SPECIES DIFFERENTIATION OF ORAL LACTOBACILLI FROM MANINCLUDING DESCRIPTIONS OF LACTOBACILLUS SALIVARIUS NOV

SPEC AND LACTOBACILLUS CELLOBIOSUS NOV SPEC

MORRISON ROGOSA, R. F. WISEMAN, JOYCE A. MITCHELL, M. N. DISRAELY, AND AssISSDBY A. J. BEAMAN

National Institute of Dental Research, National Institutes of Health, Public Health Service, Federal Se-curity Agency, Bethesda, Maryland

Received for publication December 15, 1952

It is easy to assume from the literature thatthe oral lactobacilli are a group of variantsrepresentative of the species Lactobacillusacidophilus. Indeed, results obtained by theconventional Hadley (1933) technique for theenumeration of oral lactobacilli have beenexpressed almost exclusively as the "L.aidophilus or L. A. count".

Furthermore, Rosebury (1944) in a review on"The Parasitic Lactobacilli" has expressed aprevailing view of the dental literature that"L. acidophilus is the designation used here toinclude all aerobic parasitic lactobacilli."

Bergey's Manual (1923) states that thelactobacilli are microaerophilic. In order to avoidconfusion, it seems necessary to explain that theHadley (1933) technique is one in which anappropriate dilution of saliva is streaked bymeans of a glass rod on the surface of solidtomato agar (pH 5.0) plates. The "aerobicparasitic lactobacilli" mentioned above are thosecapable of growth under these conditions.However, with the same medium and incubationunder reduced oxygen tension it is obvious thatthese "aerobic parasitic lactobacilli" grow opti-mally under microaerophilic, if not anaerobic,conditions. Rodriguez (1930, 1931) had developeda method for cultivation of the oral lactobacillibased on this property. In other words, the orallactobacilli are not unique in this respect fromthe general behavior of the members of thegenus.

Various attempts have been made to definethe taxonomic status of the oral lactobacilli.Xligler and Gies (1915) have been quoted oftensince they were among the earliest who studiedthe lactobacilli from the mouth. Although theyaccepted the designation Bacillus acidophilus(Moro) for their strains, they recognized 3types from the limited number of tests conducted.

Furthermore, they considered that "the pleo-morphic bacillus undoubtedly belongs to theacidophilus or acidific group' of organisms foundin human and animal feces...."

Dental and intestinal strains have beencompared by Morishita (1928, 1929), Rosebury,Linton, and Buchbinder (1929), Hadley andBunting (1932), Hadley, Bunting, and Delves(1930), and others. Morishita observed dif-ferences in antigenicity and the utilization ofcarbohydrates whereas the others consideredthese strains as variants representative of"L. acidophilus". Warner and Arnold (1941)also concluded that "oral aciduric bacilli do notappear to be a group separate and distinct fromLactobacillm acidophilus of Moro."2The role of L. acidophilus in dental caries,

either as an etiological agent or an index organismof expected caries experience, has been empha-sized repeatedly. At least 100 references can becited. Occasionally the terms Bacillus acidophilus,B. acidophilus, Lactobacillus acidophilus-odontyli-cus, L. odontylicus, or similar epithets have beenemployed.The authors have studied the available extant

strains of Anderson and Rettger (1937), Enright,Friesell, and Trescher (1932), Curran, Rogers,and Whittier (1933), and Morishita (1928, 1929).All the intestinal strains were typically L.acidophilus, and nearly every dental strainremaining from the foregoing investigations wasL. casei. It is not surprising therefore that,except for Curran, Rogers, and Whittier (1933)who did not study agglutination reactions, these

1 Italics ours.2 The authors have studied this strain from the

Kral Collection and have confirmed the finding ofOrla-Jensen, Orla-Jensen, and Winther (1936) thatthis is not a lactobacillus but rather Microbac-terium lacticum.

681

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

682MO-iRISON ROGOSA ET AL.

investigators had the common experience thatnone of the antisera of oral strains agglutinatedintestinal types at significant titer8.Our consistent experience has been -at the

remnants of oral lactobacillus collections fromhuman sources are almost invariably a compactgroup of L. casei. These are the hardiest of theoral lactobacilli, and unless diligent effort is madein the maintenance of collections, nearly all theremaining strains die. It is apparent that a studyof such a residual culture collection is not repre-sentative of the species distribution in themouth. It is nearly certain from the data and inretrospect that Morishita (1928, 1929) andCurran, Rogers, and Whittier (1933), for instance,almost always studied the comparative charac-teristics of L. casei as their oral strains andL. acidophilus as their intestinal strains.

Ulicny (1936) studied acid production byintestinal and dental strains and found that theywere distributed into those producing small andlarge amounts of acid. Sullivan, Still, andGoldsworthy (1939) also divided dental strainson the basis of weak acid and strong acid pro-duction. They also noted fermentative and otherdifferences between the two groups.

Curran, Rogers, and Whittier (1933) wereunable to agree with the prevailing concept of thedental literature that the dental strains wereL. acidophilu8. Over half of their dental strainsseemed to be L. casei and they were clear that,"Insofar as the evidence from this collectionindicates, the lactobacilli occurring in cariousteeth are not of one species and are not usuallyof the acidophilus type."What then may be the reason for the concept

that the oral lactobacilli are simply variants ofL. acidophilus? It is based primarily on the workof Hadley, Bunting, and Delves (1930) andHadley and Bunting (1932). These authors haddescribed 4 colonial types. Furthermore, theymaintained that they could dissociate the smoothtypes to rough ones very easily. This ease ofdissociation convinced them that the orallactobacilli were simply colonial variants ofL. acidophilus. This view has been accepted andperpetuated.An examination of their papers reveals that

they worked with two media, one a casein digestoleate medium and the other a glucose infusionmedium. The isolations also were made first onthe casein digest oleate medium. In recent years,

however, some knowledge has been gainedconcerning the influence of certain culturalconditions on the rough -* smooth growthtransformation. Dubos et al. (1946) observed thatMycobacterium tuberculosis exhibited smoothgrowth under the influence of polyoxyethylenesorbitan monooleate ("tween 80") and grewnormally in the rough phase in its absence.Rogosa and Mitchell (1950a) also described thesame tendency for smoother growth in thepresence of tween 80 with a number of species oflactobacilli and have since observed this phe-nomenon routinely. Furthermore, they clearlydemonstrated that "the alteration of colonymorphology of entire populations is not the resultof genetic changes, but rather is a difference inthe phenotypic expression of genetically similarpopulations in two different environments."McDonald and Frazier (1951) have confirmedthis general principle and have summarizedsuccinctly the experimental evidence as follows:"Thus it appears that the use of colony type asthe sole criterion for the determination of thedissociative stage of a culture could prove verymisleading if it were assumed that colony typewas accompanied by changed physiological andbiochemical properties."The authors do not wish to leave the impression

that genotypic changes do not occur among thelactobacilli. However, they appear with suchinfrequency on media not containing substancesconducive to variation in phenotypic expressionthat in nearly all instances the colonial types onstandard media are distinctive for the species.(See Anderson and Rettger, 1937; Weinstein etal., 1933; Curran et al., 1933; Orla-Jensen, 1919,1943; Orla-Jensen et al., 1936; Tittsler et al.,1947; Barber and Frazier, 1945; McDonald andFrazier, 1951.) As the foregoing authors haveobserved, there is a constant tendency forreversion to the predominating standard colonialtype of the species. Furthermore, Orla-Jensenand co-workers (1919, 1936, 1943) used caseindigest media but without substances conducive tovariation in phenotypic expression.

It seems that Rodriguez (1932) has expressedthe present status well. "For years it has been myopinion, based on personal study and observationof many thousands of strains of both dental andintestinal origin, that we are dealing with a verycomplex group... a condition comparable tothe status of the gram-negative bacilli in the

682 [VOL. 65

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SPECIES DIFFERENTIATION OF ORAL LACTOBACILLI

intestines of man and certain animals before theseparation of the typhoid, para-typhosis, and colitypes, but as in the latter instance, capable ofeventual taxonomic classification."

EXPERIMENTAL METHODS

Isolation of strains. Paraffin stimulated salivawas collected from 130 children in the elementaryschool grades 1 to 7. The salivas were streakedat a dilution of 1/50 on the surface of tomatoagar (Jay and Arnold, 1946) and incubated forfour days at 37 C.

Representative colonies in approximate relationto the differential count were selected with the aidof a wide-field microscope for isolation into amedium of the following composition, herebydesignated as medium 1: Tryptone (Difco), 20 g;tryptose (Difco), 5 g; yeast extract (Difco), 5 g;tomato juice, 200 ml; water soluble liver extract(Wilson Laboratory), 1 g; glucose, 3 g; lactose,2 g; tween 80, 50 mg; distilled water to one liter;pH 6.5.

These 500 isolates were replated at least onceand as often as was deemed necessary to ensurethe establishment of a pure culture. The colonialtypes of the reisolated strains again were noted.The following preliminary procedures wereperformed: Gram stain employing the Burkemodification, catalase tests, and tests for thereduction of nitrate to nitrite. Precautions weretaken to test for residual nitrate and to makesure that good growth occurred in the nitratemedium. Only gram positive, catalase negative,nitrate negative rods were retained for furtherstudy.

