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I m = ^^^=^ CO ^^° CO M ^ *. o "—e o o - CO =^^o .^ W 05 ■>! u o J^ Do not deface books by marks and writing. ENVIRONMENTAL STUDIES OF • STREPTOCOCCI WITH SPECIAL REFERENCE TO THE FERMENTATIVE REACTIONS A THESIS Presented to the Faculty of the Graduate School OF Cornell University for the Degree of DOCTOR OF PHILOSOPHY KY JEAN BROADHURS.T Reprinted from The Journal of Infectious Diseases, Vol. xvii, No. 2, September, 1915 ^ ENVIRONMENTAL STUDIES OF STREPTOCOCCI WITH SPECIAL REFERENCE TO THE FERMENTATIVE REACTIONS * JeanBrOAD HURST From the New York State Veterinary College at Cornell University, Ithaca, New York Four recent articles on streptococci by Winslow, Thro, Lyall, and Hopkins and Lang have included most of the historical review orig- inally a part of this paper. Additions to these historical surveys will be found in Table 17, or in the foot-note accompanying it. The investigations just mentioned cover five different phases: (1) the qualitative determination of acid and the correlation of the results in the various media (as used by Gordon, Andrewes and Horder, and Houston); (2) the constancy of the reactions of streptococci, especially in the various media originally suggested by Gordon (tested quite incompletely by most investigators) ; (3) the application of the biometric method introduced by Andrewes and Horder; (4) thexsub- stitution of quantitative for the original qualitative measures, or standards, in the biometric applications, as emphasized chiefly by Winslow and his associates; and (5)- the correlation of the reactions of streptococci with their habitats in an attempt to gain results of "practical sanitary importance." This paper gives a condensed account of the results obtained in a study of 767 strains of streptococci, of which all but twenty-three were freshly isolated for this purpose. The account is subdivided as fol- lows : I. Constancy of the streptococci (measured chiefly by fermentative activities, but also by other characteristics and powers, as, for example, colony characteristics on agar, effects upon blood agar, gelatin, etc.) (a) As exhibited at varying periods after isolation, and * Received for publication May 4, 1915. Part of tlie investigation was prosecuted with tlie aid of a grant from the Esther Herrman Research Fund of the New York Academy of Sciences. 4 Jean Broadhurst (b) As aifected by variations in ^.environment (1) within the range of possible laboratory variations in media, technic, etc., and (2) more extreme, designed to induce changes in streptococci. II. Characteristics of the streptococci on isolation from various sources or habitats. These were studied to ascertain whether strepto- cocci bear or retain sufficient impress of a given environment to yield diagnostic differences— ^differences distinctive enough, for instance, to aid in practical sanitary investigations. I. CONSTANCY Any study of the characteristics* of streptococci, morphologic or physiologic, introduces at once the question of constancy. To avoid numerous digressions and repetitions during the following discussion, it will be necessary to consider first this still open question of constancy of the streptococci. As shown in an earlier paper, constancy may be viewed in two ways: (1) Constancy may be used for the sum total of identical responses exhibited by a given strain or strains under duplicate or identical conditions (necessarily contemporary) ; or, (2) constancy may be broadened to include the power of continuing such identical responses under varying or varied conditions. And, fairly or not, that is what most of us demand of a given strain before we term it "con- stant." Constancy throughout a given period falls under this second type of constancy; for with the greatest possible care no one can ^ insure complete uniformity of all conditions during a period of several months, or even weeks. This is primarily because even the media we use are substances still too completely unknown, and therefore factors unknown ; there are, of course, other unknown influences, including the continued effects of bacterial products upon the bacteria themselves. Constancy of the first type — under identical conditions — has been vouched for by Hilliard. From about 240 strains of streptococci, he made duplicate inoculations into saccharose, lactose, etc., securing three days later similar titra- . tion results for practically all of the 240 pairs in five or six media each. In my own work, several hundred such duplicate strains were tested; a variation in acidity of more than 0.2 or 0.3 occurred in less than 0.5 percent of the tubes titrated. (Differences greater than this were usually found only in (1) * Details concerning the preparation of media, titration methods, stains, and other matters of technic are all placed together in the appendix. Environmental Studies of Streptococci S mutating (?) strains, in which a new fermenting power was suddenly acquired, evincing itself in one tube more markedly than in another; or (2) in weak strains that were dying out. Repetition in the first case invariably yielded like positive results. These exceptions were but few in number, and relatively unimportant.) The same parallelism was, as might be expected, exhibited by TABLE 1 Regional Likenesses of Streptococci Strains from Cat 3 Region Saccharose Lactose Salicin Railinose Mannite Inulin 44 Cardiac, stomach, contents 0.1 2.7 5.1 0.0 3.5 0.2 45 Cardiac, stomach, contents 0.2 3,4 5:1 —0.1 3.5 0.2 46 Cardiac, stomach, contents 0.2 1.2 4.9 0.0 3.6 0.3 47 Cardiac, stomach, contents 5.2 4.4 4.3 1.7 0.1 0.1 48 Cardiac, stomach, contents 0.2 2.2 . 4.8 0.0 3.6 0.3 49' Cardiac, stomach, contents 0.3 2.6 5.0 0.0 3.6 0.2 50 Cardiac, stomach, contents 0.2 2.6 5.0 0.0 3.S 0.3 51 Cardiac, stomach, contents 0.2 2.3 4.9 0.0 —0.2 0.1 54 Mucous membrane duodenum 0.2 2.5 4.8 0.1 3.6 0.1 55 Mucous membrane duodenum 0.0 2.3 5.3 0.0 3.4 0.2 56 Mucou^ membrane duodenum 0.2 3.1 3.7 0.1 4.1 0.1 57 Mucous membrane duodenum 0.0 2.6 6.0 —0.1 3.6 0.2 58 Mucous membrane duodenum 0.3 2.3 5.0 0.0 3.4 0.2 59 Mucous membrane duodenum 0.1 2.0 4.7 0.0 3.3 0.4 60 Mucous membrane duodenum 0.1 2.4 5.4 - 0.0 - 3.7 0.1 61 Mucous membrane duodenum 0.2 1.5 , 4.8 0.1 3.6 0.2 62 Small intestine, contents 0.0 1.7 5.2 0.2 3.5 0.2 63 Small intestine, contents 0.3 1.8 4.9 0.0 3.4 0.1 104 Small intestine, contents 0.2 2.3 4.5 0.1 2.8 0.2 101 Mucous membrane, small intestine 0.2 2.2 4.5 0.2 3.2 0.2 52 Large intestine, contents 0.1 2.5 5.7 0.0 3.6 0.2 53 Large intestine, contents 0.1 2.1 - 5.0 0.0 3.7 ■ 0.1 102 Large intestine, contents 0.2 1.6 4.5 —0.1 2.7 0.2 103 Mucous membrane cecum 4.4 0.2 3.7 0.7 0.1 LI these duplicate strains in other media not quantitatively measured: litmus milk, broth of various kinds, blood agar, gelatin, etc. This doubtless explains the marked similarity often shown by strains from the same sample; for example, Winslow and Palmer (1910). This similarity characterized strains isolated from the same sample in my own work, even tho an effort was made to select strains showing morphologic differences (in size, 6 Jean Broadhurst chain length, regularity in the plane of cell division, relative frequency' of abnor- mal cells in the chain, etc.) ; differences in the characters on agar and in broth were also considered in selecting the strains. The same detailed likenesses were often presented not only by the strains from several samples from a given locality, such as the samples from various parts of the stomach or of the intes- tine, but by those from different regions of one individual — stomach, small intestine, cecum, and large intestine (see Table 1). Becaiise of this similarity, usually but one to three strains were taken from each sample. Unless some such scheme of limiting the number from each sample is fol- lowed, as readily will be seen, one may draw entirely erroneous conclusions regarding the relative frequency of the various types of reactions in a given locality or habitat. For example, a disproportionate number of strains from Cat 3 (Table 1), in which twenty-one of twenty-four strains fermented TABLE 2 Contemporary Fluctuations in Two .Subcultures of One Strain of Streptococcus Subcultures Date of Test Saccbarose Lactose Salicin Raffinose Mannite Inulin 98a January 23 2.8 2.S 2.3 0.2 2.0 2.7 98b January 23 . 2.7 -2.3 1.7 0.0 1.7 2.7 98a February 25 3.7 2.4 2.1 2.4 2.0 0.0 98b February 25 2.8 2.6 2.0 2.4 2.1 0.0 98a April 10 3.4 3.0 1.9 2.6 2.5 2.6 98b April 10 3.9 3.1 2.4 3.1 . 2.9 2.6 98a May 16 •3.3 3.1 2.3 2.8 2.5 2.8 98b May 16 3.0 3.3 2.5 2.6 2.6 2.6 mannite, would lead to an erroneous view concerning the relative frequency of mannite fermenters in the alimentary canal of cats ; for 87 percent of the strains from this cat fermented mannite, but in the four other cats studied (a total of eighty-nine strains) only 35 percent fermented mannite. This identity of response shown in freshly isolated strains often continues to manifest itself in duplicate strains carried along independently for a given time. This is illustrated in Table 2, by the similar, contemporary fluctuations in two subcultures of Strain 98 (cat, palate). Other strains might be added confirming by such contemporary fluctuations the conclusion that a given strain at a given time under identical conditions shows an identity of response in the sum total of reactions. Such parallel fluctuations are strong arguments for constancy of the first type. But, as just shown, these responses may vary with age. Changes due to age or to more or less favorable conditions for growth belong under the dis- cussion of constancy of the second type ; that is, constancy under varying con- ditions, more or less definitely known. Environmental Studies of Streptococci 7 Constancy in Relation to Age In any laboratory the period necessary for isolation is variable, especially when feces and similar materials are used as sources. And, if one is working "to capacity," the routine can never be so well established that it is possible to use only cultures that ( 1 ) fall within a brief, fixed isolation period "and, (2) are fresh transfers, eighteen to twenty-four hours old. One often has to choose between making fresh transfers or using slants more than twenty-four hours old. To see the possible efJfects of such differences, slants (of about twenty strains) were compared with their respective subcultures, both being inocu- lated into the test media at the same time ; the results obtained from twenty- four-hour slants were also compared with those obtained from the same slants when they were five to eight days old. ' Even in the fermentative reactions the differences were negligible. While such variations in laboratory routine might make a difference with less hardy strains, allowing them to die off before the special or test media were inocu- lated, one apparently need not expect, any wide range in the final results that could be attributed to age differences of one to three days, which is as wide a range as would be found in most laboratorfes. This second view of constancy (under varying conditions) was still further tested for the effects of age on the fermentative activities : A number of strains (twenty) were kept on slants at room temperature for one year. Four sur- vived ; they were repeated in the usual test media with the results shown in Table 3. As is apparent, they were remarkably constant * in view of the uncon- genial conditions to which they were subjected; Strains 47 and 93 had both lost the power of fermenting raffinose. In this connection, it is interesting to know that Strain 93 did not, when first isolated, ferment raiifinose, but gained that power suddenly during the sixth month, just before the slant for drying was prepared. As will be shown in later studies, the fermenting power latest in development or manifestation is the one most easily lost. It was not, of course, expected that the results with these four strains would yield anything of more than passing interest. It is much more important to know how constant strains are under ordinary labo- ratory conditions — whether we can compare strains freshly isolated with older ones loaned by other laboratories, etc. With this in mind, 134 cultures, representative of the various types of bacteria, were selected as they were isolated and kept as stock cultures to be tested as often as opportunity allowed. Such strains were transferred every ten days, incubated twenty-four hours at 37 C, and stored (the remaining nine days) at room temperature in closed containers to prevent drying out. At intervals of two weeks, four weeks, two to four months, six months, and twelve months or longer, they were again tested in the usual Gordon media. Strains proving inconstant were usually dropped, tho a number of inconstants were carried on to note the trend of the changing reactions. * Complete constancy of this kind has been reported by Holman (1914) for one strain of Streptococcus faccalis, dried on a cover glass for over five months. All of my survivors were short-chained streptococci. n W u M !" M M < Q Is 9 ^ o p O CI d d r-t 29 1 mo. 20 (68%) 43 '2 mo. 33 (76%) 19 3 mo. 12 (63%) 48 4 mo. 31 (64%) 57 6-7 mo. 42 (73%) 27 12-13 mo. 21 (77%)- 6 15 mo. 4 — 3 21-24 mo. 1 — Total 258 ' not given in Table 4. In 262 retests, the results in all six of the Gor- don media were like those of the latest tests; in 83 retests, changes occurred, usually in but one, sometimes in two, suDstances, giving really a constancy of over 93 percent. My percentage of constants was higher than the percentages obtained by several workers, and lower than those given by' others. TABLE 5 Record of One Strain of Streptococci for Six Months Saccharose Lactose Salicin RafHnose Mannite Inulin January 21... February 25 . . April 1 April 15 May 16 July 21 ''}_ _ + -f X + + + + — + + Those who have tested the greater number of strains (e. g., Lyall) report more favorably regarding constancy. The percentage of constants is, of course, directly related to the number of test sub- stances used, and, as shown later, affected by the kind of media used, and by the methods of measuring acidity. Environmental Studies of Streptococci 11 In considering constancy several questions arise: (l)_7w which substances are streptococci least variable? This can be tested only in those strains which have previously, exhibited any designated power. On comparing the repetitions of the" 134 strains tested for constancy, we find that, on retesting, the changes or losses are in the following order: in salicin 2 percent; lactose 2 percent; saccharose 4 percent; raffinose 6 percent; mannite 10 percent; and inulin 21 percent. (2) Has the relative constancy in various substances any relation to the readiness ivith which these substances are fermented? This relative constancy corresponds in a general way to the order of fermentability of these 134 strains, for 97 percent fermented salicin; 96 percent lactose; 84 percent saccharose; 57 percent mannite; 28 percent rafifinose; and 20 percent fermented inulin. It also corre- sponds to the order of fermentability for the whole 767 strains'studied ; 88 percent fermented lactose ; 85 percent salicin ; 74 percent saccharose ; 46 percent mannite ; 30 percent raffinose ; and 24 percent inulin. (3) Are newly acquired powers as constant as those of earlier manifestation? Newly acquired powers seem more variable. For example. Strain 11 (originally fermenting none of the Gordon media) sQddenly began to ferment salicin ; during the next five months it lost this power twice, regaining it each time. Other non-fermenting strains have wavered similarly after acquiring salicin. Other strains, originally fermenting one or more substances, show the same fluctuations . with their latest acquired powers; mannite or raffinose, or raffinose and inulin. Ark- wright (1913), who attributes variation to a general lowering of functional activity, suggests that the power suppressed is probably more recently acquired. ' (4) Is there, with age, a greater tendency to gain or lose in fer- mentative power? There seems under ordinary laboratory conditions a greater ten- dency to gain than to lose fermenting powers. Among these 134 strains, forty (29 percent) gained one or more substances, while but twenty-six (19 percent) lost substances. There is also this decided difference: A strain that loses a power may, and usually does, regain that power later. But a strain that gains a power usually keep?, it; in this the fluctuation is strikingly less than that which attends the loss of a' power. Lyall ( 1914) reports for his 263 pathologic strains 12 Jean Broadhuest changes by addition only, stating that variants by loss were not observed. Floyd states (in a letter of 1915) that the gains in the Floyd and Wolbach series outnumber the losses ; their strains, it will be recalled, contained an unusually high percentage of non-fermenters. Differences might be expected not only in the number of substances fermented, but in the amount of acid formed. Since strains from the alimentary canal and from strictly non-parasitic sources generally yield a higher percentage of acid, one might argue perhaps that a long period on artificial media might be expected to have such an influence ; but apparently age does not affect markedly the titration results. Of these 134 strains, eighty-four (62 percent) did show a gain in the amount of acid formed in a total of 201 substances (averaging 2^ substances a strain) ; but these strains usually had other records lower than the original ones, and the gains were not at all continuous or progressive. (5) Is there any relation between constancy and the source, or origin, of the strains? The strains from hay, water, ' and milk (non-parasitic sources) seem a little less variable than most of the strains {7Z percent con- stant). They are however too few in number to be convincing. The twelve strains from the alimentary canals of dogs after feeding of streptococci are remarkably constant (91 percent) ; only one strain showed any change in the year through which they were studied. The twenty-three strains isolated from the alimentary canals and the feces of normal dogs are also relatively constant (78 percent), but this may be a mere coincidence. There would seem to be no real reason why strains from dogs should be more constant than those from other sources. (The throat strains from the same dogs were more variable; but throat strains from several other animals studied are apparently both more variable and more varied than those from any other source.) The general feeling that strains from pathologic conditions are- more variable is probably correct, tho Lyall reports less than 5 percent showing variation in his 263 strains, and Floyd and Wolbach but "few" changes in their seventy-five retests. These high proportions constant may be due to the fact that Lyall incubated his tubes for ten days, and Floyd and Wolbach incubated theirs for seven days. My own strains which were recently isolated from such sources would indicate that they are probably the most variable. The number actually tested is so small, however, that no definite statement can be made; Environmental Studies of Streptococci 13 and those obtained from other laboratories were old cultures and usually fermented at least four of the Gordon test substances used in this work. The variations that occur seem related not only to the number, but to the range of fermentative powers that the strain possesses. This will be included under (6). (6) Does the fermentative combination or complex of a strain enable one (a) to forecast with regard to its constancy or (b) to predict the trend of any changes that may occur? (a) The highest records for constancy (83 percent) were shown by the strains originally fermenting saccharose, lactose, salicin, and mannite (30 strains). Those originally fermenting saccharose, lactose, salicin, rafBnose, and mannite (20 strains) showed 75 percent constant strains. But a tabulated comparison of all the mannite strains showed but 67 percent constant through the periods tested. This is quite within the range of general constancy, so that mannite organisms as such are probably not more constant than other streptococci. (Of the inconstant mannite strains, however, one-fourth varied only in a lapse of the mannite power.) No other prominent group (such as the saccharose-lactose-salicin group) showed more than the usual number of constants. (b) The substance gained or lost by a given group is apparently in the line of other comparatively stable groups; for example, lactose- salicin strains tend to gain mannite, the lactose-salicin-mannite strains being relatively common, and varying usually in mannite activities only. Saccharose-lactose-salicin strains more often pick up raffinose (12 in 40) than mannite (2 in 40); yet saccharose-lactose-salicin- mannite strains form the largest group constant studied (25 out of 30). It seems to originate in a lactose-salicin group rather than from saccharose-lactose-salicin strains. A strain that begins to fer- ment rafHnose often later (occasionally simultaneously) ferments inulin. (Libman and Celler (1910) observed earlier that the raffinose and inulin fermenting powers are usually associated. Their percent of linkage is evidently a little higher than that shown by my tables in this paper.) On cultivation under ordinary conditions, strains that acquire the mannite power rarely acquire also raffinose or inulin; the reverse of this usually holds also. The more stable groups are the more common, not only in this series, but in the whole 767 strains studied ; for example, lactose- 14 Jean Broadhurst salicin-mannite strains and saccharose-lactose-salicin-mannite strains (mentioned as stable) are common in feces, the alimeiuaiy canal, etc. In this connection a scheme* has been worked out showing the probable relationship