British Journal of Venereal Diseases, 1979, 55, 387-393

Optimum concentration of dissolved oxygen for thesurvival of virulent Treponema pallidum underconditions of low oxidation-reduction potentialS. GRAVES AND T. BILLINGTON

From the Department of Microbiology, Monash University Medical School, Alfred Hospital, Prahran,A ustralia

SUMMARY A maintenance medium with a low oxidation-reduction (redox) potential, when gentlybubbled with 5Vo oxygen in nitrogen or with air for various periods of time, gave a range ofdissolved oxygen concentrations between 16 and 5'8 pig/l. Virulent Treponema pallidum(Nichols strain) inoculated into these media were assayed 24 and 48 hours later for motility andvirulence and were compared with samples taken at zero time. Virulent T. pallidum survived bestin the presence of 2 4,g/l dissolved oxygen over a 48-hour period, which corresponded to agaseous mixture of 3'7o oxygen in nitrogen. Higher concentrations of oxygen did not givesignificantly different results from anaerobic conditions over this period. Thus, until it can begrown in vitro, T. pallidum would appear to be a microaerophilic bacterium.

Introduction

During this century attempts to grow virulentTreponema pallidum (the causative agent of syphilis)in vitro have been unsuccessful despite claims to thecontrary (Willcox and Guthe, 1966). Growth ofavirulent T. pallidum from a virulent inoculum hasrecently been reported (Sandok et al., 1976, 1978).Since the availability of large numbers of T. pallidumwhich are relatively free from host-cell contaminantsis probably a prerequisite for the development of avaccine against syphilis, attempts to grow thisbacterium in vitro are important steps in reducing theincidence of this disease.

For many years T. pallidum has been considered astrict anaerobe with a low oxidation-reduction(redox) optimum (Metzger and Smogor, 1966). Onlyin reduced media could the organism maintain itsviability, as measured by rabbit virulence. Recentlythis opinion has been challenged. Freshly isolated T.pallidum utilises oxygen at a rate equivalent to thatof aerobic organisms (Cox and Barber, 1974), andthe presence of a cytochrome system (Lysko and

Address for reprints: Dr S Graves, Department of Microbiology,Monash University Medical School, Alfred Hospital, Prahran,Victoria 3181, Australia

Received for publication 27 April 1979

Cox, 1977), suggests that this organism may respireaerobically. Glucose oxidation, pyruvatedegradation, and protein synthesis were stimulatedunder aerobic conditions (Baseman et al., 1976).Norris et al. (1978), using a reduced culture medium,found that virulent T. pallidum survived better under3% oxygen than under either aerobic or anaerobicconditions, indicating that the bacterium is micro-aerophilic.The importance of oxygen for the survival and

metabolism of T. pallidum has also been shown intissue culture systems where the addition ofsuperoxide dismutase to infected cell monolayersprolonged the virulence of T. pallidum (Fitzgerald etal., 1975). With certain cell lines and reduced culturemedium an atmosphere of 3%7o oxygen maintained T.pallidum without loss of virulence for six days(Fitzgerald et al., 1977) and 2- 5%o oxygen enabled asurface acid mucopolysaccharide to be synthesised asa result of the cell-treponeme interaction (Fitzgeraldet al., 1978). Fieldsteel et al. (1977) established anoxygen gradient in tissue culture and found that T.pallidum survived best in a region neither fullyaerobic nor anaerobic, indicating the importance ofmicroaerobiosis for the survival of T. pallidum.Kiraly and Horvath (1976) used low oxygenconditions (7 mm Hg partial pressure) and foundthat T. pallidum survived well, but in their work

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survival was poorer with, rather than without, a

tissue culture system.In our study, an optimum dissolved oxygen

concentration of 2-4 ugll (corresponding to a

gaseous mixture of 3% 0°2 in N2) was required for T.pallidum to retain its virulence.

