STREPTOCOCCAL L FORMS

I. EFFECT OF OSMOTIC CHANGE ON VIABILITY'

CHARLES PANOS AND S. S. BARKULIS

Department of Biological Chemistry, University of Illinois College of Medicine, Chicago, Illinois

Received for publication February 6, 1959

An L form was first isolated by Klienebergerin 1935 from cultures of Streptobacillus monili-formis and classified as a symbiont of the strepto-bacillus (Klieneberger, 1935). It was subsequentlyshown by Dienes (1938, 1939) and Dawson andHobby (1939), however, that the L form was agrowth form of the bacillus and not a symbiont.Since the initial isolation, L forms have beenobtained with regularity from a number ofbacterial genera. The techniques for obtaining,handling, and cultivating this form have beenamply detailed in excellent reviews (Dienes andWeinberger, 1951; Klieneberger-Nobel, 1954; andKandler and Kandler, 1954) and in a paper bySharp (1954).A characteristic of L forms, recognized since

their earliest study, is fragility. It was thisproperty which led to the conclusion that thisform lacked a rigid cell wall. Proof for the absenceof a rigid cell wall was provided by a number ofmethods. Electron microscopic examination re-vealed the presence of a "veil-like" membrane inplace of the easily discernible thick and rigid cellwall structure (Smith et al., 1948). Penicillin,whose mode of action is purported to be inhibitionof cell wall formation, was without effect on thegrowth of this form. More conclusively, Sharpet al. (1957), in their examination of L forms fromvarious group A streptococci for cell wall con-stituents, were unable to demonstrate thepresence of rhamnose and hexosamine whichmake up the group specific polysaccharide of thecell wall. They suggested that the lack of rigidityin the L form was due to the absence of the cellwall polysaccharide.L forms from #-hemolytic streptococci were

successfully isolated recently with the aid ofpenicillin and high electrolyte concentrations(Sharp, 1954). This paper deals with the effect of

1 This investigation was supported by a grant(no. E1514) from the Institute of Arthritis andInfectious Diseases of the U. S. Public HealthService.

osmotic change on the viability of an L formfrom a group A, f-hemolytic streptococcus. Someof these results have been reported in preliminaryform (Panos and Barkulis, 1958).

MATERIALS AND METHODS

Growth of the organism. The L form employedin these investigations was obtained from Dr.Louis Dienes or was reisolated from the parentorganism, a nontypable group A, 3-hemolyticstreptococcus designated AED, as previouslydescribed (Sharp, 1954). The liquid mediumutilized for growth was composed of 2.8 per centbrucella broth (Albimi), 3 per cent NaCl, 10 percent horse serum inactivated at 56 C for 30 min,and penicillin (1000 units per ml). For the solidmedium, 4 per cent trypticase soy agar (BBL)served in place of the brucella broth. Horse serumand penicillin were added separately to themedium after it had been adjusted to pH 7.0 to7.2, autoclaved for 10 min, and cooled. In allexperiments, L form cultures and suspensionswere examined by the stained agar preparationtechnique (Dienes, 1945; Dienes and Weinberger,1951), phase microscopy, and with a double lensmagnifier (3.5 X). Optical density readings as anindex of growth were obtained with a Beckmanmodel DU spectrophotometer set at 450 my andonly 16 to 20-hr cultures with optical densityreadings between 0.465 and 0.530 were used.

All test solutions in which the L form wassuspended were prepared on a weight/volumebasis, 1000 units per ml of penicillin added, thepH adjusted to 7.2, and sterilization effected byBerkfeld filtration. MgSO4-7H20 served as thesource of Mg++ and 0.3 M phosphate buffer wasprepared with K2HPO4 - 3H20 and adjusted topH 7.2. Reagent grade chemicals were employedin all solutions.

RESULTS

Growth characteristics of the L form in liquidmedia. Figure 1 illustrates the growth curve of

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0.6

0.5-

0.4-

0.3-

c

0._0

0.2-

0.1

0

2 3 4 5 6 7 8 9

H o u r s

Figure 1. Growth curve of the streptococcalAED L form.

the streptococcal AED L form. This strain hadbeen subcultured at least 90 times on agar and inliquid media. Although visible growth occurredafter 20 hr on agar, broth cultures usually at-tained their maximal cellular mass after only 8 hrincubation. Dienes has previously described thedifficulty of initiating growth of the L form inliquid media. We have observed that once growthwas established in a 10 per cent horse serum

broth medium, subculturing became less difficult.The growth rate increased until there was no

significant difference between the rate of growthof the L form and the parent streptococcus.Horse serum as well as brucella broth was neces-

sary for growth. On omission of either constituent,no growth was observed even after prolongedincubation.

