Bacteria [Communications]

[Trader]

Thank you so much!

Thank's for the link -- I checked it out, and its concepts are very relevant and challenging at the same time. It clarifies a lot haha I love their metaphors -- this makes me want to attempt to construct that sensor strain again =P

Thanks for explaining the stationary phase/lag phase "renewal" process! Yes that clarifies a lot thanks

I've really been trying to find someone (for the AEM or any microbiology society I've found, they are in the States and I live outside of the States...) -- if I do find someone in my area (Shanghai), I think the language barrier will still be an option because I'm part of an international school and those who work in the universities won't know English too well...

Which means I'll search for English-speaking microbiologists who happen to be living in Shanghai...I shall try harder =)

Ahh, and first school day after winter break -- My science teacher is recommending that I should base a large part (if not all) in determining the optimal growth conditions for e. coli and b. subtilis (incl. effects of temperature) and determine the timetable of growth to prepare for phase II which uses the actual sensor strain later (or... I think "next year" is implied)

But I want to do more than that =P but I'm not sure if time can allow it. As a result,

I would still have my "large" research question with the QS v. distance question as part of the research question that I hand in before experimentation, and then a "mini-question" below it for determining the time table for b. subtilis right?

It's somewhat of a minor question now that I think about it, but it would be nice to have a better idea of what is expected

It really depends on what I can fit in...If I can get the answer to this, I'll continue down the path of AHL v. distance (?), but the idea of using b. subtilis sensor strains is very interesting.

Good news is, I've already got "everything" ready. Meaning b. subtilis, e. coli (my science teacher nicely resent another email for v. fischeri, but no responses yet), nothing for "special TLC", but I'll attempt to do "normal" TLC to confirm my findings with the sensor strain which means I'll attempt to find the optimal growth conditions asap!

There's also something that I want to ask -- as I have never touched microbiology tools before and have almost no idea of how long it would take,

In your experience, would it be practical to attempt to have an experiment running at the same time attempting to create a sensor strain exposing it to various mutagens that repress LuxS genes (for example for AHL that it'll eventually lose its ability to produce AHL? I'm not sure if my e. coli has that, so I'll have to test for that)?

I guess it is somewhat related to the project as I could develop understanding of the use of sensor strains, both using the timing method and the exposure to mutagens and find some way to "make your own sensor strain"! So that there will be one less of a "materials" problem when doing microbio projects. It is really interesting but I'm just not sure if it's practical

But it looks like I won't have access to any kind of a "proper" sensor strain, so I'll be seeing if the sensor strain "timing" method works (the e. coli and b. subtilis I got were actually dug up from an old school supply hidden in some storage room...in Shanghai, it's quite hard to get things because...I guess science fairs aren't that common? )=)

Thank you very much for explaining the dilution plating method! Sorry, the question below may seem a bit obvious but I was a bit confused as you mentioned both liquid broth and agar plates (which I thought were two different mediums for different purposes)

Just to confirm, I think you mentioned "A broth culture that is visibly cloudy w/ bacteria will contain more than 10 million organisms per ml..." would dilution plating with agar plated (no liquid broth at all) bacteria? (Or...should I be growing the bacteria in liquid broth?

I think I know that "Agar plates are used to detect the presence of AHL’s; broth cultures are used to identify the specific AHL" -- and that I should be using agar plates as I'm only planning to detect the presence of AHLs if I get that far...but then would that mean dilution plating won't apply? O= Sorry if I'm confusing this with "you can count plates that contain between 30 to 300 colonies" because I immediately associate "plates" with agar plates and assume that is not liquid broth...which makes me even more confused sorry!

>> I can't believe I'm actually STARTING! (after 61 posts of the experts' help! Thank you!)

[donnahardy2]

Hi Trader,

Your English is so American that I would never have guessed you were in Shanghai. I apologize for just assuming you were in the US. There is an official microbiology society in Shanghai and also one in China. Here are the websites with the contact information in English. English is used universally in scientific circles, and many Chinese scientists are fluent in English, so you may be able to find someone who can communicate with you in English. Unfortunately, they don't have a webpage of volunteers who are available to help, so you may need to learn more about the local custom in asking for help like this:

http://www.sast.gov.cn/en/1219825907d8516.html

http://www.im.ac.cn/en/new/_s.php

I have a coworker who is from Shanghai, and, although she says most of her former colleagues are plant biologists, she has promised to ask for the name of a local microbiologist who might be able to provide you with assistance.

Your teacher has an excellent suggestion and is trying to help you with a project that will help you learn the techniques you will need in the future, and will give you a project that you can actually complete this year. Of course, if a sensor strain becomes available, you can do the experiments that you really want to do. Your teacher is really trying to help you, so please be patient and spend this year learning the techniques. It will really pay off in the long run.

