Part II - Growth of Ureaplasma Urealyticum

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Morphology of Ureaplasma urealyticum (T-Mycoplasma) Organisms and Colonies

SHMUEL RAZIN,' GERALD K. MASOVER,2* MARINA PALANT, AND LEONARD HAYFLICK Department of Medical Microbiology, Stanford University School of Medicine, Stanford, California 94305

Received for publication 31 August 1976

The morphology of Ureaplasm urealyticum in broth cultures was studied by phase-contrast microscopy. Most organisms appeared singly or in pairs. Long filaments and long chains of cocci, common in classical mycoplasma cultures, were not observed. On solid medium, U. urealyticum produced "fried-egg" colonies which developed according to the scheme suggested by Razin and Oliver (J. Gen. Microbiol., 1961) for the morphogenesis of the classical mycoplasma colonies. The formation of the peripheral zone of the colonies followed that of the central zone only when growth conditions were adequate. Hence, the appear- ance of peripheral zones, and consequently the larger colony size, can be taken as an indicator of improved growth conditions. Incubation in an atmosphere of 100% CO2 resulted in significantly larger colonies than in an atmosphere of N2, 02, or air. CO2 acts as a buffer, keeping the pH at the optimal range for Ureaplasma growth (pH 6.0 to 6.5) in the presence of the ammonia produced from the urea hydrolyzed by the organisms. The addition to the medium of 0.01 M urea together with 0.01 M putrescine enabled better growth than with urea alone. Small amounts of phosphate improved growth in an atmosphere of CO2, apparently fulfilling a nutritional role. Under nitrogen, higher phosphate con- centrations were required for good growth, apparently serving as a buffer as well as a nutrient. Sodium chloride and sucrose which had been added to increase the tonicity of the medium inhibited growth above 0.1 M. An increase in the agar concentration above 2% resulted in decreased colony size. Likewise, prolonged drying of the agar plates caused a marked decrease in colony size, mostly affecting the peripheral zone. The addition of both urea and putrescine to the growth medium and incubation in a humidified CO2 atmosphere are recom- mended for improvTed growth and formation of fried-egg colonies of U. urealyti- cum on agar. It nmust be emphasized that these experiments were carried out with a laboratory-adapted strain.

The T-mycoplasmas differ from the classical mycoplasma in at least two basic properties: their ability to hydrolyze urea (5, 20, 29, 33) and their failure to grow in the conventional myco- plasma media to titers higher than 107 colony- forming units (CFU) per ml (35). Associated with their poor growth in liquid media is their tiny colony size on solid media, giving rise to the trivial name T-mycoplasmas ("T" from tiny colonies; 28). The T-mycoplasmas were recently provided with the status of a separate genus, Ureaplasma, within the family Mycoplasmata- ceae (35). Yet, our knowledge of their biology is still meager, and their phylogenetic relation- ship to the other organisms included in the

' Permanent address: Biomembrane Research Labora- tory, Department of Clinical Microbiology, the Hebrew University-Hadassah Medical School, Jerusalem, Israel.

2 Present address: Department of Surgery, Division of Urology, Stanford University school of Medicine, Stanford, CA 94305.

Mycoplasmataceae, the so-called classical my- coplasmas, is not clearly understood. One aim of the present investigation was to study the morphology of ureaplasmas by phase-contrast microscopy, under conditions minimizing arti- fact formation, and to compare it with the mor- phology of the classical mycoplasmas; another aim was to elucidate the factors influencing Ureaplasma colony shape and size and to de- termine whether the morphogenesis of Urea- plasma colonies follows the pattern proposed for classical mycoplasma colonies (24). The re- sults presented in this communication show that the ureaplasmas resemble the classical mycoplasmas in gross morphology of the orga- nisms and in morphogenesis of their colonies.

