Bacteria [Communications]

[Trader]

Data:

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As for the data, I know that the uncertainty with dilution plating is much greater than when using the spectrophotometer. If I were to get results from the spectrophotometer, would I want to shift my conclusion and analysis primarily on that obtained from the spectrophotometer because it is so much more accurate, or would it be depending on whether there is a close correlation between the values obtained for each?

IF I inoculate nutrient broth in the morning and get 80 minute intervals until late in the night, would that be enough? I'm a bit worried because it seems like the stationary phase won't come until 12 hours or so after incubation, which means that I'll have to stay very late...

Oh no... but I can't do that during the day because I'll have to leave them out... <_<.

OK, I'll have to use the Saturdays then. I wish I had separate incubators!

One more thing! For EVERYTHING, there is an uncertainty of half the smallest increment right? So even if I was using machines with electronic readings (is this what a spectrophotometer does?), there is always uncertainty right?

From what I understand, I would want to plate the samples at 30 and 37 at least...one of the first things on my to do list is to find out what conditions bacillus subtilis are used in when creating industrial enzymes. If there are certain temperatures they are usually created in, I would want to measure those right? [I'm worried about the significance of my project...sorry -- maybe I'm worrying about the wrong thing]

(For its use in probiotics, the stomach would be a microaerophilic environment right? And that would be 37 C, so that's cool)

I just want to clarify -- oxygen levels cannot or should not be measured because I won't have the tools (I don't think I do) or the time right? I was thinking that there might be some coefficient that has a linear regression with the amount of oxygen levels. If I can somehow measure the oxygen level available in my "microaerophilic condition", and then somehow do "normal" growth curves by having an incubator and stirrer working the entire time at a high heat that results in 30 or 37 given the unclosed incubator door...and then find if there's a linear relationship.

But I can already tell by the description that I'll never be able to get that done in time. Awww...

For now it looks like: growth curves (two Saturdays, or maybe one entire weekend) for 30 and 37 C, and then STOP.

[Trader]

Graphs:

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These were poorly done... I definitely know this.

I should have the axis constant, and I should have definitely somehow graphed the (hopefully) constant growth rate (especially for 30) with semilog y axis.

I should have added error bars (?) or at least indicated my uncertainty somewhere.

One of my graphs (for the aerobic conditions) was also completely based on the assumption (unsupported by any literature because it was last minute ) that the doubling time would be 20 minutes ... <_<. The problem is, when I type in Google "bacillus subtilis growth aerobic", I can't get any ASM journal type (or related) studies on its growth. I've only found a few studies mentioned a doubling time:

[link]http://www.pubmedcentral.nih.gov/articl ... tid=214739[/link]
[link]http://jb.asm.org/cgi/reprint/179/10/3371.pdf[/link]

I'm afraid I really have no support about the doubling time of bacillus subtilis. I contacted the authors for those who investigated the growth of bacillus subtilis under anaerobic conditions if that might help, but just how different the conditions my experiment was setup in (such as the special type of nutrient broth) worries me a bit.

I'll continue hunting for it -- a really cool modeling study of e. coli growth had models of its growth to compare with, and I hope that perhaps someone modelled b. subtilis growth before? It's really cool.

I really want to do the below for b. subtilis ... at least part of the below ^^:

[link]http://rms1.agsearch.agropedia.affrc.go ... 7-3029.pdf[/link]
[link]http://aem.asm.org/cgi/reprint/71/12/7920.pdf[/link]

But I can't seem to find any of the above for any other species but e. coli . I'll keep searching...

[Trader]

Discussion:

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I know that with the addition of growth curves, I would definitely be able to discuss more.

My teacher appears to be reluctant for me to continue my project -- I am pretty sure I will be able to conduct growth curves using the spectrophotometer, but (sorry if I overlooked anything) would it be "worth it" for me to continue to plate samples (even when I can access the more accurate alternative of the spectrophotometer?) -- if I can, I know I can reinforce the data that the dilution plating appears to give.

My abstract paperwork is due in approx 5 days and my teacher wants it in soon -- I know that the weekend isn't until another 5 days, so I wouldn't be able to add what I learned from the spectrophotometer in the abstract. I'll ask about the details regarding updating the abstract (though I don't think they'll like that), but in general, would it be OK if the abstracts have "vague" conclusions such as "the lag times were found to be longer, the doubling times were found to be longer, and the highest amount of viable organisms at the stationary phase were found to be approximately the same as those reported in literature" -- would that be OK?