Do you have a particular strain of E. coli? Where did it come from? Unless it has been genetically modified already, it should produce AHL's. Do you have the strain that comes with the green fluorescent protein plasmid in an educational test kit? Or, are they colorless normal varieties? If you dug them up from a storage room, have you tried growing them up to make sure they are still viable?

It seems to me that designing experiments to optimize the growth conditions for E. coli and B. subtilis would be separate from the experiments to try to induce an AHL-negative mutation. And, you can't do the mutation experiment unless you have an identifiable characteristic that will disappear when the mutation occurs. And, you can't do the TLC without an AHL-negative sensor strain. So, let's think more about this before your start. If you can't answer the question about the E. coli strain, I might be able to suggest something.

Bacterial cultures are grown on agar plates and in broth. Normally for inoculating the agar overlay used in TLC, the culture would be grown in a broth culture and then diluted in agar for the overlay. When a culture is grown on agar, you can't really quantitate the number of viable organisms because each colony will have a combination of live and dead cells; by the time a culture appear on the solid agar, it will be in stationary phase, which is after the AHL's appear. Using the broth culture allows the researcher to identify the growth phase, which is critical for AHL studies. I’ll explain more about this

I can't believe you are starting also. This is exciting. You are going to learn a lot even though you don't have a sensor strain yet.

Donna Hardy

[Trader]

Haha...well I'm in a Shanghai American School, so it's almost as if I'm in the States =P. No problem --

Thank you so much for the pages! I was Googling in "Shanghai science fair mentor" and didn't get any results that way -- this would be extremely helpful!

Thank you for asking your coworker to do that!! Also, if you can, tell her I really appreciate the help

I think it's right to take on an experiment that would be manageable and make that lead to phase II. Hopefully, after finding out the optimal conditions, if I have time ... I'll find out whether AI-2, proposed as a "universal signal" in bacteria, would work between b. subtilis and e. coli.

For now then, I think I'll find the optimal conditions for growth for the e. coli and b. subtilis so that I can properly culture them later (together) when I may be using e. coli as a sensor strain...

I'm still attempting to check out whether the "sensor strain timing method" (I think you mentioned it's "doable" but I might not see the results...would it be practical to attempt to get the timing as right as possible then see what results there might be? -- Sorry that I'm poking at a lot of ideas when I think I should be focusing on finding the optimal conditions for e. coli and b. subtilis so that I can culture them together for phase II and use a sensor strain with them.

I am really not sure about the e. coli as from what I can see, there is about a 5 cm diameter cylinder tube that's about 7 cm long and has a cotton (very short) cylinder swab -- the instructions said to put distilled water in it and put the plug in and let it "unfreeze" (though it wasn't "frozen", -- dormant?) for about one day --

Just to double check -- After the bacteria is brought out of its 'dormant' stage, it can go directly into the broth right?

My science teacher recommended "reviving" the bacteria (bringing it out of the frozen state?) with sterile water so we are trying that right now. After that perhaps I should observe (through liquid broth) whether it is colorless e. coli or with gfp.

Thanks for clarifying the agar plate v. liquid broth question and all the concepts =P. With your help I eliminated a lot of definitely unworkable research questions.

Would it be possible to combine two independent bacteria strains (one in b. subtilis and one in e. coli) but with the test strain in the log phase and sensor strain in the stationary phase in a liquid broth?

I guess another way to ask it is -- in what cases would one prefer an agar plate over liquid broth culturing method?

Thanks for everything!

Report "from the lab" (xD) -- first attempt at sterilizing water didn't go so well (didn't set up properly =P) -- attempt #2 soon! But the autoclave and the concept is so cool --

Attempt #2 is great. Tomorrow it's time to get the nutrient agar/broth set up and hopefully start growing the bac.

[donnahardy2]

Haha...well I'm in a Shanghai American School, so it's almost as if I'm in the States =P. No problem --

Thank you so much for the pages! I was Googling in "Shanghai science fair mentor" and didn't get any results that way -- this would be extremely helpful!

Thank you for asking your coworker to do that!! Also, if you can, tell her I really appreciate the help

I think it's right to take on an experiment that would be manageable and make that lead to phase II. Hopefully, after finding out the optimal conditions, if I have time ... I'll find out whether AI-2, proposed as a "universal signal" in bacteria, would work between b. subtilis and e. coli.

For now then, I think I'll find the optimal conditions for growth for the e. coli and b. subtilis so that I can properly culture them later (together) when I may be using e. coli as a sensor strain...

