T.Mycoplasmas: Some Factors Affecting Their Growth, Colonial Morphology, and Assay on Agar

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1 THE JOURNAL OF INFECTIOUS DISEASES VOL. 128, No.6' DECEMBER by the University of Chicago. All rights reserved. T.Mycoplasmas: Some Factors Affecting Their Growth, Colonial Morphology, and Assay on Agar Geoffrey Furness From the Department of Microbiology, College of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey T-mycoplasma strains no. 960 and T-McA grew optimally on T-agar, ph , containing 10% horse serum when incubated in an atmosphere containing ~ 5% CO 2, Growth was retarded by aerobic incubation but not by anaerobic or microaerobic incubation. Both size and number of colonies were affected by the volume of T-agar in the petri dish. Colony size only was below normal on T-agar, ph , containing ammonium salts, 5% and 20% horse serum, and especially at an alkaline ph, but it was unaffected by addition of hepes buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) or an extra 6 mg of urea per 100 ml. Colonies of strain no. 960, T-McA, three other laboratory and five wild strains could be changed from rough colonies averaging 43 urn in diameter to "friedegg" or smooth colonies 100 ~m in diameter. These variations were dependent on the T-strain, type and concentration of agar and of MgS0 4, and substances in Ionagar. These findings suggest further that T-mycoplasmas have different requirements for growth, and strain-to-strain differences are evident. Although most T-mycoplasmas undoubtedly are saprophytes of the human urogenital tract, there is increasing evidence that some species may be responsible for nonspecific urethritis [l] and also for female reproductive failures [2]. In patients with bacteriuria, not only the bacterial species but also the level of infection is considered significant in identification of the etiologic agent; and it is likely that the same criteria apply to infections with T-mycoplasmas. Therefore, techniques for accurately assaying the number of T-mycoplasmas in clinical specimens could be important. This paper reports some of the conditions that can affect the assay of these organisms and change their characteristic rough colonial morphology, Received for publication June 9, 1971, and in revised form June 21, This research was supported by grant no. ROI AI08282 from the National Institute of Allergy and Infectious Diseases of the U.S. Public Health Service. The author wishes to thank Dr. Denys K. Ford for his strains of T-mycoplasmas and Mrs. Maria DeMaggio Cioffi and Miss Marsha Trocola for their excellent technical assistance. Please address requests for reprints to Dr. Geoffrey Furness, Department of Microbiology, College of Medicine and Dentistry of New Jersey-New Jersey Medical School, 100 Bergen Street, Newark, New Jersey thus leading to difficulties in the recogmtion of colonies of T-mycoplasmas on primary isolation. Materials and Methods The techniques used and the source of the T mycoplasma strains have been described [3]. T mycoplasmas, strains no. 960, T-McA [3], no. 27, no. 58, and Pirillo [4], were laboratory strains while strains no. 207, 210, 213, 220, and 221 were wild strains isolated from patients with nonspecific urethritis [3]. They grew into typical rough colonies on 9-cm petri dishes containing 20 ml of T-agar. The T-agar, ph , was prepared by incorporation of Oxoid no. 2 Ionagar in 3% trypticase soy broth [3] (Baltimore Biological Laboratories, Baltimore, Md., BBL) to give a final concentration of 0.75% agar after addition of 10% unheated horse serum containing 20 mg/100 ml of urea (BBL), 10% yeast extract [5], Eagle's minimal essential medium, vitamins, and 1,000 units of penicillin per ml. All plates were incubated at 37 C in a humidified incubator in an atmosphere of 5%-10% CO 2 in air. Urea and hepes buffer (N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid) were prepared at 25 % and 23.64% aqueous solutions and sterilized by autoclaving for 10 min at 10 psi. The medium was 703

