How it works: There are two menus named "2 pigments" and "3 pigments". Underneath each menu are the author names and year of publication of a determination method. Each time you select a method, a new window will appear. You may have as many windows as you like (up to the memory available to the program). You may also have several windows open with each using the same or different formulas. Enter the absorbance readings, hit "Calculate" once, and the results are then displayed at the bottom of the window. If you had to dilute your sample before measuring the absorbances, enter the dilution factor like "5", in the box named "dilution". If you want the results expressed on a cell basis, enter the number of cells used per mL of suspension. These values are optional.
The types of formulas are separated into those for higher plants and green algae and cyanobacteria, because cyanobacteria have phycobilisomes and higher plants don't. These dividers show up in the menus as greyed out because you can't select the dividers. "Cyanobacteria (Chl & PC)" is there to indicate that the two methods below relate to cyanobacteria. For a cyanobacterial example, go under "2 Pigments", and select "Arnon et al. 1974".
Clear after Calculate Option: To speed up calculations, the program clears out the readings you entered every time you press the "Calculate" button. If you press twice in rapid succession, on the second press the results will be erased. You can turn this option Off under the File menu.
Solvents. There are pop-up menus under the solvent names in methods that allow several solvents to be used. The coefficients used in the calculation depend on the solvent you used for the extraction. Note how the wavelengths of the required absorbances change depending on the solvent selected.
Whole cell determinations: For measuring the Chl concentration in whole cells, our spectrophotometer was modified by adding Scotch (3M)"Magic Tape" No. 810 to the light path before and after the cuvette in order to correct for light scattering by the cells. This yielded a fast and reproducible assay that was linear from A=0.01 up to about A=0.5 optical units, and which was within 10% of Chl determinations performed using 80% acetone extraction.
It is helpful to measure pigment concentrations in whole cells because its fast and reproducible. The "2 pigments" methods for cyanobacteria are geared for water/growth medium as the solvent, i.e., all you need is a cell suspension. You need to correct for light scattering by adding Scott "Magic tape" or something similar that diffuses light to the light path inside the spectrophotometer.
The "3 pigment" cyanobacterial method, however, requires biochemical preparations as described in the reference.
Limitations of whole cell determinations: depending on your spectrophotometer, you may find that even with a diffuser to correct for light scattering, dense cell suspensions do not give linear readings. The practical top absorbance limit with ours is about A=0.5 (at 678 nm) of cell suspension. In addition, adding a diffuser reduces the beam intensity and therefore lowers the signal-to-noise ratio.