Science Buddies: "Ask an Expert"

Trader,

I echo Donna's comments completely. A readable board that summarizes the main points of the project is preferable to a tome of information that is poorly organized and that fails to help me see the most important points. You have spent a lot of time preparing and working for this; now is the time to relax and enjoy it. You will do your best and that is all you can do - relax and have fun. Stressing about these things at the last minute will only frazzle you.

Cool -- I was just unsure about the inconsistency between the title and the focus of the project "effect of microaerophilic environment on growth of facultative anaerobe b. subtilis" and having my independent variable as temperature, which makes it a bit weird.

I can upload a picture very soon -- as for the significance, I was wondering if the significance goes beyond helping better understand / quantitating (even though I dont know the actual oxygen level I used) the effect of less oxygen on b. subtilis. I also have stated in my purpose "to test the definition of b. subtilis as a facultative anaerobe" -- is this risky? I thought that it was shown that it was suggested/shown that b. subtilis wasn't an obligate aerobe at around 1997 which was more than 12 years ago which isn't that recent -- and mentioned in 2000 that it was a facultative anaerobe I think.

So it wouldn't be really significant because it seems like the proposal that it is a facultative anaerobie is well established? Just not sure how definite the classification of b. subtilis as a facultative anaerobe is. If it is definite, I have the time to remove that. I'm not too sure...

As for the significance of the experiment (sorry yet again) -- I know that b. subtilis is used for the fermentative/creation of industrial enzymes -- and I remember that back in the regional fair, I was asked about a specific example that it applied to. I wasn't able to give a specific example, and now I think I have one --

Under this -- "Anaerobic Growth of Bacillus mojavensis and Bacillus subtilis Requires DNA" aem.asm.org/cgi/content/abstract/70/9/5252 it says that one example is for the recovery of petroleum hydrocarbons that "remain entrapped after current recovery technologies reach their economic limit"...microorganisms produce a variety of biosurfactants, several of which generate the ultra-low interfacial tensions needed for hydrocarbon mobilization, and bacillus mojavensis produces this under anaerobic conditions

I'm not sure if this applies to b. subtilis, but is this the around the idea of what b. subtilis does? I also know w/ b. subtilis' use in probiotics -- would the stomach be a microaerophilic environment? =P Back then I wasn't sure, so I didn't use that example, but it'd be interesting to anticipate growth patterns in places where we want the bacteria

Just want to clarify:

There are only TWO ways that b. subtilis grows under anaerobic conditions
> fermentation growth (which is the opposite of cellular respiration, = does not donate electrons to nitrate but instead takes electrons? This is the process of oxidization?"
> using electron acceptors which donates electrons to nitrate and not nitrite.

Finally I was looking at the judging guidelines for the fair and noticed that it called for some things that I don't think I have:

1) creative ability and originality in analysis and interpretation of data (I'm not sure what this means -- I tried to display creativity and analysis through my graphs, with the exponential curve and the semilog graph -- anything else I can add into my presentation and/or to my research paper? I do have one more day of setup...)

2) variables clearly recognized and defined (I'm afraid this isn't the case with the temperature/microaerophilic environment messup ) because changing both the oxygen requirements into microaerophilic and changing temperature, that would be 2 variables right?

and 3) control group -- I know that a control group is to make sure that the results are actuall as a result of the independent variable (which is temperature? But I'm trying to manipulate oxygen level requirements ), so would the control group be when I plated bacteria in a microaerophilic environment in any temperature, any amount, and after dilution plating, there were colonies that occured?

Thank you once again -- last day tomorrow to prepare! I'm terribly sorry for the last minute-ness

Hi Trader,

This is good that you are focusing the details of the project. The oxygen levels in your experiment were one of the controlled variables; you grew all of the cultures under microaerophilic conditions (because you did not have a way to continuously aerate them).

There have been several references showing that B. subtilis is a facultative anaerobe, so I think it would be safe to leave this information in. Just leave your write up the way it is. Your results support the literature references.

