Aerosols as a Source of Widespread Mycoplasma Contamination of Tissue Cultures

Mycoplasma isolates were cultured from 15 antibiotic-free cell cultures obtained from a single laboratory. Complement-fixation tests showed that these isolates were antigenically related to each other but were unrelated to M. hominis type 1, M. hominis type 2, M. arthritidis, M. laidlawii type B, Mycoplasma sp. H.Ep. #2 (Barile), or M. salivarium. Examination of serum used to feed the infected cell lines revealed no Mycoplasma. Infection resulting from cross-contamination by a single Mycoplasma strain from one cell culture to another was investigated. Although the organisms were not found in the air over the work area, aerosols containing these contaminants were produced in tissue culture bottles during the trypsinization of cell monolayers. The minimal infectious dose of Mycoplasma for tissue cultures was measured, and it was determined that one organism was capable of initiating an infection in a tissue culture. The pattern of contamination and the small dose required for infection indicated that Mycoplasma contamination was spread from one tissue culture to another via aerosols. It was demonstrated that Mycoplasma can be transferred from one cell culture to another through the use of a common burette for dispensing medium.     

Immunofluorescence Identification of Mycoplasma on Agar by Use of Incident Illumination

The fluorescent-antibody method has been employed for the rapid identification of Mycoplasma colonies growing on agar plates. The method was found to be effective for detection of mixtures of Mycoplasma serotypes growing on primary isolation plates. The technique also helped to define the presence of mycoplasmas which did not produce typical colonies. It was also possible to identify Mycoplasma colonies overgrown by bacterial or fungal contaminants. Conjugates directed against 10 distinct Mycoplasma serotypes have been successfully employed in this system. One of the serotypes is a human oral isolate which has not been previously characterized.     

Cultivation of Mycoplasmas on Glass

Eight Mycoplasma species of human origin were successfully cultivated on glass. Complement-fixing (CF) antigens prepared from glass-adherent mycoplasmas were potent, specific, and free from anticomplementary activity. PPLO broth medium supplemented with 1 to 5% PPLO serum fraction (bovine), 2.5% fresh yeast extract, and 1% glucose (glycolytic species) or 1% arginine (arginine-utilizing species) supported moderate to luxuriant growth of mycoplasmas on glass. The potency of CF antigens prepared from glass-adherent mycoplasmas varied with the species of Mycoplasma tested and the duration of incubation. When the potency of CF antigens prepared from glass-adherent mycoplasmas was compared with that material sedimented from the broth phase of the same culture, three patterns of growth were observed: M. hominis and M. orale type 2 grew preferentially in the broth phase; M. salivarium, M. orale types 1 and 3, M. pneumoniae, and M. lipophilum preferentially adhered to the glass; and M. fermentans was biphasic. The growth of mycoplasmas on glass provides a simple means of concentrating and purifying such organisms for immunological and biochemical studies.     

Clonal analysis of vertebrate myogenesis. II. Environmental influences upon human muscle differentiation

In vitro procedures for obtaining the differentiation of human fetal muscle colonies were developed, and the sensitivity of clonal differentiation to environmental influences was examined. Human muscle colonies are capable of differentiating in the absence of an exogenous collagen substrate. The dependence of clonal diffeentiation upon the addition of chick embryo extract to the culture medium is determined by the serum type used in the medium and by the substrate upon which the colonies are grown. Clonal differentiation also depends upon conditioning of the medium by the colonies. The rate of medium conditioning is affected by clonal density and initial medium composition. The required medium modification is not species specific since medium conditioned by chick muscle cells also permits the early differentiation of human muscle clones. By manipulating the various environmental parameters described above it has been possible to define a number of in vitro conditions which permit a normal rate of cell proliferation but do not permit cell fusion. Results from these experiments are discussed in terms of their developmental implications.     

