 Figure 1: FISH-Based Flow Cytometric Detection of Listeria monocytogenes in Mixed Culture. A mixed culture of L. monocytogenes Scott A and Lactobacillus fermentum was grown for one hour in a non-selective medium (MRS broth). A 1 ml sample of culture was removed and fixed for 15 min in a 50:50 mixture of ethanol and phosphate buffered saline. A 100 µl portion of this sample was hybridized (55°C, 10 min, 200 pmol ml-1 probe) with a fluorescein-labeled peptide nucleic acid (PNA) probe targeting the 16S rRNA of Listeria spp. Samples were then analyzed by flow cytometry (BD FACSCalibur). In this experiment, a minority of L. monocytogenes (25.8% of 20,000 events) was easily detected against a large background of L. fermentum (74.2% of 20,000 events). Data were analyzed using FloJo software (TreeStar, Inc.).  Figure 2: FISH-Based Detection of Listeria monocytogenes in Hot Dog Wash. Hot dogs were purchased from a local grocery store, inoculated with L. monocytogenes, vacuum-packaged and stored for one week under conditions of mild temperature abuse (8°C). At sampling time, contaminated and control packages were opened aseptically and hot dogs were washed with 10 ml of a dilute, non-selective medium (1/4-strength MRS broth). Samples of hot dog wash (1 ml) were prepared for hybridization with a 10 min fixation in a 50:50 mixture of ethanol and phosphate buffered saline. Alternatively, some samples were incubated at 35°C for 70 min prior to fixation. One hundred microliter portions of either sample were hybridized (55°C, 15 min, 200 pmol ml-1 probe) with a fluorescein-labeled peptide nucleic acid (PNA) probe targeting the 16S rRNA of Listeria spp. Samples were then analyzed by flow cytometry (BD FACSCalibur). Listeria populations were readily detectable in all contaminated samples (right hand panels, top and bottom). These data indicate that L. monocytogenes remains active at refrigeration temperatures, producing sufficient rRNA to permit FISH-based detection. Also, separation of Listeria-specific fluorescence signals from background was enhanced after incubation, indicating increased production of target rRNA during this brief nutrient amendment. Data were analyzed using FloJo software (TreeStar, Inc.).  Figure 3: FISH-Based Detection of Listeria monocytogenes in Hot Dog Wash. Data from the same experiment as in Figure 2 were analyzed using WinMDI and labeled using Adobe Photoshop.   Figure 4: Effect of Gating Solution on Sensitivity of FISH-Based Detection of L. monocytogenes in Sliced Turkey Wash. Sliced turkey was purchased from a local grocery store, inoculated with L. monocytogenes, vacuum-packaged and stored for one week under conditions of mild temperature abuse (8°C). At sampling time, packages were opened aseptically and turkey slices were washed with 5 ml of a dilute, non-selective medium (1/4-strength MRS broth). Samples of turkey wash (1 ml) were prepared for hybridization with a 10 min fixation in a 50:50 mixture of ethanol and phosphate buffered saline. One hundred microliter portions of fixed turkey wash were hybridized (55°C, 15 min, 200 pmol ml-1 probe) with a fluorescein-labeled peptide nucleic acid (PNA) probe targeting the 16S rRNA of Listeria spp. Samples were then analyzed by flow cytometry (BD FACSCalibur). Because turkey samples contained a high background of particulate matter, Listeria spp. were not easily detected in ungated samples. In panel A, 99.7% of all events collected were background, and only 105 cells of L. monocytogenes (circular gate) were detected (50,000 total events collected). In panel B, the same sample was analyzed, but an internal reference culture of L. monocytogenes was used as a guide in setting a gating solution for the detection of FISH-positive Listeria cells. Here, 2,611 cells of L. monocytogenes were detected (out of 6,800 total events collected). Movie 1: Binding Positions on the 23S Ribosomal Subunit of Various FISH Probes Derived From the Scientific Literature. Still images of probe binding positions were generated in Protein Explorer (http://www.proteinexplorer.org) using the Escherichia coli-derived data on 23S rRNA structure of Mueller et al. (2000). A series of still images were then compiled into a QuickTime movie using Moover software. Probe specificities and binding positions (E. coli base numbering) are given below:
References Amann, R.I., Ludwig, W. and K-H. Schleifer. 1995. Phylogenetic Identification and In Situ Detection of Individual Microbial Cells Without Cultivation. Microbiological Reviews 59(1): 143-169. Mueller, F., Sommer, I., Baranov, P., Matadeen, R., Stoldt, M., Wohnert, J., Gorlach, M., van Heel, M. and R. Brimacombe. 2000. The 3D Arrangement of the 23S and 5S rRNA in the Escherichia coli 50S Ribosomal Subunit Based on a Cryo-Electron Microscopic Reconstruction at 7.5 Angstrom Resolution. J. Mol. Bio. 298: 35-59. Nordentoft, S., Christensen, H. and H.C. Wegener. 1997. Evaluation of a Fluorescence-Labelled Olgonucleotide Probe Targeting 23S rRNA for In Situ Detection of Salmonella Serovars in Paraffin-Embedded Tissue Sections and Their Rapid Identification in Bacterial Smears. Journal of Clinical Microbiology 35: 2642-2648. Veeh, R.H., Flood, J.A. and C.C. Davis. 2000. In situ Identification of Staphylococcus aureus by Whole-Cell Hybridization with Fluorescently-Labeled Oligonucleotide Probes. Poster Presented at The American Society for Microbiology’s 100th General Meeting, Los Angeles, CA. Acknowledgements: This work was supported in part by a grant from the North American Branch of the International Life Sciences Institute (ILSI N.A.). The opinions expressed herein are those of the authors and do not necessarily represent the views of ILSI. | ||||||
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