The use of B. subtilis to produce industrial enzymes and to recover petroleum hydrocarbons are excellent practical examples of the significance of the type of experiment. The use of B. subtilis in probiotics in the treatment of gastrointestinal disorders caused by antibiotic treatment is another good example; the spores survive the acidic environment of the stomach and germinate and grow in the microaerophilic environment of the intestinal tract. These are also excellent topics for future science fair projects. I think that a basic understanding of how microorganisms grow is significant as well, and is intrinsically interesting.

You are correct. B. subtilis has two different metabolic pathways that can be used under anaerobic conditions. If nitrate is present, an enzyme called nitrate reductase is produced and nitrate is used as the final electron receptor. If nitrate is not present, then a fermentative pathway, which requires amino acids, is used and glucose is fermented to produce end products called acetoin and butanediol. Since your medium did not contain nitrate, but did contain glucose and amino acids, your B.subtilis cultures probably were using the fermentative pathway to produce energy needed for metabolism.

Your analysis of your data is very creative and original, and I would definitely not recommend adding anything at this point. Your experiment had only one variable, temperature. The flasks were incubated under identical oxygen levels, and were aerobic initially but rapidly because microaerophilic because you did not have a way to aerate them.

You are well aware of the limitations of your experiment, and the data you were able to obtain with limited resources. However, the strong point about your project is your understanding of the subject and the background reading you have done on current literature references. This shows through on your board write up, and you should use to your advantage when you talk to judges.

Donna Hardy

Thank you very much!!! That really clears things up

One last thing -- I'm not too good at identifying a control group, but if the purpose of a control group is to determine that the results are actually as a result of the independent variable -- would my experiment that grew any amount of bacteria in broth, and then processed it through dilution plating and obtained results be an indicator of this? (My technique isn't that good =P)

Also for the microaerophilic environment, as I was unable to continuously aerate, would there be decreasing oxygen levels throughout the experiment, therefore there might have been a decrease in growth rate throughout the experiment? (then the environment would eventually be anaerobic?)

I'm also adding the last bits of my error analysis. I think that because the smallest increment of the micropipette is 0.01 mL, the uncertainty is 0.005, and the smallest increment of the pipette is 0.1 mL so the uncertainty would be 0.05 mL.

In the case where I use the pipette once, then the micropipette maybe 4 times, would the systematic error % be 0.005 + 0.05 *4? = 0.205 = up to a 20% difference from the actual population?

I also mentioned aerobic conditions of 20 minutes as well as anaerobic conditions in some of the related experiments.

If I can tell that anaerobic growth much slower than and aerobic growth is faster than that in microaerophilic environment (from what I read in the related articles), can I graph my experimental results, the "theoretical" aerobic growth and "theoretical" anaerobic growth? .

Because during practice someone asked me "so how can you just assume that the lower growth was because of some difference in the experimental setup and not because of oxygen??

Also, ONE LAST THING (last post!!! because judging is tomorrow!!) --

I am also a bit worried about the microaerophilic environment. I tried to say that after it starts at aerobic, it eventually goes into microaerophilic, ... BUT!! someone asked me "how much time do you think it'll take until the oxygen reaches microaerophilic levels?. I have no idea.

One last thing is the practical application -- someone asked me a really good question: given what you now know about b. subtilis, what would you change about its use in probiotics and the creation of industrial enzymes? I wasn't too sure, but I thought that we would be able to change the quantity of it becaues we would be able to anticipate the growth and therefore know how much we should have and when?

Thank you once again. I look forward to seeing you at the fair!!

I'm sleeping early

Thanks Donna Hardy and all the kind mentors who have helped me through the entire experience. I'm really glad to have made it this far.

Thank you ScienceBuddies for the wonderful site and the wonderful compilation of information and this forum -- it took my science fair journey far

I guess this concludes a school year of science fair .

