FAQs

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You’ll find answers to all of the most commonly asked questions about QuickFISH testing and products below. If you can’t find the answer you’re looking for, please contact us.

 

Questions relating to setting up your laboratory and equipment for QuickFISH

How do I set up my microscope / equipment for QuickFISH?

Use a high power oil objective (60x or 100x).

Clean the objective with lens paper and cleaner.

Ensure the microscope is in proper working order. If using a microscope fitted with an Hg bulb ensure that it is aligned properly and has had time to warm up. Replace the Hg bulb if it has exceeded its lifespan, typically 200hrs. Incorrect bulb alignment or use of a life expired bulb will impair the sensitivity and accuracy of the assay.

Check the temperature of the slide station before starting, to be sure it is stable at 55°C. Be sure that slides are made from fresh liquid cultures.

Ensure that fluorescence free immersion oil is used for optimal visualisation of samples

What light sources work with QuickFISH?
QuickFISH is currently widely used in laboratories employing Mercury and Metal Halide light sources, but we will check to ensure your particular lamp is compatible. If you are using an LED light source please contact us in the first instance so we can assess suitability.
Can bottles containing charcoal be used with QuickFISH?
Please inform us if you are using charcoal, so we can advise as to how best to proceed. Ideally, however, when using QuickFISH it is preferable to avoid the use of charcoal whenever possible. The original PNA-FISH protocol is compatible with charcoal bottles.
Which filter is the correct filter for visualisation 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 colour when the dual band filter is in place.
Why does the Mercury lamp have to be changed after 200 h? It still works well with our other tests.
The bulb has to be changed because the intensity of the light becomes significantly decreased.

For other test, where filters with one wavelength are used it might not make a difference but for QuickFISH we work with two different wavelengths at the same time, meaning the intensity of the light gets “divided” in to two. A decrease of light intensity will cause weak signals in QuickFISH.

Why is it important to have the bulb changed, aligned and serviced by the microscope manufacturer's representative?
The filter cube for QuickFISH contains two filters since we work with two wavelengths and three colours at the same time. To get the optimal florescence of red, yellow and green, the light/bulb has to be aligned to the centre of the two filters.

 

Questions about the QuickFISH Protocol

When do controls need to be run?
The QuickFISH slides contain integrated negative and positive controls for ease of use.

Quality control for fluorescent testing should be done each time testing is performed. The QC results should monitor appropriate testing conditions, particularly those affecting hybridisation stringency and cell wall penetration, since PNA methodology is designed to optimise cell wall penetration.

Is it important to bring the reagents to room temperature before use?
The slides and reagents should be brought to temperature before use. If it isn’t possible to bring the reagents / slides to RT before use, the slide has to be taken out of the folio bag as soon as the bag has been opened and placed on the slide station. Otherwise there is a risk of condensation that can cause a weak signal and blurry morphology during result reading.
What happens if the positive blood culture is older than 24 hours?
If the positive blood culture is older than 24 hours it can cause a weak signal.
Can a 10 µl loop be used to add blood to the slide instead of a pipette?
No, only a 10 µl, calibrated pipette can be used. The volume has to be accurate.
Can I use a pipette tip or a loop to mix blood and fix 1, instead of an inoculating needle?
No, using a loop or a pipette tip will change the volume of the dispensed blood and fix 1.
How critical is timing, volume and temperature of each of the steps?

Fix:

The 10 µl volume of blood is important. Less than 10 µl makes it difficult to find cells. More than 10 µl causes background noise that makes it difficult to read the result.

It is important to add only one drop of fix 1. Fix 1 has to be added before two drops of fix 2. If fix 2 is added before fix 1 the result gets very unclear.

Fix 2 is not a critical step. 1-3 drops of fix 2 is ok.

Fixation longer than 5 min at 55 C can cause false positive results due to non-specific binding. True positive samples can lose signal.

Hybridisation:

If more than one drop of “blue” or “yellow” is added to the cover slip, the cover slip has to be discarded.

It doesn’t make a difference if “yellow” is added before “blue” to the cover slip.

Foam or air bubbles (>3) changes the ratio between the reagents. In case of foam or air bubbles the cover slip should be discarded.

The cover slip has to be flipped into the marked area on the slide. If the cover slip is placed on the white writing area it will not be in proper contact with the smear. That can cause floating cells under the microscope. It can also cause false negative results due to insufficient hybridisation.

Hybridisation > 20 min can cause non-specific binding and false positive results.

Examination:

As QuickFISH is designed to provide rapid diagnostics it is always best to read the slides when they are freshly prepared.

However, the slides can be stored in the dark for several hours should this be required. If the slides are not kept in the dark and the results aren’t read within 2 hours there is a risk of false negative results or weak results as the controls will lose signal.

 

Suggestions for Troubleshooting

What if I see 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.
What if I can see only a few positive organisms on the slide?
Visualising 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.
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 the microscope shutter while not viewing the slides.
Why can’t I see organisms on my negative control and negative slides?
QuickFISH 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 visualised with the fluorescence microscope. Negative cells will have no or little fluorescence therefore they are difficult to visualise by fluorescence microscopy.

In some cases, negative cells can be seen with the FA scope, this is due to auto-fluorescence 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.