Cell sorting of formalin treated pathogenic Mycobacterium paratuberculosis expressing GFP

N. B. Harris, D. K. Zinniel, M. K. Hsieh, J. D. Cirillo, Raul G Barletta

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

GFP is widely used as a molecular tool for the study of microbial pathogens. However, the manipulation of these pathogenic microorganisms poses a health threat to the laboratory worker, requiring biosafety level II or III containment. Although the GFP fluorophore is tolerant to formalin, a thorough analysis of this treatment on fluorescent output in prokayotic systems has not been described. In addition, the analysis of microorganisms expressing GFP often depends on specialized equipment, which may not be housed in biosafety level II or III laboratories. Therefore, we sought to develop a safe and effective method for manipulating the GFP-expressing pathogenic bacterium Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) utilizing a formalin treatment that would permit the analysis of GFP fluorescence without requiring stringent biosafety containment. We demonstrate that formalin-treated M. paratuberculosis expresses 50% less fluorescence than viable cells, but this reduction is still compatible with spectrofluorometry and cell sorting. Furthermore, plasmid DNA that expresses GFP can be recovered efficiently from nonviable, sorted fluorescent cells. This approach is flexible, provides an additional margin of safety for laboratory personnel, and can be easily applied to other pathogenic microorganisms expressing GFP.

Original languageEnglish (US)
Pages (from-to)522-527
Number of pages6
JournalBioTechniques
Volume32
Issue number3
StatePublished - Mar 20 2002

Fingerprint

Mycobacterium avium subsp. paratuberculosis
Sorting
Microorganisms
Formaldehyde
Cells
Fluorescence
Paratuberculosis
Laboratory Personnel
Mycobacterium avium
Fluorophores
Fluorescence Spectrometry
Pathogens
Bacteria
Plasmids
Health
Personnel
Safety
Equipment and Supplies
DNA
Therapeutics

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Harris, N. B., Zinniel, D. K., Hsieh, M. K., Cirillo, J. D., & Barletta, R. G. (2002). Cell sorting of formalin treated pathogenic Mycobacterium paratuberculosis expressing GFP. BioTechniques, 32(3), 522-527.

Cell sorting of formalin treated pathogenic Mycobacterium paratuberculosis expressing GFP. / Harris, N. B.; Zinniel, D. K.; Hsieh, M. K.; Cirillo, J. D.; Barletta, Raul G.

In: BioTechniques, Vol. 32, No. 3, 20.03.2002, p. 522-527.

Research output: Contribution to journalArticle

Harris, NB, Zinniel, DK, Hsieh, MK, Cirillo, JD & Barletta, RG 2002, 'Cell sorting of formalin treated pathogenic Mycobacterium paratuberculosis expressing GFP', BioTechniques, vol. 32, no. 3, pp. 522-527.
Harris, N. B. ; Zinniel, D. K. ; Hsieh, M. K. ; Cirillo, J. D. ; Barletta, Raul G. / Cell sorting of formalin treated pathogenic Mycobacterium paratuberculosis expressing GFP. In: BioTechniques. 2002 ; Vol. 32, No. 3. pp. 522-527.
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