Science Buddies: "Ask an Expert"

Hello,

I was wondering if anyone had any advice on measuring the mortality rate of algae in small quantities, like in a Petri dish?

Thank you

Hi Amittal,

Single-celled algae such as diatoms are grown in liquid culture and the easiest way to determine their numbers is to count them using a microscope and a counting chamber called a hemocytometer. You can use this technique with any type of alga.

Some algae such as chlamydomonas will form colonies on agar in a Petri dish, but this method requires that you prepare agar plates, make serial dilutions of each of your alga cultures, spread a drop of the dilution on the agar plate then wait a day or so for the colonies to grow large enough to count. You also need to spread several dilutions of your culture to make sure you have enough colonies to get an accurate count—neither too many nor too few.

If you have access to a microscope and counting chamber I would certainly use that method. Most biology labs have hemocytometers and someone there can show you how to use it. Here’s a link that explains what it is and the correct method for doing dilutions, counting and calculating the number of cells per mL: http://www.ruf.rice.edu/~bioslabs/metho ... nting.html

We can also help you in preparing agar plates, making serial dilutions and plating the algae if you decide to do it that way. Please post again if you need additional information.

Good luck!

Sybee

Hi Sybee,

Thank you so much for your response. I have a few questions about hemocytometers.

From what I read about these counting chambers, it seemed like they are mainly used to get an average number of cells, and the cells have to be evenly distributed. If the concentration of cells is not that high, will this method still work?

Also, do you have any suggestions as of how to look at the algae cells under the microscope? It doesn't seem possible to look at an entire Petri dish under a microscope, so would i have to look at it with a magnifying glass, or just with my naked eye?

Thanks so much

Hello Amittal,

The hemacytometers will definitely be more accurate if your concentration of algae is higher since the percent deviation from the actual concentration of algae will be less if you use a higher concentration. The cells will be evenly distributed under the cover slide in the hemacytometer--you do not have to worry about that. If you need any help with the calculations to determine the number of cells you have, let us know!

As for microscopy, you would only need to look at a small area in your petri dish using the microscope and then move the petri dish around to get a sense of how your algae are doing. You do not have to look at the whole plate at once to see if they are multiplying/dying.

A few suggestions:
Since you are measuring mortality rate in algae, you can either quantify using the hemacytometer or use an assay. If you were to use the hemacytometer, you would have to determine for yourself how many algae are dead (perhaps you could look whether the chlorophyll/pigments are red/brown/green or by seeing whether the cells are intact). Then measure what percent of the total number of algae were within your parameter of death to determine your percent of cell mortality. You could also use fluorescent dye assays that allow you to distinguish between dead and alive cells or allow you to quantify them using a spectrometer. A quick Google search yielded lots of options in terms of this, though money may be a concern since some of them are relatively expensive. Perhaps some other experts may have additional suggestions for measuring mortality.

Let us know if you have any more questions!

-crystalball

Here are two links for quantifying algae using microscopes:

http://www.botany.wisc.edu/courses/bota ... ations.pdf
https://biology.mit.edu/sites/default/f ... -23-13.pdf

The first site describes several different methods for quantifying algae. Both sites feature sample calculations. I hope that these sites help.

Hi guys.

To quantify mortality effectively there are a couple options: evaluate oxygen output (p vs. I curve). Do a live / dead stain (many commercial products exist). But an easy way is to look at settling rates using an imhoff cone. Counting cells under the scope is ok, but you can not evaluate their viability.

Photosynthetically,
Matthew