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 10⁷ 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.     

Color test for the measurement of antibody to T-strain mycoplasmas

Purcell, Robert H. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), D. Taylor-Robinson, D. Wong, and R. M. Chanock. Color test for the measurement of antibody to T-strain mycoplasmas. J. Bacteriol. 92:6-12. 1966.-A metabolic inhibition technique for the measurement of antibody to T-strain mycoplasmas was developed, based upon the ability of T-strain mycoplasmas to metabolize urea with the concomitant production of ammonia, and the ability of specific antiserum to inhibit this ammonia production. Phenol red added to the medium served as an indicator of pH change resulting from ammonia production. Specific antiserum to T-strain mycoplasma T-960 was prepared. The T-strain organism was shown to be serologically distinct from the recognized large-colony mycoplasmas. Antibody to mycoplasma strain T-960 in human sera was demonstrated with the metabolic inhibition technique.     

Occurrence of urease in T strains of Mycoplasma

A previously unknown metabolite necessary for growth of T strains of Mycoplasma in artificial culture media has been identified as urea. The source of this metabolite was the mammalian plasma or serum enrichment of the culture medium. Normal horse serum was the most satisfactory native protein enrichment for cultivation of T strains of mycoplasma, and it is believed that its superior performance in agar and fluid culture media is associated with its relatively high urea content (approximately 40 mg/100 ml). T-strain urease activity was maximal at pH 6.0 +/- 0.5. This is also the optimal pH for growth of T strains. Substrate concentrations greater than 1.0% urea were inhibitory to growth and urease activity of T-strain organisms, and optimal urea concentrations in fluid media appeared to lie within the range of 0.008 to 0.01 m. This range of urea concentration permitted maximal growth of T-strain organisms without rapid loss of viability due to excessive ammonia accumulation and rise in pH to lethal levels. T strains of Mycoplasma were cultivated in a serum-free fluid medium containing urea as the only added metabolite and nitrogen source. T strains are the only known human mycoplasmas which exhibit urease activity, and this biochemical marker can be employed as an aid in the detection and identification of T strains of Mycoplasma (urease color test) and in distinguishing T strains from other members of the human Mycoplasma group.     

Influence of Urea on the Growth of T-Strain Mycoplasmas

T-strain mycoplasmas require urea for propagation, but urea metabolism also occurs in nonpropagating viable cultures. Ammonia results from this metabolism and alkalinizes the medium. Ammonium ions and an alkaline pH both inhibit the multiplication of T strains and reduce the viability of T strains in broth. These toxic effects of urea metabolism currently limit the growth of T strains in broth. Stock T-strain cultures are optimally maintained in continuous culture if the routine medium at pH 6.0 is supplemented with 0.05% urea and 0.002% phenol red, but an incubation temperature of 30 C is preferable to 37 C for subculture at 24-hr intervals.     

Urea transport in Saccharomyces cerevisiae.

Urea transport in Saccharomyces cerevisiae occurs by two pathways. The first mode of uptake is via an active transport system which: (i) has an apparent Km value of 14 muM, (ii) is absolutely dependent upon energy metabolism, (iii) requires pre-growth of the cultures in the presence of oxaluric acid, gratuitous inducer of the allantoin degradative enzymes, and (iv) is sensitive to nitrogen repression. The second mode of uptake which occurs at external urea concentrations in excess of 0.5 mM is via either passive or facilitated diffusion.     

Urea hydrogen peroxide reduces the numbers of lactobacilli, nourishes yeast, and leaves no residues in the ethanol fermentation

Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five Lactobacillus spp. (Lactobacillus plantarum, L. paracasei, Lactobacillus sp. strain 3, L. rhamnosus, and L. fermentum) from approximately 10(7) to approximately 10(2) CFU/ml in a 2-h preincubation at 30 degrees C of normal-gravity wheat mash at approximately 21 g of dissolved solids per ml containing normal levels of suspended grain particles. Fermentation was completed 36 h after inoculation of Saccharomyces cerevisiae in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with L. paracasei at approximately 10(7) CFU/ml. There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added). However, the presence of L. paracasei at approximately 10(7) CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control. The bactericidal activity of UHP is greatly affected by the presence of particulate matter. In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present. No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30 degrees C whether the bactericidal agent was added as H(2)O(2) or as urea hydrogen peroxide. NADH peroxidase activity (involved in degrading H(2)O(2)) increased significantly (P = 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H(2)O(2). H(2)O(2)-resistant mutants were not expected or found when lethal levels of H(2)O(2) or UHP were used. Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca. 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast.     

Effect of prostaglandins on cell function and multiplication of parainfluenza 3 viruses in WISH cells.