I've found some contacts that could set me up with a university level research setting (which I hope I can get to the summer)

Unfortunately, I was NOT accepted into the internship in New York -- is there any way any internships or research facilities are still looking for applicants this close to the summer? I really want to be able to start researching right away, I know that I would be able to do more if I am in the right place...

Hi Trader,

I'm glad that you had a good time at ISEF. It's exciting to have made some contacts that might help you connect with an internship! Off the top of my head I am not aware of any specific summer research programs that still have openings, but there is a fairly comprehensive listing that you can search here: http://societyforscience.org/stp/index.asp.

Congratulations on a job well done this year!

Congrats on a very productive season!

Trader wrote: I'm also adding the last bits of my error analysis. I think that because the smallest increment of the micropipette is 0.01 mL, the uncertainty is 0.005, and the smallest increment of the pipette is 0.1 mL so the uncertainty would be 0.05 mL.
In the case where I use the pipette once, then the micropipette maybe 4 times, would the systematic error % be 0.005 + 0.05 *4? = 0.205 = up to a 20% difference from the actual population?

Sorry I didn't get to this earlier. One way to tackle this problem is with error propagation analysis or the "law of error propagation."
http://ikpe1101.ikp.kfa-juelich.de/brie ... ode72.html
For starters, you need the equation for the final measurement, which is simply the sum of the individual measurements:
Mt=M1+M2+M3+M4+M5
where Mt is the total volume transferred and M1,2,3,4,5 are individual pipette measurements.
From this equation, the derivatives of Mt with respect to M1, 2, 3, and 4 are all 1. Therefore the variance of the final measurement is equal to the sums of the variances of the M1, M2, M3, M4, and M5 measurements. If we assume that your systematic error of 0.05 or 0.005 represents two standard deviations, then the standard deviation of the final volume is
stdev=sqrt(0.025^2+0.0025^2+0.0025^2+0.0025^2+0.0025^2)=0.0255
So, the systematic error (2 standard deviations) of the sum of the 5 pipette measurements is 0.051.
Depending on your level of math, that might be confusing (sorry if it is). I'm glad to explain more if you want. Basically, the end result is that the error of your pipette is so much bigger than the errors of the micropipettes that the latter hardly matters.

Chris

Hi Trader,

Welcome back from ISEF, and congratulations again for your outstanding job on a project well done! I think you did an amazing job in a short amount of time. If you can make contact with a local laboratory for your next research project, it will be so much easier to get your experiments done. You need to spend some time identifying a unique topic for next year's project. You should talk to the local lab and find out what they are working on and perhaps pick a related topic as the local researchers will be subject matter experts who will be able to help you.

It's disappointing that you did not get the New York internship. I am not aware of any openings for internships that are still open. There are always very limited openings for these positions, but I was hoping that you would be accepted.

Donna Hardy

I'll be in NYC and Boston, MA (near Emerson College) so I began asking local colleges for possible projects they are working on.

Unfortunately, it seems like most are reluctant to even reply to my email inquiries when I ask them whether any professors would be interested in offering a mentorship for a short period over the summer -- then again, I'm not too sure how this is done ^^.

Donna, do you happen to have any contacts in New York or Boston who may know some of the professors in the area? That might help. There are a LOT of colleges in Boston, so while I'll be having camp at Emerson (not known for science I believe), I can always move around the area and perhaps get some research done! )

I'll get started on related literature... Especially articles dated 2008/2009...

Hi Trader,

I will look through all my Boston and NYC contacts and see if I can find a referral for you. This sounds like a good plan. What dates will you be in NYC and Boston?

Donna

I'll be in Boston from 7/18 to 8/1 (the camp is only two weeks long)

As for New York, I have yet to finalize everything so I'm very flexible right now but I'm leaning towards 8/1 - 8/17 or so...

It's a bit weird because my home state is NY and I'll have to go to MA for two weeks before moving back.

Thank you very much!! I'm checking out many websites of many colleges in the NY but mostly Boston area... wow