Comparison of two methods for enumeration of mycoplasmas

Stemke, Gerald W.; Robertson, Janet A.

doi:10.1128/jcm.16.5.959-961.1982

Cited by 48 publications

(36 citation statements)

References 14 publications

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“…-10 6 M. pneumoniae CCU(Stemke & Robertson, 1982) was added apically to initiate infections (eight wells per strain, dilution, and time point). For some experiments, we included infections initiated with 10 3 or 10 9 CCU, as indicated.…”

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confidence: 99%

Modelling persistentMycoplasma pneumoniaeinfection of human airway epithelium

Prince

Krunkosky

Sheppard

et al. 2017

Cellular Microbiology

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Mycoplasma pneumoniae is a human respiratory tract pathogen causing acute and chronic airway disease states that can include long-term carriage and extrapulmonary spread. The mechanisms of persistence and migration beyond the conducting airways, however, remain poorly understood. We previously described an acute exposure model using normal human bronchial epithelium (NHBE) in air-liquid interface culture, showing that M. pneumoniae gliding motility is essential for initial colonisation and subsequent spread, including localisation to epithelial cell junctions. We extended those observations here, characterizing M. pneumoniae infection of NHBE for up to 4 weeks. Colonisation of the apical surface was followed by pericellular invasion of the basolateral compartment and migration across the underlying transwell membrane. Despite fluctuations in transepithelial electrical resistance and increased NHBE cell desquamation, barrier function remained largely intact. Desquamation was accompanied by epithelial remodelling that included cytoskeletal reorganisation and development of deep furrows in the epithelium. Finally, M. pneumoniae strains S1 and M129 differed with respect to invasion and histopathology, consistent with contrasting virulence in experimentally infected mice. In summary, this study reports pericellular invasion, NHBE cytoskeletal reorganisation, and tissue remodelling with persistent infection in a human airway epithelium model, providing clear insight into the likely route for extrapulmonary spread.

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confidence: 99%

Modelling persistentMycoplasma pneumoniaeinfection of human airway epithelium

Prince

Krunkosky

Sheppard

et al. 2017

Cellular Microbiology

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“…The GC ⁄ CFU ratios observed in this study for several mycoplasmas are in good concordance with the GC ⁄ CFU and GC ⁄ colour changing units (CCU) previously determined for Ureaplasma urealyticum and Myc. hominis (Stemke and Robertson 1982). The results of that study showed the ratios between genome equivalents (calculated on basis of the amount of isolated genomic DNA) and CFU determined for U. urealyticum and Myc.…”

Section: Culture With Mammalian Cells (Suspension Of Cho Cells)mentioning

confidence: 87%

Preparation of reference strains for validation and comparison of mycoplasma testing methods

Dabrazhynetskaya

Volokhov

David

et al. 2011

Journal of Applied Microbiology

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Aims: To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC) number to that of colony forming units (CFU). These stocks are required for assessment of relative limits of detection (LOD) of alternative nucleic acid testing (NAT)‐based methods in comparison to the conventional microbiological methods. Methods and Results: A kinetics study was used to assess the changes in ratios between the numbers of GC and CFU at different growth phases of six different mycoplasma cultures Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orale and Mycoplasma pneumoniae. All tested mycoplasmas demonstrated low GC/CFU ratios (≤10) within the log and early stationary growth phases. A significant increase in GC/CFU ratios was observed at the very late stationary and death phases, when the titre of cultures has declined. Similar patterns of GC/CFU profiles were observed for A. laidlawii and Myc. gallisepticum co‐cultured with suspension of Chinese hamster ovary (CHO) cells. Conclusions: Tested mycoplasma strains harvested at the exponential‐early stationary phases of growth demonstrated the lowest GC/CFU ratios and low propensity to form filamentous structures or aggregates under proposed conditions and can be used for the preparation of a mycoplasma reference panel for methods comparability study. Significance and Impact of the Study: This study shows that the preparation and use of viable mycoplasma reference strains with low CG/CFU ratios is the most reliable way to adequately evaluate the LOD of alternative NAT‐based mycoplasma testing methods.

