JOURNAL OF BACTERIOLOGY, Nov. 1973, p. 994-1000Copyright (0) 1973 American Society for Microbiology

Vol. 116, No. 2Printed in U.S.A.

New Method for the Isolation of Membranesfrom Mycoplasma gallisepticum

M. C. GOELDepartment of Microbiology, Lister Institute of Preventive Medicine, Chelsea Bridge Road, London, SWI W

8RH

Received for publication 27 July 1973

Mycoplasma gallisepticum lysed readily in carbonate bicarbonate buffer at pH9.2 to 10.5. The hemagglutination titer of the lysates was 2- to 16-fold greaterthan a cell suspension at the same protein concentration in buffered saline.Membranes prepared from cells lysed by this method at pH 10 were relativelyfree from cytoplasmic contaminants as shown by electron microscopy of thinsections. The membranes retained their hemagglutination activity, gave reac-tions in immunodiffusion tests identical to those obtained by osmotic lysis andsonic treatment, and showed a similar pattern of protein bands by polyacrylam-ide disk electrophoresis. When inoculated into rabbits, the membranes gave riseto antibodies active in growth-, metabolic- and hemagglutination-inhibitiontests. On the average, membranes obtained by lysis at pH 10 contained 44% ofthe original cell protein. The method is simple, giving high yields of membranes,and may be adaptable to other mycoplasmas.

The membrane of Mycoplasma gallisepticumis the site of antigens which elicit the produc-tion of growth-, metabolic- and hemagglutina-tion-inhibition antibodies (9, 26). Separation ofmembranes is therefore a prerequisite for theisolation and characterization of these antigens.Several methods, including lysis by osmoticshock, ultrasonic and digitonin treatments,have been used to obtain M. gallisepticummembranes. Whereas the organisms are insensi-tive to osmotic shock when transferred from0.25 M sodium chloride to water (18), mem-branes for immunological studies were obtainedfrom M. gallisepticum by injecting suspensionsin 2 M glycerol into water (9, 24). However, themajority of the organisms often remain unlysedby this method. Ultrasonic treatment leavesvariable amounts of organisms unlysed anddisaggregates the membrane into minute frag-ments which cannot be separated from thesoluble fraction (9, 16). Recently, Rottem andRazin (23) reported that M. gallisepticum canbe effectively lysed by digitonin.This investigation describes a new method for

the lysis of M. gallisepticum at pH 10, its effecton the hemagglutinating activity of the orga-nism, and the preparation of immunogenicallyactive membranes.

MATERIALS AND METHODSM. gallisepticum strain Prem C1/69 was obtained

from P. K. Uppal (School of Veterinary Medicine,

University of Cambridge). This strain was isolated inJanuary 1968 from chickens suffering from coryza andhad undergone six passages in artificial mediumbefore lyophilization.M. gallisepticum was grown in a liquid medium

consisting of pleuropneumonia-like organism (PPLO)broth (Difco) supplemented with 2.5% (wt/vol) driedyeast extract, 0.5% (wt/vol) glucose, 0.002% (wt/vol)sodium deoxyribonucleate, 125 U of penicillin per ml,0.0125% (wt/vol) thallous acetate, 0.002% (wt/vol)phenol red, and 2% (vol/vol) PPLO serum fraction(Difco). The pH of the medium was adjusted to 7.8; itwas sterilized by filtration through a 0.45-Am filter(Sartorius-Membranfilter GMBH, G6ttingen, W.Germany), and was distributed in 200-ml volumes in500-ml bottles. When organisms were cultivated forrabbit inoculation, PPLO serum fraction was replacedby 15% (vol/vol) rabbit serum. For storage at -30 C,the organisms were grown in medium supplementedwith 20% (vol/vol) horse serum because the organismstended to die in medium enriched with PPLO serumfraction.