I'm still attempting to check out whether the "sensor strain timing method" (I think you mentioned it's "doable" but I might not see the results...would it be practical to attempt to get the timing as right as possible then see what results there might be? -- Sorry that I'm poking at a lot of ideas when I think I should be focusing on finding the optimal conditions for e. coli and b. subtilis so that I can culture them together for phase II and use a sensor strain with them.

I am really not sure about the e. coli as from what I can see, there is about a 5 cm diameter cylinder tube that's about 7 cm long and has a cotton (very short) cylinder swab -- the instructions said to put distilled water in it and put the plug in and let it "unfreeze" (though it wasn't "frozen", -- dormant?) for about one day --

Just to double check -- After the bacteria is brought out of its 'dormant' stage, it can go directly into the broth right?

My science teacher recommended "reviving" the bacteria (bringing it out of the frozen state?) with sterile water so we are trying that right now. After that perhaps I should observe (through liquid broth) whether it is colorless e. coli or with gfp.

Thanks for clarifying the agar plate v. liquid broth question and all the concepts =P. With your help I eliminated a lot of definitely unworkable research questions.

Would it be possible to combine two independent bacteria strains (one in b. subtilis and one in e. coli) but with the test strain in the log phase and sensor strain in the stationary phase in a liquid broth?

I guess another way to ask it is -- in what cases would one prefer an agar plate over liquid broth culturing method?

Thanks for everything!

Report "from the lab" (xD) -- first attempt at sterilizing water didn't go so well (didn't set up properly =P) -- attempt #2 soon! But the autoclave and the concept is so cool --

Attempt #2 is great. Tomorrow it's time to get the nutrient agar/broth set up and hopefully start growing the bac.

[donnahardy2]

Hi Trader,

It sounds like you have lyophilized (freeze-dried) vials of each culture. You should sterilize the outside of the vial with 70% ethanol, let it dry, and break it open reconstitute the vials in sterile nutrient broth and incubate the cultures at 37 degrees Centigrade. Or, if you already reconstituted in water, transfer the sterile water to nutrient broth. The broth should turn cloudy within a day or two. You should then check the cultures to make sure you have the correct organisms. Do a streak plate of each culture on an agar plate and verify that the colonies are identical.

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

You should select individual colonies to transfer to agar slants in test tubes to maintain your cultures. If possible, do a Gram stain on each culture to verify the E. coli is Gram-negative (red) and the B. subtilis is Gram positive (purple). As I recall, E. coli colonies are translucent while and shiny; B subtilis colonies are white, and become wrinkled with age when the spores start to form. If your streak plate shows a variety of different colony types, then you will suspect that the original culture has been contaminated.

You should store your cultures on the slant tubes for up to a month in the refrigerator. Don’t let your original cultures dry out, otherwise they might die.

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

Now, how are your going to determine conditions for optimum growth? One way to do this is to transfer about 1000 log-phase organisms per ml to fresh nutrient broth and then do a plate count periodically during log phase to determine the time required for doubling the population. Impedance measurements have been done to measure bacterial growth and the impedance of the medium will start to change when the population increasing to about 10,000,000 organisms per ml. Or, you can grow the organisms for a certain time, and then centrifuge the cell pellet and measure the volume or weight of the cells grown in a certain volume of medium. Or, you can measure the time required to reach certain turbidity, although using turbidity measurements have a limited range. Or, you could measure total protein using a general protein assay technique like the Bradford protein assay. Did you have any other ideas?

Donna Hardy

[Trader]

WOW...xD

Thank you so much for outlining the procedure for me ... I feel somewhat like a disappointment as I am still learning the most basic of microbio techniques =P

Sadly, in my attempt to make sterile water (for the nutrient broth), I took a total of 3 days -- this is very interesting, because now I'm finally seeing how long things actually take.

Thank you for the links! This would be something I'd want to have right next to me in the lab as I go through the whole thing. Coool.

I think I'm going to use the dilution plating method (is this the first technique mentioned?) to take a weekend off and count the bacteria hourly (especially during the log and stationary phase). The centrifuge idea is cool (I was so excited [excuse me =P] when I saw a microcentrifuge in our lab storage supply! Or are those one of the 'common' things that a lab supply would have?) but I was wondering -- would the weight measurement technique include the dead cells and be somewhat inaccurate? (Though if it is log phase, I think this should be okay). I could use all these techniques and confirm my findings.