Chemically Defined Medium for Cultivation of Several Epiphytic and Phytopathogenic Spiroplasmas

A chemically defined medium, LD82, was formulated for in vitro cultivation of spiroplasmas. Medium LD82 supported good growth for four epiphytic and insect-pathogenic spiroplasmas, Spiroplasma floricola 23-6^T, Spiroplasma sp. strain SR3, Spiroplasma sp. strain brevi, and Spiroplasma sp. strain AS576, and of the phytopathogenic spiroplasmas Spiroplasma citri Maroc R8A2^T and PC1. Titers of all six strains grown in defined medium LD82 reached 2.0 × 10⁹ to 6.0 × 10⁹ CFU/ml of culture. All spiroplasma strains tested formed colonies readily on agar medium LD82. None of the spiroplasmas formed typical fried-egg colonies. All formed diffuse colonies, but the forms of colonies differed somewhat among the spiroplasma strains. In preliminary studies of nutritional requirements, phospholipids slightly enhanced the growth of the epiphytic and insect-pathogenic strains in medium LD82 and were found essential for good growth of S. citri.     

A new cost-effective, selective and differential medium for the isolation of Cronobacter spp

The objective of the present study was to develop a new selective, differential and cost-effective medium (Kim and Rhee — KR-medium) for the isolation of Cronobacter spp. In this new medium, which contained salicin as a differential agent, Cronobacter spp. generated typical colonies with characteristic violet-colored centers surrounded by a transparent to opalescent border, and the growth of other microorganisms (40 strains) was inhibited or produced visually distinguishable colonies. Using healthy and heat- and desiccation-injured cells, the quantity of nutrients was adjusted to determine the optimal recovery rate, selectivity, differentiation and cost-effectiveness. Peptone and salicin concentrations were established as 10 and 8 g/L, respectively. The KR medium was then validated using salicin fermenting organisms, including Cronobacter spp. (52 strains), Enterobacter cloacae (50 strains) and Klebsiella pneumonia (10 strains) isolated from clinical and food specimens. All strains of Cronobacter spp. produced typical colonies and other salicin fermenting organisms were easily distinguishable from Cronobacter spp. with the exception of 2 E. cloacae strains. The verification of KR medium was carried out in powdered infant formula artificially inoculated with healthy, heat-injured, and desiccation-injured Cronobacter spp. and the expected typical colonies were appeared. The KR medium had a high specificity (98%) and sensitivity (100%), with no false-negative results. Moreover, we show that the cost of the KR medium is much lower than that of other selective and differential media. The use of the KR medium for the selective isolation of Cronobacter spp. in laboratories and food industry settings may therefore lessen the financial burden of Cronobacter spp. detection.     

Improved medium for lactic streptococci and their bacteriophages

Incorporation of 1.9% β-disodium glycerophosphate (GP) into a complex medium resulted in improved growth by lactic streptococci at 30 C. The medium, called M17, contained: Phytone peptone, 5.0 g; polypeptone, 5.0 g; yeast extract, 2.5 g; beef extract, 5.0 g; lactose, 5.0 g; ascorbic acid, 0.5 g; GP, 19.0 g; 1.0 M MgSO₄·7H₂O, 1.0 ml; and glass-distilled water, 1,000 ml. Based on absorbance readings and total counts, all strains of Streptococcus cremoris, S. diacetilactis, and S. lactis grew better in M17 medium than in a similar medium lacking GP or in lactic broth. Enhanced growth was probably due to the increased buffering capacity of the medium, since pH values below 5.70 were not reached after 24 h of growth at 30 C by S. lactis or S. cremoris strains. The medium also proved useful for isolation of bacterial mutants lacking the ability to ferment lactose; such mutants formed minute colonies on M17 agar plates, whereas wild-type cells formed colonies 3 to 4 mm in diameter. Incorporation of sterile GP into skim milk at 1.9% final concentration resulted in enhanced acid-producing activity by lactic streptococci when cells were inoculated from GP milk into skim milk not containing GP. M17 medium also proved superior to other media in demonstrating and distinguishing between lactic streptococcal bacteriophages. Plaques larger than 6 mm in diameter developed with some phage-host combinations, and turbid plaques, indicative of lysogeny, were also easily demonstrated for some systems.     

