the probe`s instruction sheet
Contents
1. click Apply to accept this calibration point Click Complete View the Calibration Summary screen and QC score Click Fxit to return to the sensor calibration menu and then the back arrows to return to main Calibrate menu Rinse the sonde in tap or purified water and dry the sonde2. serial dilution of a surrogate solution of Rhodamine WT R gt 0 9999 and this should ensure relative accuracy of field BGA PE readings i e a BGA PE reading of 100 units will represent twice the algal content of water with a BGA PE reading of 50 units A significant advantage of the EXO BGA PE sensor is that its readings show less interference from turbidity and this will allow for much more accurate determination of BGA PE content during rainfall events which release both sediment and algae into the water item 603796 dwg 603796 K C EXOwater com Aug 2013 Rev A Calibration Chlorophyll Please see the EXO User Manual for detailed information on calibration of the chlorophyll channel Blue green Algae Phycoerythrin This procedure calibrates BGA RFU or BGA ug L If the user has both units selected then this procedure must be performed twice once for each unit to completely calibrate the parameter For the 2 point calibration one of the standards must be clear water 0 ug L and this standard must be calibrated first The other standard should be in the range of the suspected BGA PE content at the environmental site Two general types of standards can be used a phytoplankton suspensions of known BGA PE content and b dye solutions whose fluorescence can be correlated to that of BGA PE The user is responsible for determining the BGA PE content of algal suspensions by using standard cell counting techniques For option b we
3. Rhodamine VVT Dye Solution for Total Algae Sensor BGAPE A Read and follow all the safety instructions and MSDS documentation supplied with the dye before proceeding Remember that only trained personnel should handle chemicals Preparation Use the following procedure to prepare Rhodamine WT solutions for use as a sensor stability check reagent for the EXO Total Algae Chlorophyll and Blue green Algae Phycoerythrin sensor 1 Purchase Rhodamine WT dye in solution form which can vary somewhat in nominal concentration Recommended supplier for a solution that is approximately 2 5 in Rhodamine WT Fluorescent FWT Red Dye item 106023 Kingscote Chemicals 3334 South Tech Blvd Miamisburg OH 45342 USA 1 800 394 0678 2 Accurately transfer 5 0 mL of the Rhodamine WT solution into a 1000 mL volumetric flask Fill the flask to the volumetric mark with deionized or distilled water and mix well to produce a solution that is approximately 125 mg L of Rhodamine WT Transfer this standard to a glass bottle and retain it for future use 3 For chlorophyll a calibration accurately transfer 5 0 mL of the solution prepared in step 2 to a 1000 mL volumetric flask and then fill the flask to the volumetric mark with deionized or distilled water Mix well to obtain a solution that is 0 625 mg L of Rhodamine WT a 200 1 dilution of the concentrated solution 4 For BGA PE calibration accurately transfer 0 2 mL of the 125 mg L solution prepared in step 2 to
4. a 1000 mL volumetric flask and then fill the flask to the volumetric mark with deionized or distilled water Mix well to obtain a solution that is 25 ug L or 0 025 mg L of Rhodamine WT 5 Store the concentrated standard solution in a glass bottle in a refrigerator to retard decomposition The dilute standard prepared in the previous step should be used within 24 hours of its preparation Discard the used standard When Rhodamine standards are required in the future perform another dilution of the concentrated Rhodamine WT solution after warming it to ambient temperature Effect of temperature on fluorescence The intensity of the fluorescence of many dyes shows an inverse relationship with temperature This effect must be accounted for when calibrating the EXO Total Algae sensor with Rhodamine WT Enter the ug L or RFU value from the table below that corresponds to the temperature of the standard RFU BGA PE pg L BGA PE 37 3 Temp C RFU Chl XG Total Algae Chl amp BGA PE Sensor Overview The EXO total algae sensor is a dual channel fluorescence sensor that generates two independent data sets one resulting from a blue excitation beam that directly excites the chlorophyll a molecule present in all photosynthetic cells and a second from an green excitation beam that excites the phycoerythrin accessory pigment found in blue green algae cyanobacteria This green excitation triggers a transfer of energy from the phyc