And the Bradford protein assay -- That would be very cool! Since it measures amino acids, that would include QS signals right? Though amino acids that are non-QS are also produced, so I wouldn't be able to detect QS that way, but it would be important in determining the protein composition optimum for bacteria

I'm planning to first set up a control at 35 degrees Celsius (I happened to find one report that reported growth there -- when determining questions such as "optimal temperatures", is this usually how a control is set up? Picking a proven temperature and varying it higher and lower to see which has higher growth? -- thanks

I'll focus on finding the optimum temperature, protein amount and pH for now because I kind of want to work towards using one or both as a sensor strain after this phase I ... this would mean that perhaps both strains would be in one liquid broth (?) at the same time, and understanding the difference in optimum temperature, protein amount and pH would be useful in predicting the growth rate. (A possible RQ in the later stage might be, can e. coli detect possible AI-2 signals from b. subtilis? [will depend on whether or not the e. coli I have is in fact a "sensor strain" in the sense that it can't produce AHL or AI-2])

This reminds me:

Sorry if I skipped over the answer in some of the previous posts, but it would be practical to attempt the "timing" sensor strain method (after getting the timing as close as possible i.e. through determining the optimal temperature above) to see if e. coli would pick up any signal from b. subtilis?

Thanks.

Of course, I've definitely underestimated the length (and difficulty) of microbiology procedures before (which makes the whole experiment even cooler) that I am not sure if I might even get to the "substitute" sensor strain or if it'll even work. But I believe you mentioned it was "doable", but that the results might not be clear? Would making the timing as accurate as possible and perhaps examining a culture under the microscope partly solve the problem? (I would first check to see what the features of the e. coli strain I received are -- then I'll be looking for something such as cell division [controlled by QS])

Thank you for your patience in this -- I can't wait to get the sterilized water done, then get the nutrient agar/broth ready and cultivated (THANK YOU again for the method for culturing and storing bacteria) and THEN start finding the optimum conditions for growth, determine the time table, then see if I have time left .

[donnahardy2]

Hi Trader,

You are very welcome. I'm happy that my advice is helping you in this project. I'm also glad that your finding out how much time and work is required for this project has not affected your enthusiasm. Once you have done the techniques a few times, everything will become much easier. Even the simplest tasks seem to be challenging when you first try them.

The plate count technique with dilutions is a good choice if you are willing to do the work. You will want to start out with a broth culture that has been inoculated with a very small amount of culture and grown overnight. This culture will have between 10,000,000 and 100,000,000 bacteria per ml. If you dilute it down to 100 to 1000 organisms per ml and start the growth cycle again at 35 degrees Centigrade, the lag phase will be very short, probably an hour or so, and then the log phase will start and your cultures will double in number every 20-25 minutes. You would want to plate enough dilutions to get a countable (30 to 300 colony) plate for each sample, so probably your target number, plus or minus a dilution or two.

For this experiment, you would want to include a one plate that is not inoculated as a negative control to make sure your agar is sterile and that the colonies present are from your sample. Officially, you would want to include a positive control to make sure that your agar was made properly and would support the growth of microorganisms, but if you get growth in all of your samples, you can assume that your medium was OK.

For your growth conditions, starting with the published optimum temperatures would be the best place to start and 35 degrees Centigrade is a good temperature to start with. Generally, when you get results of one experiment, you will be able to think of 10 more experiments that would be nice to do. The key to making progress is to pick the best experiment to do next.

I'm not sure if you can develop a substitute sensor strain with this technique. You will have to see. I have not seen a reference that indicates that cell division is controlled by QS. Most of the references report that the AHL's are produced after the population reaches a certain density. So, I don't know what characteristic you would look for in an ordinary strain of E. coli. That's what so nice about the visible colors produced by the sensor strains.

I am assuming that you still haven't had a response to your e-mailed inquiries to the various authors?

Donna Hardy

[Trader]

Thank you for your clear explanations! I think I will have future experiments to test (and if time allows, I'll want to experiment more! I'm glad I'm just a sophomore, though I wish I knew that there such a thing as a science fair last year =P) and continue this project . Just in case I don't have any other experiment (though I doubt it... yet I kind of want to pursue this RQ too), I'll see if the substitution works.

On a side note regarding my enthusiasm =P -- Sometime before I was about to give up...but I don't know, quorum sensing kept me going =P. As for the "longer than expected"... that's definitely keeping me going! Because I know I'm moving to the fun part... -- I may not be experimenting w/ quorum sensing now, but that's the hope for phase II

I have a somewhat irrelevant question -- IF (I doubt it, but...) I have "reasonable" time to pursue another research question after I answered my first research question ...

I would create another research plan meaning that one year (in my case, 2 month =P) science fair projects may or usually do contain more than 1 research plan right?

