Science Buddies: "Ask an Expert"

hello,
my school's fair is in january but my regional fair is in march. most schools do their fair in march so the timing is very inconvenient for someone like me who is doing a cell culture study, which requires quite a bit of time. the lab i am working at is closed over xmas break and my materials will probably only ship during or after break. the thing is, i did a project last year when it was not required, so i feel that i should be given a bit of leeway. should i ask my teacher for an extension? i could also mention turning in my last year's project to the fair b/c i never presented it at my school fair (just went straight to the regional fair). another option is just turning in partial results, but i don't even know if i'll have results by that time.

Hi,

This is a difficult problem. It sounds like you won't be able to start your project until very close to the January deadline. With science fairs, projects are judged at the local level, and then winners are allowed to progress to the regional level. I don't know if you would be allowed to go directly to the regional level if you were supposed to enter the local fair. This would be unfair to the other participants, so I don't know if it would be a good idea to even try it this year.

You can't enter the identical project that you did last year, but many of the best projects I have seen have been new projects on the same topic. Working on the same problem in successive years can result in a really outstanding project, so I recommend that you write up this year's project as completely as possible and enter it at the local level in January. You may just have minimal results at the January deadline, but hopefully your presentation of the scientific principles, background information, analysis of data, and conclusions will compensate enough to allow you to win a placement at the regional fair in March. You can then continue working on the project until the March deadline, and hopefully this will give you a winning strategy.

Do you have any questions about the details of your current project?

Good luck!


Donna Hardy

donnahardy2 wrote:Hi,

This is a difficult problem. It sounds like you won't be able to start your project until very close to the January deadline. With science fairs, projects are judged at the local level, and then winners are allowed to progress to the regional level. I don't know if you would be allowed to go directly to the regional level if you were supposed to enter the local fair. This would be unfair to the other participants, so I don't know if it would be a good idea to even try it this year.

You can't enter the identical project that you did last year, but many of the best projects I have seen have been new projects on the same topic. Working on the same problem in successive years can result in a really outstanding project, so I recommend that you write up this year's project as completely as possible and enter it at the local level in January. You may just have minimal results at the January deadline, but hopefully your presentation of the scientific principles, background information, analysis of data, and conclusions will compensate enough to allow you to win a placement at the regional fair in March. You can then continue working on the project until the March deadline, and hopefully this will give you a winning strategy.

Do you have any questions about the details of your current project?

Good luck!


Donna Hardy

Actually, it is not the local fair in January - it is just my school fair, a pretty small event. Grades 4, 6, and 8 are required to do a project (I'm in 8th), but I did a project last year as well. The way it works is school fair ==> regional ==> state. (there's no separate local and regional fair)
Basically whoever wants to go from my school goes to the regional fair - I've already filled out the preapproval forms, so its pretty much a given that I'm going to regional. The only problem is the deadline for the school fair, which I personally think is way too early to do a good project (only two months of preparation and testing).
The main problem with the January deadline is that some of my materials will only ship after Xmas break, so the circumstances are kind of beyond my control at this point.
If you were a teacher, would you give me an extension? I'm just a bit hesitant about asking her, since I don't want her to think that I should be given special treatment or anything - it's just that I think my project would be a lot better quality and have a far better chance of winning if I was given some extra time beyond the school fair.

I do actually have a question about my project - I am doing an ELISA test to determine the level of cytokines in my cell culture supernatant. I'm doing 10 96 well plates. Do you know how many wells per test group is recommended? Right now it is about 5 wells per test group, which is 25 total b/c I have two time intervals (5 plates per time interval). Is that enough to do a proper statistical analysis? Do you know of any resources online for using statistics in biology?

Hi,

Thanks for explaining your situation. You are in 8th grade and doing a project on cell culture supernatant cytokine analysis? I'm completely impressed, and I want to encourage you to continue on this project to compete in the regional science fair. However, this does not excuse you from the local school science fair requirement. It is understood that you may not be able to complete the experimental work before the local school fair, however, you should explain the circumstances of that to your teacher (you are waiting for the cytokine kits to be delivered). And, you should still turn in a project board, which includes the title, hypothesis, summary, background information, maybe some results from last year, conclusion and bibliography and present it at the school fair. You are going to have to do these sections anyway, so it will be good to have them done early, even if you have to revise the conclusion after you get some results. You need to demonstrate that you understand the principles of doing a science fair project for the school fair. You should discuss your situation with your teacher immediately because if turning in a project with no results is not acceptable, then you will need to do a different project for the local school fair that you can complete on time. You can still continue on your topic of interest and turn in the cytokine project for the regional fair.

