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!!

[deleted-2131]

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

[deleted-71588]

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...

[deleted-71447]

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

[deleted-2131]

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

[deleted-2131]

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

[deleted-2131]

ISEF doesn't have a specific format for bibliographies; however, using a format that is standard in your particular field of research is expected. I'm sure that one that Donna suggested will work well. As she said, it is very important to have references on your board and in your write up (if you have one).

[Trader]

OK, I've received confirmation of my registration -- I think I'm all done!

Now I've started the preparation work for running just two tests for the spectrophotometer growth curves and was making petri plates, and when I incubated some of them there were contaminants! In all of them!

This is a bit unfortunate because I know that when I did my first batch probably 3 months ago, this did not happen O=. (This is especially discouraging -- I'm moving backwards! o=).

I had a roll of plastic sterilized petri plates, and I made the nutrient agar, let it cool to when its cool to handle, and then poured it into the plates. I know I did that. I think I should actually be letting the nutrient agar in the Erlenmeyer flask cool under the UV light -- this would be sufficient enough right?

Here is a photo of the board -- I'll definitely edit the right panel -- that was printed out last minute . And I could definitely add more graphs and make them more prominent in the board. Ahh, so little space to display things in!:

http://www.flickr.com/photos/tiseagles/3408261107/ > My board
http://www.tiseagles.com/extracurricular.php?id=18 > Photos of all the projects in my fair.

[deleted-2131]

Hi Trader,

I'm glad that you got all the forms and paperwork completed! It must be frustrating for you to have found contaminants in your petri dishes; I'm sure that Donna will have some advice on how to resolve that particular issue. The pictures of your display board and science fair were interesting to see. Your board looks quite nice. You will need to update it with your new data, so I would wait to make a detailed plan of how you want to alter sections of your board until after you have completed your latest batch of experiments. You are absolutely right -- you have a lot of information to display in a confined space.

Keep up the good work!

[donnahardy2]

Hi Trader,

Thanks so much for sending all of the pictures. It was very interesting to see all of the projects. The variety and presentation of the science fair projects was typical and I thought your project was outstanding. And, I'm so glad you completed all of the forms.

Welcome to the world of research. Experiments occasionally fail for some reason, and it always seems to happen at a deadline. Your experience with contamination is unfortunately, a problem that occurs occasionally. You can't do any experiments with contaminated media. You need to think about where the contamination came from. If everything was grossly contaminated, then it is likely that the agar was not autoclaved long enough. Spore formers are very heat resistant and the autoclave cycle has to be at 121 degrees Centigrade for 15-20 minutes. You have to let the agar cool before you pour it, but if the top is covered with a sterile plug, then bacteria should not be able to get in before you pour the plates. If the roll of Petri dishes had been opened previously, it's possible that some dust had gotten in and contaminated all of the plates. A drop of non-sterile water could contaminate an entire flask of medium. Before you pour plates, you should wipe the counter top with 70% ethanol or other disinfectant. If you don't have any idea what happened, then just make the medium again the same way, and hope it doesn't happen again.

It's very important not to worry too much when things like this happen. In scientific research, experiments don't always work perfectly, and the best thing to do is to concentrate on what do to next to avoid repeating the problem. A failed experiment is never the end of the world. In your case, your goal is to redo your board for the ISEF fair, so you may need to do this without getting more data. Or, you may have time to repeat the experiment with only one temperature instead of two. Plan out your time, and budget enough time to plan you layout and finish everything without having too much to do at the last minute. You do want to have time to enjoy the experience of going to ISEF.

Donna Hardy

[Trader]

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

So from what I gather, the spectrophotometer can estimate the population of a bacteria by using this standard? I'm a bit confused because my teacher says that apparently we should be able to first know what turbidity = X amount of bacteria, what turbidity = Y amount of bacteria and then we'll be able to estimate the number of bacteria.

Unfortunately, my dilution platings only give the doubling time -- would I have to inoculate broth, use dilution plating to count how many bacteria it has, and then read the optical density at 600 nm? (ALSO -- why is it at 600 nm and not at any other optical density reading?)

I checked the link, and I'm not too sure > does

In microbiology, McFarland standards are used as a reference to adjust the turbidity of bacterial suspensions so that the number of bacteria will be within a given range.

Mean that we will be able to directly estimate the population from the turbidity after preparing this McFarland standard? (Sorry, I've never used/seen a spectrophotometer before, but I have time to work on the project now )

Thanks.

[deleted-2131]

Trader,

I can't answer this particular question as certainly as I'm sure that Donna can, but I can explain a bit about spectrophotometry and how it relates to the amount of bacteria. Speaking in fairly broad generalizations, a spectrophotometer sends a beam of light through the sample that is in the cuvette and then tells you about how the light is affected by the sample. Transmittance, for example, is a measure of how much of the light makes it through to detector on the other side of the sample compared to the amount that was sent into the sample. The way that the light is affected by the sample will, in your case, depend on the amount of bacteria in the suspension. The more bacteria that is in the suspension, the more strongly the light will be affected. I can't comment on the McFarland standards, but I'm sure Donna will be able to provide a more rigorous technical answer.