Also, as the "Phase I" will involve finding the optimal conditions for e. coli and b. subtilis, I've been writing up my research plan and was wondering how I might formulate a hypothesis from it...? I've seen references support 35 and 37 degrees Celsius, though they were grown in different agar (I'll check to see which one is nutrient agar),

But in this experiment would a hypothesis with a research question "what is the optimum temperature for growth of e. coli and b. subtilis?" include an actual specific number where if the optimum temperature obtained is in fact 35, then I've supported the hypothesis, and if it is one lower or one higher, the hypothesis is incorrect?"

Sorry I'm not too familiar with hypothesis forming, and only have the school instructions of making an "If...then..." statement involving qualitative results only )=.

For the cell division/QS regulation -- I (now I think falsely?) presumed that QS did control cell division in articles such as this one and some other similar ones that I've come across. AI-2 does control biofilm production and I could test for that. I could indicate the presence of biofilm as the presence of AI-2 ... where how "strong" the biofilm (perhaps under different pH?) is is an indicator of the relative levels of AI-2.

I think the AHLs (and quorum sensing signals in general, I think?) are usually produced during the log phase (would there be any way to detect the presence of QS signals without a sensor strain? ) and there is minimal AHL production and high "consumption" of AHLs and AI-2 (I'm not sure about the other QS signals, could be another something to research are maximum at the stationary phase. At least this applies to e. coli -- let me dig up the reference and update soon.

Perhaps I inaccurately described quorum sensing to be the reason behind why any bacteria strain would stop growing when it reaches the edge of a plate? (Now that I'm thinking about it, could it possibly due to nutrient depletion instead?)

I would definitely want v. fischeri, but the supplier hasn't replied...some authors have replied but with answers of some concept questions related to their work I was interested but I'll write a reply to see if a sample of v. fischeri/v. harveyi is might be availanle. Or c. violaceum.

But somehow I also think e. coli will work because I've seen it being used as a sensor strain somewhere...? Unless that was a genetically modified one -- I have yet to find out about the traits of my e. coli...

My emails to the microbiology societies in Shanghai received no reply yet...I'll telephone them on the weekend -- it would be great if they have v. fischeri!

I shall finally get started on making the agar and broth -- just to confirm -- agar slants are a separate culturing media type from agar and broth right? I'll need to start making those too...

Thank you for everything -- from the advice to the responses to my questions to the patience with explaining all the methods =)

[donnahardy2]

Hi Trader,

Typically, because of their limited time span, science fair projects include only one experiment. However, a research plan presented in a science fair could certainly include results of more than one type of experiment. There is no limitation as long as you follow the guidelines of the science fair you are entering. It would be useful to get a list of the rules and make sure that your presentation complies with all details. In helping science fair project students, I sometimes suggest to start by planning an outline the presentation board including all of the steps, and then work backwards to make sure your experiment includes everything you will need.

For a project on determining the optimum conditions for growth of E. coli and B. subtilis, you will need to formulate a hypothesis, or prediction of what you will work. Here is the science buddies website information for developing your hypothesis and designing your experiment.

http://sciencebuddies.com/science-fair- ... esis.shtml

http://sciencebuddies.com/science-fair- ... dure.shtml

You will be doing a baseline experiment of growing the bacteria in nutrient medium at 35 degrees Centigrade. Then you will need to do experiments to compare the baseline results with media or temperature, varying one condition at a time. If you are doing growth curves, you will find out how long it takes for the population to double in number during log phase, so you will be trying for the shortest possible generation time. Or, if your growth curves go through the stationary phase, you will be looking for the maximum number of viable organisms produced under each condition. You will define what you mean by optimum growth with your experiment.

Here is an example of an experiment which determined the optimum growth for recombinant E. coli in a broth culture. The authors evaluated their results bases on total weight of the E. coli and amount of the desired recombinant protein as measure by specific activity.

http://www.athenaes.com/tech_brief_optimum_media.php

This study was done by a company and is not a published paper, but it is a good example of this type of experiment.

QS controls the expression of certain genes after a bacterial population has reached a certain population density. The biofilm references I saw were done on organisms that grow in water that drains from mines that are successful because they can attach to the surface of rocks. The bacteria produce the confluent growth or biofilm due to the presence of A1-2. E. coli doesn’t grow in acid mine water so would not exhibit this activity. E. coli is an intestinal bacterium and it swims around freely in solution. The Canadian students used E. coli that had been modified with green fluorescent protein, so this would be a good strain to get if you could find it because the GFP is A1-2 induced, like in V. harveyii.

I hope your E. coli and B. subtilis grow up soon. When did you inoculate them into the broth? If there’s no growth after 3-4 days, then I’m afraid you will need to start looking for new cultures.