Culture of blastomeres from in vitro-matured,fertilized, and cultured bovine embryos

A culture system was devised to study the differentiation of bovine blastomeres. Blastomeres (2–13 per well) from embryos produced by in vitro maturation, fertilization, and culture of oocytes obtained from slaughterhouse ovaries were cultured for 10 days in 24-well culture plates on feeder layers in blastomere culture medium (BCM: equal parts tissue culture medium 199 and low-glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum). Ovine embryonic fibroblasts and STO cells were superior to bovine and mouse embryonic fibroblasts as mitotically inactivated feeder cells. Over five studies in which four blastomeres from an embryo were added to each culture well, an average of one colony per well formed from the blastomeres. The colonies continued to grow throughout the culture period, and most colonies resembled trophectoderm in their cellular characteristics, although some cultures contained a mixture of trophectoderm and endoderm. When the number of blastomeres cultured in each well was varied from 2–8, the number of colonies formed was proportional to the number of blastomeres added. Blastomeres from day 5 and day 6 embryos produced fewer colonies than did those from day 4 embryos, perhaps as a result of differentiation and tighter blastomere adhesion resulting in damage during their separation. The absence of serum did not alter the number of colonies formed. A number of growth factors, including LIF, OM, PDGFα, and FGF4, had no effect on the number of colonies, the size of colonies, or their alkaline phosphatase staining score beyond that provided by the feeder layer or serum when present. Blastomeres did not form colonies in the absence of feeder layers. Mol. Reprod. Dev. 48:238–245, 1997. © 1997 Wiley-Liss, Inc.     

Development of a reproducible method to determine minimum inhibitory concentration (MIC) of plant extract against a slow-growing mycoplasmas organism

Mycoplasma species are fastidious bacteria that require a specialized medium for their growth, isolation and identification. There are no standardized tests to evaluate the in vitro susceptibility of mycoplasmas to medicinal plant extracts. A widely used in-broth, microtitre plate, minimum inhibitory concentration (MIC) assay was adapted and evaluated using acetone extracts of Anoigeissus leiocarpus on the isolates of Mycoplasma mycoides subsp. mycoides small colony variants (MmmSC). Several problems were encountered including the contamination of the medium by Bacillus species found in plants and the fact that the slow-growing mycoplasmas proved to be poor reducers of the indicator tetrazolium salt or resorcinol. We then examined a pH indicator-dependant technique to detect the acid production caused by the growth of the organism after glucose utilization from the broth medium. The method gives a clear cut-off point that was easy to read and interpret and was also reproducible.The MIC value for acetone extract of A. leiocarpus was 0.16 mg/ml. The development of this method now makes it possible to evaluate extracts of several plant species for antimycoplasmal activity.     

Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii

Methods for efficient growth and manipulation of relatively uncharacterized bacteria facilitate their study and are essential for genetic manipulation. We report new growth media and culture techniques for Caldicellulosiruptor bescii, the most thermophilic cellulolytic bacterium known. A low osmolarity defined growth medium (LOD) was developed that avoids problems associated with precipitates that form in previously reported media allowing the monitoring of culture density by optical density at 680 nm (OD₆₈₀) and more efficient DNA transformation by electroporation. This is a defined minimal medium and does not support growth when a carbon source is omitted, making it suitable for selection of nutritional markers as well as the study of biomass utilization by C. bescii. A low osmolarity complex growth medium (LOC) was developed that dramatically improves growth and culture viability during storage, making it a better medium for routine growth and passaging of C. bescii. Both media contain significantly lower solute concentration than previously published media, allowing for flexibility in developing more specialized media types while avoiding the issues of growth inhibition and cell lysis due to osmotic stress. Plating on LOD medium solidified by agar results in ~1,000-fold greater plating efficiency than previously reported and allows the isolation of discrete colonies. These new media represent a significant advance for both genetic manipulation and the study of biomass utilization in C. bescii, and may be applied broadly across the Caldicellulosiruptor genus.     

