growth characteristics, bile sensitivity, freeze damage...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1981, p. 1461-14670099-2240/81/061461-07$02.00/0

Vol. 41, No. 6

Growth Characteristics, Bile Sensitivity, and Freeze Damagein Colonial Variants of Lactobacillus acidophilust

T. R. KLAENHAMMER* AND E. G. KLEEMANDepartment ofFood Science, North Carolina State Uriversity, Raleigh, North Carolina 27650

Received 9 January 1981/Accepted 5 March 1981

Rough (R) and smooth (S) colonial variants were isolated from a heterogeneousculture of Lactobacillus acidophilus RL8K. R and S types were stable uponrepeated transfer on agar, but revertant colonies did appear after broth transfers.When propagated in commercial MRS broth, R and S cultures showed similargrowth characteristics, and both cell types were insensitive to freezing and frozenstorage at -20°C. Alternatively, during growth in scratch MRS broth, R culturesshifted to a reduced rate of growth during the late logarithmic phase. R cellsgrown under these conditions were susceptible to death by freezing and injury at-20°C. Microscopically, R cells were observed as long gram-positive rods withsmall nonstainable blebs protruding from the cell wall. In bile sensitivity studiesof R and S cells plated on MRS agar plus oxgall, the S culture was resistant to 1%bile, whereas the R culture was sensitive to 0.6% bile. Differences in the bileresistance and freeze damage of R and S cells suggest that colonial and cellularmorphologies are important considerations for the selection of Lactobacillusstrains as dietary adjuncts and for the development of growth conditions forpreparing frozen concentrated cultures from either cell type.

The lactic acid bacteria are composed of adiverse group of bacteria that are responsible forthe characteristic end product of most food fer-mentations, lactic acid. Numerous benefits havebeen realized due to the fermentative abilities ofthese food-bioprocessing bacteria. The wide va-riety of fermented foods is the direct result ofthe different growth characteristics, metabolicactivities, and end products of the lactic acidbacteria. Within this group, the lactobacilli par-ticipate in numerous milk, meat, and vegetablefermentations and are critical to the successfulcompletion of many thermophilic food fermen-tations. Beyond the fermentative abilities of thelactobacilli, an additional role has been attrib-uted to those species that reside in the intestinaltracts of humans and animals. It has long beensuggested that Lactobacillus acidophilus mayenhance resistance to common intestinal disor-ders through stabilization of normal intestinalmicroflora (5, 19, 23). More recently, Goldin etal. (9) reported that viable L. acidophilus cellsfavorably altered the formation of fecal bacterialenzymes that contribute to carcinogen formationin the bowel lumen. Unfortunately, studies iden-tifying the beneficial effects of Lactobacillus sp.in the intestinal tract are limited and often sub-ject to conflicting reports (25).

t Paper no. 6745 of the Journal Series of the North CarolinaAgricultural Research Service, Raleigh, N.C.

The lactobacilli have been subject to extensiveinvestigations concerning their taxonomy,growth, nutritional requirements, and metabolicactivities to clarify their roles in the intestine orenhance their fermentative activities. Thesecharacteristics are highly variable among thelactobacilli, and the differences contribute to thedifficulty in defining optimum conditions for thegrowth and activity of different species. Pheno-typically, the lactobacilli respond to alteredgrowth conditions by morphological changes ap-parent microscopically or colonially on solid me-dia (1). These morphological transitions appearcharacteristic of the lactobacilli (1) but in recentyears have been neglected during the selectionand propagation of strains for fermentativestarters or dietary adjuncts.