I think phoning the local microbiology society is a good idea. Microbiology societies are staffed by volunteers, so there is not always someone available. If you can reach someone, I’m sure you will get a helpful referral.


Donna Hardy

[Trader]

Thank you for the reference... the materials and methods are particularly useful!

I've never thought about the hypothesis from that perspective before! Sorry for not searching Sciencebuddies.org well enough before asking you in terms of hypothesis forming, my bad. )=

I just finished pouring the LB agar (so COOL! It hardened just like that!! Ok, clearly I can see that I am more of a theory person and not of a "DIY" person) but didn't have time to make the broth or slants. I didn't even reconstitute the bacteria yet )= sorry I feel like I am moving through slower than expected...

I would definitely want to find which temperature produces the highest amount of viable organisms by the stationary phase -- though, I was wondering

In what ways might the organisms resulting from a "lowest doubling time" optimum temperature be "unviable"?

There is also another concept question I am not too sure about -- since death phase occurs when the nutrients in the agar plate is depleted...

Does that mean that a thicker agar layer would mean that the bacteria would "last longer"?

I CAN'T WAIT TO GET STARTED! Though I should also continue searching for that v. fischeri... but it'll pay off in the end

THANK YOU!

P.S. -- Am I somewhat "slow" for a sophomore in terms of knowing the concepts? I sometimes feel bad for asking questions that you give references to because I know that I could have searched harder and/or searched with the right keywords in Google and went to the link myself... =)

[donnahardy2]

Hi Trader,

In doing a plate count, your can either measure the generation time required for the population to double, or measure the maximum number of organisms present. During lag and log phase, presumably all of the microorganisms are viable. At the end of log phase, when space and nutrient become limited, and waste products (like acid) are accumulating, the population will stop increasing and the population stops dividing and enters stationary phase. The viable population is then stable until the start of death phase, when the organisms start dying off. If you compare generation times at different temperatures, you might have one population that doubles in 20 minutes and one population that doubles in 25 minutes. The shortest generation time that you can obtain could be interpreted as the optimum growth conditions. Or, as you are thinking, the population that reaches the maximum number might be considered optimum. I think you should keep your options open because there might not be a difference in generation time or maximum number with different conditions. You might also consider centrifuging the broth culture and measuring the total volume of the cell pellet per ml of broth to get another measurement. I think you will understand this better after you have done an experiment.

The bacteria will live longer on the agar plate if it doesn’t dry out, so a thicker plate of agar could be slightly better. To maintain your cultures, however, you should not count on agar plates for the long term. Definitely make a number of slant tubes, transfer your culture every week to keep them viable, and store them in the refrigerator after they have grown up.

How many agar plates did you make? One good way to do a plate count is to pipet dilutions of your culture that you want to grow into an empty Petri dish, and then pour in prepared liquid agar that has been cooled to about 45 degrees Centigrade. You then gently swirl the agar plates a few time to thoroughly mix the sample, and then let the agar solidify before you turn the plate upside down for incubation. This traps the individual bacteria in the agar and makes the colonies easier to count. If you use solid agar, you have to use something like a sterile swab to spread the sample around on the surface of the agar. Either technique will work, so you should try both options to see what works better for you.

Here’s a standard method for doing a plate count on food products that has lots of good details included.

http://www.foodsafety.gov/~ebam/bam-3.html

You are definitely a thinker and not slow at all. There are so many things to think about with a science fair project, and especially an ambitious one like this. It may seem difficult the first time you go through the process, but after the first year, you will find that a science project will be a lot easier because you will have experience and knowledge to build on. For a long-term plan, I definitely think you should consider getting your PhD in microbiology or related field and plan on becoming a research director eventually. Research directors have to do lots of creative thinking, and that seems to be one of your major talents. However, one step at a time, and for the present, you need to concentrate on getting your project done without all of the resources you know you need and learning the microbiological techniques. You have your teacher and science buddies as a resource for advice and information, and hopefully an AHL-knowledgeable microbiologist will be available to provide specific advice sometime in the future.

Lab work takes more time that thinking, so your progress is normal. Just do one thing at a time as you can.

Donna Hardy

[Trader]

Cool -- thanks for the confidence boost.

Thank you for the very detailed explanation! I'll keep that in mind when I form my hypothesis for the research plan.

Since bacteria won't die if the agar plates are thicker,... (and sorry if I accidentally skipped over the answer) but...

Would that mean that I should pour the plates to a constant thickness of agar so that the nutrients depleted by the bacteria are constant (and therefore another petri plate won't show different death phase times b/c it had more nutrients to live on?

I made 20 LB agar plates and about 8 nutrient agar plates as a back up just in case LB does not work ... I have yet to test everything out )=. I'll definitely start making the slant tubes...

