Science Buddies: "Ask an Expert"

Good research often does take more than a year so it is completely understandable that you are thinking about your project over a long period of time. It is wonderful that you are thinking about your project long term. It will improve and become more advanced over time. My large research project (the one about TLC) took me more than 2 years to complete if you count the research paper and my initial study.

As for science summer programs, here is a good list to get you started:

http://sciserv.org/stp/

Hope that helps! Have a great holiday.

Hi Trader,


Happy New Year! Be sure and take some time to relax and enjoy the holiday.

I would not worry about the Gram-stain results of the Bacillus subtilis until you can get a chance to look at in the microscope. Just proceed and assume that you are working with the right organism for the time being.

If you have E. coli, then you could include this organism in your research question along with the presumed B. subtilis. When you do a Gram stain on the E. coli, it should be a Gram-negative rod.

Yes, when you are preparing the Gram stain, you should let all of the water evaporate from the smear on the microscope slide before you heat-fix it for 2 seconds. Then continue with the crystal violet step. And, yes, tap water works well for the washing steps. I’m sure you are doing this correctly; you just need to wait until you can look at the slides under a microscope to get more information about your B. subtilis.

Looking at the lawn and the streak plates, do you see any difference in the color and morphology of the bacteria that are growing? If not, then it probably it is probably not worthwhile to do a Gram stain on other portions of plate. If you do see any differences, then you may have a mixed culture and then it would be worthwhile to try to isolate individual colonies on a streak plate and do a Gram stain on each type of colony. When you get a chance, do a Gram-stain of your oldest culture; plates that are 7-days old or older should have some cells that have turned into spores.

When growing cultures for experiments, it is important to periodically check the microorganisms to make sure they are pure and correctly identified. The process to check a culture involves doing the streak plate and isolating an individual colony. Diluting the culture out until it grows with single colonies that have space in between each colony and usually ensures that you are working with a pure culture. You would then need to do a Gram stain and check under the microscope to make sure it looks like the right organism.

The identity of the isolate can then be identified by classical methods, which include colony morphology, spore morphology, ability to hemolyze red blood cells, motility, oxidase and catalase activity, sensitivity to penicillin, and the ability to ferment carbohydrates. Bacillus subtilis is Gram-positive, catalase positive, and can metabolize a wide variety of sugars. Please describe the color, shape, texture, and size of the colonies you have growing. If you can, inoculate a broth culture (a clear broth soup would work well) and let the culture grow. Bacillus subtilis is an aerobic organism and grows best with oxygen, so will form a layer on the surface of the broth. This is called a pellicle, and it is distinctive characteristic of Bacillus species.

http://www.hpa-standardmethods.org.uk/d ... sopid9.pdf

Or, identify can be confirmed by newer analytical methods such as mass spectrometry or PCR.

http://aem.asm.org/cgi/content/abstract/65/10/4313
http://www.ingentaconnect.com/content/b ... 4/art00002

Doing all the work to confirm identity of an organism is way beyond the scope of your science fair project, so if you find that you don’t have a rod-shaped spore former growing that forms a pellicle in a broth culture, then you would need to get a new culture of B. subtilis.

http://www.ebi.ac.uk/2can/genomes/bacte ... tilis.html

Applying for a summer internship is a great idea! I did an internship in microbiology at a university when I was 16 and it was a great experience. Here is some information to get started. I saw one deadline for February 1 for applying, so this is a good time to get your applications in. What state do you want to go to?

When doing an internship, you will be working in a laboratory on the research that is ongoing in the lab, and you would not be working on AHL unless you could get an internship in a lab doing this type of research. So, do check out the availability of internships at the specific institutions from your reference papers.
http://www.answerbag.com/articles/How-t ... 6acb7e12bd

http://www.drexelmed.edu/GraduateStudie ... fault.aspx

http://hpap.syr.edu/spstate.htm

Let me know

Donna Hardy

I remember looking closely at the bacteria and saw that there was no difference in the morphology -- then again, I still feel like I haven't quite gotten the "hang of" microbiology yet being unable to do a gram stain (though thanks for telling me it's not something to worry about ) -- so I can't be too sure ;P.

Mass spectrometry and PCR are probably things our school doesn't have access to. So from what I understand, since the gram stain reads gram negative, that probably means that there was some contamination in the process? (Though...then shouldn't it be a mix between red and violet?)

>< I've been searching all day but most of the deadlines are in Feb 1...and require a teacher recommendation. Chances are that the teachers aren't going to be here -- however, I've also seen many deadlines of summer programs due in March and some even in April/May, so perhaps I can add my regional fair experience onto the application.

I have an address in the state of NY which is pretty good because many summer camps and internships are located there. I've checked out ASM internships, but it seems like we need to have a mentor that has been working with us for a year...or? I remember something made me know that I wasn't eligible for an ASM internship -- that was also the one I was hoping to find a slot for.

Cool, the reference papers would be a great place to start off. That means I'll really have to define what I want.

This may seem a bit far-fetched, but even before I've narrowed down bacteriology down to "quorum sensing", I've actually been quite interested in the biomedical field. Many of the "significance" paragraphs in reference papers note that finding more about inhibiting quorum sensing signals could help lead to the development of antibiotics. THAT is cool!

Would it be somewhat disappointing if I said that I'm still not sure what I want to do in my future? ^^ I thought I remember you saying that there's still a long way to go before becoming a microbiologist. I don't know what to do -- there are just so many options open, even outside of sciences (I am tied between something in science, and something related to debating/law -- of course, those are the "top two" jobs parents want children nowadays to do). Ahh, but I digress .

I love the idea of internships! The second link http://www.drexelmed.edu/GraduateStudie ... fault.aspx is very appealing!!! BUT... it might span over too long of a time! I wish there was something just like this (microbiology internship) but shorter, perhaps a 6 week program. Though a long program would be very interesting because I will be able to get things done.

Ahh...I'm getting the "so many things to do but so little time!" feeling again ><. I'm definitely bookmarking the above site.

Hi Trader,

It sounds like you have a pure culture of something, and additional investigation when you return to school will verify if it is B. subtilis or not. You may be doing your whole science fair project on this question, and it would actually be a good subject for a project. Although I wouldn’t want you to get sidetracked away from your topic, and you will still have the known E. coli to work with.

I would definitely not expect to find a mass spec or thermocycler (for PCR) in a high school lab. This type of equipment is expensive and that’s why I said using these techniques to identify your culture would be beyond the scope of your current project. However, you could grow the organism in a broth culture and look for the pellicle; you can describe the color and morphology of the culture growing on the agar plate;
http://sciencebuddies.com/science-fair- ... ates.shtml.
And you could look for endospore formation of an older culture under the microscope. You also might be able to check the fermentation of mannitol and other sugars and perhaps check the sensitivity for penicillin if these reagents are available. These observations would at least verify if you are working with an organism that could be B. subtilis.

I recommend that you apply for the internships with the February 1 deadline. Go ahead and apply for every position that sounds interesting to you. You can explain that you won’t be able to submit a teacher’s recommendation until after the New Year. Or maybe, you can find someone you know who can submit a personal recommendation, and then follow up with a teacher’s letter later. If you apply for an ASM internship, you can diplomatically mention that it has been difficult to contact the Shanghai Society for Microbiology. You can include the fact that you read scientific journals in your spare time, and that you have a great deal of knowledge about your subject of interest (ALH), and that you just need some experience in the lab to reach your goals. I’m sure your initiative, enthusiasm, intelligence, perserverance, and knowledge will shine through in your applications. If the programs have too many applicants, your application might not be considered if it is incomplete (without the teacher’s letter), but if there are not a lot of applicants, then your application would certainly be reviewed. You won’t get accepted for any position you don’t apply for. However, please apply only to the programs that you have time to complete a very thorough and excellent application for.

It’s important to have goals, but it’s definitely OK to change direction. Microbiology can be a good background for any position in the biomedical field. It requires lots of math, chemistry, and physics, and these subjects are the basis for any career in science. It’s important to pick a career that you really like, because you will be pursuing it for many years. You should try out law/debating and continue to pursue your interest in science. Law and microbiology would be a good combination if you wanted to be a patent attorney, for example. You just need to take advantage of any opportunity that you get to pursue your goals.

Donna Hardy

Thanks for the advice!

Just to clarify -- the current direction I am heading in can be integrated as part of a "biomedical" research internship right? Because the Drexel U-C of Medicine seems very interesting now.

Though it says that the deadline for the application is Feb 1...2008. Are science internships offered by these colleges usually offered every year ...? Ahh, I asked anyways

Also, do you happen to know how long science internships last on average? I'm curious because it seems like my parents want me to "relax" and ... well not go all "scienc-ey" on them during the summer xD, so I'm afraid I don't have more than 6 weeks.

So far many science internships last 8 weeks or so, so I was wondering if this is "the norm"? I'm sure there are 6 week internships out there though.

