Questions about cryoprotectants

[acm44]

My research showed me that the most-used cryoprotectants are glycerol and DMSO. For my experiment, I am testing 4 different concentrations of glycerol, 4 of DMSO, and 4 of both together.

1. Cryoprotectants work pretty much like antifreeze, right? I'm not so clear on how antifreeze works, but I believe that, basically the antifreeze solution's molecules diffuse through the cells of the organism (in my case, pinto beans) and depress the freezing point of the water inside them. But --- how exactly does this help? The beans are being frozen in liquid nitrogen, in groups of five in vials filled with cryoprotectant. The water in the beans will freeze, it'll just take longer to freeze, right? So is it just the slower freezing that helps prevent ice formation? Or is there something I'm not getting?
2. Do cryoprotectants have a freezing point?
3. I am struggling to form a hypothesis. What makes one cryoprotectant better than the other? Its capacity of diffusing through the cell membranes? Size of the molecule, etc. Does anyone have any idea where I can find this sort of information?
4. I've read many articles that say that a mixture of both glycerol and DMSO work better than any of these by themselves, perhaps because of the decrease in toxicity. WHY is this? Cryoprotectants are toxic -- shouldn't two be even more toxic? I don't understand, and I can't find a more thorough explanation anywhere.

4. This is less related to cryoprotectants. After I take the beans out of the liquid nitrogen (they are staying in it for 3, 5, and 7 day periods), I expect that they will be stuck in the cryoprotectant solution, which will be frozen solid. Do I let it thaw at room temperature? I've checked out the Science Buddies cryopreservation of seeds experiment, but they don't work with cryoprotectants so I don't know if the procedure is different. Should I let them thaw, then put them in the incubator for germination? Or put them in straightaway? Will the de-frosting process harm the beans? Can I just plant them after they thaw, or is the germination in the incubator necessary?

[deleted-71712]

Hi acm44,

I moved this question to the Life Sciences forum where our biologist experts will see it.

For everyone's reference, the cryopreservation experiment you mentioned: https://www.sciencebuddies.org/science- ... p004.shtml

2. Do cryoprotectants have a freezing point?

All solvents have a freezing point. You might find it useful to look up the MSDSs (materials safety data sheets) for everything you're using; they'll tell you the freezing points along with other info about safety precautions. Glycerol is edible so it's fairly safe, but DMSO will go right through your skin and can carry other things along with it (perhaps a clue to its usefulness in cryoprotection?), so using the right kind of (thick) gloves is important.

http://www.siri.org/msds/
https://fscimage.fishersci.com/msds/96127.htm
https://fscimage.fishersci.com/msds/07770.htm
http://en.wikipedia.org/wiki/Dimethyl_sulfoxide

Amanda

[donnahardy2]

Hi acm44,

This will be an excellent project and you are asking very good questions before you start the experiments. Amanda has given you good information on freezing points of organic solvents. Here is some additional information and suggestions that should help with this project.

First, you need to do more background reading so you understand why freezing damages plants. Here is an article that will help you get started on this topic.

http://www.ncbi.nlm.nih.gov/pubmed/3077862

Next, you need to understand how cryoprotectants work. Here are some websites that contain more information on this.

http://en.wikipedia.org/wiki/Glycerol

http://www.plantphysiol.org/cgi/reprint/64/5/675.pdf

You also need to know if glycerol and DMSO are toxic to plants, and if so, why they are toxic. Are there any other possible cryoprotectants that are less toxic? This will help you design an experiment using the least toxic combination.

http://ww2.mackblackwell.org/web/resear ... 03025.html

http://www.gaylordchemical.com/bulletin ... in106B.pdf

Here is a website that describes how to freeze and thaw lab samples. These directions specify a fast thawing in a warm water bath. Thawing conditions should be a controlled parameter in your experiments, so you will want to select one method for thawing your samples and do it the same way every time.

http://www.immunocytometry.com/freezingthawing.html

You also need to know if the solvents will permeate into the beans. If the DMSO or glycerol can enter the plant cells, they will reduce the freezing point and protect the cells, as long as they are not too toxic.

http://books.google.com/books?id=RG-XCK ... &q&f=false

http://books.google.com/books?id=DM4xQ2 ... &q&f=false

I think that additional reading will help answer all of your questions and will help you ask a question that can be answered with a controlled experiment. You should be able to develop a hypothesis that will make sense based on your reading.

Please let us know if you have additional questions.


Donna Hardy

[acm44]

thank you for all the links! they are really helpful
i do have one question, not really about cryoprotectants--can anyone help me out with the germination part? in the example experiment, the seeds are placed in an oven in petri dishes. an oven? what kind of oven? we have an incubator at my school...also, does anyone have a suggestion on how long i should wait for the pinto beans to germinate, before assuming they are too damaged to sprout? i can't really hog the school incubator for more than a week or so. and i need a really effective method, because i need to plant them soon so i have actual results (project due at the end of the semester)
also, i am doing the liquid N part at a local uni lab. after i take them out and thaw them, i should be taking them back to school for the germination&planting part...how do you guys think i should transport the seeds? i know it seems like a silly detail, but i want to be sure.
i know there is a way of germinating&growing the seeds all in thepetri dish. if i leave them in the cotton, in the petri dishes, will they grow? for how long before i should really plant them in soil? i am thinking 5 seeds to a petri dish, is this a good number?

[donnahardy2]

Hi,

What kind of seeds are you going to be testing? The germination step for your project needs to be done within the temperature range for the specific seeds you are using. Since the percent germination will be your dependent variable, you need to try to provide optimum conditions for germination.

http://en.wikipedia.org/wiki/Germination

So you need to do more background reading to find the optimum conditions for the seeds you are using.

It's best to use as many seeds as possible. Five is an adequate number, but 10, 20, 50 or more would be better. Be sure to include a control sample that is not exposed to a cryoprotectant at all. If you purchase seeds that have a low germination rate, then you will not be able to get statistically significant results if you only use 5 seeds.

Donna Hardy

[acm44]

I'm using common pinto beans for my exp -- I've found the info about adequate temperature, watering, soil type, etc. It's just that there are a lot of pre-planting procedures to help germination, like keeping the seed in damp cotton or even just leaving it in water overnight. I'm not sure if the oven step in the example experiment is one of these procedures, or if it is really necessary because of the liquid N freeze. I was wondering if anyone has an opinion/has maybe used beans in an experiment before and can let me know exactly how helpful&necessary these germination-helping procedures are or if I can just plant them directly in prepared soil (after thawing, of course).

[donnahardy2]

Hi,

Do you know the temperature used for the oven step? Beans will germinate between 60 and 85 degrees F and have an optimum temperature range of 80 to 85 degrees F. So you will obtain good germination if you can maintain the temperature within the specified range. It will just take a little longer if the temperature is less than 80 degrees F.

http://www.aces.edu/pubs/docs/A/ANR-1061/ANR-1061.pdf

I recommend that you do a trial run and test the germination of 10 of your beans, and it would be easiest to just plant them all in soil. You will want to define the germination conditions for your experiment and make sure that all of the seeds are maintained under identical conditions, so a preliminary test to validate the germination conditions would be very helpful.

The science buddies website has some excellent information that will help with your experimental design and help you decide how many beans to use for each trial. Please read through these articles and let me know if you have questions. Planning your data analysis before you start your project is a good strategy for success.

https://www.sciencebuddies.org/science- ... sign.shtml

https://www.sciencebuddies.org/science- ... atio.shtml

https://www.sciencebuddies.org/science- ... ysis.shtml

Donna Hardy