2 704 Furness adequately buffered, and the ph did not change significantly at the concentrations of urea used in these experiments [3]. The diameter of the colonies was measured with a micrometer scale in the microscope eye piece and at a magnification of 125 X. Only well separated colonies were measured since their diameter varies with their proximity to each other [6]. Test for agar gel strength. Colab Laboratories, Glenwood, Ill., kindly supplied the following method for comparing the gel strength of lonagar with that of other agars. A 1% solution of agar was obtained by dissolving 1 g of agar in 100 ml of deionized water by boiling. Then the solution was adjusted to ph with 2 N NaOH, autoclaved at 15 psi for 15 min, and cooled to 80 C. Tubes containing agar at a final concentration of 0.25 %, 0.22%, 0.19%, 0.16%, and 0.13% were prepared by heating to 80 C in screw-capped tubes containing 7.5,7.8, 8.1, 8.4, and 8.7 ml of deionized water and then adding the 1% agar solution to give a final volume of 10m!. After the agar was mixed by inversion of the tubes several times, the tubes were cooled for 4 hr at 2-4 C. They were then inverted and placed at 24 C. The gel strength of the agar is the lowest percentage agar that has not dropped down the tube after 20 min at 24 C. Results Growth on agar. Single-cell suspensions of the T-mycoplasmas no. 960 and T-McA were assayed for the number of viable cells simultaneously on T-agar containing 0.75% lonagar, at ph , and on the experimental agar media. The number, size, and morphology of the colonies were compared after incubation in a humidified incubator gassed with 5%-10% CO 2 in air. Effect at aeration on growth on agar. A comparison was made of the effects of incubating T-agar plates in an atmosphere of 5%-60% CO 2 in air, in a mixture of H 2 and CO 2 in a Gaspak anaerobic jar that contained an indicator to confirm that the contents were anaerobic (BBL), and in an anaerobic jar through which a continuous stream of either CO 2 or N 2 was passed at a rate of ~ 300 ml/min. The results of the assays and the size and morphology of the colonies of both T-mycoplasmas were the same under all these conditions; the colonies reached their maximal size in 48 hr. However, the appearance of the mycoplasma colonies often was delayed by aerobic incubation; the colonies sometimes took up to six days to reach the size achieved in two days in an atmosphere of 5% 60% CO 2 in air. Therefore, T-agar plates were incubated routinely in a humidified incubator gassed with 5% -1 0% CO 2, Concentration of equine serum. T-agar plates, ph , were prepared with 5%, 10%, and 20% unheated horse serum containing 20 mg of urea per 100 ml. Both species grew best on T-agar containing 10% horse serum; the colonies reached maximal size in 48 hr. Therefore, this concentration was used in all media. Effect at depth at T-agar media and concentration of agar. When volume of T-agar in 9 em petri dishes was decreased from the standard 20 ml [10] to 10 ml, the colony counts and diameter of the colonies decreased significantly. Therefore, the relationship of the volume of medium to the concentration of lonagar and Noble agar (Difco, Detroit, Mich.) was investigated. The colony count was always higher on standard plates containing 20 ml of medium [10], and the increase was constant irrespective of the brand and concentration of agar within the range 0.75%-2% (table 1). Therefore, for consistent results, T-mycoplasmas should be assayed on petri dishes containing the same volume of T-agar. The diameter of the colonies also varied with the thickness of the T-agar. Only well-separated colonies were measured. The diameter of the colonies of strains 960 and T-McA averaged 29 and 36 urn, respectively, on 10 ml of T-agar and increased to an average diameter of 38 and 48 urn, respectively, on 20 ml of T-agar. Both reached their maximal size after incubation for 72 hr; therefore, standard plates containing 20 ml of agar were used routinely. Addition at urea, ammonium salts, and hepes buffer to T-agar. To elucidate the effect of NH 3 on the ability of T-agar (ph and ph ) to support the growth of T-mycoplasmas, NH-tOH and (NH4hSO-t were added at concentrations of 200 mg/i00 ml and 110 mg/100 ml, respectively, to give a concentration of NH 3 ions in excess of that which might be expected from the breakdown of the urea supplied by

3 Growth of T-Mycoplasmas on Agar 705 Table 1. Effect of concentration of agar in the T-agar medium and the volume of T-agar medium in the petri dishes on the colony counts of single-cell suspensions of T-mycoplasmas. Mycoplasma T-McA Mycoplasma no. 960 (count X 10 5 ) (count X 10 5 ) Agar Ionagar Noble agar Ionagar Noble agar (% ) Standard* Thin Standard Thin Standard Thin Standard Thin * Standard and thin plates contained 20 ml and 10 ml of T-agar, respectively, in 9-cm plastic petri dishes. horse serum, Also, the urea content of the T-agar was increased by addition of 6 mg per 100 ml of urea to ascertain whether the increased amount of NHg produced by the mycoplasmal urease was toxic. Furthermore, in some experiments the agar was buffered at the required ph with g of hepes per 100 ml. Moreover, since it was possible that the effects of these additives might be synergistic, the growth of the T-mycoplasmas on T-agar containing