Because I hope that the spectrophotometer will emphasize what my dilution plating results appear to indicate. (my 37 degrees Celsius results are not so great, and my 30 degrees Celsius results appear to be a bit weird too ...)

In response to a few things Craig said:

"microaerophilic" is a property of a bacteria or organism which by definition means that organism requires oxygen to multiply; however, its growth is NOT impeded by an atmosphere that contains less O2 partial pressure than the normal earth atmosphere at STP.

OH. Does this mean that a facultative anaerobe by definition would have growth impeded by an atmosphere that has less 02 in the atmosphere than at normal levels? [I think this is meant by STP?] I think definition of b. subtilis as something that's not a strict aerobe was established in 1998, as for it being a facultative anaerobe I thought I saw another study showing that it is a facultative anaerobe...

There are ways to measure oxygen levels but they are expensive in terms of the equipment needed and difficult in terms of controlling equipment contamination so they typically aren't used for your kinds of experiments. The two common methods for insuring an environment to prove an organism is microaerophilic are 1) introduce a competing gas such as CO2, NO2, or helium and 2) reduce the atmospheric pressure with a partial vaccum but these methods require a sealed environmental chamber. In order to classify an organism, you have to attempt to grow it in aerobic, microaerobic, and anerobic environments to compare the growth and growth rates.

OH, so an environment between aerobic and anaerobic environment would not be microaerophilic, but would be microaerobic instead (or is there a difference between the two?) -- where microaerophilic only describes the properties of the organism's growth under varying oxygen levels?

Sorry if I overlooked any of this -- so then, my condition (nutrient broth plugged w/ cotton, no stir [is no stir equal to no aeration?]) is microaerobic?

Ohh I really want to be able to measure oxygen levels...but that is for sometime else. Bacteria growth is cool!

Thanks!

[Trader]

Conclusion:

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My favorite part is explaining why!!!

The metabolizing of nitrate and nitrite is really cool. I'll definitely look into it after I analyzed the data (starting with error! I was really worried that I didn't mention anything about error, only sources of uncertainty, in my presentation) ...

To do list right now is to get growth curves in 30 and 37 C under spectrophotometer. Of course, the paperwork.

Thank you so much for everything!!! I am so excited about this. It's probably the coolest experience I'll have in my sophomore year, and definitely one of the most unforgettable ones in my high school life -- I can already tell!!!!!!!

XD

Abstract --

I've never written one of these before, and I've tried to get as much as I can from the abstract-teaching guides. I hope I did an OK job:

The experiment analyzed the growth of facultative anaerobe bacillus subtilis under microaerophilic conditions. Until recently, bacillus subtilis was characterized as a strict aerobe; studies concluding the bacterium’s metabolism of nitrate under anaerobic conditions found that this was not the case. The importance to analyze microaerophilic growth of bacillus subtilis is then apparent to test the classification of b. subtilis as a facultative anaerobe; because of the definition of this classification, it was hypothesized that the lag phase and doubling times would be between reported lag times and doubling times under aerobic and anaerobic conditions with similar experimental setup, and that the highest amount of viable organisms reached in the stationary phase under microaerophilic conditions would be lower than that under aerobic conditions. Quantitative observations to a turbidity standard were measured to record the lag time. Dilution plating and optical density measurements by the spectrophotometer calculated the doubling time and samples of the nutrient broth after 24, 36 and 48 hours of incubation were plated to record the highest amount of viable organisms at the end of the stationary phase. Recorded lag times were longer than that reported under similar experimental setups with aerobic conditions; calculated doubling times and generation times indicate a growth rate between aerobic and anaerobic conditions, supporting the hypothesis. However, the highest amount of viable organisms found at tested intervals of the stationary phase was found to be similar to that reported in related literature, refuting the hypothesis.

241 words?

I know my wording is terrible, I will definitely look into that later -- I wouldn't be penalized for vagueness right? It worries me that the judges might not even interview me if my abstract isn't good enough ... that I'll have to adjust to (I can definitely express verbally better than ... through writing) ... Kind of.



Thank you once again!!

[tdaly]

Hi Trader,

Wow! That's a lot of posts and a lot of information for us to digest, so I apologize if I miss one of your questions in my response - just let me know and I'll answer it.

