measuring bioluminescence

[deleted-32471]

I was observing my cultures bioluminesce, but they did not glow. I put them under light or dark respectively for a whole week. Should I continue with it just sitting under light or darkness for more time? Did I do something wrong?
Thanks

[deleted-71536]

Hi tapman,

Could you tell us little more about your project? What kind of bioluminescent bacteria are you using? Some species only glow for the first few days after you culture them. If you give us some more details, we may be able to help you more effectively.

Heather

[deleted-32471]

I am using a species of marine plankton called Pyrocystis noctiluca (P. noctiluca), and what I did to instigate bioluminescence was that I recreated its light-dark cycle, placing two tubes in darkness all the time, two tubes in light all the time, and two tubes in darkness for 12 hours and light for 12 hours. I kept this pattern for about a week, and when I followed the directions of observing bioluminescence, the tube didn't light up at all.

[deleted-71536]

Hi tapman,

Did you agitate the tubes? P. noctiluca glow in response to turbulence.

Here is a link to a scientific abstract that studied how agitation induces bioluminescence in P. noctiluca:
http://www.sciencedirect.com/science?_o ... 99791aa0bd

What were the instructions you followed?

If you're still having trouble, you might have better luck with Pyrocystis lunula, a related species that is commercially available.

If this information didn't help you, please let me know, and I'll try to find some more information for you!

Heather

[deleted-32471]

I agitated the tubes. Well, I didn't shake the tubes vigorously, but I turned it upside-down and back a few times. My experiment instructions are aimed towards Procystis lunula, but UTEX, the place where I bought the cultures, told me that whether I get P. noctiluca or P. lunula is chosen by the health of those cultures by the cultural manager, and that they would both bioluminesce. I have followed the science buddies experiment instructions for my project.

[deleted-71536]

Have you tried calling the company? If you followed the instructions and the cultures didn't bioluminesce, customer service should replace your cultures.

Beyond that, I am at a loss to explain why you saw no bioluminescence in any of your cultures. If you do get a new set of cultures, you may want to check one culture before performing your experiment, to make sure that they bioluminesce.

[aelin]

Hi,

I'm not a particular expert with this type of plankton, but I have done a project with cyanobacteria before to measure UV resistance. One of the problems with these sorts of marine organisms is often that they are very very hard to keep alive. So, this is a rather basic thing, but are you sure that your plankton are still alive? With the improper settings for light intensity/type, pH, temperature, etc, they very easily die... which was problematic for me at least. Hopefully not for you, so definitely contact the company like Heather suggests if the plankton are alive but nonfunctional.

Hope this helps!
Aaron Lin

[deleted-32471]

Hi
Im doing a project on Bioluminescence in the species of marine dinoflagellates, P. lunula. I put two tubes in total darkeness all the time, two tubes in total light all the time, and two tubes in darkness for 12 hours and light for 12 hours. can you please help me by explaining to me what tubes should illuminate the most.
When I measure bioluminescence, I am not getting any obvious trend in the intensity of the light.

[donnahardy2]

Hi Tapman,

I found your earlier posting on this topic. It is helpful if you continue on the same thread so everyone will see your new inquiry. Hopefully the moderator will merge this topic with your earlier posts.

Dinoflagellates have a circadian rhythm and would be expected to exhibit luminescence during dark hours after being exposed to light. If you kept cultures in the dark or light all of the time, this would probably have an adverse effect on the luminescence because it would affect the circadian rhythm. I could not find a reference that I had access to that indicated what the intensity of luminescence would be. The emission of photons will very brief from each cell. Temperature and mechanical agitation are important in the induction of luminescence.

http://pubs.acs.org/doi/pdf/10.1021/bi011651q


What were your results?

Donna Hardy

[deleted-32471]

There is no trend in my results. They are all over the place. The tubes in total darkness sometimes illuminate much more than the rest of the tubes, and sometimes illuminate well under the other tubes.
my results for one reading were:
Tube millivolts
A 7.4mv
B 3.6mv
C 9.9mv
D 7.4mv
E 5.4mv
F 7.1mv

[donnahardy2]

Hi Tapman,

I wish UTEX had not substituted cultures for you because most of the literature citations are reports for P. lunula. For the purposes of your discussion section, you will have to assume that Pyrocystis noctiluca behaves similarly. If you order from this company again, you can write on your order “no substitutions,” to avoid having your order switched again. However, you did receive a healthy culture, and it is growing, and you have obtained results, so that is very positive.

Apparently variable results are a common problem when working with this group of organisms. I am going to attach an article that was written by authors apparently having the same problem that you observed. Notice the title of the paper is, “Variability in the bioluminesence response of the dinoflagellate Pyrocystis lunula.” You could have a similar title for your project. The authors found that both shear and acceleration in the flow are needed to trigger a bioluminescent response. So it’s possible that your technique of agitating the tubes did not provide a consistent or optimum trigger for the response. The authors also mention that exact mechanism of bioluminescence is not completely known, so additional research needs to be done in this area.

The article that Heather found mentions that cell flashing can be induced with osmotic shock, pH, temperature and pressure. The cells produce flashes of light at about 480 nm in less than 20 ms and the flashed last from 50 to 150 ms. When you made your measurements, did you measure the light at the same time that you were agitating the cultures, or was there any delay?

I could not find voltage responses in the literature references; most of the graphs include light emission in photons on the y axis. Do your voltages correspond to photons? What voltage do you get when testing a non-agitated control or a tube of sterile culture medium that is being agitated? It would be helpful to have a point of reference for your results.

Were the results you posted for A-F replicate readings from one culture? If so, do you have similar sets of data for your other samples (all dark, all light, 12 hours dark, 12 hours light)? If so, you can calculated the standard deviation of your sets and compare the mean values and standard deviations. You can then use the student’s t test to evaluate whether or not there is any significant difference in your results.

http://en.wikipedia.org/wiki/Student's_t-test

Here’s a t-test calculator:

http://www.graphpad.com/quickcalcs/ttest1.cfm

It could be that the variable results you obtained are to be expected with this culture. It would be interesting to write to the authors of two papers (one in France, one in San Diego, the e-mail addresses are in the papers) and ask if they have similarly variable results.

The key to writing up results that are not clear cut is to include possible reasons for the results and hopefully some of the suggestions I have made here will give you some ideas. Let me know if you have any questions.


Donna Hardy

variable bioluminescence part I.pdf

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[donnahardy2]

variable bioluminescence part II.pdf

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[donnahardy2]

variable bioluminescence part III.pdf

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