of the various 134 strains. As stated before, several retests were made for each of these strains. Each strain proving constant for the whole period in which it was tested (whether two weeks or two years) is included in the number given in the oblong figures in the accompanying chart. Changes in the fermentative com- plex causing a strain to be placed in a new group are indicated by the lines connecting the oblong figures. The number of changes between two fermentative complexes is indicated by a figure placed in an arrow. For example, twenty-five saccharose-lactose-salicin-man- nite strains remained constant; two others changed to lactose-salicin mannite; eight others changed to saccharose-lactose-salicin-raffinose, mannite; five saccharose-lactose-salicin-rafiiinose-mannite strains lost raffinose power and fell into the sacchcirose-lactose-salicin-mannite group, and one lactose-salicin-mannite strain gained saccharose power and entered this group. With this explanation a. glance at Chart 1 will show (1) that these 134 strains included several stable groups of considerable size (e. g. saccharose-lactose-salicin) ; (2) that between these groups there are usually well-worn paths (e. g., between saccharose-lactose-salicin and saccharose-lactose-salicin-raffinose) ; (3) that the variation is more often progressive (acquiring rather than losing fermentative powers) ; and (4) that these combinations fall into three main groups, each prob- ably sharing a different ancestral history, through lactose, through saccharose, or through salicin. Note, for example:, the lower half of the chart, where a probable family-tree of the lactose line is presented. The eighty-eight titrations in this series show that it is possible to pass from a non-fermenter to a lactose f ermenter, on to a lactose-salicin f ermenter, on to a lactose- salicin-mannite fermenter and so on to a saccharose-lactose-salicin- raffinose-mannite fermenter. • This scheme includes a record focevery change in the titration records of the inconstant strains. In the preceding discussion, an inconstant strain is listed but once; here, how- ever, it is counted each time a change occurs. For example, if a strain changed from lactose- salicin, to lactose-salicin-mannite, and then back to the original lactose-salicin combination, it would be represented in connection with the corresponding arrows twice, once in each direction. No records are included for the times a strain corresponded with its immediately preceding record, the aim being to find the routes traveled in the changes that do occur. In'ulin was omitted from this scheme. It followed raffinose closely enough to add little of any value, and the several additional small groups, often of one strain only, complicated the chart and distracted the attention without yielding any apparent compensations. Environmental Studies of Streptococci 15 Saccharose and its additions (65 titration records) lead to the raffinose groups (lacking mannite). A third path of descent through salicin leads less directly either to the mannite or to the raffinose com- plexes (21 records). The raffinose and mannite complex seems to develop almost entirely from mannite strains with a lactose ancestry. Q8 Titration Records Chart 1. — The possible relationships of the common fermentative complexes. This scheme includes 174 titration records (the repetitions which proved constant are not represented in this chart). Of these, 88 fall into the lactose-mannite line, or group; 65 into the saccharose, raffinose line; and 17 into the salicin (lacking lactose) line. 16 Jean Broadhurst Note here the small number (4) of cross connections (indicated by dotted lines) between the three^" family-tree" lines in Chart 1. The strains that changed more than once during the period of observation were compared individually with this scheme to see whether they afforded any supporting evidence for this theory of descent. The following facts are of interest: ( 1 ) Several strains wavered betw^een two well-defined complexes : (a) One strain changed three times from a non-fermenter to a salicin fermenter and back again. (b) Three strains each changed from lactose-salicin to lactose- salicin-mannite and back again. (c) Three strains changed from saccharose-lactose-salicin- mannite to saccharose-lactose-salicin-mannite-rafifinose and back again; one strain made the double trip two times. (2) Several strains ranged through two or more groups, each keeping nevertheless to the line or series to which this scheme would indicate that it belonged : (a) One strain changed from lactose to lactose-salicin, and later to lactose-salicin-mannite. (b) One strain changed from saccharose-lactose-salicin-man- nite to saccharose-salicin-mannite and back to saccha- rose-salicin. (c) Another strain traveled routes as follows: Saccharose to saccharose-lactose; saccharose-lactose to saccharose- lactose-salicin ; saccharose-lactose-salicin to saccharose; saccharose to saccharose-lactose; and saccharose-lac- tose to saccharose, when the investigation ceased. ' The metabolic gradient suggested by Howe (1912) and enlarged upon by Winslow (1912) and Milliard (1913) may throw some light upon these three divisions or series of streptococci (Chart 1). RafHnose power presupposes the power to use saccharose ; out of 234 raffinose fermenters (in the 767 strains studied), but one failed to ferment saccharose (Strain 7, human throat). Mannite similarly includes lactose; out of 360 mannite fermenters, but seven failed to ferment lactose (4 of the 7 fermented salicin, and our chart indicates a possible mannite descent thrpugh salicin). Of the strains fermenting both raffinose and mannite, but one lacked the power of using saccha- rose and lactose (Strain 87, dog feces). The converse of these Environmental Studies of Streptococci 17 metabolic relationships* does not hold, as indicated by the order of f ermentability. These relations are interesting as they make plausible, at least, the lines of descent given in Chart 1. Tho anticipating soihewhat a part of a later section of this paper, it seems best to add here the following related comparisons. If this ancestral scheme is biologically sound, it might be expected that the stimulating environments would afford interesting evidence. A com- parison of Hilliard's results (1912) with duplicate strains grown at 20 C. and at 37 C. supports this theory. Selecting the strains yielding acid in at least one substance at both 20 C. and 37 C, the only dif- ference we find occurring more than once is a change also represented in our series, saccharose to saccharose-lactose ; eight strains fermented saccharose only at 20 C. and saccharose-lactose at 37 C. The induced changes obtained in my later work also offer some support for these theoretic descents ; for example, the change obtained by subjecting Strain 29 to saliva, and also to alimentary canal conditions (collie dog) occurred later in the regular stock culture of 29. It would seem in such cases as if the changed environment pro- vided the needed stimulus, but that the ability to vary and the type of variation are inherent in the organisms. Changes reported by several other workers fall in with my scheme very well. Most of them, however, used too few strains in the retests or else did not give sufficient details concerning the fermentative reactions to enable me to add to the generalizations already given. As will be shown later (Table 17), these groups have some habitat affiliations; the strongest is probably shown by my human mouth streptococci, 85 percent of the strains of which fall into the saccharose- raffinose groups ; 74 percent of Hilliard's 185 mouth strains are found here, also. That the variation occurs within the limits shown in this "family- tree" indicates that the most important factor is, after all, the organism itself. Environmental influences may hasten such changes, but the line of change seems to lie with the organism. Rettger and Sherrick (1911), experimenting with Bacillus prodigiosus, sta:te that variation does not depend solely upon changes in nutriment, environ- mental conditions, etc., but is frequently brought about through inherent proper- * It is possible that the alcohol group in lactose is definitely related to the power to use mannite, a hexahydric alcohol; the chemical likeness of saccharose and raffinose has also been pointed out by Hilliard (1913). Chamot says (letter of 1915), "There are not a few cases where a bacterial species will ferment certain carbohydrates and also hydrolize some indi- vidual_ glucosid, but so far as I am aware, no one has yet discovered the fundamental reason for this selective action." . 18 Jean Broadhurst ties within the organism itself. And Penfold (1912) states that the "inability of Bacillus coli to vary quickly in respect to certain carbohydrates would appear to be as characteristic of certain species as the power to vary." Thro (1914) emphasizes the variability of the organisms, tho he mentions additional causes of variation in the streptococci. , CHANGES WITH AGE OBSERVED IN OTHER MEDIA Besides the fermentative observations just described for the 134 strains, more general observations were made for thirty-one strains kept for one year (transferred to fresh agar every ten days) and retested in milk, blood, etc., as well as in the Gordon media. The changes are marked (Table 6). It is a surprise to find that the quantitative Gordon' results are more constant than the less definitely measurable results in such media as blood, litmus milk, and gelatin. The chain length runs much the same ; the characters on agar are relatively constant. Gelatin indicates great differences in the ease with which these strains grow in it at room temperature. (In this connection, recall that these stock strains were kept at room temperature nine of every ten days.) In twelve of fourteen strains, growth in gelatin is earlier than in the freshly isolated strains ; in four of these fourteen it is two to ten days earlier. There were "no changes with regard to liquefying power. Broth* is rather disappointing in this connection. Fourteen of twenty- five vary two to five days in the time of clearing. One, clearing on isolation, fails to do so after one year; and oiie not clearing when isolated clears in five days. There seems to be no distinct tendency to lengthen or shorten the period before clearing. Differences m this regard are often attributed to differences in the reaction of the medium. Forty-eight strains inoculated simultaneously into sugar-free broth and into plain broth of various acidities -|- 0.5, + l.S, and — O.S, and into calcium carbonate plain broth, and 126 strains inoculated into all but the — O.S broth indicate that, while there may be a difference of one to two days in the time of clearing, it is apparently due to differences of vigor and amount of growth, due, in turn, probably to the more or less favorable initial acidity. Lyall (1,914) finds broth characteristics "so largely dependent on the nature and reaction of the medium" that they seem of little value. In the various Gordon media, the three-day appearances vary greatly, but the records of 767 strains in the six kinds of media, including many hundred duplicates, totaling about 8,000 tubes, do not yield anything leading to a corre- lation of turbidity or clearing with the amount of acid formed; nor is there any definite relation, apparently, between the clearing in plain broth and that in the Gordon media. Tho strains clearing in the former usually do so in the latter, the converse is not true, and the effects in sugar broth are decidedly less differential than those in plain broth. Litmus milk is not much more satisfactory; four of the strains changed with regard to the power of coagulating milk. Blood probably offers the greatest surprise of all. On blood agar, but few non-green streptococci change to green; ten of the thirty-one strains originally green or with no color changed, forming colonies with a definite hazy area or rim; four gained the power to hemolyze blood; and fourteen (ten of them hemolyzing) strains showed no change for blood agar, even after one year on plain agar. * Moore (1893) described over thirty streptococci mainly from pathologic conditions, emphasizing the time of clearing, etc. Laabs (1910) ranked broth first (above injection) in diagnostic value. ^ d 1-10 1-t M «* CO , COIN iH CM iH . iH ^ rH CM « 3-" 00 ^ 00 T T ii t~t-i ^ rH w >-l IH tr-eo i-i (M 03 c» eo CM w CM 00 T ° ^ CON ^- C9 CM CO ma iHr-l •«* t- 00 to in m ^ so CO CO in 10 CO T =" d d -* (M tHOJ CO CO ■* CO ©4 « CM CO cj ^ iHrH CO m © iH CO r>io «D 03 CO -* Tj( CM in iH OS as r CO tH id oi mco 10 CO CO CJ •=> CO CO CO CM ^5 . a en a d m tn m Kl m 03 W ■a t. o« 1^" 2 25 hflO So 15 £a g m a m 5 5 - w S >> ■Ss>. aa§ II 5 g III s 1 'S ■ a -■a ri^l d « n.S bo bo 5 «, M bo ta ba bj) so 1; n a as a 2S d "c d •o d a a 03 as a ,!4 d a 5s II s H 6 ?5 ^ P. d Ph fM (^ fk h Litmus Milk Coagula- tion in S 03 1 a d !i 03 e8 IN CSl 03 03 CO CO ^5 iz; 9 9 IZi IZi CM CO 1 09 CM 1 13 CM £ 13 CM 03 f1 CO woo w 3 1 jq .a It a ■a j3 •a '"a (N IM 03 a 00 i ■a S £ 03 -a .1 « d >>>) S ,8 SaSa 1 rt fl 2-SST3 « >. =^ 2^ 3 a 03 SI 03 ^5 CO aj P 1 a 1 bA 1 d a 3 p| 3 5 ^ ea oj rt ea 03 d BJ d 03 A* . ' ^ s s S «.9S.S -O TD 'O -O X) -a . ■a v •0 -O 13 •o 'O -a s» (N M S g " S "= s a CO 00 8 1 1 § M ^ ssl .So* ■OS &.5 ■° 3 >> Id ■0 3 e 2 ta ^r -0 >* & 2 ■o- S 03 ■a 03 OS Si •0 3 £ CM iH k-^ t*. OJ ^ •-' ^ r-i iH iH iH '"' .H . \ rH 1 a is ^ 'Is |l a« IN w in d S3 m as ac aS 00 u a a ;a g i-i s S 8 u s rt !S CO Q ^ 20 Jean Broadhurst conclusions regarding constancy in relation to age Periods of cultivation on artificial media may produce or be characterized by changes in physiologic activities. The quantitatively measured fermenting powers — 63 to 77 percent constant for periods of one year or less— are fully as constant as the qualitatively measured results obtained with gelatin, broth, litmus milk, and on plain and blood agar plates. When fermentative changes occur, they seem sufficiently related to the former fermentative complexes to indicate three main lines of development of fermentative powers: (1) a lactose-mannite line, (2) a saccharose-raffinose line, and (3) a smaller and more variable salicin. line. The fluctuation within these three groups serves to emphasize the integrity of these groups; this, with the rarity of cross-connections between the three groups, emphasizes the probable ancestral limitations of these groups or series. Constancy as Affected by Temperature and Media The variations shown by streptococci during a long period of cul- tivation do not necessarily affect the diagnostic value of the characters they may exhibit when freshly isolated. Even if we think of the streptococci as definitely or permanently modified by the environments from which they have been isolated, that increases (and does not lessen) the possible diagnostic value of such characters. The point of real importance is, rather, are there any differences of laboratory procedure (during isolation, pending the final tests, etc.) that may modify .the final results, and so explain the differences in the results obtained by various workers. And these results are very different ; for example, 8 percent of the forty-six milk strains described by Hilliard (1913) ferment salicin, but 77 percent of my 133 milk strains are salicin fermenters. If some of the conditions attending identical or differing results in any one laboratory could be found, we might know where to look for the factors that must be considered in comparing the results obtained in different laboratories. Upon investigation, definite differences in technic came to light. Omitting age (already discussed), we may treat these under two* main topics, temperature and media. * Variation in the amounts used for inoculating the Gordon test substances has also been mentioned as possibly causing some of the. differences in these Gordon results. To test this, forty tubes were inoculated with varying amounts: three loops, one loop, and the tip of a fine stab needle. The titration results three days later showed no differences worth noting. Some- times the result with the smaller amount was 0.1, 0.2, or even 0.3, ahead of the record for the larger amount. Environmental Studies of Streptococci 21 TEMPERATURE (a) Cultures in some laboratories were stored (after incubation at 37 C.) in the ice box; in others, they were kept at room temperature. To see the effects of such different temperatures on the later (37 C.) fermentations, duplicate cultures of ten strains were kept from four to seven days at the two temperatures 20-24 C. and 6-10 C. and inocu- lated directly into the Gordon media. In the titrations at the end of the usual three-day period, no differences were found. (b) In most laboratories, practically all the strains described were grown on agar at 37 C. Gelatin was occasionally used, however. Here we have a double difference of media and. temperature, but ten dupli- cate cultures used experimentally to find possible modifications due TABLE 7 Effect of Temperature on the Fermentative- Reactions of Streptococci Strain Source Tempera- tures C. Saccha- rose Lac- tose Sal- icin Eaffi- nose Man- nite Inu- lin 38 42 Blood Abscess Original 37 40 Betest, 37. . Original.... 40 Betest Original... . 40 Betest Original.... 40 Betest Original 40 Betest 5.2 2.7 3.2 5.1 1.7 4.1 6.9 4.5 4.3 4.7 3.5 3.7 0.0 0.1 0.6 i.S 2.7 6.0 4.5 1.7 5.6 0.4 0.0 —0.1 1.4 1.6 3.6 5.4 3.7 6.7 5.3 2.9 6.8 0.2 0.0 0.2 5.7 3.6 4.3 6.0 3.6 8.6 4.7 2.8 6.0 0.7 0.2 0.3 4.6 1.9 6.2 0.2 0.0 0.1 0.5 0.0 0.2 0.1 0.0 0.3 3.4 2.4 5.2 0.1 0.1 0.1 0.2 0.0 0.1 4.0 2.7 6.0 0.5 —0.1 0.9 0.4 0.1 —0.1 0.4 29 7 4 Human throat Dog's throat Dog's stomach, car- diac 0.1 0.3 0.1 0.1 0.0 0.2 0.0 0.3 -0.1 0.2 0.2 to such differences in preliminary treatment gave practically identical results in the Gordon media (and also in blood, litmus milk, etc.). Hilliard found that milk streptococci gave the same fermentative result when grown at 20 C. and at 37 C., while throat streptococci at 20 C. did not readily use any of the sugars utilized at 37 C. (c) Differences slighter than this may affect the results appre- ciably. There is some indication in my work that 35 C. to 37 C. is a more favorable range than 37 C. to 38 C. Temperatures above 37 C. are apparently less favorable. One very hot night the temperature in our incubator room exceeded 37 C. 22 Jean ' Beoadhurst Sometime between 11 p. m. and 5:30 a. m., the temperature reached 40 C. This was about the middle of the three-day incubation period for the Gordon tubes. When titrated later, they all yielded records decidedly lower than those of earlier titrations of the original strains, and also below those obtained in later work with subcultures of the original strains. Table 7 shows for a few of these strains the marked depression due to this period of unfavorable temperature. The effects were not only marked but consistent throughout the entire set of 40 strains in all six media. Other strains (12 in all) incubated on agar at 40 C, and then inoculated into Gordon media, did not differ materially from their original records. With regard to temperature, therefore, it would seem as if the temperatures during the period of growth, observation, or measurement were most important ; differences in temperature pre- ceding these final tests are appa:rently not important, so long as they are not so extreme as to kill the organisms. (It is probable, of course, that the sugar and related Gordon media act as revivifying influences.) MEDIA Most of the differences in procedure, however, are usually ques- tions of media — of concentration, acidity or^ alkalinity, or additional nutrient substances, etc. For example, some workers used plain agar throughout for isolation; a few invigorated all cultures preparatory to the Gordon tests by dextrose agar. North's media, etc. ; others used selective media, such as lactose litmus agar, for isolation of their strains; most of the workers used meat extract instead. of meat for all the preliminary media as well as the final test or Gordon media. Special media used for isolation of streptococci seem to act selec- tively with regard to the streptococci ; for example, lactose litmus agar yields a series of strains with a non-typical proportion of lactose fer- roenters. The main problem here, however, is the effect of media upon the subsequent Gordon reactions. For these experiments duplicate cultures of ten to thirty strains (listed as follows) were cultivated, and then inoculated into the usual Gordon media: (a) On agar (all agar, broth, and gelatin throughout made from meat, unless meat extract is definitely mentioned) and on gelatin (1 to 3 days). (b) On agar and in (autoclaved) milk (1 to 10 transfers, 3 days apart) . (c) In (Arnold) milk and in meat extract broth (1 to 3 transfers). Environmental Studies of Streptococci 23 (d) On plain agar and on dextrose agar (1 to 8 days). (e) On plain agar and in dextrose broth (1 to 10 transfers, 1 day apart). (f) In plain broth, in sugar broth, and on plain agar (10 transfers, 1 to 3 days apart). (g) In serum broth and in dextrose broth (3 transfers). (h) In plain broth of varying acidity 0.5, 1.5, or calcium carbonate (10 transfers, 3 days apart). (i) On plain agar, in sugar-free broth, and in each of the six Gordon media (10 transfers, 3 days apart). (j) In meat extract broth, and in meat broth, and also in Gordon media made of meat extract and of meat. None of these first nine experiments (a-i) yielded any differences worth noting in the final Gordon titrations which followed. Experi- ments (b) and (c) were included to see whether they accounted for the high acidities obtained in my early work with milk streptococci. The differences here, also, are too slight to be of importance. Milk, as a preliminary culture medium, was therefore not entirely, if at all, responsible for the extremely high acid results reported earlier (1912) for my milk streptococci. The 174 strains studied under (h) indicate (1) that +0.5 is a more favorable initial acidity than +1.5; and (2) that calcium broth is not more favorable for growth than broth without the addi- tion of calcium. Nearly 200 tubes incubated for ten days and streaked for viability showed a lower percentage (76 percent) of viable strains than did plain broth (93 percent). This is not in accord with the common practice of bacteriologists, especially those working with strains of pathologic origin; the alkaline reaction of the body tissues and fluids may explain this accepted practice. The condition in part of the alimentary canal, at least, is decidedly higher in acidity ; Escherich reports also a difference in the acidity of infant and adult stools. All this will explain, probably, why calcium broth presented apparently no advantages for my strains, many of which came from the alimentary canal and strictly saprophytic, or not strictly parasitic, sources. My results for (i) do not indicate any real or definite influence* due to the lack of sugars or to the presence of saccharose, lactose, and * In many cases certain of the Gordon media (not utilizable) showed less indication ot growth (gross and microscopic) than sugar-free media. This is^ explained by Hilliard in st letter, 1914, in which he says that "the added organic substance has acted as a bacterio* static agent." The larger number of swollen organisms in such cases (when compared with sugar-free smears) would indicate that the effect of the Gordon substance may be something more than bacteriostatic. 