Early descriptions of L. acidophilus colonialvariation identified characteristic "X" and "Y"types. On solid media, the X type appeared as afuzzy, rough, flat, and irregular colony, whereasthe Y type was smooth, regular, and convex inappearance (17, 22). Strains were typically iso-lated from feces as rough (R) colonies and read-ily dissociated to smooth (S) colonies upon lab-oratory transfer (16). S types rarely dissociatedback to R types, and the R -. S transition wasfavored upon repeated laboratory propagation(16, 22). Kopeloff (16) also described an inter-mediate colony type that approached either the

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1462 KLAENHAMMER AND KLEEMAN

R or S types but would not stabilize at eitherextreme. Subsequent studies on numerous spe-cies of Lactobacillus, including L. acidophilus,Lactobacillus bulgaricus, Lactobacillus helvet-icus, and Lactobacillus casei, concluded thatmorphological alterations in colony types arenot the result of genetic variability but ratherrepresent differences in the phenotypic expres-sion of a population in different growth environ-ments (18, 21). Growth conditions that havebeen found to affect the colony morphology ofthe lactobacilli include the presence or absenceof oxygen (18), temperature of incubation (2),pH (18), and components of the growth medium,such as acetate (18), salt (1), fatty acid esters(21), and antibiotics with surfactant properties(1). Microscopically, R strains produce chains oflong or filamentous cells, and S strains are morecompact bacillary structures in short chains (10).Nutritional deficiencies in deoxyribonucleosides,vitamins, and divalent cations have also beenreported to induce filamentous growth in thelactobacilli (4, 12-14, 26). Dramatic alterationsin colonial or cellular morphology of entire Lac-tobacillus populations under limiting or variablegrowth conditions, however, do not address theheterogeneity that occurs from pure isolates ona single complex medium under stable environ-mental conditions. We have observed that a pureculture of L. acidophilus RL8K exhibits heter-ogeneous colony types on MRS agar. This reportdescribes the isolation of stable R and S types,their growth characteristics in MRS broth, andthe response of each type to bile salts and frozenstorage, conditions that are important in theselection of Lactobacillus strains for use in fro-zen concentrated cultures and dietary adjuncts.

MATERIALS AND METHODSCulture propagation, selection, and storage. L.

acidophilus RL8K was obtained from the dried cul-ture collection maintained by the Department of FoodScience at North Carolina State University. The cul-ture was propagated in commercial MRS broth (DifcoLaboratories, Detroit, Mich.) at 37°C and held at 4°Cbetween transfers. Unless otherwise noted, Difco MRSbroth was used to prepare all liquid and solid propa-gation media. Incubation conditions were 37°C for 48h under a flowing CO2 atmosphere (0.4 liter/min).R and S colonial variants of L. acidophilus RL8K

(designated RL8KR and RL8KS) were isolated fromthe surfaces of MRS agar plates (1.5% agar; BBLMicrobiology Systems, Cockeysville, Md.). A dissect-ing microscope was used to define colony types andassist in the selection of R and S colonies throughrepeated isolation until stable types were obtained.Working stock cultures were prepared by picking sin-gle colony isolates into MRS broth, transferring oncethrough MRS broth, and then mixing 1 ml of culturewith 2.5 ml of autoclaved 11% Matrix milk (Galloway-

West Company, Fond du Lac, Wis.) and freezing at-76°C. Stock cultures were thawed a maximum ofthree times for subculture.

Identification of the cultures as L. acidophilus wasconfirmed by Gram stain, catalase test, growth at 15and 45°C, and carbohydrate fermentation patterns (8,20).Growth studies. From frozen stock cultures, L.

acidophilus RL8KR and RL8K' cells were streakedonto MRS agar and incubated for 48 h at 37°C. TypicalR and S colonies were picked into MRS broth andincubated for 24 h at 37°C. The cells were harvestedby low-speed centrifugation (3,000 x g at 4°C for 10min) and resuspended in 2 to 3 ml of fresh MRS broth.These cells were used to inoculate 10 ml of MRS brothprepared from the commercial dehydrated medium orbroth prepared with the following components as perthe Difco MRS broth formula: protease peptone no. 3(Difco), 10.0 g; beef extract (Difco), 10.0 g; yeast ex-tract (BBL), 5.0 g; dextrose, 20.0 g; Tween 80, 1.0 g;ammonium citrate, 2.0 g; sodium acetate, 5.0 g; mag-nesium sulfate, 0.1 g; manganese sulfate, 0.05 g; diso-dium phosphate, 2.0 g; distilled H20, 1 liter; pH 6.7.This medium was designated scratch MRS broth.Growth at 37°C was followed optically at 650 nm ona Bausch & Lomb Spectronic 70. Colony types fromeach culture were confirmed on MRS agar at the startand completion of the growth experiments.