Sorry this has gotten somewhat off topic! I'll write up actual research paper soon ...

Wow, 4 months ago I never knew I'd be working with bacteria ><.

Hi Trader,

The internship program at Drexel looks very good, although the description says that it is for “area” students. So I don’t know if they are open only to local students. The information given isn’t that clear. The 2008 date probably indicates that the website has not been updated, but you should send an e-mail message to the address given to verify the program will continue this year, and also ask if they will accept someone from out of the local geographic area. Most of the internships are paid, so it’s like a summer job and it’s hard to get any research done in 6 weeks, so I do think that 8 weeks is more typical. If it will help, you can tell your parents that you will have lots of fun, in addition to gaining invaluable lab experience that you need to continue your long-term science project.

It’s interesting. I have contacted a former coworker, who is currently in Singapore, but originally from China. I asked her, of course, for any contacts in Shanghai that we could ask to be a mentor for you. She explained that volunteering and mentoring are not traditional activities for the area, so that is probably why it has been so difficult to find someone who can help you. She will make inquiries on your behalf, however, because there are lots of scientific activity and outstanding scientists in the area. Of course, she said nothing could be done until after the New Year. But this means that you may need to be even more creative in figuring out how to do this project on your own.

Donna Hardy

^^ That is very true

I used to have a list of internships, and now it's ... down to 3 of ones I am eligible. I should contact local universities too. A rather unrelated question -- in an internship, why do they pay you to pursue your own research project? --

Many of the websites probably aren't updated and/or aren't offering a summer program for this year -- I'll be awaiting their email .

My Considering List:):

*Secondary School Training Program June 21 – Aug 1
http://www.continuetolearn.uiowa.edu/SSTP/index.html

Baylor U HSSSRP May 31 - July 3
http://www.baylor.edu/summerscience/

*Rockefeller Outreach Program – June 28 – Aug. 13 – will take entire summer.
http://www.rockefeller.edu/outreach/summer.php

(1)UC Davis Young Scholars Program – June 21-August 1, 2009
but too many field trips…
http://ysp.ucdavis.edu/

*Feinstein Institute for Medical Research – Just for science fair projects http://www.nslij.com/body.cfm?id=2586&o ... inkID=5096

(1)Michigan State RSI 21 June – 8 August 2009
https://www.msu.edu/~hshsp/

(2) Summers of Discovery – Minimum of 8 weeks
http://www.niehs.nih.gov/careers/research/summers/

MDIBL -- Nomination deadline is past, but I emailed them asking them if it would be possible that they extend the deadline
http://www.mdibl.org/edu/highschool.shtml

(1) Chances are these internships/programs I can't consider unless I get a reply on email indicating that I can actually pursue an individual project. It seems like they assign us a project to pursue which might be interesting...I'm not sure.

(2) A question regarding terminology -- to what extent may my current project direction be considered part of a "biomedical" study? -- Because it seems like after applying to (2), they have specific branches which we can contact to make them aware of our research project, yet I can't seem to find one in microbiology.

Of course, there are those summer programs that can "open my eyes" to the possibilities out there, and perhaps I can take my project/restart a complete new project. ... I'm not sure -- in your opinion, which might be better 1) an "eye-opening" summer program, or a 2) research internship? (I'm actually more interested in the latter, but my curiosity prompts me to consider the former too

As for my ACTUAL project.

I've been reading the support, and I've seen that "Growth of B. subtilis Cell Surface" mentioned that the starvation of d-adanine led to altered growth/swelling of the swell so I was wondering ...

Is d-adanine something to be "added" to nutrient agar to ensure b. subtilis growth, or is it already included in there?

Also another question regarding making slides with the cells.

In the same article, it mentioned that the cells were "fixed in formaldehyde (final c/c 2%), spread on gelatin subbed slides, and dried" -- I assume dropping a drop of sterile water on the glass slide, briefly tapping the inoculating loop w/ b. subtilis and making the water evaporate from burning, then let it cool, then waving it over a flame for 2 s is also an adequate method of making a slide right? ^^

This gives a generation time for 35 C... which should be some good support. -- When the article says "Modeling Surface Growth of E. coli on Agar Plates", it makes me wonder if the surface growth of b. subtilis has been modeled? (Blah, original-topic craving again ) I'm definitely keeping

"One more idea. E. coli is one of the organisms that can utilize lactose, so maybe the addition of lactose to the growth medium for this organism would affect the optimum growth conditions. This reference is a study of glucose utilization by a Bacillus species: http://jb.asm.org/cgi/reprint/179/23/7603.pdf"

in mind...

But I don't mind if "is there a difference between the optimum temperature for highest # of viable organisms and shortest doubling time" isn't an original question (unless it hasn't been investigated before?), because I do want to find out.

If possible, perhaps the optimum temperature would also result in difference in total cell volume like you mentioned, so that's definitely something I want to find out! (Now I'm beginning to not like the holiday so much )

A final question -- for my hypothesis, I believe you mentioned "you can just predict that one of your conditions will give the optimum number of viable organisms and the shortest doubling time, and you can base the conditions that you select on the literature references that you find" -- I've only found references that give the shortest doubling time or the highest number of viable organisms (I'll dig them up somewhere) --> I could try to find two references that give the exact same conditions, but have one temperature for doubling time, and one temperature for highest number of viable organisms, but I doubt I'd find that ...

In that case, I'm not sure how I can find a "support" for my hypothesis in the way that I'm not sure whether to think that the two temperatures will be the same... I'll continue to hunt for references.

Thank you for your patience with everything again (Esp. for ... a student who ... doesn't really get much done )(and always changes her mind)(and pretty much spends...90 posts brainstorming)(and then changing her mind again)(and being unable to get a gram stain done )(but at the same time very interested and hoping that CNY is over soon)(and adding too many parentheses)

Hi Trader,

Good job of researching the summer internships! These are all outstanding institutions and you would learn a lot with any program you apply to. I hate to tell you this, but high school students don't usually know what they want to do research in, and these programs are designed to give the students an opportunity to learn how to do research and find out what they might be interested in doing. They only way that you will get to work on your research is if you can get a position in a microbiology lab. All of these institutions have labs that are microbiology labs, so do ask for a microbiology lab position since this is important to you. I agree with you that a research internship would be most helpful for your project. With a general summer program, you won’t be doing as much in the lab, but you would still learn a lot. So please go ahead and apply!

Your current research project is related to the biomedical field because it is a basic research project on bacterial communication that could be important in human diseases. For example, dental decay is an example of biofilm production, which you know from other research papers is dependent on AHL production. Pseudomonas infection in cystic fibrosis patients is another example. It is possible that understanding how bacteria communicate with each other will be the basis of future breakthroughs in medical treatment, but this is a field that is largely unexplored. Your project on E. coli and B. subtilis could provide additional understanding in this area.

The article on B. subtilis growth used a mutant strain of B. subtilis that apparently required the addition of D-alanine (an amino acid) to grow. The authors added the amino acid to nutrient agar. Your B. subtilis is not a mutant and should grow well on any type of medium, so you don’t need to worry about this. The authors were using a special phase contrast microscopy technique, so prepared the slides on gelatin. You don’t have a phase microscope, so you don’t need to worry about fixing in formaldehyde either. Your technique of air-drying and heat fixing is perfect for glass slides and regular microscopes.

I have an idea that would make your project more original, but would keep maintain the same experimental protocol that you have been considering for the past 2 weeks or so. You would still do growth curves and measure generation time and maximum viable cell number, but instead of comparing different temperatures, you would keep the temperature constant, and your independent variable would be the presence of AHL producing cultures of either E. coli or B. subtilis. You could measure the effect of AHL producing. E. coli in either E. coli cultures or B. subtilis culture in a broth culture.
You would inoculate a culture of 100-1000 organisms per ml and start the cultures growing in lag phase. Then either add a heat-killed sample, or a stationary phase culture enclosed in dialysis tubing or other porous material that would allow the diffusion of AHL’s, but not whole bacteria cells. Using a small piece of tubing with 0.45 micron filters glued on each end would be a good way to enclose the ALH—producing cells. You know that E. coli doesn’t start producing AHL’s until the end of the log phase of growth, so you would test the effect of AHL (and other molecules in the mature culture) on your non-AHL producing culture. Based on what you know about AHL’s, what do you think would happen in this experiment? Your title could be something like “battle of the bacteria.” Sorry, to be a bad influence and try to change your experiment at this point, so just consider this as an idea and let me know if you want to switch again.

I owe you a few answers, but I’m out of time. I will send more information tomorrow.

Donna Hardy

The idea does seem interesting!

In other words, it's like simulating the test strain/sensor strain environment, only seeing the effect of ONLY AHLs on b. subtilis, seeing whether it can receive it or not?