Changes in morphological characteristics alsodeveloped upon continued subculture in a liquidenvironment. Upon isolation and initial growthin broth the macroscopic appearance of this Lform resembled individual small and granular"ball-like" structures which quickly settled to thebottom of a flask after agitation. Microscopically,the intact L structure varied in size and was

composed of aggregates of intact large bodies ofdiffering size and density. In some of these dense

large bodies, the reproductive granular elementsdescribed by Dienes were evident. Very few ofthe large bodies were found free in the suspension.After prolonged subculture in liquid media, i. e.,at least 60 subtransfers, the macroscopic appear-ance of the L form became progressively typicalof most bacterial cultures. Growth no longerappeared as small ball-like aggregates but washomogeneous and showed sedimentation onlyafter prolonged incubation. Microscopically, theintact L structure was not as prevalent or aslarge as in the original broth culture. Instead, thestructure had almost completely dispersed, withthe various components floating freely in pairs,or small aggregates throughout the medium.Although reaggregation occurred upon storage at4 C, dispersion was easily accomplished withslight agitation.A photomicrograph of stained preparations of

the L form from a broth culture appears in figure2. The characteristic L form morphology dis-playing the dense large bodies is clearly dis-cernible.

Growth after suspension in various solutions ofdiffering osmolarity. A simple method for assessingthe fragility of the L form was to determine itsviability after exposure to solutions of varyingosmolarity and composition. Three-ml samples ofthe culture were centrifuged at 2200 rpm in ananglehead centrifuge (Servall, SS-1) for 20 minin 16 by 120 mm test tubes and the mediumaseptically decanted. Following resuspension andrecentrifugation, twice with 3-ml samples of therespective test solutions, the L form was re-suspended in a third sample and allowed toremain at room temperature (25 to 27 C) for 5hr. After examination by dark phase microscopyand following centrifugation, the test solutionwas decanted and the cellular mass transferredinto a 50-ml Erlenmeyer flask containing 10 mlof the liquid medium. The optical density wasrecorded after 20 to 22 hr incubation at 35 C.Incubation was continued for 72 hr for thosecultures displaying no growth after 20 to 22 hr.

Figure 3 illustrates the growth of the L formfollowing suspension in various solutions. Therewas no significant difference in the amount ofgrowth obtained after suspension in solutionscontaining equal molar concentrations of sucroseor phosphate (IX-XIII, VI). Growth followingsuspension in 0.3 M phosphate buffer (VI andXIII), however, proved to be erratic. Consistent

I --- --I

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AW;Xt' w0 ; ; S . ................ ......... .. .. ..: . . . : : :.:. .: ::.X e W. . . .... ....... ..

.:: .: :: ::

.. ...*:

....i

Figure 2. Photomicrograph of the L form from a broth culture by the stained agar block techniqueof Dienes (magnification, X980).

results were difficult to reproduce with the incubation, solutions without sucrose or phos-growth response varying from 10 to 100 per cent phate buffer (I-V) did not respond even afterof the refrigerated control after 20 to 22 hr these prolonged incubation periods. In theincubation. Although maximal growth with these various sucrose concentrations tested (0.1, 0.15,solutions usually developed after 48 to 72 hr 0.2, 0.25, 0.3 (XII), 0.5, and 0.88 M) growth

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06-

05

-~0.3-

0.2-

0.1

0

m x XE Xs x

Figure S. Growth of the L form following sus-

pension in various solutions. I. Distilled water,penicillin; II. Distilled water, without penicillin;III. Buffered saline (0.007 M Na2HPO4-NaH2PO4,pH 7.3); IV. Normal saline (0.15 M); V. Mg++(0.008 M); VI. Phosphate buffer (0.3 M, K2HPO4-3H20, pH 7.2); VII. Horse serum medium, 5 hrat 4 C (control); VIII. Horse serum medium, 5 hrat room temperature; IX. Sucrose 0.3 M, Mg++0.008 M, phosphate buffer 0.3 M; X. Sucrose 0.3 M,

Mg++ 0.008 M; XI. Sucrose 0.3 M, phosphate buffer0.3 M; XII. Sucrose 0.3 M; and XIII. Mg++ 0.008 M,

phosphate buffer 0.3 M.