Preliminary testfor gas production. Immediatelyafter the primary isolation and purification of theisolates, heavy inoculations from actively grow-ing cultures were made into 10 ml of medium 1supplemented with 2 per cent agar. The tubedmedium had been melted previously and held at45 C. After solidification, the medium was layeredwith an additional depth of approximatelyone-quarter inch of nonnutrient 2 per centagar, over which a thin layer of heavy mineraloil was added. Gas production was noted bycracks or disturbance of the agar. Very oftensufficient gas (CO2) was produced to force theplug of agar out of the tube.

Acidity in milk. Nine ml of skim milk weresterilized in 16 by 150 mm tubes, inoculated byloop, and incubated for two weeks at 37 C. The

contents of the tubes were washed into Erlen-meyer flasks (a stirring rod aiding the breakingof the clot where it was formed) and titrated withN/10 NaOH, using phenolphthalein as theindicator. The acidity values were correctedappropriately for control acidity.

Temperature limits for growh. The organismswere tested for their ability to grow in medium 1at 45 C during one week of incubation in thewater bath. The tubes were stoppered withsterile rubber stoppers to avoid evaporation andsurface cooling of the tubes which, in a numberof instances, would have led to false positiveresults.

Initiol Wte on the fermentation of carbohydrates.Fermentation tests were conducted on thefollowing substrates at a concentration of 0.5 percent: D-glucose, D-levulose, iarabinose, D-lactose,D-mannitol, D-mannose, D-melezitose, D-melibiose,D-raffinose, D-sucrose, salicin, D-trehalose,D-xylose, adonitol, D-cellobiose, dulcitol, D-galac-tose, inositol, inulin, D-maltose, i-rhamnose,D-sorbitol, L-sorbose, a-methyl-D-glucoside, anda-methyl-D-mannoside. The carbohydrate sub-strates were Pfanstiehl products whose specificrotations were in excellent agreement withaccepted values. Preliminary tests demonstratedthat arabinose, xylose, mannose, galactose,levulose, sorbose, and maltose required steriliza-tion by filtration. This was accomplished byemploying sintered glass ifiters without theapplication of heat at any time.The initial screening tests were conducted in

the following basal medium, hereby designatedmedium 2: Trypticase (BBL), 0.5 per cent;yeast extract (Difco), 0.3 per cent; beef spleenextract (Wilson Lab), 0.25 per cent; MgSO4.7H20, 0.05 per cent; vitamin supplement, 1 ml;and brom cresol purple, 0.0016 per cent. The beefspleen extract was infused with 50 ml boilingdistilled water and after some cooling wascentrifuged. The supernatant fluid was filteredby suction in a Buchner funnel through papercoated with a filter aid. The remaining ingredientswere dissolved in the now clear beef spleeninfusion. The vitamin supplement comprised thefollowing in terms of micrograms per 100 ml ofmedium: nicotinic acid, 100; thiamin hydro-chloride, 50; calcium pantothenate, 50; riboflavin,50; pyridoxamine hydrochloride, 20; pyridoxalhydrochloride, 10; pyridoxine, 20; inositol, 1,000;choline chloride, 1,000; biotin, 1; p-amino-

19531 683

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

MORRISON ROGOSA ET AL.

benzoic acid, 1; and folic acid, 1. The pH of themedium was brought to 6.6 to 6.8 and the volumeadjusted to 100 ml.The medium was dispensed in 2 ml quantities

into 10 by 75 mm tubes which were sterilized inracks. The autoclave was brought quickly to115 C at the exhaust, the steam supply closedimmediately, and the pressure allowed to returnto atmospheric pressure. This minimal heatapplication was found to be adequate when theforegoing conditions were maintained. The in-ocula generally were prepared by making twopreliminary transfers, or in the case of theslower growing strains, after a period in whichgood growth had appeared. The second transferswere washed twice with distilled water and the

Figure 1. Buffermedium.

to 12 14 1 I* 20

ML M/10 LACTIC ACID

curve of basal fermentation

inocula comprised one drop by pipette. Theimportance of employing cultures which had beenbrought to a vigorous state of activity becameevident early in this study and will be discussedlater.

Repetition of the fermentation teat8. Two yearsafter the completion of the original fermentationtests they were repeated. During this periodmedia had been studied intensively, and a highlyselective medium had been developed for theprimary isolation of lactobacilli (Rogosa et al.,1951a,b) from oral, intestinal, and vaginalsources. Therefore, the selective medium forprimary isolation was adapted for the lessrigorous requirements of pure culture study tobecome an excellent basal medium for therepeated fermentation study. The primary

selective medium (SL agar)j.Was modified byreducing the sodium acetate to 1.5 per cent,omitting the agar, glucose, and acetic acid, andby the addition of alizarine red S as indicator ata concentration of 0.004 per cent. The mediumhad a final pH after sterilization of 5.9. This isdesignated as medium 3. Sterilization waseffected exactly as described previously. Thecarbohydrates were added to this medium at aconcentration of 2 per cent except for galactosewhich was present at a concentration of 1.4 percent.The indicator was observed daily for at least

two weeks and a notation made the day itchanged color distinctly. In addition, in therepeated fermentation study, the pH of thecontents of each tube was determined after twoweeks of incubation. In order to relate the pHreadings to total acidity, as certain importantand fundamental investigations (Orla-Jensen,1919, 1943; Orla-Jensen et al., 1936; Pederson,1929a,b, 1930, 1936, 1938) have done, thebuffer curve of the basal medium is shown infigure 1.

Additional tests8. Tests not performed in theoriginal screening program were those for thehydrolysis of sodium hippurate, esculin, and theproduction of ammonia. For the hippuratehydrolysis tests medium 3 was modified by 1 percent sodium hippurate and 2 per cent glucose.After 2 weeks' incubation at 37 C, 3 ml of 50 percent H2S04 were added to 2 ml of the medium.3In positive reactions the characteristic crystals ofbenzoic acid were observed. Tests for the hy-drolysis of esculin were made in plates pouredfrom medium 4. This was medium 3 made solidby 1.5 per cent agar and supplemented with 0.3per cent esculin, 2 per cent glucose, 0.5 per centarabinose, and 0.5 per cent ferric citrate. Thetypical blackening of the medium in stronglypositive tests was distinctive. However, asufficient number of delayed and equivocalreactions was observed so that the esculin results,although suggestive of differential value, will bepresented with some reserve. Ammonia wasdetected qualitatively by direct nesslerization inmedium 1.

Optical rotation of latic acid. The mediumemployed (5) was a modification of SL agar in

8 The experience of some investigators, includ-ing us, is that the ferric chloride test for hippuratehydrolysis is unreliable.

2

684 [VOL. 65

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SPECIES DIFFERENTIATION OF ORAL LACTOBACILLI

the following respects: omission of the agar;reduction of the sodium acetate and tween 80concentrations to 1 and 0.03 per cent, re-spectively. Arabinose and xylose at concentra-tions of 0.5 per cent were added in the cases ofcertain heterofermentative strains which other-wise did not produce sufficient lactate foranalysis. The medium was dispensed in 300 mlquantities into 500 ml flasks, 10 g of calciumcarbonate added, and sterilized at 15 lb steampressure for 15 minutes. The flasks were in-oculated heavily and incubated for 2 weeks at35 C. Volatile acids were distilled off and zinclactates prepared from the ether extracts. Theanalyses were performed according to the methodof Curran, Rogers, and Whittier (1933).

Studies of nutrition. The "complete" mediumcontained the following ingredients per liter offinal strength medium: acid hydrolyzed caseinor Difco "vitamin-free" casamino acids, 5 g;L-cystine, 0.1 g; DL-tryptophan, 0.1 g; DL-

asparagine, 0.25 g; ammonium citrate, 2 g;KH2PO4, 3 g; K2HPO4, 3 g; sodium acetatehydrate, 16.5 g; adenine, guanine, uracil,xanthine, each 0.01 g; tween 80, 1 g; glucose,20 g; Difco casitone, 2.5 g; sodium thioglycolate,1 g; L-cysteine, 0.1 g; salt solution, 5 ml; cholinechloride and inositol, 20 mg each; p-aminobenzoicacid, pteroylglutamic acid, and biotin, 10 jgeach; niacin, 2,000 j,g; thiamin hydrochloride,calcium pantothenate, and riboflavin, 1,000 geach; pyridoxamine and pyridoxine, 400 ,ugeach; pyridoxal, 200,g; and crystalline vitaminB12, 0.25 pg.The salt solution was made as follows:

MgSO4.7H20, 11.5 g; FeSO4.7H20, 0.68 g;MnSO4.2H20, 2.4 g; and distilled water to 100 ml.The casamino acids or acid hydrolyzed casein

was treated as follows: 100 g were dissolved inapproximately 750 ml distilled water, pH broughtto 4 with hydrochloric acid, 25 g Darco G60added, and stirred with an electric stirrer for 30minutes. The filtrates were recovered. Four suchadsorptions with Darco G60 were made and thefinal filtrate brought to a volume of one liter.The casitone was treated in the same wayexcept that the final volume was brought to 2liters.The inocula were prepared, as was customary,

from actively growing cells previously carriedthrough at least two transfers in medium 1 or 3.The cells were washed twice in distilled water

and usually brought to a final volume in distilledwater of 10 times that of the original culture. Thefinal dilution was sometimes less depending onthe ability of the particular culture to grow inthe "complete medium". However, the inoculawere kept as constant as was possible in differentexperiments. The final inocula were added induplicate to the test media in one drop aliquotsfrom 1 ml pipettes. The same pipette was used ininoculating from an individual culture suspensionin any experiment.

