related that they are probably variants of the same species. The Orla-Jensens

Transcription

1 THE ISOLATION AND CULTIVATION OF LACTOBACILLUS BIFIDUS: A COMPARISON OF BRANCHED AND UNBRANCHED STRAINS' ROBERT F. NORRIS, THELMA FLANDERS, R. M. TOMARELLI, AND PAUL GYORGY William Pepper Laboratory of Clinical Medicine and the Nutritional Service of the University of Pennsylvania, Philadelphia 4, Pennsylvania Received for publication August 19, 1950 Lactobacillus bifidus ("B. bifidus") was first described, by Tissier (1899), as constituting almost the entire flora of the stools of breast-fed infants. In subsequent publications (1905, 1906, 1908), he found that the predominating organism in the stools of bottle-fed infants was Lactobacillus acidophilus ("B. acidophilus"), which, however, was first isolated, also from nurslings' stools, by Moro (1900). Tissier (1905) and Moro (1905) agreed that L. acidophilus was present in the feces of breast-fed infants in small numbers. Interest in the isolation and growth requirements of L. bifidus was stimulated by its apparently unique occurrence in the stools of nurslings since it was surmised that the presence of these organisms in the intestines was of physiologic significance and might prevent intestinal disorders caused by other bacteria. The original observations of Tissier concerning the predominance of L. bifidus in nurslings' stools have been amply confirmed. However, the factors responsible for the establishment and maintenance of this bacterial flora in the colon of the breast-fed infant are still unknown. As seen in smears of stools, the organisms are gram-positive, straight or curved rods that are nonmotile and that do not form spores. One end may be bulbous or racket-shaped. One or both ends may appear to be split longitudinally to give the effect of two short branches. This appearance led to the term "bifid." Short lateral branches may also be present. On repeated subculture, the organisms isolated by Tissier lost their bifid structure and became unbranched rods. This has been the experience of many other investigators. Although L. bifidus appears to be a strict anaerobe on primary isolation, it is not generally agreed whether bifidus is a strict or a facultative anaerobe on subculture. Although the similarity of L. bifidus to diphtheroids and actinomycetes has often been noted, most workers consider that the organisms are true lactobacilli. Weiss (1933) and Weiss and Rettger (1934), who thoroughly reviewed the literature, undertook a systematic study of L. bifidus, which on subculture was likewise unbranched. The writers concluded that L. bifidus and L. acidophilus are so closely related that they are probably variants of the same species. The Orla-Jensens and Winther (1936) disagreed with this claim because they could not substantiate a relationship between their "Bact. bifidum," which was a branched organism, and L. acidophilus. Most of their strains, however, were isolated from the feces of adults on an ordinary diet. Subsequently Orla-Jensen (1943) concluded 1 This investigation was supported in part by a research grant from the Division of Research Grants and Fellowships of the National Institute of Health, United States Public Health Service, and by a grant from Wyeth, Incorporated, Philadelphia. 681

2 682 NORRIS, FLANDERS, TOMARELLI, AND GYORGY [vol. 60 that Bacterium bifidum should be included among the Actinomycetales. In his studies on fermentation reactions of the intestinal lactobacilli of infants and adults, Eggerth (1935) described two types of so-called "Bacteroides bifidus": group I found principally in the stools of breast-fed infants and group II occurring normally in stools of adults. Both groups apparently exhibited bifid characteristics on subculture. In 1938 Weiss and Rettger, having studied several of the Orla-Jensen strains, concluded that the organisms were unlike either L. acidophiluts or L. bifidus isolated from nurslings' stools and found them similar to B. bifidus, group II of Eggerth. Weiss and Rettger were still convinced that their L. bifidus was a variant of L. acidophilus and was identical with that originally isolated by Tissier and called by him "B. bifidus." The latter was therefore considered to be the "true Lactobacillus bijdus." Weiss and Rettger proposed that since there appeared to be at least two types of bifdus organisms, they should be classified as Lactobacillus bifidus, type I, which usually became aerobic and unbranched following primary isolation, and Lactobacillus bifidus, type II, or Lactobacillus parabifidus, which usually remained anaerobic and branched. The first corresponded to that originally isolated from nurslings' stools by Tissier and the second to that isolated from the feces of adults by Eggerth and Orla-Jensen. A detailed description of the morphological and cultural reactions of both types was given. Although Lewis and Rettger (1940) came to the same conclusion as Orla-Jensen that certain strains of bifidus resemble the actinomycetes, King and Rettger (1942) discarded this belief and reaffirmed the position of Weiss and Rettger (1938) that all of these organisms should be grouped as lactobacilli. The classification of Weiss and Rettger was adopted by Topley and Wilson's Bacteriology (Wilson and Miles, 1946) and Bergey's Manual (Breed et al., 1948). The subject has recently been reviewed in a monograph by Olsen (1949). As a result of his own investigations, the author confirmed the findings of Orla-Jensen concerning the morphologic and cultural characteristics of Bacterium bifidum and the character of metabolites formed by the organism. He disagreed with Orla-Jensen concerning the classification of Bacterium bifidum and proposed that the name should be changed to Corynebacterium bifidum. In a review of the literature it is found that many types of media have been employed for the isolation and cultivation of L. bifidus and that not one of them has been widely adopted as satisfactory for the purpose. It appears that much of the uncertainty concerning the proper classification of the organisms has resulted from the lack of a satisfactory medium. In the present paper a chemically defined medium will be described that will support the growth of L. bifidus indefinitely in a bifid phase. Some of the differences will be discussed between bifid organisms isolated on this medium and unbranched bacilli, which appear to be identical with the organisms described by Weiss and Rettger (1934, 1938) as L. bifidus, type II, and L. bifidus, type I, respectively. MATERIALS AND METHODS For purposes of isolation, fresh stools of infants from 5 days to 3 months of age, who were exclusively breast-fed, were cultured. The specimens were ordi-