permutations of these substances was compared. On standard plates containing 20 ml of T-agar, ph , the results of assays of single-cell suspensions of both strains T-McA and 960 were the same since the number of colonies growing on the experimental media was similar to that on the control; this indicated that every viable cell grew into a colony (table 2). The size of the colonies grown on T-agar was unaffected by the addition of hepes and by an increase in the content of urea from 2 mg/100 ml to 8 mg/100 ml. Strain T-McA averaged 45 urn in diameter (so ± 5 um ), and no. 960 had an average diameter of 47 urn (so ± 5 urn). The size of the colonies of both strains T-McA and no. 960 were slightly smaller on T-agar containing NH40H and averaged 42 urn in diameter (so ± 4 um ) and 37 urn (so ± 4 urn), respectively. They were significantly smaller on T-agar containing (NH4)2S04 (average, 12 urn ± 2 urn so and 15 urn ± 2 urn so, respectively). The.addition of 6 mg of urea to 100 ml of the T-agar containing ammonium salts did not have a synergistic effect since the colonies were similar on the media with and without the extra urea. Therefore, cultures in broth of laboratory strains no. 27, no. 58, and Pi together with the wild strains no. 207, 210, 213, 220, and 221 were also assayed simultaneously on T-agar, ph , with and without (NH4hS04. The assays did not differ significantly, thus confirming that T-mycoplasmas are not killed by low concentrations of NH 3 ions in T-agar at an acid ph. On T-agar at ph , the colonies of both strains T-McA and 960 were definitely smaller than those on T-agar at ph , averaging Table 2. Effect of addition of urea, ammonium salts, and hepes buffer to T-agar at ph and ph on the assay of T-mycoplasmas. No. of minimal reproductive units per ml of suspension* Strain T-McA X 10 6 (T-agar) ph 6.0- ph 7.2- Additivest Strain 960 X 10 5 (T-agar) ph 6.0- ph Control -none Urea NH40H (NH4)2S < <20 Hepes buffer; urea NH 40H (NH4)2S < <20 urea + NH 40H urea (NH4)2S < <20 * Assay of single-cell suspensions. t Amount added: urea, 6 mg/ioo ml; NH 40H, 0.2 g/100 ml; (NH4)2S04' 0.11 g/ioo ml; hepes, g/100 ml. *Hepes = N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid.

4 706 Furness 25 11m ± 3 Ilm SD and 26 11m ± 4 11m SD, respectively, which indicated that the alkaline medium was less than optimal. The size of the colonies was not altered further by the addition of the extra 6 mg/100 ml of urea, hepes, or NH 40H. However, no growth appeared on T-agar containing (NH 4 ) 2S04' which suggested that sulfate ions may be toxic in agar. However, on those media on which there was growth, the number of colonies was similar to that on T-agar at ph , indicating that all the cells grew into colonies and that an alkaline ph decreases the amount of growth of T-mycoplasmas on agar without affecting their viability (table 2). Effect of agar and M gso) on colonial morphology. Almost all the colonies were typical rough colonies of T-mycoplasmas on thin agar plates irrespective of the concentration of agar, and only an occasional fried-egg colony was seen among the typical rough colonies of T-mycoplasma of strains no. 960 and T-McA on standard plates of T-agar solidified with 0.75 % Ionagar (figure 1a). However, their morphology changed dramatically when the concentration of agar in standard plates was increased. On plates containing 1.0%-1.25% Ionagar, the percentage of fried-egg colonies increased to 90 %. Moreover, they were larger. The "yolk" was rough and approximately the same size as the entire colonies on 0.75% agar. The smooth "white" periphery of the fried-egg colonies accounted for the increase in diameter (figure 1b ). As the content of Ionagar was increased, the "yolk" decreased in size until the colonies were smooth (figure 1c) at concentrations of 1.5%, 1.75%, and 2.0% agar. Both the fried-egg and smooth colonies had diameters of up to m. On T-agar solidified with Noble agar, fried-egg colonies did not appear until the concentration of agar reached 1.75%-2.0%. Moreover, smooth colonies were never seen, suggesting that Ionagar either contained some substance not present in Noble agar or produced a harder medium that mediated this change to smooth morphology. Ionagar is rich in Mg [6] and, since only an occasional fried-egg colony was seen on 0.75% Ionagar and none on 0.75% Noble agar, the effect of adding MgSO4 to these two media could be compared. We found that the addition of mg of MgS0 4 per 100 ml increased the per- Figure 1. Changes in the morphology of T-mycoplasma colonies on increasing the concentration of Ionagar. 8, rough colonies; b, fried egg colonies; and c, smooth colonies. The bar (lower left) = m.