Bile sensitivity. Single colonies of R and S typeswere isolated, propagated once through 10 ml ofMRSbroth, and concentrated to a small volume as describedabove. Concentrated cell suspensions were used toadjust 10 ml of MRS broth to an initial optical densityat 650 nm of 0.62 to 0.64. The cultures were immedi-ately plated in MRS agar containing various concen-trations (0 to 1%) of oxgall (BBL). Dilutions wereprepared in 0.1% peptone (Difco) and 0.01% AntifoamB (Sigma Chemical Company, St. Louis, Mo.).Pour plates and dilutions were prepared by standard

methods (7). The plates were incubated under a flow-ing CO2 atmosphere for 72 h at 37°C. Colony typeswere confirmed by preparing a streak plate on MRSagar from the adjusted cultures.

Freeze studies. Concentrated cells of L. acidoph-ilus RL8KR and RL8KS were prepared as describedabove and used to adjust 10 ml of MRS broth to aninitial optical density at 650 nm of 1.3. A 1-ml amountof this suspension was used to inoculate 100 ml oftempered (37°C) MRS broth. The culture was incu-bated for 12 h at 37°C. Colony morphology was con-firmed at the beginning and end of this 12-h incuba-tion. Cells were then harvested by centrifugation at8,000 x g at 4°C for 10 min and suspended in 100 mlof cold (40C) 11% Matrix milk. This suspension wasblended (20 to 30 s at high speed in a Waring blender)and dispensed into 2.0-ml Cryule vials (Wheaton Sci-entific, Millville, N.J.) The vials were frozen at -20°Cand stored for 35 days. The Lactobacillus populationwas determined on MRS agar and MRS agar contain-ing 0.15% oxgall (MRSO) before freezing and after 18and 35 days of frozen storage. Dilutions were preparedas described above, and the pour plates were incubatedfor 72 h at 370C under a CO2 atmosphere beforecounting.

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COLONIAL VARIANTS OF L. ACIDOPHILUS 1463

RESULTSColony selection. Original plating of L. aci-

dophilus RL8K revealed heterogeneous colonytypes on the surfaces of MRS agar plates. Iso-lation ofR and S colony types was accomplishedby repeatedly picking the roughest and smooth-est colonies until stable R and S types were

obtained. As a surface colony, the R type was

large, irregular, and flat to umbonate, with a

matte surface and mottled opacity (Fig. 1). Incontrast, the S colony was smaller, circular, witha smooth edge, and convex with a glisteningtranslucent appearance. Removal of the coloniesfrom the agar surface with an inoculating needlerevealed that the S colony was moist andcreamy, and the R colony was dry and granular.Gram stains showed that R colonies were com-

posed of a mixture of long and short rods in longchains and filaments, often observed in tangledmasses of cells. Alternatively, homogeneous andindividual short rods were observed in Gramstains of S colonies.The R and S colonies isolated from L. aci-

dophilus RL8K were homogeneous and stable,although colony characteristics varied slightlybetween experiments. This variation was prob-ably caused by the sensitive response of thelactobacilli to slight differences in culture andincubation conditions, such as dryness of agar

surfaces or total C02 atmosphere (1, 18). The Rand S types were gram-positive rods, were cat-alase negative, did not grow at 150C, and showedvariable growth at 45°C. Fermentation patterns,characterized with the Minitek system (BBLMicrobiology Systems) (8), were identical for

both types and consistent with typical fermen-tation reactions of L. acidophilus.Growth studies. In commercial MRS broth