became erratic below 0.25 M after 20 to 22 hr.However, after 48 or 72 hr incubation, maximalgrowth was always obtained with sucrose concen-

trations from 0.1 to 0.25 M. Concentrations offrom 0.25 to 0.88 M were equally effective in per-mitting maximal growth consistently with theshortest incubation time. It was always possibleto recover viable L forms up to 24 hr followingsuspension in 0.3 M sucrose. Glycerol concentra-tions of from 0.3 to 1.4 M failed to support eitherviability or structural integrity of the L form.Each suspension was examined by dark phase

microscopy prior to inoculating its sedimented Lform content into the liquid medium. Thosesuspensions which permitted growth after 20 to22 hr incubation (VII-XII) displayed the intactL structure as well as many free dense largebodies, with few vacuolated or ghost forms,similar to figure 2. Those suspensions whicheither did not support viability or permittedgrowth only after prolonged incubation alwayscontained relatively fewer dense large bodies butmore vacuolated large forms. Granular materialwas evident within some of these otherwiseempty ghost forms adhering mainly to their wall-like membrane. Various degrees of L structurefragmentation as well as considerable cellulardebris was always apparent. Examination of eachgrowing culture, after 20 to 22 hr or 48 and 72hr incubation, always revealed the typical Lform morphology.

Since viability of the L form could be main-tained for as long as 24 hr in 0.3 M sucrose sus-pensions, it became of interest to determine ifviability could be preserved by lyophilization insuch a hypertonic solution. A large broth cultureof the L form (0.8 L) was harvested at 6000 rpmafter 36 hr incubation, washed twice, and re-suspended in a third sample of 0.3 M sucrose.Following shell freezing, the cellular mass waslyophilized and stored at room temperature in adesiccator over P205 and NaOH. After 2 monthsit was still possible to obtain typical L formgrowth within 48 hr following inoculation of thedry powder into the liquid medium.

DISCUSSION

It has been indicated that initial growth of theL form in broth is difficult. That the difficulty isnot due to lack of nutrients is substantiated bythe fact that colonies of the L form continue togrow from pieces of agar submerged in broth. Ithas been suggested that the physical propertiesof the medium are at fault (Dienes, 1953). Ourresults seem partially to justify such an assump-tion. A definite tendency towards dispersion ofthe L structure was observed upon continuedsubculture in a liquid environment resulting in amore rapidly growing culture. Dispersion of theL structure into its intact component parts aftermild sonic treatment suggests that only weakforces may play a role in maintaining the integrityof the L structure (Panos and Barkulis, 1958). Apossible hypothesis for explaining the constantnecessity for large inocula in broth may be theneed of overcoming such external or internalforces before maximal growth can be achieved.The ability of the L form to survive suspended

in various solutions is probably primarily anosmotic effect, but not all of the observationsmade can be readily understood in these terms.For example, suspension of the L form in equi-molar concentrations of glycerol, phosphate, andsucrose was attended by a considerable differencein the viability of the organism as measured bysubsequent inoculation into broth. Sucrose overa wide range of concentrations, 0.25 to 0.88 M,consistently preserved the organism in an intactand viable form. On the other hand, suspensionin phosphate solutions followed by inoculationinto broth gave inconsistent growth after 20 to22 hr incubation. Suspension in glycerol was nobetter than suspension in water. Similar resultswith glycerol and sucrose have been obtained in

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the preservation of mitochondria (personal com-munication, L. G. Abood) from mammaliantissues. However, hypertonic phosphate is toxicto mitochondria. The results with glycerol mightbe due to a ready penetrability of this substanceinto large bodies and L granules. This would pre-vent the maintenance of an osmotic barrieragainst the suspending medium. The protectionof the L form over a wide range of sucrose con-centrations suggests that these forms, as has beenfound true for mitochondria and protoplasts, canadjust rapidly to various osmotic pressures with-out apparent effect on their viability and func-tion. In hypotonic solutions of sucrose or phos-phate, the microscopic picture observed was ofloss of structure and large body content as wellas considerable cellular debris. This representsan intermediate picture between complete main-tenance of form in sucrose solutions above 0.25M and the complete destruction of the L form asobserved in water suspension. Although Mg++has been reported to stabilize protoplasts ofEscherichia coli obtained by exposure to peni-cillin sucrose broth (Lederberg and St. Clair,1958), Mg++ was without effect in maintainingthis form. Suspensions in hypotonic solutionsmay result in damage or loss to some life sus-taining process which is slowly repaired or re-placed by the cell if removed to a nutritive andosmotically protective medium in time. Thiscould account for the longer incubation periodconstantly required before maximal growth wasreached with L forms from these suspensions.The structural disintegration and impairment

of viability seen after suspending L forms inhypotonic and water suspensions introduces atechnical difficulty in the preparation of L formmaterial for chemical analyses. Washing withsaline or water would result in only a residueportion of these forms remaining rather than anintact and viable form. Analyses of such residueswould be erroneous and open to serious criticismif reported in terms of the chemical constituentsof intact L forms. In a succeeding paper data willbe presented documenting this point in detail.The ability to obtain viable L forms even after24 hr suspension in hypertonic sucrose solutionaffords an obvious means of freeing the L formfrom components of the growth medium byrepeated washing in sucrose. This method is beingemployed currently in our laboratory in variousmetabolic and chemical studies of L forms.The need for preserving L forms possessing

definite cultural characteristics is obvious andwould eliminate much of the confusion existingin reports on L forms. The ability to obtain viableL forms from lyophilized preparations even after2 months is, therefore, worthy of note. Suchpreparations would allow for indefinite preser-vation and permit exchange of cultures withdefined characteristics. The extent to whichviability can be maintained in such lyophilizedpreparations is currently being investigated.