24 Jean Broadhurst such related substances. After one month on sugar-free media or on the Gordon media, the tenth transfer of each strain was again inocu- lated into its respective substance (salicin into salicin, inulin into inulin, etc.). These special media were incubated three days as usual and titrated in the customary manner. A table, is given for part of these strains (Table 8), as many workers have obtained effects interpreted as directly due to such preliminary treatment. TABLE 8 Effect of Preliminaky Treatment with Regard to Sugars Strain Source Preliminary Media Pielim- inary Period Saccha- rose Lac- tose Sali- cin Baffl- noee Man- nite 3 wk. 3 wk. 3.8 3.9 4.7 4.4 4.4 4.9 2.9 3.8 2.9 2.9 3 wk. 3 wk. 4.4 4.4 4.3 8.4 4.6 6.2 0.4 0.4 3.9 3.4 3 wk. 3 wk. 4.1 2.2 8.5 3.6 4.7 6.5 0.3 0.4 3.7 3.5 1 mo. 1 mo. 4.S 4.5 -0.1 -0.1 4.8 4.2 0.1 0.1 0.1 0.1 5 da. 5 da. 3.8 3.7 1.0 0.9 4.4 5.0 0.0 3.1 8.1 5 da. 6 da. 1.4 1.3 3.8 3.5 ti 1.4 1.1 2.9 2.9 5 da. 5 da. 1.3 1.2 3.5 3.5 4.2 4.5 1.5 1.8 2.9 2.8 1 mo. 1 mo. 0.1 0.0 . 0.3 0.8 0.1 0.1 0.1 0.0 0.2 0.1 1 mo. 1 mo. 3.5 8.3 4.1 2.8 4.4 4.4 2.5 2.6 2.2 2.9 1 mo. 1 mo. 4.1 2.3 3.4 3.8 4.6 4.6 3.2 3.1 3.2 3.0 1 mo. 1 mo. 3.8 4.0 2.9 1.9 8.4 1.9 0.2 0.8 1.8 1.4 1 mo. 1 mo. 3.8 4.1 4.0 4.4 5.2 5.6 0.3 0.3 3.4 3.8 Inu- lin 7 25 26 20 21 21 8 18 30 24 Milk Human feces... Human feces. . Human throat Milk Cat, abscess... Cat, abscess... Cat, abscess... Blood. Milk Chicken, blood Human feces... Agar Sugar free Agar Sugar free Agar Sugar free Agar Dextrose agar Agar Dextrose agar Agar Dextrose agar Agar Dextrose agar Agar Dextrose agar Agar Ee spective Gordon media Agar Res pective Gordon media Agar Res pective Gordon media Agar Respective Gordon media 0.2 0.3 0.5 0.4 0.2 0.' 0.0 0.0 0.0 0.2 1.6 1.5 1.6 1.6 0.2 0.2 3.0 2.8 0.2 —0.1 0.4 0.7 0.4 0.3 My strains showed for the substances fermented no gain in the amount of acid formed; and no strain learned to ferment any of the substances which it had not previously fermented. And the few changes apparently induced in later similar experiments often appeared simultaneously or later in agar controls. Environmental Studies of Streptococci 25 There is nothing in the worlc of Klotz (190S-07), Neisser (1906), Penfold (1910), and Walker (1910) to indicate that this is not really a question of vigor of growth rather than of the inducing of specific fermentative powers. Penfold (1912) used single-cell cultures, so that such variations or mutations cannot be dismissed summarily as due to "mixed strains." Then, too, the differences attributed to sugar-feeding are not always consist- ent; for example, their strains do not progressively gain the power to ferment a given substance, as one might expect if such gain were due to continued sub- jection to that substance; and strains usirig a given substance may apparently suddenly cease using that substance. These discrepancies or irregularities, I think, are at least partly to be explained as follows : (1) Sugar media often kill off streptococci within a short period.* Thirty different strains yielding (after three days) heavy growth in the Gordon media and characterized by high acidity (3.6 to S.3), were streaked (two to three loops each) on agar; about 61 percent failed to grow on the agar plates. The accumulated acids were probably mainly responsible for these germicidal results, shown in saccharose, salicin, and similar media, tho growth was obtained from a few of the tubes with very high acidity (4.7, S.3). (2) The explanation of discrepancies and irregularities when this work was carried on in meat extract; media, is probably found under the next experiment, (j). (j) The comparative effects of meat and meat extract have been described in an earlier (1913) paper. Some strains grew very well„ at first, in meat extract broth ; but after two or three successive trans- fers in meat extract broth, one or more strains in each lot (of six to. twelve strains) died or had to be revived by more favorable food material. If, however, the strains remained alive, they did as well as. their controls when they were inoculated into the usual Gordon media (made from meat). More striking was the difference between Gordon media made of meat extract, and Gordon media made in the usual way, from sugar-free meat broth. Here, even tho the two lots of Gordon media were inoculated from the same agar slants, a great variation was found in the titration records. Strains that gave a high acidity in Gordon media made from meat often fell to 1.0 or even to 0.8 and 0.7 in meat extract f Gordon media. This range includes the neutral point for litmus, and it readily can be seen that strains classed as fermenters when meat was used would often give negative results with litmus in meat extract media. Almost all workers on these fermentative activities of strepto- cocci have used meat extract media for these tests. This surely * In this connection it may be of interest to add that even plain broth and milk culturea (ten-day incubations) differ in this respect. Of the 85 strains, 88 percent survived the ten-day period in plain broth, and 54 percent in litmus milk. Milk usually had the higher final acidity, 2.3 to 3.0; that of the plain broth ranged from 1.2 to 1.8. Most of the Gordon media failing to give indications of live organisms ranged from 4.5 to 5.6. t This difference obtained also in a lot of nine strains compared in 1914 by Miss Alma Booth, of Teachers College. This indicates a great difference in physiologic activities, and may therefore have an important bearing upon the substitution of meat extract for meat (infusion) media in the production of toxins, vaccines, and similar preparations. 26 Jean Broadhurst accounts for many of the irregularities one finds when trying to com- pare the records of various investigators, altho several state that their results were the same, whether measured with litmus (or Anrade's acid fuchsin) or by titration with phenolphthalein. This may be true for any individual worker using only meat extract media or meat media throughout. But I believe it is impossible to compare satisfac- torily, if at all, strains grown in the two kinds of media, especially where there is also a difference in the indicators (e. g., litmus and phenolphthalein). Related to this probably are the curious results obtained by Floyd and Wolbach. Of 247 strains, mainly from pathologic conditions, over 60 percent failed to ferment any of my six Gordon media. They used neutral red as an indicator, and while their control tests indicated that this was extremely sensi- tive (to 0.25), it is hardly possible that there is not some difference in technic to explain why their strains (orte from a sample, evidently) differ so markedly from those of other investigators. Another cause of discordant results in the work of many investigators is probably the variety of media used for the Gordon tests. Hiss serum water, meat infusion broth, meat extract broth, and peptone solution are the principal foundation substances to which the Gordon test substances are ' added. One worker uses meat infusion broth without making it sugar-free, subtracting the results of control tubes of the meat infusion broth. This seems hardly reliable, as the acid formed from the muscle sugar may influence the cleavage of the Gordoii test substances. Rogers and Dahlberg add dibasic phosphates to their media; feeling that it favors materially the acid production. Their records com- pare more favorably with my meat media records than those of any other workers. Still more important in producing the lack of uniformity is the combined effect of such depressing influences as accumulating acids, and such unfavorable media as meat extract, just described under experiments (i) and (j) in the final Gordon tests. Few of my strains (on meat-infusion agar) showed any tendency to die off. Altho several workers state specifically that their strains were alive when inoculated into the Gordon test media, the conditions under which many of them carried their stock cultures were, in my experience, warranted to subject them to such depressing influences. While I gained no differences due to such preliminary treatment, it is doubtless true that prolonged suljjection to one or both of these unfavorable conditions would (1) yield less vigorous strains that might not recover equally in the various Gordon media, or (2) would act selectively on the strains under cultivation. (Bergey (1912), for example, says that one or more of each set of his strains died at each subtransf er. ) Environmental Studies of Streptococci 27 conclusions regarding constancy as affected by temperature and media Summarizing the experiments listed, we may draw two conclu- sions : Tho the life of any strain is apparently shorter, and more uncer- tain, in meat extract media, and in media of high acidities, such "previous conditions of servitude" do not appreciably affect the later fermentative activities of streptococci. The fermentative activities are markedly depressed (1) by increased temperature during the Gordon media tests, and (2) by the use of meat extract instead of meat for making the Gordon media. Morphologic Variations Tho constancy is here discussed from a physiologic standpoint, there are certain induced morphologic variations that may be of inter- est, tho they seem to be temporary only. No matter how extreme they may be, they disappear entirely with the conditions that evoked them. The most marked morphologic modifications observed in this study are produced by dextrose and the six substances (saccharose, etc.) used for the Gordon media. Of the 767 strains studied, smears were made of the Gordon media just before titration for about 600 strains. These, with the duplicates and repetitions that were made, make a total of over 8,000 Gordon slides. While the original purpose was to confirm the purity of the cultures, it soon became noticeable that there were certain differences in the morphologic characteristics that were as indicative of a given medium as of the various streptococci themselves. Most strains which grow well in these Gordon test substances tend to show the following changes : (1) Marked increase in chain length or a very marked decrease. A strain ranging normally in plain broth from 30 to 20 units to a chain usually shows occasional to many chains of 60 to 100 (or more) units, with varying numbers of shorter chains of 30, 20, .50 units, etc. ; or a chain ranging from 8 to 12 units will often, in Gordon media, have 8 or 10 as its highest limit, with almost all of the organisms in com- binations of 2 and 4 units, or even reduced to 2's and single units. (2) With heavy growth, there is also a tendency to form new organisms at right angles to the usual plane of division, so that the ordinary linear or chained arrangement is often broken by one or more cells which have divided in this way. Escherich ( 1886) pictured 28 Jean Broadhurst intestinal streptococci characterized by such division. The short- chained forms from the intestines and feces (and they are usually short-chained) are very readily induced to show this less common type of division. When this change is accompanied by a tendency to reduce the chain length, we have an accumulation of small clumps of three to six or eight organisms that present anything but the customary streptococcus slide. (3) The morphology of the individual cell may be likewise affected. In media not utilized, some of the chains are usually of full or increased length, and there is usually a small proportion of swollen organisms (rounded and elliptical) either in short chains or interposed here and therfe in chains composed mainly of normal organisms." When the media are utilized, there is, besides this swelling, a distinct tendency to abnormal shapes. The cells are often elongated, two to four times the natural length. This is probably due to a failure to divide promptly; careful focusing will usually bring out some indication of the units represented in the rod-like objects. Often, however, more varied forms are seen — organisms which may be actually pear-shaped, club-shaped, or obtusely diamond-shaped. These changes are most marked in mannite, tho they may occur in the other media. The swollen and undivided forms, without these abnormal shapes, are more often found in saccharose, lactose, or salicin; perhaps less often in raffinose. A normal appearance is effected at once, however, by transplanting a mannite culture to plain broth; the abnormal charac- ters reappear as promptly when mannite feeding is resumed. Some of the intestinal forms show on isolation a tendency on the part of the units in the occasional pairs to lie so that one side of the pair forms ^ an angle of less than 180 degrees. This tendency is increased by Gordon media which the strain can utilize. Changes in morphology have of course been previously reported for other bacteria, due to hypertonic salt solutions, phenol, etc. I have not seen any . references to such changes in streptococci with sugar media, tho it must be well known. In several instances, dextrose cultures showed decided capsules (Hiss method) and the plain broth cultures did not. This was marked in some of the strains possessing wide, hazy capsules. Strains having apparently no capsules were grown, in dextrose broth; two-day dex- trose broth ' cultures of these (24) strains showed very decided capsules. Following this lead, I made forty duplicate inoculations (1) , Environmental Studies of Streptococci 29 into serum broth and dextrose broth, and forty others (2) into plain and into dextrose broths. The results were not consistently in favor of the serum broth ; dextrose broth seemed more favorable than plain, tho not without exception. Altho dextrose and serum broth may bring back a lapsed capsule, the indications are that they do not cause a capsule, except as they encourage a more vigorous growth.* Finally, the common statement that reduction in chain length of streptococci follows cultivation on artificial media might be referred to here. I have noted no tendency of the kind in nearly 150 strains ' continued on artificial media for periods ranging from fifteen days to nearly two years. Practically every strain (including those from pathologic conditions) gives in plain broth or in the Gordon media the chain length originally given in the respective media. If meat extract had been used for my work, I feel sure this would not have been true, and I might then agree with the popular statements regard- ing not only the rediiction in chain length, but the difficulty in keeping the streptococci alive. f CONCLUSION Morphologic characters often vary greatly with the media, but such induced variations are apparently temporary only. Constancy as Affected by Variations in Environment Designed TO Induce Changes in Streptococci The wider fermenting range of the saprophytic, intestinal, and fecal streptococci when compared with those known to be of patho- genic origin, and the intermediate character of many of the mouth streptococci, suggested the following questions: What is the natural or usual habitat of the non-fermenters, the low fermenters, and of the strains fermenting but one or two substances ? Do such strains fail to get past the throat- region ? Or do such strains gain fermenta- tive power by a stay in the alimentary canal, as we have already seen that they may when kept on artificial media? In this connection, several experiments were made with strepto- cocci peculiar or limited in their original fermenting powers to see whether these powers could be materially and permanently changed or increased by subjection to various influences found in the animal * This has been confirmed by Miss Edna Twichell, of Teachers College, in a compara- tive study of about two dozen strains of streptococci. 1 1 had practically no trouble in this connection. Some from blood, etc., were exceedingly difficult to isolate, growing well in broth, but absolutely refusing to grow on agar (or vice versa). But once isolated, I have lost of the hundreds studied less than ten strains of those I desired to continue. 30 Jean Broadhurst body. These experiments fell into two lots: (1) those in vitro sub- jecting streptococci to such substances as saliva, milk, and intestinal extract; and (2) experiments with living animals in which streptococci were (a) fed by the mouth (with streptococci-free food or in celloidih capsules) or (b) inserted in capsules directly into the intestines. A complete list of these experimental environments follows: 1. Hydrochloric acid. 2. The alimentary canal, streptococci having been given (a) with food or (b) in celloidin capsules, the capsules being inserted directly into the small intestine and body cavity (regained at autopsy), or given by mouth (regained from the feces). 3. Milk, raw, and heated to various degrees. 4. Intestinal extract (a) from young kittens and (b) from fetal pigs. 5. Pure cultures of other bacteria. 