Interesting! I'm afraid the descriptions won't make sense until I actually test them out in the lab (I still have loose definitions of what the difference between agar plates and agar slants -- wouldn't it just be the shape of the container they are in? Though it is best for me to find out, I think I have asked you enough )

Thank you for the methodology in everything! I guess my progress isn't slow, but I feel that I could work harder. Hopefully I can pick up the pace after I've learned about everything . Your explanations really help because right now I feel that I can picture every step until I find myself with the optimum temperature, and then I'll see what I should continue to do if there is time.

[deleted-71490]

This will help you visualize a plate and slant culture.

An agar plate is a standard petri dish with the surface of the medium parallel to the bottom of the dish.

An agar slant is normally a test tube or bottle with the surface of the medium at a 45 degree or so angle to the sides of the tube or bottle.

Matthew W. Mulanax

[donnahardy2]

Hi Trader,

Thanks to Matthew for a great explanation on plates and slants.

Plates and slants are used for different purposes. The agar plates are used for doing steak plates to verify your culture is pure and for doing plates counts. The plates dry out fairly rapidly and it's easy to get a contaminant on an agar plate when you take the lid off, so they are not good for maintaining a stock culture for a long period of time. The slant cultures are made by tilting a 16 to 18 mm test tube at a 45 degree angle while the agar is solidifying so there is a relatively large surface area of agar in the narrow test tube. Agar slants are good for maintaining your stock culture and storing it in a small space in the refrigerator. It's good if you have a screw capped test tube to make the agar slant in to keep the agar from drying out. It's OK if you don't have agar slant, you will just need to transfer your culture more frequently, and do steak plates to make sure your culture doesn't become contaminated.

Here’s a website with pictures that will help:

http://images.google.com/images?hl=en&q ... 1&ct=title


Donna Hardy

[Trader]

Thank you very much for the link.

I've just rehydrated the bacteria (it'll take another 2 days...in the meanwhile perhaps I'll start making nutrient slants)

I've poured the LB plates and stored them upside down, and I've left them under the UV sterilizer thing over the weekend (though I think that is only temporarily as my science teacher probably closed the light afterwards?) -- according to sciencebuddies, it would be OK to store them in room temperature?

Just to confirm my understanding of the difference between LB (for example) agar, slants and broth.

Both LB plates and LB slants can be used to identify the organism. Though this does make me wonder:

Besides for allowing a plate count, what advantages do storing bacteria cultures on LB plates have that storing them on LB slants do not have?

LB slants are best for long term storage and are basically consists of what LB plate agar has, only that it is in a sealable tube where the agar solidifies at a 45 degree angle -- with the materials to make LB agar, and given the sealable tubes, one can create LB slants properly.

LB broth is most efficient for determining the population of a certain bacteria with the use of dilution plating.

Another thing that strikes me is:

It is possible to transfer bacteria from an LB plate (for identification) to an LB slant (for long term cultivation, though would it be more efficient and better to transfer it directly to an LB slant?

Interesting. I might make LB slants tomorrow so that I may culture the rehydrating strains properly.

Thanks

[donnahardy2]

Hi Trader,

You are making progress if you are growing your culture. You may not have to wait 2 days; it may grow up with overnight incubation.

It's OK to store plates upside down at ambient temperature for a while.

LB medium is a general all purpose medium using for growing a wide variety of bacteria like E. coli and B. cereus. It contains tryptone (protein), yeast extract (vitamins), and sodium chloride. For some experiments, antibiotics are added to make a selective medium. But as it is, it is not very selective and many types of bacteria can grow on it. It can be used for counting on plates, but cannot be used to identify bacteria on its own.

Yes, when your culture grows up in the broth, transfer a small amount using a sterile inoculating loop to the plate and do a streak plate (see the science buddies website). All of the colonies should appear identical if you have a pure culture. Transfer one colony to an agar slant to incubate and serve as your stock culture. For your experiments you can transfer a colony from a recently grown Petri dish plates or from your agar slant. It is best to avoid going into your agar slant too many times to avoid contamination of your stock culture. Be sure and use sterile technique whenever you transfer the bacteria. There's nothing worse than discovering you have a contaminated culture at the end of a project. I promise this will all make sense once you have started to do the experiments, but keep asking questions if something is not clear.

Donna Hardy

[Trader]

If we don't wait two days, how might we know that the culture has rehydrated? (I just want to make sure because if I contaminate the last of these strains, I would have to spend perhaps weeks to locate another source... )=)

I will prepare a "lawn" of bacteria on petri dishes -- there is a question that I am somewhat curious about;

In your experience, approximately how long might e. coli and b. subtilis "live" (without refrigeration) on an agar plate?