Materials and methods

PROPAGATION OF T. P4LLIDUMThe bacterium was grown in the testes of sero-

logically negative rabbits by inoculatingapproximately 5 x 107 viable T. pallidum per testisand harvesting it 11 days later, at which time orchitishas developed. Rabbits (of various breeds) were

housed at 18°C to permit satisfactory growth of theT. pallidum. After the rabbit had been killed withintravenous sodium pentobarbitone, the testes were

removed aseptically and minced in successive 5-mlsamples of medium (see below) under aerobicconditions. This eluted the T. pallidum from thetissue, yielding about 107 organisms/ml. Thesuspension was used without purification and 0-5 mlwas inoculated into 10-ml tubes of medium to give a

final concentration of T. pallidum of about5 x 105/ml . This concentration varied fromexperiment to experiment depending on theconcentration of treponemes in the rabbit testes. The1/20 dilution of the inoculum into experimentalmedium meant that the treponemes were onlyexposed to air, albeit rn a reduced medium, for about30 minutes.

T. PALLIDUM IN VITROAfter inoculation of the medium with T. pallidumunder a stream of sterile, oxygen-free nitrogen thetubes were tightly sealed with butyl rubber stoppers(Bellco Glass Inc., Vineland, New Jersey), placed inanaerobic jars to prevent extraneous oxygen enteringthe medium, and incubated at 35°C.

PREPARATION OF MEDIUMThe medium of Graves et al. (1975) was modified andmade in two parts (A and B), which were combinedimmediately before inoculation with T. pallidum.The compositions of medium A and B are shown inTable 1. Medium A (without buffered bicarbonatesolution or reducing agents) was prereduced byautoclaving at 121°C for eight minutes with slowexhaust. Immediately after removal from theautoclave the medium was continuously flushed withoxygen-free nitrogen until cool; the latter wasobtained by passing nitrogen through a hot coppercolumn before use. When the medium was cool,buffered bicarbonate solution and reducing agents(as powders) were added by gently stirring. The pH

Table I Composition of T. pallidum maintenancemedium

Amount/l(Final total

Component medium)

Medium ABalanced salt solution concentrate (10 x )* 60 ml(NH4)2SO4 (50 g/l) 2 mlGlucose 1-60 gFructose 1 -60 gH20 400 mlBuffered bicarbonate solutiont 10 mlGlutathione (reduced) 2-38 gCysteine 1 -58 gSodium thioglycolate 1.00 gResazurin solution (0-25 g/l) 4 ml

Medium BGlutamine (29-2 g/l) 10 mlAmino acid concentrate A* 10 mlAmino acid concentrate B§ 10 mlAmino acid concentrate Cl 1l0 mlVitamin concentrate¶ 10 mlSolution D** 10 mlFoetal calf serum (heat inactivated) 250 mlH20 220 ml

*NaCl (40 g/l), KCI (4 g/l), CaC12 (1-4 g/l), Na2HPO4 (0-6 g/l),KH2PO4 (0-6 g/l), MgSO4.7H2O (2 g/l)tNaHCO3 (50 g/l), K2HPO4 (40 g/l) and KH2PO4 (8 g/l)*Composition per 10 ml: arginine HCI, 105 mg; histidine HCI H20,31 mg; isoleucine, 52 mg; leucine, 52 mg; lysine HCI 58 mg;methionine, 15 mg; phenylalanine, 32 mg; threonine, 48 mg;tryptophan, 10 mg; and valine, 46 mg§Composition per 10 ml: cystine, 24 mg and tyrosine, 36 mgIlComposition per 10 ml: alanine, 8-9 mg: asparagine H20, 15 mg;aspartic acid, 13 -3 mg; glutamic acid, 14-7 mg; proline, 11 5 mg;serine, 10-5 mg; and glycine, 7-5 mg¶Composition per 10 ml: choline chloride, mg; folic acid, 1 mg;1-inositol, 2 mg; nicotinamide, mg; calcium pantothanate, mg;pyridoxal HCI, I mg; riboflavin, 0- 1 mg; and thiamine HCI, I mg**Composition per 10 ml: sodium pyruvate, 250 mg; adenine, 5 mg;cocarboxylase, 3 mg; coenzyme A, 50 Mg; isobutyric acid, 10 mg;a-lipoic acid, 5 mg; p-amino benzoic acid, 5 mg; biotin, 50 Mg;nicotinic acid, 500 Mg; pyridoxine HCI, 2-5 mg; pyridoxamine HCI,5 mg; putrescine 2HCI, 10 mg; and cyanocobalamin, 50 mg

