Mycoplasma detection in cell culture by concomitant use of bisbenzamide and fluoresceinated antibody.

Conditions are presented for application of both bisbenzamide (Hoechst 33258) stain and a specific fluoresceinated anti-Mycoplasma hyorhinis IgG to a single cell culture preparation. This allows the same field on a slide to be viewed for presumptive diagnosis of any cell culture contaminant mycoplasma by bisbenzamide staining and for definitive diagnosis of M. hyorhinis strains using fluoresceinated antibody. The use of this method plus a cultural procedure will permit identification of the "noncultivable" M. hyorhinis strain DBS 1050.

Cultivation of Ureaplasma urealyticum in continuous culture.

Continuous culture of Ureaplasma urealyticum is reported with a steady-state cell biomass of greater than 10(6) cells per ml. Thus, large cell numbers can be easily obtained; in addition, the system provides a powerful means for exploring what nutrients(s) limits the growth yield of this organism. Urea is shown not to be the growth-limiting nutrient in conventional media, although when provided in excess it appears to be completely hydrolyzed.

Some characteristics of Ureaplasma urealyticum. Urease activity in a simple buffer: effect of metal ions and sulphydryl inhibitors.

Urealytic activity of the cytoplasmic fraction of Ureaplasma urealyticum prepared by digitonin lysis was assayed in a simple buffer system (HEPES plus EDTA) by measuring the release of 14CO2 from [14C]urea. The Km of this preparation agreed with our previous observations of the same activity measured in a more complex reaction mixture. The substrate concentration at which maximum velocity occurred was approximately 20 mM. The activity was sensitive to heavy metals and inhibitors which react with sulphydryl groups such as N-ethylmaleimide and p-chloromercuribenzoate. It was not inhibited by Ca2+ or Mg2+ or by the reaction products, ammonia and carbon dioxide.

Effects of carbon dioxide, urea, and ammonia on growth of Ureaplasma urealyticum (T-strain mycoplasma).

By use of a simple device for continuous CO2 gassing of Ureaplasma urealyticum cultures growing in a liquid medium, we have been able to separate some of the effects of urea, CO2, ammonia, and pH on growth. The CO2 acted as a superior buffer in the pH range 5.7 to 6.8, which is optimal for Ureaplasma growth. It was, therefore, possible to observe the effect of repeated additions of urea to the culture without alkalinization of the growth medium. We found that the repeated additions of urea did not enhance Ureaplasma growth, and the resultant accumulation of ammonium ions (greater than 2,000 microng/ml) did not cause more rapid death under these conditions. By abruptly changing the gaseous environment from CO2 to N2, it was possible to cause a rapid pH change in the culture to a value above 8.0. This resulted in a more rapid death of the organisms.

Localization of enzymes in Ureaplasma urealyticum (T-strain mycoplasma).

Ureaplasma urealyticum cells were lysed by osmotic shock or by digitonin. The membrane fraction contained four to ten times as much protein as the cytoplasmic fraction. These values are in large excess of those reported for classical mycoplasmas, suggesting that the Ureaplasma membrane fraction was heavily contaminated with proteins derived from the growth medium. The U. urealyticum urease activity was localized in the cytoplasmic fraction, whereas the adenosine triphosphatase activity was localized in the membrane fraction. Significant urease activity could be detected also in nonviable cells. Urea, at concentrations above 0.25 M, was mycoplasmastatic to Acholeplasma laidlawii, Mycoplasma hominis, and U. urealyticum, so that the Ureaplasma urease did not afford preferential protection against urea toxicity. The intracellular localization of the urease would be expected to release ammonia from urea in the cytoplasm. The ammonia will take up protons to become ammonium ions. It can be hypothesized that the intracellular NH4+ plays a role in proton elimination or acid-base balance, which might be coupled to an energy producing ion gradient and/or transport mechanisms.

Morphology of Ureaplasma urealyticum (T-mycoplasma) organisms and colonies.

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 appearance 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, O2, 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 concentrations 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 recommended for improved growth and formation of fried-egg colonies of U. ureaplyticum on agar. It must be emphasized that these experiments were carried out with a laboratory-adapted strain.