Do you have the instruction manual for the cytokine kits you have ordered? Please let me know the manufacturer and I'll take a look at it and point out some important details. Cytokines are very unstable proteins, and the samples are usually tested fresh, or frozen at minus 70 degrees Centigrade. How are you going to store the standard? What cytokines are you planning to assay for? What is the purpose of your project?

Cytokine test kit results are variable and good results depend on how you handle the standard that is included in the test kit. In order to obtain really good results, you should test control and test samples on the same plate with the same standard curve. It is best to run samples and each dilution of the standard duplicate, so you will be able to do one standard curve and about 40 samples on each plate. Cytokine assays are really expensive, so sometimes real researchers run their samples singly, but it's better science to run duplicate samples.

Donna Hardy

Hi,
Thanks for taking the time to respond to my posts. I really appreciate it.
I emailed my teacher and I explained to her about the materials shipping lag and how the lab is closed over xmas break. Hopefully she will understand, but if not I'll just have to limit the number of days I can culture the cells. I have exactly 9 days between the end of break and the project deadline. And I'm not sure how long the data analysis will take me, so it's cutting it really close.
Either way, I'm definitely going to create a project board with the appropriate sections - I have already finished most of the sections aside from Results and Conclusion. And the graphs, etc. of course.
here is the cytokine kit i am using: http://www.ebioscience.com/ebioscience/ ... 8-7324.pdf
The samples will be tested fresh, as I can't control for the effect of freezing. The standard is currently being stored at -20 C - the cytokine kit was already shipped, i am just waiting for some other materials. I know it says for the standard to be stored at -80 C on the datasheet, but the facility I am using only has a -20 C freezer. I emailed the tech service at eBioscience to double check, and they said it was fine.
Purpose: to determine the effect of beta amyloid on tnf-a production in j774.2 macrophages. i'm also hoping to identify some novel inhibitors of tnf-a production. it's an alzheimers' disease related project. if i do identify novel inhibitors, then it could lead to a possible treatment for alzheimer's, as inflammation may be a significant factor in the disease.
yes, I am running 5 plates per time interval (10 plates total) - luckily I was able to get the kit donated, otherwise i might not have been able to run multiple tests.
About 16 wells/plate are in use for the the standard dilutions; thus I have 80 wells open. I'm using the assay diluent as the negative control, and that is 2 wells. As I'm using DMSO to dilute my inhibitors, I have a DMSO vehicle control which is 1 well/plate. I'm not sure if I should have more wells for this vehicle control - what do you suggest?
And then I have 2 controls, and 10 test groups. That amounts to a little more than 6 wells per group per plate. So 30 wells total.
Also, I have one more question:
How many concentrations of each inhibitor would you recommend? I'm currently planning 2 concentrations, but I'm pretty sure I would need a lot more if I'm going to make a dose response curve. But then again, that would decrease my test size. What do you think?

Thanks again for all your help,
Anna

Hi,

Please don’t try to do your entire project in 9 days; that’s just not enough time, even if you were to spend 24 hours a day on this. You are going to have lots of data, so you need to plan at least a week on the data analysis. Lab work is very time consuming, and there is a lot of pipetting required to set up these assays. You need at least a week to grow the tissue culture cells and it takes an unbelievable amount of time just to set up and run one Elisa plate, and I noticed you have an overnight incubation, so you are going to have to plan your time line carefully. You need to plan everything out, and preferably to a trial run without using your kits. And you should plan to run just one plate the first time, very slowly and carefully so you won’t make any pipetting mistakes. Your primary objective is to get results for the regional fair, and you were right that you don’t have time to do this before the school fair. If your teacher will not accept a project without results, can you think of a simple qualitative experiment you could do so that will give you some results to present for the school fair? Maybe you could grow up some tissue culture samples and do a trial run with 2 dilutions of your inhibitors and look at the cell morphology to see if can see if there is a visual effect on the macrophage cell growth. This experiment will help you select dilutions for the definitive experiment. You can spend all of January conducting your real experiment, and all of February analyzing the results. That’s a much better time frame for this type of project. You will want to be able to write back to the individual/company who donated the reagents and report a successful project to reassure them that their contribution was worthwhile. This is a wonderful project, and I want you to be successful as well.

