (1 to 1000) of crystal violet (Hochst B.). (i) Bile Salt (Rebipel) Agar (After Savage). Sodium tauro- cholate 5 grams, Witte's peptone 20 grams, and distilled water 1 liter, are boiled up together, 20 grams of agar are added and dissolved in tlie solution in the autoclave in the ordinary way. The medium is cleared with white of egg and filtered. After filtration, 10 grams of lactose and 5 117 c. c. of recently prepared 1 percent neutral red solution are added. The medium is then tubed and sterilized for 15 minutes on three successive days. (j) Aesculin Agar. (After Eyre) (B. coli and allied organisms give black colonies surrounded by black halo.) Measure out 400 c. c. distilled water into a tared 2-liter flask. Weigh out Agar 15 grams Peptone 10 grams Sodium taurocholate 5 grams and make into a thick paste with 150 c. c. distilled water. Add this paste to the distilled water in the flask. Dissolve the ingredients by bubbling live steam through the mixture. Weigh out Aesculin 1.0 gram Ferric citrate 0.5 gram and dissolve in a second flask containing 100 c. c. distilled water. Mix the contents of the two flasks — adjust the weight to the calculated medium figure (in this case 1031.5 grams) by the addition of distilled water at 100° C. Clarify with egg and filter. Tube and sterilize as for nutrient agar. (k) A Simplified Eosine Methylene Blue Agar. (Levine) Distilled water 1000 c. c. Peptone (Difco) '.. 10 grams Dipotassium phosphate (KjHPCi) 2 grams Agar 15 grams Boil ingredients until dissolved and make up any loss due to evapora- tion with distilled water. Place measured-quantities (100 or 200 c. c.) in flasks or bottles, and sterilize in the autoclave at 15 pounds pressure for 15 to 20 minutes. Just prior to use add, to each 100 c. c. of the melted agar prepared as above, the following: Lactose, sterile 20% solution 5 c. c. or 1 gram dry substance Eosine yellowish 2% aqueous sol 2 c. c. Methylene-blue 0.5% aqueous sol 2 c. c. Pour medium into perti dishes, allow them to harden, and inoculate by streaking on the surface. There is no adjustment of the reaction and filtration of the medium is not necessary. V. SPECIAL MEDIA FOR DIFFERENTIATION OF THE COLI AND AEROGENES SECTIONS. A. Clark and Lubs Medium. (Standard Methods A. P. H. A., 1920). 1. To 800 c. c. of distilled water add 5 grams of Proteose- Pep- tone Difco., or Witte's Peptone (other peptones should not be substituted), 5 grams c. p. dextrose, and 5 grams dipotassium hydrogen phosphate 118 (K2HPO4). A dilute solution of the KjHPO^ should give a distinct pink with phenolphthalein. 2. Heat with occasional stirring over steam for 20 minutes. 3. Filter through folded filter paper, cool to 20*^ C. and dilute to 1,000 c. c. with distilled water. 4. Distribute 10 c. c. portions in sterilized test tubes. 5. Sterilize by the intermittent method for 20 minutes on three suc- cessive days. B. Synthetic Medium. {After Clark and Lubs). (Standard Methods A. P. H. A., 1920). 1. NajHPO^ (anhydrous) 7 grams or Na^HPO,. 2H,0 8.8 grams KHphthalate 2 grams Asparatic acid 1 gram Dextrose 4 grams Warm distilled water 800 c. c. 2. When solution is complete, cool and make up to 1 liter at room temperature. 3. Heat in an autoclave for 15 minutes after the pressure has reached 15 pounds, provided the total time of exposure to heat is not more than one-half hour. 4. The hydrogen-ion concentration of the medium is fixed bv the composition. It should be very close to PH 7.0, slightly red with phenol red. All materials should be re-crvstallized or if used from stock fur- nished by manufacturers, should be carefully examined. The di-sodium hydrogen phosphate may be used either as the anhydrous salt obtained by dessication in vacuo at 100° C. or else as the salt containing two mole- cules of water of crystallization. This is obtained by exposing the re- crystallized Na2HPO,12H,0 for two weeks. Use 0.88 percent of Na,HPO, 2H2O. " ^ " C. Uric Acid Medium (Koser) Distilled ammonia-free water 1,000 c. c. NaCl 5.0 gm. MgS04 0.2 gm. CaCl., 0.1 gm. K^HPO, 1.0 gm. Glycerol 30.1 gm. Uric acid 0.5 gm. This combination gives a colorless and clear medium. It is filled into ordinary test tubes and sterilized in the autoclave at 13 to 15 pounds extra pressure for 15 minutes. A slight turbidity may be apparent after auto- claving, due, presumably, to a finely divided precipitate of calcium sul- phate. On cooling, the solution becomes clear. On the addition of 1.5 percent of washed shred agar to the solution mentioned in the foregoing an agar medium was obtained on which the same distinction between the two types may be brought out. 119 REFERENCES 1. Archibald, R. G., 1917. Lactose fermenting bacilli in surface water, feces, etc. Wellcome Tropical Research Laboratories, 4tli Report, 319. 