Keep up the good work!

[donnahardy2]

Hi Trader,

Thanks to Terik for the explanation about the spectrophotometer.

The McFarland standards are suspensions of barium sulfate, a white insoluble salt that has an appearance that is similar in appearance to a growing culture of bacteria. Different concentrations of barium sulfate will give a visual substitute for the spectrophotometer; you compare the density of the bacterial culture to the McFarland standards to estimate the number of microorganisms per ml. A spectrophotometer at 600 nm will give more accurate results, and your teacher is right; this is the best way to do this. I had just suggested the McFarland standards in case the spectrophotometer was not available for you to use, so don't worry about this option.

I think you should stop doing experiments, and concentrate on writing up your board. Getting some additional results will not be worth it if you sacrifice the time you need to redoing the board. Are you in the middle of an experiment? If so, then finish it, but I would not recommend continuing experiments until the last minute. What do you have left to do?


Donna Hardy

[donnahardy2]

Hi Trader,

I didn't answer one question. 600 nm is used to estimate microbial numbers because it works best for this application, and was determined empirically (it works). If you have a spectrophotometer that does not have a 600 nm filter, you could substitute a 570 or 620 filter, if those are available.

Donna

[Trader]

OK. So just to make sure:

I'll need the following to make a growth curve with the spectrophotometer.

1) Need to have taken the optical density reading of two nutrient broths with known amounts of population BEFORE
2) I can test for growth curves with the spectrophotometer?

If thats the case then I don't think I've started on the experiment yet A bit disappointing.

[donnahardy2]

Hi Trader,

I'm so sorry I was not clear. You should just take a sample and measure the OD at 600nm periodically as your culture grows; the OD 600 will increase as the population increases. The plate count is the definitive result on a growth curve; the OD 600 nm just gives another measurement to monitor the increase in population. I had suggested this because the plate count results on your original experiment were unexpected. If you had had an OD 600 reading at the same time points as the plate count, it would have helped confirm results, or helped explain why the generation time was so short. However, it's a lot of work for one person with limited lab access to do a plate count and do an OD 600 with a growth curve at the same time, so don't worry if you don't get this done.

If you do have any results, please post the results. If you have not done any more experiments, just start on the revisions of your board and post the copy, and we'll make suggestions. You want to have the best possible presentation of the results you have obtained; it's not worth it to continue experimenting. You have plenty of data now!

Donna Hardy

[Trader]

I think I'll be walking in with the same data that I had for the regional fair --

I attempted to add to the analysis of the data because that is what I think I'm lacking the most in. For graphs, I wanted to graph the exponential curve of the 3 doubling points I obtained from various temperatures, and I wanted to compare the curves (and coefficients of the curve fit) between the experimental data and the data obtained from aerobic growth.

Graphical analysis allows me to use two types of curve fits: a logarithm base 10 curve and an inverse exponent curve. Both seems to fit the curves really nicely, but they're two separate formulas -- would it make a difference as to which one I use? Given the formula that I mentioned in my materials protocol above, since it mentioned log 2 and that is in base 10, I am leaning towards using a logarithm base 10 curve?

Perhaps for the 600 OD readings I will continue that after the fair... onto next year

Also, I'm worried that the judges might see me testing two different temperatures the wrong way. Since I'm testing the effects of microaerophilic environments on the growth of bacillus subtilis, I wouldn't want to vary temperature, but the levels of oxygen -- would the judges see this the wrong way?

Another thing is that while I like my left panel and my center top, I think that in addition to my graphs problem (I don't know whether to graph the exponential curve and/or the constant growth rate (hopefully) on a log y axis, I'm not sure what to do with the conclusion.

A lot of people say that when I'm going from the regional to the big Intel ISEF I want to "improve" my board. I attempted ot understand more of the reason why, but I'm not sure if I have the space to communicate the "why" through the board -- would the judges think that this is a weakness? I'll aim to show that this isn't true through the presentation, but I think the board has a big part of the judging process...

I'm literally leaving the hypothesis, background research, materials and data untouched going into the Intel ISEF and this somewhat worries me. I'll take a picture of it tomorrow as tomorrow Sunday is one of the first setup days.

As for the concepts, I think I know the using of nitrate as an electron acceptor and not nitrite becuase it cannot be reduced by the structure of b. subtilis? (Does the term redox system have to do with this?) --

I've also seen that in addition to using nitrate as an electron acceptor for anaerobic growth b. subtilis also undergoes fermentative growth. Does b. subtilis use ethanol, lactic acid and hydrogen and convert them into energy through fermentative growth?