Cytotoxic effect of prostaglandins E2 and F2alpha on cells grown in vitro and the influence of these compounds on multiplication of myxovirus parainfluenza 3 were investigated. The prostaglandins were added to culture medium (0-01-10 mug/ml) 24 hr before virus infection, or for 2 and 48 hr after inoculation with viruses. WISH cells and monkey kidney cell cultures were used. No direct cytotoxic effect of prostaglandins at concentrations 0-01-1 mug/ml was found (viability, supravital staining, phase-contrast system, Nitro-BT reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to partial injury of the cell population with symptoms of damage to mitochondria. Prostaglandins E2 and F2alpha inhibited multiplication of parainfluenza 3 virus at concentrations 0-1-10 mug/ml. The inhibitory effect was most pronounced if prostaglandins were added to medium for the whole period of virus multiplication i.e. for 48 hr but little or no effect was found if they were added prior to inoculation or for 2 hr after it. Inhibitory effect of prostaglandins on replication phase of viruses is suggested.     

Studies on the Physiology of Bacillus fastidiosus

Bacillus fastidiosus was grown in a minimal medium that contained uric acid or allantoin, aerated by vigorous stirring. A constant, optimum pH of 7.4 was maintained by controlled addition of sulfuric acid. Washed cells converted both urate and allantoin into carbon dioxide and ammonia, simultaneously assimilating part of the available carbon and nitrogen. Urate oxidase (formerly called uricase) was present in extracts from urate-grown but not allantoin-grown cells. The formation of urate oxidase was apparently induced by urate. Urea was detected as an intermediate in some but not all of these experiments. However, the high urease activity observed in cell-free extracts may have prevented accumulation of urea in many of the experiments. The presence of glyoxylate carboligase and tartronic semialdehyde reductase activities indicates that the glycerate pathway may be involved in urate and allantoin catabolism in this organism.     

Enterotoxin B Synthesis by Replicating and Nonreplicating Cells of Staphylococcus aureus

Although 95% of the enterotoxin B produced by Staphylococcus aureus appears during the latter part of the exponential phase of growth, growth per se is not necessary for toxin synthesis. A procedure is described whereby a concentrated suspension (at least 6 x 10(10) cells per ml) of a 16-hr culture of S. aureus was found to be capable of producing toxin, without replication, when air and glucose were present. This technique allows the growth requirement to be separated from toxin formation. Although higher (100 mug/ml) concentrations of toxin appeared in the medium when nitrogen was present, lower levels (30 mug/ml) were produced in the absence of N-Z-amine A. Toxin production proceeded without any net increase in deoxyribonucleic acid, ribonucleic acid, or protein. Chloramphenicol did not inhibit toxin formation in a nitrogen-free medium. The optimal pH for toxin production in a nitrogen-free medium was 8.0 to 8.5; for synthesis in a medium where nitrogen was available, the optimal pH was 7.0 to 7.5. Increasing the rate of aeration increased toxin release during growth, but decreased the amount of toxin subsequently produced when the bacteria were resuspended. These results suggest the presence of a precursor pool in the cells collected after 16 hr of growth.     

Synergistic effect of caffeine and heparin on in-vitro fertilization of cattle oocytes matured in culture

Frozen-thawed spermatozoa obtained from six different bulls were suspended in Brackett and Oliphant's (BO) medium (14), with or without 10 mM caffeine, after washing. A 50-mul aliquot of the sperm suspension was added to the 50-mul BO medium supplemented with bovine serum albumin (BSA, 20 mg/ml) and heparin (20 mug/ml) in which the bovine follicular oocytes matured in culture had been introduced previously. The proportion (35%) of oocytes penetrated in the presence of heparin alone 20 to 24 h after insemination was not significantly different from those (32%) penetrated in the presence of caffeine alone as reported previously (1). When heparin was added to the caffeine in the fertilization medium, the penetration rate of oocytes increased significantly to 68% (P < 0.001), indicating that both chemicals act sinergistically to induce capacitation and/or acrosome reaction of spermatozoa and stimulate in vitro fertilization of cattle oocytes. However, great variation in penetration rates (35 to 96%) was observed among the different bulls. The optimal concentration of heparin in the suspension medium in which the highest rate of oocyte penetration took place was 10 mug/ml.     

The urea requirement and urease production of some human species of T-mycoplasmas.

Seven species of human T-mycoplasmas that grow in Fraction A and 20 mug urea/ml died when the urea was omitted. Two species would not grow in Fraction A broth containing 10 mug/urea/ml. The other five strains grew in broth containing 10 mug urea/ml and were adapted by serial passage in broth containing decreasing concentrations of urea to grow in broth containing 2.5 mug/ml urea, but not in broth containing 1.25 mug/ml. Therefore the minimal urea requirement is not the same for the growth of all strains of T-mycoplasmas. In exponential phase broth cultures, urease was detected only intracellularly, none being found in the medium.     