RESULTS

For purposes of orientation a summary of thecharacteristics of the homofermentative lacto-bacilli is presented in table 1. This summary wasprepared originally by R. P. Tittsler and isbased on the data of Tittsler, Geib, and Rogosa(1947) and Rogosa, Tittsler, and Geib (1947),which previously have been presented in abstractform only.A study of the descriptions of the hetero-

fermentative lactobacilli in Bergey's Manual(Breed et al., 1948) makes it apparent that clear-cut differentiation of these species is mostdifficult.

Lactobacillus acidophilus. The concept ofL. acidophilus stems from the descriptions ofMoro (1900a,b), who described organisms ofintestinal origin, forming irregular and fuzzycolonies. This type of colony was termed the Xtype by Rettger and his associates. Infrequently,some cultures develop some colonies whose edgesare entire (the Y type of Rettger). Nevertheless,these smooth or Y colonies, during 15 years ofobservation, always have reverted in time to theX type or mixtures in which the X type ishighly predominant. It seems strange to us thatthe colonies of L. casei should be confused withthe variant smooth colonies of L. acidophilus.Although older colonies of L. casei may exhibit

local areas in which thread-like projections mayappear, there is a constant tendency for reversionto a smooth, relatively large, disc, or lens-shapedcolony in deep culture. There are other dif-ferences in the colonies themselves, such as size,rate of development, color, physical appearance,and texture. Variant colonies of L. acidophilusand L. casei are shown in figures 2, 3, and 4,respectively.The tacit assumption that oral and intestinal

sources are mutually exclusive for the same

19531 685

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

I I I +1-H

I +

II -HI

I I I +1-H

I I I ++

I -H II I w

I

I

040

1 o

I +

+

+ +

+ +

+ +

I +H-

+++I

I +,I

+ I +1

+ 1.

II I I +H

I I I ++-

I I I w +++

I

I I

I I

+ I

+ I

+ I

++

+ I

+1I

++

++

++

++

++

++

+ + ++ ++ +++

E%A]11 1 1 11 ++ +++I I I I I ++ +++Q+~~~ ~~++ ++ ++l++ + $ I I ++ +++-'0~~~~~~~~~~~~~~00 -~~~~~~0

C4CX 14 C; P- V-wm w

III

-4- +- - ---

0~~~~~4

PC) 00 P o P00 0000 P0000 O0Po

..a a -a e *s.2.2

686

4) "

o .. 00"0

00

00

.

. 00 .o

. .2.0000

0N.0

4)0 -.0N '004o0..04)'0o4. '.00 '0-.

0 0.

0 4)4)00..0U

*'. Oo04)

"000 -00. 040

U00

.0ON

00.0 ,.o.

.0

.00 -

,04)

04"00

0 0N

0

,0

04 4)

05 z

CD 0 0

++I3.:, gr

IcI!SOUI

lol!q°oS

lolPina

asoqzoS

0so;Tz0lj0j

I ulinuj

asolqx

asoutqs.tV

I

I

I

I

I

I I

I I

I I

0soUIqsI I I

asotq!o]K

osoutumuq

IO1!UUWIN

asozqolla )

a0so.Ions

asouonS

asomaI

I I

I

I I

I+

I I

I I

III+

go0I-H

10

0a

.v4t

PC

¢

t3b

*::.

+ + -H

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SPECIES DIFFERENTIATION OF ORAL LACTOBACILLI

Figure 2. Typical X or rough colony of Lactobacillus acidophilus in the depth of tomato agarMagnification 46X.

Figure 3. Smooth phenotypic variant colony ofLactobacillus acidophilus. This reverts in one

transfer on tomato agar to the rough type shownin figure 2. Magnification 46X.

organisms has incited a futile controversy.Personal habits, diet, etc., and similar ease ofimplantability in different yet related anatomical

Figure 4. Typical sub-surface colony of Lacto-bacillus casei illustrating disc types. Note differ-ence in size from phenotypic variant of Lacto-bacillus acidophilus in figure 3 and differences inreflection of light. Magnification 46X.

regions should make it not surprising thatL. acidophilus, originally isolated from theintestine, should be found also in the humanmouth. It is a common experience to isolateabundant numbers of fecal streptococci from themouth. In the present study, L. acidophilus wasfound in varying numbers in about 21.5 per centof the 130 samples but constituted only 11 percent of the 500 isolates. The most prevalenthomofermentative species, however, was L. casei.Table 2 presents typical data differentiating thespecies.

68719531

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

MORRISON ROGOSA ET AL.

TABLE 2Distinguishing characteristics of Lactobacillus

acidophilus and Lactobacillus casei

Lactose......Maiinitol....Melezitose ...

Melibiose....Raffinose....Sucrose...Xylose ......

Adonitol....Dulcitol .....

Inositol .....Inulin .......

Maltose.....Rhamnose...Sorbitol.....Sorbose ......

Hippurate ...

Glucose .....a-Methyl-

glucoside..a-Methyl-mannoside.

Basal .......Nutritional

pattern*...Lactate......Colony.Acidity-milk-%....

Growth45 C.......

Growth16 C.......

Lactobacillusacidophilus

4.0+15.5-5.3-5.7-5.7-4.0+15.5-5.8-5.3-5.4-5.7-4.1+15.5-5.8-5.7-

4.1+1

5.7-

5.8-5.8-

2IRI

0.26-1.43Mean =

0.5

Lactobacillus casei

var. var.csi alac-

losus

4.1+1 5.9-4.1+1 4.3+14.0+' 4.0+15.4- 5.4-5.9- 6.0-4.5+4 4.6+45.3- 5.5-5.11 4 6.0-5.7- 4.0+'5.24-14 5.8-5.9- 5.9-4.5+3 4.7+35.5- 5.6-4.1+2 4.1+'4.0+2 4 .0+2+ +

4.1+' 4.1+'

4.1+14 4.2+'o 4.6+3

5.6-6.0-

5.9-6.0-

1

DL

s0.09

+

1iDL

s.51,

+

* Refers to nutritional data of table 3.All strains ferment mannose, trehalose, salicin,

cellobiose, galactose, and levulose. None fermentsarabinose and glycerol, or hydrolyzes esculin.

Superscripts in all tables signify day of incu-bation at which indicator was observed to changecolor distinctly.

i indicates weak, incomplete, or variable re-

action.DL indicates that D-lactic acid is the predomi-

nant optical form.S = smooth colonies; I = intermediate or ir-

regular; and R = rough colonies..

In the notation of fermentative data, super-

scripts have been included to indicate the first

definitely observed change. This has beenconsidered important since one determination ofacidity after extended incubation may yield noinformation concerning the probable adaptivenature of certain fermentations. Many caseshave been observed in which significant quantitiesof acid have accumulated with extended incuba-tion, but in which the apparent initiation offermentation was delayed significantly and oftenconsistently. The species have been evaluated bymeans of a correlation of characteristics in whatis believed to be a natural taxonomic approach.The authors have recognized 4 variant types of

L. acidophilus. More than 80 per cent of thestrains formed a compact unit of identity(group 1). About 2 per cent varied from themodal behavior of the species in fermentingmelibiose slowly and weakly (group 2), 4 per centvaried in fermenting sorbitol and being derivedfrom a smooth colony on isolation (group 3), and14 per cent in fermenting raffinose very slowlyand very weakly (group 4). It is evident that thevariations were largely in terms of slow and weakreactions. To conserve space only the typicalreactions of group 1 are shown in table 2.

Lactobacillus casei. Previously Orla-Jensen(1919) observed 2 varieties, one of which fer-mented rhamnose and grew at 45 C, and sug-gested that they might well be regarded asseparate species. Orland (1950) has shown thatthese 2 varieties possess dissimilar antigeniccomponents.The authors recognize 3 varieties: L. casei

var. casei var. nov. which does not fermentrhamnose but ferments lactose; L. casei var.alactosus var. nov. which does not ferment eitherrhamnose or lactose; and L. casei var. rhamnosusvar. nov. which ferments rhamnose.

Despite these variations, all the strains wereidentical positively in the fermentation of 12substrates. They also were identical in notfermenting 8 substrates or in fermenting themonly very weakly and slowly. In addition, all thestrains possessed some fundamental enzymaticidentities. The lactate accumulating in cultureswas invariably a mixture of the D and L formswith the optically active D-lactic acid always inexcess. The strains also behaved alike in nu-tritional tests and in growth at certain extremesof temperature (16 C). The colonial type also wasidentical.Only data for group 1 of L. casei var. casei are

shown in table 2. The detailed data for groups

688 [VOL. 65

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SPECIES DIFFERENTIATION OF ORAL LACTOBACILLI

2, 3, and 4 are omitted. The groups within thisvariety differed among themselves in eitherweakly fermenting or not fermenting adonitol,inositol, and very infrequently, xylose. Group 2(15 strains) differed from group 1 (73 strains) infermenting inulin, while group 3 (17 strains)differed from group 1 in fermenting inulin andin not fermenting melezitose and sorbose. Group4 (6 strains) varied from group 1 in fermentinginulin and feebly fermenting dulcitol.Groups 1 (26 strains), 2 (2 strains), and 3 (2

strains) of L. casei var. alactosus did not fermentlactose. This was surprising to us, but it shouldbe remembered that previously described isolatesof L. casei have been obtained almost exclusivelyfrom dairy products. The detailed data forgroups 2 and 3 are omitted from table 2. Group 2differed from group 1 in fermenting raffinose andinositol weakly and slowly, in fermenting inulin,and in not fermenting sorbose. Group 3 variedfrom group 1 in questionable fermentations ofinositol and rhamnose, and in not fermentingsorbose.Only data for group 1 (45 strains) of L. casei

var. rhamnosus are shown in table 2. Group 2(2 strains) was different from group 1 in fer-menting dulcitol, slowly fermenting inulin, andnot fermenting sorbose. Group 3 (8 strains) alsofermented dulcitol, did not ferment sorbose, andwas weak and variable in hippurate.