MATERIALS AND METHODS Organisms and growth medium. Ureaplasma

urealyticum (strain 960) was originally supplied by M. C. Shepard (Camp Lejeune, N.C.). Sterile basal

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broth prepared from 50 g of beef heart infusion and 10 g of peptone (Difco Laboratories, Detroit, Mich.) per liter was supplemented with 5% (vol/vol) un- heated horse serum (Microbiological Associates, Inc., Bethesda, Md.), 10% (vol/vol) fresh yeast ex- tract (10), 1% (vol/vol) phosphate-buffered saline (lOx concentrate; Grand Island Biological Co., Grand Island, N.Y.), 1,000 U of penicillin G per ml, 0.002% phenol red, 0.01 M urea, and 0.01 M putres- cine hydrochloride (Sigma Chemical Co., St. Louis, Mo.). The pH was adjusted with 1 N HCI to a value between 6.0 and 6.5, and the medium was filtered through a membrane filter (0.22-gm average pore diameter; Millipore Corp., Bedford, Mass.). Agar medium was prepared by the addition of 1.4% (wt/ vol) agar (Difco) to the basal broth. Ten-milliliter volumes of the agar medium were dispensed into polystyrene petri plates (60-mm diameter; Falcon Plastics, Oxnard, Calif.). For some experiments pol- ystyrene petri plates (100-mm diameter) divided into four compartments were used; in this case, 8-ml volumes of agar were added to each quadrant of the plate. Growth conditions. The agar plates were dried

for 10 min with their lids ajar in a laminar-flow hood. A 24-h culture of U. urealyticum (containing about 106 CFU/ml) was centrifuged at 18,000 x g for 15 min at room temperature, and the sedimented organisms were resuspended in an equal volume of basal broth. The cell suspension was then diluted 1:100, 1:200; 1:400, and 1:800 in basal broth. By use of a calibrated pipette, 0.01-ml drops from the various dilutions were carefully placed on the surface of the dried agar plates. Immediately after the drops had dried, the lids were replaced and the plates were transferred to anaerobic jars (Anaerobic Systems, BBL, Cockeysville, Md.) containing a moist gauze pad. The atmosphere in the jar was then changed to C02, N2, or 02 by flushing the jars for several min- utes with the desired gas taken directly from gas tanks. The jars were then sealed with silicone grease and incubated at 37°C for 5 days.

Determination of colony number and size. The number of colonies was determined after 5 days of incubation at 37°C by using a Leitz inverted micro- scope (x 3.5 objective and x15 eyepiece). For best results, drops producing between 30 and 100 colonies were selected, and the diameters of all of the colo- nies in the drop area were measured with a cali- brated Filer micrometer eyepiece. Colonies were photographed with type PB Polaroid camera (binoc- ular model Mic-1625) using Polaroid black and white film type 107.

Phase-contrast microscopy. Small drops of broth cultures were put on glass slides, covered with cover slips, and examined with a Zeiss Photomicroscope II, using a x 100 neofluor-phase objective, a x 1.6 Opto- var, and a x 12.5 eyepiece, giving a total magnifica- tion of x 2,300. The organisms were photographed as soon as they settled on the slide, using Kodak Tri-X pan (ASA400) film.

RESULTS Morphology of organism. Figure 1 shows

photomicrographs of U. urealyticum growing in

MORPHOLOGY OF UREAPLASMAS 465

filtered broth. To minimize artifact formation, the culture was not centrifuged, fixed, or treated in any other way before its examination in the phase-contrast microscope. Filtration of the medium before its inoculation with U. urealyticum was essential in order to remove particulate matter which could resemble Urea- plasma cells. Figure 1 shows the morphology of the organisms in broth and the various forms of cell associations which closely resemble those seen in Mycoplasma and Acholeplasma cul- tures (2, 23). The only difference is that far fewer organisms could be seen in the micro- scopic field, a finding which agrees with the much lower number of viable organisms in U. urealyticum broth cultures (106 to 107 CFU/ml as compared to more than 109 CFU/ml in the classical mycoplasma cultures). In addition, we did not observe long filamentous forms or long chains of cocci, which are characteristic of loga- rithmically- growing cultures of many classical mycoplasmas (23).