Effects of Trace Metals on the Production of Aflatoxins by Aspergillus parasiticus

Certain metals added as salts to a defined basal culture medium influenced the level of aflatoxin production by Aspergillus parasiticus in the low micrograms-per-milliliter range of the added metal. In many cases no change or a relatively small change in mat weight and final pH of the medium accompanied this effect. With zinc at added levels of 0 to 10 mug/ml in the medium, aflatoxin increased 30-to 1,000-fold with increasing of zinc, whereas mat weight increased less than threefold. At 25 mug of added zinc per ml, aflatoxin decreased, but mat weight did not. At an added level of 25 mug or less of the metal per ml, salts of iron, manganese, cooper, cadmium, trivalent chromium, silver, and mercury partly or completelyinhibited aflatoxin production, without influencing mat weight.     

Urea and water permeability in dogfish (Squalus acanthias) gills

We used a perfused gill preparation from dogfish to investigate the origin of low branchial permeability to urea. Urea permeability (14C-urea) was measured simultaneously with diffusional water permeability (3H2O). Permeability coefficients for urea and ammonia in the perfused preparation were almost identical to in vivo values. The permeability coefficient of urea was 0.032 x 10(-6) cm/sec and of 3H2O 6.55 x 10(-6) cm/sec. Adrenalin (1 x 10(-6) M) increased water and ammonia effluxes by a factor of 1.5 and urea efflux by a factor of 3.1. Urea efflux was almost independent of the urea concentration in the perfusion medium. The urea analogue thiourea in the perfusate had no effect on urea efflux, whereas the non-competitive inhibitor of urea transport, phloretin, increased efflux markedly. The basolateral membrane is approximately 14 times more permeable to urea than the apical membrane. We conclude that the dogfish apical membrane is extremely tight to urea, but the low apparent branchial permeability may also relate to the presence of an active urea transporter on the basolateral membrane that returns urea to the blood and hence reduces the apical urea gradient.     

Initiation and Termination of Bacterial Deoxyribonucleic Acid Replication in Low Concentrations of Chloramphenicol

Escherichia coli 15T(-) can initiate a cycle of deoxyribonucleic acid replication with equal efficiency in the presence of 25 or 50 mug of chloramphenicol/ml. However, these replication cycles are not completed in the presence of these drug concentrations, and the amount of replication decreases with increasing drug concentration.     

Urea as sole source of nitrogen for plant growth

Summary Spirodela oligorrhiza grown in sterile culture was able to use urea as sole source of nitrogen but only when the pH of the culture medium was below 4.3. Plants inoculated into urea media at pH 6.4 initially made little growth and became nitrogen-deficient in appearance and composition although they contained about 100 grams of urea per gram fresh weight of tissue. After a period the pH of the medium usually fell below 4.3 and growth commenced. Growth with other compounds, e.g. ammonium, nitrate or allantoin, as sources of nitrogen was not similarly affected by the pH of the culture medium.Urease activity could always be detected in the tissues of Spirodela oligorrhiza growing on urea. Plants with little or no urease activity soon developed significant activity when inoculated into urea media at pH 4.0. When the pH of the medium was higher there was no increase in urease activity and no growth ensued. Plants growing on urea possessed an activity of about 50 milliunits per gram fresh weight of tissue, but if the pH of the medium fell to 3.5 or lower, the activity present rose to 10 times this level.Urease activity also appeared, in the absence of supplied urea, as plants became increasingly nitrogen-deficient.     

Urease Color Test Medium U-9 for the Detection and Identification of “T” Mycoplasmas in Clinical Material

A urease color test fluid medium (U-9) for the detection and identification of T (T-strain) mycoplasmas in clinical material is described which is sensitive and specific for this group of mycoplasmas. The medium was prepared from commercially available components and contained 95% half-strength, tryptic digest broth (pH 5.5), 4% unheated horse serum, 0.05% highest-purity urea, 0.001% sodium phenolsulfonphthalein, and 1,000 units of potassium penicillin G per ml. The final reaction of medium U-9 was pH 6.0. The overall agreement (positive and negative) between urease reactions in U-9 urease color test medium and culture findings in a standard agar primary culture system among 686 clinical specimens was 98.1%. The disagreement consisted of 13 false-positive urease reactions which were recognized visually as false-positive reactions due to other microorganisms. For specimens from the female genitourinary tract, the inclusion of 2.5 mug of amphotericin B (Fungizone) per ml of medium U-9 is recommended for the suppression of growth of Candida species and filamentous fungi.     