I’m not sure I have followed the plate set up. I need to understand exactly what you are planning to do. I think the following set up will work:

16 wells standard curve
2 wells negative control
2 wells positive control
1 well blank
1 well DMSO control
That will leave 74 wells per plate for your samples.
How many tnf-alpha kits do you have (5 or 10)? Do you have enough reagents to set up your entire experiment twice? How many vials of the standard and control do you have? Cytokine samples can be thawed and used just one time, and the time interval between thawing and testing the standard will affect the quantitation.

Have you been able to find any literature references for any of the inflammation inhibitors you are planning to test? That would help you determine what dilutions to make for the inhibitors. I think you will want to test at least two dilutions of each inhibitor, but maybe 3. A dose response curve would be ideal, but maybe you need to save that project for next year. I think it's good you are taking the time to carefully plan out the number of tests you can do on each sample.

Donna Hardy

Donna has given you lots of good advice.

You are doing a very advanced project as an 8th grader. I'm not sure what the standards are for the school project, but you could do a simpler and faster experiment for the school fair (but still a very good 8th grade project). We have tons of projects on this website that you could do in a shorter time frame and still end up getting a great grade. Or, like Donna suggested, talk to your teacher about using the research you did last year, but expanding on it for the regional fair.

If you do end up doing a simpler experiment for the school fair, I know it might be frustrating for you to end up doing two projects, but sometimes that's just life . If you choose something that sounds really cool, hopefully you'll enjoy both projects.

Good luck!

Good news - my teacher has been kind enough to allow me to submit my last year's project for the school fair. So I have until march. Thanks for your input about that though.
My materials should be shipping around break time, so I plan to start around January, latest early February with the testing.
Question: Are both negative controls and blanks necessary? I'm using the assay diluent for the negative control. As for the positive control, I contacted the tech support of the assay and they said that the standards would suffice for the positive control, to show that the assay is working.
My original plan was:
16 wells standard
2 wells negative control - assay diluent
1 well DMSO control
Leaving 77 wells for samples. I'm still deciding how many inhibitor dilutions to use, so the wells per test group could vary.
I have 10 kits. Yes, I have the reagents to do all the experiments. I'm not exactly sure how many vials I will be receiving - it is aliquoted at 20 uL/vial, and I only know that after dilution it is 100 uL/well (16 wells) per plate. I will contact eBioscience about it.

Yes, I have found some concentrations of the inhibitors, in different conditions however, in literature. They have been used in very different concentrations - one at more than 200 uM, and but most at 10-30 uM. I am testing 5 different inhibitors. To find a middle ground I was planning on using 50 and 100 uM of each, but now I may be doing a dose response curve which needs 6 different concentrations. I have about 3 months (exactly, actually - the fair is on march 16-17) so I think I would have enough time to figure out how to do this. I think it would make identification of a novel inhibitor more credible, so it is definitely worthwhile.
The only problem is that using 6 different concentrations would reduce my test size to 12 wells total per inhibitor (over 5 plates, 2.4 wells per plate). I'm not sure if this is enough or not. I have seen studies that use only 4 wells per group total though - http://www.informaworld.com/smpp/conten ... 075&db=all

Thanks so much to both of you,
Anna

Hi Anna,

This looks like its shaping up pretty well and is definitely quite an important project.

A 6 point dose curve of course will have very high power, but given the limitation you have on the number of wells, so this may not be the best idea (alternately, it might still be, but I guess those are the two options...). I've gotten away with 4 before for cytokine response (different cell line and different stimulus), so it will depend on how you sensitive you expect the difference to be. More explicitly put, if you expect there to be a large difference in response between the different doses, it would be wiser to go with the larger number of doses so that you can pick up an accurate curve fit. If you expect the difference in response to be larger, fewer doses might be easier. In my own experience, I have worked with mouse IL-2 data, and the response counts get exponentially larger only between 0 and 4 uM, so 4 doses was plenty fine to generate reasonable numbers between 0 and 20 uM (I was less concerned with the lower concentrations at the time). I doubt that TNF-a and IL-2 data will correlate perfectly, so try and find data in the literature from previous dose response curses to gauge how sensitive you need to make your scheme to be to get an accurate curve fit.

Hope this helps!
Aaron Lin