of the solution was adjusted to 8-6 with 10 mol/lNaOH. The medium was maintained under a streamof nitrogen until it was added to nitrogen-flushed,anaerobic tubes (Bellco Glass Inc.) using a pipettepreflushed with nitrogen (4-7 ml/tube). The tubeswere tightly sealed with butyl rubber stoppers andautoclaved in a press at 121PC for 15 minutes usingfast exhaust. Medium A was never prepared earlierthan one day before use. After medium A had beencooled, medium B was added to the tubes of mediumA under sterile, oxygen-free nitrogen. To make 10 mlof complete medium, 5 - 3 ml of medium B was addedto 4- 7 ml of medium A. All components of mediumB were previously sterilised by filtration. The finalpH was 7-2-7-4.

OXYGEN BUBBLING INTO MEDIUMCylinders of 5% oxygen in nitrogen (CommonwealthIndustrial Gases, Melbourne, Australia) or of airwere used to add oxygen to the medium. Whenmedium A had been prepared, and immediately

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before it was added to the nitrogen-flushed anaerobictubes, it was bubbled with the appropriate gasmixture for the predetermined time. Foi example,5010 02 x 15 minutes means 15 minutes' bubblingwith 5'7o 02 for every 10 ml of medium beingprepared, that is, 150 minutes for 100 ml finalmedium. The bubbling rate was approximately 2bubbles per second from a 21-mm Pasteur pipette(orifice diameter approximately I mm). Afterbubbling, medium A was dispensed in 4-7-mlvolumes into nitrogen-flushed anaerobic tubes andsterilised by autoclaving in a press; medium B wasthen added as before.

MEASUREMENT OF OXYGEN IN MEDIUMAn oxygen meter (Model LR-30, Titron Australia,Melbourne), fitted with a Clark-type oxygenelectrode modified to allow measurement in staticsolutions, was used. The meter gave readings in 'partper million' (jug/l) dissolved oxygen in water. Theelectrode was calibrated at 22°C with air (being thesame as air-saturated water at 22°C), 507o 02, 3bo 02,and 1% 0°2 in nitrogen and 10007o nitrogen (oxygen-free). The exact oxygen concentration in the gasmixtures was determined by the suppliers(Commonwealth Industrial Gases, Melbourne) andwas accurate to 0- 1%'o. The relationship betweenoxygen-meter readings (u.g/l) and 02 concentration inthe gas was linear (Figure); this was the standard

Ea8-/

0.

go -

c1

'-3

?

x0

0 1 2 3 4 5 6 18 19 20 21Gaseous 02 in N2 mixture (0/O 02)

Figure Relationships between oxygen meter reading andconcentration of oxygen in the gaseous 02-N2 mixture.This standard curve was used to determine the oxygenconcentrations in culture media as equivalent gaseous

oxygen concentrations.

curve from which all subsequent 02 concentrations inmedia were determined. We appreciated that thecalibration curve, based on dissolved oxygen con-centration in water, was not strictly applicable to theculture medium. Chemical determinations ofdissolved oxygen concentrations in the culture mediawere not done. However the method used enabledthe most appropriate gaseous 02-N2 mixture foroptimum retention of T. pallidum virulence in theculture medium to be determined.