Inhibitory Effect of Fatty Acids on the Entry of the Lipid-Containing Bacteriophage PR4 into Escherichia coli

Various unsaturated fatty acids (notably palmitoleic acid and oleic acid) interfered with plaque production by the lipid-containing bacteriophage PR4 on lawns of Escherichia coli. Addition of fatty acid to give 50 mug/ml ( approximately 0.2 mM) at the time of infection prevented phage replication. If, however, the fatty acid was added after infection, normal amounts of phage were produced. If the fatty acid was added (to 50 mug/ml) to the host cell culture a long enough time before infection such that the fatty acid concentration in the growth medium at the time of infection was reduced to less, similar5 mug/ml (due to fatty acid incorporation by the host cells), normal phage replication occurred also. Neither palmitoleic acid nor oleic acid prevented PR4 attachment to E. coli. Several types of experiments indicated that it is the entry process of the virus that is inhibited by these fatty acids. Specifically, if the fatty acid was added at the time of infection, the host cells were not killed by the virus and no detectable amounts of viral protein were synthesized. In addition, experiments using purified radioisotope-labeled virions showed directly that entry is inhibited. Mutants of PR4 that did replicate in the presence of oleic acid arose spontaneously at a frequency of 10(-6). Three of these mutants that have been further characterized have protein and phospholipid compositions indistinguishable from those of wild-type PR4.     

Degradation and utilization of exogenous allantoin by intact soybean root

Allantoin is produced by soybean [ Glycine max (L.) Merr. cv. Harper] nodules during nitrogen fixation. Decomposed nodules, therefore, may release allantoin into the surrounding soil. If the released allantoin were to be taken up by the plant without degradation, it is possible that the exogenous allantoin might repress subsequent nodulation. Using a hydroponic growth system, degradation of exogenous allantoin by soybean root was studied. In the presence of intact soybean root exogenous allantoin was rapidly degraded, yielding ca 2 mmol of urea per mmol of allantoin. Hydrolysis of urea to ammonia proceeded very slowly. Instead, the urea seemed to be taken up by the intact soybean root. The enzyme(s) required for the production of urea from exogenous allantoin could not be detected in the aqueous rooting medium. Therefore, these enzymes seem to be attached to the exterior surface of the intact soybean root. This study shows that exogenous allantoin can be readily degraded and assimilated by the growing soybean plant.     

Urea Production and Putrescine Biosynthesis by Escherichia coli

Cultures of Escherichia coli B and K-12 produce urea during growth in minimal medium. Results of isotopic labeling experiments were consistent with the sole source of urea being from the conversion of arginine to putrescine. Since E. coli itself has no demonstrable urease activity, the rate of urea production is a measure of the flux through the arginine to putrescine pathway.     

Urea Hydrogen Peroxide Reduces the Numbers of Lactobacilli, Nourishes Yeast, and Leaves No Residues in the Ethanol Fermentation

Urea hydrogen peroxide (UHP) at a concentration of 30 to 32 mmol/liter reduced the numbers of five Lactobacillus spp. (Lactobacillus plantarum, L. paracasei, Lactobacillus sp. strain 3, L. rhamnosus, and L. fermentum) from ~10⁷ to ~10² CFU/ml in a 2-h preincubation at 30°C of normal-gravity wheat mash at ~21 g of dissolved solids per ml containing normal levels of suspended grain particles. Fermentation was completed 36 h after inoculation of Saccharomyces cerevisiae in the presence of UHP, even when wheat mash was deliberately contaminated (infected) with L. paracasei at ~10⁷ CFU/ml. There were no significant differences in the maximum ethanol produced between treatments when urea hydrogen peroxide was used to kill the bacteria and controls (in which no bacteria were added). However, the presence of L. paracasei at ~10⁷ CFU/ml without added agent resulted in a 5.84% reduction in the maximum ethanol produced compared to the control. The bactericidal activity of UHP is greatly affected by the presence of particulate matter. In fact, only 2 mmol of urea hydrogen peroxide per liter was required for disinfection when mashes had little or no particulate matter present. No significant differences were observed in the decomposition of hydrogen peroxide in normal-gravity wheat mash at 30°C whether the bactericidal agent was added as H₂O₂ or as urea hydrogen peroxide. NADH peroxidase activity (involved in degrading H₂O₂) increased significantly (P = 0.05) in the presence of 0.75 mM hydrogen peroxide (sublethal level) in all five strains of lactobacilli tested but did not persist in cells regrown in the absence of H₂O₂. H₂O₂-resistant mutants were not expected or found when lethal levels of H₂O₂ or UHP were used. Contaminating lactobacilli can be effectively managed by UHP, a compound which when used at ca. 30 mmol/liter happens to provide near-optimum levels of assimilable nitrogen and oxygen that aid in vigorous fermentation performance by yeast.