JOUR-NAL OF BACTERIOLOGY, Mar., 1965Copyright C 1965,- American Society for Microbiology

Vol. 89, No. 3Printed in U.S.A.

Interaction of Viscid Material of Staphylococcusaureus with Specific Immune Serum

STUART MUDD AND SAMUEL J. DECOURCY, JR.U.S. Veterans Administration Hospital, Department of Clinical Pathology of the Philadelphia General

Hospital, Department of Public Health and Preventive AMedicine, and the Henry Phipps Institute,School of Medicine. University of Pennsylvania, Philadelphia, Pennsylvania

Received for publication 30 October 1964

ABSTRACTMUDD, STUART (U.S. Veterans Administration Hospital, Philadelphia, Pa.), AND

SAMUEL J. DECOURCY, JR. Interaction of viscid material of Staphylococcus aureus withspecific immune serum. J. Bacteriol. 89:874-879. 1965.-Re-examination of the phenom-enon of Price and Kneeland and of Wiley revealed the following. (i) The prototype"wound strain" of Wiley, and viscid-colony strains obtained by aging and selection oflaboratory or field strains, differed in growth characteristics in liquid and solid mediafrom the Smith encapsulated strain and from ordinary, unselected laboratory and fieldstrains of coagulase-positive staphylococci. (ii) The wound strain and ordinary un-selected strains, unlike the Smith encapsulated strain, did not exhibit capsules whenexamined in thin films of Pelikan Waterproof Drawing Ink. (iii) The phenomenon ofPrice and Kneeland and of Wiley is exhibited when the wound strain and other viscid-colony strains interact with anti-Wiley immune sera or various human sera. In our ex-perience, this phenomenon was not exhibited by the Smith or by ordinary, unselectedstrains. (iv) The staphylococcal polysaccharide antigen previously characterized as thecapsular substance of a Smith-like strain was completely different chemically and sero-logically from extracellular material prepared from the Wiley wound strain. We con-clude that the viscid-colony strains are not, in fact, encapsulated, and that the phe-nomenon in question is a precipitation of extracellular material about the periphery ofthe cells.

Price and Kneeland (1954, 1956) demonstrateda phenomenon dependent upon the interactionof specific immune sera with mucoid strains ofStaphylococcus aureus, which they interpreted as"Quellung" or capsular swelling. The work ofPrice and Kneeland has been confirmed andextended by Wiley (1961, 1962, 1963, 1964).The interpretation of this phenomenon as"capsular swelling" or "specific capsular reac-tion" has stood in the literature almost unchal-lenged for 10 years. In the present communica-tion, we re-examine this phenomenon andquestion its interpretation as a capsular reaction.

Capsular swelling was first demonstrated byRoger (1896) with Oidium albicans and by Neu-feld (1902) with pneumococci, as dependentupon the interaction of immune serum withcapsulated cells:

"Mischt man nun gleiche Theile vonagglutinirendem Serum und Pneumo-kokkenbouilloncultur ... so beobachtetman in hangenden Tropfen deutlicheQuellungserscheinungen..." (Neufeld,1902, p. 57).

This reaction has since been used extensivelyfor the specific recognition of many species andtypes of encapsulated bacteria. Etinger-Tulezyn-ska (1933), a colleague of Neufeld, distinguishedtrue bacterial capsules ("Kapseln") from themucoid, viscid material ("Schleimhiulle") pro-duced by many bacteria. Various strains mayhave true capsules or may secrete extracellularslime; in the case of pneumococcus III, the truecapsule may be surrounded by an excess ofviscid material, which may be precipitated byspecific immune serum around the bacterial celland its enveloping capsule (Mudd, Heinmets,and Anderson, 1943a, b). Whether capsular"Quellung" actually represents swelling, ormerely the impregnation of a preformed capsulargel with specific and nonspecific components ofthe immune serum, has been debated (Kliene-berger-Nobel, 1948; Tonmcsik, 1956) and neednot be further discussed here.The question under discussion in this paper is

whether the mucoid staphylococci of Price andKneeland and of Wiley possess true capsules(a capsule we define operationally as an envelop-

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ing structure, outside the bacterial cell wall,which is regularly present under ordinary condi-tions of culture, and which can be demonstratedby negative staining) or merely viscid extra-cellular material in excessive amounts. Thisquestion has more than semantic importance,because true capsules ordinarily are criticalfactors in determining virulence of invasivebacteria, and are specific sites for interactionwith protective immune sera.

