Bacteria [Communications]

[Trader]

Thanks.

I have 4 plates at 37 C, stationary phase (24 h). One of them I'm sure is good plate, and it has around 200 at 10^-6 ... From my understanding, this is ... 200,000,000 per mL? The original was 20 mL, so that was interesting. I have 3 other plates at 10^-6, 10^-7 and 10^-8 b/c I wanted to see further dilutions, and originally I couldn't find any colonies so I incubated them for quite a long time. The 10^-6 I'm still not sure what to make of it, but I can clearly see that there are 3 "big" colonies in the 10^-8, and by the looks of it 25 "big" colonies in the 10^-7. Do these results make sense? I'm thinking that some of them seem like they're off... a lot. I'm not sure.

I do have a lot of TNTC...they might come in handy.

Sadly I have yet to get the results back on my doubling time for 37 C.

I've also gathered samples of 3 points of 30 C. I'll be able to get those results very soon. As for other possible "results", I have a few other "mini-experiments" that I conducted during the big experiment, such as time it took for the nutrient broth to actually get turbid (perhaps I'll find some other support that shows when the "normal" lag phase would be?) -- I've also jotted down notes of the differences in duration of the lag phase w/ colony that is originally from agar, and another with a colony that was inoculated in nutrient broth, and then put in the fridge... would this be relevant at all?

If tomorrow works out well, I'll be able to get the max number in 24 h incubation w/ 30 C as well as the doubling time for 37 and 30 C.

If only I had one more week .

Aii.

I'm writing up my paper, and I'm a bit stuck on what the significance for finding the lag phases/doubling time or stationary number for 30 and 37 C (where support says b. subtilis' optimum temperature is from 30 - 37 C)...

I know that I originally set out to do growth curves, and didn't manage to do this. Perhaps if I find a difference in 30 and 37 C doubling time (chances are I will... IF the plates turn out OK), ... well, doubling time will help me increase the efficiency for my hope of a future experiment w/ accurately predicting bac growth as a substitute for a sensor strain for AHLs. ... That seems a bit insignificant =P.

IF I find a difference in 24 h, I think that the use of b. subtilis as industrial enzymes is generally an anaerobic process? If more of the b. subtilis is required to increase efficiency, in the given conditions (of that nutrient broth and temperature), this difference in temperature would help. But then, I have no idea whether the creation of those industrial enzymes would be under 'nutrient-broth' like conditions .

I wouldn't really have any chance at anything at the fair, but I'll go for the experience and see what others in microbiology are doing and how they're successful =P.

Thank you for cheering me up and encouraging me through this whole way -- I'll do better next year.

[donnahardy2]

Hi Trader,

Good job! You have results. And the numbers make perfect sense. If you have 200 colonies on the 10^-6, then you would expect to have 20 colonies on the 10^-7 and 2 colonies on the10^-8 dilution. The numbers between 30 and 300 colonies are considered the most accurate, so the 25 and 2 colonies are actually very close to the expected numbers. If these numbers are accurate, then it means that the bacteria only can get to about 10^8 when grown under non-aerated conditions. This is a very reasonable result. The TNTC's will be good to use because you know that the numbers are above a certain number. You should be able to see lots of dots on the TNTC, not a continuous lawn, which would indicate smearing due to excess moisture.

You can include all of your results and observations about the difference in lag time between agar plate and broth-started cultures. Even if you didn't record the exact times, you should be able estimate the times and make a reasonable conclusion about these results.

I would be very nice to have 30 and 37 degree C results, but don't worry if you don't. Just present whatever real data you have. If you had another week, you could have a perfect project. It's always this way with science projects.

Donna Hardy

[Trader]

It looks I'll be able to have a doubling time for the 37 C growth, as well as the 30 C growth. I still have to finish up counting b/c it seems like many of the colonies are quite small, though it looks like its below 300.

There are some plates that have only 3 colonies or something -- I've made a slide from one of them and they look like b. subtilis -- should I accept these results?