6. Saliva, (a) normal and (b) heated. HYDROCHLORIC ACID Hydrochloric acid is listed here because it is one of the conditions suggested as probably determining the type of bacteria found in the intestines, either by acting as a selective agent or by modifying the streptococci during their stay in the stomach. Ten strains were subjected (1) to water plus 0.2, 0.4, and 0.6 percent hydro- chloric acid, and (2) to broth, originally neutral to phenolphthalein, to which the same amounts of acid were added. The broth plus acid strains lived longer, often surviving fifty minutes in 0.6 percent, seventy minutes in 0.4 percent, and 120 minutes in 0.2 percent. The broth plus acid medium represents prob- ably more nearly the condition in the stomach than does the water plus acid. Strains regained frofti these broth-acid conditions were not impaired in their power to ferment dextrose. Since streptococci survived* 0.2 to 0.4 percent HCl for from ten to seventy minutes, and since water alone is immediately passed through the pylorus, and carbohydrates sometimes begin to leave the stomach ten minutes after such food is taken, these results show that the effect of hydrochloric acid on the streptococcic flora of the intestine must be much less than is usually implied. It seems wise, therefore, not to accept too readily current statements concerning the germicidal action of the gastric juice on ingested bacteria. * Miss Elizabeth •Selden, of Teachers College, confirmed thfe results just reported for several of my strains. Miss Edna Twichell, of Teachers College, has since subjected 17 strains of streptococci to hydrochloric acid. The water-acid medium ranged from 0.2 to 0.7 in acidity; the broth-acid medium from 0.2 to 1.1. Nine of the 17 strains were able to survive the effect of 0.7 acid in water from ten to forty minutes; and all the strains were able to withstand 0.7 to 1.1 acid in broth from twenty to forty minutes. Environmental Studies of Streptococci 31 A STAY IN the- ALIMENTARY CANAL Streptococci were also fed to several dogs kept on streptococci-free food (milk, bread, oatmeal and water). A litter of nine hound puppies about two months old were studied for a period of nearly five months. First, an effort was made to find the range oi throat and fecal streptococci characteristic of these dogs. In this work, seventy strains of streptococci were isxslated and studied in htmus milk, gelatin, blood agar, Gordon media, etc. The dogs were then kept for nearly three months on streptococci-free food. No attempt was made to free the air of streptococci, tho various precautions were taken- to control the introduction of new streptococci (see appendix for other details). After six weeks on streptococci- free food, one puppy was examined after death to determine the characteristic, persisting streptococci. The remaining eight puppies were divided into three groups. Three puppies kept in one run were fed Strain 29, two in another run were fed Strain 11, and the other three were kept in a third as controls. Strain 11 was a non-fermenting strain of not very pronounced character; it was chosen to see whether it might not persist as a non-fermenter in the stomach, intestines, etc. Strain 29 was an "unusual one, chosen because it was not at all like the alimentary streptococci of the animals previously examined (six cats, three dogs, one pigeon, two chickens), nor like any fecal streptococci found in the 160 odd strains previously isolated from man, horse, cow, cat, and dog feces.' It differed from all stomach and intes- tinal streptococci in that it formed rather long chains (thirteen to forty units on plain broth, 100 to 200 in sugar media), had an unusually broad, hazy cap- sule (Hiss stain), cleared rapidly in plain broth (with numerous fine, soft granules), did not affect litmus milk, and (in my experience) had an unusual fermenting combination, saccharose and salicin only. A throat strain often shows one or more of these characters, but not the whole complex; none of my earlier studies of the alimentary and fecal bacteria of cats and dogs (about 200) showed any streptococci at all resembling this strain. The streptococci were poured on sterile (170 C.) crusts, which were fed to the respective dogs. E^ight days after the third and last feeding (seven-day intervals), the dog was examined after death and samples (thirty to forty-five) taken from various parts of the alimentary canal (mouth to large intestine). From these, isolations were made, and from twenty to twenty-five strains selected for study in the various media — milk, blood agar, gelatin, Gordon media, etc. It was found impossible to keep the outdoor cages entirely free of flies, and so but six of the dogs were examined at autopsy (two for Strain 11, two for Strain 29, and two controls). No non-fermenting strains were recovered from the dogs fed with Strain 11. The two fed with Strain 29 were each killed as usual eight days after the last dose of streptococci. Strains which I feel sure were the original 29 were gained from one or more samples from the esophagus, cardiac sphincter, fundus, and duodenum. Strain 29 had apparently retained its medium chain 32 Jean Broadhurst length (often 20 to 30 units), its broard capsule, and its way of clear- ing in broth — all of "which are unusual for alimentary streptococci, at least below the cardiac end of the stomach. It had changed however in several striking ways, having now gained the power of coagulating milk, of fermenting lactose, and of hemolyzing blood — the latter not being considered common in intestinal strains. I realize perfectly that many would feel that it was very unlikely that I had ever regained my original strains. I have no doubt of it myself, however, having experimented with Strain 29 for over four months in an endeavor to induce certain changes and knowing how much unlike the usual alimentary streptococci its retained characters are. Most striking of all was its appearance on agar, a curious, thick- ened to hardened or branching center, with a thin margin, and, with age, a concentric-ringed appearance, the margin finally becoming (under certain conditions) thicker. This is not at all characteristic of intestinal streptococci. The next summer similar feeding experiments were made with two ten- day kittens. They were kept wider much more carefully controlled conditions, but they need not take space here, as, after autopsy, the control cat yielded the usual range of streptococci, while the streptococci-fed cat (also Strain 29) did not yield streptococci in any of the twenty samples taken, even tho/the cat was fed a 1 c.c; dose twenty-four hours before it was killed. This absence of strepto- cocci is most unusual, if judged from the eight other cats previously examined. In the later work, known streptococci were grown in little celloidin capsules (or parchment-tipped, glass capsules) and inserted in the intestines of two healthy dogs. Duplicates were at the same time placed in the peritoneal cavity. Dr. Max Pickens generously performed the operations. One dog died in two days. The other was killed eight days later. In both cases, the capsules had been invaded by foreign organisms and showed no streptococci. Later, a better method of preparing the capsules was found, and the next year a collie dog was used in another feeding experiment. This dog was fed three meat-wrapped celloidin capsules, each contain- ing dextrose cultures of the same Strain 29. After three days, the capsules were recovered "from the feces, opened, examined microscop- ically, and streaked on agar plates. The contents of the three capsules when titrated showed four times the acidity of control capsules ; exchanges with some of the alimentary canal substances were therefore assured. Two capsules were found to be sterile ; the third contained a pure culture of streptococci. Environmental Studies of Streptococci 33 The streptococci regained from this capsule had gained* (Table 9) the power to ferment lactose, raffinose, and inulin. They also turned litmus milk pink and coagulated it. These recovered Strains 29 still cleared in broth, and, on dextrose feeding, showed the very broad, hazy capsules. Even more striking was the retention of the peculiar character of the agar colonies. There is no room for doubt as to the verity of the strain in this case. And it must be admitted that the changes claimed for the regained Strain 29 in the original feeding experiment described are at least probable. Seven fishings were made from the celloidin capsule streak plate, and all agreed in the characters described. Two of these fishings were continued on agar in the usual manner for ten-day transfers and when tested (5 months later) in their Gordon activities were constant in all the characters acquired during their stay in the alimentary canal. Streptococcus 29 Before AND After TABLE 9 A Stay (xn Celloidin Capsules) IN TriE Alimentary Canal Agar Colony Broth Litmus Milk Blood Saccha- rose Lac- tose Sali- cin Raffi- nose Man- nite Inu- lin Strain 29 originally and at Time of Exper- iment Strain 29 Radiate center; thin margin; con cen trie ringed with age Radiate center; thin margin; concentric- ringed char- acter empha- , sized Clearing in 2 days No change Acid, co- agulated No color Hemo- lyzed + 4.7 + 4.6 0.1 -1- 4.5 -1- 3.5 -1- 4.5 , 0.1 -1- 4.3 0.0 0.0 0.1 -1- Regained from Capsules 1.1 This increase by Strain 29 in fermentative range is in line with the greater range of fermentative activity shown by intestinal forms. milk Meanwhile, Rosenow had described a transformation produced on Streptococcus mucosus by milk baths of raw, unheated, sterile milk. Strains after "one soaking" in sterile, unheated milk regained "disap- pearing characters" (such as the capsule) characteristic of septic sore throat streptococci. These effects were less marked with incubated milk, still less when pasteurized milk was used, and absent with auto- claved milk. * In this line, it is interesting that five months after this experiment and twenty months after isolation, the stock culture of this Strain 29 also began to ferment raffinose and inulin, tho not lactose (see Table 11). 34 Jean BROADHUiRST I secured a few dozen samples of sterile milk, milked directly into sterilized tubes, from the Walker-Gordon Dairy at Plainsboro, N. J. Two sets were kindly given me, one set of 16, in which 14 remained sterile, and a second set of 70, including 57 sterile tubes. With these I tried out several duplicates of Strain 29 to see whether this strain, which in many ways resembles the septic throat streptococci described by several workers, could be induced to change in any way. The only effect was upon the agar plate characters, more colonies showing the concentric, thicker-ringed type instead of the original curiously centered type. INTESTINAL EXTRACT To test still further the effect of various conditions or substances found in the alimentary canal, seven strains were subjected to intestinal extract from young kittens, and, later, two other strains to intestinal extract from fetal pigs. No changes were obtained in the first experi- ment. Tho freshly made, the extract reduced lactose but feebly. TABLE 10 Streptococcus 26 Before and After Subjection to Intestinal Extract Odndition Agar Colo- Dies Gelatin Litmus Milk Blood Agar Saccha- rose Lac- tose Sali- cin Kaffl- nose Man- uite Inu- lin Before Aug. 21 Usual No growth in 10 days No change No color + 2.5 0.0 0.1 -1- 2.1 + 2.0 ■f 1.8 Alter Sept. I Usual In 18 hours Slightly acid; not coagu- lated Slightly greenish 0.2 -1- 2.0 -1- 4.2 0.1 + s.o 0.1 The pig extract, obtained from five fetal pigs sent by Jacob Dole of Buffalo, reduced lactose readily. Several tubes of this extract were made and examined for bacteria. In none were streptococci ever seen ; large cocci occurred in a few tubes, and one or two contained rods. Capsules containing twenty-four-hour dextrose growths of the two different streptococci (29 and 26) selected wefe suspended in this extract. After two days, these capsules were examined and streaked as previously described. Each one yielded a pure culture on the stained slide, and apparently streptococci only on the agar streak plates. Strains were isolated from these streak plates and tested in the usual media — gelatin, blood agar, litmus milk, Gordon media, etc. In one only,^ Strain 26, were changes found (Table 10). These were so peculiar that an unused tube of fetal extract was examined for sterility. Environmental Studies of Streptococci 35 new capsules of Strain 26 prepared, and the experiment repeated, with the same result. All of the eleven fishings tested had lost two of their original powers, saccharose and inulin, and gained two new ones, lactose and salicin. The complex thus became lactose-salicin-mannite, a common intestinal complex. SALIVA Following this, experiments with saliva were tried in a similar manner. Table 11 gives the results for two of these strains after subjection (in celloidin capsules) to saliva. TABLE 11 Titration Records of Strains of Streptococcus (in Celloidin Capsules) Subjected to Saliva Saccharose Lactose Salicin RafEnose Mannite Inulin Strain 29 originally + + 4.7 0.1 3.5 0.1 0.0 0.1 + + + 3,9 0.0 4.8 0.2 2.5 0.1 + + + + Strain 29 subjected 4.0 0.1 4.3 3.4 0.1 4.7 to saliva (1 'da.). + + + showing various 3.4 0.1 4.6 0.0 0.2 4.9 resulting types + + + 2.8 0.1 2.5 0.1 0.1 4.8 , + + ■ + ■ 3.4 0.0 2.4 0.1 0.1 5.0' Clianges appearing in Strain 29 after + + + H- 5.4 —0.1 4.7 2.7 0.2 4.9 20 mos. constancy + + + + ■ 5.4 0.2 4.6 3.1 0.3 4.8 Strain 29 subjected + + + to another sample 0.1 0.1 4.5 3.3 0.2 51 of saliva, while + , + + + still constant to 5.5 0.1 4.5 3.2 0.3 4.2 original fermenta- + + + + tive complex. 5.3 0.3 4.5 3.1 0.0 4.8 yielding + + + + 5.2 —0.2 4.3 3.0 0.2 3.4 Strain 42 originally + + + - 2.2 2.5 0.1 2.6 0.5 0.2 + + ■ + + + 3.8 3.0 2.7 3.3 0.4 1.3 + + + Changes appearing 3.5 3.0 3.8 0.1 0.1 0.1 in Strain 42 after + + + , similar subjection 3.3 2.6 2.9 0.1 0.1 0.1 to saliva + + + 3.2 3.0 3.1 0.1 0.1 0.2 + + -r , 2.9 2.5 2.7 0.1 0.2 ' 0.1 In Table 11, it will be noted that in both strains often more than one type resulted, a circumstance suggesting that the buccal conditions were sufficient to initiate change — to upset the original character com- plex, without fixing one set of characters upon the subjected strepto- cocci. 36 Jean Broadhurst Duplicate capsules of several different strains of streptococci were treated with heated and unheated saliva (same sample), to ascertain whether the results obtained by Rosenow with heated and unheated milks could be paralleled. Some differences were found, but they were mainly negative, such as the loss of lactose in the unheated saliva strain, while the heated saliva strain retained its lactose-fermenting character. In most of the cases, neither the heated nor the unheated saliva straitjs varied at all from the stock control. This was probably due to the fact that most of these strains were old stock cultures. I had difficulty about this time in isolating or obtaining elsewhere fresh cultures with limited fermentative complexes; with such strains I should expect differences in the heated and unheated experiments. OTHER BACTERIA About a dozen other cultures in similar capsules were then sub- jected to the influences of other known bacteria (pure cultures). The ■results were sometimes negative, sometimes a mixture of the charac- ters ; and they sometimes showed a character or power not character- istic of either original strain! (Strain 42 subjected to 29, Table 12). I failed to induce or to inhibit hemolysis in these experiments. TABLE 12 Titration Records Showing Effects of Streptococcus 29 (in Capsules) on Streptococcus 42 Saccharose V Lactose Salicin Raffinose Mannite Inulin Strain 42 ^- + + 3.1 2:7 0.1 2.7 0.5 0.1 Strain 29 -1- + 4.7 0.1 3.5 0.1 0.0 0.1 + -1- + ' 3.1 2.0 0.1 2.7 0.5 0.4 Strain 42 after sub- + -1- + jection to 29 3.6 3.3 3.1 0.1 0.1 0.0 (various fishings) + -1- + 2.7 3.1 0.2 2.9 0.7 0.3 4- + + + 3.4 3.2 3.5 0.1 3.6 0.3 • Summary The experimental work described was undertaken to test the con- stancy of the streptococci, or to induce changes in their reactions in various media. The experiments fall, as indicated, into two main divi- sions : The first division includes those dealing with relatively simple physical or chemical factors or influences, such as temperature, Environmental Studies of Streptococci 37 acidity, and various artificial niedia. When these did affect the organ- isms, the effects were usually of a depressing or inhibitory type ; these changes, which were usually dififerences in amount rather than in kind, . were apparently temporary only. The second division includes the effects of such influences as raw milk, saliva, intestinal extract, and other bacteria; these might be described, by way of comparison with those of the first division, as vital, not merely physical or chemical. The changes produced by these environmental factors differ, not only in being constant, but in being active, rather than merely passive, and in initiating previously unexhibited powers, instead of merely depressing or inhibiting former activities. They may arise as "muta- tions," when the conditions have been apparently (but probably only apparently) unchanged, as in the changes appearing with age. Or they may appear as responses to varying environmental conditions, to various "vital" stimuli, such as saliva, intestinal extract, substances in the alimentary canal, etc. The very short time necessary in Rosenow's work with milk gives increased interest to this view of environmental factors as "vital." In his work the amount of change in the streptococci was inversely proportional to the amount of heat to which the milk was subjected. In connection with these milk changes, it may be of interest that Houston of London (as he told me in 1910) uses .very low temperatures for sterilizing* his milk media, because he found that high temperatures gave, in a given set of cultures, a lower percentage showing coagulation. Granting that both of these workers were really working with sterile milk, we may point out that in both cases it is what might be called the "liveness" or degree of "liveness" of the milk that seems to be directly associated with the increased activity of the organisms, or the availability of the media. . In this connection, it is interesting that the positive, active changes produced in the foregoing experiments are all due to similar "live" substances — saliva, intestinal secretions, and other bacteria. Unfor- tunately, the ultimate causes of these changes, whether they imply the inhibition or the acquisition of specific fermenting powers, are in that most speculative field of enzyme action, and beyond the scope of thi.s paper. It has been shown by various workers (1) that pathogenic strains are more limited in their range of physiologic activities than related 38 Jean Broadhurst non-pathogenic strains, ahd (2) that strains may become so limited* by animal injection (e. g., Klotz (1905) for B. coli). My work, on the contrary, emphasizes the broadening of the range of physiologic activities (1) during periods of cultivation under ordinary saprophytic conditions, and (2) during subjection to the influences to be found in the alimentary canal. Since hydrochloric acid (in the percent characteristic of animal stomachs) is ineffective as a germicidal agent, and since strains of limited physiologic ability may persist when bathed in the animal secretions '(saliva, intestinal extract) and take on the wider characters of saprophytic streptococci, it is at least probable that such modified, or transformed, pathogenic streptococci may be not uncommon inhabitants of the alimentary canal,f the alimentary canal forming what might be loosely called an "alternate" habitat. This cyclic theory for streptococci of such varying fermentative powers is supported by the occurrence of hemolyzing strains in normal intestines and feces. Conclusions Two types of induced changes are found. The first type embraces quantitative, temporary changes, in which unfavorable temperatures or the presence or absence of suitable food materials affects directly the physiologic activities of the organisms. With sufficient depression of a physiologic power or powers, a quantitative change may appear as a qualitative one, especially when qualitative measurements are applied, as in the litmus test for acids formed. These changes disappear with the condition that evoked them. The second type of change is not so mechanical, nor so passive, as the first. It is, on the contrary, active, initiatory, and not to be prophe- sied. It is illustrated, in my experiments, by the marked changes often produced by saliva, intestinal secretions, and other substances found in the animal body. These changes are more varied, qualitative (and often quantitative, also), and apparently rather constant. (All tested ( 1 to 2 months) were constant in their acquired characters.) * Holman (p. 293) thinks that the natural invasive streptococci are not sufficiently con- sidered in some of these animal passage experiments. His "regained" streptococci usually fermented salicin only. t The streptococci of the alimentary canal are usually much shorter-chained than the accepted form for Streptococcus pyogenes. Yet two normal dogs yielded throughout the alimentary canal strains that were very like S. pyogenes in morphologic characters. These strains had, however, a wide fermenting range, fermenting all six of the Gordon substances, and they did not hemolyze blood. Environmental Studies of Streptococci 39 II. STREPTOCOCCAL CHARACTERS AS RELATED TO ORIGIN OR HABITATS The work described in the first part of this paper on constancy of the fermentative reactions has two bearings on the possible dififeren- fiation of streptococci according to their origins. ( 1 ) Such differences, if they exist, might be expected to show after isolation and in the various test media. (2) Differences induced (e. g., by means of saliva, intestinal secre- tions) do not necessarily affect the diagnostic value of the Gordon tests and other differential media. If a stay in the alimentary canal, for instance, can induce changes in the Gordon records of streptococci, the possible diagnostic value of such tests is not impaired, but TABLE 13 Human Fecai- Types op Streptococci Isolated from the Same Individual at Varying Intervals fTHau One Year Human Feces Sample 1 Sample 2, yielding A and Sample 3, yielding B and Sample 4, yielding D and Sample 5 Sample 6, yielding E only Type B C D E Saccharose + + ■ 4- b Lactose + 6 + + + + + Salicin + + + + Raffinose Mannite Inulin + + + ■ + + increased. That is, of course, unless all saliva, all intestinal secre- tions, etc., act in exactly the same way, yielding (1) the same types therefore in any given animal at all times and (2) the same types from all animals. The experiments already described, with saliva, intestinal extfact, etc., contradict such an assumption. Altho there may be a great similarity in the strains isolated from the various regions in a given animal. Table 13 of strains from one individual through a period of several months also contradicts this assumption. Similar variations were found in seven other animals (throat and feces). The one given covers the longest period (12 mo.), and includes seven different fermentative types. We also know that in disease, and with age, various foods, etc., the ifora of the alimentary canal changes very rapidly and completely. If, therefore, as seems undoubted, saliva and other body secre- tions have the power evidenced in this experimental work, the ease 40 Jean Broadhurst or rapidity with which they afifect the streptococci, will influence the phase at which we catch throat, stomach, intestinal, and fecal strepto- cocci. But having isolated them in a definite way (e. g., with extract or with meat media, or with or without selective media, such as lactose litmus agar) we may expect the results in various labora- tories to be comparable. With the range in a given individual as varied as in the case described in Table 13, it looks as tho the normal ranges of the strepto- cocci (mouth, fecal, etc.), from different species (cat, dog, horse, cow, and man) must necessarily overlap. Still, hoping to verify the predictions of earlier workers, I have studied 767 strains of streptococci isolated from various sources. Usually one to three were taken from each sample; in animal autop- sies, in which about twenty strains were usually studied from each animal, but one to three were taken from any one locality (e. g., fundus end of stomach), and often but one from any sample from a given region. ^ * PLANT, SOIL, AND WATER STUDIES An effort was first made to widen the range of known habitats. German bacteriologists (e. g., Puppel, 1912) speak very casually of "hay" and "fodder" as among the sources of the streptococci found in milk. Prescott (1906) reports streptococci as occurring on hay and grain (8 rye, 6 oats, 3 buckweat, and 1 wheat). Streptococci are known to be present in appreciable numbers in sewage- polluted water. Soils are also mentioned as habitats. Dr. V. A. Moore tells me that he frequently found streptococci in garden soil in Washington. The gardens, however, had b^en heavily and frequently manured. Besides this, there is very little definite information available. Andrewes (1906) states that, in nature, streptococci cannot grow and multiply for any length of time apart from the human body. Eighty-four samples of hay, grass, and leaves from country roadsides, pastures, park, and garden paths were examined.