(Difco), both RL8KS and RL8KR cells showedexcellent growth when examined turbidometri-cally (Fig. 2A). However, a slight reduction inthe growth rate during the later stages of loga-rithmic growth was consistently observed for theRL8KR culture but was not detected in theRL8K' culture. Gram stains of the RL8Ks cul-ture revealed short rods found individually or inshort chains, whereas the RL8KR culture grewas longer rods found in short or long chainsmixed with filamentous cells (data not shown).The late log reduction in the growth rate for

RL8KR cultures in MRS broth was enhanced bypropagation in scratch MRS broth preparedfrom individual components by the Difco recipe(Fig. 2B). After 4 h of growth in the scratchbroth, the R culture showed a temporary cessa-tion of growth, with subsequent growth proceed-ing at a reduced rate. The L. acidophilus RL8KSculture showed identical growth in either thecommercial or scratch MRS broths. Microscopicanalysis of cells from the RL8KS and RL8KRcultures in the scratch MRS broth revealed adramatic difference in cell morphology beyondthe typical short versus long rods. Blebs pro-truding from numerous points about the cellwall were detected for RL8KR cultures examinedduring the reduced growth phase (Fig. 3). Theblebs did not retain crystal violet stain and wereobserved as red protrusions from the gram-pos-itive cell wall. Bleb formation was common inthe RL8KR cells propagated in the scratch broth,occasionally observed in RL8KR cells from com-mercial broth, and rare in RL8Ks cells fromeither broth formulation.Bile sensitivity. The bile sensitivity ofR and

- 0.1

- u.30

o - 0.5

- 0.7

FIG. 1. Appearance of L. acidophilus RL8KR andRL8Ks colonies on the surface ofMRS agar. Objec-tive magnification, x3.2, reflected light.

o 2 3 4 5 6 7 0 1 2 3 4 5 6 7 6 9 10 11 12 13

time of incubation (h, 37 C)

FIG. 2. Growth ofR and S cells in commercial (A)and scratch (B) MRS broths. Symbols: I, RL8KRcells; 0, RL8Ks cells. O.D., Optical density.

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FIG. 3. Photomicrograph of R cells with blebs (indicated by arrows) after 5 h of growth in scratch MRSbroth at 37°C. Cells were Gram stained. Objective magnification, xlOO.

S cells was tested by pour plating broth cultureson MRS agar containing increasing concentra-tions of bile (Fig. 4). RL8Ks cells were resistantto 1% bile and maintained the original popula-tion level through all tested concentrations. Al-ternatively, RL8KR cells were more sensitive tobile, and a significant reduction in colony-form-ing ability was observed as the bile concentrationwas increased from 0.6 to 1.0%.

In MRS pour plates, the differences betweenR and S colonies were apparent but were lessstriking than forms observed on the agar surface(Fig. 5). However, in MRSO, R and S colonialmorphologies were dramatically different whencompared with each other and with their re-spective morphologies in MRS agar withoutoxgall. In MRSO, the S colony was disk to roundin shape and much smaller than that in MRSagar (Fig. 5A and B). No difference in colonysize was observed between the R type in MRSagar and that in MRSO. However, in the pres-ence of 0.15% oxgall, the R type formed a veryrhizoid colony (Fig. 5C and D). The results dem-onstrate a significant difference between R andS types of L. acidophilus RL8K in the level ofbile resistance and the types of colonies formedin the presence of 0.15% bile.Freeze damage. In early literature, reports

suggested that exposure of the lactobacilli tostress favored the development of the S colonytype, generally considered more resistant to en-

9

E"I.-U

0CT-0-i

7

6

5

4

0 Q2 Q4 0.6 08 ID

% BILEFIG. 4. Sensitivity of RL8KS (0) and RL8KR (0)

cells to increasing concentrations of bile. CFU, Col-ony-forming units.