Fragility has been reported to be a character-istic of L forms as well as a criterion for theiridentification. Sonic disruption investigationscurrently being conducted in this laboratory havedemonstrated that the L form remains viableeven after 23 min of exposure to 9 kc. The resultsof such treatment on this streptococcal L formwill be presented in a subsequent publication.

ACKNOWLEDGMENT

We are grateful to Drs. L. Dienes and J. T.Sharp for providing cultures and making availablethe facilities of their laboratory to one of us(C. P.), and also to Dr. J. R. Ward and Miss S.Madoff of the same laboratory for their kindassistance. We are also indebted to Dr. J. 0.Alberts, Head, Department of Veterinary Science,University of Illinois, for our constant supply ofhorse serum, to Mr. N. Bartly for his assistancein obtaining photographs of the L cultures, andto our colleague Dr. J. A. Hayashi, for his helpfulsuggestions during the preparation of this manu-script.

SUMMARY

Upon prolonged subculturing in a liquidmedium the macroscopic growth characteristicsof the L form of the AED strain of f-hemolyticstreptococcus resembled those of the parentstreptococcus.The effect of solutions of varying composition

and osmolarity on structural integrity andviability has been investigated. Although phos-phate and glycerol solutions were either erraticor without effect, 0.25 to 0.88 M sucrose solutionswere found to be osmotically protective. Theseresults afford a means of obtaining the viable Lform intact and free from components of thegrowth medium and suitable for chemical andmetabolic studies.

Successful preservation of viability of this Lform for a period of 2 months by lyophilizationin 0.3 M sucrose has been achieved.

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REFERENCES

DAWSON, M. H. AND HOBBY, G. L. 1939 Pleuiro-pneumonia-like organisms as a varianit phaseof Streptobacillus moniliformis. Intern.Congr. Microbiol., 3rd Congr., Abstr. Com-mun., New York, pp. 21.

DIENES, L. 1938 Further observations on theL-organisms of Klieneberger. Proc. Soc.Exptl. Biol. Med., 39, 365-367.

DIENES, L. 1939 "L" organisms of Klienebergerand Streptobacillus moniliformis. J. Infec-tious Diseases, 65, 24-42.

DIENES, L. 1945 Morphology and nature of thepleuropneumonia group of organisms. J.Bacteriol., 50, 441-458.

DIENES, L. AND WEINBERGER, H. J. 1951 TheL-forms of bacteria. Bacteriol. Revs., 15,245-288.

DIENES, L. 1953 Some new observations onL-forms of bacteria. J. Bacteriol., 66, 274-278.

KANDLER, 0. AND KANDLER, G. 1954 Unter-suchungen uber die Morphologie und dieVermehrung der pleuropneumonie-iihnlichenOrganismen und der L-Phase der Bakterien.Arch. Mikrobiol., 21, 178-201.

KLIENEBERGER, E. 1935 The natural occur-rence of pleuropneumonia-like organisms inapparent symbioses with Streptobacillus monil-iformis and other bacteria. J. Pathol.Bacteriol., 40, 93-105.

KLIENEBERGER-NOBEL, E. 1954 Microor-ganisms of the pleuropneumonia group. Biol.Revs. Cambridge Phil. Soc., 29, 154-184.

LEDERBERG, J. AND ST. CLAIR, J. 1958 Proto-plasts and L-type growth of Escherichia coli.J. Bacteriol., 75, 143-160.

PANOS, C. AND BARKULIS, S. S. 1958 Some bio-chemical and morphological characteristicsof a group A streptococcal L form. Bac-teriol. Proc., 1958, 51.

SHARP, J. T. 1954 L colonies from hemolyticstreptococci: new technic in the study of Lforms of bacteria. Proc. Soc. Exptl. Biol.Med., 87, 94-97.

SHARP, J. T., HIJMANS, W., AND DIENES, L. 1957Examination of the L forms of group A strep-tococci for the group-specific polysaccharideand M protein. J. Exptl. Med., 105, 153-159.

SMITH, W. E., HILLIER, J., AND MUDD, S. 1948Electron micrograph studies of two strains ofpleuropneumonialike (L) organisms of humanderivation. J. Bacteriol., 56, 589-602.

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