Six of 196 strains of L. casei, originally fer-menting inulin, were unable to do so on furthertesting, and 2 strains, originally negative insucrose, acquired a slow fermentation. However,a slow, adaptive fermentation of sucrose byL. casei var. rhamnosus characteristically is notuncommon.

The great majority of the variations within thevarieties of L. casei was in the cases of substanceswhich were attacked either weakly or not at all.

Distinguishing characteristics of L. acidophilusand L. casei. In the experience of the authorswith about 1,000 strains of L. casei and 200 ofL. acidophilus, L. casei has always fermented,and L. acidophilus has never fermented, mannitol.This finding for L. acidophilus agrees with Orla-Jensen, Orla-Jensen, and Winther (1936),Harrison and Hansen (1950c), and Tittsler,Geib, and Rogosa (1947); and the observation forL. casei is in agreement with Orla-Jensen (1919,1943). A strain of L. acidophilus has not beenencountered yet which is capable of hydrolyzingsodium hippurate. In contrast, 98 per cent of all

strains of L. casei tested hydrolyzed sodiumhippurate readily. All strains of L. casei fer-mented a-methyl-D-glucoside slowly, whereas nostrain of L. acidphilus fermented this substrate.These 2 species differed fundamentally in theproduction of lactic acid. In repeated tests allstrains of L. acidophilus accumulated primarilyopticallv inactive lactic acid, and no strain ofL. casei did this. No strain of L. acidophiluscould grow at 20 to 22 C. This observationagain agrees with Weinstein, Anderson, andRettger (1933), Orla-Jensen (1943), and Orla-Jensen, Orla-Jensen, and Winther (1936). Indeed,

TABLE 3Nutritional patterns of the lactobacilli

HOMOFERMEN- HETEROFER-TATIVE MENTATIVE

1 2 3 45 6 7 8

Complete....... + + + +4+Niacin ... ...... - _Thiamin......... .-+ + + + +_Pantothenate ............ - - .Riboflavin........ -- -+- - + + -

VitaminB6........ F±4-+++ ±+ +4Folic acid.......-- + -_i-Vitamin B12....... + -++ + + _4_

1. + equals growth in the absence of addedsubstrate.

2. :i equals significant limitation of growth inabsence of added substrate.

3. F equals severe limitation of growth inabsence of added substrate.

4. In the absence of vitamin B12 but in thepresence of desoxyribosides Lactobacillus acidoph-ilus exhibits growth.

all strains of L. casei grew at 10 C and a greatnumber at 6 C. The two species also differedfundamentally in their nutritional behavior, ascan be seen in table 3. Finally, we wish to repeatthat the modal colonial types were entirelydifferent.

Lactobacillus plantarum. Among the homo-fermentative types there were 16 strains whichseemed to be related to L. plantarum. Theseisolates resemble organisms previously studied inother investigations by Tittsler, Geib, andRogosa (1947), Rogosa, Tittsler, and Geib(1947), Orla-Jensen (1919, 1943), Harrison andHansen (1950b,c), and Pederson (1929b, 1936).There has been some lack of clarity in the

6891953]

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

MORRISON ROGOSA ET AL.

definition of L. plantarum. Orla-Jensen (1919) hasdefined a group which produces primarilyinactive lactic acid and ". . . prefers as a rulemaltose and cane sugar to lactose..." Thelatter condition, however, seems a somewhattenuous basis for the definition of a species. Insummarizing his own further studies Orla-Jensen(1943) has delineated the species more clearly. Itwas found, for instance, that all strains of L.plantarumn fermented melibiose. Thus, they

TABLE 4Lactobacillus plantarum group

GROUP ....................... 1

No. strains................... 5

Arabinose .............

Melezitose ............

Melibiose .............

Raffinose ..............

Rhamnose ............

Hippurate ............

Glucose ...............

a-Methyl-D-glucoside..a-Methyl-D-manno-

side.................Esculin ...............

Basal .................Nutritional pattern*.Lactate ...............

Colony ................

Acidity milk-%......Growth 45 C..........Growth 16 C..........

4.2+'5.6-4.2+'4.2+'5.3T

4.1+14.1+7

5.7-

5.7-

3IS

0.50

2

6

4.2+24.0+'4.0+14.1+15.4T+

4.1+'5.4-

4.4+2+

5.7-4IS

0.50

3

5

5.5-4.0+14.1+'4.3+25.1++

4.1+'4.7 ±"I

4.1+3

5.7-3I

S

0.50

* Refers to nutritional data of table 3.All strains ferment lactose, mannitol, mannose,

sucrose, trehalose, salicin, cellobiose, galactose,maltose, sorbitol, and levulose. None fermentsadonitol, dulcitol, inositol, glycerol, iilulin, andxylose. One strain variable in sorbose.

= negative or weak.i = slow.

could be differentiated in this respect (as wellas in others) from L. casei, whose strains did notferment melibiose. Neither could the latter be so

adapted. Nevertheless, the data presented byOrla-Jensen (1919, 1943) for L. plantarum isclearly for a group of varieties within a species ifnot for different species.The summary of typical data obtained for the

L. plantarum group in this study is presented intable 4. Three sub-groups were found. In cnn-

firmation of Orla-Jensen (1943), Tittsler, Geib,and Rogosa (1947), and Harrison and Hansen(1950c), all the strains fermented melibiosereadily. Previously, Rogosa, Tittsler, and Geib(1947) had identified two types, one requiringriboflavin for growth and one not requiring it.The same division of types was found in thepresent study.

Although groups 1 and 3 are alike nutritionallyin requiring riboflavin for growth, they aredifferent from each other in four major respects.Group 3 hydrolyzes hippurate, fermentsa-methyl-D-glucoside and melezitose, and doesnot ferment arabinose, whereas group 1 behavesoppositely. Groups 1 and 3 are identical withvarieties previously studied as L. plantarum syn.Streptobacterium plantarum by Orla-Jensen (191]9)and others.

Orla-Jensen (1943) stated that Streptobacteriumplantarum grew well in the absence of riboflavin.This conclusion is based on work done before1936. The authors have tested some of Orla-Jensen's strains and have found that they dorequire riboflavin for good growth in adequatelycontrolled tests. Harrison and Hansen (1950c)agree with the present authors that L. plantarumdoes not grow in an appropriate medium unlessriboflavin is added. There is no reasonable doubtthat the limitations of Orla-Jensen's earlytechnique led him to an erroneous conclusion.Group 2 is distinctly different from the other

groups of L. plantarum in growing well in theabsence of riboflavin and hydrolyzing esculin. Inaddition, it is different from group 1 in hy-drolyzing hippurate, fermenting a-methyl-D-mannoside strongly, and in fermenting melezitose.Also, it is different from group 3 in fermentingraffinose on primary isolation and in fermentingarabinose. Thus, the strains in group 2 differnutritionallv and in 4 other respects from thosein group 1. They also differ nutritionally and in 2other majoor characters from those in group 3.The group 2 strains are identical with strainsdesignated as Lactobacillus arabinosus by Fred,Peterson, and Anderson (1921) and also studiedby Tittsler, Geib, and Rogosa (1947). Orland(1950) has discovered a new major antigen (G)in L. arabinosus. In the judgment of the presentauthors L. arabinosus Fied et al. is a distinctspecies.The antigenic structure of groups 1 and 3,

L. plantarum, is not yet known, but it is probable

690 [VOL. 65

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SPECIES DIFFERENTIATION OF ORAL LACTOBACILLI

that specific antigenicity may exist for eachgroup.

Lactobacillus 8alwarius nov. sp. The finalhomofermentative group seems to us un-questionably a hitherto undescribed species, andwe designate it as Lactobacillus aliwarius.Obviously, this species is a gram positive, non-sporeforming, nitrate negative rod, producingmainly lactic acid from the fermentation ofavailable hexoses. Although chiefly inactive lacticacid is produced by the majority, some strainsproduce considerable quantities of a rotatorylactic acid. Growth is good at 37 C.Two varieties are recognized. The first is

designated as Lactobacillus salivarius var.salivarius. It produces significant acidity inglucose, levulose, melibiose, trehalose, lactose,mannitol, mannose, raffinose, sucrose, galactose,maltose, rhamnose, and sorbitol. It does notferment arabinose, xylose, cellobiose, melezitose,a-methyl-D-glucoside, a-methyl-D-mannoside, sal-icin, glycerol, and sorbose. Esculin and hip-purate are not hydrolyzed.The second variety we name Lactobacillus

8alivarnus var. salicinius. It differs from the typevariety in fermenting salicin and not fermentingrhamnose.