This is VERY COOL. This can answer a lot of the questions I have about b. subtilis and e. coli QS, because I've only seen that b. subtilis produces ComX and CSF QS signals, and some e. coli produce AHLs and AI-2, but I could see whether AI-2 is truly a "universal language" between bacteria! (I hope I didn't misunderstand what you're proposing?)

This allows me to dig up my old research! I'm pretty sure that e. coli's AHLs won't have an effect on b. subtilis (it seems like only ComX and CSF can be detected by b. subtilis?)

BUT I have seen AI-2 proposed as a "universal language", and if I understand all the QS research done on this, the AI-2 produced by e. coli should have some kind of an effect on b. subtilis -- notably its subtilin (sp?) production. At least it controls the biofilm production in some bacillus species, I'm not sure about b. subtilis.

I'm looking at http://aem.asm.org/cgi/content/abstract/72/1/937 for how AI-2 affects some species of bacillus biofilm production while here http://www3.interscience.wiley.com/jour ... 1&SRETRY=0 is confirmation that b. subtilis in fact does produce AI-2, while http://jb.asm.org/cgi/content/abstract/188/1/305 says that AI-2 controls biofilm production in e. coli.

I really hope no one has put it together to test if there is any possible variation between the AI-2 that b. subtilis produces/receives and the AI-2 that e. coli produces/receives! Do you think there could be a difference in terms of the reception and production of AI-2 being successful/ineffective because it's produced from a different species?

Here's also a general question -- I know that e. coli is found in the human intestine and that b. subtilis is found in the soil.

Since there seems to be a minimal chance of the two bacteria interacting, would that make the project less significant?

THANK YOU SO MUCH FOR THE IDEA! I think as SOON as I get back... (darn, I'll only have a month ), I'll first test out my question "is there a difference between the two temperatures that blah blah", so I can have something, and THEN I'll see if I can move onto see if the above project works.

AND I LOVE how AI-2 controls biofilm production in e. coli and b. subtilis! That is a greater link to biomedical sciences

Now my key concern is ... hmm... if it's a "valid question" whether or not AI-2 produced by any bacteria can/not be received by another bacteria just because it's produced by a different species.

I'm thinking this because AI-2 has been received by one species, that is produced by a completely different species...

Now I'm also worried that I misunderstood what you were saying (because now I realize that I can't see why this would be a battle ^^)

Wait...unless I do something that has b. subtilis and e. coli in (the same culture? or same environment so they can battle over it?) a culture that receives b. subtilis-created AI-2, and then the same culture with the same bacteria that receives e. coli AI-2, and see if there's any possible "translation" the "non-native" bacteria of the signal has to do before receiving the signal.

Sorry this post has been one great brainstorming pad. I hope the above paragraph is a valid start? (Unless it's not what you were saying? ^^)

Hi Trader,

You are good at brainstorming. Your questions are all excellent. I think you do understand the proposal perfectly. My only hesitation with this suggestion, and one that the science fair judges will ask about, is that with the tools you have available, you will only be able to test the effect of one bacterial culture on the growth of another culture. You won’t be able to prove that any effect is due to AI-2 or Com-X. The science fair judge will ask if there is something else in the culture medium that caused the effect. However, with your background research you will be able to propose a possible mechanism for the result, and explain what you would need to do to prove it. But this is a more original project compared to determining the optimum temperature for growth of the two species. I can almost guarantee you that there won’t be anything else like it submitted at the local science fair, because I’ve never seen a project like this.

You have probably noticed that all journal articles report results of a few specific experiments; no one article or experiment can answer all of the questions. So you just need to concentrate on answering one question. E. coli represents a typical Gram-negative organisms and B. subtilis represents a typical Gram-positive organism. Maybe next year, you can switch to organisms are present in the normal flora or humans.
http://www.textbookofbacteriology.net/normalflora.html

I could not open up the second link you sent from Wiley. Can you resend it? I will print out the other two and read them later. I noticed the Univ of Conneticut/Storrs and Univ of Maryland/College Park addresses for the authors of one paper. Are there any high school internships at these institutions?

I need to reread some papers before I can comment on your other questions, but why don’t you see if you can predict what will happen when you add either Com-X producing B. subtilis or AI-2-producing E. coli with lag and early log phase cultures of each of these organisms. And, do plan your experimental details. With only a month to go, you will need to produce results of some sort, so you will need to start the experiments as soon as possible.

Donna Hardy

Some updates!

*-- with regards to that article, I can't open it myself either due to some cookies problem -- I've found an alternate source which appears to imply that b. subtilis produces AI-2 under some conditions (I'll have to analyze to see what those conditions would be) --*

Some articles I have yet to read (I'm just going to list them out here so I'll know there here -- I'm not sure if they're relevant)

http://www.pubmedcentral.nih.gov/articl ... tid=383170

Here is what I have for support so far:

According to some quick articles (some are repeat of the above, but just for organization ),
AI-2 controls biofilm production in e. coli
AI-2 appears to control biofilm production bacillus
E. coli produces AI-2 (new link)
B. subtilis produces AI-2 (I think?)
Interesting article about AI-2 AND AHL activity v. growth in e. coli

>> About the last article -- oh no...I'm not sure and I only briefly went over it, but I think its results indicate that "AI-2 activity is not related to the growth of e. coli", though "as expected, there was no AI-2 activity during incubation" ...

I'm going to dig up my previous article supporting "e. coli has minimal/negligible AHL production during stationary phase" statement then... ><

I briefly viewed over them though I'm not sure about the last one. Thank you for taking the time to check the first two ^^.

As for ComX and AI-2...(I'm very excited when I'm typing this ) -- ComX I am 99.9% sure cannot be received by e. coli, and AHL 99.9% cannot be received by b. subtilis because there are numerous reference articles that support the proposal (and I believe it so far is an "undefeated" proposal) that AI-2 is a "universal language" between bacteria. It would be OK if I test if there's any possible "translation issues" when bacteria receive their "non-native" source of AI-2?

*-- Update -- Oh no... It looks like b. cereus can degrade AHLs in some media -- this is interesting. Let me see if it might influence the above...

Perhaps there might be something in the culture medium -- I think I should first use the experiment I am doing now (that is, understanding their growth curves so I can predict their growth) so I'll know what to expect in terms of biofilm production in e. coli or something in b. subtilis (I emailed the author finding that AI-2 affects biofilm production in b. cereus).

What I'm worried about is,

1) the articles above are actually the only ones I managed to find, so if one of them used a mutant strain or something, then that's not good for me . Though I must thank you a lot again for going over them! (I'll check out the later two...the last one looks a bit suspicious, because it explicitly stated that b. subtilis does not produce AI-2 ... under "these" conditions)

2) How would I be able to measure biofilm production? I think this could be possible, but it seems like one of those things that require fancy tools...might have to wait until the summer, but the key question that is gnawing at me right now is ....

3) What are AI-2's effects on b. subtilis? (Unfortuntely, I am not much of a google expert myself, but I did go through 10 pages of Google results of "b. subtilis AI-2 "controls"" and found much of the same one article (of the author I emailed) as well as ... other not so relevant results)

4) A minor concern since it's a bit far away now, but since e. coli starts producing AHLs only in log phase, and receives them in the stationary phase... I'm concerned if a) it would be different for AI-2 production though i think that QS production in general has that schedule because they wouldn't release QS until their population reaches a certain population density.

This leads to another question that I'm a bit confused about ^^

Determining whether or not a bacteria is in the "log phase" or the "stationary phase" depends (only?) on the amount of nutrients in the area of the bacteria right? IF we had a petri plate that expanded forever, and b. subtilis for example was grown in its optimal conditions, after about 48 hours, the center bacteria would be in the stationary phase because it ran out of nutrients there, while the bacteria at the edge would be in the lag phase?

I'm most doubtful about that last statement, because somehow I have a feeling it's not true =P.

BACK TO brainstorming : It looks like there aren't any experiments finding out what AI-2's effects on b. subtilis are (I really hope the author replies!) because it seems like AI-2's effect on b. subtilis would be something that's already known?

If it isn't, I think I'll have to focus on growth curves for the science fair, and for the summer, find what b. subtilis/AI-2 does (based on how the author of AI-2's effect on biofilm production in ... b. cereus found out about b. cereus' reception of AI-2 and its effects), and THEN continue onto the project.

I'M SO EXCITED ;D

Returning to my ... old unoriginal (just kidding ) -- cool, slightly original (I hope?) question regarding the "difference between temperatures?" experiment. (I'll continue this while possibly pursuing preparation for the other )

I'm finalizing my research plan and I just want to confirm: By "modeling", does a mathematical formula need to be included? -- unless only observations of the growth (based on population) can conclude a experiment titled "modeling growth of e. coli under 37 degrees Celsius" for example?

Also, just to confirm -- plate count shows the population for agar plate cultures (I've been looking for possible sources of slant tubes, but not any luck so far...I'm sure there are "normal" screw tubes somewhere) and does not require the liquid broth right? (In other words, is it possible to "accurately" determine the population without a liquid broth culture?