* Streptococci were seen in but two cases, and three strains were isolated from the recently cut side of a hay stack. Soil and water from wood edges, moist roadside banks and brooks, woodroad humus, and near tie posts at mills,„ etc., were examined ; of these eighteen samples, one sample of water (country roadside overflow) yielded a short-chained coccus organism (six to eight cocci in favorable media). The other strains studied during the past four years were obtained from the following sources: (1) Human throats, chiefly diphtheria suspects; (2) alimentary canals (mouth to large intestine) of various animals — ^hen, pigeon, cat, and dog; (3) feces of cat, dog, man, cow, and horse; (4) milk, including a few mastitis suspects; and (5) blood (glanders and anthrax specimens), antisera, and pathologic conditions. A few pathogenic strains (fifteen) were obtained from other laboratories. * Miss Bernice Replogle, of Teachers College, also examined 25 plant samples and 23 soil samples, finding streptococci but twice, once on a blade of grass near a Bronx Park path, and once on a clover leaf from the Columbia Campus; and Miss Henrietta Lisk, also of Teachers College, examined 75 samples from plants and 7^ from soils, finding strepto- cocci but two times, once on a house fern, and once on a dandelion near a college entrance. Environmental Studies of Streptococci 41 When I began my work in 1911, the types of streptococci found in milk had been sought for in 'human throats and in the feces of the horse, the cow, and man. Bovine mouth streptococci are doubtless also important. And dogs, cats, and other small animals contribute also to the streptococcic flora of milk, even on many of the "inspected" farms. In the hope of confirming the diagnostic value of streptococcic reac- tions, 752 distinct strains were isolated and studied ; and fifteen strains from other laboratories were compared with these. One hundred milk strains (Broadhurst, 1912) were studied in meat media for the Gordon reactions, and in litmus milk and neutral red broth (anaero- bically) ; and 113 in meat extract media for the six Gordon reactions only (Table 14). The remaining 554 were studied more fully : ^first, for staining qualities or differences with stains — methylene blue, carbol- f uchsin, Jenner's stain. Gram's stain, and Hiss's capsule stain ; secondly, in the six Gordon media, as described (arabinose also tried, but dropped as evidently not of value), smears for microscopic exami- nation being made from each tube just before titration; thirdly, in nine other media (Table 15), in the hope that one or'more would be itself definitely of value, or aid by making distinctive complexes — for example, yielding certain Gordon combinations plus litmus milk changes, but without blood hemolysis, and without the power of grow- ing at room temperature in gelatin. The other nine media used for these 567 strains included agar plates for colony characters ; agar stabs plus paraffin oil for anaerobic growth; gelatin for liquefaction and ihe ability to grow at room temperature ; plain broth varying in acid- ity, plus 0.5, plus 1.5, and plus calcium carbonate; a variation of Todd's neutral-red medium; blood agar (horse blood) ; and litmus milk. This made a total of five stains and fifteen media. Sugar-free broth and fermentation tubes of dextrose were also used for a small part of these, but discontinued later, as hot of value. Observations of these media were made after a lapse of eighteen hours, twenty-four hours, two days, and three days ; the litmus milk, gelatin, and broth observa- tions were also examined after five days, and ten days, and the gelatin usually after fifteen days, also. After these 554 strains were completed, it was decided to drop all routine stains except carbolfuchsin, and the Hiss capsule stain. Media observations were continued for Ijtmus milk, blood agar, gelatin, plain broth (+0.5), agar plates', and the six Gordon media, the others* * Neutral-red has been discarded as net diagnostic by almost all workers. Todd (1910) and Crowe (1912^13) described distinctive colony characters on neutral-red agar. I tried 100 milks strains in neutral-red broth anaerobically and' about 400 from other sources aerobi- cally on two modifications of Todd's neutral-red agar without securing anything of differential value. Anaerobic conditions, fermentation tubes, and oil-covered agar stabs were also d-s- continued after our 400 strains had been studied. TABLE 14 Titration Records for 113 Strains of Streptococci Tested in Meat Extract Media Milk Strain , Sariiple Saccharose Lactose Salicin Eaifinose Mannite Inulin 1 1 2.2 2.2 2.8 0.2 2.0 1.9 2 2 1,2 ~ 1.5 0.4 1.2 0.0 0.5 3 3 —0.2 • 3.3 0.2 0.0 0.2 0.1 4 4 —0.1 1.2 0.2 —0.2 0.0 0.1 5 5 —0.3 0.1 0.0 —0.1 —0.2 —0.1 6 6 —0.2 0.2 ■ —0.2 —0.1 —0.2 0.0 7 7 —0.2 3.0 —0.1 0.0 0.0 0.0 8 8 2.2 1.8 2.6 0.1 2.0 . 1.8 9 9 2.3 1.9 2.6 0.4 1.8 2.1 10 10 2.1 1.9 2.2 0.4 1.9 1.9 11 11 1.7 1.8 1.9 0.2 1.5 1.8 12 12 2.7 1.8 2.9 0.5 2.1 1.7 13 12 2.0 2.9 1.9 0.5 1.4 1.6 Bovine Feces 1 1 3.6 3.5 3.4 0.4 0.0 0.1 2 2 2.9 3.3 3.2 3.3 0.0 2.2 3 3 3.0 3.2 3.0 3.2 0.0 2.6 4 4 2.9 2.6 3.5 3.1 0.0 3.1 5 5 2.8 3.2 3.2 3.1 0.1 3.0 6 6 2.6 2.9 3.7 3.6 0.2 0.3 7 6 2.7 3.0 3.9 3.2 0.2 0.4 8 7 1.8 3.3 4.2 0.4 2.2 0.3 9 8 2.6 3.5 4.1 0.4 2.4 0.2 10 8 2.5 3.5 4.2 0.5 2.3 0.2 11 9 3.1 2.7 2.9 3.1 0.1 3.6 12 10 2.8 2.8 2.8 3.4 —0.1 2.9 13 11 3.6 3.7 3.2 0.2 0.0 0.1 14 12 0.1 0.3 0.0 0.5 0.0 0.1 15 13 4.1 3.5 ' 3.4 1.9 0.0 , 16 14 1.8 2.2 2.3 1.7 1.4 0.9 17 15 2.2 1.7 2.7 0.9 1.9 0.1 18 16 3.6 4.8 3.4 0.3 1.3 0.0 19 17 2.7 2.4 2.9 3.0 0.0 4.0 20 18 2.3 2.3 2.5 1.4 —0.1 3.9 21 18 2.4 2.5 3.3 2.8 0.0 4.1 22 19 3.4 2.8 3.« 0.2 2.2 0.1 23 20 0.1 0.0 -0.1 0.0 0.1 —0.2 24 21 0.0 —0.1 0.1 — 0.1 0.1 —0.1 25 22 2.8 2.8- 2.9 2.4 0.2 4.8 26 22 2.4 2.3 0.5 2.6 0.0 4.0 27 20 —0.1 —0.2 —0.1 0.0 —0.2 28 20 —0.2 —0.3 —0.1 —0.1 — 28 20 0.1 — 0.1 —0.1 —0.2 .^ 29 21 —0.2 —0.3 —0.2 —0.1 30 21 —0.1 —0.2 —0.2 —0.1 — Equine Feces . 1 1 0.3 0.0 1.0 0.1 0.2 . 0.2 2 2 2.6 2.8 3.2 0.2 1.5 0.2 3 3 2.8 2.9 3.5 0.5 1.7 0.1 4 4 0.9 0.5 0.6 —0.1 -0.2- 0.0 5 5 1.9 1.8 2.0 1.5 —0.2 1.7 6 6 —0.1 0.1 —0.1 —0.1 —0.2 0.0 7 7 1.0 0.1 0.9 0.0 —0.2 —0.1 8 8 0.5 0.3 0.8 —0.2 —0.1 0.1 9 9 —0.2 0.2 1.3 ,—0.1 —0.4 0.1 10 9 2.6 2.9 3.0 1.4 1.5 0.2 11 10 1.5 0.4 2.0 0.2 0.2 0.2 12 11 ).S 2.0 0.9 1.5 1.6 0.0 13 12 1.8 2.2 1.9 1.4 1.5 0.8 14 13 1.4 ■ 0.2 0.5 0.0 —0.1 0.2 15 14 —0.1 0.3 0.2 0.0 0.1 0.1 TABLE 14 — Continued Equine Feces — Continued Strain Sample Saccharose Lactose Salicin RafRnose Mannite Inulin 16 14 3.3 3.5 3.4 1.3 2.1 0.3 17 IS 3.4 2.7 3.2 0.5 2.0 0.1 18 16 3.4 3.3 3.3 0,4 1.9 0.2 19 16 3.5 3.5 3.4 1.5 2.0 0.2 20 17 0.9 0.1 1.0 O.O —0.1 0.2 21 18 1.4 0.2 0.5 2.0 —0.1 0.2 22 18 —0.1 0.1 0.5 0.0 —0.2 0.1 23 19 4.8 5.3 CO 4.9 0.8 3.4 24 20 2.0 2.6 2.4 2.1 0.0 1.3 25 21 1.8 2.4 2.5 1.5 1.8 1.1 26- 22 2.9 0.1 3.7 2.4 —0.1 2.8 27 23 2.9 2.6 3.1 0.5 1.7 0.0 28 23 2.3 0.2 2.6 0.2 0.0 2.4 29 24 2.1 1.8 3.1 1.9 -0.1 2.8 30 24 2.5 2.5 1.7 2.0 —0.1 0.2 31 25 1.5 1.8 2.4 1.4 1.4 1.2 32 26 2.9 2.7 3.3 0.5 1.8 0.1 33 27 1.6 1.7 2.1 1.5 1.6 1.5 34 28 1.5 1.6 1.0 1.4 2.0 1.2 35 29 1.1 0.2 1.2 . 0.5 0.0 0.0 36 29 2.1 0.1 1.9 0.3 —0.1 2.6 37 30 1.6 1.8 1.2 1.6 1.6 1.4 38 30 1.5 2.2 1.4 1.4 ■1.6 1.4 39 31 2.8 1.6 3.1 2.6 0.0 3.2 40 32 1.0 1.9 2.6 0.9 . 1.7 0.9 41 33 1.5 1.9 1.0 1.1 1.7 1.7 42 34 3.3 0.2 2.7 '2.3 0.0 1.7 43 34 0.1 2.6 4.2 0.2 0.1 0.2 44 35 1.7 1.3 2.4 0.5 0.0 2.6 Human Feces 1 1 3.0 3.3 4.2 0.4 2.6 0.3 2 1 0.0 3.5 4.0 0.1 1.1 0.6 3 2 1.5 2.7 3.3 0.2 2.0 0.0 4 2 0.0 2.6 2.7 0.0 1.4 0.1 5 3 0.0 3.2 2.9 —0.1 0.0 0.0 6 4 4.0 2.8 3.5 0.2 0.0 0.2 7 5 2.2 1.4 3.6 0.2 0.3 0.0 8 6 0.0 3.0 3.1 0.1 0.0 0.1 9 • 7 3.8 2.6 2.7 0.1 0.0 0.1 10 8 2.2 0.5 3.9 0.3 1.6 0.3 11 9 2.7 3.1 3.1 0.0 1.5 0.3 12 9 3.5 3.2 3.2 0.0 2.2 . 0.4 13 9 3.1 3.5 2.5 0.1 1.9 0.0 14 10 0.1 2.6 3.3 0.0 1.2 0.1 15 10 0.0 2.6 3.3 —0.1 1.6 0.1 16 11 1.1 3.2 3.3 0.1 1.5 0.2 17 12 2.3 3.3 3.1 0.2 1.5 0.2 18 12 0.1 3.7 4.0 0.0 —0.1 0.1 19 13 4.0 2.7 4.0 ' 0.3 1.1 0.0 20 , 14 3.2 2.6 2.9 0.2 1.7 0.1 21 14 0.2 2.3 3.4 ■ 0.1 2.0 0.0 22 15 0.2 2.0 2.0 0.2 0.3 0.0 23 16 3.4 2.9 3.2 0.1 1.8 0.0 24 17 1.4 1.9 1.6 1.1 1.6 1.4 25 17 1.6 2.5 3.4 0.3 1.2 0.2 26 18 2.7 3.4 3.7 0.4 2.5 0.0 27 19 0.1 2.5 3.1" —0.1 1.8 0.0 28 19 0.1 3.0 3.2 0.1 1.7 0.0 29 20 0.1 1.7 0.8 0.5 1.5 0.2 30 20 2.6 . 2.5 '3.3 0.4 2.2 0.2 31 21 1.7 2.2 3.8 0.5 1.9 0.2 32 21 2.0 2.4 3.3 0.3 2.5 0.2 33 22 1.6 2.6 3.5 0.3 1.8 —0.1 34 22 2.2 2.2 3.5 0.4 1.2 0.2 35 23 3.4 3.0 2.7 0.2 2.2 —0.1 36 23 1.7 1.8 3.0 0.2 2.1 0.1 37 24 2.1 2.2 3.6 ■ 0.4 1.7 0.0 38 23 0.1 — 3.2 0.1 1.2 —0.1 39 25 2.6 2.6 0.1 2.4 0.2 0.5 44 Jean Broadhurst having proved of doubtful value. Some of the details for these 554 strains are given in Table 15. Many records have been omitted as apparently not important; some of those given are of doubtful value, but they are included, as other investigators may find them confirma- tory or otherwise useful. From the work outlined above there has resulted a mass of details the comparison of which is an appalling task. Even after excluding the several special series (such as the meat and meat extract compara- tive series, and the constancy tests in Gordon media), there is for over 500 of the strains an average of eighty-five morphologic and physio- logic records. To select the significant ones is very difficult. In the first place, it is almost impossible to bring such a mass of data into a form compact enough for a just consideration of the details leading to any real correlation of morphologic and physiologic characteristics. Secondly, morphologic terms are probably ultimately but an expression of physiologic powers and changes which are often unknown or immeasurable. Distinctions based upon modifiable morphologic char- acters (such as the presence of a capsule and the chain length) are at best uncertain as morphologic distinctions and may be very misleading in physiologic correlations. Thirdly, it is difficult to tell whether the sources from which the strains were isolated are themselves of classi- fying value, or whether they should be viewed only as "previous (and often temporary) conditions of servitude." Much study and many groupings and re-groupings of the data will be necessary to determine the importance (or the insignificance) of the sources from which the streptococci have been isolated. Several conclusions, however, may be drawn. Conclusions Streptococci occur less commonly in soils and water than most text-books imply. Hay, grass, etc., are apparently but temporary and accidental bearers of streptococci. Streptococci are apparently as constantly a part of the flora of the mucosa and contents of the alimentary canal in cats, cows,* dogs, and horses as in man. Streptococci vary greatly in the comparative easef with which they may be isolated from various animal sources; for example, readily * Kinyoun found streptococci in all but 2 of 2,000 calf autopsies. Andrewes (1906) reports them in the feces of fox, stoat, etc. fEase in isolation depends both on the relative number of streptococci and on the type of bacterial companions in that sample ("spreaders," etc.) ; but from repeated examinations ot broth tubes of the samples it seems probable that the numeric relation is usually the most important factor. The optimistic statements of various workers regarding the dominance of streptococci in 18 to 36 hours are certainly not true of the streptococci of the wide range of habitats covered by this investigation. TABLE 15 Selected Details of 538 Strains of Streptococci from Various Sources [Meat Media Throughout] .Strains from Plants Long- est Chain Length Seen Com- mon Cliain Lengths Clearing in Broth (10-Day Period Ob- servation). Days Agar Colonies * Visible Growth in Gelatin. Days Litmus Milk t (10-Day Period Observation) Colonies I on Blood Agar (S-Day Period Observation) Gordon Reactions strain Saccha- rose Lac- tose Sali- cin Eaffl- nose Man- nite Inu- lin 36 40 40 8 12, 8 12, 8 2, 4 3 3 None —1 —1 —1 No change No change A/2, —10 G-1- haze 0.2 0.1 3.1 0.5 0.2 8.0 4.5 4.2 3.9 0.3 0.1 2.4 0.4 0.6 2.6 0.2 41 G -J- haze 0.1 18 Usual ... ... 2.4 f 1 I 8 I 9 J 10 ill 12 (13 \ 14 16 16 (17 j 18 27 28 19 I 68 -1 64 I 55 ( 60 (61 12 12 10 40 40 40 15 12 8 8 IB 60 15 30 26 6 6 6 40 40 4,2 4, 2 2, 10 20, 8 12, 8 12, 4 4,8 2 2,8 4,8 3 4 4,8 12, 8 8, 12 8, 12 8, 12 Strains from Water (Strains from the same sample included in braces) f 48 12 12 12 12 12 4, 2 4, 2 4,2 4,2 4,2 10 10 10 10 10 —1 —1 —1 ~1 —1 A, —5 A, C-5 A, C —5 A, C~5 A, C— 5 ? Color 5.7 5.8 5.5 4.7 5.5 4.4 4.6 4.9 4.6 4.6 4.8 4.8 4.9 4.9 5.2 0.5 0.5 0.4 0.6 0.4 8.2 3.2 8.0 3.8 3.0 0.3 49 ? Color 0.2 60 ? Color 0.0 61 ? Color I 52 ? Color 0.3 Strains from Milk 5 5 None None None None None None None None None None None None None Usual, D Usual, D Usual, D Usual, D Usual, D Concentric rings Usual, D Usual, D Usual, D Usual, D Usual, D Usual, D Usual, D Usual, D Muggy Usual Usual Usual Usual Usual —1 —1 —1 —1 —1 —1 —1 —1 —1 —1 —1 —1 —1 —1 None —1 - -1 —1 1 —1 A, C— 1 A, C~l A, C— 1 A, CI A/2, 1 A, H2O, C 1 A, C— 1 A, H2O, -1 A, O ~1 A, C— 1 A, H2O, C ~1 A, H2O, C —1 A, — 1 A, — 1 No change —A, C —A, —A, A, C— 1 A, C— 1 Green Green (jrreen Green G -1-haze. Green G -\ haze. Green G -J- haze. Green (rreeii Green G -[- haze. G- -1- haze. ? Color... Green (jreen Green Green GJreen 3.8 4.7 4.4 2.9 2.9 3.6 4.1 4.5 2.8 3.5 4.0 3.9 2.8 2.2 3.1 0.0 0.2 O.S O.B 0.1 0.0 0.2 0.1 0.0 0.2 8.9 8.9 0.2 0.2 0.0 4.0 4,3 4.2 2.4 3.1 8.8 8.7 8.1 3.2 2.8 3.6 3.9 4.3 8.5 2.9 3.8 4.1 4.3 2.2 2.8 3.5 3.7 3.5 3.1 2.8 3.3 4.1 4.7 3.2 2.8 0.3 4.8 3.2 0.1 0.3 3.8 3.S 0.4 0.2 0.1 0.5 1.2 4.8 0.2 1.0 4.5 4.6 5.0 0.3 3.2 4.8 4.7 4.8 O.S 3.2 1 4.4 4.4 6.0 0.6 3.2 0.1 3.8 .-oa 0.0 0.0 0.2 4.0 0.1 -^>.2 —0.1 0.2 0.3 0.2 0.1 0.0 0.3 0.0 0.4 0.2 -0.2 0.8 0.4 0.4 0.1 0.3 0.3 0.2 0.3 0.0 -0.8 Strains from Cat Throat 67 68 69 70 71 72 (■ 85 ( 86 5 80 ) 81 ' 82 88 84 87 88 89 90 91 92 95 96 12 10 10 10 12 10 10 10 10 6 12 6 12 8 15 16 15 12 40 12 97 , 8 98 6 99 20 105 30 > 100 8 106 12 28 36 ■ 29 86 : 1 30 80 : /81 20 ( S4 200 J \ 85 80 i ,36 40 37 SO .88 20 39 20 40 20 41 42 SO 200 48 80 44 60 ( 45 80 ( 46 50 47 60 48 60 49 40 50 80 2p3 80 \ 2pll 30 J 4,8 4,8 4,8 4,8 4,8 4,8 4,8 4,8 4,8 2, 8 4 2, 4 2,' 4 2, 4 8, 4 8, 4 8,4 8,4 8, 4 8, 4 2, 4 2,4 2,4 2, 4 2, 4 8, 12 4, 2 2 12, 4 12, 4 12, 8 8, 4 8, 20 8, 20 12, 8 , 4 ,4 8,4 8, 4 8, 4 12, 8 12, 8 8,4 12, 8 40, 20 8, 12 8,4 8, 12 12, 8 20, 12 12, 20 None None None None None None None None None 5 None None None None None None None None None None 6 5 None None 5 None None None Very slight Very slight Very slight 5-10 6-10 5 10 3 3-5 3-5 3-5 10 Very slight Very slight Very slight 5 5 5 Very slight Very slight 5 Very slight Very slight Usual . Usual . Usual . Usual . Usual . Usual Muggy ? Harder ? Harder ? Harder ? Very pale Very pale Usual Usual Usual Usual Thin, irregular margin.. Thin, irregular margin.. Thin, irregular margin.. Thin, irregular margin.. Pale Thin, irregular margin.. Usual Thin, irregular margin.. Thin, irregular margin.. Thin, irregular margin.. Muggy Usual Muggy Muggy Usual ? Usual? Usual? Thin, irregular margin.. Thin, irregular margin.. Thin, irregular margin.. Muggy Muggy Muggy Concentric rings Muggy None 10 None None None None None None None 3 None None Muggy Muggy Muggy Muggy Usual, floceulent . Usual, floceulent . 3 None None None None None None None None None None None None None None None None None None None None None None 10 A/2, C 2 A/2, C 2 A/2, C 2 A/2, C 2 A/2, C 2 A/2, O 2 A/2, O 2 A/2, O 2 A/2, C 2 A/2, C 2 A/2, C 2 A/2, C 2 A/2, C 2 A/2, C 2 — A, OC — A, OO Yellow. A/2, 2 H2O top, O 2 HaO top, 2 A/2, C 3 A/2, C 3 A, —1 —A, C —A, C — A, A/2, C 2 A/2, —1 A, 00 A, OO A, C2 A, OC A, 1 A, 01 A, O A, A, A, O A, No change A, 1 A, 1 A, 1 A, OC A, 1 A, 1 A, CI A, 1 A, 1 A, 3 A, 3 Green (. Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green GJreen Green. Green Green Green G -1-haze Green ? Green ? Green ? Green ? Haze ? Haze Haze No color No color No color No color Haze Haze -}■ no color . .. No color Haze -(- hemolysis. Haze ~\ hemolysis. Green ? No color Green No color No color No color No color -I" haze ?. No color No color -(■ haze ?. No color Green Green 8.6 3.7 8.5 3.6 8.6 8.1 3.8 3.2 3.7 4.2 4.4 2.4 2.4 1.4 3.0 3.0 3.7 4.8 0.8 0.2 4.4 4.2 2.5 2.8 2.6 2.7 3.8 4.2 2.8 3.0 2.8 2.8 2.6 2.7 2.7 2.7 2.7 2.6 2.7 2.8 2.9 2.9 3.4 2.8 8.1 3.0 2.9 2.9 2.7 1.7 2.5 8.7 3.6 8.8 4.2 8.7 3.4 8.5 8.4 8.7 4.6 4.6 2.5 2.6 2.5 2.9 2.7 1.0 4.0 1.7 2.2 2.8 8.0 2.8 2.6 2.1 2.8 8.9 2.9 2.4 2.5 2.7 2.6 2.8 2.4 2.1 2.5 2.7 2.7 2.5 2,9 2.8 2.8 2.9 2.6 2.8 2.8 2.8 2.8 2,9 2.4 2.7 4.1 4.8 4.2 4.1 4.3 3.6 3.7 8.7 4.1 2.8 3.0 0.2 0.1 0.1 8.0 3.2 4.7 4.1 4.6 4.8 4.2 4.4 3.9 2.8 2.1 1.7 8.7 2.9 1.9 1.9 2.6 2.0 2.8 2.7 1.8 1.9 2.0 1.7 1.9 2.0 2.7 2.9 2.6 1.7 2,7 8,0 2,8 2,6 8,0 2,7 2,7 0,0 0,1 -0,1 0,0 0,0 0,1 0,0 0,1 0,0 2,7 2,5 0,0 0,0 0,0 0,2 0.1 0.2 O.S 0.2 0.2 8.4 3.4 4.7 0.2 0.3 0.0 0.0 4.3 0.2 0.1 2.3 0.0 2.4 2.1 0.2 0.1 0.2 0.2 0.2 0,1 2,4 2,6 2,4 0,1 2,4 2,4 2,7 2,2 2,8 0,0 0.2 2.9 3.0 2.8 2.4 8.0 2.8 2.8 2.8 3.0 0.1 -0.1 1.8 2.0 1.9 0.0 0.0 3.4 2.6 3.4 8.1 0.2 0.8 0.0 2.0 0.0 1.7 3,2 0.0 2.5 2.0 0,0 2,0 —0,1 0,0 1,9 1,8 2,1 1,9 1,8 2.2 —0.3 0.0 0.0 2.0 0.0 0.1 —0.1 0,0 —0,1 0,1 Strains from Cat Esophagus and Stomach 0,1 -0,1 0,0 -0,1 0,0 0,1 0.2 0.0 0.2 1.2 1.1 0.0 0.1 0.0 0.1 0.2 0.2 0.1 0.2 0.1 1.0 1.7 2.3 2.7 0.3 1.6 —0.1 —0.1 3.1 -0.1 2,9 2,8 0.1 0.0 0.2 0.1 0.1 —0.2 2.7 2.7 8.0 0.1 —0.1 5.7 2.9 3.1 2.8 —0.1 0.1 13p2 20 7 20 9 60 14 80 146 20 7 16 8 16 10 40 141 8 3 8 12 8 44 8 45 10 46 8 47 12 48 8 49 8 50 8 51 4 98 12 94 12 12 25 13 20 14 80 41 8 42 8 43 8 32 40 38 40 151 60 163 60 2, 6 2, 6 12,20 20,12 12,20 2,8 2,8 12,20 2 2 2 2, 4 2, 4 2, 4 2,4 2,4 2,4 2 - 2, 4 2,4 8 8, 12 20, 12 2,4 2, 4 2, 4 8, 12 8, 20 20,16 20, 16 Very slight Very slight Very slight Very slight Very slight Very slight Very slight Very slight Very slight Very slight Very slight None None None None None None None None 5 5 None None None None None None S 3 ?2 ?2 Muggy Muggy Pimbriate, pale . .. Pimbriate, pale Pimbriate, pale — ,. .. Thin, irregular margin. Thin, irregular margin, Pimbriate, pale Usual ~\ floceulent Ploceulent, heavy Ploeeulent Usual ,. Usual Usual Usual Usual Muggy ? Usual Usual Whiter ? Usual -- Usual Usual Usual Usual Usual Usual Usual Usual Muggy ? Usual None None None None None None None None None None None 2 2 2 2 2 2 2 2 1 1 1 1 —1 2 2 2 —1 —1 None None A, 0—1 A, 1 A, 2 A, C2 • A, 2 A, OC A, OC A, 2 A, 01 A, 01 A, 1 A/3, HaO top, 2 A/3, H2O top, C 2 A/3, HaO top, 2 0, 0-1 A/8, HaO top, C 2 A/3, HaO top, 2 A/3, HaO top, O 2 A/8, HaO top, 2 —A, —A, A/2, O —1 A/2, —1 A/2, —1 —A, C —2 —A, O —2 —A, O —2 No change No change " A, — 2 A, C— 2 Green Green Green Green Green Green -\ haze — Green Green No color -(■ haze No color Green GJreen Green Green GJreen Green GJreen Green Green (Jreen Green GJreen Green Green Green Green Green Green Green G -1-haze ? G -1-haze ? 4.1 4.3 2.5 2.6 2.6 2.4 2.6 2.6 4.1 8.9 4.2 0.1 0.2 0.2 6.2 0.2 0.3 0.2 0.2 6.0 4.8 0.1 0.0 —0.1 4.5 4.2 4.2 —0.1 2.2 2.7 2.7 2.8 2,6 2,3 2,4 2,4 2,2 2,1 2.3 2.8 2.8 2.8 2.7 3.4 1.2 4.4 2.2 2.6 2.6 2.3 3.4 3.8 4.6 4.5 8.7 3.7 8.7 4.2 0.0 2.0 2.6 2.5 8.9 3.6 2.4 2.4 2.5 1.4 1.6 2.4 S.3 3.8 3.5 5.1 5.1 4.9 4.8 4.8 5.0 5.0 4.9 4.4 4.1 4.2 4,3 4,0 4,7 4,4 6.5 0.1 -0.2 2.7 2.7 8.0 3.0 2.0 1.9 2.1 0.1 0.1 2.1 3.0 2.9 2.7 0.0 -0.1 0.0 1.7 0.0 0.0 0.0 0.0 0.2 0.2 -0.2 -0.2 -0.2 0.3 0.0 0.0 0.0 0.0 0.0 0.1 2.1 3.3 0.0 0.0 0.0 1.9 1.7 -0.1 8.3 2.6 2.9 8.5 3.5 3.8 0.1 3.6 3.6 8.8 -0.2 0.1 0.0 -0.1 -0.1 0.1 0.0 -0.1 0.0 -0.1 0.0 0.0 0.0 3.0 3.1 2.6 2.5 2.6 —0.1 0.1 2.4 2.9 3.0 3.0 0.2 0.2 0.8 0.1 0.8 0.2 0.3 0.1 0.1 0.0 —0.2 —0.1 0.0 0.0 0.2 4.9 2.6 0.0 —0.1 0.0 Strains fro.m Cat Inte,stine f 1 12 2 12 3 20 4 25 5 100 6 25 7 45 8 IB 9 20 10 20 11 20 15 600 16 40O 17 80 18 15 19 100 20 200 21 15 22 8 28 8 24 6 25 6 26 6 27 6 28 6 29 6 80 • 6 81 6 32 6 84 6 35 4 86 6 37 6 , 38 6 1 89 6 12 8, 12 4, 12 8, 12 4 4, 12 8, 12 8, 12 12, 100 20, 8 8, 12 8 50,12 4, 12 2,4 2, 4 2,4 2,4 2,4 2, 4 2, 4 2, 4 2, 4 2,4 2,4 2,4 2, 4 2 2 2 2 2 None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None Thin, irregular margin. Thin, irregular margin. Thin, irregular margin Usual Usual , Usual Usual Muggy Muggy Usual Usual Usual, D Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual U^ual Usual Usual Usual Usual Usual Usual Usual Usual Usual — 1 1 1 1 1 — 1 — 1 , tiny 1 1 1 1 — 1 1 1 1 1 2 2 2 a 2 2 2 2 2 2 2 2 2 2 2 2 2 2 A/2, O —1 Ac, C —1 Ac, O —1 Ac, C —1 Ac, 1 Ac, —1 Ac, —1 —A, C —1 A, 0-1 A, C— 1 A, 0—1 A, 0—1 A, —1 A, C— 1 A, 0—1 A, 0—1 —A, 1 —A, C 1 —A, 01 —A, 1 —A, 1 —A, CI —A, CI —A, 1 —A, 1 —A, 1 —A, 1 —A, 1 —A, 1 —A, 1 —A, 00 A, 2 A/2, C 2 A/2, C 2 4/9 n9 Green, Green . jGJreen. Green. Green. Green. Green. Green. Green. Green. Green. Green. Green. Green. Green. Green, (^reen. Green . Green. Green. Green. Green. Green . Green Green. Green Green Green Green Green Green Green Green Green Green GJ-reen 0.1 0.1 —0.2 0.0 —0.1 0.0 0.0 5.8 0.1 —0.2 —0.2 0.0 0.0 0.0 0.0 0.1 0.3 4.3 4.8 4.5 2.3 6.4 4.3 4.5 4.2 4.5 4.2 4.6 4.4 4.8 4.6 4.3 4.4 4.6 4.6 4.6 3.6 4.0 —0.2 —0.1 4.5 4.2 —0.8 —0.1 4.8 4.0 —0.2 0.0 4.4 8.7 —0.2 —0.2 4..T 4.0 —0.1 —0.1 4.4 3.9 -0.2 0.0 '4.0 4.0 -0.2 —0.1 5.2 3.5 3.3 —0.1 4.6 4.1 —0.2 —0.1 4.5 3.9 —0.1 —0.1 4.4 4.2 —0.1 0.1 4.6 4.1 —0.2 —0.2 4.8 4.0 —0.2 —0.1 4.4 8.9 —0.2 0.1 4.7 8.8 0.0 —0.1 4,8 8.8 —0.2 —0.2 4,9 3.9 0.0 —0.2 3,6 4.7 0.2 -0.2 3.6 4.6 0.0 0.0 3.8 6.0 0.1 0.2 3.6 4.5 0.0 -0.1 8.7 4.6 0.0 0.1 8.7 4.6 0.1 —0.1 3.9 5.2 0.1 —0.1 4.0 4,0 0.0 0.1 3.8 4,4 0.0 0.0 3.7 6.3 0.3 0.0 3.6 4.5 0.1 0.0 8.7 4.6 —0.1 0.1 3.6 4.6 0.1 0.0 8.8 6.4 0.0 —0.1 8.5 4.1 0.0 0.1 4.1 4.4 0.0 —0.1 4.1 5.8 0.1 —0.1 4.2 4.9 0.0 0.0 4 4 .6 1 n n 0.0 -0.3 0.0 -0.1 —0.1 -0.1 -0.1 l.U -0.2 —0.1 0.3 -0.1 -0.1 — oa 0.0 -0.2 0.0 0.3 —0.? 