B. B. Wiley has generously received in hislaboratory and demonstrated his phenomenon toan emissary from this laboratory, Theodore Sall,and has given us transplants of his "woundstrain" and other strains, and high-titer immuneserum. The reproducibility of the phenomenonof Price and Kneeland and of Wiley is unques-tionable.

MATERIALS AND METHODS

Strains of coagulase-positive Staphylococcusaureus used. Three prototype strains were used.

Encapsulated strains. The Smith strain wasoriginally obtained from Ren6 Dubos of theRockefeller Institute and carried in this labora-tory for 4 years without animal passage. The Smithstrain may be taken as the prototype of trulyencapsulated S. aureus (Hunt and Moses, 1958;Morse, 1962; Lenhart et al., 1962; Koenig, Melly,and Rogers, 1962a).

Strains kindly furnished us by Ralph Tompsett,designated Adams, Welwood, K6, K93, and SV,grow diffusely on serum soft-agar, and, like theSmith strain, are pathogenic for mice (Koeniget al., 1962a, b). We have demonstrated capsuleson these strains by negative staining with PelikanWaterproof Drawing Ink, although they are notas prominent as those associated with the Smithstrain (Li, Mudd, and Kapral, 1963).

Viscid, tenacious colony strains. The Wiley 1961"wound" strain (Wy) is the prototype. Variantstrains with viscid, tenacious growth habit weredeveloped from nonviscid field strains by theaging and selection procedure of Bigger, Boland,and O'Meara (1927). Certain strains of Inter-national Serotypes I-XIII received from S. T.Cowan after similar treatment also fell into thiscategory.

Ordinary strains of S. aureus. The great ma-jority of strains isolated from human infectionsand grown on standard laboratory media withoutselection form nonviscid, nonmucoid, nonstringy,creamy-yellow colonies on agar plates, and, inso-far as gross morphology is concerned, are geneti-cally stable. All of the International Serotypes,unless treated by the aging procedure of Biggeret al. (1927), also fall into this category. Becauseof the considerable experience in this laboratorywith the "parent" strain 18Z (Kapral and Li,1960; Sall, Mudd, and Taubler, 1961; Lenhart etal., 1963), this was taken as the prototype strain

for the commoni or ordinary pathogenic staphylo-cocci.

All three of the prototype strains, Smith, Wiley,and 18Z, are coagulase-positive, ferment mannitol,and possess deoxyribonuclease activity, as de-termined on Di Salvo deoxyribonuclease medium.

Vaccine and antisera. Hyperimmune aniti-Wileyrabbit and rooster sera were obtained from NewZealand white male rabbits (4 to 5 lb) and Plym-outh Rock roosters which had been immunizedaccording to the Wiley (1961) modification of theprotocol of Alexander, Leidy, and MacPherson(1946) with 0.5% formalin-killed, Wiley woundstrain organisms. Anti-Wy hyperimmune y-globu-lin was prepared from corresponding high-titerrabbit antiserum, according to a modification ofthe fluorescent antibody techniques outlined byCherry et al. (1960). In brief, this involves a tripleprecipitation in the cold with 50% saturated am-monium sulfate followed by exhaustive dialysis infrequent changes of isotonic saline, reduction ofvolume by dialysis against polyvinylpyrrolidone(PVP), and lyophilization of the purified product.In addition to homologous anti-Wy rabbit and

rooster hyperimmune sera produced both in thislaboratory and in the laboratory of B. B. Wiley,many samples of human sera obtained from pa-tients convalescing from staphylococcal infec-tions, including chronic cystic fibrosis, staphylo-coccal cellulitis, chronic furunculosis, suppurativeosteomyelitis, postoperative infections, and lobarpneumonia, as well as a number of "normal"human sera and pooled human -y-globulin, wereused to challenge the Wy indicator strain.Sources of these sera included the Hospital of theUniversity of Pennsylvania, Boston City Hospital,the hospital at Milledgeville, Ga., and the U.S.Veterans Administration Hospital in Philadelphia.