5. conds click Apply to accept this calibration point Click Complete View the Calibration Summary screen and QC score Click Exit to return to the sensor calibration menu and then the back arrows to return to main Calibrate menu Rinse the sonde in tap or purified water and dry the sonde RFU l or 2 point RFU is a percent full scale output it outputs relative fluorescence from 0 100 This calibration procedure is recommended if you are also using grab samples to post calibrate in vivo algae readings Pour the correct amount of clear deionized or distilled water into the calibration cup Immerse the probe end of the sonde in the water In the Calibrate menu select BGA PE Chlor then select BGA RFU Select either a 1 or 2 point calibration Enter 0 for first standard value and 45 for second standard value Click Start Calibration Observe the readings under Current and Pending data points While stabilizing click the Wipe Sensors button to activate the wiper to remove any bubbles When data are Stable or data shows no significant change for approximately 40 seconds click Apply to accept this calibration point Next place the sensors in the Rhodamine WT standard Click Proceed on the pop up window Observe the readings under Current and Pending data points While stabilizing click the Wipe Sensors button to activate the wiper to remove any bubbles When data are Stable or data shows no significant change for approximately 40 seconds
6. entration of Rhodamine WT dye The sensors can then be deployed and generate i n ay en data that is relative to all other sensors Once a sensor is retrieved it can BG M mee exe be checked against that same standard to assess sensor performance i dilution of Rhodamine WT drift or the potential effects of biofouling i solution from 0 280 pg L PE equivalents Sensor Type Optical fluorescence Chl R2 gt 0 999 for serial dilution Linearity The ug L output generates an estimate of pigment concentration The relationship between ug L and sensors RAW signal should be developed through following standard operating procedures of sampling the water body of interest collecting sensor data from sample and then extracting the pigment to establish a correlation The higher the temporal and spatial resolution of the sampling the more accurate this estimate will be Optics a Chl Excitation 470 15 nm Emission 685 20 nm Pigment concentration ranges of algae sensors were determined in monocultures of specific algae species This range will vary depending h h on algae assemblage and environmental conditions For accurate C orop y pigment concentration estimates at particaular sites or samples the Please see the EXO User Manual for detailed information on chlorophyll user must determine the RFU to pigment concentration relationship on a site by site basis Blue green Algae The EXO BGA readings show excellent linearity on
7. oerythrin to the central chlorophyll a where photosynthesis is initiated Although blue green algae contain chlorophyll a the chlorophyll fluorescence signal detected by in situ fluorometers is weaker than in Units eukaryotic phytoplankton This results in an underestimate of algae Chlorophyll RFU pg L Chl biomass when using a single channel chlorophyll sensor when blue RFU ug L PE green algae are present The EXO total algae sensor generates a more aera ssddeudssebasaiaadedevadoasbtacecd obdvadbeggeassagelasiacedbvadsasotaeie accurate total biomass estimate of the planktonic autotrophic community Temperature by exciting chlorophyll a and phycoerythrin Operating 5 to 50 C Storage 20 to 80 C The sensor generates data in three formats RAW RFU and an estimate m gas gn of the pigment epneenttation in ug L 3 a ae CERN The RAW value is a value unaffected by user calibrations and provides a sssssveesssecsnscssnsccssnecsasefossecsnecourecsnsesnsesenrecsnscoasscserecanscenscoasscancensssensetass range from 0 100 representing the per cent of full scale that the sensor detects in a sample Specifications Chl n RFU 0 01 pg L Chl RFU stands for Relative Fluorescence Units and is used to set sensor n D CAPE 0 01 RFU 0 01 pg L mere output relative to a stable secondary standard such as Rhodamine WT dye This allows users to calibrate sensors identically for example calibrating all sensors in a network to read 100 RFU ina conc
8. recommend using a 25 ug L Rhodamine WT dye solution for detailed instructions see next pages and the solution is used in the calibration steps below ug L 1 or 2 point This procedure will zero your fluorescence sensor and use the default sensitivity for calculation of phycoerythrin containing BGA in ug L allowing quick and easy fluorescence measurements that are only semi quantitative with regard to BGA PE However the readings will reflect changes in BGA PE from site to site or over time at a single site Pour the correct amount of clear deionized or distilled water into the calibration cup Immerse the probe end of the sonde in the water In the Calibrate menu select BGA PE Chlor then select BGA ug L Select either a 1 or 2 point calibration Enter 0 for first standard value and 126 for second standard value Click Start Calibration Observe the readings under Current and Pending data points While stabilizing click the Wipe Sensors button to activate the wiper to remove any bubbles When data are Stable or data shows no significant change for approximately 40 seconds click Apply to accept this calibration point Next place the sensors in the Rhodamine WT standard Click Proceed on the pop up window Observe the readings under Current and Pending data points While stabilizing click the Wipe Sensors button to activate the wiper to remove any bubbles When data are Stable or data shows no significant change for approximately 40 se
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