FAQ bbe BenthoTorch

The BTo differentiates greens, diatoms and bluegreens separately for chlorophyll. Additionally it has integerated GPS that record the position of measurement.

BenthoTorch is not designed for moss or macrophytes measurement. It will probably detect “green algae” chlorophyll and display high numbers. But the numbers cannot be used to determine the content.

• Limit of Detection is 0,006 µg/cm²,
• Detection Capability is 0,012 µg/cm²
• Limit of Quantification (maximal relative uncertainty 33%) 0,072 µg/cm²
• Limit of Quantification (maximal relative uncertainty 20%) 0,121 µg/cm²
• Limit of Quantification (maximal relative uncertainty 10%) 0,242 µg/cm²

Those parameters are device specific, but I would expect they are in this range.

There is only one range: 0 to 15µg/cm². All results above 15 are not very reliable and should be considered only as an indicator. Results above 15 are an effect of overcorrection that often occurs when measuring over the range. If you do not want results over 15 to show, you can block the results by adjusting the maximal allowed total concentration in the Parameter window at the bottom of the "Fit parameter" tab.

It is required only for reflection calibration. You are right it is standard for 100% reflection and in principle it is not necessary to do that because we already calibrated it.

Never make reference measurements with extracted chlorophyll! not true. It is just difficult and has a lot of pitfalls. It is only if somebody feels that standard calibration of the BT not perfectly fit to their local algae fingerprints then they can proceed with a calibration on living algae.
Validation is not necessary - it is up to the costumer if he feels like he needs it.

The instrument is delivered pre-calibrated. It is relatively complicated to calibrate this specific instrument due to the corrections therefore we recommend to send it to bbe factory for calibration. There are three factors to consider:

• One is the fingerprints of algae - meaning characteristic fluorescence patterns that allow us to differentiate between green algae, diatoms, and cyanobacteria. This could be changed if you have any specific algae that for some reason is not properly classified.
• The second factor is the quantification of algae - this is done by adjusting the intensity of the fingerprint to reflect the fluorescence of 1µg chl-a/cm². This was calibrated against our reference instrument, which in turn was calibrated against a DIN 38412-12L method. We regularly participate in ring tests to validate our chlorophyll extraction method. So, if you use any other method of chlorophyll extraction, the results can differ significantly, but it should be possible to recalculate by multiplying the results by an estimated factor.
• The third factor is the background correction based on reflection measurement. (it is mostly relevant on brighter stones or backgrounds) For more info see the paper of Carpentier "The influence of hard substratum reflection"

The measurement is performed on a surface of only 1cm², so a relatively flat surface of around 2cm² would be good. Unless there are deep indents and cavities the measurement should be possible, but the smoother the better.

Due to a shadowing sponge element it is possible to measure in all possible directions.

Yes, this is possible.

No! Leave it in the water! The probe is constructed to deliver almost the same values inside and outside of the water!

Experience shows that measuring biofilms grown on microscope slides is possible, but subject to certain limitations. The resulting biofilm layers do not grow particularly thick, meaning the underside of the microscope slide is usually visible through them. However, depending on how the slide was exposed, the underside may also be covered with biofilm. Consequently, when measuring the upper layer with the bbe BenthoTorch, the lower biofilm layer is also excited, thereby influencing the fluorescence signal from the layer on the top surface.

This results in an overestimation of the biofilm density on the top surface—the side that is actually of interest. Furthermore, since the density of the biofilm on the underside cannot be accurately determined (as the measurement is taken through the biofilm layer on the top surface), the total result (combining both biofilms) is also incorrect. Removing the biofilm from the underside of the slide provides a solution, allowing for a reliable area-based measurement. It should be noted that the glass surface is highly reflective and can affect the measurement signal. Regarding the background surface, the best results are obtained using a matte black base (e.g., black poster board). Background fluorescent lighting should be turned off, as the 50 Hz flicker frequency interferes with the excitation light and affects the fluorescence signal.

It is also advisable to shade the edges of the slide to prevent ambient light from reaching the measurement area. Ceramic plates and cleaned stone surfaces have proven to be effective artificial substrates for growing biofilms. However, one must bear in mind that the chemical properties of mineral surfaces influence biofilm composition. On the other hand, background interference is minimal with these substrates; there is essentially nothing present that the BenthoTorch’s reflection offset correction cannot handle.

The estimation of filamentous algae density is always problematic independent of the method due to the 3-dimensional structure. The bbe BenthoTorch was design as a none-invasive method to measure the photoactive upper ~100µm of algae biofilms. The advantage of the BenthoTorch is the possibility to revisit the same measurements areas to monitor the trend of biofilm growth. Also, the speed and therefore the possibility to measure high numbers of samples as well as observe the patchiness of the biofilm is an advantage that has to be taken in account.

The BenthoTorch measures chlorophyll, independent from the number of cells in which this chlorophyll is. There is a possibility to convert the chlorophyll concentration to cells/sqcm - but this is based on a fixed ratio (chlorophyll/cell). So, in the mentioned case the chlorophyll concentration measurement is fine the conversion only an approximation.

Yellow substances are not relevant in the measurement of algae layers, because the signal from the layer is much higher than from the YS in the small amount of water. The reference signal is a reflection measurement (no fluorescence) to get a algae concentration measurement independent from the more or less bright surface.