As I'm writing up the research paper I was also wondering -- would a varying nutrient broth size be something that would be an uncontrolled variable? Its a different worry from the previous "wouldn't it result in less bacteria per mL" question. Since I'm partly investigating how much longer lag time and doubling time is under anaerobic conditions, ... wouldn't a 500 mL broth in an 1 L Erlenmeyer flask be exposed to more air than a 250 mL broth in a 500 mL Erlenmeyer flask, and a 10 mL nutrient broth test tube w/ much less exposure to air? Or is the entire process going to be the same "degree" of anaerobic-ness because there is a cotton plug and it is pretty much air tight?

I've seen some pertinent literature (a long time ago) that had growth curves and doubling times for b. subtilis and I can see that their lag time and doubling time is much shorter. Is it "common sense" that the lag time and doubling time will be longer that I shouldn't state that in my conclusion, or is it worth mentioning?

It's also a bit confusing because in these articles, it seems to say that b. subtilis anaerobic growth requires this and that to be possible. I didn't add any of the below, yet it still worked...or at least, there was growth, but very little.

http://aem.asm.org/cgi/content/abstract/70/9/5252
http://arjournals.annualreviews.org/doi ... o.52.1.165

Does this mean that my incubation was semi-anaerobic? (Is this possible?) ... it'd be interesting to see if doubling time increased among the results I got from the 500 mL (in the very beginning) and the results I got from the 250 mL. I obtained lawns from the 500 mL first 5 hours, with up to 10^-6 dilutions, while I obtained countable colonies in 10^-6 250, for 37 C at least. Didn't have the time to do 30 C. If I wanted to include this possible difference b/c of anaerobic/semi-anaerobic conditions, is it possible to incorporate it without adding "another independent variable"? It's more like two separate projects though ...

The big day is on Tuesday! I hope I'll do OK. I guess I somewhat found that anaerobic growth increased the lag time and doubling time by X amount in these temperatures ... what would be the significance?

I think the use of b. subtilis in industrial enzymes is largely anaerobic. Perhaps if they can make the process aerobic they'll be much more efficient? (Then again, I don't think this is a practical suggestion)...

One last thing! For the research paper, what would be "common sense"? I find myself writing out the procedure for pouring plates and making streak plates -- is this necessary?

And another technicality issue -- is it necessary to write out a materials list if I've already listed out all that is needed in the procedures section?

Ahhh!

[donnahardy2]

Hi Trader,

You are amazing. I think you are going to actually have a complete project with data and excellent analysis for your project. And your questions and concerns about the final presentation are very perceptive. Your observation that the lag time and doubling time are slower than published results in the literature, and the analysis and explanation of this may just carry your project to prize-winning status. But that, of course, will depend on the quality of the other projects that are entered in the science fair. However, I think that your knowledge and understanding of the science behind your results will give you a definite advantage in the competition.

Here are some answers.

If you have 3 colonies on the lowest dilution you plated, and the result seems to make sense based on your expectation of numbers, you can use this result, but with a note that you know 3 colonies is not an ideal number for a plate count. 3 colonies could easily be a contaminant, so you can disregard the results if it seems appropriate. If this were a continuing research project, you would of course be planning to repeat the experiment and verify your results. So a plate with 3 colonies could be considered a tentative result.

Varying the volume of nutrient broth in the flask would be a minor uncontrolled variable because the surface area exposed to oxygen and the depth of the nutrient broth would be different with 250 and 500 ml in a 1 L Erlenmeyer flask. The conditions you used for your growth curves with either volume of broth would be considered microaerophilic in either container, so the availability of oxygen was a limiting factor in the growth rate of the B. subtilis. (Anaerobic is no oxygen, microaerophilic is about 5-7% oxygen, and aerobic would be 20% oxygen). You know from the literature that B. subtilis is an aerobic organism, so would grow better with unlimited oxygen. I wouldn’t spend too much time discussing this in your paper because you didn’t measure the oxygen levels. You might just want to point out that it is a possible source of error.