India ink negative stain techniquze. A smallloopful of the appropriately grown suspension oforganisms was thoroughly mixed with an equalvolume of black Pelikan Waterproof Drawing Ink(Guinther, Wagner) on a cover slip, overlaidwith a microscope slide, and the excess of themixture was squeezed out by gentle pressure onthe cover slip and blotted up. Because of the veryfine particle size of solids in this ink, the thicknessof the wet-mount film proved to be critical.Optimal contrast was obtained when a thin filmof undiluted ink was used rather than a thickerfilm of diluted ink. After teasing out all the bub-bles, the edges of the cover slip were sealed withcolorless nail polish (Cutex).EPPR technzique. Since we believe that the

interaction of immune serum with the Wileywound straini and strains of similar vis -id colonvhabit is a precipitation, and not a "capsularreaction," we refer to this phenomenon as extra-cellular peripheral precipitation reaction (EPPR).The technique employed to induce the EPPR isidentical with that reported by Wiley (1961).One loopful of a 6- to 8-hr culture of organismsgrown in buffered glycerol nutrient broth, threeloopfuls of anti-Wy hyperimmune rabbit or

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rooster sera (or one loopful of corresponding -y-globulin), and a small loopful of a 1:5 dilution ofa 1% aqueous stock solution of methylene bluewere well mixed on a Vaseline-rimmed cover slip,overlaid with a microscope slide, bubbles andexcess mixture teased out, and the edges of thecover slip sealed with gentle pressure. Overflowwas blotted away with filter paper. In practice,the EPPR is demonstrable almost immediatelywhen the indicator strain is clhallenged withhomologous hyperimmune antiserum. However.when checking serum of unknown activity, aperiod of 48 hr was allowed for the reaction tooccur. If, at the end of this period, there was stillno evidence of a reaction, the slide was discarded,and the serum was considered to be negative.

RESULTS

The encapsulated, the viscid, and the ordinarystrains of coagulase-positive S. aureus exhibitcharacteristic differences when cultivated inliquid or on solid media and when examined bynegative staining or in interaction with immunesera.

Growth in liquid media. When grown in bufferedglycerol nutrient broth, each of the three proto-type strains possesses its own peculiar growthcharacteristics. In stagnant culture, the Smithencapsulated strain grows profusely in overnightculture with cells settling to the bottom of thetube, the supernatant fluid being homogeneouslyturbid. If agitated, the settled cells rise in amucoid swirl. The Wy strain gives rise to smallaggregates of clumpy growth, which tend tocling to the side of the culture tube, leaving arelatively clear supernatant fluid. When agitated,this culture becomes a suspension of flocculantaggregates of cells. The 18Z culture presents aconventional picture of homogeneous turbiditywith a pellet of settled cells which, upon agita-tion, goes into suspension quite readily.

In roller tube culture, both the Smith and 18Zstrains give homogeneous suspensions. The Wystrain, as in stagnant culture, grows in clumpyaggregates with a heavy ring of growth adheringto the glass at the air-liquid interface. In makingan EPPR preparation, a homogeneous suspensionof cells is required. This can be achieved with theWy strain if the culture is briskly agitated(foamed) every 30 min over a growth period of6 to 7 hr.

Growth on solid media. The Smith encapsulatedstrain grows characteristically as cream-colored,highly mucoid colonies on standard laboratorymedia, e.g., nutrient agar and Trypticase SoyAgar (BBL). These colonies string out whentouched with an inoculation needle, but lack theextremely tenacious and rubbery consistency ofcolonies of the Wiley strain.

The Wiley "wound strain," when grown onbuffered nutrient agar containing 2% glycerol(Wiley, 1961), forms yellow-orange compacttenacious colonies which string out when teasedwith an inoculating needle. However, after agingfor apl)roximately 3 weeks in a sealed petri dish,the colonies lose their sticky consistency, be-coming rather hard and firmly anchored to thesurface of the agar, and can be removed onlywith difficulty. Cells from such colonies, whenrevived by several serial passages through liquidand solid buffered glycerol nutrient medium, forthe most part revert to producing colonies pos-sessing the original sticky character. It is notuncommon to find a few nonsticky colonies onsuch plates, samples of which fail to elicit theEPPR. By reason of such relative genetic in-stability, frequent transfer of selected coloniespossessing the desired characteristics has beenfound to be essential to guarantee the final result.The 18Z "parent" strain forms nonviscid,

nonmucoid, nonstringy, creamy yellow colonieson standard laboratory media, and, insofar asgross morphology is concerned, is geneticallystable.

