Viable cell enumeration of luciferase-marked strains can be achieved using traditional dilution plate counting and luminescent colonies can usually be detected by the unaided eye in a darkened room.32 Greater sensitivity can be achieved using photographic film (with long exposure), X-ray film or photon imaging (see below). The majority of luciferase-marked strains are also marked with antibiotic resistance (see Chapter 2), enabling selectivity against indigenous populations by enumeration on media supplemented with the appropriate antibiotic. In the absence of antibiotic resistance markers, selective enumeration depends on the competitive ability of the marked strain on enumeration media, the nutrients required for luminescence and the sensitivity of detection. Nevertheless, visual detection of a single luminescent colony of lux-marked Erwinia carotovora is possible in the presence of 3000 nonluminescent colonies.32 Similar techniques may be used for in situ detection of luminescent strains. Photography has also been used to compare in vivo activities of firefly and click
Fig. 5.4. Luminescence emitted by two strains of Pseudomonas putida containing plasmids pAP2 and pACR3 with PR::luc and PR::lucOR fusions, respectively. Cells were incubated on nitrocellulose filters placed on solid medium, incubated for 30 h and photographed under normal illumination (A) and after 30 min exposure in the dark. Reprinted with permission from Cebolla A, Vázquez ME, Palomares AJ. Appl Environ Microbiol 1995; 61:660-668.
beetle luaferase in E. coli cultures on nitrocellulose filters, with exposure for 10-15 minutes.30 Similar examination of E. coli streaks on solid media demonstrated the four colors of bioluminescence emitted by different click beetle luciferases, with 2 s exposure time.33 This approach has also been used to determine regions of plants infected with lux-marked pathogenic bacteria34 and for detection of luciferase activity in alfalfa nodules induced by R. meliloti containing a nifH::luc fusion.35
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