Frequently Asked Questions
Please refer to the package inserts for complete instructions for use. The information provided below are recommendations only. Check with your internal regulations and guidelines for procedures and requirements specific to your institution.
Can PNA FISH be used with all types of blood culture systems?
Yes, PNA FISH has been validated on the 3 major blood culture systems (bioMerieux BacT/ALERT, BD BACTEC, and Trek ESP). This includes the FAN BacT/ALERT bottles and the BACTEC bottles with Resin. Occasionally some of the resin beads will be present in the smears preventing the coverslip to seal the well. When this occurs, slide the coverslip away from the well, remove the bead with a sterile loop, and slide the coverslip back into place over the well.
Air bubbles under the coverslip during hybridization.
If air bubbles are present after placing the coverslip on the slides gently push down on the center of the coverslip. This action will force the air bubbles out of the well and allow for even coverage of the PNA probe solution.
Which filter is the correct filter for visualization of the slides?
If the fluorescence microscope is used for multiple applications with multiple filter sets it is important to return the dual band filter supplied by AdvanDx to the correct position before scoring the slides. The light that is emitted from the objective should be yellow in color when the dual band filter is in place.
How are the controls prepared?
Pre-fixed, kit specific controls slides are commercially available for each PNA FISH kit. If not using PNA FISH control slides, refer to the control slide guide or package insert for detailed instruction. It is important to make control slides from fresh liquid cultures only.
The fluorescent signal fades while viewing the slides.
This is a common occurrence with fluorescence microscopy known as bleaching. Excess exposure to the light source will cause bleaching of the signal. To see the fluorescence, move to another area of the slide. Always close microscope shutter while not viewing the slides.
Why can’t I see organisms on my negative control and negative slides?
PNA FISH is a presence/absence test, meaning positive is determined by presence of fluorescence and negatives are determined by its absence. Only positive organisms will be clearly visualized with the fluorescence microscope. Negative cells will have no or little fluorescence therefore they are difficult to visualize by fluorescence microscopy.
In some cases, negative cells can be seen with the FA scope, this is due to auto-flourescence of the cells. Because auto-fluorescence varies from cell to cell and it is weak in comparison to positive signal it is not always possible to see it by eye.
There is variability in the fluorescence from cell to cell.
Occasionally the fluorescence will vary from cell to cell. This is a normal aspect of FISH based tests. This can occur for several reasons, most commonly due to the variability of target (rRNA) concentrations from one cell to the next.
I can see only a few positive organisms on the slide.
Visualizing a few positive cells in each field of view is sometimes indicative of a mixed culture. When this occurs, one can switch to light microscopy and try to find other non-fluorescent cells. If the slide does have both fluorescent and non-fluorescent cells, the sample is most likely a mixed culture.
Can I reuse the wash solution?
No, regardless of how many slides were washed in the wash solution, it must be disposed of and replaced after each run.
The fluorescent signal is weak or decreased from previous runs.
Be sure that your microscope has had time to warm-up, typically 15 minutes.
Use a high power oil objective (60x or 100x).
Clean objective with lens paper and cleaner.
Replace the Hg bulb if it has exceeded its lifespan, typically 200hrs.
Check temperatures of slide heater and water bath to be sure they were used at 55°C.
Be sure that slides were made from fresh liquid cultures.
Control slides appear weaker than actual patient samples.
Actual samples sometimes appear brighter than control slides when control slides are prepared from a liquid broth medium. In order to equal the fluorescence, control slides can be prepared from spiked (negative) blood culture bottles which are incubated on the blood culture instrument and used when the instrument has signaled positive.
How critical is the timing and temperature of each of the steps?
The hybridization and wash are both time and temperature critical, with a +/- timing of 5 minutes and +/- temperature of 1°C.
Can other microscope slides be used for PNA FISH?
No. Slides supplied by AdvanDx are the only validated slides for this application. These slides have a proprietary coating which ensures the sample will remain intact during the FISH procedure. Any substitution of slides is considered a variation of the procedure.
How long after the procedure can the slides be visualized?
Slides should be stored in the dark if they are not going to be visualized immediately after processing. Slides must be visualized within 2 hours after the end of the wash step for reporting.
When do controls need to be run?
Quality control for fluorescent testing should be done each time testing is performed. The QC results should be able to monitor for appropriate testing conditions, particularly those affecting hybridization stringency and cell wall penetration, since PNA methodology is designed to optimize cell wall penetration. Once prepared, control smears may be stored for up to 1 month at room temperature. Refer to the “Quality Control” section of the package insert for more details.
How should I report PNA FISH results?
Refer to the Results Reporting Guide in the Procedure Guides and Information section of this Product manual.
PNA FISH tests allow the user to run multiple samples and different tests together at the same time. For example, a user can run two GPCC’s using the S. aureus/CNS PNA FISH kit together with yeast using the C. albicans/C. glabrata PNA FISH kit.