3 1950] ISOLATION AND CULTIVATION OF LACTOBACILLUS BIFIDUS narily emulsified in about 10 volumes of sterile physiologic solution of sodium chloride before being streaked on agar plates, which were incubated at 37.5 C. If the cultures were incubated anaerobically, either the Fildes-McIntosh jar (1921) or the Brewer ( ) modification of the Brown jar was employed in accordance with the technique of the respective authors. A solution of methylene blue was used as an indicator of anaerobiosis. When the amounts of 02 and CO2 were varied, the jars were first evacuated with a vacuum pump followed by the addition of measured amounts of 02 and CO2 to bring the total concentrations to the desired quantities. Atmospheric pressure was restored with N2. Measurements of the vacuum and amounts of gases added were made with a barometric gas manifold. Media were prepared as broth and agar. The first media employed for the isolation of L. bifidus were the Blaurock (1937, 1939) and Boventer (1938) cystine liver infusion, tomato juice (Difco), and the Weiss and Rettger (1934) tomato juice. These media were modified in several ways by the addition of fruit and vegetable juices, by the addition of whole human and cow's milk, and by the addition of various fractions of milk. None of these combinations proved satisfactory. The medium finally adopted was a modification of that employed for the assay of folic acid by Teply and Elvehjem (1945) hereafter called "bifid medium." The composition of the basic medium was reported by Tomarelli, Norris, Gy6rgy, Hassinen, and Bernhart (1949). Folic acid was added in the amount of 200,ug in each liter of double-strength medium. This medium was modified further in the present study by the addition of pancreatin ("viobin"), 200 mg; sorbitan monooleate, 2 ml; ascorbic acid, 1 g; and vitamin B12, 10 jug, to each liter of double-strength medium. The final ph of the medium was adjusted to ph 6.8. In experiments with the bifid medium, unless otherwise indicated, the results were read after 48 hours' incubation at 37.5 C in an anaerobic atmosphere to which 10 per cent C02 was added. When used as a liquid medium for the study of the optimal ph for growth and for measuring the quantity and character of the acid produced, 10 ml of medium were added to each broth tube, 18 by 150 mm. The inoculum of test organisms was prepared by washing the sediment of a 48-hour broth culture with an equal volume of a sterile physiologic solution of sodium chloride; the organisms were resuspended in 10 ml of saline; 0.1 ml of this suspension served as the inoculum. The final ph of the medium was varied by the addition of sterile 0.1 N NaOH or HC1 after autoclaving. For measuring the amounts of acid formed by the various strains of organisms, the sodium acetate of the bifid medium was replaced by sodium citrate, 50 g, and calcium phosphate,2 20 g, and the amount of lactose was reduced to 7 g for each liter of double-strength medium, hereafter referred to as "bifid citrate medium." Uninoculated broth tubes incubated simultaneously served as blanks. Total acid was measured by titration with 0.1 N NaOH to the ph of 2 Calcium phosphate was added to the medium as a freshly prepared suspension; 65 g of CaCl2 (anhyd.) and 45 ml of phosphoric acid (85 per cent) in 500 to 600 ml of H20 were precipitated by adjustment of the ph to 8.0 with concentrated NaOH. The ppt. was washed once and suspended in 1 liter of H

4 684 NORRIS, FLANDERS, TOMARELLI, AND GYORGY (vol. 60 the blank. The content of each tube was then diluted with distilled water to 50 ml. In one aliquot of 25 ml, lactic acid was measured by the method of Barker and Summerson (1941). Volatile acid was measured by adding to the second aliquot 1 ml of concentrated phosphoric acid (85 per cent). The mixture was steam-distilled, and 125 ml of distillate were collected in an excess of 0.1 N NaOH. The distillate was titrated back with 0.1 N HCl to an end point with phenolphthalein. Duclaux constants were measured separately by the method of Gillespie and Walters (1917) following acidification of the culture and collection of volatile acids by steam distillation. For the measurement of the water of crystallization and the optical activity of lactate salts, zinc lactate was prepared from 64-hour cultures in the bifid medium in which phosphate was substituted for four-fifths of the acetate in order to minimize the amount of volatile acid to be removed before extraction of the salts, according to the method of Pederson, Peterson, and Fred (1926). The presence of catalase, the formation of indole, and the reduction of nitrates were determined in 48- and 96-hour cultures in the bifid medium according to the methods given by Topley and Wilson (1946). Uninoculated medium served as negative controls. Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa were employed as positive controls for catalase. These organisms were grown on tryptose phosphate media (Difco). When reduction of nitrate was not detected in cultures, quantitative nitrate was measured in cultures and uninoculated medium by the method given in Official and Tentative Methods of Analysis of the Association of Official Agricultural Chemists (1945) to verify the lack of reduction of nitrate. RESULTS When stools were cultured on the Blaurock-Boventer cystine-liver-infusion agar plates anaerobically, or with the addition of small amounts of 02 and CO2 to an anaerobic atmosphere, small porcelainlike colonies were usually present after 48 hours' incubation; these colonies were composed of gram-positive rods having a typical bifid morphology. Subculturing on plates was not successful for more than one or two transfers. If colonies from the original plates were transferred repeatedly every 48 hours in liver infusion broth anaerobically, the result frequently was growth of straight or slightly curved rods, which soon became aerobic, grew well on plates, and did not revert to a bifid morphology. When filtered skimmed breast milk was added to the liver infusion medium in 10 per cent concentration, colonies of bifid organisms were larger on the original plates, but subculturing was no more successful in maintaining bifid organisms than it was without the addition of the supplement. Employing whole breast milk digested with pancreatin, which was supplemented with 1.0 g of ox bile (Difco) per liter, bifid organisms were regularly isolated as rounded, glistening, buff-colored colonies. On this medium the organisms could ordinarily be kept alive from 4 to 6 weeks, but they died out almost at once when transferred to a liquid medium of this composition. By contrast on digested cow's milk medium, the organisms did not grow at all. The addition of varying quantities of several