You should definitely state that the reason that the log phase and doubling times are longer with a microaerophilic environment. Your literature references cite the difference in metabolism and nutrient requirements for growth in anaerobic conditions compared to aerobic conditions. B. subtilis grows well with just about any carbon and nitrogen source in an aerobic environment, but requires a sugar source and extra nucleosides with anaerobic conditions. When you take biochemistry in college, you will learn about all of the biochemical pathways that organisms use for metabolism. B. subtilis gets a lot more energy per sugar molecule with aerobic metabolism. The organism can grow in microaerophilic and anaerobic conditions, but metabolism is much slower. The growth curves from the literature were all done on a shaker flask with aerobic conditions; you used static conditions so growth was slower. The references you have cited provide documentation to support this statement.

The significance of your results is in the basic understanding of how microorganisms grow in different conditions. You have quantitated the doubling time of B. subtilis under microaerophilic conditions. I don’t this information has been published in the scientific literature, so this may indeed be a new fact to add to our scientific knowledge. This information could be important, for example, if you were planning to do additional studies on the nutrient requirements of this organism under microaerophilic conditions, or if you were designing a fermenter to produce industrial enzymes with microaerophilic conditions. Basic facts about microorganisms, like doubling times, are always useful and highly significant.

With science fair projects, teachers and judges expect students to write out all of the materials used and all of the procedures. So you would need to include a list of all materials, and the procedure you used for pouring plates and doing streak cultures. It would be acceptable to use a copy of a published method if you had followed the exact method. You don’t need to include every tiny detail like how you autoclaved the nutrient broth and disposed the used agar, so use your judgement on what to include. What you want to do is describe your method with enough detail that someone would be able to follow your method and obtain exactly the same results that you did. Making streak plates is a standard, common technique, so you don’t have to describe this if you are out of time. In the future, you can refer to earlier publications that describe the technique you are using, so you don’t have to rewrite everything every time you publish new results.

Since you have your research paper with all of the details, make sure your board just has the basic information, and is very clear and easily readable. You have so much information to present that it would be easy to make it too complicated. You will want the judges to be able to understand the basic project within about 30 seconds after glancing at the board.

You are doing great! Don’t forget to get some sleep this week-end.

Donna Hardy

[Trader]

Thank you for this.

Wow it's so exciting typing my first real research paper . I find that the procedures section takes up so much, but there WAS a lot of preparation work ...

I am a bit worried about the control group. In the experiment I've noticed that lag times are longer (just by measuring the time from inoculation to turbidity), the doubling time is longer, though the stationary phase population appears to be the same.

But how can I claim that the lag phase and doubling time is longer than the same conditions, only w/ aerobic growth? . I have various sources displaying growth curves of b. subtilis and it shows that their lag time is around 2, but I have no idea whether that is from agar to broth, or broth to broth, and I don't know if whatever their setup is (their addition of chemicals/use of different broth) would have impacted the results enough to make their lag time shorter). I did record that there was approx. 7 hour lag phase (or at least before turbidity) for agar to broth, and around a 5 hour phase to first sign of turbidity, from broth to broth, and I'm pretty sure b. subtilis really takes less than that under aerobic growth -- but I can't make a comparison if the experimental setup is different right?

Something interesting I found: I was previously worried about the different colonies that I found that all turned out to be b. subtilis. It seems that they were all b. subtilis (probably) for a reason!

http://www3.interscience.wiley.com/jour ... 6/abstract

It mentions the ridges that I found! It looks like that it was b. subtilis after all, and was because of the variations in nutrient in the agar itself. Cooool.

If I'm not mistaken, it seems like b. subtilis can survive anaerobically (with the aid of so and so), microaerphilicly and aerobically. Would this mean that its an facultative anaerobe?

I love the history of the oxygen requirements of b. subtilis growth. It seems like until 2004, b. subtilis was also thought of as an obligate aerobe. And then research for its anaerobic conditions were investigated and it was found that they could survive anaerobic conditions, given the addition of nitrite, nitrate, etc. I can't find any characterizing the growth of b. subtilis and its microaerophilic growth on places such as Google, so it seems like additional nitrite, nitrate, etc is not required in the presence of oxygen.