2. Ayers, S. H., Rupp., 1918. Simultaneous acid and alkaline bacterial fermenta- tion from dextrose and the salts of the organic acids, respectively. J Infect Dis., XXIII., 188. 3. Barber, M. A., 1908. The rate of multiplication of B. coli at different tempera- tures. J. Infect. Dis. V., 379-400. 4 Bartow, E., 1916. 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III., 231-252. 168. Rogers, L. A., 1918. The occurrence of diiferent types of the colon-aerogenes group in water. J. Bact. III., 313-28. 169. Russell, H. L. and Fuller, C. A., 1906. The longevity of Bacillus typhosus in natural waters and in sewage. J. of Infect. Dis. Supplement No. 2, 40. Also Trans, of the Am. Pub. Health Assn. 1905 Meeting 31, Part II. 170. Salter, Raymond C, 1919. Observations on the growth of B. coli. J. Infect. Dis. XXIV., 260-84. 171. Savage, W. G., 1905. Bacteriological examination of tidal mud as an index of pollution of the river. J. Hyg. V., 146. 172. Savage, W. G., 1916. Bacteriological examination of food and water. University Press, Cambridge, England, 2nd Ed. N. Y., G. P. Putman's Sons, 200 pp. 173 Savage W G. and Wood, D. R., 1917. The vitality and viability of Streptococci in water. J. Hyg. XVI., 227-239. 174. Sawin, L. R., 1907. Experience with lactose-bile medium for the detection of B 'coli in water. J. Infect. Dis. Supplement No. 3, 33. 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IV., 927-946. 188. Twort, F. W., 1907. The fermentation of glucosides by bacteria of the typhoid- coli group and the acquisition of new fermenting powers by bacillus dysen- teriae and other microorganisms. Proc. Roy. Soc. Series B, LXXIX., 329. 189. Voges and Proskauer, 1898. Beitrag zur Ernahrungs physiologic und zur differ- ential diagnose der Bacterien der hamorrhagishen septicamie. Ztschr. f. Hyg. XXVIII., 20. 190. Wagner, E. A., and Monfort, W. F., 1921. Lactose broth for isolating Bact. coli from water. 191. Walpole, S. G., 1910-11. The action of B. lacds aerogenes on glucose and man- nitol. The effect of free oxygen on their production. The action of B. lactis aerogenes on fructose. Proc. Roy. Soc. (B), LXXXIH., 272. 192. West, F. D., 1909. Notes on the Voges and Proskauer reactions for Bacillus coli communis. Am. J. Pub. Hyg. XIX., 227. 193. *Whipple, G. C, 1903. On the practical value of presumptive tests for Bacillus coli in water. Technol. Quart. XVI., 18. 194. Winslow, C. 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Studies on the classi- fication of the colon typhoid group of bacteria with special reference to their fermentative reactions. J. Bact. IV., No. 5, 429. 201. Wolman, Abel, 1917. The quality of water and confirmatory tests for B. coli. J. Am. Water Works Assoc, IV., 200-5. 127 202. Wolman, A. and Weaver, H. L., 1917. A modification of the McCrady method of the numerical interpretation of fermentation-tube results. J. Infect. Dis. XXL, 287-91. 203. Wood, Denys R., 1919. Studies in the bacteriology of water. Inst, of Water Engin., London. 204. Wood, Denys R., 1919. Recent advances in the differentiation of lactose fer- menting bacilli with special reference to water and food products. J. Hyg. XVnL, 46-58. 205. Yule, U., 1916. An Introduction to the Theory of Statistics. London. *Origiiial articles not available. PRESS OF THE NEWS PRINTING CO. NEWTON, IOWA BULLETINS OF THE ENGINEERING EXPERIMENT STATION *No. 1; The Iowa State College Sewage. Disposal Plant investigations. *No. 2. Bacteriological Investigations of the Iowa State College Sewage. *No., 3. Data of Iowa Sewage and Sewage Disposal. *No. 4, Bacteriological Investigations of th^ Iowa State College Sewage, Disposal PJant. *No, 5 , The Chemical' Composition of the Sewage of the Iowa State College Sewage Dis- - posal Plant. ' ■ , *No. 6. Tests of Iowa. Common Brick. ' , , *No. 7. Sewage Disposal in ' Iowa. , . ' *No. 8. Tests of Dry Pressed Bricjc Used in -loWa. ' *No. 9. 