Factors influencing colony morphology and size. (i) Urea, putrescine, and the gas phase. Previous results from this laboratory (19) showed that putrescine can replace urea in liq- uid Ureaplasma media, following the adapta- tion of the organisms to this amine. The effects of putrescine and urea were, therefore, assessed in our experimental system, in which Urea- plasma growth was estimated according to col- ony number and size. Previous reports (5, 30) indicated a beneficial effect of CO2 on Urea- plasma growth; consequently, we combined the examination of the effects of the gaseous envi- ronment on colony size with those of urea and putrescine. Figure 2 shows that incubation in an atmosphere of 100% CO2 resulted in signifi- cantly larger colonies compared to incubation in an atmosphere of N2, 02, or air. The figure also shows that supplementation of the medium with a mixture of urea and putrescine improved growth under C02, though a reverse effect was seen under 02 (Fig. 2). The pH of the agar plates incubated under CO2 remained acid, whereas that of the plates incubated under N2, 02, or air became alkaline after the develop- ment of colonies. The removal of the plates from the CO2 jar resulted in an alkaline shift of the pH of the medium within 10 to 30 min, depending on the number and size of the colo- nies on the plate. Since horse serum is expected to contain both urea and putrescine, the effects of urea and putrescine were also tested in a medium containing dialyzed calf serum, which is essentially free of these compounds (17). Ta- ble 1 shows that the medium containing di- alyzed calf serum without urea or putrescine failed to support the growth of U. urealyticum.

A B

C D

*..

E F

G H

FIG. 1. Phase-contrast micrographs of U. urealyticum organisms growing in broth. (A) A microscopic field ofa 20-h culture. The scarcity oforganisms in the field is noticeable. xl ,075. (B through H) Various cell associations including: a diplococcus form (B); a short chain of cocci (C); a branching chain of cocci (D); a short and branching filamentous form (E); a group of organisms (F); and "budding" forms consisting of a "normal-looking" cell associated with a swollen cell or ghost (G and H). x2,560.

466

MORPHOLOGY OF UREAPLASMAS 467,uu =L M UREA + PUTRESCINE

1 0 WITHOUT UREA OR W 125 - PUTRESCINE 2 0 -J so100 L

0 U- 0 cc 75- w

50 w

0 CO2 N2 02 AIR

GAS PHASE FIG. 2. Effect of the gas phase and added urea

plus putrescine on colony size. Organisms were grown on the solid medium containing 5% horse serum as described in Materials and Methods.

TABLE 1. Effects of urea and putrescine on the growth of U. urealyticum on agar containing

dialyzed calf serum or horse seruma

No. of colonies (mean di- ameter [,m])

Urea (0.01 Putrescine M) (0.01 M) 5% Di- 5% Horse

alyzed calf Horseserumserum

0 0 0 72 0 (105)

+ - 9 55 (124 ,um) (140)

0 + 0 90 (110)

+ + 73 87 (125 ,m) (235)

a The atmosphere was 100% CO2. The strain was not adapted to grow with putrescine alone.

The addition of 0.01 M urea alone supported very poor growth, as evidenced by the small number of colonies. Putrescine by itself did not support growth, but the combination of urea and putrescine supported good growth, as indi- cated by the larger number of colonies that developed on the agar. The beneficial effect of putrescine could also be seen in the medium containing the nondialyzed horse serum, where the addition of putrescine together with urea improved growth considerably, as evidenced by the much larger colony size (Table 1). Subse- quently, a mixture of urea and putrescine was routinely added to all our growth media.