0.1 0.0 0.2 0,2 0.0 0.4 -0.1 0.2 0.0 0.0 0.8 0.1 0.0 0.2 0.2 0.1 0.3 12 12 20' 25 100 25 45 15 20 20 20 600 40O SO 15 100 200 15 12 10 6 8 8 8 10 6 6 6 8 8 8 12 8, 12 4, 12 8, 12 4 4, 12 8, 12 8, 12 12, 100 20, 8 8, 12 8 50, 12 4, 12 2, 4 2, 4 2, 4 2,4 2,4 2, 4 2, 4 2, 4 2, 4 2, 4 2, 4 2,4 2, 4 2 2 2 2 2 2 2 2 2, 4 4,2 4,2 4, 2 4,2 2, 4 2, 4 » 2,4 2,4 2,4 2 2 2 4 4 2,4 None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None None ? Clearing None Thin, irregular margin.. Tliin, irregular margin.. Thin, irregular margin.. Usual Usual .» Usual Usual Muggy Muggy Usual Usual Usual, D Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual ,. . Usual Usual Usual Usual Usual Usual Usual Usual Muggy Muggy Muggy Muggy Usual Usual Usual Usual Usual Usual ..,..' Harder, tiny Harder, tiny Usual Usual Usual Usual Thin, irregular margin.. Usual —1 1 1 1 1 —1 —1 2, tiny 1 1 1 1 —1 1 1 1 1 . 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 1 2 None 1 A/2, —1 Ac, G —1 Ac, —1 Ac, —1 Ac, CI Ac, —1 Ac, —1 —A, C —1 A, 0—1 A, C— 1 A, 0—1 A, 0—1 A, —1 A, 0-1 A, C— 1 A, —1 —A, 1 —A, 1 —A, 1 —A, 1 —A, 1 —A, 1 —A, 1 —A, 1 —A, 1 —A, 1 —A, 1 —A, CI —A, 1 —A, 1 —A, C A, 2 A/2, 2 A/2, 2 A/2,0 2 A/2, H2O, 2 A/2, H2O, 2 A/2, H2O, 2 A/2, H2O, 2 A/2, H2O, 2 A/2, H2O, C 2 A/2, H2O, 2 A/3, H2O, 2 A/3, H2O, C 2 A/8, H2O, 2 A/3, H2O, C 2 A/3, H2O, C 2 —A, 2 —A, 2 —A, 2 -A, 2 A/2, C 2 —A, 2 Grreen Green gGrrGen. Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green Green. ... , Green Green Green Green Green Green Green Green Green Green Green Green Green G -(- haze, Green Green G -]- haze 0.1 3.6 4.0 —0.2 —0.1 0.1 4.5 4.2 —0.3 -0.1 —0.2 4.8 4.0 —0.2 0.0 0.0 4.4 8.7 —0.2 —0.2 —0.1 4.7 4.0 —0.1 -0.1 0.0 4.4 3.9 —0.2 0.0 0.0 ' 4.0 4.0 —0.2 -0.1 5.3 5.2 3.6 3.3 —0.1 0.1 4.6 4.1 -0.2 —0.1 —0.2 4.5 3.9 —0.1 —0.1 —0.2 4.4 4.2 —0.1 0.1 0.0 4.6 4.1 —0.2 —0.2 0.0 4.8 4.0 —0.2 —0.1 0.0 4.4 3.9 —0.2 0.1 0.0 4.7 3.8 0.0 —0.1 0.1 4.3 3.8 -0.2 —0.2 0.3 4.9 8.9 0.0 —0.2 4.3 3.8 4.7 0.2 -0.2 4.3 3.6 4.6 0.0 0.0 4.5 3.8 5.0 0.1 0.2 2.3 3.8 4.6 0.0 —0.1 6.4 8.7 4.8 0.0 0.1 4.3 3.7 4.6 0.1 —0.1 4.5 3.9 5.2 0.1 —0.1 4.2 4.0 4.0 0.0 0.1 4.5 3.8 4.4 0.0 0.0 4.2 8.7 5.3 0.3 0.0 4.6 3.6 4.5 0.1 0.0 4.4 3.7 4.6 —0.1 0.1 4.3 3.6 4.6 0.1 0.0 4.5 3.8 5.4 0.0 —0.1 4.3 3.0 4.1 0.0 0.1 4.4 4.1 4.4 0.0 —0.1 4.6 4.1 6.3 0.1 —0.1 4.6 4.2 4.9 0.0 0.0 4.6 4.4 6.0 0.1 0.0 0.1 2.5 6.7 0.0 3.6 0.1 2.1 5.0 0.0 8.7 0.2 2.5 4.8 0.1 3.6 0.0 2.3 6.3 0.0 3.4 0.2 3.1 8.7 0.1 4.1 0.0 2.6 6.0 -fl.l 8.6 , 0.3 2.3 6.0 0.0 3.4 0.1 2.0 4.7 0.0 3.3 0.1 2.4 6.4 0.0 8.7 0.2 1.5 4.8 0.1 3.6 0.0 1.7 5.2 0.2 8.5 0.3 1.8 4.9 0.0 8.4 4.4 8.3 3.9 0.0 —0.1 4.3 3.8 4.4 —0.1 0.1 0.2 2.2 4.5 0.2 8.2 0.2 1.6 4.5 —0.1 2.7 4.4 3.0 0.0 3.7 0.0 0.2 2.3 4.5 0.1 2.8 Strains from Cat Feces r 3 30 4 80 5 12 6 12 73 6 74 6 75 6 76 6 77 6 78 6 I 79 6 8, 12 8. 4 12, 4 4, 8 2, 4 2,4 2, 4 2, 4 2,4 2, 4 2, 4 3-5 8-6 (?) None None None None None None None None None Usual Usual Usual to yellow white. . . Usual Usual Usual, irregular margin Usual Hard Usual Usual Usual —1 1 1 2 2 2 2 2 2 2 2 0-1- Alk. -1 Alk. 0-l-Alk. —A, —1 A/2, 2 A/2, 2 A/2, O 2 . A/2, 2 A/2, J 2 A/2, 2 A/2, 2 Green Ore en Green Green Green Green Green 2.4 2.7 3.9 0.1 2.0 2.6 2.9 3.9 0.1 1.7 4.7 8.9 4.6 0.0 0.1 0.0 4.1 4.2 0.1 - 0.2 4.5 3.8 4.7 0.1 0.0 4.2 3.9 4.2 0.0 0.1 4.8 3.9 4.4 0.1 0.1 4.1 3.9 3.7 0.1 —0.1 4.6 3.8 4.7 0.0 0.0 4.4 8.7 ' 4.4' 0.2 0.0 4.3 3.9 4.1 0.0 — oa Strains from Dog Throat 12 16 15 40 50 40 40 26 30 8 12 16 16 8 16 8 18 10 8 8 20 12 12 12 10 40 40 20 20 8 '.(8 20 30 40 12 8 40 40 40 20 50 20 20 12 8 20 16 40 40 40 40 100 80 100 200 200 200 300 500 20 80 80 12 40 70 12 30 20 20 4,2 4,2 4 4,8 4,8 8, 16 12, 20 8, 12 8, 12 2,8 4, 12 4, 12 4, 12 2,4 2, 4 2,4 4,8 2, 4 4,8 2,4 2, 4 2,4 2,8 2, 4 2,8 8,4 8,4 12, 8 12, 8 2, 4 2, 4 2,4 2, 4 4,8 4,8 2, 4 2, 4 2, 4 2, 4 4,8 8, 12 8, 20 2,8 2, 4 2, 1 2 2,4 8, 12 8, 12 12, 8 2,8 8, 12 R, 2 4, 12 4,8 2, 4 4,8 4,8 8, 12 5, 12 8, li 12, 8 40, 12 12, 8 12, 8 8, 20 40,60 40,60 200, 60 40, 200 8, 12 20, 8 20, 12 2,8 8 20. 8 20, 8 20, 8 20, 8 4,8 None None 10 None None None None None None None None None None None 10 10 10 10 10 10 10 10 10 10 10 10 ., 1«. None None None None None None None None None None None None None None None None None None None None None 10 10 None None None 10 10 None 10 5 10 None 10 3 2 2 Algal, clear 10 None 10 10 1 5-10 5-10 5-10 6-]0 None ^ irregular margin.. ^ irregular margin.. > heavier . \ heavier . Concentric rings, muggy ?.. j Muggy Muggy Muggy Muggy Muggy ? Usual Usual -} heavier Usual -[ heavier Usual Usual ; Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual Usual -[ heavy margin Usual Usual Muggy Muggy Heavier margin Heavier margin Muggy Usual Muggy Muggy Heavy rim Muggy Heavy rim Heavy rim Muggy Usual Very pale Usual Thin, irregular margin Thin, irregular margin Thin, irregular margin Usual -|- muggy Flocculent Flocculent Usual Usual Muggy USUul Usual Usual Usual Usual Usual Concentric rings, thin marg Usual Usual Usual Usual, irregular margin Usual I'locculent Usual Usual Usual Usual Usual Usual Usual Usual -|- muggy Usual Usual Usual -. 1 1 1 1 1 1 —1 —1 —1 —1 —1 —1 —1 —1 —1 —1 —1 —1 —1 —1 —1 None None None None None None 1 1 1 1 Liq. 1 Liq. 1 2 2 Liq. —1 Liq. —1 Liq. —1 Liq. — 1 2 3 3 3 Liq. 1 1 1 1 6 5 10 1 2 —1 -1 Liq. 15 15 15 5 15 16 5 10 10 10 15 3 None 10 None 3 3 2 None None 1 None None None —A, — A, C — A, OG — A, OC — A, C A, 5 A/2, 5 A/2, 5 A, O 2 A, 2 A, O 2 A/2, O 2 A/2, 2 A, H2O, 2 A, 6 A/2, 5 — A, C —A, —A, A/2, 2 A/2, 2 —A, 00 —A, 00 —A, OC —A, 00 —A, — A, OC — A, OC —A, 00 A/2,0 A/2, C A/2, A/2, O A, O A, A/2, O A/2, C A/2, C A/2, O — A, OC — A, O —A, C —A, C A/2, 3 A/2, O 8 A/2, 3 A/2, C 3 A, O 8-6 A, 3-5 A, OC A, 2 —A, OC — A, OC A, C A, -A, H2O, — A, 00 A, 00 A, A, C A, C A, A, 1 , H2O, 2 , H2O, O 2 , H2O, 2 —A, 00 , H2O, — 1 —A, 00 A, 3 A, 2 A, 3 A, C3 A, 2 —A, O —A, O A, A, A, A, 3 No change A, C3 Green ? Green ? Green ? Green Green ? Green No color G-brown No color Green Hemolysis Hemolysis Hemolysis Hemolysis ? Green Green Green Green No color No color Green No color No color Green Green Green Green ? G -f haze G -\ haze G -|- haze No color Green Green G -|- haze G-brown No color ? No color ? No color ? G -} haze Green G -[- hemolysis... Green ? Color ? Color Green Green Green Green Green G -[- haze G -^haze G -\ haze G -!■ haze No color No color Green Green Green Green Green Green G -^ haze Hemolysis Hemolysis Hemolysis Hemolysis G -1- Hemolysis. G -f- Hemolysis. Pale Hemolysis G -[ haze Hemolysis Hemolysis No color ? Color ? Color Green ? Green V No color No color 4.7 1.4 6.0 0.5 4.0 4.5 1.4 6.0 0.5 4.2 4.7 1.6 5.9 0.6 4.2 4.7 1.5 5.8 0.6 4.0 4.5 1.3 6.0 0.6 4.0 6.3 4.9 4.2 0.3 0.9 0.1 3.2 3.5 0.2 1.4 4.1 4.2 8.5 0.2 1.4 3.8 3.6 3.8 0.1 8.2 3.8 3.8 3.8 0.2 8.0 2.7 3.9 3.9 0.2 3.0 0.0 4.2 4.7 0.3 2.7 0.2 4.1 ■ 4.8 0.2 2.9 0.2 4.1 4.6 0.3 3.0 0.0 8.4 4.3 0.1 0.1 0.1 3.7 4.4 0.5 0.1 3.0 2.2 3.0 0.7 2.5 3.2 2.8 3.1 0.7 2.5 3.1 2.4 3.1 0.6 2.8 0.1 8.8 4.4 0.2 2.7 0.1 3.6 4.5 0.1 8.0 8.9 2.2 3.1 8.4 2.7 8.0 2.2 8.1 1.1 2.7 3.0 1.9 3.0 1.2 2.6 2.9 2.2 3.0 1.1 2.5 3.1 2.1 3.0 0.9 2.6 8.0 2.1 8.0 0.9 2.1 0.2 0.1 2.5 0.2 1.8 0.2 0.1 2.5 0.2 1.9 2.8 2.7 8.2 2.2 2.4 2.7 2.7 3.3 2.8 2.1 8.1 2,9 8.6 0.1 2.6 3.7 2.9 2.9 0.1 2.7 0.1 2.0 2.5 0.1 1.8 0.2 1.8 2.8 0.1 1.8 3.0 2.9 8.4 0.2 2.7 3.2 3.1 3.5 0.1 2.6 2.8 2.9 3.8 0.8 2.5 2.9 2.9 8.2 0.1 2.7 1.1 1.8 2.9 0.1 1.9 0.3 2.1 2.6 0.2 1.8 1.4 2.2 2.2 0.2 1.9 2.6 2.1 3.2 1.9 2.1 3.2 2.9 8.8 0.3 8.1 8.2 2.9 2.9 2.8 2.7 3.0 2.9 2.9 2.0 2.7 3.0 2.7 2.7 2.2 2.4 J 2.2 1.7 1.4 1.9 1.9 2.2 1.8 1.6 1.8 1.8 2.2 1.7 1.6 1.3 1.7 2.4 3.0 3.1 0.2 2.8 0.6 0.1 2.6 0.4 2.0 0.5 0.2 2.6 0.5 2.0 2.6 1.7 1.6 1.6 2.0 2.8 1.9 1.6 1.6 1.8 ' 3.3 8.2 3.2 0.2 3.2 2.3 1.8 1.5 1.4 1.7 2.4 1.8 1.6 1.7 2.0 2.3 1.5 1.5 1.8 2.1 2.3 1.6 1.8 1.8 1.9 2.3 1.4 1.7 1.5 1.8 2.6 2.5 2.0 2.1 ■ 2.1 3.0 2.7 2.7 0.3 0.7 1 2.9 2.8 2.4 0.1 0.1 1 2.8 2.9 2.4 0.2 0.0 i 2.6 2.8 2.8 0.2 0.2 ! 3.0 2.8 2.9 2.0 2.4 8.2 2.7 2.9 2.0 2.8 2.8 1.0 0.0 0.1 0.2 8.0 2.4 2.1 2.3 0.0 2.7 2.6 1.5 0.1 1.7 3.0 2.7 2.2 —0.1 0.1 2.7 2.7 2.2 0.0 0.1 5.8 6.2 5.8 4.8 0.0 4.2 3.5 3.2 0.2 —0.1 4.6 3.4 8.8 0.8 0.0 4.0 3.8 8.7 3.1 2.9 3.5 4.0 8.7 2.8 2.8 0.2 0.1 0.1 —0.1 —0.1 4.7 4.0 4.6 0.8 0.0 Strains from Dog Esophagus and Stomach [215 20 216 100 217 100 218 100 219 80 220 80 '221 60 "222 60 228 60 224 80 225 80 226 100 227 20 228 100 240 40 '241 80 i242 30 '243 40 244 SO 245 100 248 80 247 100 [248 80 8, 4 12, 40 12, 40 12, 40 8, 12 40, 12 20 20 " 20 20, 8 20, 40 20, 12 40, 12 20, 12 4, 20 12, 20 8, 4 20, 12 8,4 12, 40 8, 4 40, 8 12, 40 None —1 —1 10 —1 —1 2 2 2 2 2 2 None 2 None 10 6-10 10 3 3 5 10 Usual Concentric rings, thin marg. Concentric rings, thin marg. Concentric rings, thin marg. Concentric rings, heavy Concentric rings, heavy Concentric rings, heavy Concentric rings, heavy Concentric rings, heavy Concentric rings, heavy Concentric rings, heavy Concentric rings, heavy Usual Concentric rings, thin marg Usual Usual Usual Usual Flocculent Usual Usual Usual Heavy margin 15 15 15 15 15 15 15 16 16 15 15 15 15 15 None 10 10 None None 30 10 10 8 A, C 2 A, H2O, C 1 A, H2O, 1 A, H2O, C 1 A, 1 A, O 1 A, 1 A, C 1 A, 01 A, CI A, 2 A, 2 A, C A, 01 A, C A, 2 A, C A, OC A/'. C 3 A2, 2 A2, 2 A2, 2 Allc.?, C G -1-haze.. Hemolysis. Hemolysis. Hemolysis. Hemolysis. Hemolysis. Hemolysis. Hemolysis. Hemolysis. Hemolysis. Hemolysis. Hemolysis. No color. . . Hemolysis. G-yellow . . . Hemolysis. Green G-yellow.. . Green Hemolysis. Hemolysis. Hemolysis. G-brown.. . 2.4 2.3 1.9 2.0 2.8 3.1 2.4 8.0 0.8 0.0 8.0 2.7 2.9 0.3 0.1 3.1 2.7 2.7 0.2 0.0 3.0 2.8 2.9 0.3 —0.1 2.9 2.9 3.0 0.2 0.0 3.0 3.0 3.0 0.2 0.1 2.8 2.5 3.0 0.3 0.0 2.9 2.9 2.8 0.0 —0.1 ( 8.1 3.0 2.8 0.2 0.1 ! 3.2 2.7 3.2 0.2 0.0 2.9 2.7 2.7 0.2 0.0 2.6 2.4 1.9 2.1 1.9 3.1 2.8 2.7 0.3 0.2 ! 2.2 2.2 1.8 0.8 1.6 I 3.1 2.9 2.5 0.3 0.0 2.1 2.4 1.8 1.8 0.9 ! 2.2 2.3 1.8 1.8 1.1 6.0 5.5 5.8 4.8 0.0 3.1 2.6 2.4 0.8 0.0 2.9 2.8 2.2 0.2 0.1 2.5 2.9 2.7 0.1 0.1 1.8 0.2 2.7 0.2 0.1 * "Usual" means semi-transparent, without concentric rings, or thinner edges; "D" means tiny dot center. Thin, irregular margins are found more com- monly in pathologic samples. Some workers limit their fishings to colonies of this type. ^ t "A" means acid, the denominator referring to proportion of tube found acid (pink) on tenth day, the absence of a denominator indicating that the whole tube was pink, 1 '*C" means coagulated; the figure following, the day it was first observed. H^O means water expressed. I *'G" means green; G-Brown, green-brown; G — ]■ haze, that green colonies developed hazy areas during the 3-day period of observation. '-A /yvxcci-'Vtx^ JU^Ui^ dLlJL^f h Ik. (LLuJA, JIJL^ i,^yu..MAy4Zi4n^,A^nX'<^>^). TABLE 15— Continued Long- est Chain Length Seen Com- mon Chain Lengths Clearing in Broth (10-Day Period Ob- servation). r>ays Agar Colonies ^ Visible Growth Gelatin. Days Litmus Milif t (10-Day I'eriod Obseryation) Colonics + on Blood Agar (3-Day Period Observation) Gordon Reactions Saccha- Lac- rose i tose Sali- ! Raffl- Man- cin I nose nite Inu- lin -STi^AlNS FROM Do(; ESOPJIAGUS AND StOMACH 6 6 12 20 12 SO 500 25 12 150 150 600 12 12 16 20 40 40 40 40 60 60 60 60 60 60 30 40 60 60 200 100 12 300 SOO 300 500 2 2 2 8, 4 2, 12 2, 4 4, 12 200, 12 2, 4 4,8 4,8 8 8, 20 8, 20 4, 20 2 4 2,8 8, 12 8, 12 8, 12 8, 12 8, 12 8, 12 8, 12 8, 12 8, 12 8, 12 8, 12 8, 12 8, 12 12, 4 12, 4 20, 40 20,40 8, 40 2v4 40, 80 40, 80 40, 80 4P,20C —1 —1 —1 10 10 10 10 5 None 10 None 5 10 5 6 None 10 1 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 None —1 —1 —1 Algal, clear Usual Usual Usual Usual -!- flocculent Usual -h muggy Usual -} muggy Usual -I- muggy Usual Usual Concentric rings, muggy — Concentric rings, muggy — Muggy Muggy, concentric rings Muggy, concentric rings Muggy, concentric rings Muggy ? Muggy Muggy Tiny Tiny Tiny Tiny Tiny Tiny Tiny Tiny , Tiny Tiny Tiny Tiny Tiny Usual, muggy Usual, muggy Usual -|- irregular margin, concentric rings Usual -'i irregular margin, concentric rings Usual -}■ irregular margin, concentric rings Usual -|- muggy Usual Usual Usual I'locculent None None None 2 2 2 2 None 1 Liq. 1 1 1 1 2 1 1 2 2 —1 Liq. 2 10 2 None Alk.?, OC Alk.?, OC Allc.?, C A, OC A/2, O 10 A/2, C 10 A, C 10 Allc.?,0 A, C ~1 —A, C 10? —A, C 10? -A, OC —A, C 10 A, 5 A, C 5 A, 5 A, C6 • A, O 5 A, A, A, OC A, OC A, OC A, OO A, OC A, C A, OC A, OC A, OC A, OC A, OC A/2, H2O, C ~1 A/2, H2O, C —1 A, C 2 A, 2 A, 2 A, H2O, O —1 A, 2 A, 2 A, 2 A, 00 Haze Haze Haze White White G-brown G -j- Hemolysis Green 6 --l-h Green No color No color No color No color No color G -Miaze G -(- haze G -1- haze Green O -} haze G -[- haze G -[ haze G -(- haze G -|-haze 3.8 0.0 i 2.7 0.0 -0.1 0.3 2.2 0.1 —0.1 0.0 0.3 -0.2 0.0 0.0 0.1 0.1 0.1 0.0 0.1 0.0 0.1 0.2 -0.1 0.0 0.2 0.0 0.0 0.1 2.4 2.7 2.6 2.6 2.7 2.6 2.0 2.9 2.6 2.6 2.7 2.6 2.6 O.C 0.1 0.2 -0.1 0.1 0.0 -0.1 0.2 Strains from Dog Intestine 30 40 12 8 60 30 100 100 40 100 200 200 200 80 12 6 100 80 50 12 8 20 10 4, 8 4, 8 2,8 2,8 2, 8 12, 4 8, 20 8, 20 8, 20 2,8 40, 20 40, 20 2,4 12,40 20, 12 20, 12 2, 4 2,4 2, 4 2, 4 20, SO 12,40 12, 8, 40 12, 8, 40 2, 4 2,4 2,8 2 2 20, 40 20, 40 8, 12 2, 4 2 2, 8 2,8 2, 4 10 5 10 None None 10 2 None 10 None 2 2 1 5 5 10 None None 10 10 3 10 10 None None None —1 None None Concentric rings, muggy.. Concentric rings, muggy.. Concentric rings, muggy.. Concentric rings, muggy.. Muggy Muggy, concentric rings... Muggy, concentric rings... Usual Usual Usual Concentric rings, thin marg Concentric rings, thin marg. Usual Concentric rings, thin marg Concentric rings, thin marg Concentric rings, thin marg. Muggy ? Muggy ? Usual Usual Usual Usual Usual Usual Thin, irregular margin Tliin, irregular margin Usual -j- muggy Usual ~y muggy Usual-!- muggy Usual -! muggy Usual -[ muggy Usual -j- muggy Muggy Muggy UsujI ? Muggy Muggy 16 15 2 15 15 15 2 2 3 3 None 10 10 10 None None 1 1 —1 5 2 —1 —1 ~1 2 —1 —1 — A, OC A, 5 0, yellow —A, C 10 —A, C 10 A, O 5 A, 5 A, A, OC A, OC A, 1 A, 2 A/2, C 5 A, C— 1 A, 01 A, 2 A/3, O 6 A/3, O 5 A, 2 A/2, C 2 A/2, C 2 A/2, O 2 A/2, 2 A/2, C 2 A, 5 A, 3 A, OC . A, OC A, 00 A, 2 A, 2 A, 00 A, H2O, C —1 A, H2O, O —1 A, 2 A, H2O, C —1 A, H2O, —1 No color Green No color Green Crreen No color Green (rreen Gfreen (Sreen — Hemolysis Hemolysis No color Hemolysis Hemolysis Hemolysis Green Gtreen ? Color ~|- yellow. Green Hemolysis Hemolysis Hemolysis Hemolysis Green ? Green ? G -Miaze G -i- haze G -f- haze Hemolysis Hemolysis G~Miaze G -I- haze 0- -I- haze G -\ haze No color-!- haze.. No color -!- haze.. —0.1 5.6 8.9 0.1 0.7 0.0 5.2 4.0 0.1. 0.1 4.6 1.4 6.0 0.5 4.7 4.9 1.3 5.7 0.4 3.9 4.7 1.2 6.8 0.4 4.0 0.0 6.5 4.4 0.2 0.0 0.1 5.7 3.9 0.0 0.2 0.2 S.2 2.8 0.1 1.1 0.1 3.5 2.9 0.1 1.7 0.1 3.5 2.9 0.0 0.9 ^ 3.0 2.8 2.6 0.1 0.1 S.l 2.6 2.9 0.3 —0.1 4.6 2.9 3.8 2.6 0.1 2.9 2.8 2.7 0.3 0.0 3.0 2.4 2.7 0.1 0.1 3.1 2.7 2.7 0.2 0.1 4.4 2.8 8.8 2.3 —0.1 4.7 2.9 3.7 2.5 0.2 , 4.6 3.5 4.8 0.2 2.0 2.6 3.6 . 4.1 0.4 2.6 ■ 3.0 2.6 2.6 0.2 0.2 2.9 2.9 2.6 0.2 0.0 2.9 2.5 2.4 0.2 0.0 2.8 2.9 2.5 0.2 0.1 6.2 6.3 5.9 4.6 0.1 6.2 5.1 6.0 4.4 0.1 0.1 3.4 4.7 —0.1 —0.1 0.1 3.8 4.5 -0.1 0.0 4.6 3.7 4.8 0.0 0.0 3.2 3.0 2.8 0.0 0.1 3.3 3.0 0.2 0.2 0.0 0.0 8.8 3.0 0.0 1.1 3.8 3.8 4.0 0.1 2.9 3.6 3.7 4.0 0.1 3.6 6.4 6.5 6.2 4.9 0.0 2.8 3.2 3.6 0.1 3.2 3.6 3.3 3.7 0.2 3.1 Strains from Dog Feces 0.1 0.1 0.5 0.3 0.3 0.2 0.2 0.2 0.1 0.2 0.0 0.2 0.0 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.1 0.0 0.1 1.3 1.2 0.0 0.0 0.0 0.0 0.0 —0.1 0.1 —0.1 1.8 —0.1 0.1 20 80 60 30 00 12 8 12 200 100 80 60 20 40 60 60 15 15 20 50 15 80 80 20 16 116 30 8 115 40 116 60 117 60 118 60 119 40 18411 8 186 60 186 10 137 SO 138 120 139 40 |_140 30 12 8, 12 8, 12 12, 4 8, 12 4 2 4 12, 20 40,20 12,20 12, 8 12,20 12, 4 12, 4 2, 20 8 8 4, 12 4, 12 4, 20 15, 12 12, 4 4, 10 4, 12 2,4 12, 20 12, 20 6, 12 20, 8 20, 12 . 2 4,8 2 8, 12 12, 20 12,20 12, 20 2 2 2 2 2 2 2 2-5 2-5 2-5 10 10 10 None 5-10 6-10 None None 5 None 5 None 6? 5 None None —1 None None 3 None None None None None None Muggy ? Muggy ? Usual Usual, D Whiter ? Muggy ? Muggy ? Usual Usual Usual Usual Usual, flocculent Usual, flocculent Usual Usual, flocculent Usual Muggy Muggy Usual Usual Usual ; Usual Usual Muggy Whiter, D Muggy ? Usual Usual Usual Usual Usual -!■ concentric rings . . Usual -j- irregular raargm.. Usual Usual Usual Usual Usual Usual 2 2 2 2 2 2 2 2 2 2 o —1 —1 3 2 2 1 1 None None None None None None None None -1 —1 —1 —1 —1 —1 2 —1 2 2 —1 2 A, H2O, 2 A/2, C 5 A —A, O —A, O A — A, OC A/2, 2 A/2, 5? -A, C No change No change? A/2, O 6 A/2, O 5 — A, —A, 1 10 —A, O ? 10 —A/2, O —1 —A/2, —1 —A/2, O —1 —A/2, —1 —A/2, O —1 —A/2, C —1 —A/2, —1 —A, 5 A, 3 A, 3 — A, OC A, 3 A, 3 — A, OC A A, 0—1 A, 8 A, 3 A, OS A/2, 3 (Sreen Green Green Whitish Green Green Green G -|-haze Green Green (Jrecn G-f- Hemolysis No growth ? . . . No color -I- G ? No color -\g 1 No color -i-G ? Green... Green. .. Green. .. Grreen . . . Green. . . No color No color No color No color ? Color. ? Color. ? Color. 5.4 0.1 0.1 4.3 4.7 6.2 6.3 3.8 0.1 0.0 4.6 0.2 0.0 0.0 0.0 4.6 8.8 3.7 4.1 4.1 4.9 4.5 4.9 4.8 6.0 4.5 0.2 0.0 3.8 0.1 0.0 5.2 0.0 3.5 0.0 0.0 0.0 0.0 4.9 5.8 5.2 3.9 8.9 6.5 6.6 8.7 6.4 5.6 4.6 0.4 0.3 4.6 4.8 3.4 1.0 0.9 4.6 4.6 5.2 4.8 4.7 4.7 4.6 3.2 4.7 4.9 4.5 4.7 5.0 4.7 4.6 3.9 4.7 4.8 4.7 4.8 S.O 4.S 4.1 3.5 4.S 3.3 6.0 5.2 8.9 3.9 3.4 -0.1 0.0 3.6 8.3 8.8 5.1 6.0 2.4 2.7 8.4 3.2 4.8 3.5 3.5 S.O 3.6 3.4 -0.1 8.3 4.1 3.4 3.3 3.4 3.4 0.4 0.2 0.1 0.8 0.2 6.2 6.0 0.4 0.1 0.1 0.2 0.9 1.2 0.1 0.2 0.5 0.0 0.0 3.4 3.6 2.3 1.4 1.8 2.1 8.2 3.7 0.0 0.0 0.1 0.2 0.2 3.6 0.3 0.3 0.4 0.2 0.3 0.1 S.O 1.3 1.2 0.1 1.4 —0.1 0.0 4.2 1.4 1.9 -0.5 2.4 2.3 1.4 1.1 0.1 3.6 3.1 —0.2 —0.1 0.1 0.0 0.0 —0.1 0.0 1.7 1.4 1.4 0.1 1.5 1.4 0.1 1.5 3.4 1.5 1.2 1.4 1.4 I Longer* Vjf-frS Strains from Horse Feces 69 70 71 72 73 2 8, 20 2, 4 2, 6 2, 4 2,4 10 2 6 5 6 None Muggy ! —1 Muggy -1^ thin irreg. marg. 1 2 Usual i — 1 Usual -I- muggy j —1 Usual-! muggy ] —1 Usual-! muggy ; —1 A, H2O, C 10 No change A, C ? 10 A/2, 5 — A, OC A, H2O, O 2 Green Green (jreen Green Green Green Strains from Pigeon — Esophagus to I.ntestine (141 12 (142 12 |143 8 iiu 12 U45 •12 fl46 12 147 12 -1148 12 149 12 [I6O 20 2,4 2, 4 2, 4 2,4 2,4 2,4 2, 4 2,4 2,4 2, 4 10 10 10 10 None None None 10 None None Usual Usual Usual Usual Concentric margin Usual Usual Heavier ? .. Usual, D . . . Usual rings, heavy 2 Liq. 6 2 Liq. 5 None None None 2 Liq. 10 2 Liq. 10 None 2 Liq. 5 2 Liq. 10 A/2, O 2 A/2,0 2 —A, 00 —A, O —A, A, H2O, 2 A, H2O, C 2 —A, C A, H2O, O 2 A, H2O, 2 Hemolysis Hemolysis ? Color. . . ? Color... ? Color... Haze Haze Green Green Green .Strains from Hen — Stomach and Intes'i-ine 22 23 r 24 i 25 26 27 2 2 2 2, 4 2,4 2, 4 10 10 10 None 10 10 Usual Usual Muggy, tending to concen- tric rings Usual Usual Kft'use to muggy 1 None None 1 Liq. None 10 A/2, 3 A/2, O 8 00 A, H2O, O 18 00 A, OC No color . . Haze No color. . Hemolysis No color. . No color. . 0.1 C.l 0.0 0.0 0.0 2.1 0.0 0.1 0.1 0.0 0.0 0.2 0.1 0.0 -0.1 -0.1 0.2 0.2 1.1 1.0 1.3 1.0 1.0 1.1 1.1 8.5 0.0 0.0 0.1 0.0 0.2 0.0 0.2 0.0 0.1 0.0 0.0 -0.1 5.4 6.3 4.3 2.7 0.0 6.6 0.3 0.0 0.1 —0.1 6.8 4.6 4.9 1.7 0.1 3.8 4.1 4.5 2.8 2.9 6.6 3.6 4.8 0.4 0.0 4.6 2.7 5.4 0.5 8.4 0.1 —0.1 0.1 2.4 0.0 0.6 4.6 1.1 4.5 0.2 3.4 4.9 1.7 4.6 0.2 3.2 4.5 S.8 3.6 3.8 0.0 4.3 8.7 8.9 3.9 0.0 4.2 3.7 3.6 8.8 0.1 3.6 1.4 4.9 0.8 3.1 8.6 1.7 4.7 0.4 3.1 4.3 8.6 3.4 3.8 0.0 3.6 1.7 4.9 0.2 3.0 3.8 1.6 4.7 0.8 3.0 0.1 0.1 8.7 3.8 8.5 0.0 0.1 8.4 0.1 0.0 6.4 4.1 4.5 0.1 2.6 5.0 3.4 3.9 0.4 0.4 4.7 0.1 O.S 4.1 4.1 4.9 4.3 4.3 4.5 e.4 4.8 0.3 0.0 4.1 4.0 4.5 3.2 4.6 S.S 2.9 0.1 3.7 3.6 3.2 8.5 2.9 Strains from Human Throat r 68 6 69 60 70 6 71 6 72 6 73 6 2 8, 20 2,4 2,6 2,4 2,4 10 2 5 5 5 None Muggy ! —1 Muggy "I- thin irrcg. marg.j 2 Usual ; —1 Usual -(^ muggy —1 Usual -!■ muggy —1 Usual-I- muggy i —1 A, H2O, 10 No change A, C ? 10 A/2, C 5 —A, 00 A, H2O, O 2 Green Green Green Greeu Green Green B.4 5.8 4.3 2.7 0.0 B.6 O.S 0.0 0.1 —0.1 6.3 4.6 4.9 1.7 0.1 3.8 4.1 4.5 2.8 2.9 c.a 3.6 4.8 0.4 0.0 4.6 2.7 5.4 0.5 3.4 Strains fhom Pigeon — Esophagus to Intestine J141 (142 (143 n44 iU5 ri46 1 147 h48 149 (160 12 12 8 12 ■12 12 12 12 12 20 2,4 2,4 2,4 2,4 2,4 2,4 2,4 2,4 2,4 2, 4 10 10 10 10 None None None 10 None None Usual Usual Usual Usual Concentric rings, heavy margin Usual Usual Heavier ? Usual, D Usual 2 Liq. 5 2 Liq. 5 None None None 2 Liq. 10 2 Liq. 10 None 2 Liq. 5 2 Liq. 10 A/2, 2 A/2,0 2 — A, OC ~A, G — A, OC A, H2O, O 2 A, H2O, C 2 —A, OC A, H2O, C2 A, H2O, O 2 Hemolysis Hemolysis ? Color. . . f Color. . . ? Color... Haze Haze Green Green Green 4.6 1.1 4.5 0.2 3.4 4.9 1.7 4.6 0.2 3.2 4.5 3.8 8.6 3.S 0.0 4.S 3.7 3.9 3.9 0.0 4.2 3.7 3.6 3.8 0.1 3.6 1.4 4.9 0.3 8.1 3.6 1.7 4.7 0.4 8.1 4.3 8.6 3.4 3.8 0.0 3.6 L7 4.9 0.2 3.0 8.8 1.5 4.7 0.3 3.0 .Strains from Hen — Stomach a.^d Intesiine 22 23 r 24 1 25 28 27 2 2 2 2,4 2,4 2,4 10 10 10 None 10 10 Usual Usual Muggy, tending to eoneen trie rings Usual Usual I'Ml^use to muggy 1 None None 1 Liq. None 10 A/2, 3 A/2, 3 OC A, H2O, O 18 00 A, OC No color Haze No color. . . . Hemolysis ? No color. . . . No color. . . . 5.4 4.1 4.5 0.1 2.6 5.0 8.4 8.9 0.4 0.4 4.7 0.1 0.8 4.1 4.1 4.9 4.3 4.3 4.5 8.4 4.S 0.3 0.0 4.1 4.0 4.5 3.2 4.8 8.3 2.9 Strains from Human Throat r 1 J 2 I 83 I 34 20 21 47 48 ■ 49 50 51 52 43 44 45 4«. 47 68 67 74 1 2 3 4 5 6 7 8 9 1 2 8 (159 heo 25 26 27 28 ( 29 60 60 60 60 12 12 40 80 160 30 100 100 40 40 40 80 200 60 SO 40 60 50 50 200 30 100 200 40 100 200 200 200 200 40 40 40 40 40 20 20 20 20 20 12, 20 12, 20 12,20 12,20 4 4,8 4 80,12 20,40 8, IB 12, 40 15,40 8, 12 8, 12 8, 12 8, 12 ■ 8, 12 12, 8 12, 8 12, 8 12,20 8, 12 8, 12 20,40 8, 12 40, 20 100, 20 8, 20 12,60 12,60 12,60 40,80 40, 80 8, 12 8, 12 8, 12 20, 12 20, 12 8, 12 8, 12 8, 12 8, 12 12, 8 None None None None None None None None 2 None 2 2 2 2 2 2 2 1-2 1-2 1-2 1-2 1-2 (?) 