Of course, I still need to perform a gram stain ><. SORRY -- this is the last question!

If the morphology of the sample after viewing it under the microscope appears to be b. subtilis, what might be the likeliness that the sample (and therefore the petri plate) is still contaminated because of the gram stain's negative result?

I apologize for the huge wave of questions ><

*-- It looks like the available summer programs are for undergrads, but I asked an email seeing if they would make an exception anyways.
*-- Yes, the details are very important! I remember how preparation kind of "swerved" off course from my anticipated course from my first draft of my research plan ...

THANK YOU so much

Hi Trader,

You are hard to keep up with. I did not get much information from the articles from yesterday. It would be nice if we had access to the entire article with data so we could answer the questions you have. It looks like you have found some complete articles this time, so I’ll print them out and read them next.

From your analysis, it sounds like you will have the following assumptions:

1. Com-X is produced by B. subtilis
2. Com-X has no effect on E. coli, but does have an effect on B. subtilis
3. ALH’s are produced by E. coli
4. ALH’s do not affect B. subtilis and do not alter the growth rate of E. coli.


We need to pin down exactly when Com-X and ALH’s are produced. My impression from previous articles was that these are not produced in lag phase, but are produced as cultures reach stationary phase. But I will have to go back and reread the articles and pay attention to this detail.

I don’t think there will be anything in the culture medium. Culture medium contains hydrolyzed protein, salt, and maybe some glucose. ALH’s are very specialized lipid/pheromone type molecules that would not be present unless a microorganism had produced them. So you should assume that the culture medium you use will not contain AHL or COM-X.

The papers with the mutants are interesting, but you would not expect similar results because your cultures are probably more like wild-type bacteria that can grow on regular culture medium.

You can’t measure biofilm because E. coli does not produce biofilm, as far as I know. And, B. subtilis also swims around freely and does not attach to a surface either. Other organisms have this type of activity (dental plaque, bacteria in mines). And you are correct, you would need a way to measure biofilm production, if it occurred.

There haven’t been a lot of papers on this subject, so the area is open for more research. I’m not certain either if B. subtilis is affected by AI-2.

Microorganisms start growing in lag phase, then double in number every 20-30 minutes when grown under optimum conditions during log phase, and then slow down when stationary phase starts. Since the AHL effect of the organisms is not seen until after the organisms have reached a certain number, it seems that there would not be any AHL’s produced until the end of log phase.

You will need to focus on growth curves for your science fair project because you don’t have a control AHL sample or a sensor organism.

A mathematical formula is not required for a science fair project. Just a question, hypothesis, an experiment, data and conclusions. Math formulas are always great if they fit with the data, but I’m not sure it would add anything to this project. You need to focus on getting some results; otherwise your project will be all theory.

Your data will be obtained from plate count results. The slant cultures won’t help for your data collection. You will be making dilutions of your cultures and growing the bacteria on plates; you will be counting plates with colony numbers between 30 and 300 colonies (or zero or TNTC (too numerous to count)), if you make the wrong dilution. Have you thought of how you will make a dilution of a culture that may contain 10,000,000 cells per ml (close to the end of log phase), and end up with only 30-300 colonies per plate? This is usually done by making 1:10 and 1:100 dilutions in sterile water. Do you have pipettes available that you can use for this? If you don’t have the equipment to do plate counts, then we need to identify an alternative method for measuring the bacteria.

You will have to do growth curves in a broth culture; you can’t do this directly on a plate. You make a dilution of a culture into a flask with nutrient broth so the count is low, maybe 100-1000 bacteria per ml into new medium. The bacteria sit around for a while getting their metabolism adapted for the nutrients available and start making biomolecules necessary for growing and dividing, but they do not increase in number for a while. This is lag phase. Then the bacteria start dividing in two every 20-30 minutes, which results in a doubling in number for every generation until the population reaches about 10 to the 9th organisms per ml. This is log phase. Then the population dies and divides at a steady rate for a while and the total population number does not change. This is stationary phase. For your project, whether you do temperature or presence of AHL or Com-X, you will be comparing the generation time (doubling time) during log phase, and the total number of bacteria at the end of log phase.

Yes, do the Gram stain first as this will help verify that you have an organism that could be B. subtilis. If the morphology and color of the colony, the shape and size of the cells under the microscope, the appearance of endospores, the formation of a pellicle in a broth culture all match B. subtilis, then we’ll ignore the Gram stain results.

It was good that you asked for an exception on the summer program. You never know until you ask.

I’m sorry I have not answered all of your questions; I’ll try to answer more tomorrow.

Donna Hardy

Thanks for answering my lengthy post =P. I think my posts are too long, and that's not good. I shall now focus on being concise. And no extraneous comments, save this one .

First thing -- I think we're talking about different experiments o=...because what I'm describing doesn't involve AHLs and Com-X...only AI-2 which is a special/different quorum sensing signal.

I was thinking that -- since b. subtilis and e. coli can both produce and receive AI-2, I could compare the rate at which they receive their "non-native" AI-2 (that is, b. subtilis receiving e. coli AI-2, and vice versa) and compare it with the control of them receiving "native" AI-2, and see if there is a possible difference between the two.

This article and many many others support how AI-2 is a universal signal >> where

"In this report, we also provide evidence that the biosynthetic pathway and biochemical intermediates in AI-2 biosynthesis are identical in Escherichia coli, Salmonella typhimurium, V. harveyi, Vibrio cholerae and Enterococcus faecalis. This result suggests that, unlike quorum sensing via the family of related homoserine lactone autoinducers, AI-2 is a unique, 'universal' signal that could be used by a variety of bacteria for communication among and between species."

E. faecalis is gram positive, but all the others are gram-negative and I was hoping to expand this, seeing if the same AI-2 also applies to the bacillus species.

I think I deviated from your proposal -- or was I on the right track and then swerved off?

This article has

"...Based on previous reports and confirmed in this work, the signal of the reporter strain is minimal between 5 and 5*5 h after inoculation (i.e. interference by endogenous AI-2 of the reporter is < 1% of the bioluminescence signal)"

Would 5 and 5*5 (the asterisk is the same as the "dot" sign one uses to indicate multiplication? From this I'm assuming that most AI-2 production occurs after the 5 - 5.5 h, that is, the log phase? I'll find a better article to pinpoint the exact timing later.

As for e. coli and biofilm, did I misunderstand this article... ? It's titled "Effect of AI-2 on biofilm production of e. coli ... something something" -- perhaps the 'something' indicates a mutant strain involved?

After knowing when AI-2 is produced in both, I'll make sure the time both strains are not producing AI-2 (and this time will be known from my growth curve experiments -- knowing when the lag, log phase etc) to be inoculated into a culture w/ AI-2 (somehow isolated, one sample from b. subtilis, another from e. coli), and then somehow test for biofilm (if the above holds) and however AI-2 affects b. subtilis. ... Very theory-based

As for my not-so-theory-based experiment...

OK, so let me outline a procedure to test my understanding: Things to do:

1) The lawn of bacteria should be ... "renewed", which means that I'm going to transfer them all to nutrient agar plates now.

One quick question with pouring plates -- after I poured the agar in and leave the cover tilted so it can cool....I close the cover. And then as soon as the agar has solidified, I turn it up side down and leave the plates overnight under the UV sterilizer...and THEN I move them into the fridge the next day -- that's OK right?

2) Inoculate sample of e. coli and transfer them to 3 plates to start with, and incubate overnight.
3) Inoculate individual colony of b. subtilis streak plate and make slide out of it -- gram stain and observe morphology.

Quick question:

In theory, if the morphology observed in step 3 does not appear to be b. subtilis, would be "worth it" to continue making streak plate from source A (which streak plate was made from) and make more and more individual colonies until verified that it is b. subtilis?

4) If correct morphology, ignore gram stain (though it still bugs me...why gram negative?)
5) Would further check (checking that it is catalase positive -- How can we check if a certain bacteria is catalase-positive and can metabolize sugars?)
5) Prepare nutrient broth (materials should be here after school starts)
6) Inoculate confirmed individual colony to liquid broth.

Quick questions regarding this:

To identify under the microscope I inoculate half of an individual colony on the petri plate right? And after it's confirmed, I would take the remaining half and move that to the liquid broth?

7) Is it better for me to first inoculate the confirmed colony into agar plates to make sure that they are "stocked", and then move it to broth?)
8. When the samples of the e.coli are grown as well, confirm with above steps until both b. subtilis and e. coli are at this step. Now they will be referred to as "bacteria"

Quick Question:

If we transfer an individual colony to a liquid broth, after overnight incubation does the number of colonies stay the same, but the number of bacteria increase?