(ii) Serum concentration. In the experi-

ments shown in Table 1 and Fig. 2, the concen- tration of serum in the medium was 5% (vol/ vol). This concentration was found to be ade- quate, since an increase of the serum content above 5% did not increase the number or size of the colonies. In fact, when the growth medium was supplemented with urea + putrescine and incubated under 100% CO2, the serum content could be reduced to 2.5% without affecting growth. However, no growth occurred when the serum was totally excluded from the medium. In view of these results, 5% (vol/vol) horse se- rum was included in all experiments. It has to be emphasized that all our experiments were carried out with a laboratory-adapted strain, so that 5% (vol/vol) of horse serum may not be optimal for the growth of fresh U. urealyticum isolates.

(iii) pH and phosphate concentration. Fig- ure 3 shows that the maximum colony size was obtained at pH 6.15, though the size of colonies at pH 5.75 or 6.70 was not much smaller. Phos- phate was added to the growth medium to serve as a pH buffer. While our work was in progress, we became aware of a report by Romano et al. (25) showing that the incorporation of phos- phate buffer into the growth medium of U. urealyticum considerably increased colony size. Therefore, we were interested in attempting to differentiate the effect of phosphate as a buffer from its effect as a nutrient. Figure 4 shows that

o 60- 0 0 LL 0 cc 40

LU 20-

pH OF AGAR

FIG. 3. Effect ofpH on colony size. The medium, as described in Materials and Methods, was buffered with 0.05 M K2HPO4 and adjusted with NaOH or HCl to the different pH values. Incubation was under N2 instead of C02 to prevent the acidification of the medium by the CO2. The colonies were measured after only 3 days of incubation.

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468 RAZIN ET AL.

the effect of phosphate depended on the gas phase: under nitrogen, its addition, at least up to 100 mM, improved growth, only at low con- centrations, whereas higher concentrations in- hibited growth.

(iv) Tonicity. The tonicity of the growth me- dium may considerably influence the growth of mycoplasmas and spiroplasmas (12, 27). Table 2 shows that the addition of NaCl or sucrose to the growth medium did not enhance the growth of U. urealyticum and, at concentrations of 0.10 M and higher, the added solutes inhibited growth. Also, from this table it can be seen that NaCl or sucrose did not act as substitutes for urea and putrescine, as evidenced by the poorer growth in the medium not supplemented with urea and putrescine. Agar concentration and moisture. As had

been found for other mycoplasmas (24), the agar concentration enabling the best growth of

CO2 ATMOSPHERE

e~ I I\

0~ ~ ~ 0

U-

200

100 N2 ATMOSPHERE wcc\ w

25 50 75 CONCENTRATION OF K2HPO4 (mM)

FIG. 4. Effect of the phosphate buffer concentra- tion of the growth medium on colony size. The solid medium used was that described in Materials and Methods.

J. BACTERIOL.

U. urealyticum was about 1.4% (wt/vol). An increase in the agar concentration above 2% resulted in decreased colony size, and with 3% agar the inhibition of growth was almost com- plete. Prolonged drying of the agar plates in the laminar-flow hood also affected their ability to support growth. Thus, lengthening the drying time for more than an hour caused a marked decrease in colony size, mostly affecting the peripheral zone, and plates dried for over 2 h completely lost their ability to support growth of U. urealyticum.

(v) Other factors influencing colony shape and size. U. urealyticum colonies grown under N2, 02, or air, but not under CO2, on agar containing urea produced small, dark granules found mostly at the periphery of the colony. The granules appeared only after the growth medium turned alkaline, so that colonies on plates kept under 100% CO2 were devoid of granules. However, the granules were pro- duced when the plates were taken out of the CO2 jar and kept in air so that their pH became alkaline (Fig. 5). Another interesting observa- tion, for which we have no explanation as yet, was a marked increase in the size of the periph- eral zone of colonies developing in areas where an unidentified precipitated material covered the agar surface (Fig. 6).