1-2 (?) 2 10 10 3 10 None 5 None 3 None None None 2 Usual Usual Usual, D? USQal, D Concentric rings Usual Usual Usual Usual, D,? Usual to concentric rings.. Usual to concentric rings.. Some concentric rings Usual None ? None ? None None None None Usual Usual Usual Usual Thin margin . Usual Usual Usual Usual Usual ;. . Usual ? Usual ? Usual 1 Usual ? Usual, heavy margin . Usual Radiate center Radiate center Usual Usual Thin concentric rings . Thin concentric rings . Usual Tiny Tiny \ Tiny Tiny Crinkled center, thin margin: None 10 ? 10 ? None None None None None None None None None None None None None None None None IJone 1 1 None None None None None None None None None None None None A, C— 1 A, C— 1 A, C— 1 A, C ~1 A, 1 A, 1 No change —A, 2 ~A, 2 A, H2O, 3 A, M2O, O Z A, H2O, C 8 A, 6-f- A, H2O, C 3 A, 2 A, H2O A, Co A/2, O 2 A/2, 2 A/2, 2 A, OC —A, A/2, O 5 A, 2 No change A, C 2 A, H2O A, H2O A, H2O, O 1 A, H2O, O 1 A, A, A, OC No change No change A, 2 A, 2 A, 2 A, 2 No change Haz& Haze Haze Green (jf -1- haze None No color No color G -f Hemolysis. Green Green. ..:v. . ... . , Green Green (Jreen G -j- haze G ■-(■ haze G - (■ haze G -}haze G -I- haze (t -\ haze None G -^haze G --[ haze G ~1 haze G -!■ haze G -1- hemolysis... No color No color G ~\ haze G -\ haze G -I- haze G -Jhaze G -1-haze (Jreen Brown-green No growth No growth No growth No growth No color 4.8 0.0 0.3 3.7 0.3 4.7 2.8 0.1 3.7 0.0 8.8 5.2 0.1 3.8 0.1 4.3 4.2 0.1 3.9 0.2 0.0 0.2 O.S 0.1 0.1 4.5 4.1 -0.2 4.4 0.0 0.1 0.2 0.5 0.0 0.0 0.0 0.2 0.1 0.0 0.0 2.8 0.2 0.1 4.0 0.0 -0.1 0.0 0.0 —0.2 0.1 8.7 4.3 0.0 4.9 0.2 0.0 0.0 0.1 4.7 0.1 4.5 5.0 4.7 8.0 0.6 4.7 4.6 5.2' 3.6 0.2 4.8 4.4 4.8 3.7 0.1 4.5 4.8 4.1 4.0 0.0 4.3 4.5 4.2 3.4 0.0 5.0 B.l 0.2 4.7 0.2 5.3 4.5 0.2 0.3 0.1 4.4 4.3 8.4 0.0 0.1 5.8 5.2 -0.5 2.0 0.0 6.1 5.7 1.7 5.5 0.1 4.7 5.5 0.1 5.4 0.1 8.8 4.0 3.1 0.1 0.1 1.7 2.7 1.6 O.S 0.2 2.9 2.9 2.3 0.1 0.0 2.2 1.6 0.2 0.2 0.2 3.1 2.1 0.8 0.0 0.0 3.4 2.6 0.1 2.9 0.1 3.7 3,6 3.4 2.1 0.0 0.5 3.8 8.6 2.0 0.0 8.9 3.2 0.3 3.0 0.0 3.3 3.2 0.1 2.8 0.2 5.7 B.3 0.4 . O.S 0.4 5.3 5.0 1.5 0.2 0.2 5.4 B.O 0.4 O.S 0.2 3.7 3.7 2.6 0.1 0.2 4.0 3.7 0.1 3.8 0.2 3.6 3.0 3.1 3.0 2.8 4.0 8.3 8.4 3.5 3.0 4.1 3.1 8.2 8.5 3.1 3.6 3.2 2.9 3.3 3.0 3.3 —0.2 4.5 0.4 —0.1 Strains from Human Feces " 22 23 24 25 26 1 2 39 40 41 42 104 105 106 107 108 109 110 111 fll2 ■jus (^114 59 57 58 17 9 30 20 r 3 \ i \l i I 32 ( 30 I 81 J 44 ( 45 17 19 ( 20 ( 21 ) 22 ( 23 40 48 259 260 261 262 263 264 266 (266 (267 268 289 (270 80 20 80 12 8 40 12 50 20 25 25 12 200 25 60 10 10 10 10 10 12 6 50 20 12 40 25 12 12 40 12 50 10 10 12 10 12 20 12 12 20 20 20 24 20 40 100 40 40 10 SO 60' 20 25 8 8 500 500 15 12 200 BO 200' 200 30 40 100 60 12 8 12. 8 8,4 4,2 4,2 4,2 4, 12 4,8 20, 12 4 8, 12 15 8,2 12, SO 8, 12 20, 8 2,8 2,8 2,8 , 2,8 2,8 2,4 4,8 2,4 2, 4,8 2 8, 12 2,4 2,4 2,4 2,4 2, 4 None None None None None 5 5 —1 None i None —1 None None 10 10 10 B 10 10 10 10 Usual Usual Usual Usual Usual Thin, irregular margin.. Usual Usual Usual Usual Usual Usual Usual Muggy Usual -1- muggy Usual -J muggy Usual -1- muggy Usual -\ muggy Usual -!- muggy Usual -(- muggy Muggy ? Usual Muggy ? Muggy ? Muggy ? Muggy ? Muggy ? Muggy Usual Muggy Muggy —1 —1 8 —1 —1? None None —1 —1 —1 —1 —1 —1 None 1 2 —1 —1 —1 —1 —1 —1 10 10 —1 —1 —1 —1 —1 —1 —1 A, 01 A, C— 1 A, C— 1 A, 0—1 A, 0—1 A/2. C 2 A, C —1 —A, O —1 —A, O —1 —A, O —1 —A, O —3 -A, —1 —A, —1 No cliange A/3, 2 A/3, 2 A/3, 2 A/3, O 2 A/3, 2 A/3, 2 — A, C A, 3 No change No change A, H2O, — ] —A, C —A, O A, O 2-3 —A, A, C 2-3 A/2, O 10 Green (Jreen Hemolysis Hemolysis Hemolysis No color -1- haze No color ~i- haze Green G -{- haze G -I- haze Haze ? G -1-haze ? Hemolysis Hemolysis Hemolysis Hemolysis Hemolysis Hemolysis No color -j- haze No color ~\ haze No color -[ haze No color -[ haze No color -I- haze No color -}- haze G -} haze G -j-haze G ~l haze G -(- haze Strains from Blood, Pathologic Conditions, Etc. 4, S 4,8 2,8 4, 2 4,8 12, 8 2,4 8, 12 4,8 "2, 4 2, 4 4,8 8, 12 2 2,4 2 2,4 4,2 8,4 8,4 4, 12 8, 12 2,4 2,4 2,8 40,20 20, 2 12,15 2 2,4 2, 4 4,8 8, 12 2, 4 2, 4 12, 40 12, 40 4 8,4 60,40 20, 8' 40,12 8, 40 8, 12 2,4 12,30 2,8 8,2 8, 20 8,4 2,4 3 3 5 None None None None None None None None None None 1-2 10 1 None None None Muggy Muggy, some cone, rings.. Heavy margin Thin, irregular margin.. . . Thin, irregular margin.. . , Floceulent Muggy Usual ..' Muggy Usual Usual Usual Irregular margin Usual Muggy Usual Usual Usual, heavy margin —1 None None None None 1 Usual -|- tiny. Usual Heavier ? . . . . Usual None —1 Liq. 1 5 —1 None Whiter None None None ? None ? None 1-2 None None 10 10 Concentric rings, irregular margin i Centered ? Centered ? Unfavorable Unfavorable Usual Usual, irregular margin.. Usual Usual Usual Usual Usual Concentric rings, radiate.. Usual Usual Usual Usual Usual Concentric rings Usual Usual Usual Usual Usual Liq. Liq. None 2 2 None None None 15 5 5 15 None None 5 2 2 2 2 2 A, H2O, O 3 —A, —.1, C A, 3 A, H3O, No change No change —A, O —A, —A, 00 —A, C —A, —A, 00 No change A, H2O, 3 No change A/2, 3 —A, C A, H3O, 2 A, O 18 No change No change —A, O A, 1 A, 1 A, O 1 A, C 1 A, 1 Alk.,0 C A, O 1 A 3, H2O base, 3 A 3, H2O base, 3 No change No change A, C 2 A, 2 No change No change No change No change A, 1 A, 1 A, 0-1 —A, A/2, 10 —A, A, H2O, O —1 A, H2O, O 1 A, H2O, —1 —A, 00 A, H2O, C -1 A, H2O, C —1 4.3 O.S 3.2 0.2 0.2 0.1 4.2 3.3 0.8 0.1 4.8 8.7 4.8 —0.1 3.6 4.4 4.8 4.6 0.4 3.9 4.1 S.6 4.7 0.3 3.7 0.1 4.0 —0.1 0.1 -O.l 4.3 4.2 4.5 0,0 O.S 8.9 2.9 3.4 0,2 0.1 0.2 3.8 B.l 0,3 3.2 3.9 0.4 3.6 0,0 0.3 4.1 0.5 3.8 0,1 0.1 0.3 3.6 5.6 0.3 3.7 2.3 3.1 3.6 0.1 0.1 3.9 0.6 3.8 0.6 0.1 0.4 4.5 B.S 0.5 2.9 0.3 4.4 5.3 0.3 2.8 0.4 4.7 5.2 0.4 s.s 0.4 4.S 6.2 0.4 8.1 0.4 4.7 5.1 0.2 8.2 0.4 4.7 B.S 0.4 3.1 4.0 3.7 4,8 4.6 0.0 0.1 8.8 2,8 0.1 0.3 4.3 2.9 4.3 4.4 0.0 4.1 2.7 4.5 4.9 0.1 3.1 8.5 S.9 0.3 l.B 8.7 2.5 4.0 4.4 0.1 2.4 2.8 4.4 4.B 0.0 1 5.0 4.0 3.7 0.0 0.1 4.8 3.4 3.8 —0.1 2.5 4.8 3.9 8.2 0.0 0.0 4.6 3.6 8.9 -0.1 2.S Green. ItemolysiiS-. Haze -\ hemolysis . Haze -f hemolysis . Green, haze Brown No color No color ? No color ? No color ? (Jreen ? Green ? Brown ? Brown, hemolysis... ? Color Green Green Hemolysis, whitish. Green Brown -(-haze Yellow -I- haze Haze Green (Jreen Haze (Jreen No color Haze -i- hemolysis. Haze ? Color ? Color Hemolysis Green (Jreen Green Green Green. ? Color Yellow-brown... Green Hemolysis Hemolysis (Jreen Green Hemolysis G-} hemolysis . G-l- hemolysis , (J-Miemolysis , G -j-haze G-l- haze S.2 s.d 3.4 5.4 5.1 8.4 3.3 3.6 3.6 3.6 3.5 3.6 1.8 2.3 8.8 8.6 5.7 8.2 4.9 0.2 2.2 1.5 4.3 3.7 8.3 3.7 3.5 0.0 4.0 5.6 1.9 1.7 3.1 2.9 1.6 1.3 2.3 2.6 0.2 0.5 3.0 3.0 3.2 2.7 -0.1 3.8 2.4 -0.1 0.0 0.0 -0.1 0.0 2.8 4.5 4.5 0.2 8.5 3.4 3.3 3.3 3.4 3.7 S.l 0.4 2.1 3.6 4.8 3.3 8.8 8.5 -0.1 1.8 2.1 8.6 3.6 3.7 2.6 3.0 4.1 3.0 3.0 2.9 0.1 3.7 4.1 2.2 2.5 0.0 1.2 3.0 3.4 3.5 0.3 0.4 ^.4 2.8 3.6 3.7 0,1 S.4 3,5 2.7 2.8 0.1 0.2 4.7 3.7 3.6 8.7 3.8 8.2 3.1 4.7 3.5 B.l -0.1 B,6 0.0 1.6 1.5 2.6 4.4 4.3 4.0 0.5 0.1 2.7 4.6 4.7 5,6 3.9 0.0 4.3 4.3 3.0 1.9 0.4 4.3 2.8 0.0 0.4 2.6 0.1 5.4 0.0 0.1 0.1 1.3 0.1 0.1 0.7 3.4 1 0.4 0.2 ; 0.6 0.2 i 4.8 0.2 4.6 0.1 0.4 0.2 0.1 3.0 0.2 1.5 0.1 2.4 0.2 3.1 0.2 3.3 0.2 3.1 0.2 2.1 0.2 2.1 0.4 2.5 0.2 1.9 0.2 3.7 3.9 0.1 0.7 3.6 0.1 0.0 1.7 0.1 0.3 0.4 0.3 0.4 3.1 2.8 2.6 3.0 i 8.2 3.0 2.9 0.1 0.1 0.1 0.2 1.1 2.6 0.2 0.4 8.5 O.B 3.7 0.0 2.6 0.3 0.0 1.8 2.9 1.1 2.7 2.8 2.3 2.2 2.7 0.1 -0.1 0.2 1.0 0.1 0.1 0.1 0.1 0.2 0.0 0.1 0.0 0.8 0.0 i 6.3 0.0 ! 0.1 0.0 i 0.2 0.2 0.1 0.1 0.0 1.2 0.1 0.0 0.1 O.S Strains of Uncertain Origin (184 1185 31 83 34 38 37 103 80 30 8 100 30 4 2, 12 2, 12 2,4 20, 15 8, 12 2 2,8 4, 12 None I Muggy 10 I Muggy 1-2 Usual None Muggy 10 i Muggy None \ Muggy, yellowish None j Muggy... 2? j Thin, irregular margin warty center 3 3 2 -1 —1 Liq. 1 1 None A, OC A, —A, O 10 ? A, OC A, A/4, C 10 —A, 00 G-|- haze (J -f hemolysis Green (Jreen Green (Jreen G -|-haze Brownish. .... 2.7 2.2 2.2 0.3 1.9 2.4 2.3 1.8 0.1 1.8 5.3 4.4 4.5 0.4 8.5 4.9 4.8 5.4 0.8 S.S 5.2 5.1 5.3 0.7 3.2 1.6 1.1 0.2 0.6 0.9 4,9 4.2 4.1 ' 0.2 3.1 3,8 4.1 3.8 3.4 0.0 Environmental Studies of Streptococci 45 from horse feces and from throats (of cats, dogs, and man), rather easily from normal human feces, and with difficulty from other fecal material (of cats, dogs, and especially cows). Strains from a given region of any one individual are markedly alike, morphologically and physiologically; strains from the various regions of a given individual may all resemble each other very closely, Sample specimens from a given throat yield strains which usually vary more, physiologically and morphologically, than those from any other single sample. Human throats and pathologic sources yield approximately 50 percent of the strains clearing in broth in ten days. Cat and dog throats yield 20 to 30 percent; for the other regions of the alimentary canal of animals, the averages are much lower; clearing strains are not uncommon in the feces. Pathologic samples yield the greatest proportion of hemolyzing strains. They may occur in throat samples, and in those from the ali- mentary canal. A high percentage of hemolyzing strains was found in dogs — 32 percent of 101 strains from the stomachs and intestines of eight dogs. Hemolysis does not seem to be correlated with the results in litmus milk, gelatin, or with any of the Gordon reactions or complexes.* * This lack of correlation is illustrated by the fact that the seventy-six hemolyzing strains isolated fall into fourteen different fermentative complexes, many of but one or two strains each. The most common complexes for these hemolyzing strains are given as follows: (a) Saccharose-lactose-salicin for 31 strains (40%) from dog stomachs; 6 strains (7%) from dog throats; and 2 strains (2%) from blood, etc. (b) Saccharose-lactose-salicin-mannite for 3 strains (3%) from Jalood, etc.; 3 strains (3%) from human feces; 2 strains (2%) from pigeon intestine; 1 strain (1%) from cat throat, and 1 strain (1%) from dog throat. (c) Lactose-salicin-mannite for 6 strains (7%) from human feces, and 3 strains (3%) from dog throats. (d) Saccharose-lactose-salicin-raffinose-mannite-inuHn for 2 strains (2%) from dog throats, and 1 strain (1%) _from hen intestine. As to sources: (a) is not what one would expect of Stfeijtococcus pyogenes; (b), which has to do with faecalis, is more within the expected range of habitats or sources; (c) is gracilis, except that it lacks the ability to liquefy gelatin, and its contributing sources support its position as a variety of faecalis; (d) is not the fermentative complex usually attributed to hemolyzing strains. The question of relation between hemolysis and pathogenicity is entirely outside my investigations. But the correlation of hemolysis with the saccharose-lactose-salicin complex is so generally accepted (e. g., Lyall finds that 74 percent of his hemolyzing strains fall into this complex) that it seems worth while to point out that less than SO percent of my hemolyzing strains are limited to the saccharose-lactose-salicin complex. Floyd and Wolbach (1914) state that hemolysis is, in general, a characteristic of pathogenic streptococci, but add^ that the property of hemolysis is not characteristically associated with the fermentative com- plexes. Often both hemolytic and pathogenic strains have a wider fermenting range than is generally- believed, or than the figures of Lyall, Davis, and Floyd and Wolbach would indicate. Heidelck (1913), for instance, reports for his 21 pathogenic strains fermentation of all six of the Gordon substances discussed in "my paper. Six of Holman's eleven strains fer- mented mannite; over half of Thro's thirty strains fermented raffinose or mannite, or both; Hiilpers (1911) reports raflfinose and mannite both foi* streptococci pathogenic to rabbits. See Lyall for the relative values of blood agar and blood broth. I found on blood agar a distinct type — causing neither hemolysis nor green color, but a very fine delicate growth, seen only on the upper surface. It occurred in long-chained streptococci, and, while definite, the growth was so delicate as to be unobserved in pour plates, tho plainly present in streak plates. These constituted about 11 percent of the strains tested on blood agar; while more common from the mouth than from any other source, they are not limited to that habitat. They cover a wide range in their fermenting powers, and they usually ferment raffinose or mannite. 46 Jean Broadhurst Litmus milk does not seem to be very closely correlated- with the origins of the streptococci or with their fermentative reactions. This is noticeably true with regard to litmus milk and lactose.* Strains from pathologic conditions and from throats often fail to develop in gelatin ; strains from all other sources usually grow readily at room temperature. Only twenty-one of the 554 strains liquefy gelatinf; tho short-chained forms, they have apparently no correlated characters of any value. It is rather strange that among my numerous fecal and intestinal strains there is not one strain conforming to the gracilis type. (If the gelatin character is ignored, this type is well represented.) . A comparison of the amounts of acid formed yields little of value. Saprophytic strains (including those from the feces and the alimentary canal) are usually high fermenters (3.0 to 5.0 or even 6.0). But a careful analysis of my strains does not warrant for any habitat com- parative quantitative statements similar to that made by Hilliard (1913) when contrasting throat and milk strains : "Throat streptococci, on the other hand, seldom yield over 2.5 percent acid in any substance." Morphologic characteristics are not, independently or in connec- tion with other characteristics, definifely differential.! Classification The six species recognized by Andrewes and Horder (one of the original seven has been placed under Pneumococcus) have been used as a starting pointy by several later workers. Winslow (1908) recog- * Floyd and Wolbach state that in certain instances the media showed acid production where no change was produced in the milk, and occasionally acid was produced in milk with- out the fermentation of lactose in serum water. Shorer (1912) states that the milk acid formed is usually lactic acid, but that coagulation may occur as the result of the formation of amino-acids (and possibly other causes). In my own work, of about 400 strains tabulated in this connection 6 percent failed to ferment lactose and 30 percent to coagulate milk. Half of this 30 percent fell below 2.0 in their lactose titration records, and over two-thirds below 2,6; yet strains very often coagulated milk with lactose-acidity records below 1.9; and at least twenty-seven tubes failed to coagulate milk (in ten days) with high lactose records for acidity (ten tubes ranging from 3.1 to 3.5, nine from 3.6 to 4.5, and eight from 4.6 to 5.5). t They do not conform however to the Winslow fermentative limitations of Streptococcus gracilis, as all of them fermented saccharose. They were distributed as follows: dog throat, 8 strains; dog alimentary canal, 2 strains; pigeon and hen alimentary canals, 7 strains; blood, etc., 3 strains; uncertain origin (stock culture from another laboratory), 1 strain. t In the sources examined, wide, hazy capsules (such asare usually attributed to Strepto- coccus mucosus) were rare, and they were practically limited to throat and to pathologic conditions. Intestinal streptococci were usually larger and shorter-chained than throat forms. The conglomerate character was most common in throat strains. Cell-division at right angles to the chain length was more common in intestinal strains. Tf It must be admitted that the classiiication of Andrewes and Horder does not work out very satisfactorily when all of the Gordon test substances are used. The number of variants is too large. In Gordon's paper on the scarlatina strains, for instance, anginosus is represented by 54 strains, 3 conforming to the type and 51 variants; and salivarius by 37 strains, 4 typical strains and 33 variants! With fewer substances, however, the matter is less hopeless. Every method of classification has its troublesome intermediates; and they are as numerous and as troublesome in the biometric method as in any other. In this connection-, it will be helpful to recall Bergson's statement, written, of course, in an entirely different connection: "The group must not be defined by the possession of certain characters, but by its tendency to emphasize them." Environmental Studies of Streptococci 47 nized also gracilis of Escherich and others. Bergey, Hilliard, and others have modified somewhat the physiologic attributes of one or more of these species. For example, Bergey drops salicin from the pyogenes characters, and Hilliard raffinose from those attributed to anginosus. Here, as in other fields of biologic nomenclature, it is a question how far subsequent modification of described species may go. I have indicated in Table 16 the larger groups into which my 767 strains fall, using the Winslow classification plus two fermentative combinations common in my own strain's. The number of strains from the various habitats or sources varies so greatly that the per- centage is given, because the percentages are more nearly comparable than the actual numbers of strains would be. A glance at this table shows that the main representatives of a species are usually from the habitat designated by Andrewes and Horder ; for example, 28 percent of the human throat stra,ins fall under salivarius, and the only large group under equinus came from equine feces. On the other hand, strains from a selected habitat are scattered through a number of species; for example, human throat strains are also found under mitis ( 14 percent) , still leaving 42. percent unplaced; It will be seen that several other habitats are barely represented in these seven groups ; for example, but 22 percent of the bovine fecal strains appear in these seven species. It will be noticed however that the combinations most frequent here are also those occurring most often in the "family-tree" worked out earlier (Chart !)• ^ Altho these fermentative complexes are not definitely diagnostic, there are interesting relations suggested. For example, most of the mouth strains, 85 percent, belong to the saccharose-raffinose branch. (Ten percent of the mouth strains fermented none of the six sub- stances. This makes a possible total of 95 percent of the human mouth strains.) And 74 percent of Hilliard's mouth strains belong here also. The other branches are less characteristic. In the lactose-mannite branch we find 53 percent (meat extract) and 86 percent (meat) of the human fecal strains, 64 percent of the blood strains, and 77 percent , of the dog throat strains. The large number of bovine fecal strains which yield the saccha- rose-lactose-salicin-raffinose combination led Prof. C.-E. A. Winslow to suggest Streptococcus bovinus as an appropriate name. This pro- 48 Jean Broadhurst portion of bovine fecal strains fermenting raffinose is even more marked in the work of other investigators. Another nameless and important combination is the saccharose-lactose-salicin-rafRnose-man- nite one. Dog throat strains form the most prominent contributions, TABLE 16 Principal Sources Contributing the Common Fermentative Complexes of Streptococci Names of Species of Streptococci Vicla, Equinus Mitis 4' \ Pyogenes j Salivarius Anginosus Gracilis . Fsecalis . Versatilis Hovinus . Gordon Substances Fermented Saccharose, salicin, but not lactose Saccharose, lactose, and salicin Saccharose, lactose, and salicin. Hem- olysis Saccharose, lactose, and raffinose Saccharose, lactose, , and raifinose. Hemolysis Lactose and salicin? and m a n n i t e? Gelatin liquefied Lactose, salicin, man- nite, but gelatin not liquefied Saccharose, lactose, salicin and man- nite Saccharose, lactose, salicin, raffinose, and mannite Saccharose, lactose, salicin, and raffi- nose Largest Group Found in My 767, Strains 15% equine feces... 70% feline feces 31% canine alimen- tary canal 28% human throat.. 39% canine feces. 45% feline throat. 34% canine throat. % bovine feces. Group Second in Rank S% human feces. . . , 2% feline alimen- tary canal 7% canine throat. 29% feline throat. 24% feline alimen- tary canal 36% human feces. 29% equine feces. 