5 1950] ISOLATION AND CULTIVATION OF LACTOBACILLUS BIFIDUS IQ5 fruit and vegetable juices to either digested breast or cow's milk medium did not enhance the growth of bifid organisms. On agar plates of the bifid medium, incubated for 48 hours anaerobically in an atmosphere of 10 per cent C02, luxuriant growth of bifid organisms was regularly obtained from the feces or intestinal content of breast-fed infants. The only other organisms that grew regularly on this medium were enterococci. Coliforms were almost invariably absent, although abundant growth of the latter wn-as obtained wi-hen blood agar plates were inoculated simultaneously. Six strains of bifid organisms from five infants have been maintained continuously in stock culture on the bifid medium for purposes of study. Strains "Perrish (L)," "Perrish (S)," and Timberlain" were originally isolated on digested breast milk agar and were subsequently transferred to the bifid medium. The remaining strains e-ere isolated and have been maintained on the bifid medium. Strain "Timberlain" wvas isolated by intubation and aspiration from the lower ileum of a living infant. The data concerning the strains are given in table 1. It is seen that the age of the infants ranged from 5 days to 2' months. At the time of writing the oldest culture has been maintained for 18 months. The growth of all strains on subculture has continued luxuriant. The colonies and organisms of the various strains have remained morphologically indistinguishable from one another. The bifid character has been maintained. On streaked agar plates, although somewhat smaller and less opaque on primary isolation, the colonies of bifid organisms vary between 0.5 and 1.0 mm in diameter and are hemispherical with an entire border. They are buff-colored and moderately opaquie to transmitted light. The surface is alvays smooth or even mucoid (figure 1). The organisms are pleomorphic and resemble those seen in films of stools. Curved or bent rods, which may or may not have bulbous ends, are numerous. M\ost of the organisms, however, show varying degrees of branching. The Y-form is the most common, and occasionally branching may be seen at both ends. Lateral knobs or short stubby lateral branches are often present, but only wi-hen there is also terminal branching. Central or subterminal bulbous expansions of the bacilli aie also seen. In addition, there arc a few short coccobacilli, the appearance of which suggests their origin from branches of longer bacilli (figure 2). On agar plates smallei' colonies, wi-hich may be mutants, rarely appear. These colonies can be readily differentiated and isolated from those of the bifid organisms. The diameter of the small colonies averages only about one-fifth that of the bifid organisms and is therefore from 0.1 to 0.2 mm. The colonies are not so elevated as those of the original organism, are gray rather than buff, and are more translucent. The surfaces remain smooth and the edges entire on repeated subculture (figure 1). MIorphologically, the organisms are unbranched and are straight or slightly curved rods, which often showv palisading Coccobacillary forms are also present, an(l rarely there are short chains of bacilli. Carried in repeated subcultures uinder identical conditions with the bifid strains, the small colonies have not reverte(l to the bifid morphology nor to the original type of colonv. Tlhe uinbranched strains employed for comparison were strains "Lock-