This species requires pteroylglutamic acid andniacin for good growth. Riboflavin is markedlystimulatory. Good growth does not take place innutritional test media in the absence of poly-oxyethylene sorbitan monooleate (tween 80).Eleven isolates from 8 different human

subjects have been studied in detail. In addition,106 strains from 57 oral samplings of Cricetusauratus have been investigated extensively. Thisspecies is the predominating homofermentativeoral lactobacillus in the hamster.The characteristics of this organism will be

described more fully in a subsequent publication.Lactobacillusfemeni. The data for 148 strains

describing 12 variant types of L. fermenti arepresented in table 5. Like all other hetero-fermentative lactobacilli tested, this speciesproduces inactive lactic acid. The majority (56per cent) did not ferment arabinose and xylose.Nearly all the strains grew well at 45 C fromsmall inocula, and about 95 per' cent behavedidentically in nutritional tests. These strainsrequired niacin, thiamin, and pantothenate butdid not require extrinsic riboflavin, pyridoxine,pyridoxamine, or pyridoxal, pteroylglutamic

acid, and vitamin B12 for growth. Equivocalnutritional results were obtained with theremaining 5 per cent either because of inhibitingsubstances in the nutritional test medium orbecause they may really be different.Groups 1 and 2 are easily recognizable as

identical with types designated as Betabacteriumlongum Orla-Jensen syn. L. fermenti Beijerinck.Twenty-eight tests which had been conducted onthe 12 groups of freshly isolated strains wererepeated nearly three years later. Sixty-eight of83 strains in group 1, and groups 3, 5, 6, 9, 10,and 12, or 66 per cent of the total number ofstrains, were identical in all characters in bothtests. The chief variation in repeated testingwas in the fermentation of 1 subetrate, namely,mannose. Some strains gained and some strainslost the capacity to ferment it; but mannosewhen fermented originally was attacked veryslowly and weakly. It may be noted from thedata that the fermentation of mannose byL. fermenti in the more recent tests also wasdelayed. Eleven strains in group 4 and 4 strainsin group 11, originally negative in lactose,adapted to this fermentation. The 26 remainingfermentative characters of groups 4 and 11,however, were the same in both tests. The 3melibioseless and raffinoseless strains of group 9may be considered as probable mutants sinceotherwise they were repeatedly identical withthe typical strains of group 1.Some variants have been included in this

species which ferment either arabinose or xylose.Later, it will be seen that certain other species,particularly Lactacillus buchneri and Lacto-bacillus brevis, are characterized by their abilityto ferment both arabinose and xylose, orarabinose only. It seems appropriate, however, todefine these species by what seems to be a naturalsystem, permissive of minor variation about amodal type of behavior. The remaining variationsfor L. fermenti were chiefly in the consistentfermentation of trehalose by less than 15 percent of the strains.

Lactobacilus buchneri. Twenty-five strainsdesignated as L. buchneri were isolated from 13individuals. In addition to these 25 oral isolates,65 strains from other sources were studied. Thelatter group was identical with L. buchnerifrom oral sources. The characteristics of variantgroups of L. buchneri are shown in table 6.A definition of L. buchneri is most difficult

691j1953]

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

MORRISON ROGOSA ET AL.

since the original incomplete description was consistently. This is a unique property forbased on one strain which is not available for heterofermentative lactobacilli within the scope

study. However, this species is defined by of the present investigators' experience or theBergey (Breed et al., 1948) as synonymous with previous literature.Bacterium mannitopoeum, Lactobacillus mannito- Although Pederson's strains fermented bothpoeum, or Lactobacillus mannitopoeus. Com- arabinose and xylose, only 14 per cent of theparisons were made with strains studied by present strains fermented both these substratesPederson (1929a,b, 1930) and isolations from and were identical with Pederson's in all respects.sources other than that from which the 25 oral The remaining 86 per cent fermented arabinosestrains were obtained. In the foregoing studies but not xylose. However, organisms from spoiled

TABLE 5Lactobacillus fermenti and variants

GROUP........... 1

No. strains....... 3

Arabinose. . . 5.5-Lactose...... 4.5+'Mannose.... 4.8+10Melibiose.... 4.5+1Raffinose.... 4.6+1Trehalose.... 5.5-Xylose.... 5.5-Galactose.... 4.7+1Glucose....4.5+1a-Methyl-D-

glucoside.. 5.5-Basal ..... 5.7-Nutritionalpattern* ... 6

Colony...... I-SAciditymilk-% ... 0.15

Growth 45 C0. +

5.8-4.6+'5.9-4.6+14.7+15.9-4.1+14.9+14.6+1

4.6+24.6+15.1:41114.8+14.7+15.9-5.7-5.0+14.7+1

4.3+25.24-105.1:4104.6+'4.7+14.7+15.8-4.8+24.6+1

4.6+10 5.34-14 5.7-5.9- 6.0- 5.9-

6I-S

6I-S

0.20 0.23

6I-S

0.03+

5.6-

4.6+24.9+74.9+'5.0+25.1 &;45.6-5.24-104.5+1

5.8-5.8-

6I-S

0.28

4.1+14.5+'4.6+104.6+14.6+15.8-4.1+14.8+ '

4.6+1

4.6+75.8-

6

I-S

0.10+

5.8-4.6+75.7-4.6+14.6+14.6+14.1+34.7+ 44.5+1

4.5+115.8-

6I-S

0.07+

5.7-4.6+74.7+14

4.6+14.6+14.6+14.2+'4.86+44.6+1

5.5-4.5+14.6+105.8-5.4-5.6-5.6-4.8+14.6+1

4.1+14.6+4

4.6+14.6+44.6+14.2+14.8+14.6+1

5.8-4.9+'6.0-4.9+54.9+'5.9-5.6-5.1+'5.1+'

4.5+11 5.34t14 4.6+"1 5.9-5.9- 5.8- 5.9- 6.1-

6I-S

0.06+

6I-S

0.18+

6I-S

0.27+

I-S

0.06

+1

+1+2

+1I

+1+1

I-S

0.034i

* Refers to nutritional data of table 3.All strains ferment sucrose, levulose, and maltose. None ferments mannitol, melezitose, salicin,

adonitol, cellobiose, dulcitol, inositol, inulin, rhamnose, sorbitol, sorbose, glycerol, and a-methyl-Dmannoside. None hydrolyzes hippurate and esculin. None grows at 16 C. All strains produce inactivelactic acid.

= weak, incomplete, or questionable reactions.

Pederson thoroughly investigated a group ofheterofermentative lactobacilli obtained fromspoiled tomato products and sauerkraut which hedesignated as L. mannitopoeum or L. buchneri. Ofall the heterofermentative lactobacilli only L.mannitopoeum syn. L. buchneri fermentedmelezitose. This is clear from Pederson's dataalthough he omitted to stress this fermentationas a distinctive characteristic for the differentia-tion of L. buchneri (1938). All 90 strains of table6 were tested repeatedly during a three year

period and were found to ferment melezitose

tomato products or certain natural fermentationseasily may have been subjected to the naturalpresures of competition and selection so that a

single type only may have been isolated. EvenPederson's strains formed only approximatelyhalf as much mean acidity in xylose as comparedto arabinose.

It may be noted in table 6 that although thegreat majority of the strains was unable tohydrolyze hippurate, variable results are reportedfor this character as well as their behavior innutritional tests. A few strains did not grow well

2 3 4 5 6 7 8 9 10 11 12

15 11 11 2 7 4 2 3 2 4 4

[VOL. 65692

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SPECIES DIFFERENTIATION OF ORAL LACTOBACILLI

in the medium used in the nutritional studies, butin all cases extraneous pteroylglutamic acid wasnot required for growth when the growth wasgood and unequivocal in the control completemedium. There is little doubt from continuingwork in this laboratory that the variants ofL. buchneri are in reality fundamentally identicalin their nutritional behavior and that sensitivityto inhibitory substances in the nutritional test

fermented or could be adapted to fermentmelezitose. If group 4 is excepted, all the strainsfermented arabinose and xylose readily. None ofour strains fermented mannose. This species mayferment lactose, raffinose, sucrose, and mannitolslowly and weakly. Occasionally, some strainsmay fail to ferment even one or more of thesesubstrates. All the strains hydrolyzed hippuratereadily and required pteroylglutamic acid. They

TABLE 6Lactobacillus buchneri and variants

GROU'P................................. 1 2 3 4 5 6 7

No. stramins................................ 23 48 1 5 2 8 3

Arabinose........................ 4.1+1 4.2+1 4.3+1 4.2+1 4.2+1 4.3+1 4.2+1Lactose......................... 4.5+2 4.7+3 4.6+9 4.7+3 5.34 5.310 4.6+10Mannitol......................... 5.3X14 5.4t14 5.8- 5.4414 5.8- 5.2-10 5.1'14