DISCUSSION Phase-contrast microscopy of unfixed urea-

plasmas in liquid culture confirms their mor- phological similarity to classical mycoplasmas, which were observed by electron microscopy (1, 26). A rough estimate of the diameter of the unswollen coccoid Ureaplasma cells, based on measurements of their image on the phase- contrast micrographs, gives values ranging be- tween 0.3 to 0.8 Am, resembling those found for the classical mycoplasmas (23). However, long filaments and long chains of cocci, common in fast-growing classical mycoplasma cultures, were not found in the Ureaplasma cultures. This may be taken in support of the thesis (21,

TABLE 2. Effects of changes in tonicity of the medium on U. urealyticum growth

No. of coloniesa Concn (M) of sol- NaCi Sucrose ute added to the NaCl Sucrose

medium Urea + putrescine Without addition of Urea + putrescine Without addition of added urea + putrescine added urea + putrescine

0 32 (165 um) 14 (85 ,um) 43 (151 Am) 13 (70 ,um) 0.05 35 (126 ,Mm) 41 (70 Mm) 41 (122 Mm) 3 (77 Mm) 0.10 32 (75 Mm) 38 (78 Mm) 17 (101 Mm) NG 0.17 NG 25 (72 Mm) NG NG 0.25 NG NG NG NG

a The number in parentheses is the average diameter. NG, No growth.

MORPHOLOGY OF UREAPLASMAS 469

B 4 a, ** 6.w.

6I'. .

54. .,.,

"--'9'.

FIG. 5. U. urealyticum colonies. (A) Five-day colonies from a medium supplemented with urea plus putrescine and incubated under 100% CO2. The central and peripheral zones of the colonies are discernible. x240. (B) A colony from the same medium showing convoluted borders and dark granules appearing after alkalinization of the growth medium. x240. (C) Similar colonies but with heavier granules. xlOO.

23) that filaments appear only under conditions enabling rapid growth, when cytoplasmic divi- sion lags behind genome replication. A similar phenomenon has also been recorded for wall- covered bacteria. Arthrobacter sp. (14) and other bacteria (11) growing in continuous cul- ture produced long rods when their growth rate was at a maximum and coccoid bodies when their growth rate was lower. Our finding that most of the cells in Ureaplasma cultures appear singly or in pairs supports the suggestion of Fumess (9), based on sonic oscillation and ultra- violet inactivation curves, that the CFU of Ureaplasma cultures mostly consists of single organisms. Our study indicates that the morphogenesis

of Ureaplasma colonies follows the same pat- tern as that suggested for the classical myco- plasma colonies (24). This pattern consists of an initial spherical growth inside the agar (the cen- tral zone), followed by spreading of the orga- nisms into the thin free-water film on the agar, forming the peripheral zone. Factors that re- tard growth were shown to inhibit the forma- tion of the peripheral zone. Razin and Oliver suggested in 1961 (24) that the tiny Urea- plasma colonies are only composed of the cen- tral zone embedded in the agar, as a result of the inadequacy of the then available media to support good growth of ureaplasma. Ford's fail- ure in 1962 (3) to transfer ureaplasmas on solid media by the conventional inverted agar block

VOL. 130, 1977

v ...

.!P-t.'WA 1, . V.Pt

-t16

470 RAZIN ET AL.

FIG. 6. U. urealyticum colonies developing in areas where unidentified material has precipitated on the agar surface. The large dimensions and fried- egg shape of these colonies can be contrasted with the minute and irregular shape of the colonies growing on the adjacent uncovered agar surface. x50.

technique can also be taken to indicate the absence of peripheral zones in the colonies. Subsequent improvements in the growth media for ureaplasmas, including the addition of urea (6, 33), strengthening the buffering capacity of the medium by incubation under 20% CO2 [4, 29; by inclusion of HEPES (N-2-hydroxyethyl- piperazine-N'-2-ethanesulfonic acid) buffer (16) or by phosphate buffer], and simply increasing the volume of the agar in the plates (8, 13), resulted in larger colonies and the appearance of peripheral zones. Our finding that an atmosphere of CO2 most