First - TURN IN YOUR PAPERWORK ASAP! Completing your abstract, the online finalist questionnaire, and mailing in your completed forms should be your most important priority right now. ISEF is incredibly strict about paperwork and deadlines. If you don't get things done on time, you will be disqualified - no ifs, ands, or buts about it. You've worked incredibly hard to get this far, so please, please get your paperwork done. You don't want to wait to the last minute to do it, either. I strongly suggest that you write your abstract and post it here for feedback before submitting it. Many judges - especially for special awards - will read your abstract and use it to decide whether to look at your project. If your abstract isn't superior (in content, grammar, style, spelling, etc.) then some of these judges might decide not to even look at your display, let alone interview you. It's OK if your conclusions are, to use your phrase, "somewhat vague". I think that the sample sentence you gave in your post would be just fine. Deadlines for abstracts are always far ahead of the competition or conference, so we can't always be as specific as we might want to be. There are also sections of the online finalist questionnaire that require writing and you should make sure that these sections are quality as well.

Second - I think that the time line Donna suggested for your project during this next 5 weeks is a reasonable one. Getting more data is important to helping you be successful; you also need to have ample time to analyze that data and put together your board. Stopping two weeks before ISEF to do the latter is reasonable. I can understand why your teacher would like you to stop experimenting and focus solely on the board and presentation, but I would recommend Donna's suggested timeline instead.

Third - Donna's suggestion about printing your board out on paper and laying it out to look at it is an incredibly important one. When I did my displays for ISEF, I would usually do a dry run about 4-5 times before I was pleased with it and then finally printed it for real. Her comments on using flowcharts, diagrams, and pictures are also excellent. I've attached an example of one of the panels from one of my old ISEF boards that shows one approach to integrating text, images, and flowcharts in the methodology section, which can hopefully give you some ideas to spark your imagination.

Keep up the hard work and keep the questions coming!

Terik

[donnahardy2]

Hi Trader,

Thanks for everything. It's great to see the whole project put together. Your writing is excellent. I have printed everything out, and will read it tonight and post comments tomorrow. I have just a couple of suggestions on your proposed lab project. It's very ambitious and if you do 30 degrees C on April 4, and 37 on April 11, this would leave you with final data to analyze in less than a month before May 10. There is a definite advantage of stopping now and concentrating on the presentation, but redoing the experiment would offer the advantage of having duplicate results, with two experiments at each temperature You do have plenty to discuss with the results you have now.

Two comments on the abstract. Your abstract should refer to results from your first experiment, and you should not include, for example, "highest number," and optical density measurements in the abstract because these results don't exist yet. You can definitely add them to your presentation if you obtain more results, but you have to submit your abstract before the second experiment, and you don't know what will happen or if the second experiment will happen. The other comment is that Bacillus subtilis is a genus/species name so should be spelled with a capital B and printed in italics. You can make your abstract a little vague withand intriguing to leave room to include the second experiment results and inspire the judges to want to come and look at your project. How many words can be included in the abstract?

Microaerobic is a new term for me. I looked it up, and it seems to be used to describe organisms that preferentially grow in low oxygen environments, which is not Bacillus subtilis, which prefers to grow aerobically. Go ahead and use the term microaerophilic, and we'll make sure you have enough information to defend your use of terms in case you meet a judge who is an expert at microbiological semantics.

I'll post more comments tomorrow. Keep up the hard work.


Donna Hardy

[Craig_Bridge]

Trader,
Sorry if I confused you...

A "facultative anaerobe" is by definition a microorganism that grows equally well under aerobic and anaerobic conditions which means it will grow equally well with an excess of oxygen to an environment where there is no free oxygen.

STP (Standard temperature and pressure) based on NIST (National Institute of Standards and Technology) is 20 degrees C (68 F) and 101.325 kPa (14.696 psi). The only reason I introduced this term was that you can reduce the amount of oxygen available below that of a nominal earth atmosphere by creating a partial vacuum.

As Donna points out, your "Bacillus subtilis" prefers to grow aerobically (which means it prefers to grow in the presence of oxygen) so at some point between a normal earth atmosphere oxygen content decreasing down through a microaeophilic environment to one devoid of free oxygen, the growth rates will decrease significantly. A "microaeophilic" environment is one of those terms that is loosely defined and covers a broad range of oxygen levels.