Melezitose ....................... 4.6+2 4.6+2 4.7+2 4.7+2 4.6+2 4.7+2 4.6+6Melibiose........................ 4.6+5 4.7+2 4.7+' 4.7+2 4.6+4 4.7+2 4.6+5Raffinose......................... 4.6+'2 4.7+3 4.7+7 4.7+4 4.6+6 4.7+6 4.7+10Sucrose......................... 4.6+2 4.6+1 4.8+2 4.7+2 4.9+6 4.7+4 4.6+'Xylose......................... 5.7- 5.7- 5.8- 5.7- 4.2+2 4.2+2 4.2+1Galactose........................ 4.8+2 4.9+' 4.9+2 4.9+2 4.8+' 4.9+4 4.8+9Hippurate ....................... _ T + 4 + + +Glucose......................... 4.6+2 4.7+2 4.7+2 4.7+2 4.6+2 4.7+2 4.6+6Levulose......................... 4.8+' . 4.7+2 4.7+4 4.7+2 5.0+6 4.7+3 4.6+7a-Methyl-D-glucoside............. 4.6+10 4.7+7 4.6+10 4.7+10 4.6+ 4 4.7+B 4.6+10Esculin.......................... - - - - - - +Basal.......................... 5.9- 5.9- 5.9- 5.9- 5.9- 5.9- 5.9-Nutritional pattern* ............. 6,8 6,8 6,8 6,8 6,8 6,8 6,8Colony......................... I-R I-R I-R I-R I-R I-R I-RAcidity milk-%................. 0.07 0.10 0.01 0.07 0.04 0.03 0.24Growth 45C ..T - - - - _4_ +Growth 16 C.........- -

* Refers to nutritional data of table 3.None ferments mannose, trehalose, salicin, adonitol, cellobiose, dulcitol, inositol, inulin, rhamnose,

sorbitol, sorbose, glycerol, and a-methyl-D-mannoside. All strains ferment maltose.All strains produce inactive lactic acid.d equals incomplete and slow reactions in fermentation tests.For hippurate data only F denotes most strains negative and d equals most strains positive.

medium probably resulted in the poor growth ofa small minority of the cultures.Except for one strain, originally negative in

lactose but later adapting to this fermentation,unvarying results were obtained for all strains inall characteristics in repeated tests.

Lactobacillus brevis. Thirty-one strains whichwere designated as L. brevis were isolated in thisoral sampling from 22 individuals. The data arepresented in table 7. Four variant groups areincluded, but it is conceded that group 4 may bedifferent from L. brevis. None of the strains

also hydrolyzed esculin strongly in the greatmajority of cases and to some extent in allcases. Pederson (1929a,b, 1930) has studiedthoroughly a number of strains which hedesignated as L. brevis and which seem to beidentical with Betabacterium breve Orla-Jensen.Some of these available strains were compareddirectly with the groups of oral strains in table 7and were found to be identical. There were novariations of any character of any individualstrain of L. brevis in repeated tests.

Lactobacillus cellobiosus nov. sp. Six strains of a

6931953]

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

MORRISON ROGOSA ET AL.

new heterofermentative species were among the500 isolates. Organisms of this type have beenfound in small numbers in an extending series oforal samples of wide geographic distribution.The colonial morphology, fermentative and othercharacteristics are distinctive from any hithertodescribed heterofermentative lactobacillus.Therefore, we do not hesitate to describe it as a

TABLE 7Lactobacillus brevis and variants

GROUP .................

No. strains.............

Arabinose........Lactose...........Mannitol.........Melibiose.........Raffinose .........Sucrose .........Salicin ...........Xylose...........Galactose.........Maltose..........Glucose ..........a-Methyl-D-

glucoside.......Basal ............Nutritionalpattern* ........

Colony...........Acidity milk-%..Growth 45 C.Growth 16 C.

1

19

4.2+15 .3-t145.7-4 .7+44.8+4

4.7+44.7+1

4.97+75.9-

7R-I

0.04

2

7

3 4~~I4

4.2+1 4.0+2 4.2+24.5+6 5.14'44 .5+25.3414 51414 45.04144.6+5 14.5+2 4.6+25.1+6 4.5+2 5.14144.7+8 4.5+2 15.7-5.7- 5.8- 15.9-4.3+1 4.1+1 5.7-4.8+7 4.8+2 4.9+34.6+2 4.5+1 4.6+24.9+' 4.5+2 4.6+3

4.8+7 4.7+7 5 7-5.8- 5.8- 6.0-

7 7 8?R-I R-I I

0.01 10.00 0.02

I± 4

* Refers to nutritional data of table 3.None ferments mannose, melezitose, trehalose,

adonitol, cellobiose, dulcitol, inositol, inulin,rhamnose, sorbitol, sorbose, glycerol, and a-methyl-D-mannoside.

All produce inactive lactic acid and hydrolyzehippurate and esculin.i equals slow and weak in fermentation data

and questionable growth at 45 C and 16 C.

new species and designate it as Lactobacilluscellobiosus nov. sp.

This organism possesses the common charac-teristics of the heterofermentative lactobacilliand therefore produces from the fermentation ofavailable carbohydrates considerable quantities ofvolatile products, particularly CO2, in additionto inactive lactic acid. It ferments arabinose,melibiose, raffinose, sucrose, trehalose, cellobiose,

galactose, maltose, glucose, and levulose.Although one strain fermented xylose onlyslightly, this substrate generally is fermentedrather well. Mannose is fermented slowly andoften weakly. Lactose and salicin are fermentedslowly and occasionally are not attacked.Mannitol, melezitose, adonitol, dulcitol, inositol,inulin, rhamnose, sorbitol, sorbose, glycerol,ca-methyl-D-glucoside, and a-methyl-D-mannosideare not fermented. Esculin is hydrolyzed but

TABLE 8Frequency distribution of oral lactobacilli

SAMPLES INISOLATES WHIICH SPECIES

SPECIES APPEAR

No. IPer cent No. IPer cent

Homofermentative

Lactobacillus casei.. ... 196 39.2 77 59.2Lactobacillus acidoph-

ilu.55 11.0 28 21.5Lactobacillus salivariusnov. sp .. 11 2.2 8 6.2

Lactobacillus plan-tarum ..10 2.0 3 2.3

Lactobacillus arabino-us.. 6 1.2 4 3.1

Unidentifiable .. 2 0.4 2 1.5

Heterofermentative

Lactobacillusfermenti. 154 30.8 59 45.4Lactobacillus buchneri. 25 5.0 13 10.0Lactobacillus brevis. 31 6.2 22 16.9Lactobacillus cellobio-

sus nov. sp.......... 6 1.2 2 1.5Unidentifiable.4 0.8 2 1.5

Total. 500 100.0 130 -

hippurate is not. There is little or no acidity inmilk. No growth takes place at 45 C. Its nu-tritional behavior conforms to pattern 6 as shownin table 3.

This species will be discussed more completelyin a subsequent publication.

UnideWifiable strains. A small group of 4heterofermentative lactobacilli was isolated from2 individuals. We are frank to state that thesestrains were oddly individual and could not beplaced in any group of which we had any knowl-edge. Two other strains, in some ways resembling

694 [VOL. 65

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SPECIES DIFFERENTIATION OF ORAL LACTOBACILLI

L. plantarum, but producing dextrolactic acidby means of a homofermentation, also aretermed unidentifiable.

Species distribution. Table 8 summarizes thefrequency distribution of the species found in thepresent sampling. L. acidophilus comprised 11per cent of the total number of isolates but wasfound in 21.5 per cent of the samples. L. caseiand L.fermenti were the predominating organismssince they constituted 70 per cent of the isolates.L. casei was present in 77 and L. fermenti in 59of a total of 130 samples.

DISCUSSION

It has been demonstrated that the lactobacillifound in a sizable sampling of salivas compriseda spectrum of species of which 56 per cent of theisolates were homofermentative and 44 per centwere heterofermentative. Also, homofermentativespecies were isolated from 94 per cent andheterofermentative species from 75 per cent ofthe positive samples. At least two species werepresent simultaneously in 60 per cent and threespecies in 23 per cent of the samples.

Curran, Rogers, and Whittier (1933) foundthat more than half of their dental strains wereL. casei and that the oral lactobacilli availableto them were not usually L. acidophilus. Pederson(personal communication) has emphasized to usthat many of the oral strains he was able to studyare heterofermentative. Harrison (1939) observedgas in 33 per cent of his cultures. Although Bergey(1923) had divided the genus on the basis ofhomo- and heterofermentation and Orla-Jensen(1919) had even created separate genera for thetwo types, dental investigators before 1950 didnot take advantage of this natural approach forthe differentiation of species.

Tilden and Svec (1950) in a preliminaryreport identified 58 of 101 oral strains. Forty-fivewere L. casei and 13 were L. ferinenti. Theystated that none of their cultures resembledL. acidophilus Bergey (Breed et al., 1948) and asalso defined by Anderson and Rettger (1937),Weinstein, Anderson, and Rettger (1933), Orla-Jensen (1919, 1943), Orla-Jensen, Orla-Jensen,and Winther (1936), and Tittsler, Geib, andRogosa (1947). Their special medium for isolationmay not have been especially favorable for thegrowth of the latter species. Nevertheless, it issignificant that their results are in substantialagreement with those reported in this paper in

that L. casei and L. fermenti comprised the bulkof the cultures.The concept of certain investigators that the

oral lactobacilli are uniquely indigenous to theoral environment merits reexamination in view ofpresently available evidence.

L. acidophilus has been found in the intestinaltract of animals and man. L. fermenti andL. plantarum have been found in fecal materialfrom turkeys. The oral lactobacilli from thehamster are present in large numbers in the feces.L. buhneri, L. fermenti, L. plantarum, andL. arabinosus have been found in a wide varietyof fermenting materials such as sauerkraut,pickles, beets, tomato products, silage, potatoesand cheese. L. casei is found commonly incheeses and dairy products. Some of the men-tioned species have been isolated from grains,mash, yeast, and sour dough. It is clear thereforethat the lactobacilli in the mouth are identicalwith species found ubiquitously in fermentingmaterials in nature.