9) After overnight incubation (I've seen terms like "shaking" and "rpm" associated with liquid cultures -- does this apply?) Cloudy liquid broth means that the cultures are probably in the stationary phase already (?) -- refrigerate.
10) Diluting the culture (in this case, I will do two separate dilutions for each culture) into new broth means that it is "reset" into the lag phase.
11) Take one sample (through sterile pipette?) and make a slide out of it, and confirm that it is pure culture? Sorry -- not sure how to verify that the liquid broth culture is a pure culture
12) After confirmation that the liquid broth culture is pure, transfer to fresh broth culture and begin to model growth curve.

Quick question:

Would it be OK if I skipped above 4 steps and immediately, after verifying pure culture from streak plate, go to the below step and model growth curve?

13) Be ready to spend one entire day for measuring the growth curves for one temperature. So both bacteria were just transferred to a fresh broth culture. Incubate at 37 degrees and every 1 h measure the population via dilution plating.

>> I know step #13 would actually require a lot more steps, but I first want to make sure that the beginning is correct.

Thank you so much for all this -- I think I'm causing the most annoyance when comparing my post lengths with all the others I hope I'm not too much of an inconvenience! Thank you for making my science fair experience what it is

Hi Trader,

I do apologize; I had not had a chance to read all of the articles, and I still haven’t read them all, but I’m still trying to get through them. AI-2 is possibly the universal signal used by both Gram-positive and Gram-negative organisms, and E. coli does produce biofilm, but I didn’t know this until I read the latest reference. So thank-you for finding that reference.

I was suggesting an experiment that you could do that would yield measurable results. Doing a biofilm assay is certainly a possibility, and here a procedure for doing one:

http://media.wiley.com/CurrentProtocols ... leUnit.pdf

You would need broth cultures, a solid surface, crystal violet, ethanol, and a spectrophotometer to do the experiment. You don’t have any purifying AI-2 or a sensor organism, so you will have to do an experiment with the cultures based on your background reading, and make any conclusions based on information from the references. You can assume that the E. coli does produce AI-2, so you could test the effect of E. coli and B. subtilis and measure the result (by biofilm assay or growth curve, whatever you have available to measure).

It seems reasonable that 5*5 means 5.5 hours, but another reference would be helpful. Or, send an e-mail message to the author to verify the information.

In the AI-2 reference the authors describe procedure for making copies of the mqsR+ gene by PCR, cloning it into a plasmid, and then expressing the resulting protein in E. coli. For this part of the experiment, the E. coli is just used to make a quantity of enzyme that synthesizes AI-2. You won’t be able to do anything like this until you can get a job in a molecular biology lab.

What is the exact experiment that you are thinking about doing?

Question on transferring bacteria to new plates. Your procedure for tilting the cover of the plates and allowing the agar to solidify and then waiting for 10-15 minutes before turning them upside down should result in drier plates than you obtained the first time. Leaving them out under the UV lamp should be OK, but you can put them in the refrigerator immediately if you want to.

Question about morphology of B. subtilis. The culture should be long, thin rods about 0.5 micron x 3-5 microns. If the culture you are testing is several days old, you should see a mixture round endospores and some rods. If you don’t see this, then go back to your original plate and do more streak plates to see if you can isolate any colonies with a different appearance. Please let me know the description of the colonies and the appearance under the microscope.

A catalase test is easy to do. You just need to mix some of the culture with hydrogen peroxide on a slide:

http://ftp.ccccd.edu/dcain/CCCCD%20Micr ... e_test.htm

Bacillus species are aerobic, so should be catalase positive and you should see bubbles when the hydrogen peroxide (H2O2) is converted to water and oxygen gas by the catalase enzyme. Fermentation of sugars is done by adding a sugar to nutrient broth with a phenol red indicator. If the sugar is fermented, acid will be produced and the phenol red will turn from red to yellow. Bacillus subtilis will ferment the sugar mannitol . Here’s a key to show you how bacteria can be identified:

http://faculty.ivytech.edu/~bsipe/UNKN/ukkey.htm

Don’t worry if you can’t do the mannitol fermentation. Everything else will confirm that it’s at least a Bacillus species.

Yes, using half of the colony for a microscopic analysis and the other half to inoculate the broth culture would be a good procedure.

If you have a confirmed a colony on a plate by microscopic observation, and all of the colonies on the plate appear to be identical, you can assume you have a pure culture, so you could use any of the colonies.

I’m not sure I understand what you are asking on #8. If you have isolated a colony from a pure culture on a streak plate, you can label your cultures as E. coli and B. subtilis, unless you find out otherwise.

If you transfer a small amount of an individual colony to a broth culture and grow it overnight, the bacteria will not be growing in colonies in the broth. They will multiply and the broth will become turbid due to the high numbers of bacteria growing in a uniform suspension. After a while, dead cells will sink to the bottom, but a newly grown culture will consist of free-swimming individual cells, though the B subtilis does tend to grow in chains.

Shaking and rpm refers to putting the flask with the broth culture on a rotating shaker that aerates the culture. If the culture is grown without shaking, the growth rate will be slower because the amount of oxygen will be limited. Both E. coli and B. subtilis are aerobic organisms and will grow much faster with plenty of oxygen available. Overnight cultures would be in the stationary phase. If you don’t have a shaker available, then you will have to do static cultures, which is OK, but a shaker would speed up the results.

Yes, when you take a small amount of the stationary phase culture and transfer it to new broth, you do reset the growth cycle to the lag phase again. You want to make a high dilution so the numbers start low enough to get a full growth curve.

You will have started the experiment in the broth culture with bacteria from a single colony, so you assume that you have a pure culture at this step. Frequently, during the course of your experiments, you will recheck the purity of your culture by doing a streak plate.

Yes, after verifying the pure culture on the agar plate, go directly to the broth culture experiments.

If you want to check multiple points on the growth curve, you would need to devote an entire day and doing plate counts periodically. Once every hour would require a lot of plates. You could plan the number of data points to match your supply of plates and energy. Once you’ve done a growth curve, you will become familiar with the process, and it will be a lot easier.

So, will it be growth curves or biofilm assay for your experiment?

You are welcome. I’m happy to provide whatever assistance I can with your project. I do want this to be a successful project, and I think it will be. It’s an exciting topic, and definitely worthwhile spending some time and effort discussing.

I have good news. My coworker talked to her friend in Shanghai again, who has provided the information for microbiology institutions in Shanghai that might be able to provide you will local information and assistance.

Institut Pasteur of Shanghai
86-21-6385-0152
[email protected]

The Institute of Microbiology
86-10-6480-7462

Institute of Biochemistry & Cell Biology
86-21-5492-0000
[email protected]

Of course, you won’t have to dial the 86. After CNY, do try to contact these institutions and ask if they have any suggestions to help you learn more about microbiology techniques, or if they know of anyone doing AHL research in Shanghai. If your teacher or a friend is native Chinese, you might ask for suggestions on the best way to ask for help. You probably won’t make a connection in time to help for this year, but local help would be very beneficial for next year’s project.

Good luck!


Donna Hardy

Wow...I'm just reading through the link for making a biofilm assay...and I understand it! (That probably means it's doable! ) This is great!

I've received a reply from an author and apparently not much is known about AI-2's role with b. subtilis. In this case, I was thinking that I could do a biofilm assay comparing the amount of biofilm produced by e. coli with b. subtilis AI-2 (if I can get a stronger support that b. subtilis actually produces AI-2, how can I "transfer" this AI-2 to the e. coli culture? -- sorry this proposal sounds very unscientific) and the amount of biofilm produced by e. coli, with e. coli AI-2.

>> Is this what you're saying? (This is so close to what I've wanted to do before, only it's doable! ... I think )

It looks pretty clear now that "AI-2 is produced until the stationary phase" where the "transition into the stationary phase begins the consumption of AI-2" -- [1] and [2]

For filtering the AI-2 ... I know that ComX won't affect e. coli and AHLs won't affect b. subtilis, so it may be that AI-2 would be the only thing that can be read by both e. coli and b. subtilis.

That means that I'll need to quickly do the growth curves to make sure I can find out when the e. coli goes into the stationary phase.

Thanks for the information! I'll definitely keep that in mind when making the plates and analyzing the b. subtilis.

I think the subsequent paragraph answered # 8 (sorry for the awkward wording) -- I was just asking whether the numbers of colonies grow (because I wasn't too sure under what conditions would 1 colony turn into 2 colonies)

Ooh. I like lots of plates . Thank you so much for explaining everything ><! This would be great. I can spend one Saturday one this! Now all I have to do is confirm that it is b. subtilis and e. coli I am working with. If I continue the project above, then what I really do need are e. coli growth curves, but it'd be interesting to compare between the two.

I'm going to do the growth curve experiment first because it seems like I should really pin down the exact time when e. coli makes the AI-2. If I can pinpoint within a 10 - 20 minute range...then I can make sure that when I put the AI-2 in (somehow),...that the e. coli itself wouldn't be making AI-2 and would only be consuming it.