Hope this clears up any confusion I added.

[Trader]

Thanks!

There is sure a lot of paperwork with ISEF...

Just to double check, "ISEF Questionnaire and the abstract" are the ONLY things that are required to be sent within 12 days of the fair, and is this only the electronic copy -- meaning we don't need to send the hard copy of forms within those 12 days?? I've asked the fair director and haven't had a reply yet -- it would be best to make sure, because the checklist tells us to have a hard copy (one to send to the society apparently), and another to send electronically.

Attached is final copy of the abstract -- some things edited

Until recently, Bacillus subtilis was characterized as a strict aerobe; studies concluding the bacterium’s ability to metabolize nitrate under anaerobic conditions found that this was not the case. This experiment analyzed the growth of Bacillus subtilis under microaerophilic conditions established by broth without aeration to test the establishment of the bacterium as a facultative anaerobe. Based on the definition of a facultative anaerobe, it was hypothesized that lag phase and doubling times under microaerophilic conditions would be between the lag times and doubling times reported under aerobic and anaerobic conditions, and that the highest amount of viable organisms reached in the stationary phase would be lower than that under aerobic conditions. Similar sized individual colonies from a streak plate were inoculated into nutrient broth and the time it took for the broth to reach turbidity as established by a turbidity standard were recorded. The doubling time was calculated through dilution plating and colony counting. Samples of nutrient broth after 24, 36 and 48 hours were plated to record the highest amount of viable organisms at the end of the stationary phase; two temperatures of 30 and 37 degrees Celsius were tested. Recorded lag times and doubling times were longer than times reported under similar experimental setups. The stationary phase population reached levels similar to experiments carried out under aerobic conditions

With abstracts -- is the idea to be as concise as possible, or to be concise as possible under the given word limit? There are definitely things I could add...

[ChrisG]

Hi Trader,
I like the abstract. Is this actually "final" or are you still looking for suggestions?
Chris

[donnahardy2]

Hi Trader,

I'm glad Terik alerted you to the importance of the ISEF deadlines. Did you receive a copy of all of the rules for this fair, including size of the board, etc.? You should review all show rules and make sure you follow all of the requirements.

I like the new version of your abstract. It is straightforward and clear. I prefer the end of your first abstract, however, because it summarizes your conclusion about the doubling time under microaerophilic conditions. So if you have not submitted this, try to make the last sentence kind of a summary or conclusion of the whole project. If you have already sent this, it's very good and perfectly acceptable. If I were a judge, I would definitely want to come and see your detailed results.

I will post other ideas as I have time, one at a time. I know you are busy getting ready to set up your experiments and submitting the paperwork.

Here is information on McFarland standards, which are used as turbitity standards to estimate concentrations of bacteria. You do not have to make these standards, so this is for your information. If it turns out you can't use the spectrophotometer at 600 nm, however, you could use these to estimate numbers of bacteria, if barium and sulfuric acid are available. And if a judge happens to ask you about this, you'll understand the question.

http://en.wikipedia.org/wiki/McFarland_standard

Terik's procedure section that he posted was excellent. No wonder he won. Did you notice that you can look at the page and "see" how the protocol was done? And the very conservative border of color enhanced the presentation, without being distracting. Can you think of a way to present your method that is more visual? Just think about this for a few days; I'm sure you'll think of something.

Donna Hardy

[tdaly]

Hi Trader,

EVERYTHING NEEDS TO BE TO SSP WITHIN 12 DAYS OF YOUR FAIR. All the online things need to be submitted and all your paper forms sent in.

I'm sorry I didn't get to check this earlier to answer your question; I just finished up presenting at a conference, chairing the judging at a regional ISEF-affiliated fair, and finals are just around the corner. Whew! I'll try to check everyday, but there's no guarantees.

Donna's point about reading through everything to make sure you are in compliance is very important. I know that in the packet you received when you qualified for ISEF there is a lot of material and I know that you're extremely busy, but PLEASE read through all of it. In addition, I would suggest looking at the "Creating and Effective Project Display" PowerPoint, which can be found here: http://societyforscience.org/isef/index.asp (scroll partway down the page; it's under the STUDENTS & TEACHERS heading). You might also want to look at the last two pages of the official ISEF student handbook which can be found here: http://societyforscience.org/isef/document/hbk2009.pdf. Pages 8-10 of the official ISEF rules, which can be found here: http://societyforscience.org/isef/docum ... es2009.pdf.