vironmental stress (2, 15). Lactobacillus strainsare commonly incorporated into frozen concen-trated cultures, and therefore, it was of interestto determine the stability ofR and S types of L.acidophilus RL8K under frozen storage condi-tions. As shown in Fig. 6, both R and S cellscultured in commercial MRS broth were rela-tively stable during freezing and frozen storageat -20°C. Over the 35-day storage period, little



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FIG. 5. Appearance ofRL8Ks and RL8KR colonies in MRS andMRSO plates. (A) S colony in MRS agar;(B) S colony in MRSO; (C) R colony in MRS agar; (D) R colony in MRSO. Magnification, x3.2, transmittedlight. Precipitated bile appeared as small specks in the background ofMRSO plates.

death or sublethal injury was detected. How-ever, R cells prepared from a scratch MRS brothculture were highly susceptible to freeze damage.A 90% reduction in viable cells was observedafter 35 days of frozen storage and, of the re-maining cells, approximately 60% were freezeinjured and unable to form colonies on MRSOplates. S cells prepared in the scratch broth werestable at -20°C. The data indicated that R andS cells were not significantly different in theirresistance to freezing unless the scratch brothwas used to propagate the cells.

DISCUSSION

Bergey's Manual of Determinative Bacteri-ology (20) describes homofermentative Lacto-bacillus colonies as normally R, becoming Supon laboratory transfers in the presence ofTween 80 or sodium oleate. Use of various com-pounds in the growth medium can induce thetransition of an entire Lactobacillus populationfrom one type of colonial morphology to another(21). However, it must also be recognized thatheterogeneity can occur within a single popula-

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9

_ 8EN

0

0

cx 7o70

6

0 10 20 30 0 10 20 30

days at -20C

FIG. 6. Effect of the growth medium on the freezestability of L. acidophilus RL8KR (squares) andRL8KS (circles). (A) R and S cultures were grown inand plated on scratch MRS (L and 0) and MRSO(U and 0). (B) R and S cultures were grown in andplated on commercial MRS (L and 0) and MRSO(U and 0). MRSO was used to evaluate the degree ofsublethal injury ofsurviving cells. CFU, Colony-form-ing units.

tion under a single set of environmental condi-tions. In this study, it was observed that purifiedR and S colonies of L. acidophilus RL8K werestable on MRS agar, but dissociation would pe-riodically occur at low levels. It is not knownwhether this dissociation results from pheno-typic variability or represents the beginning ofa gradual population transition caused by envi-ronmental stimuli. In either case, L. acidophilusstrains may be heterogeneous during growth inMRS-based media. As a result of this heteroge-neity, strains, media, and incubation conditionsshould be evaluated to assure homogeneous cell

suspensions when studying the characteristicsand function of this genus.During this study, differences were apparent

upon comparison of the level of bile resistanceand freeze damage exhibited by R and S cul-tures. RL8KR cells were more sensitive thanRL8KS cells to increasing concentrations of bile.The elongated cells may expose more sites sus-

ceptible to agents with the surfactant and deter-gent properties of bile salts. Additionally, differ-ences in the susceptibility of RL8KR and RL8Kscells to freezing were manifested only aftergrowth in scratch MRS broth. In this medium,RL8KR cells displayed increased bleb formationand an altered rate of growth during the loga-rithmic phase. The reason for the differenceseffected by the two MRS broth media is not

known. However, it is apparent that minorchanges in the preparation of the growth me-dium can dramatically alter the multiplicationand resulting characteristics of R cells.Morphology differences in the cells of R and

