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effect of temperature on the survival of yeast cells

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Postby blcr11 » Mon Apr 23, 2007 3:39 pm

I don't really know what the lowest temperature should be here, but, barring any mistakes, your data suggests that it's higher than 70 C. Don't know if that's reasonable or not, but I think it's not impossible. Do you have a control where you know with some degree of certainty that all your cells are not viable? (try boiling them for 15 minutes or putting them in an autoclave for 10 min--something like that). If that kind of sample clears, then I'd say there's something wrong with your indicator solution. It's possible you're not using enough indicator, but I wouldn't overdo it, either. You did add the methylene blue, right?

Some ideas:

Make sure you heat the samples long enough. 1-2 minutes is probably not long enough to heat the sample completely. You may have pockets of cells that never get hotter than a few degrees warmer than room temperature. You probably need at least 5 minutes and 10-15 minutes sounds about right to me.

Do the controls--one that will certainly remain clear, and one that will almost certainly not clear (you'll have to make an educated guess as to what that condition should be). That way you'll have the two extremes to use for comparison.

There is a remote possibility that the sugar itself can reduce methylene blue, but normally that requires the addition of alkali, which I'm assuming you didn't do (that is, you didn't add NaOH). Try shaking your samples to see if you can see any blue color. If you can, that suggests that the dye is OK. Possibly check the pH of your solution. If it is very high (like 10 or greater) then one of your solutions may be off.

I should also say that, unless everyone uses the same exact protocol with the very same equipment and dilutions of reagents, etc. your answers can differ. So long as you can support your findings with your data, I would think you should be fine.
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Postby Star12 » Mon Apr 23, 2007 3:43 pm

Thankyou...i did a room temperature one and that obviously turned clear. I didnt consider doing one at 100c but i will try that. I definitely added the meth in as i heated the yeast, water (to activate the yeast) and meth in one test tube and the sucrose in a dif. tube then mixed them together? am i on the right track here?
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Postby blcr11 » Mon Apr 23, 2007 3:57 pm

I've never actually done this experiment, so I can't be certain, but it sounds OK to me. That isn't the only way to do the experiment, but it should work. You heated the yeast/water/dye mix for a fixed amount of time and then mixed it with sugar solution (that was at room temperature, I'd guess)--if I understand what you did.
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Postby Star12 » Mon Apr 23, 2007 4:02 pm

Yes. But i heated the sucrose in an alternate test tube at the same time as the yeast/meth/water. As i assumed if the sucrose was at room temp. it would cool the yeast down?
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Postby blcr11 » Mon Apr 23, 2007 4:39 pm

Once you've denatured the critical protein(s), I doubt very much you need to worry about cooling effects, unless you cool things very slowly, in which case you might get some renaturation.

One thing occurs to me though. I'm assuming that methylene blue is stable towards heat treatment. I don't acutally know that for a fact. If I'm wrong about that, maybe the heating is destroying the dye. If you take just water and a few drops of the dye solution (i.e, leave out the yeast) and put that in boiling water, what I would expect to see, if the dye is stable is, first the solution is blue; then maybe it decolorizes on heating (but maybe not) as the dissolved oxygen is driven off; then, as the solution cools, I would expect the blue color to return slowly, if it ever decolorized. If a total water blank can give you the blue color, but it goes away permanently when you heat it (in the absence of either cells or sugar), then probably you can't heat up the dye without damaging it. I don't think that's the problem, but if it is, just mix in the dye after you mix the treated cells with sugar solution.
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factors to control?

Postby sebbyvegas » Tue Apr 24, 2007 2:43 pm

right here it is.
im sitting here the day before my plan is due in.
and im stressing over the factors to control.

the only ones i can come up with are the simple factors like
-water bath temp.
-equal amounts of yeast.
..and then the block.
:shock:

anyone wanna lend a hand?


[/i]
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Postby blcr11 » Tue Apr 24, 2007 4:46 pm

“Control” in what sense? There are a number of variables you should probably keep fixed—which is what you mean by “controlled” I’m guessing—like the number of yeast cells used per test, or the amount of dye and sucrose/glucose used, or how long you do the heat treatment, or how long after the heat treatment you wait before reading any color changes—these should be the same for all samples, including controls. There should be only one variable that changes from trial to trial and that will be the temperature of the water bath or heating block. That you will set at a particular temperature for each trial. But then there are controls that can (ought to be?) done where you have a pretty good idea what happens to the yeast. At room temperature I would think all the yeast cells survive, while at some elevated temperature (and I’m guessing boiling water is high enough) none of the yeast cells will survive. Your experimental color changes should fall somewhere between those two extremes, at least. If you expose yeast to those two conditions at the same time you put yeast at your experimental temperature (probably between 40-100 ºC) you will have some basis to relate any color changes you may see in your experiment to the color changes you see in the controls where you (most likely) know what happened to the yeast. Is that clearer (or bluer, as the case may be)?
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Postby leaping badger » Tue Apr 24, 2007 4:57 pm

lleyew_Acissej wrote:Right... im writing this up as well and i havent a clue what im ment to do with the glucose or surcrose.... why do i need it??
Im not the brightest of people so please put it as simply as you can. :D

and has anyone any idea what temp they should denature at cos i thought most enzymes denature at roughly 42 degrees C

Thank you


yes you certainly are pretty slow, the glucose/sucrose is to provide it with 'fuel' so it can ferment, without it there would be no activity.

anyone got any idea of the best way to do the results table, just a simple temperature vs does the methyl blue become clear? or does anyone have any better ideas?
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Well...

Postby Steen » Tue Apr 24, 2007 5:14 pm

Mmmm....seems everyone's had the same ideas about this damn plan...anyone know if there's any difference between using the sucrose and the glucose? And any guides on the amounts/ratios of yeast to sugar solution to use? :shock:
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Re: Well...

Postby Jacob » Tue Apr 24, 2007 6:01 pm

Steen wrote:Mmmm....seems everyone's had the same ideas about this damn plan...anyone know if there's any difference between using the sucrose and the glucose? And any guides on the amounts/ratios of yeast to sugar solution to use? :shock:


That's what I was thinking, I wouldn't have thought that there would be much difference between sucrose and glucose though because they use sucrose to make bread and it's baker's yeast that the experiment uses.
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Postby emma_h » Tue Apr 24, 2007 8:07 pm

Which is better sucrose or glucose or does it not matter :?:
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Postby Jacob » Wed Apr 25, 2007 8:31 am

I haven't done the experiment but both of them should work. I'm doing a plan of the experiment and I've used sucrose because it's the same sugar as baker's use when they make the yeast.
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