Simple method for quantitation of viable mycoplasmas

The detection of mycoplasma in milk can be performed by either culture techniques or polymerase chain reaction (PCR) based methods. Although PCR can reduce the average diagnostic time to 5 h in comparison with the several days for the isolation of the agent, there is still a need to develop methods, which could give earlier results. For this purpose, we tested the ability of flow cytometry (FC) to detect mycoplasmas in milk samples. Milk samples inoculated with four different mycoplasmas, Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. Capricolum, or Mycoplasma mycoides subsp. mycoides large-colony type, known to cause contagious agalactia in goats, were stained with the DNA stain SYBR Green I and analyzed by FC. Three goat milk samples, from which mycoplasmas have been isolated in broth medium were also analyzed. All mycoplasmas were easily distinguished from debris of milk samples, but it was not possible to distinguish between the different mycoplasma species. In our conditions, the detection limit of the technique was of the order of 10 3 -10 4 cells ml 21 . Furthermore, mycoplasmas were also distinguished from Staphylococcus aureus. FC together with SYBR Green I was able to distinguish between mycoplasma cells and debris present in milk samples and gave results in 20-30 min. This is an important first step in developing a robust, routine flow cytometric method for the detection of mycoplasmas in milk samples. '

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“…had been detected using culture‐based techniques, were also analyzed by FC. The number of CFU was determined as described elsewhere (13).…”

Section: Methodsmentioning

confidence: 99%

Detection of mycoplasmas in goat milk by flow cytometry

et al. 2007

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“…Other field strains used in this study (2193, Emi 2, 207/93, 153/93, B93, IU, ELZ, TR, N5, AC 33) were isolated from clinical cases (milk, ear swabs) submitted to the Department of Epidemiology and Preventive Medicine of the Las Palmas de Gran Canaria University (Spain) between 1993 and 2002. All mycoplasmas were propagated at 37°C in PH broth medium under aerobic conditions (Kirchhoff and Rosengarten 1984) and time-matched samples (0-120 h, depending on the type of experiment) were taken to perform CFU as described elsewhere (Albers and Fletcher 1982) and FC analysis (see below). All measurements were performed in triplicate and experiments were repeated at least three times on different days.…”

Section: Strains and Culture Conditionsmentioning

confidence: 99%

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

Assunção

Antunes

et al. 2006

J Appl Microbiol

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Aims: The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green‐I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large‐colony type (MmmLC) in broth culture was examined. Methods and Results: The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green‐I (SYBR), and the results were compared with plate count method (colony forming units, – CFUs). There was a good correlation (linear regression, r2 = 0·93) between mycoplasma counts determined by FC (cells ml−1) and by traditional plate count method (CFU ml−1). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 104 cells ml−1 and 103 CFU ml−1, respectively. FC method allowed results in 20–30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU). Conclusion: Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method. Significance and Impact of the Study: These findings suggest that FC could be a good alternative to replace other time‐consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).

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“…A stock culture was harvested at late log-phase, aliquoted to 1 ml stocks and frozen at Ϫ70°C without the addition of cryoprotective agents. Determination of growth and survival of the mycoplasma cells was performed by the colony count method (microtiter system) suggested by Albers and Fletcher (1982).…”

Section: Bacterial Strain and Growth Conditionsmentioning

confidence: 99%

A model of the deciliation process caused by Mycoplasma fermentans strain incognitus on respiratory epithelium

Stadtländer

2006

Scanning

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The aim of this study was to investigate by light microscopy as well as by scanning and transmission electron microscopy the deciliation process which takes place on the respiratory epithelium of tracheal explants after experimental infection with Mycoplasma fermentans strain incognitus. Time-point photography allowed distinguishing five phases which occurred during the infection on the epithelial cell surface: (1) Attachment of M. fermentans to the cilia causing clumping of the cilia tips; (2) matting of cilia into bundles; (3) formation of abnormally shaped and shorter cilia; (4) collapse of cilia onto the epithelial cell surface; and (5) widespread loss of cilia. Based on the photographic images, a schematic model of the deciliation process was developed. Various potential factors contributing to the cilia destruction are discussed, including the release of mycoplasmal toxins, the physical presence of a high number of M. fermentans cells attached to the cilia, and the depletion of culture medium components by the mycoplasmas. This model of M. fermentans strain incognitus infection of respiratory epithelium is important for understanding mycoplasmal pathogenicity on a comparative level.

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Simple method for quantitation of viable mycoplasmas

Cited by 56 publications

References 6 publications

Detection of mycoplasmas in goat milk by flow cytometry

Detection of mycoplasmas in goat milk by flow cytometry

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

A model of the deciliation process caused by Mycoplasma fermentans strain incognitus on respiratory epithelium

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