TITLE: Reading Sample Fluorescence (PDMPO) via Fluorometer
AUTHORS: Brittany Zepernick, Matthew Saxton, Steven Wilhelm

[STEPS]
?. [Calibration]
From the home screen of the device, perform the following:
?. [Calibration]
Select the 1. Button for “set-up”
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Select the 1. Button again for “mode”
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Confirm machine reads “simple multi-operational” (which is the mode) press ENTER.
?. [Calibration]
Select the 2. Button for “calibration procedure”
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Use   arrow buttons to select “Direct concentration” (as the type of measurement to be taken/read)  press ENTER.
?. [Calibration]
Select the 3. Button for “units” Then select mg/L, mg/mL, ng/mL etc.
Depending on your own concentrations selected for your standards. We utilized mg/mL or ng/mL for our standards, depending on whether they are cultures or environmental samples.
?. [Calibration]
Press ESCAPE and return to the main home screen.
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Select “max range”
Which is the maximum approximate concentration range you expect, you will have to perform a little trial and error depending on your samples and their concentration. *This will vary on your cell concentration, incubation conditions, and the volume filtered.
?. [Calibration]
You will then be prompted to enter the number of standards you are using (5) and press ENTER.
?. [Calibration]
The machine will ask you to insert your highest standard first:Pipet  of the standard into a disposable glass 10 mL tube and wiping the outside prior to insertion.
4 ml
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Enter the concentration (in your prior selected units) from your standard. (This is your highest standard). Select 1. “OK”. Then press “*” to read the standard.
?. [Calibration]
Machine will prompt you to repeat the same steps for your other standards (Order does not matter).
?. [Calibration]
Follow prompt to insert a blank, press ENTER.
of NaOH and  1M HCl
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Once blank stabilizes, press "0".
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Screen will revert to home screen when reading is complete.
?. [Calibration]
Once calibration is complete and displayed on the computer screen that is hooked up to your fluorometer, You will use this equation to convert your sample readings (FSU) to actual concentrations of PDMPO and then silica.
It is recommended to record your fluorescence readings (FSU) of your standards as you go through the calibration process and plot them manually to validate and compare the program’s results (actual concentration, x-axis) vs. (fluorescence, y-axis).
?. [Calibration]
The linear regression equation must have an R2 value higher than 0.95 (R2 > 0.99 preferred).
?. [Reading Samples]
Carefully pipette the top  of matrix from your recently centrifuged samples into a clean 10 mL disposable glass tube.
4 ml
Do not to disturb the bottom of the 15 mL Falcon tube where filter fragments and unlysed/digested cells may be, as it will interfere with fluorescence readings.
?. [Reading Samples]
Press the “*” button and begin sample reading which will be visible on computer screen.
The end number is an average of a variety of fluorescence measurements.
?. [Reading Samples]
After reading all samples, enter the fluorescence readings into your linear regression equation (y) and solve for the actual concentration of PDMPO (x).
The actual concentration must be converted from mL/nL to mol. Then converted to silica using the conversion factor 3230:1 for Si: PDMPO (mol:mol).
?. [Reading Samples]
Normalize to either cells/mL or chlorophyll-A data, for each sample, to obtain Si (mol)/cell.
?. [Reading Samples]
Information can be graphed to show silica deposition rates over time.