With regards to biofilm production, under what conditions would e. coli make biofilm, or is it something that all e. coli go through during the stationary phase? If it is something they go through then I wonder if adding more AI-2 (when I say this I feel very unscientific =P) would result in more biofilm production --... if not, I'll have to check out the environment under which they make biofilm (is it when they are under extreme temperatures? Or is that going into a dormant phase?)

THANK YOU so much for the email addresses and the telephone numbers!! The links are very very useful! If this takes just a quick Google search, please let me know -- I think I'm enough of an annoyance already =P ... though, I've never been good with Google.

Hi Trader,

I’m glad you like the biofilm experiment. I liked it too because it does not require complicated equipment and I think you should be able to do it with your available resources. Which biofilm assay are your going to use? It would be good to do the first option where results are read at 500 to 600 nm with replicates so you will obtain quantitative results, and your will be able to calculate the mean and standard deviation of the results. I have forgotten. Do you have a spectrophotometer that will read at OD 600 at your school? Also, do you have a microscope with a camera? It would be great if you could take pictures of the bacteria growing on a cover slip. Having actual photographs or lab drawings of the biofilms on your project board would a powerful visual addition to the science fair board?

That’s exciting that you received a reply from an author. There is a lot of information that not published and a personal communication from someone who is involved in the research is invaluable.

You can’t find out if B. subtilis produces or responds to AI-2 this year because you don’t have any pure AI-2. Why don’t you do a very straightforward experiment and find out if your E. coli and B. subtilis produce biofilms and find out how long it takes? The protocol suggests a 4-day incubation time, but perhaps you could test for biofilms at 1, 2, 3, 4, and 6 days. Or, you could try different media (with and without a usable sugar source, for example) and incubate all cultures for 4 days. You would not need the data from growth curves, which are a lot of work, for this year’s project. You just need to grow up broth cultures overnight and dilute them 1:100 to inoculate them into the test medium and incubate them. The E. coli will be a kind of positive control because we know that it’s supposed to produce a biofilm. If you include any other independent variables other than time, perhaps you could include extra samples for the 4 day data point on B. subtilis, and try using 80% ethanol/20% acetone, which is recommended for solubilization of E. coli biofilms to 100% DMSO, which is a recommendation for one of the Gram-positive organisms on the list (from the biofilm protocol). If you started your experiment right away, you would have time to repeat it if there is any uncertainty about the results. You need to be finished with your experiments at least one week before deadline so you will have time to write up your board properly. Look at the calendar and see how many days you have left to complete the experiment before deadline minus 7 days, and try to realistically decide how much you can do.

You can do the growth curve experiment, but I think you only have time to do one experiment before deadline. However, the biofilm experiment would allow you to answer the question about whether or not the cultures you are working with produce biofilms and how long it takes to produce a measurable biofilm. It would be helpful to verify that your B. subtilis is correctly identified, but this would be a side issue. This experiment would definitely be a good background experiment for your future projects

Do not worry. You are definitely not an annoyance. I just want you to be able to complete a successful science fair project. The next step is to review your question and hypothesis again. Then write the materials and methods section, and then write a detailed protocol.

Donna Hardy

Since the actual fair is March 24, and we have another holiday at that time, (and March 24 is in the middle of it), I'm thinking that I can do some work then -- if it won't bother my science teacher. Well, chances are, by that time I should be working on the board.

SO we're looking at about 1.5 months.

As for the spectrophotometer and the microscope with the cover slip camera (cool! That's how they take the cool pictures! xD) ... if they are things that are normally found in a lab, I think so =P. I'll find out on Wednesday -- I thought our school didn't have much (not saying that it has a lot), but appears to surprise me every now and then. Unless a centrifuge is a "common lab material"?

Why don’t you do a very straightforward experiment and find out if your E. coli and B. subtilis produce biofilms and find out how long it takes?

I agree . I should conduct more straightforward experiments xD.

"You just need to grow up broth cultures overnight and dilute them 1:100 to inoculate them into the test medium and incubate them."

>> Quick question: Why is dilution required? (Unless there would be too many bacteria to start with if we inoculate it straight into the test medium?)

This is perfect! Just to be sure, this is the Microtiter Plate Biofilm Assay right? (I just searched up microtiter -- never knew those were called that! xD)

I could test for the independent variable time, and if I have the time to fit it in, keep time constant and on the 4th day see the effect of different media on biofilm production ... Oooh!

"If you include any other independent variables other than time, perhaps you could include extra samples for the 4 day data point on B. subtilis, and try using 80% ethanol/20% acetone, which is recommended for solubilization of E. coli biofilms to 100% DMSO, which is a recommendation for one of the Gram-positive organisms on the list (from the biofilm protocol)."

I briefly know what the solubilization of e. coli biofilm involves, but I'm wondering -- what's the purpose of solubilizing e. coli biofilm? Would it one of the "organic solvents" that can dissolve the biofilm while other normally very "harmful" peroxides and other chemicals won't dissolve it? (cool)

About the growth curve experiment -- I really want to somehow involve the growth curve experiment...But if I do include it, I probably wouldn't be able to do this.

Since I have a bit longer than a month left (and I am definitely leaving things to the last minute when I say that I'm hoping to finish the board in the holiday), is it possible that I see what I can finish in the growth curve experiment? It's all the "real stuff" I've done, and I kind of don't want to throw it away. A bonus would be somehow relating this biofilm experiment to depend on the results from the growth curve, this way (if everything works out -- but then again, I'm usually not realistic with timing and science experiments, xD) I wouldn't have two separate experiments on the board.

I have no idea of time, so there are definitely things I want to do but I'm not sure if it can happen.

In the meanwhile, I might first type up my research plan for both. For the growth curve experiments,

Would a hypothesis of "The temperature that produces the highest amount of viable organisms is the same as the temperature that yields the lowest doubling time during the log phase" an 'OK' hypothesis'?"

>> I've seen many many references saying how e. coli's optimal temperature is 37, and that it yields the lowest doubling time -- but does this mean that the exponential growth lasts the longest? Would it be at all possible that one temperature yields the lowest doubling time, but that fast exponential growth only lasts for a shorter amount of time, while another temperature yields a longer doubling time, but it lasts longer?

Hmm... but it wouldn't make senses since if the latter temperature yields a longer doubling time, wouldn't that mean that it isn't optimal for growth, and therefore wouldn't last longer? Unless there is something during e. coli exponential growth that after it's doubling time is too little, it overworks itself?

That's my reasoning for the above hypothesis, but I couldn't find any similar experiments. ... Is the experiment results "that obvious"? (But sometimes I feel bad when I leave things unfinished -- that's assuming that I have truly "started" on this experiment) ... >> From your experience, do you think I should go straight to the biofilm?

I think the "are these two temperatures which yield different things, the 'same one'?" is one of those...things that "make a question" out of something that people don't really care about, though from what I understand, it is not known whether to not the temperatures would yield the same thing and I want to find out. (Is that usually enough to get a sci proj going? =P) .. Unless the "it is not known whether or not these temperatures are the same" is not true? o=.

As for not having pure AI-2 (I know bringing back the AI-2 project probably wouldn't be realistic) -- is it true that if the only common signal b. subtilis and e. coli can understand is AI-2, would that still mean that we would need a pure AI-2 sample? >>Then again, even if that's true, we don't know what AI-2 to b. subtilis does .

Since right now I don't know which project or hopefully projects to pursue, I'm going to brainstorm for hypothesis

>> Please don't read through all of these! I've just compiled a list for hypothesizing --

If we're going to keep time as the independent variable and measure biofilm formation over period of 6 days, I'm not sure how I can form a hypothesis -- if it involves numbers, this means taking directly from a previous similar experient right?

I think the hypothesis for the sugar/non-sugar media is straightforward

Just to make sure -- a pellicle is a type of biofilm, or is it "the type" of biofilm formed by b. subtilis?

Useful
More on b. subtilis biofilm formation
About an endospore -- is it the same thing as a biofilm? (I have a feeling no)
This one is a bit confusing -- I thought the title implied that the experiment would discuss the materials required for "normal" biofilm production in b. subtilis, but it involves mutant strains which I think we do not need...so what is this article saying?

E. coli biofilm formation there is the old article here
As well as this one.

I like the topic of biofilms because it's very related to quorum sensing ^^. And biofilms are a serious problem and if there was only some way to inhibit QS so that we can inhibit biofilm formation...

Hi Trader,

You have just enough time to get this project done/ You should plan to set it up at least twice. Do a small trial run the first time, and then a definitive experiment after you have learned the technique.

On Wednesday, let me know what equipment you do have available. With this experiment, you can improvise if the exact equipment is not available.

With a timed experiment, you want to start with the same population in each tube or well, and have all of the bacteria growing at the same rate. It’s kind of like a race. If you transfer from a colony growing on a plate, you won’t be able to inoculate a uniform number of bacteria into each sample, and the bacteria will be in stationary to death phase, so will have a long and possibly variable lag time. If you grow up a fresh culture overnight, it will be actively growing and you will transfer a uniform amount to each sample, and lag time will be minimal and more uniform. You’ll be able to repeat the experiment and get the same results. And someone following your method would be able to reproduce your results.