Dissolved oxygen measurements on triplicate tubeswere performed at daily intervals. Fresh tubes wereused for readings at each time interval becauseinsertion of the oxygen electrode biologicallycontaminated the medium. The electrode wasinserted into the bottom of the 10-ml column ofmedium and stable readings were obtained withinone to two minutes. Exposing the medium to air forthis short time did not affect the oxygen reading, asconfirmed by flushing control tubes with nitrogenwhile taking oxygen readings.

MEASUREMENT OF REDOX POTENTIAL INTHE MEDIUMA platinum calomel combination redox electrode(Model 96-78, Orion, Cambridge, Massachusetts)was connected to an Orion model 701A 'digitalionalyser' and inserted directly into the medium forfive minutes. Measurements were made atapproximately 22°C and pH 7 3. The redox potentialwas read directly from the millivolt scale of themeter. The efficacy of the electrode was confirmedwith a standard redox system of potassiumferricyanide and potassium ferrocyanide asrecommended by the manufacturer. The readingsobtained, being based on a calomel referenceelectrode, are designated Ecal; to convert them to Ehvalues, approximately 241 mV must be added to thereading (Jacob, 1970)-that is, -276 mV (Ecal) isequivalent to - 35 mV (Eh).

DETERMINATION OF T. PALLIDUM VIRULENCESamples (0 6 ml) of culture medium were takenimmediately after inoculation with T. pallidum andat 24, and 48 hours thereafter. Each sample wasmixed with 0 6 ml of sterile deoxygenated 20%1oglycerol in saline and stored at - 70°C until allsamples were available. Samples were thawedimmediately before inoculation into the rabbit. Thebacks of male rabbits were shaved and marked into a10-cm x 10-cm grid, which was subdivided into 16equal areas. Into each site 0- 1 ml of culture wasinjected intradermally. For each time sample,quadruplicate samples were injected into each rabbit(4 x 0 h; 4 x 24 h; 4 x 48 h). Rabbits were keptshaved and cool (18°C) and examined daily for

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development of lesions as determined by thedetection of induration at the site of inoculation,which subsequently developed into a typical syphiliticlesion. Some of these lesions were abraded andshown to contain treponemes. The time the lesionstook to develop for the 24-hour and 48-hour sampleswas compared with that for the zero time samples.Testing all samples from one experiment on the samerabbit overcame, the problem of variability betweenrabbits in their susceptibility to T. pallidum.

DETERMINATION OF T. PALLIDUM MOTILITYDarkfield microscopy was used to examine 100 T.pallidum chosen at random in a drop of culturemedium on a microscope slide. Each bacterium wasscored as motile or non-motile and the percentagemotility of the culture determined. Only activelymotile T. pallidum were considered motile-that is,those with snapping, spinning, or flexing motions.For each experiment motility determinations werecarried out in triplicate from three identical tubes ofmedia.

Statistical comparisons were made by Student's ttest.

Results

The anaerobic medium (Table 1) used for the main-tenance of T. pallidum could be made partiallyaerobic by bubbling 5% oxygen in nitrogen or airinto it for various periods of time. Maximumoxygenation obtained in this series of experimentswas 5 - 8 Mg/l, equivalent to an atmosphere of 12 6%oxygen (Table 2) with a series of lower values downto 1-6 ,ug/l (equivalent to an atmosphere of 0- 7%oxygen), the anaerobic medium.Redox potentials of the anaerobic medium were

not affected by bubbling the medium with 5%0oxygen in nitrogen (data not shown), but bubblingwith air did cause the redox potential to becomemore positive (Table 3). Bubbling the medium withair for 60 minutes produced the highest redox

S. Graves and T. Billington

Table 2 Measurement of dissolved oxygen concentrationin medium* following bubbling with 50So oxygen innitrogen or air for specified periods

Dissolved oxygen concentrationin ,g/l (%02)