markedly improves the growth of U. urealyti- cum can be explained by the excellent buffering capacity of CO2 in the acidic pH range, between 6.0 and 6.5, which had been found by Shepard and Lunceford (31) to be optimal for Urea- plasma growth. This optimal pH can be kept with C02, even in the presence of excessive amounts of ammonia derived from urea hydrol- ysis (19). Although isolated U. urealyticum colonies

developing under C02 very frequently exceeded 200 ,um in diameter, the mass of organisms included in them was apparently much smaller than that in classical mycoplasma colonies of the same diameter. This was evidenced by the thinness of the Ureaplasma colonies, a factor which made their observation by the naked eye very difficult. Our data (19) showing that, un- der CO2, the maximum titer of viable ureaplas- mas (CFU per milliliter) in liquid culture is not much higher than that under N2, though the stationary phase under CO2 is longer, appear to be in accord with the smaller mass of cells in Ureaplasma colonies. Our results confirm those of Shepard and

Lunceford (33) and Ford and MacDonald (5) by showing that the dialysis of the serum compo- nent of the growth medium abolished its growth-promoting activity. Working with di- alyzed horse serum, the above-mentioned au- thors were able to restore its growth-promoting activity by the addition of urea. Our results with dialyzed calf serum indicate that the addi- tion of urea alone is not as effective as the addition of a mixture of urea and putrescine. Although in the experiment shown in Table 1 putrescine by itself could not support growth in the dialyzed calf serum medium, we were able to adapt the U. urealyticum strain to grow with putrescine alone, without the addition of urea, confirming the previous results of Masover and Hayflick (18). The fact that our experiment was carried out in an atmosphere of CO2, combined with observations of Ford et al. (4) and Masover et al. (19), rules out the possibility that the growth-promoting effect of urea is derived from the CO2 produced during urea hydrolysis. Our experiments appear to differentiate be-

tween the effects of phosphate as a buffer and as a nutrient in Ureaplasma growth. In the presence of a CO2 atmosphere, where the buffering effect of the phosphate was not neces- sary, low concentrations of phosphate improved growth, possibly indicating a nutritional effect (Fig. 4). Under a nitrogen atmosphere, the marked improvement in growth by increasing concentrations of phosphate may be due to the increased buffering capacity of the medium in addition to the nutritive value of the phos- phate. It is not clear why high concentrations of phosphate inhibited growth under CO2 but not under N2. Growth inhibition by high phosphate concentrations has been recorded for classical mycoplasmas (22) as well as for ureaplasmas (33). An increase in the tonicity of the growth

medium by adding NaCl or sucrose not only failed to improve the growth the U. urealyti- cum but inhibited growth above certain concen- trations of these solutes. This may be correlated with the findings of Makki (15) that the addi- tion of 5% (wt/vol) of glucose, fructose, fucose, or maltose to the growth medium completely inhibited Ureaplasma growth. The nature of the dark granules appearing on

the periphery of the colonies, when the medium becomes alkaline due to the ammonia released from urea, is uncertain. The granules may rep- resent amorphous ammonium magnesium phosphate, a compound shown to be produced during Ureaplasma growth (7, 26, 36), or insol- uble metallic oxides of Mg2+ or Mn2+ produced by the ammonium hydroxide accumulated in the growth medium (31, 34). As a result of the present study, we would

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VOL. 130, 1977

recommend the addition of both urea and pu- trescine to the growth medium and the incuba- tion of the cultures in an atmosphere of CO2. An atmosphere rich in CO2 can be produced either by flushing the jar containing the plates with CO2 from a tank or by placing a small piece of dry ice in the jar. Incubation under CO0 will considerably increase the size of most colo- nies and could, thus, facilitate the isolation and identification of U. urealyticum from clinical material.

ACKNOWLEDGMENT

This work was supported by Public Health Service grant A111805 from the National Institute of Allergy and Infec- tious Diseases.

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MORPHOLOGY OF UREAPLASMAS 471

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