Sorry for the last-minuteness. Thanks

Sorry for the last minute-ness.

[donnahardy2]

Hi Trader,

I think you are setting up today, so I recommend making as few changes as possible. Your project was very good at the regionals, so all of the ideas we had were just suggestions. There was nothing at all wrong with the presentation, and I think you are comfortable with it, so please don't worry about changing anything.

The graphs for the growth curve should be log base 10. This should give a linear curve during the log phase of growth.

Yes, redox reactions refer to the gain and loss of electrons in chemical reactions. Reduction refers to gaining electrons and oxidation is the loss of electrons. So B. subtilis has the ability to give electrons to nitrate, thereby reducing it to nitrite. I’m not absolutely certain, but I think that the medium you used does not contain nitrate.
B. subtilis would convert the glucose to various metabolic products to produce energy. B. subtilis has been shown to use two major pathways to metabolize glucose, the Emden-Meyerhof pathway, in which glucose is converted to pyruvic acid and the hexose –monophosphate pathway, in which glucose (a 6-carbon sugar) is converted to 5 carbon sugars.

http://www.pubmedcentral.nih.gov/articl ... tid=278424

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


http://www.scribd.com/doc/5424827/Hexos ... ay-Pathway

B. subtilis would probably not be able to metabolize ethanol or lactic acid, or oxidize hydrogen.

The biochemistry is really outside the scope of your project, so don’t spend too much time on this. Please don’t put any of these reactions in your write-up, otherwise the judges will ask you questions, and since you haven’t had biochemistry yet, you aren’t quite ready to discuss this topic. You have plenty of information presented in your project, so just focus on that.

Please don’t worry about what the judges will think at this point as you can’t control this. You are jet-lagged, so do make sure you get enough rest so you can focus on what you can control, and present your project as positively as possible.

It’s good you are thinking about next year. You really have developed a good base of knowledge and experience with this project, and what you will need for next year is a really spectacular project that will build on this one, and that you will be able to do with the resources you have available. I’m sure you will think of something.

Donna Hardy

[chiazuohui]

Good luck for your ISEF!!

[Trader]

Thanks Chiazuohui! Are you going to the fair as well?

Donna -- I'm finishing up last minute touches now -- the thing I see with a lot of boards is that they have a LOT of text. Its almost like 12 pt Times New Roman on there, and I have to squint to see their long essays on the background, analysis, etc etc. Though it is printed (I should do that next year)

I know there'll probably be no use worrying or anything, but do you happen to know if the judges would prefer essay boards, or a brief summary of a board that looks like a very simple experiment -- buuut I'm hoping to explain everything in the presentation? It's probably too late to change anything, but it'll be nice to know.

I was just looking over the judging rubric, and a "are all variables correctly identified" is part of the criteria. I'm afraid all I know are dependent and independent variables, and controlled variables -- and I think that for this experiment, there isn't really an independent or dependent variable because I am unable to measure how much oxygen is in the environment to produce the change I see in the growth of bacteria?

As for the changing temperature thing -- should a judge ask "OK, so if you're testing the effect of a microaerophilic environment on b. subtilis, why did you change temperatures through the experiment?" I'm not sure how I would answer -- I hope its not going to be too bad on the judging process (though I shouldn't focus too much on that and have fun ) if I say that I originally planned to check out each growth rate, but I was unable to have the resources to continue that many trial (including time), so I ended up not being able to do it... if I had just 2 more weeks (though even after the regional fair where I did have 2 more weeks... I didn't do anything because it would just take too long to prepare everything, ...) I would be getting growth curves for 37 and 30 C.

For my experiment, would a 2 graphs showing the exponential growth (log) for 37 C, and 30 C as well as a semi-log graph showing the constant linear growth rate be the best representative? That is what I have ...

Thank you for bearing with me

It's already been very fun -- the pin exchange was great! Unfortunately I didn't bring enough pins ... =P so I ended up having to trade some of the ones I have away -- of course, only the ones that I don't like haha

[donnahardy2]

Hi Trader,

I can't imagine boards in 12-point, but I can understand why it was necessary to fit into the space. Your board must be clear and readable, so please don't reduce the type size. A summary that looks like a simple experiment would be preferable to one that is unreadable.

I've never judged at ISEF, but I always prefer a presentation that briefly presents the project, followed by details to support the outline. You have to imagine the difficulty of the judges who have to review many projects. Even at ISEF, I imagine they would want to be able to understand the project after 30-45 seconds of looking at the board, or by reading the abstract.

You really know all you need to know, and more about the topic that some of the judges. The temperature (30 and 37 degrees C) is your independent variable; the number of bacteria (your growth curves) are your dependent variable. All other parameters (medium, starting number of organisms, etc.) are controlled, or at least as well as possible, as you know from your experiments.

Good luck at the judging!

Donna