Method ofoxygen addition Day 0 Day I Day 2tNo oxygenation 16 (0 7) 1*6 (0-7) 1*6 (0 7)507° 02 in N2 x 5 min 1 8 (1-3) 1 8 (1 3) 1-7 (1 0)5%o 02 in N2 x 15 min 2-4 (3 0) 2-2 (2 4) 2-1 (2 1)Air x 30 min 2-95 (4 7) 2 6 (3-6) 2-4 (3 0)Air x 40 min 3 g(7 3) 3 6 (6 5) 3.4 (5*9)Air x 50 min 5-0 (10-4) 3 6 (6 5) 2 0(1 9)Air x 60 min 5-8 (12 6) 4-2 (8-2) 2 1 (2 1)* Mean of five independent samples. Measurements madeimmediately after inoculation of medium with T. pallidum (day 0)and on days I and 2tThe fall in oxygen concentration over the two-day period isprestimably due primarily to removal of the added free oxygen byreducing agents in the medium, as very little would be due totreponemal metabolism

Table 3 Oxidation reduction (redox) potential (m V) ofmedium after bubbling with air for specified periods

Redox potential (m V)*Method ofoxygen addition Day 0 Day I Day 2

No oxygenation - 276 - 272 - 254Air x 30 min - 271 - 271 - 265Air x 40 min - 263 - 263 - 261Air x 50 min - 259 - 258 - 227Air x 60 min - 243 - 240 - 229* Mean of four independent samples. Measurements madeimmediately after inoculation of medium with T. pallidum (day 0)and on days I and 2

potentials, but even so the medium was still 'reduced'relative to a calomel reference electrode.The oxygenation of the medium did not have a

marked effect on the reduction in percentage motilityof the T. pallidum culture (Table 4); by day 2 themore oxygenated cultures were slightly less motilethan the anaerobic controls.Using the intradermal inoculation test on the

shaved back of the rabbit, a trend was observed whenthe cultures were tested for the presence of virulentT. pallidum. In anaerobic medium the latent periodof infection was shortened by only 0 2 day over thefirst 24 hours. The only oxygenated media to

Table 4 Determination of motility of Treponema pallidum cultures (%) maintained in medium bubbled with 5%1ooxygen in nitrogen or air for specified periods

Time of motility estimation*%o decrease % decrease

Method of oxygen addition Day 0 Day I Day 2 (day 0 to day 1) (day 0 to day 2)

No oxygenation 100 94 80 6 205070 02 x 5 min 100 96 91 4 95'10 02 x 15 min 100 91 77 9 23Air x 30 min 100 88 67 12 33Air x 40 min 100 91 73 9 27Air x 60 min 100 85 62 15 38

*Mean of three samples from each of three identical tubes of media

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alter significantly the difference in the latent periodwas after being bubbled with 5%7 0°2 for 15 minutes(Table 5). After bubbling with 5% 0°2 for 15 minutes(that is, 2v4 jg/l dissolved 02 concentrationequivalent to a 3% oxygen atmosphere; Table 2), thedifference in latent period was 1-4 days (Table 5).

Table 5 Latent period of syphilitic lesions afterinoculation of T. pallidum into the shaved backs ofrabbits*

DifferenceLatent period of lesions between day 0(days)t and day I

Method of latento.Kivgen addition Day 0 Day I periods* (days)

No oxygenation 10'7 10 5 -025% O2inN2 x 5min 9-6 9.1 -0'55% O2inN2 x 15min 10 4 9 0 -1-4§Air x 30 min 9.1 7 9 -1 2Air x 40 min 9'1 8-75 -0 35Air x 50 min 12'8 119 -0'9Air x 60 min 12'8 12 4 -0'4

