Author: Bill Nelson, April 2014 Title: Lake algae sample processing using the Flow Cam Laser safety key Fine focus Procedure: SETUP 1. The 10x (4x) objective lens and 100um (300um) flow cell should already be attached. 2. Plug in the FlowCam and the LCD monitor. The keyboard and mouse are connected to the USB ports on the back of the FlowCam. Remove the dust cover from the filter and replace it with the 80um mesh filter. 3. Turn on the FlowCam (big black button on the front of the machine) and turn on the LCD monitor. - The software will boot up automatically (computer is in the FlowCam itself) and if everything is working properly, the screen should show a typical Microsoft XP desktop. - Since these samples are preserved in Lugol’s, there isn’t a need to use the laser. Make sure it is turned off by turning the safety key to the left (see picture above). 4. Start the ‘VisualSpreadSheet 10x”. Start the software (double click) that corresponds to the objective lens that’s installed (10x in this case). 5. Open a window of the camera. From the ‘Setup’ menu, select Setup and Focus. This will bring up a window showing the image of the flow cell through the microscope. 6. Focus the image. Turn the pump on at full speed (‘reverse’ direction). Put about 1ml of stock algae into the funnel and allow that to pass into the flow cell. If there are bubbles, squeeze the tube below the flow cell gently (but don’t pull on the tube—the flow cell is fragile!). Stop the pump once you have a good flow without bubbles in the image window. Adjust the fine focus by turning the white knob on the front of the machine (see picture above) to get the best image of the cells in the field of view. 7. Set camera gain. Under the Setup tab select Context à Camera and set the Gain to 5 for the 100um FC and 3 for the 300um FC. 8. Clear the flow cell. Turn the pump on in the reverse direction with fast setting and the speed dial at 5; let it run to clear the sample of stock algae. Once clear put a pipette full of DI to clean the flow cell and start AutoImage Mode No Save from the Setup menu. This will let you see how clean the flow cell is. Run this until the flow cell has no algae in it. RUNNING A SAMPLE 1. Load sample. The flow cell will have air in it from the previous cleaning. Mix your sample well, load one full pipette (~2.5mls) into the funnel through an 80um mesh. It’s important to filter the sample first through 80um so that the flow cell doesn’t become clogged. Turn the pump on at full speed. Start the AutoImage Mode No Save (Setup menu) to see what the sample looks like. Once the sample is running through nicely with no air bubbles, then you are ready to analyze the sample. It’s important to minimize the time between loading and analyzing the sample because of issues with particle settling. 2. Run the sample. From the Analyze menu, select the AutoImage Mode. Select two stop conditions: 1) stop when 5000 particles have been counted and 2) stop if run goes longer than 10 min. Save the analysis in the following format: The folder should be under project (e.g., ‘Lake Survey 2014’), then the lake (e.g., ‘Elbow Lake’), then the date (e.g., ‘May5-2014’), and finally the sample details (e.g., ‘Sample_0to5m’). The file name should be include the location, sample date and depth (e.g., ‘Elbow lake-May5_2014-0to5m_depth’). After selecting the folder, the FlowCam will run the analysis. Do not move the field of view once the sample is running, as this will negate the background calibration. 3. Store remaining sample for further analysis. Each sample will be run twice: through 300um and 100um flow cells with 4x and 10x objective lenses respectively. This is to accurately count particles in the 10 um to 50 um range with the 100um flow cell and 50 um to 300 um range with the 300um flow cell. Flow cells should be changed as rarely as possible and therefore all samples should be run at 100um, the remainder of the sample preserved, and then run once again at 300um. Remember to clear the flow cells between samples (Step 4). 4. Clear the flow cell. Once the sample is clear of the flow cell, put a pipette full of lugol-DDW and start AutoImage Mode No Save from the Setup menu. This will let you see how clean the flow cell is. Run this until the flow cell has air in it. Note that if you are running replicates you don’t need to clean between runs. But always clean between different samples. RECORDING THE ANALYSIS 1. Open the file. The information from each run is stored in the file folder created. At the end of each day, you will need to record the main summary statistics from these files. Load an analysis by going to the File menu and opening the appropriate list. 2. Check for incorrect particles. From the Edit Window, select Open View Window. Look through each page of images for any bubbles. Take care as only pieces of larger bubbles may be counted as particles (they will appear as curving black lines). If found, highlight the bubble (or bubble piece), right click and then delete the particle. Once complete, close this screen and click on the figures in the main summary window (they should be salmon in colour and return to their original green once adjusted and clicked). This will cause a refresh and the data will have been adjusted for the removed particle. 3. Summary statistics From the main window, record into the master Excel file: i. Particle count (PC), Particles / ml (P/mL), and P/mL adjusted to the calibration factor. ii. ABD diameter in um (mean & sd) iii. ESD diameter um (mean & sd) 4. Summary histogram Click the x-axis of the figure until it is in Diameter units of ‘ABD’. From the main window, record: i. Size histogram. Right click on the Frequency by Diameter (ABD) figure (Upper left plot) and select Export Histogram Data… Copy the first 3 columns (‘BinLow’, BinHigh’,’Value’) and paste them into the main spreadsheet. Adjust the scale of the x-axis to have the largest particle present at its maximum. 5. Save data from 100um and 300um runs to spreadsheet, distinguished by date and separated into sheets by Lake. SHUTTING DOWN THE MACHINE 1. Clean the flow cell. Clean the flow cell by putting ~10mls of DI water through it. Exit the program, and shutdown Windows through the software. Then turn off the machine and dispose of waste appropriately. 2. Dust cover. Make sure to replace the dustcover immediately after use so that the flow cell doesn’t become plugged with dust fibres.