6 Co86) 66NORRIS, FLA.NDEIS, TOMAR ELIA, AND GYORMGY [VOL. 6)0 hart (m),," '"St,' 'A,''a,nd 'I." On tlhis me(litumi Americani Type Culture Collection strains L. binfidols nos and (deposited by Bettger an(l now classified,as L. aci(dophiulhs) and L. acl(1op1lii108 Inos. 4355, 43571, and 9857 closely resemble t1e unbranched strains in the character of the colonies and in the morphology of the orgainisms. At times, however, colonies of the ltatter as wvell as the unbranclied strainis become roiagh wn-itlh slighjtly serra'ted etiges, especially in ol(d cultures. By the metho(d of Barber (1908), with a D)eFonbrune micromanipulator (Aloe),' a single cell clone Of the hifid strlin.jaickson was isolated in the bifid Fiqolre 1. Stralini"Ic)khart" (hifid St rain) a(l "Lockhart (M)" (unihranched strain tolught to b Il muant Ut of h ifid St laiii pul)) rpose Ivlinxell for llc h(ait ionii. Two -day -old culture on1 acgar plate of imio(lifi(d mediumi of Teplv ande1vehijem. (ni lef't a're large glistening coloiiies of piarent bifid( slain St that Show varliatioi in Size. OIl right are smiilall gray I ralslicellt colonlies of tlnl)blallihe(l St raill. X 1(0. medium. supplemented iy the a(l(lition of a Scitz flilt iate ofia previous clultlure in a concentration of 1 per cent. T'lhereafter in subctulturcs the clone was carried in unsupplenenttecl btifidnme(liuim. After several \Nveks of subculturing every see- On1(ldav (111uing wh}ich time the orgallisms remained btifid, a few colonies of typical rods weie isolate(l aerob)ically(tn agar plates of t l ifie(l m(liudlu in which gllucose 3.5 per cent was substit lite(d for lactos. These orgallisills Were readily subeuiltured on th1e (irdinarv bifid medium and corresponcle(1to Ihe straight-rod strains previously (lescribe(l. Similar resuilts were 0111taine(l withi otlher bifid strains, which wvere not, however, derivedl from simolte cell isolates. Since the Kindl1 Ioaned to s1\ii)b W ( I llitchilsoll, halahmtaov of l icol )iologv lbotalnyv Depart m0nt1.

7 1950] ISOLATION AND CULTIVATION OF LACTOBTACILLUS BIFIDUS 687 occurrence of straight-rod colonies under these conditions has been unpredictable, the factors responsible are still unknowvn. Further attempts to isolate single cell clones from other bifid strains have been unsuccessful. Employing agari plates that w-ere observed for the presence of growth after 48 houirs' incubation at 37.5 C, the amount of (2 in the atmosphere for incubation wvas varied from none demonstrable with methylene blue to 5 per cent by the addition of increments of 1 per cent by v-olume. The ('02 was kept constant at 10 per cent. All bifid strains grew wn-ell in the presence of 2 per cent 2., or less; they grew poorly w-ith 3 per cent 2.. an(d not at all wn-ith more than 3 per cent 02. In an atmosphere containing no measurable 02, the C(2. content was varied 4 jf t4 h~~ 1 4'0" Figure 2. Strain ''lperrish (1)." Photomicrograph of organisms from two-day-old culture oii agar plate of imio(lified( medium of Teply aind El vehijemi shows coceol)acillary forms, shiorit cuirve(i Irods, and IIumerous brianclied formins. X 1,280. similar-ly from none to 10 per cent by voluime at 2 per cent steps. No growth of any strain occurr--ed in the absence of (22),, but all grew wn-ell withi 2 peri cent and with the higher concentrations of T()2Ṫhe addition of 2 per cent 02 to the various atmosplheres of (20 didl no( alter the results. Wlhen broth tubes w-ere inoculated wnith the bifid strains, lowevei, some growth occurred unider all conditions, althoughl uncler aerobic con(litions the amount of growth was variable as r medium the addition of C02 mecasured by the amount of acid formed. In liquid( to the atmosphere, ther efore, appeared unne(essary for growth. If ascorbic acid an(d vitamin 1112 Welre omitte(l from t he mediuim, grown-th did not occur aerobically. T'he addition of ascorbic aci(d 0.01 g or cysteine HC g per tube, withliout v-itamin B12, r'esultedi in growth aerobically. This wn-as twice the concen-

8 688 NORtitIS, FLANDERS, TOMIARELIA, AND GYORGY [VoIl. 6O tration of ascorbic aci(l ordinarily employed in the me(liulm for plate cultures, butlt wlhen the concentrationw1-as increased to this level in the solidl medium, none of the bifid stirains grewv aerobically on agai plates. (GrOw-th aerobically occurred MIconstantly in broth when v-itamin B12, 0.1,Ig, wn-itlhotut ascorbic acid was added to each ttube. On tile othfer hand, the unbralnlched Str-ains on agar plates an(l in broth tubes grewv aerobically and un(lei all conditionis of the experimenlt. T'lhis w-as likewise trutie of ATCC strains of L. bifi(/ids nio and Ino and L. acidophilits no The effect of different temperatures was stt(lie(d for each straiin on agai plates of the bifid medium. The results are recorded in ta)ble 1 as "4" when growth was as great as at 37.5 C. Lesser degr,ees of growvth are indicated by smaller numbers. It xvill be seen that all strains grew wsell at 35 C. At 40 Cl all TABLE 1 Comtiparisinor of br-a ached( arnd oar2bra ached strains of L. 1ilf(i(Gs a ad L. acilophilas (Bifid) "I'errish (L )' 3 weeks ± "Perrish (S')".. 3 weeks ± "Tiloberlain".. 2 iiioitits "Birchl"... I I oll()iltl "Jackson. 2 2I' monlthls d(las "Lockhart. 5 (Unbranched) "Lockhiart (51) "St" "A" ATCC 4962 ATCC ATCC ATCC ATCC TEMPERA- ph of AGAR MEI)IUM AT T'IlME OPTIMAL TURES OF I\C)(ULATI()ON ph.\(;e GF D)ATE RANGE IN FAN'TI' ISOLATED IN 40 C 35 C 30 C ( LIQIUID MEDIUM _ S CONFIGTU- RATION 0OF LACTIC ACID unbranched strains, including ATCC st'ainis of L. I41/idls I1os and 4963 and L. acidophilhs no. 4357, grew w-ell, but except foi' slight growth in the case of "Birch" none of the bifidi strains giewv. At 30 (C the (lispalrity between the two groups w-as less narked, but on the whole the inbl)allche(l strains showed better growth than the b)ifid sti'ainll As will be seen in table 1 heie the relative alllomiouts of growth aie indicatedl bv the symbols ''4,'' '3,'' et(c., good growth of all ilm)branched strains of L. bijiduis on agar plates wvas observed wi-ithin the limits of ain initial ph railge of 5.1 to 7.3 inlusive. CC L. acidophilhs no griewv less well alt p'l 5').1 thlan at 5.6. Althouighl all of the bifid stiains grewv wvell at 1)II 7.3 and 7.0, less growth occurrecl at pli 6.3 anid growth was minimal at pi Only "Perrish" showed growvth, which vas barely (detectable, at ph 5.6). The optimal ph ranige for the bifid strains and for uninbranched strains "A" and "I" (v1e'e measuredl by gronwing C02 A\S GAS 0 _ 0-0 _ 0 0 L(+) 0 I,(+) + + 0± 1)1. 1)1, DI, I)L 1)1