*Samples for rabbit inoculation were taken from medium eitherimmediately after additon of T. pallidum (day 0) or 24 hours later(day 1) after incubation at 34°CtLatent period defined as the time between rabbit inoculation andappearance of syphilitic lesion as indicated by induration in the skin.The differences in latent periods of samples taken on day 0 is due todifferences in starting concentration of T. pallidum from differentharvests and to slight differences in susceptibility of rabbits used indifferent experiments. In each case the data presented are the meanof 16 lesions from four rabbits. Degree of oxygenation of mediumwould have no effect on latent period of day 0 samples* - means that day I samples had shorter latent periods than day 0

samples§When compared by Student's t test, the difference was significantP<O' 05

When the latent periods at day 0 and I were

compared by Student's t test, the value of 1 4 dayswas significant at P<0 05. In five repeatedexperiments using 5% 02 in N2 for 15 minutes, thedifference in latent period from day 0 to day I wasalways greater than for anaerobic medium,indicating better survival in the slightly oxygenatedmedium compared with the anaerobic medium.Over a 48-hour incubation period in vitro, the

latent period of infection always lengthened (Table6), indicating a loss of. virulent T. pallidum duringthis time. However the lengthening of the latentperiod with 5% 02 in N2 for 15 minutes was only 4- 8days compared with 6-6 days in anaerobic medium.Differences in latent period for media with otherlevels of oxygenation were not significantly differentto the anaerobic control.We conclude that bubbling the anaerobic medium

with 5% oxygen in nitrogen for 15 minutes (per 10-mlmedium volume), resulting in a dissolved oxygenconcentration of 2-4 .g/l (equivalent to a 3%1o 02 inN2 atmosphere), significantly enhanced the survivalof virulent T. pallidum over a 48-hour periodcompared with anaerobic controls. Media with

Table 6 Latent period of syphilitic lesions afterinoculation of T. pallidum into the shaved backs ofrabbits

Time at which samplestaken for rabbitinoculation *t

Method ofoxygen addition Day 0 Day 2

Differencebetween day 0and day 2latentperiods* (days) e

No oxygenation 10'1 16 7 +6-65% 02inN2 x 5min 9-6 15-9 +6-35/oO02inN2x 15minlO-4 15-2 +4-8Air x 30 min 9'0 16-2 +7-2Air x 40 min 9'0 16-3 +7-3Air x 60min 14-5 21-3 +6-8

*Samples for rabbit inoculation were taken from medium either'immediately after additon of T. pallidum (day 0) or 48 hours later(day 2) after incubation at 34°CtSee Table 5* + means that day 2 samples had longer latent periods than day 0samples. In each experiment the day 0 and day 2 samples wereinoculated into the same rabbit so as to overcome any variabilitybetween rabbits

higher concentrations of dissolved oxygen (up to5-8 jg/l, equivalent to a 12-6% 02 in N2atmosphere) behaved no 'differently from anaerobiccontrols regarding retention of T. pallidum virulenceduring this time.

Discussion

From many earlier observations (Willcox and Guthe,1966) we expected that a dissolved oxygen con-centration would be detected above which oxygenwas toxic to T. pallidum. This is because T. pallidumhas always retained its virulence best in vitro underanaerobic conditions, implying that an atmosphericconcentration of oxygen (21 %) was toxic to thebacterium. In this study, although we found anatmospheric equivalent of 3% 02 to be the optimumconcentration for the survival of virulent T. pallidum(Table 5), higher concentrations (up to 12 * 6%; Table2) were no more detrimental to survival thananaerobic conditions. This suggests that T. pallidumdoes have inherent mechanisms for dealing with thetoxic end-products of oxygen reduction (Fridovich,1975, 1978; Haugaard, 1968), a necessaryrequirement for aerobic metabolism.Oxygen at atmospheric concentrations (21 %) may,

however, overwhelm these mechanisms. Since T.pallidum is an obligate human parasite it may havepartially lost these mechanisms as a result ofdegenerate evolution associated with specialisationfor a parasitic existence. The guanine plus cytosine(G + C) content of T. pallidum indicates that it maybe closely related to Spirochaeta zuelzerae andSpirochaeta litoralis (Miao and Fieldsteel, 1978),free-living anaerobic spirochetes. Spirochaetaaurantia, which is a facultative anaerobe, may be aclose (but non-parasitic) relative of T. pallidum.