Thanks for all this!

[donnahardy2]

Hi Trader,

Yes, I can imagine that your procedures section was a lot of work. But it’s very important for anyone reading your report to know exactly what you did, so I’m glad you took the time and effort to include the details.

You should not be worried about the control group results. Your results are empirical, or in other words, they are what they are. This is why we do controlled experiments. Results can’t be wrong because they are what actually happened. If your hypothesis doesn’t match the results, then it’s the hypothesis that is incorrect or something was wrong with the experimental design, and you need to revise the hypothesis or the experimental design and try again. This is how progress in science is made. (Does this help?)

So it’s definitely possible that you will obtain the same maximum number of organisms at different temperatures. I’m sure there is a significance to this. Maybe it’s the AHL’s that are produced at a certain population density that inhibit doubling. Whatever your result is, I’m sure it’s significant and it would be possible to think of an experiment to explain why.

I’m not sure what data you have, but you should be able to make a general comparison between the generation time reported in the literature and your results, and I was trying to encourage you to compare your results with previously published results. The time between inoculation and the first sign of turbidity includes the lag time (no doubling) and early log phase. You don’t know exactly what conditions were used in the literature, but the standard method for this type of study is to put the flask of broth on the shaker and incubate under aerobic conditions. I believe that the literature sources show a doubling time of about 20-25 minutes for Bacillus species at 30-37 degrees Centigrade (but you could verify that with a specific reference). If you have 3 data points in a row from log phase, you can estimate the doubling time for your experiment, and I am guessing from your description that the doubling time is about twice as long as the published data. You probably don’t have enough data points to know how long lag time was in your experiment, so it would be difficult to make a comparison about the length of the log times between published studies and your data. So, if you have data from your experiment, you can compare results to the published references and offer reasonable explanations for the similarities and differences. .

Excellent! You have carefully documented observations about the variation in colony morphology and a literature reference that explains why this organism can have ridged colonies. Be sure and include this information in your report.

Yes! Bacillus subtilis is a facultative anaerobe because it normally grows aerobically, but does have the ability to grow anaerobically. Because you did not aerate the broth cultures in your experiment, it is likely that your cultures were growing under microaerophilic conditions (it would be reasonable to say this even though you didn’t measure oxygen levels because you know from the literature that an aerobic organism would use up the oxygen, and you weren't adding it back into the culture). The statement that your cultures were growing under partially anaerobic conditions is a reasonable explanation for your results, which showed a slower than expected generation times. Your use of literature references to show that the organism can grow anaerobically helps support your statement. Another possibility would be that the bacteria were still growing aerobically, but growth was just slower due to limited oxygen availability.

http://www.sciencedirect.com/science?_o ... a2ee1b3d3d

You are going to love biochemistry when you get to it, because it helps explain everything. Bacillus species need a place to park electrons after they metabolize nutrients and they prefer to use oxygen because, metabolically, this is more efficient. However, if oxygen isn’t available, they can use nitrate, and probably other electron acceptors. Nitrite is already reduced, so it can’t be used. You are correct, if oxygen is available, nitrate is not needed.

I agree that it’s very interesting that Bacillus species are no longer obligate aerobes, as I had learned when I studied microbiology. And, we had to wait until 2004 to find out about this. I wonder what other “facts” will change in the future.

Donna Hardy

[Trader]

Attached

[The extension doc has been deactivated and can no longer be displayed.]

is how I organized the data I had. Would this be the most accurate way to display the data that I have?

I'm using a formula that can find doubling time with just two points (is this the wrong approach?) -- I've based my discussion on this, and instead found the average of the two doubling times obtained from the three points for more accuracy. ...

Eeek! As for graphs, I only am graphing the growth curves as well as a linear relationship between growth rate and time (unless I got my axes wrong?) --

I don't think I'll be able to graph my observations of lag time and highest amount of viable organisms in stationary phase?