In the biofilm assay, the bacteria are grown in a small well or tube and at first the bacteria are free-swimming. After a few days after the quorum sensing molecules are released, the gene expression will be altered, and the bacteria will adhere to the surface and form the biofilm. Next, crystal violet is added to the well that may have a biofilm; the crystal violet will bind to the bacterial cell walls. Then, the free-swimming bacteria and non-bound crystal violet are rinsed from the wells. After the rinsing step, the only crystal violet left in the well bound to the bacteria that form the biofilm. The crystal violet is then extracted from the cell wall with ethanol/acetone. The quantity of the purple crystal violet is proportional to the number of bacteria that are in the biofilm.

If you complete the biofilm experiments with time to spare, you can then do the growth curve studies to complement the biofilm experiments. The biofilm experiments are more unique than the growth curves in a science fair project. Your growth curve hypothesis is good.

A short doubling time will lead to a faster exponential growth curve because the maximum number of bacteria possible is limited. A longer doubling or generation time will give a longer exponential growth curve.

If you had pure AI-2, you could test its effect on B. subtilis if you had an activity to measure. If your B. subtilis produces a biofilm, that would be something that you could test and obtain a measurable results.

A pellicle is a thick layer of cells that forms on the surface of a broth culture and is not a biofilm. It is characteristic of Bacillus species because they are aerobic, so grow better where there is more oxygen. A biofilm is a layer of bacteria that attach to a surface and grow. According to the article you referenced, the ability to form a biofilm is a characteristic of bacteria growing in a natural environment and is associated with the production of secondary metabolites, such as antibiotics.

An endospore is a dormant structure that is formed by Bacillus species when nutrients in the growth medium become depleted. The spores are resistant to heat and drying, and allow the bacterium to survive unfavorable conditions for long periods of time. The cultures that you have growing at school will contain endospores. The spores have a round or oval shape compared to the long, thin rod shape of the vegetative cells. Here is some more information with pictures:

http://advancespacemonitor.com/endospores.pdf

http://www.google.com/imgres?imgurl=htt ... hl=en&usg=

I hope this was enough explanation for all of the terms, but let me know if anything is not clear.

Yes, I think you should go straight to the biofilm experiments and do the growth curve experiments later.


Donna Hardy

Thank you for the explanation! So it looks like I will be measuring the biofilm growth in each day, and comparing this between e. coli and b. subtilis. I wonder if there is a difference between "optimal" growth for biofilm and optimal temperatures for growth?

(One can see that I am just a bit reluctant to give up my growth curve experiment =P)

For my hypothesis for the biofilm experiment, would it once again involve numbers, or can it be an answer to the above question (so that I can link it to the growth curve experiments?) -- though if so, that means that I will have to do growth curves before, and endanger my time for biofilm afterwards.

Aww... OK here's what I'm going to do. It looks like materials won't be a problem (75% sure that nutrient broth is available ... I had ordered it before the holiday, but I wonder if anything does deliver over the holiday?) -- tomorrow I will plan to make nutrient broth, and then I will check the b. subtilis, as well as inoculate new e. coli (hopefully this time it's viable! Though I do have ... 28 samples from our school's biology class =P).

-- If everything works out tomorrow, and IF I confirm that the individual colonies are "like b. subtilis" under the microscope, then would it be OK to "move on" by transferring the other half of the colony or doing a catalase test to make sure and/or incubating the nutrient broth overnight (without any bacteria) to test if there are contaminants in there?

Quick question: If one individual colony is verified to be b. subtilis, does doesn't mean the entire plate is b. subtilis right? (There could be contaminants in there, but that individual colony is b. subtilis?)

In that case, by "inoculating" the other half of the confirmed individual colony to a fresh agar plate, I am essentially using the loop, taking the sample and then tapping it on a fresh petri plate? (I think this isn't it because my "non-scientific" alarm is going off in my head =P)

Thanks! Time to start tomorrow...

Hi Trader,

The growth curve and biofilm experiments are both good and both are related to your larger quorum sensing project. However, I think you should do one of the experiments completely before doing the other. Six weeks sounds like a lot of time, but it’s not really when you are trying to get lab work done. Since you have already ordered the supplies for the growth curve, it would be fine to stick with the growth curve and delay the biofilm experiment. Just don’t try to do everything at once. I do apologize for encouraging you to switch at the last minute. It’s your project and you will be doing all of the work, so the final decision is definitely yours to make.

For the biofim experiment, you will plot the optical density measurements of the crystal violet solutions at 600 nm using the spectrophotometer vs. time. You will include control wells with no bacteria for a negative control that should start close to zero OD (optical density). The intensity of the crystal violet solutions should increase and be proportional to the number of bacteria in the biofilm. Biofilm bacteria will grow to a density of 10 to the 12th organisms/square cm, so this number of bacteria will adsorb a significant amount of crystal violet, which is then extracted into ethanol/acetone.

Can you find the source of the E. coli from your science class? You should be able to track the source and find out the name of the specific strain.

The entire plate should have colonies that have an identical appearance. If you see any variation in colony shape or color, then the culture is probably not pure. Yes, definitely to another Gram stain of the “B. subtilis” and see if endospores have formed in the vegetative cells. Endospores are resistant to staining, so will not be Gram positive purple. Do the catalase test and transfer the culture to a fresh agar plate.

Here is a picture of B. subtilis colonies from the science buddies website. Your colonies should match this appearance:

https://www.sciencebuddies.org/science- ... ates.shtml

Donna Hardy

I'll have to report back tomorrow <_< (sorry! ... especially when I need every day as the time to the science fair closes)

I really like it when you help me think of more original ideas! (it's my fault actually, because I was complaining about my project being unoriginal =P) -- I think I'll finish the growth curve experiment first. I know that if I do a cost-benefit analysis (=P who does that anyways, but...), doing the biofilm experiment would be better, but I just feel like I want to finish what I "started" -- thank you for understanding.

My hypothesis is pretty much set straight but I'll still continue planning for the biofilm experiment.

Is there any way to tie the biofilm experiment with the growth curve experiments, becuase I'm wondering if there is a difference between "optimal" growth for biofilm and optimal temperatures for growth? (I think there is actually, because it says to grow e. coli at 25 degrees C)

For my hypothesis for the biofilm experiment, would it once again involve numbers, or can it be an answer to the above question (so that I can link it to the growth curve experiments?) -- though if so, that means that I will have to do growth curves before, and endanger my time for biofilm afterwards.

Thank you so much for bearing with me.

Hi Trader,

I think you have made a good decision to start with the growth curves, and you will benefit from the experience of doing these. Do you have a detailed protocol written for doing the growth curve?

The growth curve experiments are definitely related to the biofilm experiments. E. coli and B. subtilis will grow through lag and log phase, and when the population density reaches a certain level, quorum sensing begins as AI-2 and ALH's are produced and this activates expression of genes, which change the phenotype of the culture. The cells go from just a phase where they grow and divide, to a phase where they produce biofilms and produce antimicrobial agents, for example. The change in phenotype gives the culture a survival advantage in the wild.

Your growth curve and biofilm experiments will tell you about the growth rate and the timing of quorum sensing of your specific cultures, and will provide the background for your future experiments.

Your hypothesis for the biofilm experiment could be the answer to one of these questions:

1. When do E. coli and B. subtilis start to produce biofilms?
2. What is the cell density in biofilms produced by E. coli and B. subtilis?
3. How long does it take to produce a complete biofilm
4. Do both E. coli and B. subtilis produce biofilms?

Does this give you any ideas for the biofilm hypothesis? There are lots of possibilities. What information about biofilms are you interested knowing about? Is there any question from your reading your references that you would like to answer? You have some time to think about this, and you need to find out if materials and equipment are available for this experiment. This consideration may limit the question you can ask about biofilms.

Best regards,


Donna Hardy

Yes .

(this weekend I have a tournament for debate >< -- which means AFTER this week, I will no longer have debate practice and will have 2-3 more free days every week!)

--> Nutrient broth was ordered over the holidays, but it seems like it has not arrived it for some reason...The lab assistant kindly offered to check it for me, but I have the formula to "make my own" nutrient broth.

As for the protocol, I've typed up to step #13, but I've also read over various protocols for dilution plating (which I think is so cool!!) and I think I can handle that part of it.

Wow! I've never thought about the hypothesis that way (I ... can't hypothesize =P). I'm sure I can get part of the answer for #1 and the whole of #4 as part of the growth curve experiments as a way to directly lead into my biofilm experiment.

A quick question -- is biofilm only "created" when the bacteria are in an environment that does not have optimal conditions? In this case since my growth curve tests the growth in optimal temperatures, would there be no biofilm?