16% human throat. Group Next in Rank 4% blood, etc. 20% milk; 14% hu- man throat; 12% human feces; 10% canine feces; 8% blood, etc.; 7% fe- line throat; 6% bo- vine , feces; 3% canine throat 9% human 6% blood feces; 23% human feces; 12% canine throat; 4% blood 34% blood, etc.; 31% canine throat; 22% milk; 18% feline feces; 17% canine alimentary canal; 16% bovine feces; 13% equine feces 16% bovine feces; 14% canine ali- mentary canal; 14% blood; 11% milk;- 5% feline alimentary canal; 5% feline throat 11% ejiuine feces; 5% canine alimentary canal; 4% feline alimentary canal but, tho eighty strains were studied, they represent only about a dozen dogs (studied at varying intervals). If a name is given to this com- bination, it should indicate the as yet unnamed fermentative combina- tion, raffinose and mannite. Since we have already several names based Environmental Studies of Streptococci 49 mainly on fermentative activities,* it might not be out of place to suggest also as a name for these strains, representing nearly 10 percent of rny 767 strains, Streptococcus versatilis. If the fermentative limita- tions stand, these names will be needed ; if .they are eventually dis- carded for agglutinating or other characteristics, the reclassification will be such a wholesale readjustment that one or two names more will be immaterial. To prove or disprove the value of these fermentative limitations of species of streptococci, we must have some way of designating the main groups, and the two names suggested here supply that lack. Nearly 700 of my 767 strains are included in the seven old species and the two new ones in this paper. About a dozen failed to fer- ment any of the six Gordon substances. Non-fermenters have been reported by many working with meat extract media. The differences obtained with meat media and meat infusion media might make it questionable whether the Andrewes and Horder and the Winslow classifications can be used for my meat results, even tho the groups are apparently well represented. Floyd and Wolbach give a large number (over 60 percent) which do not ferment the six Gordon substances discussed here and 6 percent that fail to ferment any sub- stance, even dextrose and milk. In view of the fact that they used Hiss serum water, these results are difficult to explain. My own work does not warrant making a new species for these non-fermenters, and therefore it seems wiser to leave this question for the present. Compared Gordon Reactions of' Various Investigators My earlier work with meat extract media and with meat media indicates that it would be almost impossible to compare the work of the various investigators, especially when we have the added difficulty of qualitative vs. quantitative determination of acid. Nevertheless, I have prepared a table listing the various investigators who have dealt with the streptococci and their fermentative reactions, giving the total number of strains each added to this work, and the relative availability of saccharose, lactose, raffinose, and mannite. The second half of the table includes the fermentative groups most promi- nently represented in the strains. (Note that inulin has been omitted from the compari^ns so as to bring most of the strains upon the same basis ; Winslow. and * No attempt is made to pass upon such species as mucosus and viridans; in the latter, for example, green pi^ent is associated with so many", different fermentative complexes that it cannot be considered in this connection. TABLE 17 Comparison of Fermentative Complexes of Various Investigator^ Worker Strains Samples Gordon Media Foundation Acid Estimated \ ^ ^ Percentage Fermenting Source 1 Lac. Sac. Man. Raf. Andrewes and Horder 94 - Meat ?.. Extract Litmus Litmus S 33 68 66 8 0? 22 0? Air 107(200) 46(52) "'23' Water Houston Extract Litmus Most Most Most Millf Savage Houston Hilliard, Stowell and Schlesinger Rogers and Dahlberg Broadhurst Broadhurst 21- 172 55(46) 42 120 13 ' 'i66(?) 25 112 12 Meat Extract Extract... Extract (phos.) Moat Extract... Litmus Litmus Titration Titration Titration Titration 100 97 91 100 78 84 60 90 69 50 66 62 44 20 69 33 63 23i 19 1 4 31 7 Mouth: Human. . Gordon Bergey Hopkins and Lang... Broadhurst HiUiard, Stowell and Schlesinger Gordon Oumpston Sogers and Dahlberg Broadhurst Broadhurst S00(?) 63(65) 10 43 185(163) 165 80 39(40) 80 51 22 17 10 14* ?» 38* 26* 21 31 26 Extract Litmus Litmus Litmus' Titration Titration Litmus Litmus Titration Titration Titration 60? 87? 100 81 73 100 100 100 94 100 100 75? 100 86 69 9T 96 89 80 97 '"6 1 18 87 86 56 ? Hiss; pept . . Meat Meat 60? 90 62 Extract Extract 44 27 10 Bovine... Canine... Feline.... Extract (phos.) Meat Meat 43 32 30 to large intestine: Canine... Feline..., Fowl Broadhurst Broadhurst Broadhurst 101 85 16 40 34 8 Meat Meat..... Meat.... Titration Titration Titration 95 97 87 80 60 100 63 32 68 39 14 60 Fecal: Human.. Human (sewage) Human.. Bovine... Houston Houston Winslow and Palmer Fuller and Armstrong Clemesha Broadhurst Broadhurst Houston Winslow and Palmer Fuller andArmstrong Rogers and Dahlberg Broadhurst Clemesha Broadhurst Broadhurst Winslow and Palmer Bergey Fuller andArmstrong Broadhurst 300(229) 100 116 123 115 38(39) 31 100 86 97(98) 114 30 39 38 11 100 11(14?) 129 44 15-20 100 15 ?■ U? 24 10 10? 21 ■? 66 23 11 • 11 2 12 ••■•j- 36 Extract Extract Extract Extract Extract Extract Meat Extract Extract Extract Extract (phos.) Extract Extract i Litmus Litmus Titration Titration Litmus Titration Titration Litmus Titration Titration Titration Titration Litmus Titration Titration Titration Litmus Titration Titration 100(76) 100 62 94 92? 100 90 100 62 77 100 76 99 94 100 8 100 24 61 86 45? '96 92? 65 64 94 ■75 98 76 ^ 65 90 '42? 46 84 24(29) 4 28 65 "si 40 6 3 18 23 "63 20 2 '"h. 45 32 100 6 92? 2 28 73 96 63 99 Canine... Feline.... Equine... Meat Meat Extract Hiss; pept Extract Extract 30 4 28? 12 47 Blood, path- ologic con- ditions (see mouth above) : Andrewes and Horder Rogers and Dahlberg North,Av:e>'.5r.-*>l''.^ft< Floyd and WOlbach. . Hopkins and Lang. . . Thro Lyall Ruediger Oumpston ;.. Beattie and Yates Broadhurst Heidelck Buerger Davis 228(300) 61 19 , 247 87 30 246 124 20 42 52 21 33 88 200? 19 14? 247? 87 "i24? 20? 42? 35 21 ? ? Extract Extract (phos.) Hiss Hiss Meat Meat (agar) Ser. and pept. .. Extract Extract Extract Meat Litmus Titration Litmus Neutral red Litmus Litmus Litmus Litmus ' Litinus Litmus Titration Litmus Litmus Litmus 90? 94 100 34 95 166 100 92 85 100 90? 94 90? 78 100 '98 ioo 100 97 79 100 90? 20? 27 "6 11 60 15? 50 10 19 38(48) 100 27? 12 20? 45 15 20 40 22? 60 37 26 100 Meat; serum.... Extract "6 ■h * Several records are omitted or left incomplete; for example, " Bergey (92 strains) and Saito (22 strains), Davis (88 strains), and Buerger (33 strains), whose reports lack the necessary details for comparison here. No in Sections 1 to 4, no percentage lower than 3 is given, nor any percentage based on less than three strains; in Gordon and Andrewes and Horder split their fermentative groups with so many variants that their totals, as an overlapping in percentages, etc., so that an accurate division here is not possible. Hiss means Hiss Serum Media; "phos." means that dibasic phosphates were added. In some cases, the figures available do not allow separation of the strains according to origin; that is, "normal *Pt. patholo^t'cal TABLE 17 ^vE Complexes or Various Investigators * Section 1 • Section 2 Section 3 Section 4 Sac. Sac. Lac. Sac. Sac. Sac. Lac. Sac. Sac. Sac. Lac. Lac. Sac. Lac. Mac. Lac. Sac. Sal. Lac. Sal. Lac. Lac. Sal. Lac. Sal. Lac. Lac. Lac. Sal. Sal. Lac. Sal. Sal. Bat. Rat. Bat. Sal. Eat. Kal. Man. Man. Man. Sal. Man. Bat. Man. Bat. Man. 20+ 3 8? •• i 9 20 57? 12 34? 6 9 6 •• 7 •• [+] •• 28 12 23 29 ... "i :; 14 7 23 7 24 33 42 10 3 16 - 40 4 e 6 .. 8 4 20 9 3 19 4 15 2!i 63 11 27 14 5 26? 12? 30 30 12? 11? 13?(24) 22? 30 " i -. 11 14 io 16 17 9 io 16 28 6 7 41 ' 61 io 12 9 5 5 30 16 "s 12 42 16 si 10 22 34 •• 7 29 3 45 10 33 6 17 14 •■ 22 42 4 26 ■ 24 43 6 •• •• /•I . 17 26 2 49 21 44 •■ 8 ■■ 24 si 33 5 23 L4J ... 'yi [86] .. [3] 10 7 ... • • > • .• SI ' 66 12 16 9 . •• 19 25 16 3? IS 75 45 9 26 io "i 6 io 70 [21] "s 111 'to '69 '46 99 25 39 'e '3 ie 16 18 L3j io 6 3 16 88 5 /3 66? 26 « io 10 31 4 ... 8 7 7 6 •• 15 6 11 - 4 13 29 19(46) 3(45) 2(8) ib 62 21 • 3i '26 '21 •• 21 61 12 2 ii 68 n 3 9 6 6 161 f231 • ■< 101 [2)1 [26] 117J •• " i4 60 70 28 20 139J 16] 'ii '19 .. 8 60 10 11 [7] 4 i 12 6 8 4 ■ 6 ■• 4 84 14 .. 100 * who omitted either manmte or raffinose; and Salomon, who used 10 percent solutions of the test substances- also habitat groups of less than ten strains (e. g., North, White and Avery, 9 milk strains) are included in this table- small or very varied lots, therefore, the percentages do not total 100. given here, do not always approach 100 percent. These workers have also later regrouped their results, with often and infected throats," Hilliard, "scarlatinal throats and lesions," Ruediger. 'fir^^tt- tn ^toniitteiv ^ A^tustviAO OyiruLL^^JL, LuiLirnA *"- ttuo AAlvCie-M- 52 Jean Broadhurst Palmer used only lactose, raffinose, and mannite ; Thro and Lyall used salicin, raffinose, and mannite only). This last part is arranged in four longitudinal divisions or sections: (1) including those strains fermenting none of the five substaaces or only the disac- charids (s^ccharose^and lactose) or salicin; (2) those fermenting raffinose and one or more of the three substances in the preceding group ; (3) those ferment- ing mannite and not raffinose, but fermenting one or more of the substances in Group 1 ; and (4) those fermenting both mannite and raffinose with one or more of the Group 1 substances. The strains in brackets belong in that main longitudinal division or section in which they occur, but of which subdivision is uncertain because of the smaller number of media used. This totals over 5,200 strains. If strains isolated, cultivated, and tested in such different ways (as those already noted in the first part of my paper) can be compared, this array of strains ought to be helpful. CONCLUSIONS The most striking observation is that by all methods human throat strains practically fail to ferment mannite. Note, in contrast to this, the large number of mannite fermenters in human feces. Raffinose fermenters are more characteristic of human throats than of the throats (and alimentary canals) of cats, cows, and dogs. Raffinose fermenters are more prominent in bovine feces than in the feces of other animals. They are strikingly lacking in milk. Mannite fermenters are most often found in bovine mouths, human feces, and in milk. Mannite and raffinose have been emphasized by most workers as the Vocks of dififerentiation. Milk strains are conspicuously not raffinose fermenters and rather commonly mannite fermenters. Note here that a large number of fecal strains fail to ferment raffinose or mannite. It is noticeable that the throat and . fecal strains from the same animal species may differ markedly; for example, compare human throat and fecal strains with regard to mannite, or bovine throat and fecal strains with regard to raffinose. Many of the strains from pathologic conditions fail to ferment raffinose and mannite. But the saccharose-lactose-salicin combination is as commonly the limit of throat strains as of pathologic strains; and 40 to 70 percent of various intestinal and fecal strains fall within this limit, tOo. This is not in accord with current statements. The non-lactose character of equine fecal strains ' seems to have been founded upon an insufficient number of strains (Andrewes and Horder, thirteen colonies ; number of contributing samples not given). My percent of non-lactose strains is much lower; it was obtained with meat extract Gordon media. These streptococci are Environmental Studies of Streptococci 53 often very large and in very long chains — larger and ranker than most streptococci, apparently. The large percentage of non-fermenters in the equine strains of Winslow and Palmer is doubtless due to the fact that they did not use salicin and saccharose ; in that case their present non-fermenters would be found 1 to 7 columns to the right. There are many striking (and erratic?) differences, due prob- ably to differences in media and indicator chiefly. The large number of non-fermehters of Floyd and Wolbach is not easy to explain, since it evidently represents almost as many samples as strains. (As noted elsewhere, it may be due to the indicator.) Heidelck finds that all his pathologic streptococci ferment both rafifinose and mannite. Clemesha's figures seem too concentrated. He used few strains, from few sam- ples. Fuller and Armstrong do not indicate the number of samples contributing to their strains. These fermentative tests do not seem to be definitely helpful in indicating the origin of a given streptococcus ; to illustrate, mannite streptococci in milk may imply either a bovine buccal origin or human fecal one. A raffinose strain (rare in milk) may indicate human or bovine fecal pollution. It would seem therefore impossible to determine positively the real origin of a strain isolated from milk, water, etc. Interesting as these comparative physiologic results may be, they are not adapted for direct sanitary application. Appendix details of technic not included in the text Acidity Division Line. — ^While 1.2 and 1.5 determined biometrically were used in my earlier papers as the division line between fermenters and non- fermenters, a careful lanalysis of 4,000 of the tubes selected from the low fer- menters shows that arbitrarily adopting the accepted neutral point for litmus, 0.8, as a standard does not shift more than two dozen strains out of the whole lot. Capsules. — (A) For abdominal insertion, these were made (1) by tying on a small glass tub^ one drum end of parchment (cultures grown in these tubes were later sealed in with celloidin) ; (2) by the usual method of forming cap- sules, namely, rinsing the inside of a clean tube with celloidin, tying top, and then sealing as above. This was unsatisfactory; they were evidently too thin, for invading organisms were found. (B) For feeding by mouth and for the extract experiments, the capsules were made as described by Brown (1914). The capsules were suspended by a string in tubes of dextrose broth, and sterilized. Inoculations were made into the capsule with a stab needle. After incubation for twenty-four hours, these capsules were examined microscopically and sealed with celloidin while still protected by the test tube. Saliva, intestinal extract, etc., were then added to the dextrose surrounding the capsule and the tube was incubated for one to two days. In the feeding experiments, the sealed capsules were wrapped in 54 Jean Broadhurst fresh meat, and fed by hand to the dog. Later, long capsules (3-4 inches) were made over gelatin-covered rods; these were left unsealed, the base touch- ing the saliva, etc. They were not inoculated until the outer surface of the capsule had dried. Smears for purity were satisfactory in all but one case. After recovery, the surface of the capsules was first air dried. In the beginning of this work, they were opened with sterile scissors, but later a hot iron rod was used to burn an opening in the top, while the capsule was held horizontally in the forceps. Loops for smears and streaks were taken through this opening at once. The contents of the capsules were then titrated with controls to confirm the supposed exchanges through the capsule. On opening the capsules, streaks were made at once on plain agar plates. In all cases (by the Brown method and by my open tube modification of it), the plates appeared to be pure cultures. Several fishings made from each plate confirmed this. Care of Animals Used for Feeding Experiments. — The dogs were kept in sunny runs, of wire, covered with mosquito netting. They were fed and watered from the outside. The runs were entered to clean them, lysol being sprayed over the ground, sides, etc., freely at such times. Milk, water, and oat meal were heated to 90 C, for two minutes, or longer. The kittens were kept in netting-covered cages, in an immense room. The dishes, milk, and soil pans were all made streptococci-free or else sterilized. The hands, aprons, etc., of the attendant were washed in carbolic acid. No windows were opened as the experiment covered less than two weeks. Intestinal Extract. — This was made by Mendel's method (grinding entire gut in sand, adding sodium fluorid, etc.) and tested for lactose by Barfoed's reagent to make sure that live enzymes were present. All extracts were pre- pared in sterile vessels, etc., and examined for streptococci. Isolation. — Plain meat agar and plain meat broth were used for isolation throughout, except in the 113 strains mentioned. Occasionally, dextrose broth was used also, as a control condition. No selective media were used. The samples were transferred to broth, incubated eighteen to twenty-four hours, examined microscopically, and the likely tubes streaked on plain agar. In the unfavorable tubes, the examinations were repeated in two days and if necessary, in three days. After three days, isolation in such cases was usually very difficult The streak plates were found to be much more satisfactory than the pour plates. Small, semi-transparent colonies were fished to plain broth, incubated for eighteen to twenty-four hours, and examined microscopically. Then, if apparently pure, second streak plates were made, and a second set (1-3 tubes) of fishings made. This was to insure purity. These second or subfishings were examined, transferred to slant agar, and incubated for eighte'en to twenty-four hours, and then used to inoculate the Gordon media, etc. No organisms were used which did not show chains containing at least six organisms. Four is too low, I think. Capsule stains were used for the last S50 strains. The a large percentage of my organisms fermented inulin, the chain length, shape, and non-encapsulated condition of most of the organisms would seem to indicate that I was dealing with streptococci. Litmus Milk. — To milk LS acid, a S percent litmus solution was added until a pastel pink-purple color was obtained. This was sterilized in the way described for the Gordon media. Media. — Broth from meat infusion was used, except in the 113 strains where meat extract was used. Broth of 1.0 and later of 0.5 was used for isolation, for Environmental Studies of Streptococci 55 determining broth characteristics, and for making the agar (l.S percent) and gelatin (IS percent.). The agar and gelatin were not corrected for acidity. Both were cleared with egg-white. Slants were not used if the water of con- densation had disappeared from the tubes. Special Media. — Blood agar : horseblood, about 1 c.c. to each plate. Calcium broth: small pieces of white marble were added to plain broth. Gordon media: sugar-free broth plus 1 percent of the test substances. These were sterilized one-half hour on two successive days, and incubated for one day before using. (Less than ten tubes of over 12,000 thus made had to be discarded.) Plain meat broth has been used by some for making the Gordon media, subtracting acids in inoculated control tubes. The effect of acids on the cleavage of some of the carbohydrates would indicate that this is not a wise procedure here. Sterilization. — All media, except for the first 213 strains, were sterilized in the streaming steam, not in "the autoclave. All media, except milk, gelatin, and the Gordon media, were sterilized for one hour on two successive days. Stock Cultures. — These were kept on plain agar, in closed tin pails, at room temperature. Transfers were made every ten days, incubated for one day, and stored the intervening nine. Titration. — In the cold, as described in my (1912) paper. BIBLIOGRAPHY Andrewes: Lancet, 1906, 2, p. 1415; 1913, 2, p. 1239. .Andrewes and Horder: Lancet, 1906, 2, pp. 708, 775, 852. ^rkwright: Jour. Hyg., 1913, 13, p. 68. Beattie and Yates: Jour. Path, and Bacteriol., 1911, 16, p. 246. Bergey : Jour. Med. Research, 1912, 27, p. 67. Broadhurst: Jour. Infect. Dis., 1912, 10, p. 272; 1913, 13, p. 404. Science, 1914, 39, p. 798; 1915, 41, p. 618. Brown: Science, 1914, 40, p. 176. Buerger: Jour. Exper. Med., 1907, 9, p. 428. Clemesha: Bacteria of Surface Waters in the Tropics, 1912. Crowe: Proc. Soc. Med., 1911-12, 5, p. 159; 1912-13, 6, p. 117. Cumpston: Jour. Hyg., 1907, 7, p. 599. Davis: Jour. Infect. Dis., 1912, 10, p. 148. Jour. Am. Med. Assn., 1912, 58, p. 1852. Escherich : Die Darmbakterien des Sauglings, 1886. Floyd and Wolbach : Jour. Med. Research, 1914, 29, p. 493. Fuller and Armstrong: Jour. Infect. Dis., 1913, 13, p. 442. Gordon : Lancet, 1905, 2, p. 1400. Rept. Investig. Com. House Commons, 1906. Rep. Med. Officer Loc. Govt. Bd., 1910, 40, p. 302. Jour. Path, and Bacteriol., 1911, 15, p. 323. Harris: Jour. Infect. Dis., 1907, Suppl. 3, p. 50. Heidelck: Inaug. Dissert., Berlin (Leipzig), 1913. Hilliard and Stowell : Am. Jour. Dis. Children, 1912, 3, p. 287. Hilliard, Stowell, and Schlesinger : Jour. Infect. Dis., 1913, 12, p. 144. Holraan : Jour. Infect. Dis., 1914, 15, pp. 209, 227, 293. Hopkins and Lang: Jour. Infect. Dis., 1914, 15, p. 63. Houston: Rep. Loc. Govt. Bd., London, ■^898-9; 1903-4; 1904-5. Rep. London County Council, 1905 ; 1908. Fifth Research Rep., Metropol. Water Bd., 1910. ■ Howe : Science, 1912, 35, p. 225. 56 Jean Broadhurst Hiilpers : Abstract, Jahr. Vet. Med., 1911, 31, p. 99. Jensen (and Holtli: Bull. Acad. Roy. d. Sc. et. d. Let. Danemark, 1910, p. 155. ^ Klotz: Jour. Infect. Dis., 1906, Suppl. 2, p. 35. Laabs : Inaug. Dissert., Bern, 1910. LeGros : Monographie des Streptococcques, Paris, 1902. Libman: Jour. Med. Research, 1901, 6, p. 84. Libman and Celler: Am. Jour. Med. Sc, 1910, 140, pp. 516, 527. Lingelsjieim, von : Ztschr. f. Hyg. u. Infections-krankh., 1910, 10, p. 331. Lyall: Jour. Med. Research, 1914, 35, p. 487. Moore : Dept. An^. Ind. Bull., 1893, 3, p. 9. Neisser: Centralbi: f. Bakteriol., 1,0., 1906, 38, p. 98. North, White, and Avery: Jour. Infect. Dis., 1914, 14, p. 124. Page: Jour. Boston Soc. Med. Sc, 1899, 3, p. 323. Penfold: Jour. Hyg., 1911, 11, p. 30; 1912, 12, p. 195. Prescott: Biological Studies by the Pupils of W. T. Sedgwick, 1906. Puppel: Ztschr. f. Hyg. u. Infections-krankh., 1912, 70, p. 447. Rettger and Sherrick: Jour. Med. Research, 1911, 24, p. 265. Rogers and Dahlberg: Jour. Ag. Research, 1914, 1, p. 491. Rosenow: Jour. Infect. Dis., 1912, 11, p. 338; 1914, 14, p. 1. Rosenow and Davis: Jour. Am. Med. Assn., 1912, 58, p. 1852. Ruediger: Jour. Infect. Dis., 1906, 3, pp. 183, 755. Sachs : Ztschr. f . 'Hyg. ii. Infections-krankh., 1909, 63, p. 463. Saito: Arch. Hyg., 1912, 75, p. 121. Salomon: Centralbi. f. Bakteriol., 1,0., 1908, 9, p. 428. Savage: Jour. Hyg., 1906, 6, p. 123. Schorer: Jour. Infect. Dis., 1912, 11, p. 295. Schottmiiller : Munchen. med. Wchnschr., 1903, 50, p. 909. Thro: Jour. Infect. Dis., 1914, 15, p. 234. Todd: Jour. Infect. Dis., 1910, 7, p. 73. Twort: Proc. Roy. Soc S. B., 1907, 79, p. 329. Walker: Jour. Path, and Bacteriol., 1911, 15, p. 124; 1912, 17, p. 140; Proc. Roy. Soc S. B., 1911, 83, p. 541. Winslow: Jour. Infect. Dis., 1912, 10, p. 258. Winslow and Palmer: Jour. Infect. Dis., 1910, 7, p. 1. Winslow and Rogers: Jour. Infect. Dis., 1906, 3, p. 485. Winslow and Winslow: A Systematic Study of the Coccaceae, 1908.