(Also, I originally hypothesized that the number of viable organisms in stationary phase would be lower b/c of lowered oxygen levels -- it turns out that the number is approximately the same as that in literature, so oxygen levels do not affect the amount of viable organisms -- I'm not sure why though ...?)

I wish I learned biochem right now -- I would be able to know the answer.

Also, from my understanding microaerophilic is 2-10% oxygen -- how am I sure that the conditions I've tested the b. subtilis is actually microaerophilic? Or is it a, if its not aerobic and if its not anaerobic, its everything else in between = microaerophilic?

Thank you so much! Second last day ...before fair. Travel tomorrow...

[donnahardy2]

Hi Trader,

Data! I love data because it’s always so interesting. Your data is very good, but is going to require some interpretation. You did a very nice job of putting the data into the tables.

If you graph your results using a semi-log scale during log phase, you would expect a linear curve as shown in this example:

http://www.splammo.net/bact102/102gcurve.htm

So you could also include a simple line plot of time (x-axis, linear) vs number of organisms (logarithmic scale) with each table of results, so the judges can “see” what your data shows.

Please check your calculator formula because the average doubling time seems off. For example, on the 37 degree date, if the population increased from 2575 to 2,900,000 from hour 1 to hour 2, this would represent 10 generations in one hour, or a doubling time of 6 minutes. The increase of 2,100,000 to 140,000,000 from 1-2 hours on the 30 degree data represents 6 generations or 10 minutes doubling time. Try the formula from the above website and see if you don’t get a different answer. I wish we had one more day to work on the data, but you are at deadline, so do the best you can.

Now, we know that data is data, so how do you explain this? The fastest generation time published anywhere in the literature is about 20 minutes, so generation times significantly less than this are impossible. One likely cause of the unexpected results is that the bacteria kept multiplying after you took the samples and before you could plate them. Another possibility is that the reuse of the pipettes and Petri dishes allowed contamination and affected your results. Your trend in your numbers is from low to high, so I doubt you made a major mistake in making dilutions or mixing up samples. Since you did the experiments, perhaps you could offer another possibility.

So, recheck your calculations, print the tables (highlight or make bold the “average population” and the “average doubling time” values so your viewers will know to focus on those values and won’t have to read through the whole table trying to figure it out. And add a graph if possible, and offer an offer a logical explanation of the results. Since the results appear to be a little unexpected, also offer a description of what you would do differently next time. One item that I am regretting now, is that I didn’t suggest that you include OD readings at 660 taken at the same time as the plate counts. That information would have helped clarify the plate count results, so that’s something that you could plan to do next time.

One advantage you have with this data is that the judges are not going to think you made it up. Sometimes, when judging projects, data is too good to be true, so one wonders if the experiment was actually performed. No one will have any doubt that your data is real.

Microaerophilic can be 2 to 10%; I don’t think there is an absolute number. It would be conditions that are less than atmospheric conditions, but more than zero% oxygen.

If you are planning to take pictures of your project, I would love to see your board, if you can post it. If you can do this, be sure to follow internet safety rules, and make sure there is no way to identify you or your school from the pictures.

Good luck at the judging! This is an excellent project, and I’m sure you will do well in the fair, depending on the competition, of course. One thing that all judges are expected to do is ask a challenging question that is difficult to answer. If this happens to you, just relax, and think. You know more about this subject than anyone else, so you will have a good response available in your vast knowledge of this topic. I’m anxiously waiting for a report. Be sure to take some time and look at all of the prize-winning projects to get ideas for next year for presentation and analysis of results.

Donna Hardy

[Trader]

Sorry for the abrupt reply: Can you give me an alternate website? I can't seem to be able to load it ><.

I've got pictures of my semi-finished board...big day is tomorrow

As for finishing up the graphs, would there only be a linear relationship b/w population (units: Log N?) and time, as well as an exponential growth using x = N0 * 2^(t/d) formula? with x axis population and y axis population? Oh no, this is a bit confusing. Does this mean that are two possible graphs to be obtained form the same axes?

According to the formula that I've used (I found out my calculation error) -- yes, I do know that I left out the bacteria in the fridge overnight b/c I didn't have time. They must have multiplied then.