S Lactobacillus cultures included filamentousand short bacillary rods (2). Close examinationof the RL8KR cells revealed bleb-like structuresprotruding from the bacterial cell wall. In Rcells, these structures occurred at regular inter-vals along the cell and could represent protru-sions of the cytoplasmic membrane through thebacterial cell wall. Bleb formation in the outermembranes of Escherichia coli and Salmonellatyphimurium has been reported in septation-di-vision mutants (6, 24). These protrusions occurat the suspected sites of cell division duringgrowth of elongated cells. In L. acidophilus Hig-gins et al. (11) observed that during autolysis ofstrain 63AM Gasser, gaps in the cell wall oc-curred at major polar and septal sites, allowingthe extrusion of membrane and cytoplasm insmall evaginations. By analogy, bleb formationin RL8KR cells may occur at sites of defectivecross wall formation during growth of these long,rod-shaped cells.The freeze damage and bile sensitivity of bac-

terial membranes has been well established. InRL8KR cells, bile sensitivity and freeze damagesuggests that the bleb-like protrusions from thebacterial cell wall are, in fact, evaginations ofthe cyotplasmic membrane. This defect in thecellular structure may be inherent to R cells dueto the growth of elongated cells that is charac-teristic of this colony type. Bleb formation wasrarely observed in RL8K' cells. Previous reportshave addressed the more resistant state of SLactobacillus colonies to environmental stress(2). Our results substantiate the resistant stateof bacillary or S cells, but only on the basis thatS cells are less sensitive to alterations in thegrowth environment that may selectively affectR cells and render them susceptible to stress.The roles of L. acidophilus in the intestinal

tracts of humans and animals are not well de-fined. In the selection of strains for use in defin-ing these roles, it is important that the bacteriumsurvive and establish itself under the conditionsencountered in the intestinal environment. Suc-cessful entrance of L. acidopilus into the intes-tinal tract would be dependent on a physiologi-cal state of the organism that is compatible withthe environment. Curran et al. (3) found that73% ofknown intestinal isolates ofLactobacillusexhibit the X or R colonial morphology andsuggested that the R colony is characteristic ofthese strains. Whether R colonies represent themorphological state of the lactobacilli foundwithin the intestinal environment is unknown.


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However, all the available evidence suggests thatin vitro filamentous growth of the lactobacilliresults from growth conditions that are not sup-portive of proper cell multiplication (4, 12-14).Bleb formation, bile sensitivity, and freeze dam-age in RL8KR cells support this conclusion. Onthis basis, it appears that the media and envi-ronmental conditions employed during the iso-lation and propagation of intestinal lactobacillimay be somewhat inappropriate. Heterogeneityof Lactobacillus populations resulting in R, S,or intermediate dissociants may reflect adaptivetendencies of the cells either to the laboratoryenvironment (R - S) or to another physiologicalstate compatible with the intestinal enviromnent(S -. ?). For this latter tendency, however, theenvironment established for the growth of thebacterium may be inadequate, resulting in theappearance of R colonies under the standardlaboratory conditions. Therefore, although theS type is more stable in vitro, it is unclearwhether this type represents the in vivo form ofthe intestinal lactobacilli or a form that is incom-patible with the intestinal environment that hasadapted to laboratory conditions.

ACKNOWLEDGMENTThis work was supported by the North Carolina Dairy

Foundation.

LIT1ERATURE CITED1. Anonymous. 1968. Type strains of the Lactobacillus. A

report by the Taxonomic Subcommittee on lactobacilliand closely related strains. American Type CultureCollection, Rockville, Md.

2. Barber, F. W., and W. C. Frazier. 1945. Dissociants oflactobacilli. J. Bacteriol. 50:637-649.

3. Curran, H. R., L. A. Rogers, and E. 0. Whittier. 1933.The distinguishing characteristics of Lactobacillus ac-idophilus. J. Bacteriol. 25:595-621.

4. Deibel, R. H., M. Downing, C. F. Niven, Jr., and B. S.Schweigert. 1956. Filament formation by Lactobacil-lus leichmannii when deoxyribonucleosides replace vi-tamin B,2 in the growth medium. J. Bacteriol. 71:255-256.

5. Fuller, R. 1977. The importance of lactobacilli in main-taining normal microbial balance in the crop. Br. Poult.Sci. 18:85-94.