Selection of tRNA charging quality control mechanisms that increase mistranslation of the genetic code

Mistranslation can follow two events during protein synthesis: production of non-cognate amino acid:transfer RNA (tRNA) pairs by aminoacyl-tRNA synthetases (aaRSs) and inaccurate selection of aminoacyl-tRNAs by the ribosome. Many aaRSs actively edit non-cognate amino acids, but editing mechanisms are not evolutionarily conserved, and their physiological significance remains unclear. To address the connection between aaRSs and mistranslation, the evolutionary divergence of tyrosine editing by phenylalanyl-tRNA synthetase (PheRS) was used as a model. Certain PheRSs are naturally error prone, most notably a Mycoplasma example that displayed a low level of specificity consistent with elevated mistranslation of the proteome. Mycoplasma PheRS was found to lack canonical editing activity, relying instead on discrimination against the non-cognate amino acid by kinetic proofreading. This mechanism of discrimination is inadequate for organisms where translation is more accurate, as Mycoplasma PheRS failed to support Escherichia coli growth. However, minor changes in the defunct editing domain of the Mycoplasma enzyme were sufficient to restore E. coli growth, indicating that translational accuracy is an evolutionarily selectable trait.     

Investigating the Diversity of Pseudomonas spp. in Soil Using Culture Dependent and Independent Techniques

Less than 1 % of bacterial populations present in environmental samples are culturable, meaning that cultivation will lead to an underestimation of total cell counts and total diversity. However, it is less clear whether this is also true for specific well-defined groups of bacteria for which selective culture media is available. In this study, we use culture dependent and independent techniques to describe whether isolation of Pseudomonas spp. on selective nutrient-poor NAA 1:100 agar-medium can reflect the full diversity, found by pyrosequencing, of the total soil Pseudomonas community in an urban waste field trial experiment. Approximately 3,600 bacterial colonies were isolated using nutrient-poor NAA 1:100 medium from soils treated with different fertilizers; (i) high N-level sewage sludge (SA), (ii) high N-level cattle manure (CMA), and (iii) unfertilized control soil (U). Based on Pseudomonas specific quantitative-PCR and Pseudomonas CFU counts, less than 4 % of Pseudomonas spp. were culturable using NAA 1:100 medium. The Pseudomonas selectivity and specificity of the culture medium were evaluated by 454 pyrosequencing of 16S rRNA gene amplicons generated using Bacteria- and Pseudomonas-specific primers. Pyrosequencing results showed that most isolates were Pseudomonas and that the culturable fraction of Pseudomonas spp. reflects most clusters of the total Pseudomonas diversity in soil. This indicates that NAA 1:100 medium is highly selective for Pseudomonas species, and reveals the ability of NAA 1:100 medium to culture mostly the dominant Pseudomonas species in soil.     

Clonal analysis of vertebrate myogenesis: IV. Medium-dependent classification of colony-forming cells

The cell lineage of chick leg muscle between 3 and 12 days of development has been studied by use of an in vitro clonal assay. The assay permits distinctions to be made among various types of muscle-colony-forming cells (MCF cells) on the basis of their medium requirements and clonal morphology. Results suggest the sequential occurrence of at least four types of MCF cells, three of which require conditioned medium for their differentiation and one of which can form differentiated colonies in fresh medium.The nature of the “conditioned medium effect” was further investigated by the use of medium-switch experiments. By this process it was shown that the same populations of colony-forming cells attach and grow in fresh and conditioned medium and that the differentiation of colonies derived from conditioned-medium-requiring myoblasts is permitted by brief exposure to conditioned medium followed by culture in fresh medium. Further investigation indicated that during brief exposure to conditioned medium the gelatin-coated petri plate surface is altered such that differentiation of conditioned-medium-requiring colonies is allowed. We conclude that the conditioned medium effect involves a surface-mediated interaction between myoblasts and one or more conditioned medium components.     

