Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA).
A method is described to discriminate between live and dead cells of the infectious fungi Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Mucor racemosus, Rhizopus stolonifer and Paecilomyces variotii. To test the method, conidial suspensions were heat inactivated at 85 degrees C or held at 5 degrees C (controls) for 1 h. Polycarbonate filters (25 mm diameter, 0.8 microm pore size) were placed on "welled" slides (14 mm diameter) and the filters treated with either PBS or PMA. Propidium monoazide (PMA), which enters dead cells but not live cells, was incubated with cell suspensions, exposed to blue wavelength light-emitting diodes (LED) to inactivate remaining PMA and secure intercalation of PMA with DNA of dead cells. Treated cells were extracted and the live and dead cells evaluated with quantitative PCR (QPCR). After heat treatment and DNA modification with PMA, all fungal species tested showed an approximate 100- to 1000-fold difference in cell viability estimated by QPCR analysis which was consistent with estimates of viability based on culturing.
Vesper
Stephen
S
National Exposure Research Laboratory, United States Environmental Protection Agency, 26 West Martin Luther King Ave., Cincinnati, OH 45268, USA. vesper.stephen@epa.gov
McKinstry
Craig
C
Hartmann
Chris
C
Neace
Michelle
M
Yoder
Stephanie
S
Vesper
Alex
A
D000391:Air Microbiology; D001386:Azides / Q000494:pharmacology*; D015169:Colony Count, Microbial; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005658:Fungi / Q000187:drug effects* / Q000235:genetics; D050296:Microbial Viability / Q000187:drug effects*; D016533:Mycological Typing Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives* / Q000494:pharmacology; D014871:Water Microbiology
D001386:Azides; D004271:DNA, Fungal; D011419:Propidium; C064136:monoazidopropidium