For lysis experiments or the preparation of mem-branes, the organisms were propagated in liquidmedium from a 1% inoculum of a 24-h culture byincubating at 37 C for 24 to 28 h. When the pH of theculture had fallen to approximately 6.8, the organismswere harvested by centrifugation at 9,000 x g for 30min in the cold. The organisms were washed threetimes with a volume of 0.15 M phosphate bufferedsaline (PBS), pH 7.4, equivalent to one fifth of thevolume of the original culture.To determine the degree of lysis in carbonate-bicar-

bonate buffer (CBB), washed organisms were sus-pended in PBS to give a concentration of 10 mg of

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protein per ml. The suspension was then added in0.1-ml amounts to 4-ml volumes of CBB at pH valuesfrom 9.2 to 10.5, prewarmed at 37 C. As a control, 0.1ml of cell suspension was added to 4 ml of PBS. Afterincubation for 5 min at 37 C, the optical density (OD)at 500 nm was measured and samples were titrated forhemagglutination activity.

For the preparation of membranes, washed myco-plasmas were suspended in PBS to a concentration of4 to 8 mg of protein per ml. A 5-ml amount ofsuspension was then added to 100 ml of 0.05 M CBB,pH 10, at 37 C. After incubation for 5 min, 2.2 g ofboric acid (Analar) was added to reduce the pH to 7.4,the mixture was shaken vigorously for 3 to 5 min todissolve the boric acid, and then was held for 15 minat 37 C. Deoxyribonuclease (BDH, Laboratory Rea-gent; 2.5 mg in 1 ml of PBS) was added to the mixtureand incubation continued for another 30 min. At theend of this period, ribonuclease (Bovine pancreas, 5 xcrystallized, 60 K units per mg, Sigma Chemical Co.;1.5 mg in 1 ml of PBS) was added and allowed to reactfor 30 min at 37 C. The membranes were harvested bycentrifugation at 9,000 x g for 20 min, and washed sixtimes with PBS, the first four times in a volume equalto that of the CBB and then with half the volume. Themembranes were designated CB/M and stored at-30 C.

For comparison with membranes prepared by lysisat pH 10, a batch of membranes was prepared byosmotic shock and ultrasonic treatment, and a solublecell fraction was prepared by osmotic shock. Orga-nisms were first lysed by osmotic shock after treat-ment with glycerol (24). Unlysed cells were removedby centrifugation at 8,000 x g for 5 min. Thesupernatant fluid was centrifuged at 40,000 x g for 30min to deposit membranes and the resultant superna-tant fluid was concentrated 100-fold by pressuredialysis and then dialysed against PBS for 48 h. Thisconstituted the soluble cell fraction. Unlysed orga-nisms deposited at 8,000 x g were suspended in 10 mlof PBS and treated in an ice bath for five periods of 1min each with a Branson S 75 sonifier (BransonInstrument Inc., Stamford, Conn.) at 20 kc/s. Aftereach period of sonic treatment the suspension wascentrifuged at 9,000 x g for 20 min and the depositwas suspended in 10 ml of PBS. The five supernatantfractions were pooled and centrifuged at 100,000 x gfor 30 min. These membranes were mixed with thosedeposited at 40,000 x g during the preparation of thesoluble fraction. The pooled membranes were washedsix times with PBS and stored at -30 C. Thispreparation was designated GS/M.

Protein was estimated by the Folin phenol methodof Lowry et al. (13), by using crystalline bovinealbumin as a standard.

For raising antisera in rabbits, the organism wasadapted to grow on rabbit serum agar and furtherpassaged 2 to 3 times in liquid medium containingrabbit serum. Washed cells from a 500-ml culturewere harvested in the late logarithmic phase of growthand were used for immunizing one rabbit according tothe schedule of Hollingdale and Lemcke (7). Forantiserum to membranes prepared by lysis at pH 10,two rabbits were used. One received a suspensioncontaining 6 mg of protein inoculated intravenously