Keep up the hard work, and keep the questions coming!

[Trader]

Thank you Terik for all the information! It clarifies a lot.

I was just out the past few days too! Went to an Asia-Pacific Activities Conference for a Quiz Team competition

Right now the Finalist login page has a complete set of ticks (complete, green)...and I followed the checklist and the Abstract, Finalist Questionnaire and ISEF Form copies are all in . I hope that's everything ^^

For the questionnaire, I kind of regret not putting in more, because from what I understand the judges will be using this information to base their judging on? The fair director gave me a contact email and I asked if I could add additional information.

There was a section asking us if the project involved anything in the following areas:

Resource Conservation
Sustainable Development
Pollution Prevention
Energy Conservation
Sustainable Development Explanation

I'm not sure about the first 3, but would the use of bacillus subtilis in industrial enzymes forming and fermenting be "energy conservation"? If they can still accept edits, I'll email them and hopefully they will add onto it?

As for the use of b. subtilis in the creation of industrial enzymes -- I can see what their use is in places such as;

http://www.rakuto-kasei.net/enzyme/natto.index.html
http://www.scialert.net/qredirect.php?d ... linkid=pdf
http://books.google.com/books?id=4VpZfx ... t&resnum=5
http://books.google.com/books?id=4VpZfx ... 5#PPA77,M1

There are many things I think b. subtilis does -- but I'm not sure if they are microaerophilic environments. It seems like many of the environments are actually anaerobic (according to my teacher), and I'm not sure I can find what environments the uses above are in -- is the process of using bacteria to create industrial enzymes usually anaerobic, or does it vary? Sorry I don't know much about the actual environments they're in, only what happens.

[donnahardy2]

Hi Trader,

Congratulations on getting the application turned in! It sounds like it was a major project to get the paperwork done.

I will think on it, but your project does not appear to be directly related to any of the conservation/energy conservation topics. It's more of a basic research project. Terik and Craig could probably explain the reason for the list of questions, but I suspect that it may be related to special awards. I'm sure the American Society for Microbiology will be judging for special awards, and your project will certainly be considered in this category.

I'll have time to post more tomorrow.

Donna Hardy

[tdaly]

Hi Trader,

Yes, the Finalist Questionnaire, the abstract, and forms are all the things you needed to do.

The information in the Finalist Questionnaire is used for a variety of purposes: collecting demographic information for statistical purposes, helping Special Awards judges determine if your project qualifies to be judged for their awards, and for the preparation of press releases should you win an award. If you can still update your information, then you are welcome to. However, if you have already submitted the online questionnaire then I'm not sure there is much you can do. You are welcome to email the contact person your fair director suggested, but I wouldn't to be able to add material to your responses if you have already submitted the questionnaire.

I would concur with Donna that your project does not fall into any of the environmental-related divisions you listed. But don't worry about it--you will still be considered for all the awards your project is eligible for.

[donnahardy2]

Hi Trader,

Yes, I agree with Terik. You should not worry if your project does not fall into an environmental category; it's a perfectly good project in its own category. It's just not an environmental project. However, when you go to ISEF, do pay attention to the awards that are presented, and if you find that there are more scholarship awards in a specific category, then you could consider doing a project next year that would give you a better chance of winning a scholarship. For example, in our local science fair, there are always lots of physical science projects and hardly any math/computer projects, so I always recommend to students looking for a project to do a math/computer project because there's one blue ribbon given in each category and it's better to be one of two, rather than one of 67, or so. So, this is a possible strategy for winning. However, you should always do a project that you are really interested in, rather than one because you think it is a better way to win. Your project won't be successful unless it's intrinsically interesting to you. I hope this makes sense.

One comment on your write up. Your references should be cited in a bibliography section at the end, and indicated with numbers in sequential order with a superscript number. I don't know if ISEF has a standard bibliography format that is suggested. If not, then pick a standard format and use it with all of your references. For example, this is the format that I usually use: "Hunter, et. al. J. Chromatog. A, 897, 65-80, 2000." The J. Chromatog, of course should be in italics. It is acceptable, and I think preferable if you have room to include the title of the reference. Having a perfectly done bibliography will enhance your project.

Donna Hardy