The effect of growth and urea concentration on ammonia production by a urea-hydrolysing mycoplasma (Ureaplasma urealyticum).

The rate of accumulation of ammonium ion in cultures of Ureaplasma urealyticum was independent of the growth rate and of the initial urea concentration above 0-025% in the medium, although the quantity of ammonium ion accumulating did depend on the initial urea concentration. Ammonium ions accumulated at a similar rate in U. urealyticum cultures of both rapidly and slowly growing organisms. Viable but non-growing ureaplasmas also produced ammonia in complete medium at a lower temperature than usual (25 degrees C) or in an inadequate growth medium at 37 degrees C. The rate of ammonium ion accumulation in a dying culture depended on the number of viable organisms present; this is relevant to diagnostic methods for ureaplasms which depend on detecting ammonia colorimetrically.

Urea-hydrolyzing activity of a T-strain mycoplasma: Ureaplasma urealyticum.

The urea-hydrolyzing activity of a T-strain mycoplasma was studied in experiments using whole cells and cell-free enzyme preparations by measuring the release of 14CO2 from [14C]urea. Under the conditions used, the urea concentration optimum is approximately 5.6 X 10(-3) M urea. The activity is soluble and not membrane bound. It is stable at -70 C for several weeks but is more labile at higher temperatures. The pH optimum is between 5.0 and 6.0. The effect of several inhibitors on the activity was tested and revealed similarities, as well as differences, between T-strain mycoplasma urease activity and the urease activity of other organisms and plants.

Some effects of growth medium composition on the antigenicity of a T-strain mycoplasma.

T-strain 960 was passaged through 24 serial 10-fold dilutions in media without added urea and with porcine serum albumin fraction V as the only protein enrichment. The organism, either grown in this manner or passaged an additional three times in medium containing horse serum and 0.1 per cent urea, was inoculated into rabbits. Resultant antisera were tested for activity against T-960 growing in these different media by: (i) growth curve analysis in the presence of antiserum, (ii) metabolic inhibition in the presence or absence of complement (fresh guinea pig serum), (iii) complement-dependent killing curves, (iv) double diffusion in gel (Ouchterlong), and (v) a new visual method for the detection of antigen-antibody reactions on glass slides coated with a thin film of indium metal. Our results indicate that the reactivity of the antisera, as assayed by the above methods, is significantly affected by the composition of the growth medium used for preparation of the antigen. In addition, it was possible to determine that the guinea pig serum-dependent killing of T960 was not affected by the presence of ammonium ion.

Dialysis culture of T-strain mycoplasmas.

Using dialyzing cultures of T-strain mycoplasmas, it was possible to make some observations relevant to the growth and metabolism of these organisms which would not be possible in nondialyzing cultures due to growth inhibition of the organisms by elevated pH and increased ammonium ion concentration in media containing urea. The rate of ammonia accumulation was found to be related to the initial urea concentration in the medium and could not be accounted for by any change in the multiplication rate of the organisms. More ammonia was generated than could be accounted for by the added urea alone, suggesting that an ammonia-producing activity other than urease may be present in T-strain mycoplasmas. Titers above 10(7) color change units per ml were achieved in dialysis cultures of a T-strain mycoplasma in the presence of urea, and such titers were maintained for approximately 60 h during dialysis culture in the absence of added urea.

Growth of T-strain mycoplasmas in medium without added urea: effect of trace amounts of urea and of a urease inhibitor.

Urea is currently considered to be a requirement for the propagation of T-strain mycoplasmas. We report here the replication of T-strain 960 (ATCC 25023) in media prepared from dialyzed components with added putrescine and allantoin but without added urea, or in dialyzed medium containing small amounts of added urea. The least amount of urea which allowed growth in the medium without allantoin was above 10 mug/ml. The amount of urea estimated to contaminate the added allantoin or putrescine was 5 mug/ml or less, which is insufficient to support T-strain replication. T-strain 960 was grown in the presence of urea and the urease inhibitor acetohydroxamic acid AHA where the organisms multiplied at a slower rate in the presence of AHA than in its absence. Urea hydrolysis occurred with concomitant ammonia accumulation and pH increase in cultures with AHA added.