Light Enhances Survival of Dinoroseobacter shibae during Long-Term Starvation

Aerobic anoxygenic phototrophs (AAPs) as being photoheterotrophs require organic substrates for growth and use light as a supplementary energy source under oxic conditions. We hypothesized that AAPs benefit from light particularly under carbon and electron donor limitation. The effect of light was determined in long-term starvation experiments with Dinoroseobacter shibae DFL 12^T in both complex marine broth and defined minimal medium with succinate as the sole carbon source. The cells were starved over six months under three conditions: continuous darkness (DD), continuous light (LL), and light/dark cycle (LD, 12 h/12 h, 12 µmol photons m⁻² s⁻¹). LD starvation at low light intensity resulted in 10-fold higher total cell and viable counts, and higher bacteriochlorophyll a and polyhydroxyalkanoate contents. This coincided with better physiological fitness as determined by respiration rates, proton translocation and ATP concentrations. In contrast, LD starvation at high light intensity (>22 µmol photons m⁻² s⁻¹, LD conditions) resulted in decreasing cell survival rates but increasing carotenoid concentrations, indicating a photo-protective response. Cells grown in complex medium survived longer starvation (more than 20 weeks) than those grown in minimal medium. Our experiments show that D. shibae benefits from the light and dark cycle, particularly during starvation.     

Mycoplasma tauri sp. nov. isolated from the bovine genital tract

Since 2006, a Mycoplasma species unidentifiable to the species level has been regularly isolated from the semen and prepuce of apparently healthy bulls, and occasionally from cattle displaying inflammatory disease of the genital tract. Seven of these Mycoplasma isolates were subjected to a comprehensive taxonomic study. The strains investigated grew well in modified Hayflick’s medium and colonies on agar exhibited typical fried egg morphology and produced ‘film and spots’. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas with spherically shaped cells bounded by a bi-layered cell membrane. The strains studied neither produced acid from sugar carbon sources nor did hydrolyse arginine or urea, and genome annotation indicated that organic acids (pyruvate, lactate) are used as energy sources. Phylogenetic analyses of 16S rRNA gene sequences, the 16S-23S intergenic spacer region, and partial rpoB gene and protein sequences placed the strains within the Mycoplasma (M.) bovis cluster of the Hominis group with M. primatum, M. agalactiae, and M. bovis being their closest relatives. Genomic information including whole-genome similarity metrics (ANIb, ANIm, TETRA, dDDH, AAI) and phylogenomics, proteomic features revealed by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry as well as serological reactions and polar lipid profiling strongly indicated that the strains examined were representatives of a hitherto unclassified species of genus Mycoplasma, for which the name Mycoplasma tauri sp. nov. with type strain Zaradi2^T (=ATCC BAA-1891^T = DSM 22451^T) is proposed.     

Size-dependent growth of Microcystis colonies in a shallow,hypertrophic lake: use of the RNA-to-total organic carbon ratio

Microcystis was cultured under standard conditions in BG-11 and M-11 media. Using results of an analysis of RNA and total organic carbon (TOC) content, a significant logarithmic relationship between Microcystis growth rate and the RNA/TOC ratio was described to measure the growth rate. Colonial Microcystis samples collected in a shallow, hypertrophic lake (Lake Taihu, China) during May–November 2012 were divided into six size classes (<75, 75–100, 100–150, 150–300, 300–500, and >500 μm), and the RNA/TOC ratio of each class was analyzed to evaluate differences in growth. The growth rate of colonies in the 150–300-μm size class was highest from May to August, but the growth rate increased along with the increase in colony size from September to November. Our results also indicated that water temperature did not change the relationship between growth rate and colony size, but the growth rate of larger colonies was higher than the growth rate of smaller colonies at conditions of low total nitrogen, low total dissolved phosphorus concentration, and high light intensity. Taken together, these results suggest that large colonial Microcystis possess an advantage that is a consequence of this faster growth at lower nutrient concentrations and high light intensities.     

Dormancy, Turion Formation, and Germination by Different Clones of Spirodela polyrrhiza

Some clones of Spirodela polyrrhiza form dormant bodies called turions which require several weeks of chilling treatment before they proceed to renew growth and develop into vegetative fronds. The individual fronds of Spirodela are less than 5 mm long and can be grown aseptically in liquid culture. Turion formation and germination can serve as a bioassay for the various compounds involved in dormancy development.

Turion formation can be induced by manipulation of light intensity during the day, photoperiod, night temperature, day temperature, and concentration of nitrate in the culture medium. Different clones of Spirodela from northeastern United States, Puerto Rico, and Argentina had different requirements for turion formation. The clones from Argentina and Puerto Rico did not form turions under any of the experimental conditions imposed. Turions of some clones required chilling treatments for renewed vegetative growth while others did not. Both gibberellic acid and long photoperiods were required to bypass the chilling requirements of some clones, but not others.