5 Growth of T-Mycoplasmas on Agar 707 cent age of fried-egg colonies to 90% on lonagar and that double the concentration of MgS0 4 was required to obtain the same percentage of friedegg colonies on Noble agar. However, on further increasing the concentration of MgS0 4, not only did smooth colonies fail to appear, but the percentage of fried-egg colonies decreased. This indicated that there was an optimal concentration of MgS0 4 for growth into fried-egg colonies and that another substance in lonagar was necessary to obtain smooth colonies. These observations were extended to the laboratory strains no. 27, no. 58, and Pi together with the five wild strains. The three laboratory strains were similar in their requirement for MgS0 4 However, the wild strains differed significantly. Strain 207 was similar to the laboratory strains on Ionagar but never produced fried-egg colonies on Noble agar, suggesting that lonagar contained another unknown metabolite which this strain needed. In contrast, strain 220 not only produced fried-egg colonies on the control media and at every concentration of MgS0 4 but did not revert to rough colonies, an indication that wild T-mycoplasmas have different metabolic requirements (table 3). Although no smooth colonies were seen in these experiments, with a concentration of 0.75 % agar they were present on 1.5%-2.0% lonagar but not on 2.0% Noble agar. The Colab-gel test showed that 1.7% Ionagar had the same gel strength as 2.0% Noble agar, and this indicated that smooth colonies were not artifacts produced by the hardness of the agar. The effect of the concentration of agar also could be tested bio- logically by preparation of 2.0% Noble agar plates without MgS0 4 and with mg of MgS0 4 per 100 ml, which was the optimal concentration for the production of fried-egg colonies on 0.75% Noble agar. Strains no. 960 and T McA grew into fried-egg colonies on both media but not into smooth colonies, indicating that the hardness of the T-agar and MgS0 4 alone were not adequate for growth into smooth colonies and that another factor present in Ionagar was involved. Some of the other strains did grow into smooth colonies both on 2.0% Noble agar and on 2.0% Noble agar containing Mg, again confirming that strains of T-mycoplasmas differ in their growth requirements for the production of smooth colonies (table 4). Discussion While media containing 20% unheated horse serum are recommended for the primary isolation of T-mycoplasmas from clinical material [6], the two laboratory strains of T-mycoplasmas, 960 and T-McA, grew better and achieved typical rough colonies of T-mycoplasmas of maximal size on T-agar (ph 6.n-6.5) containing 0.75% Ionagar and 10% unheated horse serum. The content of urea in the serum was 20 mg/100 ml, and the addition of extra urea did not improve growth. Therefore, these concentrations of lonagar and horse serum were used. The rate of appearance of the colonies was similar when the plates were incubated in an atmosphere of 5% 60% CO 2 in air, in CO 2, in N 2, and anaerobically Table 3. Transformation of rough T-mycoplasma colonies to fried-egg colonies typical of the classical mycoplasmas by addition of MgS0 4 to T-agar containing either 0.75% Ionagar or 0.75% Noble agar. Mycoplasma strain T-McA Pi * The remaining colonies were rough. Ionagar (rng of MgS0 4 / 100 ml) o o o o o o Percentage of fried-egg colonies* Noble agar (mg of MgS0 4 / 100 ml) o o o o o o o

6 708 Furness Table 4. A comparison of the colonial morphology of T-mycoplasmas grown on T-agar containing either 2% Noble agar or 2% Noble agar and mg of MgSO4/ 100 ml. Mycoplasma strain T-McA Pi Morphology on 2% Noble agar + 2% Noble agar MgS0 4 Rough and fried egg Rough and fried egg but not aerobically. Aeration did not affect the results of the assays but it did retard growth significantly, as it does in broth [3]. Therefore, plates should be incubated routinely in a humidified incubator gassed with at least 5%-10% CO 2 Even though urea is essential for the growth of T-mycoplasmas, it has been reported that they die in broth from ammonium ions and the rise in ph resulting from the hydrolysis of the urea [7]. The concentration of 6-8 mg of urea per 100 ml of T-media was selected because it was insufficient to alter the ph of T-broth on hydrolysis to NH 3 [3]; this facilitated investigation of the effect of NH 3 in acidic media. Under these conditions, it was not expected that the addition of hepes buffer would have any effect, and this was confirmed. The colonies did not grow to a larger size, neither did they change their morphology to.fried-egg colonies as has been reported for other T-mycoplasmas [8]; it is likely that these differences