(i.v.) in six doses; five on alternate days and the sixth7 days later. The second was given 2 mg of proteinsubcutaneously (s.c.) with adjuvant and 3 mg i.v.,according to Hollingdale and Lemcke (8). The rabbitswere bled 10 to 13 days after the last injection and thesera were separated and stored at -30 C.Growth inhibition (GI) tests were carried out by the

method of Clyde (4). The method of Taylor-Robinsonet al. (25) was used for metabolic inhibition (MI)tests. The immunodiffusion test was that describedby Lemcke (12). Whole cells, membranes, and solublefraction were adjusted to 2 mg of protein per ml, thefirst two being lysed with Triton X-100 (10 mg/mg ofprotein) (7). For hemagglutination (HA) and hemag-glutination inhibition (HI) tests, the techniques ofRoberts et al. (21) were adapted to the Microtitre(Cooke Engineering Co., Arlington, Va.) system,using 0.5% chicken erythrocytes. For HA titration,whole cells and membrane suspension were treated for1 min in an ultrasonic bath (Millipore ultrasoniccleaner, Millipore Corp., Bedford, Mass.). A glycerin-ated commercial antigen (Wellcome Research Labo-ratories, Beckenham, England) was included as apositive control in all tests. For HI tests the sera weretreated with receptor-destroying enzyme (WellcomeResearch Laboratories, Beckenham, England) to re-move nonspecific inhibitors (21). Preimmunizationsera were included in all tests as controls.

For polyacrylamide disk electrophoresis of wholecells and membranes ofM. gallisepticum, the methodof Rottem and Razin (22) was used. The proteins wereextracted from cell or membrane pellets by using 0.1ml of phenol-acetic acid-water (2:1:0.5, wt per vol pervol) per mg of protein. To each gel 0.1 ml of proteinextract was applied. The gels were stained with amidoblack lOB and decolorized in a mixture containing10% (vol/vol) acetic acid and 10% (vol/vol) methanol,at 110 V for 2 h.

For electron microscopy, pellets of washed mem-branes obtained by lysis at pH 10 were fixed with 2.5%(vol/vol) glutaraldehyde in 0.02 M sodium cacodylatebuffer for 40 min, and then with 2% (wt/vol) osmiumtetroxide for the same period. They were subsequentlytreated with 0.5% (wt/vol) uranyl acetate for another40 min, dehydrated in increasing concentrations ofisopropanol, and embedded in Araldite. Thin sectionsstained with Reynolds lead citrate (20) were examinedwith a Philips EM 200 electron microscope.

RESULTSAfter incubation at 37 C for 5 min, M.

gallisepticum suspensions in CBB at pH 9.7 to10.5 had OD of 0.15 to 0.2 at 500 nm, comparedwith an OD of 0.6 for the same concentration oforganisms in PBS (Fig. 1). Although suspen-sions were incubated for a standard time (5min), visual inspection showed that lysis wasalmost instantaneous upon the addition of themycoplasmas to CBB. In addition to the reduc-tion in OD, the HA titer of the lysates was 2- to16-fold greater than the control suspension inPBS. The rise in HA titer was greatest betweenpH 10 and 10.5 (Fig. 1). The high pH of the

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GOEL

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64 w'32 ,168 <

42

9.1 9A 9.7 10 10.3

P B SpH

FIG. 1. OD and hemagglutination titer of M. gal-lisepticum after 5 min of incubation at 37 C in CBB atdifferent pH. Control suspension in PBS, pH 7.4.Symbols: *, OD at 500 nm; 0, reciprocal hemaggluti-nation titer.