Often it is forgotten that the determinationsmade in this and similar studies are of thecharacteristics of bacterial populations. In theabsence of special techniques for the isolation andselection of mutants or special variants, what isdetermined is the resultant mass character of thetotal bacterial population.Much has been written concerning the varia-

bility of the lactobacilli, particularly in fermenta-tion tests. However, gross variation of individualstrains in repeated testing has been uncommon inour experience. In the rare instances in whichvariation has occurred, it has been nearly alwaysin the cases of substrates which were fermentedvery slowly or weakly in the first test on primaryisolation, or in substances of doubtful purity,composition, or heat stability. This also has beenthe experience of Orla-Jensen (1919, 1943),Tittsler, Geib, and Rogosa (1947), and Harrisonand Hansen (1950a,c). Indeed, Orla-Jensen (1943)has retested strains after 30 years and hasobtained remarkably similar results in repeatedtests in nutritionally adequate media. Rogosa(unpublished data) has tested 11 strains ofL. acidophilus, first studied in 1933, and retestedin 1938, 1943, 1947, and 1950. The same type oflactic acid, the same homofermentation, and areplication of the fermentative behavior wereobtained in all cases. A similar repetition ofdeterminations first made in 1939 was performed

6951953]

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

MORRISON ROGOSA ET AL.

in 1947 with 38 strains of L. casei, 40 of Lacto-bacillus bulgaricu-s syn. Thermobacterium jugurtOrla-Jensen, 17 of Lactobacillus lactis, 10 ofLactobacillus helveticus, 1 of Lactobacillus del-brueckii, 3 of Lactobacillus leichmannii, 6 ofL. plartarum, and 25 diverse heterofermentativelactobacilli. With one exception, the results ofthe different determinations were the same.L. delbrueckii originally fermented maltose butfailed to do so in later tests. The foregoingrepeated testings are independent of the repeatedtests on the present sampling of oral lactobacilli.

Twenty-eight thousand tests of individualcharacteristics were made in this study. Dis-crepancies between the first test on isolation andtests performed at least two years later occurredin 74 instances. This is at a rate of approximately0.5 per cent. It should be noted also that nearlyall the cultures were transferred in a variety ofstock media and stored in the refrigerator forvarying periods. Only a very small number ofthe cultures in the later tests were derived fromlyophilized material.

It is inconceivable that the fermentativevariability claimed by some previous workerscannot be explained. Harrison (1939) observednearly 100 per cent changes in the fermentationof arabinose and xylose in different tests. Thesecarbohydrates had been sterilized with themedium at 10 lb for 20 minutes. Even with thesepentoses sterilized by filtration, it has been ourexperience that certain indicators changemarkedly in their presence in uninoculatedmedia during incubation. This is true also forcertain other substrates. What has sometimesbeen reported as a fermentation has been merelya degradative change of an indicator in poorlybuffered and sometimes inadequate media. Inthe absence of measurements of acidity, it hasbeen the general experience of some outstandinginvestigators of the lactobacilli, already citedherein, that it is highly doubtful practice toassume positive fermentation reactions fromreadings of degraded indicators. This has beenproved to our unconditional satisfaction inthousands of individual instances. Moreover,some highly questionable choices of indicators,peculiarly unsuitable for the pH range of bacterialactivity and the buffering capacity of themedium, have been made in some investigationsof the oral lactobacilli.

It need not be labored at this time that the

nutrition of the lactobacilli is complex. Orla-Jensen (1919) and Orla-Jensen, Otte, andSnog-Kjaer (1936) recognized this early. Hisfermentation results are reported for a varietyof basal media. Even casual inspection of hisdata shows that fermentation of some compoundsdepended critically on the nutritional adequacyof the basal media. Evans and Niven (1951) havedemonstrated that certain infusion media, widelyused previously as basal media in the study offermentations, are deficient in manganese for theheterofermentative lactobacilli found in certainmeat products. They also described a simul-taneously favorable effect of citrate. One of us(M. R.) observed this some years ago and hassince used citrate with salt supplements routinely.This citrate-salt relationship was exploited in thedevelopment of a highly selective medium forcertain lactobacilli by Rogosa, Mitchell, andWiseman (1951a,b). The repeated fermentationresults reported in this paper were obtained in amedium containing citrate and required salts.Orla-Jensen (1919) discovered that certainmedia were deficient in magnesium and added itroutinely to his casein digest media. This ex-perience of Orla-Jensen has been confirmed byRogosa and Mitchell (1950b). The presentinvestigators have indeed obtained markedlyvariable results in some substrates with individualstrains of lactobacilli in basal media deficientin magnesium, manganese, or other factors. Insuch cases certain substrates only may befermented, and a variety of materials normallyfermented in an adequate basal medium may notbe fermented at all. For example, in deficientmedia certain strains of L. fermenti may fermentsucrose only, or L. brevis may produce acid fromarabinose and xylose only.

Unless transfers were first made in favorablemedia to stimulate good growth and the ex-pression of the reultant mass character of theculture, such erratic results also have beenobtained occasionally with cultures held forextended periods in the refrigerator.

In concluding, it seems profitable to mentionsome general properties of the lactobacilli. NVehave not yet studied a lactobacillus whichferments glycerol actively or does not requireniacin for good growth. All of the heterofer-mentative species studied are incapable offermenting adonitol, dulcitol, inositol, inulin,rhamnose, sorbitol, sorbose, glycerol, and

696 [VOL. 65

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SPECIES DIFFERENTIATION OF ORAL LACTOBACILLI

a-methyl-D-mannoside. Except for L. cellobiosus,no heterofermentative strain fermented cel-lobiose. The fermentation of trehalose waseffected only by L. cellobiosu and certain strainsof L. fermenti, and mannose was fermented veryslowly and generally weakly, if at all. Salicinwas not fermented by the vast number ofheterofermentative strains, and if fermented itwas degraded slowly.The cleavage of the genus Lactobacillus into

homofermentative and heterofermentative speciesdeserves some comment. It may be argued thatthe heterofermentative lactobacilli should assumethe rank of a separate, intermediate genus.Morphologically, the heterofermentative organ-isms may assume bi-polar or barred forms closelyresembling some members of both the generaCorynebacterium and Propionibacterium. This hasbeen demonstrated by various techniques in-cluding electron microscopy. The heterofer-mentative strains tested differ from the homo-fermentative species in producing significantquantities of CO2 from the dissimilation ofcarbohydrates, in requiring thiamin for goodgrowth, and in producing ammonia. Except forthe lack of production of propionic acid, thefermentation products of these heterofermenta-tive organisms resemble to a considerable extentthe carbon distribution of the propionibacteria.Tests made by Orla-Jensen (1943) indicate thatthe heterofermentative types may utilize lactateto some degree and probably similarly to thepropionibacteria. The final pH of the homo-fermentative organisms is significantly differentin glucose. Generally, the pH produced by thehomofermentative strains was at least 0.5 unitlower than that for the heterofermentativespecies.

These differences between the homo- andheterofermentative groups are fundamental, andvery probably more basic differences will bediscovered with further study. These differencesare also greater than those existing betweencertain other genera now recognized. Therefore, itis recommended that the heterofermentativespecies, which are now placed by Bergey (Breedet al., 1948) in a sub-group of the genus Lacto-bacillus, be separated from the homofermentativespecies and placed in a separate genus for whichthe name Betabacterium Orla-Jensen (1919) isavailable. This arrangement would keep the

name Lactobacillus Beijerinck for the homofer-mentative species.The authors are depositing appropriate cultures

in the American Type Culture Collection.

SUMMARY

Five hundred representative lactobacilli wereisolated from fresh saliva specimens obtainedfrom 130 children in the elementary schoolgrades 1 to 7. The concept held previously, thatthe oral lactobacilli are the one species Lacto-bacillus acidophilus, is not substantiated by theevidence. Instead, they comprised a spectrum ofspecies of which L. acidophilus was only oneconstituent. At least two species were presentsimultaneously in 60 per cent and three speciesin 23 per cent of the samples.The percentages of the isolates and percentages

of the samples in which they appeared were,respectively: L. casei, 39.2 and 59.2; L.acidophilus, 11 and 21.5; L. salivarius nov. sp.,2.2 and 6.2; L. plantarum, 2.0 and 2.3; L. arabi-nosus, 1.2 and 3.1; and homofermentativeunidentifiable species, 0.4 and 1.5. Among theheterofermentative species the distribution was:L. fermenti, 30.8 and 45.4; L. buchneri, 5.0 and10.0; L. brevi8, 6.2 and 16.9; L. cellobiosus nov.sp., 1.2 and 1.5; and heterofermentative un-identifiable strains, 0.8 and 1.5.The above distribution has been confirmed

largely in isolates from other groups of childrenand from adults in different geographic areas. Inall cases, L. casei and L. fermenti constituted thebulk of the strains.The characteristics of the foregoing known

species are described. The two new mentionedspecies are described briefly.The ecological relationships of the oral lacto-

bacilli are discussed also, and it is shown that theknown species of oral lactobacilli are identicalwith organisms previously found in a widevariety of fermenting materials in nature.New and more extensive tests, including data

of nutritional requirements, have been applied tothe study of significant numbers of strains. Thespecies have been conceived by means of acorrelation of characteristics in which data ofadaptive fermentations have been considered.

Repeated tests on fermentative and othercharacteristics in nutritionally adequate mediademonstrated that variation of individual strainswas insignificant.

6971953]

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

MORRISON ROGOSA ET AL.

