I am culturing Chlorella vulgaris and want to establish a relationship between optical density and biomass concentration, both dry cell weight and cell density. I will use OD 680 and dilute samples to between 0.1-1.0 absorbance. I will then use a five-point calibration using a dilution factor of two. My question is as I am culturing the algae on wastewater with high colour, what should I use as a blank as distilled water would not be representative. Should I filter each day my samples to remove the algae and use this as a blank? Also, would the blank need to be diluted in the same way as my samples with algae present i.e. dilute to 0.1-1.0 and then factor two dilutions x 5?
I am not fond of OD with algae. However, when completing OD with algae it is better to use a wavelength of 750 to eliminate any error caused by chlorophyll. Typically there is a better correlation between OD and DCW than cell density. This is because algal cells can greatly vary in size depending on the stage of the cell and culture. Filtering would be a viable option if you are also going to be completing nutrient analysis of the medium. It should be diluted in the same way the non filtered culture is. Depending on how dense the culture is I have had to dilute it by a factor of 10. It is also important if you are doing batch experiments to verify that your correlation works at different points in the experiment (i.e. exponential, stationary, late stressed phases).
Dear James,
It is very important aspect in Algal biology. Because based on the OD one is going to conclude that the growth of Algae. As you know simple turbidity also will absorb the light. Now there are three ways you can take near actually growth of Algae.
1. Having Right Blank: Please have a blank everything except that microalgae, (it should be prepared afresh if growth progress is monitored over the days). Which means whatever the backgrownd absorbance is coming it will be normalised and the resulting OD is only for the algal biomass. This wil reflect the actual growth.
2. There is another method of determination of algal growth by measuring the total Chlorophyl A measurement. Since chl mo are green colour and it generally will not come from other debris/solid waste from you municipal wastes. (Search literature, if did’t find, pl. Inform me i will send you)
By having proper control you can justify your result finding.
3. At the end of experiment (For Example the removal of any toxic or excess nutrient) removed by the algae. Measure those elements by appropriate analytical method over time or various initial algal inoculm concentrations. The amount of “toxic or excess nutrient” removed wil indicate the consumption by algae and in turn its growth over the days. Again proper control needs to be kept without algae, what is the status of removal. This will be confirmation one.
4. Microalga cell count under microscope also possible but it is time consuming and labour intensive work (reports are there).
(I am enclosing my paper, it has monitoring algal growth by OD)
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Everett E.'s answer echoes all my experience. Dilution is critical for accuracy. Also much information can be gleaned from ratios of Absorbance at different wavelengths. E.g. 750/680 is a proxy for chlorosis and hence related to oil content. Find the Amax for the brown color and monitor that ratio w 750 as well.
I suggest you to study the fitting of linear regression between OD and biomass using wastewater as medium. you can look at the average errors and R2.
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