lysate had no effect on the chicken erythrocytesand titrations could be carried out withoutlowering the pH. The lysis of the organisms andthe increase in HA titer were not affected bytemperatures of incubation up to 37 C. How-ever, the pH at which the cultures were har-vested had a direct influence on the lysis of cellsat pH 10 (Fig. 2). Cells harvested at pH 6 wereresistant to lysis even when incubation at pH 10was continued for 1 h, whereas suspensions ofcells harvested at about pH 6.8 became clear,viscous, and ropy after lysis at pH 10.The reduction in pH produced by the addi-

tion of boric acid, together with the agitation,increased the OD of the lysate. Phase-contrastmicroscopy and electron microscopy of prepara-tions negatively stained with 2% potassiumphosphotungstate showed that there wasclumping and aggregation of membranes whenthe pH was reduced.On the average, washed membranes obtained

after lysis at pH 10 contained 44% of the proteinin the original cell suspension. In thin sectionsof membrane pellets examined by electron mi-croscopy, very few unlysed cells were seen andthe membranes were relatively free of cytoplas-mic contents such as ribosomes (Fig. 3). Inaddition to membrane profiles which were simi-lar in size to the original cells, smaller vesicularforms, small membrane fragments, and longcorrugated sheets were also present, togetherwith some amorphous material. All structuresexcept the last two had a typical triple-layeredmembrane structure.Membranes prepared by lysis at pH 10 and by

osmotic shock and sonic treatment gave similar

J. BACTERIOL.

patterns of protein bands when subjected topolyacrylamide disk electrophoresis. The differ-ences were mainly in the relative intensitites ofthe common bands. The patterns given by themembrane preparations were different fromthat given by whole organisms.To determine HA activity, the membranes

were suspended in PBS at 1 mg of protein perml. Clumps were dispersed by 1-min ultrasonictreatment in an ultrasonic bath before testing.Such membrane suspensions usually had higherHA titers per milligram of protein than suspen-sions of whole cells treated in the same way,although there was some variation betweenexperiments (Table 1). The geometric mean ofthe HA titers of six different CB/M preparationswas 254 compared with a mean of 97 for thesuspensions of whole organisms from which themembranes were prepared. Thus, HA titers ofthe membrane preparations were greater by afactor of 2.6 than those of whole organismstested at the same protein concentration.

In immunodiffusion tests with antiserumagainst whole organisms, membranes preparedby lysis at pH 10 (CB/M) gave four precipita-tion lines which sometimes fused to give twoheavy bands (Fig. 4). These lines were identicalto those given by membranes prepared byosmotic shock and sonic treatment (GS/M), andwere also given by whole organisms (Fig. 4). Thelines given by the soluble fraction were distinctfrom those given by membrane preparations.

.6

.5

.4EC00

0

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2

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0 15 30 45 60

MINUTE SFIG. 2. Lysis in CBB, pH 10, of M. gallisepticum

harvested at pH 6.8 (0); pH 6.0 (A); and pH 5.0 (U).The turbidity of the suspension of washed organismsin PBS was adjusted so that a 1:10 dilution in PBShad an OD500 of 0.51. One volume of this suspensionwas added to 10 volumes of CBB, pH 10, and theOD500 was taken at intervals.

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I

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FDG..ElctronmicrgraphoflMg m d y s at pFIG. 3. Electron micrograph ofM. gallisepticum membran7es prepared by lysis at pH 10. Bar equals 0.2 tim.

Sera from rabbits immunized with CB/Mcontained GI, MI, and HI antibodies. Titers ofthese antibodies were the same in sera preparedwith or without adjuvant, but were slightly

lower than those of an antiserum against wholeorganisms (Table 2). In immunodiffusion tests,antisera to membranes produced similar pat-terns of lines with the two membrane prepara-

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TABLE 1. Hemagglutination titers of M. gallisepticum membranes prepared by lysis at pH 10 comparedwith those of suspensions of whole organisms

Hemagglutination titersa

Antigen Expt. no. Geometricsuspension _______________________________ mean titer

1 2 3 4 5 6

Whole organisms" 32-64 256 256 32 128 64 97Membranesb 128-256 512-1024 256 128-256 128-256 128-256 254Commericalc antigen 32 32 32 32 32 32 32

a Reciprocal of titers.Titrated at 1 mg of protein per ml.Glycerinated antigen (Wellcome Research Laboratories, Beckenham, Kent, England).