Attached is what I'm putting on for data...as well as pictures

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Oh no! I forgot the camera cord! I really wanted you to see this because I thought the layout was very cute ><.

Right now its one day before the fair and I'm still typing out the graphs and discussion and conclusion.

This reminds me: for the conclusion, my teacher tested me before asking me exactly how "oxygen" is substituted under anaerobic conditions (making b. subtilis a facultative aerobe) ... I didn't know how to answer (I wasn't too familiar with the literature) -- as for how b. subtilis uses oxygen under microaerophilic conditions, all I know is that it is crucial in metabolism to get more energy per glucose... I hope this is OK?

One last thing! ... Iiiif I can't find any specific articles that state the doubling time of b. subtilis, would it be a problem? It seems like one of those "assumed and true" things ...

I've calculated the times for 30, the trial that I did right before leaving, with what I know. I think the results can be called "good"?

Now to graphing! And I hope to leave in 2 hours to sleep...

[donnahardy2]

Hi Trader,

I'm sorry I didn't see your request yesterday. Here it is.

Donna

bacterial growth curve.pdf

(208.89 KiB) Downloaded 390 times

[donnahardy2]

Hi Trader,

You can see from the plots how to plot bacterial growth curves. It's possible to plot the data on semi-log graph paper, but not linear graph paper. If you plot both the 30 and the 37 degree data the same way, you can compare results. But if you are out of time, don't worry about it. Your tables look great, and you have lots of data to present. And, since there was a delay in sample collection and plating, you can explain your results. That's all you need! I'm sure you'll do fine! I can hardly wait to hear your post science-fair report.

Donna Hardy

[Trader]

Thank you for the link! From what I understand now, for a linear relationship it is

y axis = "bacteria population" and increments are 10^1, 10^-2, 10^-3?

and x axis = time.

For an exponential relationship y axis is still "bacteria population" and increments are "normal".

Is there anything else I can graph? Though I find that a lot of my centerpiece board has results taking up the space. There is so much to talk about in the discussion!

I included a formula to calculate doubling time, but not generation time ... on the board. I guess I'll add it verbally. I like formulas

ONE LAST worry: for the data tables, the final population that I obtain -- is that the population in the 1 mL that I took from the broth? In that case it would be "OK" if I base all "bacteria population" as not the bacteria that resulted from that one individual colony inoculated, but just as how the bacteria population in 1 mL changes over the course of time?

I have also seen CFU used a lot -- is there any way I can apply this term to my graphs? (I'm not too sure what it is).

Sorry for all these questions!!

[donnahardy2]

Hi Trader,

I like your teacher; he asks very good questions, but does not supply answers, even if you haven't had biochemistry. When bacteria use sugars for metablism and there is no oxygen available, there are some extra electrons left at the end of the electron transport chain. If oxygen is not available to accept the electrons, then the electrons can be given to another molecule like nitrate (N03) which is thereby reduced to nitrite (NO2). Bacillus subtilis cannot use nitrite as an electron acceptor, but some organisms can use it and reduce it to NO. An obligate aerobe would not be able to use an alternate molecule, so would only be able to grow if oxygen was available.

http://en.wikipedia.org/wiki/Fermentati ... chemistry)

Here's a reference that shows generation time, or doubling time of Bacillus subtilis in a defined growth medium with ammonium sulfate as the nitrogen source. The authors found a doubling time of 40 minutes; you were using a medium contained tryptone, which contains preformed amino acids and other biomolecules and your medium contained glucose as an easily usable energy source. You would expect a faster doubling time with your medium because the bacteria would not have as much work to do to synthesize proteins.

http://jb.asm.org/cgi/reprint/104/2/762.pdf

Generation time and doubling time are two ways of saying the same thing; it's the time required for all of the bacteria in the medium to divide in half and thereby double in number.

I'm not sure I understand you concern about the 1 ml sample. The bacteria growing in the nutrient broth are a like homogenous suspension so a 1 ml sample represents the population density of the entire sample. Hopefully your two hours of sleep will help this worry disappear.