9 19501 ISOLATION AND CULTIVATION OF LACTOBACILLUS BIFIDUS 689 the organisms in broth. Maximal acid production was considered as indicating maximal growth, and the initial ph at which maximal acid was produced was therefore considered optimal. Since the results variedfrom experiment to experiment, the spreads are shown in table 1. It will be seen that the range is not significantly different for the various strains. TABLE 2 Amounts of volatile and lactic acids produced by bifid strains and unbranched strains of L. bifidus and L. acidophilus STRAIN (Bifid) MILITERS OF 0.1 N ACID PER 10 ML OF MEDIUM Undetiid Volatile actic nonvolatile Total novltl "'Perrish (L)" (a) "Perrish" (b) "Perrish (S)" (a) "Perrish" (b) "Timberlain" (a) "Timberlain" (b) "Birch" (a) "Birch" (b) "Jackson" (a) "Jackson" (b) "Lockhart"(a) "Lockhart" (b) (Unbranched) "Lockhart (M)" "A" (a) "A" (b) "Af" (c) "I" (a) "I" (b) "St" (a) "St" (b) ATCC 4962 (a) ATCC 4962 (b) ATCC 4963 (a) ATCC 4963 (b) ATCC ATCC ATCC (a)(b)(c) indicate replications of experiment on different days. When Durham tubes were employed for the detection of gas in tubes of bifid medium, it will be seen in table 1 that none of the bifid strains formed gas. All of the straight-rod strains, except for strain I, produced large amounts of gas, which completely filled the Durham tubes. In fact, bubbles of gas floating to the surface of the medium were clearly visible even without the use of Durham tubes. Comparable amounts of gas were formed by ATCC no. 4962, no. 4963, and no. 4355, but not by no and no In all cases gas was formed

10 690 NORRIS, FLANDERS, TOMARELLI, AND GYORGY [VOL. 60 aerobically and anaerobically with or without addition of 10 per cent C02. Since the gas was completely absorbed from the Durham tubes when the cultures were made alkaline with NaOH, it is presumed that the gas was almost if not entirely CO2. In the case of those organisms that did not produce visible gas, it is assumed that little if any CO2 was formed since the ph was below 4.5. At a ph in this range, no bound CO2 should be retained in solution. When the amount of lactose was reduced from 7 per cent to 2 per cent and to 1 per cent, the quantity of gas formed on the average was sufficient to replace only twothirds and one-third of the fluid in the Durham tubes, respectively. The gasproducing strains were then grown in the Weiss and Rettger tomato juice medium and in the same medium with 3.5 per cent added lactose, anaerobically with 10 per cent CO2 and aerobically. Traces of gas were formed by no under all conditions, by strain A in the medium with 3.5 per cent added lactose, aerobically and anaerobically, and by the others only in the latter medium anaerobically. As will be seen in table 1, dextro-rotatory lactic acid was formed by the two bifid strains tested, but optically inactive lactic acid was formed by strains "A" and "I" and by ATCC nos. 4962, 4963, 4357, and In table 2, it will be seen that the bifid strains of L. bifidus produced much greater quantities of volatile acids than the unbranched strains and the ATCC strains of L. bifidus and L. acidophilus. The amounts of lactic acid formed by the two groups, however, were comparable. Consequently the total acid elaborated by the bifid strains was about twice that formed by the group of unbranched rods except for no The Duclaux values for strains "Jackson" and "A" corresponded to those found for acetic acid. Since there was no odor of butyric acid and the qualitative test for formate using mercuric oxide was negative, it may be assumed that the volatile acid was essentially acetic acid. The odor of the volatile acid from all other strains was that of acetic and not of butyric. DISCUSSION When the study of L. bifidus was undertaken in this laboratory, it seemed reasonable to assume that the presence of milk or derivatives of milk in the culture medium might enhance the growth of the organisms. This idea was not original with us since studies of this kind were made by Adam (1921 to 1927), Stransky and Maslowski (1927), and the Orla-Jensens and Winther (1936). Although bifid organisms were readily isolated either on liver infusion medium to which human milk was added as a supplement or on digested whole human milk to which only agar was added as a base, the organisms soon died out following repeated subculture or became aerobic rods, presumably by mutation, which did not revert to the original branched character. Several of these unbranched strains were kept in stock cultures and studies of their nutritional requirements were reported by Tomarelli et al. (1949). A growth factor associated with the fatty acid fraction was found to occur in milk, but was present in about five times greater concentration in human milk than in cow's milk when the bifid medium was used as the base. Unsaturated fatty acids, such as oleic, and related compounds, such as sorbitan monooleate, could be substituted for this factor. After