Exam.ination in Pelikan IVaterproof DrawingInk. The cells of the Smith strain, examined in athin film of undiluted Pelikan Ink, appear as darkspherules surrounded by well-defined whitecapsules and ill-defined whitish amorphous mate-rial (Fig. IA). Cells of the Wiley wound strain(Fig. 1B) and of 18Z (Fig. 1C) appear as brightspheres in a dark background.

Exanmination in anti-WTiley hyperimmune serum.In anti-Wiley immune serum plus methyleneblue, the Smith strain (Fig. 2A) and 18Z (Fig.2C) lpresent the usual picture of stained staphylo-cocci, whereas the wound strain (Fig. 2B) pre-sents the picture made familiar by Wiley. Theresults were obtained both with animal immunesera and with a variety of human sera fromhealthy and from infected persons.Examination by simple staining. Cells from 6-

to 8-hr cultures in Wiley broth stained by methyl-ene blue present the usual picture of stainedstaphylococci. Cells of 18Z are appreciablysmaller than those of the other strains. (Fig.3A, B, C).

Examination of field strains. None of our fieldstrains showed capsules. The Smith strain andstrains obtained from Tompsett show capsules.None of the unselected field strains in our experi-ence showed the positive EPPR when challengedwith anti-Wiley hyperimmune rabbit or roosterserum or with the sera of healthy or infectedhuman persons. However, viscid colony variantsof 18Z and of several of the international sero-types and field strains derived by the aging and

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selection procedure of Bigger, Boland, andO'Meara (1927), as well as the wound strain, didshow the EPPR when challenged with anti-Wileyserum and with some human sera. When ex-

FIG. 2. Methylene blue plus anti-Wy hyper-immune rabbit serum (2,000 X). (A) Smith capsu-lated strain, (B) Wiley "wound" strain, and (C)18Z "parent" strain.

amined after simple methylene blue staining,all strains showed the conventional picture (Fig.3).

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Smith capsulated strain, (B) Wiley "wound" strain,and (C) 18Z "parent" strain.

Examination in anti-Smith serumti. AMorse (1962)has reported that no Quellung reaction of theSmith encapsulated cells could be demonstratedwith homologous antiserum. Our experienceconfirms this. We have observed the encapsulated

Smith cells to form compact colonies in serum-soft-agar (Finkelstein and Sulkin, 1958; Shimizu,Griffiths, and Greenberg, 1963) containing normalrabbit serum in dilution of 1:10, and in rabbitantisera versus Smith encapsulated strain, Wileywound strain, and 18Z in dilutions of up to1: 1,000. However, capsular swelling was notobserved when the Smith encapsulated strain wasexposed to these same antisera, even undiluted.

Chemical characterization of the extracellularmaterial of the uound strain. Chemical purificationand characterization of the extracellular materialof the Wiley indicator strain is being carried outby Kazuhito Hisatsune and will be reported indue course. For the purposes of this communica-tion, it is relevant to report that both chemicaland serological charaeteristies indicate that thismaterial is entirely distinct from the staphylo-coccal polysaeeharide antigen (SPA) whichconstitutes the capsule of the Smith strain(Haskell and Hanessian, 1964). (T. H. Haskellhas kindly furnished us with a puie sample ofSPA.)

DIsCU.SSION

We find that th-e )rototype strain of Wiley(1961) and cther viscid-colony-forming strains ofS. aureus differ in many respects from the trulyencapsulated strains, of which the Smith strainmay be taken as prototype. The viscid-colonyformers, we believe, do not, in fact, possesscapsules. The terms "Quellung," "capsularswelling," and "specific capsular reaction,"applied to the phenomenon of Price and Knee-land and of WViley, we therefore believe to beunfortunate, first, because they are inaecurate,and second, because the term "capsule" connotescharacters of virulence and induced immunity,which in this case do not correspond to experi-mental findings (Price and Kneeland, 1956). Inthis article we have used the purely descriptiveterm "extracellular peripheral precipitation reac-tion" (EPPR) without, however, making anyparticular point about its use by others.

Rogers (1953, 1954, 1956) showed a decadeearlier, as Sall et al. (1961) and Sall (1962) showedsubsequently, that the amounts of specific extra-cellular secretions of staphylococei might varysignifieantly with cultures of different geneticconstitution, as well as under different growthconditions. The Wiley wound strain would seemto be a variant in this category.

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