REFERENCES

ANDERSON, T. G., AND RETTGER, L. F. 1937Acidogenic and aciduric bacteria of the mouthand their possible relation to dental caries.J. Dental Research, 16, 489-505.

BARBER, F. W., AND FRAZIER, W. C. 1945 Dis-sociants of lactobacilli. J. Bact., 50, 637-649.

BERGEY, D. H., HARRISON, F. C., BREED, R. S.,HAMMER, B. W., AND HUNTOON, F. M. 1923Bergey's manual of determinative bacteriology,lt ed., 442 pp. The Williams & Wilkins Co.,Baltimore, Md.

BREED, R. S., MURRAY, E. G. D., AND HITCHENS,A. P. 1948 Bergey'8 manual of determinativebacteriology, 6th ed. The Williams & WilkinsCo., Baltimore, Md.

CURRAN, H. R., ROGERS, L. A., AND WHITTIER,E. 0. 1933 The distinguishing character-istics of Lactobacillus acidophilus. J. Bact.,25, 595-621.

DUBOS, R. J., DAVIS, B. D., MIDDLEBROOK, G.,AND PIERCE, C. 1946 Effect of water solublelipids on the growth and biological propertiesof tubercle bacilli. Am. Rev. Tuberc., 54,204-212.

ENRIGHT, J. J., FRIESELL, H. E., AND TRESCHER,M. S. 1932 Studies of the cause and natureof dental caries. J. Dental Research, 12,759-827.

FVANS, J. B., AND NIVEN, C. F., JR. 1951 Nu-trition of the heterofermentative lactobacillithat cause greening of cured meat products.J. Bact., 62, 599-603.

FRED, E. B., PETERSON, W. H., AND ANDERSON,J. A. 1921 The characteristics of certainpentose-destroying bacteria, especially as con-cerns their action on arabinose and xylose.J. Biol. Chem., 48, 385-412.

HADLEY, FAITH P. 1933 A quantitative methodfor estimating Bacillus acidophilus in saliva.J. Dental Research, 13, 415-428.

HADLEY, F. P., AND BUNTING, R. W. 1932Further studies on the recognition of B.acidophilus. J. Am. Dental Assoc., 19, 28-36.

HADLEY, F. P., BUNTING, R. W., AND DELVES,E. A. 1930 Recognition of Bacillus aci-dophilue associated with dental caries. J. Am.Dental Assoc., 17, 2041-2058.

HARRISON, A. P., JR., AND HANSEN, P. A. 1950aThe bacterial flora of the cecal feces of healthyturkeys. J. Bact., 59, 197-210.

HARRISON, A. P., JR., AND HANSEN, P. A. 1950bA motile Lactobacillus from the cecal feces ofturkeys. J. Bact., 59, 444-446.

HARRISON, A. P., JR., AND HANSEN, P. A. 1950cLactobacilli from turkeys. J. Bact., 60, 543-555.

HARRISON, R. W. 1939 Studies on lactobacilli.I. Fermentation reactions of strains isolatedfrom human mouths. J. Infectious Diseases,65, 142-146.

JAY, P., AND ARNOLD, F. A., JR. 1946 Dentalcaries and fluorine. Lancaster, Pa., SciencePress, p. 46.

KLIGLER, I. J., AND GIES, W. J. 1915 Chemicalstudies of the relations of oral micro-organ-isms to dental caries. 3. A biochemical studyand differentiation of oral bacteria with spe-cial reference to dental caries. (continued)(II). J. Allied Dental Soc., 10, 282-330.

MCDONALD, I. J., AND FRAZIER, W. C. 1951Variation in morphology of colonies of lacto-bacilli. J. Bact., 61, 627-637.

MORISHITA, T. 1928 Comparative study of cer-tain aciduric organisms of intestinal anddental origin. Proc. Soc. Exptl. Biol. Med.,25, 654-656.

MORISHITA, T. 1929 Studies on dental caries,with special reference to aciduric organismsassociated with the process. J. Bact., 18,181-198.

MORO, E. 1900a Ueber die nach Gram farb-baren Bacillen des Siuglingsstuhles. Wien.klin. Wochschr., 13, 114-115.

MoRo, E. 1900b Ueber den Bacillus acidophilusn. spec. Jahrb. Kinderheilk., 52, 38-55.

ORLA-JENSEN, S. 1919 The lactic acid bacteria.M6m. acad. roy. sci., Danemark, Sect. sci.,8 s6r., 5, 81-197.

ORLA-JENSEN, S. 1943 The lactic acid bacteria.Ergiinzungsband. M6m. acad. roy. sci.,Danemark, Sect. sci. biol., 2, (3), 1-145.

ORLA-JENSEN, S., ORLA-JENSEN, A. D., AND WIN-THER, 0. 1936 Bacterium biftdum und Ther-mobacterium intestinale. Zentr. Bakt. Para-sitenk., II, 93, 321-343.

ORLA-JENSEN, S., OTTE, N. C., AND SNOG-KJAER,A. 1936 The vitamin and nitrogen require-ments of the lactic acid bacteria. M6m.acad. roy. sci., Danemark, Sect. sci., 9 s6r.,6, 1-52.

ORLAND, F. J. 1950 A correlation of antigeniccharacteristics among certain bacteria of thelactobacillus group. J. Infectious Diseases,86, 63-80.

PEDERSON, C. S. 1929a The fermentation ofglucose, fructose, and arabinose by organismsfrom spoiled tomato products. Bull. N. Y.State Agric. Exptl. Sta., 151, 1-22.

PEDERSON, C. S. 1929b The types of organismsfound in spoiled tomato products. Bull. N.Y. State Agric. Exptl. Sta., 150, 1-46.

PEDERSON, C. S. 1930 Floral changes in the fer-mentation of sauerkraut. Bull. N. Y. StateAgric. Exptl. Sta., 168, 1-37.

698 [VOL. 65

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

SPECIES DIFFERENTIATION OF ORAL LACTOBACILLI

PEDERSON, C. S. 1936 A study of the speciesLactobacillus plantarum (Orla-Jensen) Bergeyet al. J. Bact., 31, 217-224.

PEDERSON, C. S. 1938 The gas producing speciesof the genus Lactobacillus. J. Bact., 35,95-108.

RODRIGUEZ, F. E. 1930 A method of determining,quantitatively, the incidence of Lacto-bacillus acidophilus-odontylicus in the oralcavity. J. Am. Dental Assoc., 17, 1711-1719.

RODRIGUEZ, F. E. 1931 Quantitative incidenceof Lactobacillus acidophilus in the oral cavityas a presumptive index of susceptibility todental caries. J. Am. Dental Assoc., 18,2118-2135.

RODRIGUEZ, F. E. 1932 Comment on paper byEnright, J. J., Friesell, H. E., and Trescher,M. S. in the same issue of J. Dental Researchcited above. J. Dental Research, 12, 838-842.

ROGOSA, M., AND MITCHELL, J. A. 1950a In-duced colonial variation of a total populationamong certain lactobacilli. J. Bact., 59, 303-

/ 308.ROGOSA, M., AND MIrCHELL, J. A. 1950b The

indispensability of magnesium for Lactobacil-lu8 helveticus and its unavailability in a mag-nesium-azide complex. Bact. Proc., 1950,130.

ROGOBA, M., MITCHELL, J. A., AND WISEMAN,R. F. 1951a A selective medium for theisolation and enumeration of oral and fecallactobacilli. J. Bact., 62, 132-133

ROGOsA, M, MITCHELL, J. A., AND WISEMAN,R. F. 1951b A selective medium for theisolation and enumeration of oral lactobacilli.J. Dental Research, 30, 682-889.

ROGOBA, M., TITTSLER, R. P., AND GEIB, D. S.1947 Correlation of vitamin requirementsand cultural and biochemical characteristicsof the genus Lactobacillus. J. Bact., 54, 13-14.

ROSEBURY, T. 1944 The parasitic lactobacilli.Arch. Path., 38, 413-437.

ROSEBuRY, T., LINTON, R. W., AND BUCHBINDER,L. 1929 A comparative study of dental aci-duric organisms and Lactobacillus acidophilus.J. Bact., 18, 395-412.

SULLIVAN, H. R., STILL, J. L., AND GOLDSWORTHY,N. E. 1939 Acid production as a basis forclassifying lactobacilli from carious teeth.J. Dental Research, 18, 513-524.

TILDEN, E. B., AND SVEC, M. 1950 Types oflactobacilli isolated from saliva. J. DentalResearch, 29, 659.

TITT5LER, R. P., GEIB, D. S., AND ROGOSA, M.1947 Taxonomy of the genus Lactobacillus,with special reference to correlations of dif-ferential characteristics. J. Bact., 54, 12-13.

ULICNY, H. P. 1936 Comparative studies ofLactobacillus acidophilus associated with ca-ries. J. Dental Research, 15, 326.

WARNER, M. E., AND ARNOLD, L. 1941 Morpho-logical, biochemical, and serological study ofaciduric bacilli. J. Dental Research, 20, 49-58.

WEINSTEIN, L., ANDERSON, T. G., AND RETTGER,L. F. 1933 An attempt to dissociate certainoral and dental strains of lactobacilli and somenotes on differentiation of the rough variantsfrom intestinal lactobacilli. J. Dental Re-search, 13, 323-328.

19531 699

on April 25, 2020 by guest

http://jb.asm.org/

Dow

nloaded from