There are a lot of questions regarding biofilm such the previous AI-2 experiment which links with "what does AI-2 control in b. subtilis?" If I somehow find out that it is biofilm formation, it would be cool to test the interference/battle b/w the creation of the biofilm (of course, that would have to take place later). Biofilm is especially cool because it makes the bacteria a lot deadlier which makes it more important to study them ^^. Quorum sensing's role in biofilm formation is another thing I want to approach someday...

BUT I definitely want to know how to understand the growth of biofilm before attempting to address all those questions ><.

As for the internship, I'm interested in a program offered by NIH -- it seems like the professors there would be choosing their students to be their intern and it would definitely help if I contact them directly and make them be aware of my application -- I was wondering which of the sections in the link above would "biofilm formation/inhibition of quorum sensing for immunology" would fit in? There are fancy terms I'm afraid I'm not sure about ... would it be OK if you briefly check to see which branch would be most appropriate?

I typed in "quorum sensing" in the search box, but it seems like there are currently no labs on it (which is a bit confusing and disappointing), but it'll be just as cool to research about biofilm formation, or search harder for quorum sensing .

Hi Trader,

Good luck on your debating tournament this week-end!

Homemade nutrient broth will be good to use. These are garden variety bacteria and will be happy with anything that you feed them. It's helpful to have a clear broth so you can see the turbidity of the culture as it growth. The broth will turn turbid when the bacteria reach levels of about 10 to the 7th per ml.

Biofilms are created in response to limited food and space, but I'm not sure when the process starts. That's what you will be able to find out with your experiments. Your growth curve will follow the growth curve from lag to stationary phase. The cultures will reach levels of 10 to the 9th to 10 to the 10th bacteria/ml at the beginning of stationary phase, so the biofilm production probably starts about that time. The biofilm experiments suggest 4-day incubation, so the biofilm production probably continues after the stationary phase. The two experiments will tie together very nicely.

I am not familiar with the organization of the NIH. I have written to several colleagues who might have some information on this topic, and I'll hopefully have some information for you by tomorrow. An internship at NIH would be a great opportunity for you. Were there any NIH authors on any of the papers you have read?

Donna Hardy

Hi Trader,

Here is the information I received on finding a researcher at the NIH:

"The website attached is for NIEHS - which is one of the branches in NIH - I am not sure if it's the right branch though? If he is looking at the pathogenicy of bacterial infections - I would actually recommend NIAID.

He might want to start here....http://www.nih.gov/icd/index.html

but I would definitely look into:http://www3.niaid.nih.gov/


NIAID is one of the best funded institutes at NIH - which doesn't hurt.

There's a division within NIAID - Laboratory of Human Bacterial Pathogenesis - might be a good place to start:

http://www3.niaid.nih.gov/labs/aboutlabs/lhbp/"

Unfortunately, neither of the contacts I have heard from has any personal contacts who are researching your topic, bute hopefully, this information will get you into the right section of NIH.


Donna Hardy

I'm back! My partner and I got second place ^^ -- it was awesome.

For the internship, I emailed the professor -- really what I want is research in the area, it'll be just as fun if it's really entirely biomedical

Based on the information in the references (and from your explanations ), it looks like that biofilm are a defense mechanism, and once they do become biofilms, they are much "deadlier". I'll base a hypothesis predicting the time that it'll create the biofilms -- probably at late stationary phase, when the conditions no longer are "optimal".

Just curious though -- IF they start producing biofilms, and then we transfer the bacteria to fresh cultures, would they "stop" making the biofilms? This should be interesting! xD

Though according to some research, it seems like they first need to attach themselves to semi-liquid or liquid surface, which means that that probably won't do.

What really worries me is how much my experiment is based on theory.

Hi Trader,

Congratulations on your second place win in the debate tournament! You have an amazing range of talents.

I'm hoping you will hear back from the professor about your internship. It may be a while before you hear anything, however, because the professor will be sorting through lots of applicants.

The biofilm phase of bacterial growth provides a survival mechanism for bacteria when growth conditions become less than ideal. Unfortunately, the successful survival of the bacteria can result in disease if the bacteria happen to be growing in a host.

That's an interesting question. If you had a test tube with a culture that had progressed to the biofilm-phase of growth, and is you then poured off the depleted nutrient broth, and added fresh, sterile medium, I'm sure that at least some of the bacteria would revert to the free swimming form and grow in the nutrient broth, but I don't know if all of the biofilm bacteria would disappear from the biofilm. That would be an interesting question to answer with an experiment sometime in the future.

Your growth curve and biofilm experiments are based on your extensively reading of the literature, and your hypothesis is a guess of what will happen. Scientists have a saying, “results are empirical,” which means that you really can’t answer a specific question until you do a controlled experiment. With experimental results, however, you can definitely answer your question. That’s how progress in science is made; one experiment at a time.


Donna Hardy

Cool! Thank you.

I used the oil immersion and found that the bacteria was rod-shaped, and did look like the b. subtilis pictures that you gave the link of. It looks like it's b. subtilis ^^.

I was looking at the petri plates, and I'm not sure if it's because of how long last time I saw them was, but it seemed like they looked more "faint", and not as "brown" as the pictures of the b. subtilis pictures in Sciencebuddies.

As I'm pretty sure that I have b. subtilis...should I incubate them (perhaps to make them "unfreeze", though I think they are in biofilms already by now? or...), or should I directly inoculate them to a fresh plate, incubate, streak plate once again for final confirmation, and then start preparing the liquid broth?

Thank you!

Hi Trader,

That's good news that you saw the long-thin rods under the microscope. I'm sure you have B. subtilis. Don't worry about the color, but can you confirm that the colony colony is opaque (not translucent) and kind of wrinkled? Are the edges of the colony irregular(wavy) or perfectly smooth (round)?

Since you are keeping your cultures on plates, you should do a new streak plate since it has been so long since you transferred the cultures. The B. subtilis will survive drying out because it will form spores. The E. coli will die if it is allowed to dry out. The day before you do the growth curve, you should transfer a colony from either an old or new plate and grow it overnight in a broth culture to start the culture for the growth curve. The overnight culture should be very turbid and will contain at least 1,000,000,000 bacteria per ml. You should dilute it to 1,000 to 10,000 per ml, or so to start the growth curve. Have you thought about how you will make this dilution?

Donna Hardy

The colonies are definitely not translucent -- they were somewhat dark, yet I could see some darker spots within each cell than others, that is opaque right? ^^

At X1000 magnification I can only see that they were rodshaped, and I believe their edges weren't "perfectly round" -- as for what it means to be "wrinkled", I wouldn't describe it as smooth. ^^

The previous cultures of b. subtilis looked a bit faint, and I inoculated a sample to a fresh agar plate anyways -- but I'm not sure if I did the technique right. Is it "OK" if I take a sample of one colony from a streak plate and make sure the material touches the agar plate surface of the fresh plate, and then incubating it overnight?

What worries me is how it appears to be very faint.

I've gathered the beef extract and everything for the nutrient broth, so it's looking like tomorrow I could assemble the nutrient broth, see how the new cultures are doing (unless...they aren't viable anymore?), and then create a culture of e. coli.

As for dilution plating, I am gathering most from what I currently understand from here: protocol

After I take one colony and incubate overnight, I would have approx. 1 billion bacteria per ml. That means that I should roughly do dilutions of 1:1000 and 1:10,000 (unless I'm getting the numbers wrong...) so I can make sure that there isn't "too much" bacteria per ml (that would be around 30-300 colonies).

I'm not too sure about the following steps -- I would then take the dilution, incubate it overnight (best with shaking so that the sample will be properly distributed and/or will grow better? -- unless I have completely messed up my understanding of nutrient broth), and then transfer it to plates to count?

>> I've briefly looked over your explanations for dilution plating and I hope I've understood well enough and apologize if I went over anything, but as for "counting" (after transferring the diluted sample of bacteria onto the plate), would that be pouring 1 ml of the diluted sample onto the plate to create a mini "lawn" of bacteria, and then literally counting the number of colonies that result?

Though... if it's a lawn, how can we count the number of colonies?

*-- Update -- I still haven't heard from the lab assistant on the order of the nutrient broth, though I know the order was placed a long time ago. I've checked for beef extract and peptone and it seems like those materials that would "make" a nutrient broth are not available...I will know the status of the order by tomorrow, but do you happen to know of any possible "substitute" for beef extract and peptone for nutrient broth? Yeast extract seems to be out too (I was hoping to make LB broth instead)...

--> Also, I do have a nice stock of agar plates, but was wondering if it would be "worth it" to incubate them at 35 C overnight just to see which ones have contaminants. Is this usually how it goes? After the plate is made, it is incubated (w/o any bacteria) to see if it is contaminated, then the ones that are are removed, and the ones that aren't are refrigerated while being stored in a ziplock bag?

Thank you very much! I think I might be able to get the preparation work done by Friday, and perhaps devote this Saturday on growth curves?