     

Changes in extracellular polysaccharide content and morphology of Microcystis aeruginosa at different specific growth rates

The cyanobacterium Microcystis mainly exists in colonies under natural conditions but as single cells in typical laboratory cultures. Understanding the mechanism by which single cells form small and large colonies can provide a deeper insight into the life history of Microcystis and the mechanisms of Microcystis bloom formation. In this paper, Microcystis aeruginosa cultured under varying light intensities and temperatures exhibited different specific growth rates. Correlations were found between the specific growth rate, extracellular polysaccharide (EPS) content, and morphology of M. aeruginosa. Under low light intensities and temperatures, M. aeruginosa formed small colonies (maximum colony size approximately 100 μm) and exhibited low specific growth rates. By contrast, standard culture conditions yielded single or paired cells with high specific growth rates. Moreover, the EPS content decreased dramatically with increasing specific growth rate. A significant positive linear relationship was observed between the EPS content per cell and colony size. High EPS content and colony formation were associated with low specific growth rates. The specific growth rate in laboratory cultures was higher than the in situ growth rate under natural conditions. This result may explain why Microcystis normally exists as single cells or (more rarely) as paired cells in axenic laboratory cultures after long-term cultivation, but forms colonies under natural conditions.     

Biofilm Dispersal of Neisseria subflava and Other Phylogenetically Diverse Oral Bacteria

Polystyrene petri dishes containing liquid medium were inoculated with single-cell suspensions of a fresh clinical isolate of Neisseria subflava and were incubated under conditions of low vibration. N. subflava colonies grew firmly attached to the surface of the dish, while the broth remained clear. Growing colonies released cells into the medium, resulting in the appearance of 10² to 10⁴ small satellite colonies attached to the surface of the dish in an area adjacent to each mature colony after 24 h. Satellite colonies grew in patterns of streamers shaped like jets and flares emanating from mature colonies and pointing toward the center of the dish. This dispersal pattern evidently resulted from the surface translocation of detached biofilm cells by buoyancy-driven convection currents that were generated due to slight temperature gradients in the medium. Streamers of satellite colonies ranged from 2 to >40 mm in length. Satellite colonies in very long streamers were relatively uniform in size regardless of their distance from the mature colony, suggesting that mature colonies released single cells or small clusters of cells into the medium and that the detachment, surface translocation, and subsequent surface reattachment of released cells were a transitory process. Incubation of N. subflava single cells in a perfused biofilm fermentor resulted in a large spike of the number of CFU in the perfusate after 9.5 h of growth, consistent with a rapid release of cells into the medium. Biofilm colonies of several other phylogenetically diverse oral bacteria, including Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Streptococcus mitis, and a prevalent but previously uncultured oral Streptococcus sp., exhibited similar temperature-dependent dispersal patterns in broth culture. This in vitro spreading phenotype could be a useful tool for studying biofilm dispersal in these and other nonflagellated bacteria and may have physiological relevance to biofilm dispersal in the oral cavity.     

A comparison between coral colonies of the genus Madracis and simulated forms

In addition to experimental studies, computational models provide valuable information about colony development in scleractinian corals. Using our simulation model, we show how environmental factors such as nutrient distribution and light availability affect growth patterns of coral colonies. To compare the simulated coral growth forms with those of real coral colonies, we quantitatively compared our modelling results with coral colonies of the morphologically variable Caribbean coral genus Madracis. Madracis species encompass a relatively large morphological variation in colony morphology and hence represent a suitable genus to compare, for the first time, simulated and real coral growth forms in three dimensions using a quantitative approach. This quantitative analysis of three-dimensional growth forms is based on a number of morphometric parameters (such as branch thickness, branch spacing, etc.). Our results show that simulated coral morphologies share several morphological features with real coral colonies (M. mirabilis, M. decactis, M. formosa and M. carmabi). A significant correlation was found between branch thickness and branch spacing for both real and simulated growth forms. Our present model is able to partly capture the morphological variation in closely related and morphologically variable coral species of the genus Madracis.