11 1950] ISOLATION AND CULTIVATION OF LACTOBACILLUS BIFIDUS 691 this work was started, however, it was found by Tomarelli, Norris, Rose, and Gy6rgy (1950) that the unsaturated fatty acids, oleic and linoleic, were inhibitory to bifid strains but not to unbranched strains grown in the bifid medium and that sorbitan monooleate neither inhibited nor stimulated the growth of bifid strains. Some unbranched strains required an additional factor that was present in pancreatin and in enzymatic digest of casein, which could be replaced by compounds having a desoxyriboside structure. Accordingly, when the use of the Teply and Elvehjem medium was instituted for the isolation of L. bifidus, sorbitan monooleate and pancreatin were incorporated as supplements. Ascorbic acid was added to the medium with the thought in mind that the presence of this reducing agent might favor an oxidation-reduction potential suitable for the growth of L. bifidus. Since we found no evidence that vitamin B12 stimulated the growth of the aerobic unbranched strains, its addition to the medium was an insurance against its possible need. The use of 7 per cent lactose was empirical in order to ensure an excess of this sugar. Although it was subsequently learned that the concentration of lactose may be reduced from 7 per cent to 3.5 per cent, we have continued to employ the medium with 7 per cent lactose for the isolation of new strains and for carrying old strains in stock culture. The inhibition of other common intestinal organisms except for the enterococcus makes the bifid medium highly selective for the isolation of bifid organisms. Preliminary experiments indicate that the high concentration of acetate (2.5 per cent) in the medium is largely responsible for the inhibition of coliform organisms. With concentrations of less than 1 to 2 per cent (1.12 to 0.24 N), growth of coliforms occurred. These observations appear to be consistent with the report of Bergeim (1940) that, at ph 6.5, 0.2 N acetate will not prevent the growth of E. coli, since this was the highest concentration employed by him. Although in our experience unbranched lactobacilli were not so consistently isolated on the bifid medium from the stools of breast-fed infants as were bifid organisms, they were ordinarily isolated in large numbers from the stools of formula-fed infants. From the results obtained, the medium described appears to be adequate for the isolation and continued subculture of bifid organisms from the stools of breast-fed infants. Since aerobic unbranched strains may appear, stock cultures should be plated periodically in order to ensure the purity of bifid strains. For the study of nutritional requirements, this medium is superior to that of the Orla-Jensens and Winther and presumably to that of Olsen since most of the constituents are chemically defined. Since proliferation of L. bifidus occurs on the chemically defined medium on surface cultures in an atmosphere of 3 per cent 02 or less, the organisms are to be considered microaerophiles rather than strict anaerobes. The previous uncertainty as to whether L. bifidus is a strict anaerobe may have resulted from the lack of a method for the critical measurement of small amounts of 02. The necessity for the addition of small amounts of CO2 to the atmosphere of incubation when agar plates are used confirms the statement of Boventer (1938) that this factor is essential for growth. Since the bifid strains did not grow on agar plates

12 692 NORRIS, FLANDERS, TOMARELLI, AND GYORGY [VOL. 60 in the presence of greater concentrations than 3 per cent 02, it appears that the reducing action of ascorbic acid effected a sufficiently low oxidation-reduction potential in the depths of broth tubes for growth of the bifid strains to occur in liquid media under atmospheric conditions. This phenomenon corresponds with that reported for Lactobacillus lactis Dornler by Koditschek, Hendlin, and Woodruff (1949). In their experiments, however, the presence of added atmospheric CO2 was essential for growth, whereas in the present experiments the addition of CO2 was not required by the bifid strains in liquid bifid medium. That the growth range for bifid organisms at varying temperatures is more restricted than for the unbranched strains and ATCC strains of L. bifidus and L. acidophilus constitutes another important difference between the two groups of organisms. Although the optimum ph for growth of both bifid and unbranched strains appears to be similar, bifid strains will not grow under the conditions of the experiment at as low a ph as will the unbranched strains. Since the lower limit for the bifid strains appears to be at about ph 6, they can scarcely be considered "aciduric." Sherman (1921) reported that intestinal strains of L. bifidus and L. acidophilus produced CO2. Curran, Rogers, and Whittier (1933) also found that many strains of L. acidophilus produce significant amounts of C02, but they did not specify that molecular gas was visible in the medium they employed. Eggerth (1935) and Olsen (1949) also reported formation of CO2 by Bacteroides bifidus and Bacterium bifidum, respectively. It is interesting that such large amounts of CO2 as gas were formed by our unbranched strains of L. bifidus, except strain I, and by ATCC nos. 4962, 4963, and It will be recalled that 4962 and 4963, now classified as L. acidophilus, were deposited by Weiss and Rettger as examples of L. bifidus and that 4355 was isolated from the intestine of a rat by Kulp in Rettger's laboratory. It seems likely that these three organisms belong to the same group or type as our unbranched strains, which are thought to be derived from the branched strains. Since large amounts of gas were not produced in the Weiss and Rettger tomato juice broth even with added lactose, it appears that this medium is not optimal for gas production. The lack of a favorable medium for CO2 production may be responsible for the fact that a marked difference in the ability of various strains of lactobacilli to produce CO2 has not been widely recognized. The Orla-Jensens and Winther (1936) found that as much as 50 per cent of the acid produced by Bacterium bifidum was volatile acid, which was mainly acetic, and that the lactic acid was dextro-rotatory in contrast to Thermobacterium intestinale (L. acidophilus), which formed inactive lactic acid and much smaller amounts of volatile acid. Pederson (1945) reported that two of three strains of Bacteroides bifidus obtained from Eggerth also produced over 40 per cent volatile acid; the third yielded only about 5 per cent. In each instance, the volatile acid was identified by Duclaux constants as almost if not entirely acetic. The lactic acid was found to be dextro-rotatory. Olsen (1949) reported similar findings. In the present study, likewise, the bifid strains formed dextro-