'Notes on Steam Generation ,with lowa-^ Coal. *No. 10. Dredging by the Jlydraulic Method. *No. 11. , An Investigation i of Some Iflwa Sewage Disposal Systems. *Vol. II, Nfl. 6. The Good Roads Problem in Iowa. *Vol. Ill, No. 1. Tests of Cement. \*Vol.JII, No. 2. State Railroad Taxation. • .,_ , *Vol. Ill, No. 3. Sl;eamv' Generation with Iowa Coal. *'Vol. Ill, ^No. 4. Incandescent Lam^ Testing. )■ 'Vol. Ill, No. 5. Steam Pipe Covering Tests. *Vol. Ill, No.' 6. The Assessment of Drainage. Districts. *Vol. IV. No, 1. Testa of Iowa Limes. *Vol. IV, No. 2. Holding Power of Nails in Single Shear. , < *Vol. IV. No. 3. Miracle Contest Papers for 1908. (Theses on Oemefit and Concrete.) *VolT rv. ^N'o. 4. Miracle Prize Pa-pers' for 1909. (Theses on Cement and Concrete.) *Vol. IV, No. 5. Sanitary Examination of Water Supplies. * Vol. IV, No: 6 Sewage Disposal JPlamts -for .Private Houses. No. 25. Electric Power- on, the Farm. .^-^ No. 26. The PrSd'uctlon' of' ExcesslvefHydrogen- Sulfid in Sewage Disposal plants and Consequent Disintegration of the 06norete. No. 27. A Study of Iowa ^Population as Belated to Industrial Conditions. No. 28. History -of Road Legislation in Iowa. No. 29. Cost of Producing Power with Iowa Ooali. No. 30. The Determination of Internal Temperature Range in Concrete Arch Bridges. *No. 31. — ^The Theory of Loads on Pipes in Ditches, and Tests of Cement and Clay Drain 1 , , Tile and Sewer Pipe. Mo. 32. -A Toporaraphieal Survey of the Spirit and Okoboji L^kes Region. No. 33. - SottSe Heating: Eu^l Tests. ■ /: No. 34. The Use of Iowa Gravel for Concrete. ' ' Noi 35. the Iowa Engineering Experiment Station and its Service to the -Industries ol ■ the Stajte. , " ""' :.'""'•' No. 36. Report of the Investigations on Drainl Tile of Committee 0-6, Americaii Society lor Testing Materials. - No. 37. Illuminating Power of Kerosenes. No. 38. Slectric Ceptral Station Operation in Iowa. No. 39. ^Gbod Roads" 4ind Community Life. ^ ^ *No, 40. An Investigation of lowafPire Clays. Nq. 41. Sewage Disposal for Village^ and Rural Homes. rS'o^ 42. A Study of: Oil Engines In Iowa Power Plants. - No. 43. - Practical Handling of low^ Clays. V No. 44. Locomotive Tests with' lowi and Illinois Coals. %_ .«^- No. 45. Inyestigations of^ Gravel for Road Surfacing.' .-- i .j - No. 46. Electric Pumping/ with Results of Tests and Operating Reeprds. No. 47. The Supporting Strength of Sewer Pipe in Ditches, and Methods of Testing 'Sewer^ Pipe in Laboratories, to Deterniine their Ordinary Supporting Strength. No. :48i- The Early Purchase and Storage lit Iowa Coal. '~ . No. 49. An Investigation of Tests of Iowa Shale Drain Tile. , " No. 50. The -Theory of Underdraina«e. ' No. 51. -Recommendations for Farm Drainage. ' - No. 52. The Sjiacing and Depths of Laterals in Iowa Underdrainage Systems and the ' - ' ' '. Bite of Runoft from Them. ' '' - No. 53. Load Concentrations- on» Steel Floor-Joists 6t Wood-Floor Highway Bridges. No. ,54. ' Ah Investigation *tof the Proteptive Values' bf Structural Steel Faint's. No. 55. Lighting for Country Homes and Village Communities. I I No. 66. Traffic on Io*a Highways. . No. 57, , Supporting Strength of Drain -Tile and Sewer Pipe Under Different Pipe- Laying' Conditions. - , ' ^ , ■ No. 58. - Possibilities of Pottery Manufacture 'from Iowa Clays. ; No. 59. Effects on -Concrete of -Immersion in Boiling Water and Oven Drying. No. 60. Method of Proportioning, Concrete Materials — Screened and Unscreened Gravel. No. 62, Bacterid Fermenting Iiactose and Their Signifiosnco in Water Analysis. *-Out of print. _.- Bulletins not out of print ipay be obtained free of charge- upon, request addressed to The Director, Engineering Sxperime'nt Station,. Sta. A., Ames, Iowa. The College The Iowa State College of Agricultuie and Mechanic Arts con- ducts woik along five maj'oi lines: AGRICULTURE ENGINEERING HOME ECONOMICS INDUSTRIAL SQENCE VETERINARY MEDICINE The Graduate College' conducts advanced- research and instruc- tion in all these five lines. - Four, five and six-year collegiate courses are offered in differ- ent divisions of the College. Non-collegiate courses are offered in agriculture, engineering and home economics. Summer Sessions in-./ elude graduate, collegiate and non-collegiate~ work. Short courses are j)ffered in the winter. Extension courses are conducted at various points throughout the state. Research work is conducted in the Agricultural and Engineering Experiment Stations and in the Veterinary Research Laboratory. Special announcements of the different branches oj the work are supplied, free of charge, on application. The general bulletins will "^be sent on request. Address, The Registrar, ■ IOWA STATE COLLEGE, Ames, Iowa