Detection of Legionella in Various Sample Types Using Whole-Cell Fluorescent In Situ Hybridization
3.3. Detection and Quantification of Legionella in Water Samples 1. Sterilize the filtration surface of each unit of the filtration system with 100%
EtOH.
2. Place a filter membrane (with the shiny side on top) on each filter unit.
3. Put on a sterile funnel.
4. Poor 100 mL of each Legionella-contaminated water sample into a funnel, turn on the filtration system at –5 kPa and refill the funnels if necessary.
5. After filtration, turn off the pump and close each filter unit, overlay each mem- brane with 10 mL of 4% PFA buffer for 1 h.
6. Discharge the PFA buffer by turning the pump on.
7. Wash the filters twice with 1X PBS.
8. Dehydrate through a 50 to 96% EtOH series.
9. Remove the funnels and let each filter air dry.
10. Before staining, cut the filter in four pieces by using the scalpel.
11. Bring each piece on a precleaned microscopic slide.
12. Apply 20 µL of the probe solution and hybridize (see Subheading 3.1.).
13. Remove the hybridization buffer by putting the filter pieces on tissue and wash with washing buffer.
14. Put the pieces into plastic jars filled with washing buffer and incubate for 20 min at 46°C.
15. Remove the washing buffer by putting the filter pieces on tissue and rinse them one last time with double distilled water.
16. Let the filters dry thoroughly and analyze them or store them at –20°C.
17. For the microscopic analyses put each filter piece on a cleaned slide (100% EtOH) with a few drops of Citifluor.
18. For the quantification of the cells, scan each filter piece and count each field using an objective with a counting grid (see Note 18).
4. Notes
1. In the current protocol, the Cy3 and FITC staining are described. However, there are many other possibilities to use other direct labels (e.g., like Cy5, TexasRed;
consult http://www.probes.com/handbook/). The choice of the labels frequently depends on the available filter sets of the fluorescence microscope.
2. In environmental samples, the detection of cells is achieved more easily with Cy3 or Cy5 fluorochromes because these labels are much brighter and more stable than the classic fluorescein and rhodamine-derivates.
3. Kept at –20°C, the probe stock solutions, like the working solutions, are stable for years. It is recommended that one divide the working solutions into small aliquots to prevent frequently thawing and freezing of the oligonucleotide probes.
4. When analyzing fluorescent samples, it is important to choose immersion oil, which exhibits a very low or virtually zero background fluorescence.
5. Always prepare this solution under the hood. Once divided into aliquots and fro- zen at –20°C, the PFA solution is stable for up to 1 yr. When thawed, the PFA has to be stored at 4°C. At this temperature, the PFA is stable for a few weeks.
6. Hybridization buffer and washing buffer need to be freshly prepared every time.
7. Fluorescence filters are always a compromise between selectivity and through- put. Single-band filter sets provide the best compromise between these two crite- ria, whereas multiple-band filter combinations can be used for the simultaneous observation of several dyes.
8. It is recommended that one always air dry the slides in a flow to prevent the sticking of dust particles onto the slides. This sticking can disturb the micro- scopical analysis because of the possible auto-fluorescence of some of those par- ticles.
9. Gram-negative bacteria like Legionella are normally fixed in PFA buffer and need no additorial permeabilization before hybridization. Gram-positive cells can be fixed in 50% EtOH and permeabilized by exposure to lysozyme (12).
10. At this point, the dehydrated slides can be hybridized immediately or stored free of dust at room temperature for up to 3 wk until staining. If longer storage is needed, slides are stable at –20°C for several months.
11. From this step forward, avoid light as much as possible.
12. For double staining using both the LEG705 Cy3 and the LEGPNE1 FITC probes, add 10 µL of each probe solution, mix gently in the well and proceed the hybrid- ization as described for single staining.
13. The hybridization of the samples must always be performed in a moist chamber to minimize the evaporation of water, which would otherwise alter the hybridiza- tion conditions in an uncontrolled manner.
14. During the washing steps, it is important to prevent slide surfaces from drying out; otherwise, background problems may arise.
15. One significant problem when using fluorescent dyes is bleaching of the fluores- cence signal while being analyzed over time. Exposure times of minutes or even several seconds may have a critical effect on the signal destruction. This problem can be reduced by mounting the sample in an antifade solution such as Citifluor.
Other suggestions to avoid the rapid destruction of the fluorescence signal are the use of narrow band filters and photostable dyes.
16. For the analysis of FISH stained Legionella in environmental samples, such as in biofilms or in filtered water samples, disturbing auto fluorescence signals can be present. These signals can originate from micro-organisms like moulds, yeasts, or bacteria such as Pseudomonas but also from the surrounding biofilm materials like biological and inorganic debris or algae. It is recommended that one use narrow-band filters and that one check first for auto fluorescence when dealing with environmental samples.
17. After the fixation step, biofilms can be stored at 4°C in PBS for a maximum of 3 d.
18. Although normally highly abundant, the rRNA content of bacterial cells may vary considerably, not only between species, but also between cells of one strain according to their physiological state, which is directly correlated with their growth rate (13). Low physiological activity can thereby result in low signal intensity or false-negative results. This problem can be solved by placing the filters on a growth medium that stimulates the rRNA production in the bacteria cells. Another solution is to use two specific probes that each target a different position of the 16S rRNA and that are labeled with different fluorochromes.
However, this approach is restricted by the limited availability of specific tar- get sequences for the respective micro-organism.
Acknowledgments
The authors gratefully thank Joost Vanoverbeke for his critical review of the manuscript and his valuable comments. This work was funded by the IWT (GBOU no.° 20153).
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From: Methods in Molecular Biology, vol. 345: Diagnostic Bacteriology Protocols, Second Edition Edited by: L. O‘Connor © Humana Press Inc., Totowa, NJ