13 1950] ISOLATION AND CUTLTIVATION OF LACTOBACILLUS BIFIDUS 693 rotatory lactic acid and large amounts of volatile acid, whereas the unbranched strains and ATCC strains produced inactive lactic acid and much smaller amounts of volatile acid, which in each instance also appeared to be acetic acid. From a review of the literature and from the results of the present study, three problems arise. The first is whether the aerobic unbranched bacilli are derived by mutation from branched organisms. It is recalled that many, including Tissier and Weiss and Rettger, were unable to maintain branched organisms isolated from the stools of breast-fed infants in a bifid phase on subculture and that if the cultures remained alive the organisms soon became aerobic unbranched rods. As previously indicated, our experience was similar to theirs when certain media were employed. The stock cultures of the bifid strains and the single cell isolate of "Jackson" carried in the bifid medium have also occasionally yielded a few colonies of typical straight-rod organisms when subcultured under varying but unpredictable conditions. This evidence supports the hypothesis that the aerobic unbranched bacilli are in fact derived by mutation from microaerophilic branched organisms and are not simply contaminants of the original cultures. However, the biologic differences between the branched and unbranched organisms are so great as to cast doubt on the validity of this hypothesis. The second question to be answered is whether the bifid organisms and the unbranched strains should be classified in the genus Lactobacillus. Since the organisms do not appear to form butyric acid and differ in other respects from Butyribacterium, as described by Barker and Haas (1944), they are not appropriately placed in this genus. On purely morphologic grounds, the bifid strains particularly might be classified as either Corynebacterium or Actinomyces, but it appears to the writers that a more intensive study of the comparative microbiology of these organisms is required before they are to be related to either of these genera. Accordingly, since both bifid and unbranched strains are alike in producing no catalase, indole, or nitrites from nitrates, and since they produce nonvolatile acid, which is largely lactic, together with varying amounts of volatile acid, which is principally if not exclusively acetic, it is believed that these organisms should be retained in the genus Lactobacillus. If it is conceded that the organisms are lactobacilli, the third problem is whether the branched or unbranched organisms are to be considered the true L. bifidus. So far as the presented evidence goes, it is seen that the unbranched bacilli isolated by us and the ATCC strains of L. acidophilus are similar to one another with the exception of CO2 production. It will be recalled that nos and 4963, likewise producing C02, were considered by Weiss and Rettger as L. bifidus. According to them these are L. bifidus, type I (Tissier). The branched organisms isolated by us, which differ so greatly from the unbranched strains, appear to resemble those isolated by Orla-Jensen, Blaurock, and Boventer and according to Weiss and Rettger would be classified as L. bifidus, type II, or L. parabifidus. It is our belief, however, that the branched organisms that we have isolated from the stools of breast-fed infants are the actual organisms first observed and named by Tissier as L. bifidus in films of stools and that he

