Science Buddies: "Ask an Expert"

Hi!

My research topic is "Spectral sensitivity of cell shape and cell division in Euglena gracillis." I'm going to use blue, red, and white LED to culture them and observe their cell division.

The problem is

1. What cultures do I need to use?
I heard that Euglena grow well in just water. But I don't know it's quite right. Additionally, I'm a student, so I don't have much money to afford expensive cultures.

2. How bright the intensity of LED should be?
I found many LED from Internet, but it says that the same 3W LED can have different light intensity because its spectrum differs. If I want to culture Euglena with 3W LED, how many LED do I need to use? (I'm going to make 13*13*13 box and put a petri dish on its bottom, make sure that no external light can penetrate into the box by wrapping it with aluminium foil. So the LED will be on the top of the box and it will be about 12cm above Euglena. And I don't know how to calculate the intensity of light with lumens, lx, and foot candles....

3. How can I count the number of cells?
I'm going to count the number of cells to compare its reproductivity. And when I asked about it last time, they recommended me to use hemocytometer but I cannot afford that.. Is there any other way? Can I extract a certain amount of culture and then count the cells repeatedly and regard their average as its number of cells?

Hello there!

Interesting research topic--just out of curiosity, how did you come up with this question?

1. What cultures do I need to use?
You can certainly obtain Euglena gracillis cultures from Carolina Biologicals. http://www.carolina.com/algae/euglena-g ... /152800.pr
They have instructions on this page on how to grow these algae--the medium is soil water and optimal temperature is 22 degrees Celsius, and it also requires 50 to 100 ft candles of fluorescent light. I've also found other Euglena cultures sold on this site, but they're all at affordable prices. What I would suggest you doing is to call Carolina Biologicals, let them know the experiment you're planning to do, and see if they have any suggestions on which culture is the best to purchase for your experiment. They may also be able to tell you how to culture them normally in light, and also give you suggestions on how to culture them for your experiment with the blue, red, and white LED lights.

2. How bright the intensity of LED should be?
This I would also ask Carolina Biologicals since they should know a little more about how to culture these organisms. Are you trying to use the same intensity of blue, red, and white LED, but see whether the different colors affect their growth rate? Unfortunately, I cannot help you with calculating LED intensities because I personally don't know much about that. Do any other experts here have any experience with this? If no experts in this forum are able to answer this question, I suggest posting your question in the physical sciences forum because there should be physicists there that would be able to help you with this!

3. How can I count the number of cells?
A hemocytometer is the most standard way of counting cells, but like you said, it may be a bit expensive for a student to afford. Are you going to be doing your experiments at home or at a high school lab? If you are doing it at a high school lab, it may be worth asking your teacher if he/she has a hemocytometer available, or if they know there may be one that you could borrow elsewhere. If not, one way that microbiologists count bacterial cell numbers is by plating serial dilutions of the cultures on agar plates and counting the number of colonies that show up. I don't know whether this could be done for Euglena though. I would do a little research on how people work with Euglena and count cell numbers and see if there are easier alternatives than a hemocytometer if you can't acquire one. I would also ask Carolina Biologicals if they have any suggestions on how to do this.

In addition, here's a link to a Science Buddies thread that discussed working with Euglena: https://www.sciencebuddies.org/science- ... 28&t=13455. See if you can find anything there that may be helpful!

I hope this helps! Let us know if you have anymore questions!

Best,
Connie

Thanks for a nice answer! It really helped me a lot.

And, about how I came up with this idea, I am actually interested in interaction between light and living organisms. You know, light involves in quite many things than we usually think, like they regulate gene expression, or takes part in morphogenesis, and obviously phototropism or phototaxis. And I found a paper about Blue light regulation of cell division in Chlamydomonas reinhardtii (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1080634/) and found out that some microalgae needs light in their cell cycle (we still don't know whether light directly controls cell cycle or it regulates expression of cell division factor) and their cycle can be differed by characteristics of light such as intensity, wavelengths or frequency. So, I chose Euglena because it's relatively easy to culture and it's very useful to our lives. I thought if we know more about conditions of its cell cycle, it would be far easier to culture them indoor which will increase the supply of it.

And actually... the post that you pasted related to this topic.. is actually written by me... What a coincidence, right?
And yes, I'm going to do my experiment in my high school lab but we don't have hemacytometer.. So, about the dilution and counting colony, I don't know what dilution means actually, so can you explain a little bit more about it? Is it just putting more culture medium into part of original culture?

Again, thanks for your answer!!

Hi,

Connie gave you a great list of information and advice. I would just add that it is possible to count the number of Euglena without a hemocytometer but you do need a microscope. The one thing a hemocytometeer does is provide a visual space on a special slide that has squares of known dimensions etched on the glass. Since you don't have a hemocytometer, what you need to do is count the number of Euglena in the SAME area each time. There is a way to do this without a hemocytometer and that is by using a round glass reticle with a grid pattern etched onto it that fits inside the microscope eyepiece - http://www.microscope-microscope.org/ad ... ticles.htm

These are not as expensive as a hemocytometer and you might be able to find a used one on ebay for a lot less.

I don't think that Euglena, which are a single-celled, free-swimming algae can be grown on agar to form colonies, but do ask the folks at Carolina Biologicals. They love to help young scientists with their experiments!

The only other alternative that might work would be to simply count ALL the Euglena in the entire microscope field. You would have to try different magnifications (100x, 200x, etc.) until you found the one where you have 100-200 Euglena per field. You can't count 1000 per field and 10 or 20 is too few. Since the field would be the same for each slide, you will be able to directly compare the numbers of Euglena in your cultures after each different light treatment. The only thing that you can't easily do is calculate how many Euglena you have per milliliter of culture which is what the hemocytometer allows you to do. The other thing you have to do is stop the Euglena from moving into or out of the field while you are counting them. Again, ask the experts at Carolina how they would do this.

I would still try to borrow a hemocytometer. Nearly every university molecular biology lab has several stuck away in a drawer somewhere and I'm sure that they could let your school borrow one for the duration of your experiments.

I'm looking forward to hearing about the results of your light experiments. Good luck!

Sybee