are due, at least in part, to the strains being investigated. T-broth, ph 6.5, to which ammonium salts have been added, permits growth of T-mycoplasmas, but an alkaline ph is inhibitory [3]. Therefore, the effect of NH 3 in alkaline broth cannot be ascertained. We found that every viable cell in single-cell suspensions formed colonies on T-agar at ph and on T-agar at ph containing added NH 40H. The concentration of urea was not critical within the range employed, but there is no doubt that broth containing high concentrations is toxic when it becomes alkaline [9, 11]. Therefore, it was concluded that, although these T-mycoplasmas grew best on media of ph , colony counts would not be affected by variations in the urea content of the horse serum or by moderate changes in the ph of the T-agar. Colonial morphology and size is related to the concentration and brand of agar and to the volume of T-agar in the petri dish. Both the results of colony counts and the size of well-separated colonies were affected by the volume of the medium in the petri dish, and this volume of media is critical if reproducible results of assays are to be obtained. As was reported previously, magnesium ions do affect the growth of the colonies [6]. Strains that normally grew into typical rough colonies produced fried-egg colonies, double the normal size if a suitable concentration of Mg was added to the T-agar at ph or the concentration of either Ionagar or Noble agar was increased. On 1.5%-2.0% Ionagar medium only smooth colonies were formed, but they were not formed on 2.0% Noble agar medium, which was equal in gel strength to 1.7% Ionagar, indicating that smoothness was not dependent on the hardness of the T-agar. Moreover, smooth colonies were not obtained by an increase in the concentration of Mg above that necessary for growth into fried-egg colonies. Excess Mg caused a reversion from fried-egg to rough colonies. Therefore, it must be concluded that Ionagar contains another metabolite required for the growth into smooth colonies of both strains 960 and T-McA. The three other laboratory and five wild strains examined also grew on T-agar (ph ) containing ammonium salts. Strains varied greatly in their ability to produce fried-egg and smooth colonies, suggesting that they had varied requirements for growth. There was evidence that strains not only required different metabolites but also needed different concentrations of the same metabolite. The nature of these growth requirements remains to be determined. Without further data on the effect of the constituents of the media, it is impossible to decide whether the rough colonies are rough variants and that the smooth colonies are, in fact, the typical colonies of T-mycoplasmas, with friedegg colonies intermediate between rough and smooth. Mycoplasma pneumoniae, the only clas-

7 Growth of T-Mycoplasmas on Agar 709 sical mycoplasma proven pathogenic for man, characteristically has smooth colonies. T-mycoplasmas are considered potentially pathogenic for the urogenital tract, and it may be that the ability of wild strains to grow into smooth colonies is indicative of their pathogenicity as it is for many bacteria. Irrespective of the correct explanation, it is important that microbiologists appreciate that T-mycoplasmas grown from clinical specimens may form small fried-egg colonies as well as the tiny rough colonies considered typical of these microorganisms. References 1. Shepard, M. C. Nongonococcal urethritis associated with human strains of T-mycoplasmas. J.A.M.A. 211: , Kundsin, R. B., Driscoll, S. G. The role of mycoplasmas in human reproductive failure. Ann. N.Y. Acad. Sci. 174: , Furness, G. T-mycoplasmas: their growth and production of a toxic substarce in broth. J. Infect. Dis. 127:9-16, Ford, D. K. Relationship between mycoplasma and the etiology of nongonococcal urethritis and Reiter's syndrome. Ann. N.Y. Acad. Sci. 143: , Hayfiick, L. Tissue cultures and mycoplasmas. Tex. Rep. BioI. Med. 23: , Shepard, M. C. Cultivation and properties of T strains of mycoplasma associated with nongonococcal urethritis. Ann. N.Y. Acad. Sci. 143: , Ford, D. K., McCandlish, K. L., Gronlund, A. F. Metabolism of HC-urea by T-strain mycoplasma. J. Bacteriol. 102: , Manchee, R. J., Taylor-Robinson, D. Enhanced growth of T-strain mycoplasmas with N-2 hydroxyethylpiperazine-n'-2 ethanesulfonic acid buffer. J. Bacteriol. 100:78-85, Ford, D. K., MacDonald, J. Influence of urea on the growth of T-strain mycoplasmas. J. Bacteriol, 93: , Shepard, M. C. T-form colonies of pleuropneumonia-like organisms. J. Bacteriol. 71: , Shepard, M. C., Lunceford, C. D. Occurrence of urease in T-strains of mycoplasmas. J. Bacteriol. 93: , 1967.