FIG. 4. Immunodiffusion reactions of M. gallisep-ticum membranes, soluble fraction, and whole orga-nisms with antiserum against whole organisms (cen-tral well). The peripheral wells contain (1) mem-

branes prepared by lysis at pH 10; (2) me. ibranesprepared by osmotic shock and sonic treatment; (3)whole organisms; (4) soluble fraction, all at 2 mg ofprotein per ml. Membranes and whole organisms were

lysed with Triton X-100, 10 mg per mg of protein.

tions, CB/M and GS/M, and with whole orga-nisms (Table 2). In reactions with whole orga-

nisms, the antiserum prepared against CB/Mwith adjuvant gave a line additional to thosegiven by membranes. Since an identical line wasgiven by the soluble fraction, the reaction was

probably due to a cytoplasmic antigen. Themembrane antiserum prepared by i.v. inocula-tion did not give this line. This suggests that themembrane preparation CB/M was minimallycontaminated with cytoplasmic material whichwas sufficient to generate antibody when in-jected into a rabbit together with adjuvant.

DISCUSSIONLysis of M. gallisepticum in carbonate-bicar-

bonate buffer at pH 10 offers a simple and rapidmethod of preparing membranes from this orga-

nism. The majority of cells are lysed and a highyield of membranes is obtained.

It was first demonstrated in 1923 that lyso-zyme-resistant bacteria could be lysed at pH 10if the cells were first exposed to lysozyme (14).These observations were later extended to 135species of 11 genera of gram-negative bacteria(15). Digestion of the cell wall mucopeptide bylysozyme exposes the cytoplasmic membranesto lysis at high pH (5, 6). The common effects ofexposure to pH 10 were swelling of the cells,ghost formation, disruption of the cells, andincreased viscosity (5). Similar characteristicsalso marked the lysis ofM. gallisepticum at pH10. The sensitivity of the cytoplasmic mem-branes to alkali was confirmed by Razin andArgaman (19) who reported the lysis of Achole-plasma laidlawii, M. mycoides var capri, M.hominis, L-forms of Streptobacillus monilifor-mis, and spheroplasts of Escherichia coli withsodium hydroxide.The change in OD of the lysate when the pH

was reduced from 10 to 7.4 was due to theclumping and aggregation of membranes. Theformation of clumps was probably due to theremoval of electrostatic repulsion between nega-tively charged groups at pH 10. The aggregationof membranes into the long corrugated sheetsseen in electron micrographs of thin sectionspossibly represents interfacial film formation.Ultrasonically disrupted erythrocyte mem-branes are known to aggregate into very largefilms in the presence of cations at an air-waterinterface (10).Organisms harvested at or below pH 6 were

very resistant to lysis at pH 10. However, suchorganisms are also very poor in HA activity; formaximum HA titers cells harvested at about pH6.8 were best (Goel, unpublished data). Myco-

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plasmas harvested in the stationary phase ofgrowth are also resistant to lysis by osmoticshock (17) and to a lesser extent to lysis bydigitonin (23). Higher internal osmotic pres-

sure or the presence of weak areas in themembranes of mycoplasmas undergoing activegrowth was thought to be responsible for theirosmotic fragility in the logarithmic phase (17).Another possible reason for the resistance tolysis of cultures harvested below pH 6.0 is theadsorption of serum proteins from the culturemedium during incubation. Adsorption in-creases as the pH of a culture drops and this isalso thought to depress the HA activity oforganisms harvested at low pH (2). Such pro-

tein adsorption could block the access of posi-tively charged ions to mycoplasma membranes.The rise in HA titer of M. gallisepticum when