CFU, or colony forming unit, is the same thing as organisms or bacteria per ml. It's a standard term for this application and your judges would understand it if you put in on your graphs. But, I would not change your graphs or tables at this point; it would not add anything to the presentation.

Good luck!!!

Donna

[Trader]

The brief version, or the fancy version?

Brief version: (Highlight text below)

I got 1st place for the regional science fair and qualified for ISEF in Reno Nevada! That one...

Fancy version:

Night before I was hyperventilating xD because I kind of didn't have my results and discussion typed out yet. Today morning (of the fair) I printed out 1/3 of my board (pictures soon...sponsor said all pictures would be uploaded soon: http://www.tiseagles.com/extracurricula ... .php?id=18)

And approx 3 minute to deadline I was still pasting my board ... . Sorry for that.

1st round of judging. They were somewhat intimidating, but nice at the same time. Just asked w/ "tell us about your project", and I went onto talking ... and talking . They asked about the practical value of my research ... and more about the project itself, but amazingly not one of those "so if you had more resources..." or other questions.

They just left... I thought I didn't do so good, so I just said 'thanks' and sat down awkwardly. Buuut then, the fair manager came to me and said "pssst, the judges that just interviewed you -- they said you did an exceptional job...they were the only judges I've had mention this about anyone"... and then he started talking about "if you get to qualify as an ISEF Finalist..." -- I stopped listening cuz I didn't want to get my hopes up =P.

2nd round of judging. I loved this group! I thought I did same as previously, but this pair of judges nodded and listened, and said "really good job" in the end . I somewhat stopped hyperventilating. They asked quite a lot of questions regarding how I controlled the variables, and where I would see the biggest problem be in duplicating this research.

Then there was break, and they were to announce the first place winners of the 5 categories. We were told that of these 5 individuals, 2 to go to ISEF?

One final round of judging. 4 JUDGES! Stuttering quite a bit...even though I'm much better in debate <_<. Don't know why. I felt that this round was the worst... and the judges were relatively non-responsive. One nodded though. I really liked that .
They asked for how I made sure the environment was microaerophilic, and I said that the nutrient broth w/o aeration would be microaerophilic, but I felt that something was missing there.

Then final judging was over! (It turned out that the final judgings were only made by the judges who didn't see our project yet). We waited a whole 30 minutes ... in anxiety.

Right! Our school had 3 people and all 3 were part of the 5 first place winners. But by then it was a bit awkward b/c we know that best case scenario, one of us wouldn't make it.

I was already "okay, you guys tell me how it is in the states..." because I was very grateful about getting a prize already ><

Then there was the awards ceremony...and when they came to announce the two "best of show" winners...THEY SAID MY NAME O=.

I can't believe this <_<.

Anxious and very excited about being able to go... but I'm worried that I'll not do so well there. There'll be so many finalists competing!!

But first I must MUST thank you for sticking with me this whole time. I can't believe I made it here ... definitely wouldn't have done it without you!!!!

Now... I'll want to recheck those doubling times... and get my board "non-last-minute" looking.

WILL YOU GO TO THE FAIR IN MAY? I may see you there!!!!

[donnahardy2]

Hi Trader,

Congratulations!! You won!! I'm so happy for you. Your knowledge and understanding of your topic more than compensated for the data and your hard work was worth it. I'm really glad you are going to the next step. It will be a really good experience for you. Thanks for sending all of the details of the judging; it sounds like it was a well organized fair, with decisions coming from a consensus of several judges. And, it sounds like you handled the questions very well.

It's also good that you can look at the data again, and see if there's something else to interpret. And it will be nice to have the time to polish the appearance of the board. Did the judges write out any comments about your board?

Before this, I was not planning to go to ISEF, but I will see if it possible, now that there would be an excellent reason to do so. I've worked with so many students in the past, but I've never provided advice for an ISEF final project before, so this is very exciting for me as well.

I will look at the data again as well, and see what can be done about that.

Donna Hardy