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“…Mycoplasmas are the smallest and simplest free-living parasitic organisms implicated in a variety of plant and animal diseases . Although several techniques have been proposed to assess the number of cultured Mollicutes cells (Snell 1981;Albers and Fletcher 1982;Stemke and Robertson 1982;Meur et al 1989;Robertson and Stemke 1995;Lin et al 2001) accurate determination of Mycoplasma growth still remains a difficult task (Robertson and Stemke 1995). For example, light microscopy determination is hampered by the small size of these organisms (0AE2-0AE7 lm) and viable cell estimation is additionally complicated by the complex nutritional requirements of Mollicutes cells (Stemke and Robertson 1982;Robertson and Stemke 1995).…”

Section: Introductionmentioning

confidence: 99%

“…Although several techniques have been proposed to assess the number of cultured Mollicutes cells (Snell 1981;Albers and Fletcher 1982;Stemke and Robertson 1982;Meur et al 1989;Robertson and Stemke 1995;Lin et al 2001) accurate determination of Mycoplasma growth still remains a difficult task (Robertson and Stemke 1995). For example, light microscopy determination is hampered by the small size of these organisms (0AE2-0AE7 lm) and viable cell estimation is additionally complicated by the complex nutritional requirements of Mollicutes cells (Stemke and Robertson 1982;Robertson and Stemke 1995). In this context, while protein quantification is a simple and a widely used method to determine cell number, cellular proteins constitute only a small fraction of the total protein concentration of the highly supplemented media required for Mollicutes growth, thus supporting the notion that this approach is unreliable to estimate the growth of these organisms (Robertson and Stemke 1995).…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, increases in turbidity do not always correlate with growth of Mycoplasma species and may simple reflect the precipitation of medium components as a result of pH changes resulting from substrate utilization (Stemke and Robertson 1990). In addition, colour-changing units (CCU) have been used to determine the growth of several Mycoplasma species as an indicator of pH changes of the medium (Stemke and Robertson 1982). While CCU or CCU 50 (colour-change unit 50 ) determination provides an alternative assay to estimate total Mollicutes cell number (Stemke and Robertson 1982) it is worthwhile noting that not all Mycoplasma cause a medium colour change and, similar to the turbidimetric method, the accuracy of this assay is additionally hampered by the fact that colour changes do not always coincide with growth (Stemke and Robertson 1990).…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of Mycoplasma hyopneumoniae growth by flow cytometry

Assunção¹,

Rivera²,

Comas³

et al. 2005

J Appl Microbiol

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Aims: In the present study we evaluated the potential application of the flow cytometry (FC) technique to determine the growth rates of Mycoplasma hyopneumoniae in a broth medium. Methods and Results: The FC analysis was performed using the fluorochromes Syto 9, propidium iodide (PI) or a combination of both dyes and results were compared with those obtained by colour-changing units (CCU) and pH measurements. While CCU counts ml )1 were higher than those obtained from the FC technique, a good relation between M. hyopneumoniae growth rates was observed in the different phases of the growth curve (logarithmic, stationary and senescence phases). Labelling with Syto 9 alone was sufficient to differentiate M. hyopneumoniae cells with different amounts of nucleic acids, in the stationary and senescence phase of the M. hyopneumoniae growth curve. PI labelling did not detect cell death in the end phase of M. hyopneumoniae growth. Conclusions: These data show that FC is a very useful, practical and fast technique to study the growth rates of M. hyopneumoniae in broth medium. Significance and Impact of the Study: This is to our knowledge the first application of FC to the study of M. hyopneumoniae growth in broth culture.

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Comparison of two methods for enumeration of mycoplasmas

Cited by 48 publications

References 14 publications

Modelling persistentMycoplasma pneumoniaeinfection of human airway epithelium

Modelling persistentMycoplasma pneumoniaeinfection of human airway epithelium

Preparation of reference strains for validation and comparison of mycoplasma testing methods

Evaluation of Mycoplasma hyopneumoniae growth by flow cytometry

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