6. Fung, J. C., T. J. MacAlister, R. A. Weigand, and L.I. Rothfield. 1980. Morphogenesis of the bacterial di-vision septum: identification of potential sites of divisionin IkyD mutants of Salnonella typhimurium. J. Bac-teriol. 143:1019-1024.

7. Gilliland, S. E., F. F. Busta, J. J. Brinda, and J. E.Campbell. 1976. Aerobic plate count p. 107-131. In M.L. Speck (ed.), The compendium of methods for themicrobiological examination of foods. American PublicHealth Association, Washington, D.C.

8. Gilliland, S. E., and M. L. Speck. 1977. Use of theMinitek system for characterizing lactobacilli. Appl.Environ. Microbiol. 33:1289-1292.

9. Goldin, B. R., L. Swenson, J. Dwyer, M. Sexton, andS. L Gorbach. 1980. Effect of diet and LactobaciUusacidophilus supplements on human fecal bacterial en-zymes. J. Natl. Cancer Inst. 64:255-261.

10. Hadley, F. P., R. W. Bunting, and E. A. Delves. 1930.Recognition of Bacillus acidophilus associated withdental caries: a preliminary report. J. Am. Dent. Assoc.17:2041-2058.

11. Higgins, M. L, J. Coyette, and G. D. Shockman. 1973.Sites of cellular autolysis in Lactobacillus acidophilus.J. Bacteriol. 116:1375-1382.

12. Holden, J. T., and J. Holman. 1957. Abnormal cellularmorphology associated with a vitamin B6 deficiency inLactobacillus arabinosus. J. Bacteriol. 73:592-593.

13. Jeener, H., and R. Jeener. 1952. Cytological study ofThermobacterium acidophilus R26 cultured in the ab-sence of deoxyribonucleosides. Exp. Cell Res. 3:675-680.

14. Kojima, M., S. Suda, S. Motta, and K. Hamada. 1970.Induction of pleomorphy and calcium ion deficiency inLactobacillus bifidus. J. Bacteriol. 102:217-220.

15. Kopeloff, L. M., J. L. Etcheils, and N. Kopeloff. 1934.Bacteriological changes in acidophilus milk at room andice-box temperatures. J. Bacteriol. 28:489-500.

16. Kopeloff, N. 1934. Dissociation and filtration of Lacto-bacillus acidophilus. J. Infect. Dis. 55:368-379.

17. Lynch, F. B. 1928. Some observations upon certain meth-ods of administering acidophilus organisms and uponthe value of X and Y strains. Am. J. Med. Sci. 176:547-560.

18. McDonald, L. J., and W. C. Frazier. 1951. Variation inmorphology of colonies of lactobacilli. J. Bacteriol. 61:627-637.

19. Metchnikoff, E. 1907. The prolongation of life. G. P.Putnam's Sons, New York.

20. Rogosa, M. 1974. Genus I. Lactobacillus Beijerinck 1901,212, p. 576. In R. E. Buchanan and N. E. Gibbons (ed.),Bergey's manual of determinative bacteriology, 8th ed.The Williams & Wilkins Company, Baltimore.

21. Rogosa, M., and J. A. Mitchell. 1950. Induced colonialvariation of a total population among certain lactoba-cfii. J. Bacteriol. 59:303-308.

22. Roos, C. 1927. On the cultural characteristics of L. aci-dophilus. J. Lab. Clin. Med. 12:1053.

23. Sandine, W. E. 1979. Roles of Lactobacillus in the intes-tinal tract. J. Food Protect. 42:259-262.

24. Schwartz, U., A. Asmus, and H. Frank. 1969. Autolyticenzymes and cell division of Escherichia coli. J. Mol.Biol. 41:419-429.

25. Speck, M. L 1980. Preparation of lactobacilli for dietaryuses. J. Food Protect. 43:65-67.

26. Stamer, J. R. 1979. The lactic acid bacteria: microbes ofdiversity. Food Technol. 33:60-65.

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