If the solids are not organic, then it may be possible to quantify biomass based on ash free dry weight. This is becoming the standard for algal cultures that are grown outside to help account for any particulates that are blown in to the ponds or reactors. To accomplish this you need to use glass filters usually GF/Cs, place them in a blast furnace at 500°C for four hours. Remove and place in a desiccator for one hour to cool. then weight the filters using tweezers to minimize contamination. Filter samples of algae depending on concentration between 5 and 20 mL. dry in an oven at 105°C overnight. weigh the dried algae+filters and record the value. Then place the filters into a blast furnace at 500°C for four hours to burn off all organic matter. Place filters in desiccator for one hour to cool. Weigh filters one last time. To calculate ash free dry weight = [(algae w/ filter) - (ashed algae w/ filter)]/volume filtered

While work intensive, direct microscopic cell counts are a reliable way to determine cell concentrations. You will obtain a cell concentration that might or might not be correlating to dry weight measurements as described in Everett's reply since the specific dry weight (dry weight per cell) can change during algal growth.

and....Why do not you measure chlorophyll concentration????

I agree with Maria - there are meters that can measure chlorophyll content or you could filter a known volume onto a glass fiber filter and extract it with acetone and read the absorbance at 645 and 663 nm. Total chlorophyll in ug/mL = (20.5 * A645) + (8.0 * A663). Correct for volume filtered and you can tell relative amount of algae in your system.

Using ash free dry weight could potentially be a problem, as the ash component is not entirely due to extrinsic factors such as dust. Certain components of the algae (eg. silicate, if a diatom) contributes to the ash component but is intrinsic to the cellular components.

I advocate the use of cell counts although time consuming, it is the best way. The alternate is to identify what the particulate matter is and try to chemically dissolve this before measuring cell weight traditionally.

Chorophy measurements is another excellent semi-quatitative indication of cell content.

If you have access to a flow-cytometer, you can discriminate algal cells from non-algal particles by chlorophyll fluorescence of individual particles and obtain counts from cytograms: http://www.sciencedirect.com/science/article/pii/S004484860900708X

I agree with Robin as well. Direct cell counts do not always provide a direct correlation to dry weight due to variation in cell size depending on the point in the cell cycle. However, the time spent looking at the culture under a microscope gives valuable information on the health and status of the culture throughout the experiment that can help explain variations due to contamination or other factors.

The chlorophyll fluorescence is a good approximation. You can make a calibration curve, for example with direct count (microscopic count) and fluorescence at 380-500 to 680 nm. But the absortion/emission depend of your algae sample. In the next paper you can find information related with this topic (Photosynthesis Research 72: 39–53, 2002). Furthermore, you could make a test, washing the samples before read the concentration.

Microscopic cell count is probably the most accurate method, although time consuming. Another less time consuming possibility that no one has indicated is using a Coulter Counter, probably nobody has indicated it because it would measure both microalgae and your particles, if you just looked at the total count number. But, the suggestion is to look at individual size distribution graphs (count versus size), depending on sizes of microalgae and your particles and their size distributions it is possible that you have two distinct nonoverlapping peaks, one for microalgae and one for particles. However, if the distribution of the two peaks overlap then it will be hard to separate, but still may be possble mathematically depending on the degree of overlap.

Try Suyspended Solids and then, for a more accurate measure try Volatile Suspended Solids

Unless particle sizes do not overlap (see Miguel's anwer) or you have a flow cytometer handy, you can't avoid direct cell counts (in fact, if you use a flow cytometer, you would still be using a direct cell count).

Hello everyone, I quite appreciate your well-thought of responses to my question.

Undoubtedly, cell count seems to be the most accurate though it can be subjective as well. I am already working on using the chlorophyll extraction as an indirect method but I am employing 96% ethanol as solvent for extraction rather then acetone.

I will find out on the flow cytometer if it is available as I will prefer to use an equipment that measures the chlorophyll.

Is anyone familiar with cellometer as an automated cell counter? I will appreciate your comment on this equipment. My reservation for this method is that the cell slides can not be reused!

Lastly, I would like everyone to consider the solid as an adsorbent that leaches thereby leaving its fines in the medium. Maybe this information may help your responses. Many thanks to everyone.

the choice of solvent is yours (personally, I would prefer DMSO), and depends largely on the type of cells you are dealing with. just take into consideration Diana's and Erika's comments on the changing chlorophyll cell content. I still go for direct cell counts (and consider also Everett's comment on the additional info you get when using a microscope.

If you want to avoid chlorophyll extraction you may use

measurement at two wavelength to correct for the solid fines, alternatively

use differential spectroscopy, preferably 4th derivative to eliminate for not specifically adsorbing matter.

You could also try to eliminate the contribution of the fine pareticles from the suspension spectrum to obtain the 'true' absorption by the pigments (Chl in the case of the red region of the spectrum) and make a calibration curve 'scattering corrected OD vs. cell N or DW'. The method of spectral measurements is in attached paper.

You can measure the total organic carbon with a TOC analyser, provided you do not have significant bacterial biomass in your algal biomass. You can measure COD, (there will be intereference if your are algae suspension is in seawater). You can measure total suspended solids and total volatile suspended solids to get a fix on the inorganic content. You can correlate all the above these with chlorophyll concentration to form an picture of the phototrophic biomass content. Be aware that chlorophyll content of phototrophs varies with cell cycle and light cycle.

Field Cycling NMR is a quick way to get a measure of total lipid content and could be done in-line.

Assuming that your suspended solids are

you may try to use a method based on fluorescence (best: Flowrescence of Chl a), There are comercial probes for that available but a fluorometer in the lab is also enough for doing that.

Dear Akinlabi,

The answers above are very good; I will just add my opinion.

I would use microscopy, it is tedious but, in the end, relatively fast - and what you see is what you have, at least in terms of cell population. That would be impractical only if you want to process a lot of samples in paralell.

As for other methods, if you can not separate the cells of the samples, then the measure will always be indirect - a problem that appears also in solid fermentation. The content of what you are really measuring (chlorophyll, protein, organic carbon, membrane lipids) in the cell will depend on the physiological state of the culture. That being said, chlorophyll seems to be a good bet.

As for the adsorbent, perhaps its influence in absorption/scattering can be isolated from that of the cell population, as Alexei suggested. You will invest some time developing the method (absorbance spectra for the adsorbent, for the culture, for mixtures, and data correlation), but that should enable you to do direct absorbance or chl fluorescence readings withouth further sample preparation.

Is there any formula to calculate the cell count /ml of algae from the OD at 680 nm and dry cell weight (mg/ml)? Please let me know

You will need a calibration curve for your algal species o strain. Orientatively, here are some values of dry weight, mg per litre per OD unit for some known species:

Tetraselmis sueccica 593

Chaetoceros gracilis 423

Chaetoceros calcitrans 401

T-ISO 321

Nannochloropsis occulata 297

Chlorella japonnica 289

All obtained al 675 nm!