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I am doing an lab for IB Bio and don't know how to use a colorimeter to measure growth in algae. The experiment I am doing will measure the effect of pH on algae growth. The algae will be taken from a fish tank and will include cyanobacteria, diatoms, and algae bloom. I don't know how the transmittance/absorption will relate to growth. I also don't know which wavelength of light to use. Help would be appreciated, thanks!

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  • $\begingroup$ What is the color of the algae? What is the complementary color of that? $\endgroup$ – TanMath Nov 9 '15 at 1:36
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    $\begingroup$ There is/was no lab manual providing you with instructions/recipes/protocols to complete this assignment? You received no instruction or demonstration on how to use the equipment/instruments? There was no lecture material on the theory behind the experiment? That strikes me as rather unusual. What if you electrocute yourself trying to turn on the colorimeter? What if you accidentally ingest one of the solutions or the growing cells? That could potentially be dangerous. $\endgroup$ – mdperry Nov 9 '15 at 1:38
  • $\begingroup$ You can measure the turbidity of water using turbidimeters, however, that would not say whether it is caused by algae or something else. Best way, IMO, is to take samples and calculate biomass but there is no straightforward way to do this optically. $\endgroup$ – WYSIWYG Nov 9 '15 at 5:33
  • $\begingroup$ The color of the algae is a brownish green. $\endgroup$ – sousheel Nov 9 '15 at 23:45
  • $\begingroup$ No, I did not receive any lab manual. We must design it ourselves. $\endgroup$ – sousheel Nov 9 '15 at 23:46
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Let's be clear, you are using a colorimeter, not a spectrophotometer?

Can you provide the brand name and model number of this colorimeter?

Have you learned about the Beer-Lambert Law? It states that A = Ecl where

A is the Absorbance

E is the molar extinction coefficient

c is the concentration of the unknown

l is the length of the light path through the sample (the standard path length is 1 cm in spectrophotometers).

The first thing you need to do is measure lambda Max, the wavelength of light that is absorbed the most (or transmitted the least) by your sample. The Beer-Lambert law is linear when A is between approx. 0.1 and 1.0.

So take a dilute solution of the algae and starting at the smallest wave length of light that the device can emit (you can vary the wavelength?). Take readings of the Absorbance and then increase the wavelength by 10 nm. Starting at deep purple, and ending at far red, write down the A values. Plot this data with A on the Y-Axis and wavelength in nm on the X-axis. There should be a distinct maxima in the curve, that is the most sensitive wavelength, and is the one you should use for all your experiments. If the algae is green, then the maxima should be over in the red end of the spectrum (like chlorophyll).

Next you need to make a standard curve that correlates the Absorbance at lambda max with the number of algal cells in the cuvette (the test tube you put in the colorimeter). That means you need an independent method of counting the number of algal cells. If they were bacteria or yeast then you could plate them on agar-filled Petri dishes and count the number of cells per mL (for example). For some cells if you have a high powered microscope and a hemocytometer then you could visually count the number of cells per mL. If neither of these methods are available then you could measure the mass of algal cells by weighing them on a balance.

So by some method you will be able to plot the number of cells in the broth vs. the Absorbance (plot A on the Y-axis).

From this you can empirically determine the molar extinction coefficient of these algal cells. Then you just need to measure the Absorbance at lambda max on dilute aliquot of the cell culture and you will have an accurate measurement of the number of cells per mL.

With this assay working then you can inoculate a bunch of identical cultures, that vary by the pH of the growth medium. By removing small aliquots of the cultures at the same time points you will be able to plot the number of cells (on the Y-axis) versus the time they have been growing, and you will see if varying the pH alters the shape or magnitude of these growth curves.

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