the organisms are lysed at pH 10 is probablyrelated to the surface location of this antigen.Surface projections seen in negatively stainedpreparations of M. gallisepticum are believed tobe the hemagglutinin of the organism (3).Since electron micrographs indicated thattreatment with bicarbonate buffer at pH 10breaks up membranes into smaller pieces, thenumber of particles which can form bridgesbetween erythrocytes is probably increased.The increase in HA titer therefore probablydepends on the extent to which the membranesare fragmented. A similar increase in HA titer isalso observed when viruses with surface he-magglutinins are disrupted (1, 11).The major antigens of M. gallisepticum

which stimulate the formation of GI, MI, andHI antibodies are known to be present in themembrane (9, 26). Membranes isolated by lysis

of cells at pH 10 were shown to possess theseantigens and to react in immunodiffusion testsin a manner identical to membranes obtainedby osmotic lysis and sonic treatment. Thisdemonstrates that the antigenicity and immu-nogenicity of the membranes was retained.Contamination with cytoplasmic antigens ofmembranes prepared by lysis at pH 10 wasminimal; very little contamination was revealedby electron microscopy, immunodiffusion, orthe immune response of rabbits inoculated bythe i.v. route without adjuvant. Only whenmembranes were inoculated into rabbits to-gether with adjuvant was precipitating anti-body to a cytoplasmic antigen evoked. This is inaccordance with the observation of Lemcke(Lemcke, R. M., Ann. N.Y. Acad. Sci., in press)that immunization with adjuvant gives rise toantibodies to an increased range of soluble cellproteins. Moreover, membrane antisera pro-

duced with or without adjuvant possessed thesame titers of GI, MI, and HI antibodies. Thisconfirms Lemcke's contention that it is possibleto produce antisera with satisfactory titers ofantibody to membrane antigens without the use

of adjuvant.Besides retaining their antigenicity and im-

munogenicity, membranes prepared by lysis atpH 10 had higher HA titers than suspensions ofwhole organisms tested at the same proteinconcentration. The HI test is the most reliablemethod for detecting M. gallisepticum anti-bodies in poultry (21), and antigens with highHA titers are required. At present, preservedsuspension of whole organisms are generallyused. One commercially available antigen ofthis type, which comprises a 1% suspension of

TABLE 2. Serological actiuities of antisera against M. gallisepticum membranes prepared by lysis of organismsat pH 10, compared with those of antiserum against whole organisms

No. ot gel

Protein Titer diffusion lines withAntiserum inoculated GI (mm)against (mg) GI_(mm)_Whole Membranesb

MI Hla organism' GS/MC CB/Md

Whole organisms NDe 10,240-20,480 160 4.0 7' 4 4Membraneslg 6 2560 80 2.5 2-3 3 32h 5 2560 80 2.5 4-5 4-5 4-5

a Titrated against four HA doses of whole organisms or membranes.b Lysed with Triton X-100 (10 mg per mg of protein).c Prepared by osmotic lysis and sonic treatment.d Prepared by lysis at pH 10.e Not determined.' Minimum number observed; too complex to resolve.9 Prepared by i.v. inoculation.h Prepared by s.c. (+adjuvant) and i.v. inoculation.

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1000 GOEL

organisms, had an HA titer of 32 to 64. Thus,membrane preparations with titers of 256 to1024 per mg of protein compared favorably withthis antigen. If membranes prepared by themethod described here can be preserved so asto retain their HA activity, a useful method ofpreparing a high-titer HA antigen is available.Moreover, since membranes of M. gallisepti-cum prepared at pH 10 retained their antigenic-ity and immunogenicity, lysis in bicarbonatebuffer may represent a simple method for thepreparation of membranes of other mycoplas-mas for immunochemical studies.

ACKNOWLEDGMENTS

This investigation was carried out during the tenure of ascholarship from the Commonwealth Scholarship Commis-sion. United Kingdom.

I am grateful to Ruth Lemcke for advice during the projectand help in preparing the manuscript. The valuable discus-sions with A. M. Lawn and the technical assistance of AnnMartin of the Electron Microscopy Unit of this Institute aregratefully acknowledged. Lindsay Hitchcock and Hilary Tal-bot also provided valuable help.

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