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Under aerobic conditions it incompletely oxidisescarbohydrates mainly to acetate and carbon .dioxide.It does not possess a tricarboxylic acid (TCA) (Krebs)cycle but has developed mechanisms of oxidativephosphorylation and a rudimentary electrontransport chain involving one or two cytochromes(Canale-Parola, 1977).By comparison, T. pallidum degrades glucose and

pyruvate to CO2 and acetate (Nichols and Baseman,1975) under both aerobic and anaerobic.conditions,with better conversion under aerobic conditions(Baseman et al., 1976). It has a functional electrontransport system probably driven by the oxidation ofreduced nicotinamide-adenine dinucleotide (NADH)and reduced nicotinamide-adenine dinucleotide-phosphate (NADPH) (Lysko and Cox, 1977) andcoupled to oxidative phosphorylation with theproduction of adenosine triphosphate (ATP) (Lyskoand Cox, 1978). The production of ATP in cell-freeextracts was stimulated by air. The presence of-catalase was also inferred by the release of 029presumably from H202 (Lysko and Cox, 1978). LikeS. aurantia, T. pallidum lacks a TCA cycle, therebyprobably explaining its slow growth rate (Schiller andCox, 1977). The most likely origin of T. pallidumtherefore is from an ancestoral spirochete, somethinglike Spirochaeta aurantia, capable of growing in andutilising oxygen to a limited extent. Consequently, itis not surprising that T. pallidum should have arequirement for an optimum oxygen concentration.

Norris et al. (1978), comparing the effect ofanaerobic, aerobic, and 3% oxygen conditions in thepresence of reducing agents on the survival ofvirulent T. pallidum, found 3% oxygen to besuperior.We have come to the same conclusion after

comparing anaerobic conditions and a range ofdissolved oxygen concentrations in reduced culturemedium. It is probably no coincidence that ourdetermined oxygen optimum of 3% is within therange of normal rabbit tissue (2 6-4% oxygen;Campbell, 1931)-i ol5 in the testis (Cross andSilver, 1962)-and human subcutaneous tissue(2 0-3 -6% oxygen; Seevers, 1936).

Using cell-free extracts, glucose and pyruvate weredegraded under aerobic conditions (Nichols andBaseman, 1975). Baseman et al. (1976) foundoptimum substrate degradation and amino acidutilisation with 10-20% °2' The synthesis of highmolecular weight proteins and incorporation ofuridine into RNA was detected under normalatmospheric conditions (Baseman and Hayes, 1977;Nichols and Baseman, 1978), incorporation beingpoorer with lower oxygen concentrations. Howeverthere is no evidence that atmospheric oxygenconcentration is compatible with the maintenance of

T. pallidum viability as measured by the retention ofvirulence.Under anaerobic, reduced conditions amino acids

have been incorporated into protein (Baseman andHayes, 1974) and serine and uracil taken up by T.pallidum (Sandok and Jenkin, 1978).

It appears likely that when T. pallidum issuccessfully grown in vitro, it will grow in quite awide range of oxygen concentrations, but from ourpresent state of knowledge studies on the in-vitrogrowth of this bacterium should be conducted undermicroaerophilic conditions.

It is interesting that a human malaria protozoon(Plasmodium falciparum), another human parasitewhich defied cultivation in vitro for many years, hasrecently been grown in a culture system with 1% or5% oxygen (Trager and Jensen, 1976).

We wish to thank Mr Ian McLean for his excellenttechnical assistance and Dr J. Watterson of MonashUniversity, Department of Mathematics, forvaluable help with the statistical analysis.

This work was supported by grants from theNational Health and Medical Research Council, theUtah Foundation, the Danks Trust, the estate of the.late George Adams, the Ian Potter Foundation, andMonash University, from whom funds are gratefullyacknowledged.

References

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Baseman, J. and Hayes, N. (1977). Anabolic potential of virulentTreponema pallidum. Infection and Immunity, 18, 857-859.

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