FlowCam Standard Operating Procedure
Turn on FlowCamPush silver button on the left of machine face to turn on computer and machine .
Instal flow cell into the flow cell holderSelect desired flow cell tubing based on application .
( In general we use the 100 um x 1 mm flow cell ) . Use a kimwipe or lens paper to gently clean the glass portion of the flow cell . Insert glass portion of the flow cell into the flow cell holder . Secure flow cell by gently screwing the male threaded holder until the flow cell is stationary .
Be careful not to screw too tightly at the risk of breaking the fragile flow cell .
Note : Since there are multiple projects using the FlowCam be sure to select the flow cell labeled for each specific project .
Instal the flow cell holder into the FLowCamMake sure that the objective lens and collimator are compatible with each other .
For example , when using the 10X lens , use the corresponding 10X collimator . Use a kimwipe or lens paper to gently clean the objective lens and collimnator . Install flow cell holder ( screw pointing up ) into the flow cell holder mount located in the middle of the camera apparatus .
Tighten screw on top of holder to secure it to the mount . Insert tubing on top of flow cell into the bottom of sample funnel .
Insert bottom tubing into tip of syringe .
Insert syringe - out tubing into small beaker for collecting waste .
Flush Flow CellMake sure to flush the flow cell before beginning to prevent any previous cellular material or debris from showing up in your sample . 1 .
Load the funnel with ddH2O . 2 .
On the top menu bar , click Setup - > Pump - > Flush 3 .
Set to 4 - 5 cycles . 4 .
Click Start Load Sample1 .
Use pipet to draw sample from your container .
Usually about 0.2 - 0.5 mL of homogenized sample is sufficient .
For dense cultures , a dilution may be appropiate ( Be sure to note this in your files ) .
Sieving is great for raw samples that might have detritus or zooplankton .
2 .
Lower tip of pipet into bottom of P1000 sample funnel .
Forcibly squeeze pipet to ensure that sample ends up as one liquid mass and doesn't stick to the walls of the funnel .
3 .
On the top menu bar , click Setup - > Pump - > Prime - > Aspirate 0.500mL from Flow Cell 4 .
Click Stop Pump when the sample volume has reached the flow cell Run Context Click on Context .
A window with several tabs will pop up .
Notes Tab - any relevant metadata .
There is a file on the Desktop that can be pasted and modified as needed .
This file includes lines for date , experiment , strain , species , magnification , flow cell , syringe , dilution , etc . Fluidics tab - Adjust here the sample volume ( usually 1 mL ) , relative to the stop rule ( see below ) .
Also , make sure the efficiency is near 25 % . Flow cell tab - Adjust the tube length below the flow cell .
By default it is at 0 cm .
Our flow cells are cut to have 10 cm tubing above and 20 cm tubing below .
Stop tab - Stop rule for terminating the run .
Usually after 100 uL ( 0.1 mL ) of sample has been imaged .
This works well with 1 mL of sample and a short priming step ( see Setup and Focus below ) . Reports tab - Check the export data and export summary data checkboxes .
This saves the measurements of all captured particles .
The list file with collages of images are saved by default upon starting a run . You can save these settings and load them as needed .
Setup and Focus1 .
Click Setup / Focus .
A window will pop up with a live camera view of the flow cell . 2 .
Use the X - axis and Y - axis adjustment knobs to center the flow cell . 3 .
Use the Coarse and Fine adjustment knobs to bring the sample into focus .
Run Sample1 .
Click Autoimage . 2 .
A pop - up with your Notes and Stop rule ( Context ) will show up .
You can either enter a value to automatically stop the run ( e . g . # of events , volume or time ) or leave the 'Stop when user terminates . . . ' button checked and manually stop . 3 .
A window to create a folder for the run will pop up next .
Make an informative folder name with sample info in the title ( date , strain info , magnification , flow cell size , dilution , etc . ) and click save .
Once folder has been named , the sample run will begin . 4 .
The sample will run until the criteria of the set Stop Conditions are reached or the user terminates the run by pressing the Autoimage button which will now display a red stop symbol .
Data The data will be displayed for each filter on the Visual Spreadsheet page . Select your data ( either by clicking on the desired filter or highlighting portions of the graphs and selecting Open View to evaluate the images . Adjust for any dilution used in Context - > Fluidics and checking the Sample Dilution box .
Enter in the ratio of the dilution used ( volume of sample / total volume ) Particles / mL is the cell count Make sure the Efficiency is within the range of the flow cell being used .
Note : Be sure to flush the flow cell between samples .
Flush Flow CellMake sure to flush the flow cell between samples to prevent any previous cellular material or debris from showing up in your sample . 1 .
Load the funnel with ddH2O . 2 .
On the top menu bar , click Setup - > Pump - > Flush 3 .
Set to 4 - 5 cycles . 4 .
Click Start Continue running or Shut down To continue running samples repeat steps 5 - 10 ( skip steps 6 & 7 as the settings are already saved ) . To shut down continue to step 12 .
Dissasembly1 .
Remove the tubing from the syringe tip and the sample funnel .
2 .
Loosen the flow cell holder screw and remove the flow cell holder and flow cell .
3 .
Remove the flow cell from the flow cell holder . 4 .
Put the flow cell back in its protective tube . 5 .
Close the program and shut down computer .