14 694 NORRIS, FLANDERS, TOMARELLI, AND GYORGY [VOL. 60 and many others were not successful in maintaining these organisms as stable strains in subcultures. Consequently we suggest that the branched strains should be considered L. biftdus (Tissier). With respect to the unbranched strains, these organisms will be regarded by many as L. acidophilus. It may be logical, however, to remove the CO2-producing organisms from this species, especially if it should be established that these organisms are derived by mutation or variation from bifid strains. We would call these organisms L. parabifidus. In effect this classification would transpose the designations of Weiss and Rettger. Acknowledgment. The authors wish to express their thanks to Dr. Catherine Rose for measuring the Duclaux constants and the optical activity of lactic acid, and to Mrs. Carrie Attmore and Miss Fern Drexel for valuable technical assistance. SUMMARY The Teply and Elvehjem medium supplemented with folic acid, sorbitan monooleate, pancreatin, ascorbic acid, and vitamin B12 is satisfactory for the isolation and propagation of Lactobacillus bifidus as a branched organism from the stools and intestinal content of breast-fed infants. The morphology of colonies and organisms is described and illustrated. Unbranched strains probably derived by mutation from branched organisms occur frequently but can be readily differentiated on agar plates and on stained preparations from the branched organisms. The bifid organisms grown on a solid medium require for growth small amounts of atmospheric CO2 and will tolerate up to 3 per cent of atmospheric O2. When grown in broth tubes the organisms will tolerate atmospheric 02 and do not require the addition of CO2. The unbranched strains and American Type Culture Collection strains of L. bifidus nos and 4963 (now classified as Lactobacillus acidophilus) and L. acidophilus nos. 4355, 4357, and 9857 in either solid or liquid media do not require C02, will grow aerobically, and are facultative anaerobes. The growth range of bifid organisms is more limited at varying temperatures than is the case for the unbranched strains and ATCC strains of L. bifidus and L. acidophilus no Growth of the bifid strains is poor at ph 6 and is usually absent at ph 5.6. Maximum growth of unbranched strains and ATCC strains of L. bifidus and L. acidophilus no occurs at ph 5.6 and, except for the latter, as low as ph 5.1. None of the bifid strains produce CO2; all but one of the unbranched strains and three of five ATCC strains produce large amounts of molecular CO2 under the conditions of the experiment. Dextro-rotatory lactic acid is formed by the bifid strains and inactive lactic acid by the unbranched strains and ATCC strains. The relative amount of volatile acid produced by the bifid strains is greatly in excess of that formed by the unbranched and ATCC strains. In both instances, the volatile acid appears to be acetic.

15 1950] ISOLATION AND CULTIVATION OF LACTOBACILLUS BIFIDUS None of the organisms listed produce catalase or indole or reduce nitrates to nitrites. Evidence is presented that suggests that unbranched strains are mutants of branched organisms, but the validity of the hypothesis has not been proved. It is proposed that the branched organisms that we have isolated from the stools of breast-fed infants are the same as those described by Tissier and that the name Lactobacillus bifidus should be applied to them rather than to the unbranched organisms of Weiss and Rettger. It is also suggested that the unbranched strains that produce CO2 should be called Lactobacillus parabifidus. REFERENCES ADAM, A. 1921a tiber Darmbakterien. II. Zuchtung des B. bifidus auf Hamatinnahrboden. Z. Kinderheilk., 29, ADAM, A. 1921b tjber Darmbakterien. III. tber den Einfluss der H-Ionenkonzentration des NAhrbodens auf die Entwicklung des Bacillus bifidus. Z. Kinderheilk., 29, ADAM, A. 1921c tjber Darmbakterien. IV. tyber das H-Ionenoptimum der Kopfchenbakterien des Mekonium. Z. Kinderheilk., 30, ADAM, A. 1922a tber Darmbakterien. V. Grundlagen der Erniihrungsphysiologie des Bacillus bifidus. Z. Kinderheilk., 31, ADAM, A. 1922b Ueber die Bedeutung der Eigenwasserstoffzahl (des H-Ionenoptimum) der Bakterien. Zentr. Bakt. Parasitenk., I, Orig., 87, ADAM, A Zur Bakteriologie des Sauglingsstuhle. Bemerkung zu Arbeit. Zeissler und Kaickel. Jahrb. Kinderheilk., 101, ADAM, A Die Entstehung der Bifidusvegetation. Jahrb. Kinderheilk., 110, ADAM, A Die Entstehung der Bifidusvegetation. Jahrb. Kiaderheilk., 117, ADAM, A., AND KIssoFF, P. 1922a tyber Darmbakterien. VII. Zur Biologie der Darmflora des Sauglings. Ernahrungsphysiologie des B. acidophilus im Verhailtnis zu der des B. bifidus. Z. Kinderheilk., 34, ADAM, A., AND KISSOFF, P. 1922b tyber Darmbakterien. VIII. Zur Biologie der Darmflora des Sduglings. Das quantitative VerhAltnis von B. bifidus zu B. acidophilus. Z. Kinderheilk., 34, BARBER, M. A The rate of multiplication of Bacillus coli at different temperatures. J. Infectious Diseases, 5, BARKER, H. A., AND HAAs, V Butyribacterium, a new genus of gram-positive, nonsporulating anaerobic bacteria of intestinal origin. J. Bact., 47, BARKER, S. B., AND SUMMERSON, W. A The colorimetric determination of lactic acid in biological material. J. Biol. Chem., 138, BERGEIM, Toxicity of intestinal fatty acids for yeast and E. coli. J. Infectious Diseases, 66, BLAUROCK, G Zur Physiologie der Bifidusbakterien. Monatsschr. Kinderheilk., 68, BLAUROCK, G Bifidusziichtung auf zystinhaltigen Nahrboden. Zentr. Bakt. Parasitenk., I, Orig., 144, BOVENTER, K Untersuchungen uiber das Bacterium bifidum. Zentr. Bakt. Parasitenk., I, Orig., 142, BREED, R. S., MURRAY, E. G. D., AND HITCHENS, A. P Bergey's manual of determinative bacteriology. 6th ed. Williams and Wilkins Co., Baltimore, Md. BREWER, J. H A modification of the Brown anaerobic jar. J. Lab. Clin. Med., 24, CURRAN, H. R., ROGERS, L. A., AND WHITrIER, E The distinguishing characteristics of Lactobacillus acidophilus. J. Bact., 25,

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