The reason I ask this is because I am completely unsure about whether or not the e. coli/b. subtilis will grow on the LB agar that I made -- I've prepared nutrient and LB agar and hopefully will be able to see whether or not there is growth and then prepare the slants for the type of media that the bacteria do grow on.

This also brings me to question:

Bacteria can be transferred from a petri plate to an agar slant, and an agar slant to liquid broth right?

I'd also like to clarify just to make sure -- when we talk about agar slants, that mean storing them in a refrigerator to minimize their growth right?

If only I can control time and reduce the waiting time =), find out which media they grow on, culture them on the appropriate slant and store them away, and then incubate them at the appropriate temperatures, find the "optimal temperature" and then predict the times of their stationary phase for b. subtilis so that I can attempt to create a sensor strain out of it. ...

I hope it'll make a good project?

Thank you for all your help and I really really want be able to start reporting back "results" soon!

(An interesting related research question I was thinking about would be asking whether or not the e. coli in the human gut rely on a) largest number of viable organisms or b) lowest doubling rate because that might give some clues about the processes they undergo in the intestines? That would certainly be more interesting, though I don't think it would be original as perhaps the processes they go through in the human intestines are already very well known... just a thought )

[donnahardy2]

Hi Trader,

If the culture grows in the broth, it will turn cloudy. You will be able to see it very clearly. If the broth remains clear, then no growth has occurred. And, yes, you do want to be careful and not contaminate the stock culture.

Instead of a "lawn" of bacteria, which is usually used for antibiotic resistant studies, I recommend doing a streak plate as describes in the science buddies website:

http://www.umsl.edu/microbes/pdf/streakplates.pdf

Do you have an inoculating loop to use to make streak plates? If not, you might have to improvise with sterile toothpicks or cotton swabs. The idea of a streak plate is to isolate individual colonies of your stock culture to look at it and make sure all of the colonies are identical. If you have different types of colonies, then you have a contaminated culture. Hopefully, it will be a pure culture, and you can transfer one colony to the agar slants to grow up for your stock cultures, and use other colonies to transfer for your experiments. E. coli will live for at least a month on agar plates at room temperature as long as they don’t dry out; B. subtilis will form spores, so will remain viable for much longer, even if the plate dries out. You can leave your plates out at room temperature, but it would be good if you could store the agar slants in a refrigerator after overnight incubation. Once you have your culture, you can transfer it to any type of medium, slants, agar plates, broth and it will start growing again. The agar slants are used to maintain viability of the stock culture, and storing them in the cold keeps them alive for a longer period of time.

I think your project idea is good and will give you the lab experience you need for the long-term project. It will be very important for you to focus on this project, and not try to think of all of the other possible projects, so you will have some results for your project board this year.

I don’t think everything is known about E. coli growing in the human intestine. This bacterium is well-adapted; it has an optimum temperature of about 37 degrees C., it has cilia so can swim around, and it is normal flora, so doesn’t cause disease (except a few strains). But, I wonder if AHL’s are used to regulate its function in the intestine. Maybe you will be the one to find out about this in the future.

Let me know if you see any signs of growth in your culture.

Donna Hardy

[Trader]

Oh no...

I kind of ... already made a lawn of bacteria using the liquid (in the cylinder that previously had the bacteria in it) in four different plates, 2 LB and 2 nutrient to test whether it was alive/could grow on LB and/or nutrient, and then make the appropriate nutrient slants.

Would it be possible to make a streak plate after I see colonies growing in the LB/nutrient plates (if I do?) -- I could treat the colony as a source and use the same technique as I would, only instead of the liquid in the cylinder I would have the colony? (I hope this is OK!)

In your experience, would it be a "good idea" if I switch my research question (I'm sorry! I'm very indecisive this way) to

Does the 37 degrees Celsius for e. coli produce the largest amount of viable organisms and/or the shortest doubling time under intestine-like environments (and what does this show about e. coli in the intestines?)

I'm sorry, it's kind of in my personality to know that what I'm working at is as original and the best that I can do...

Right now I've already made a lawn (sorry! I really should have read the thread first!!) and am now incubating them at 35 degrees Celsius -- if I understand correctly, I should first keep them in 35 degrees Celsius until I see a colony, and then, if the answer to the above is yes, then move onto to make streak plates, which would let me make agar slants, which would then lead to me making a stock culture, which could then lead to a source of bacteria I could use, which means I could "officially" start?

Thanks!

[donnahardy2]

Hi Trader,

You didn't do anything wrong. Hopefully you have obtained some growth by now. Have you seen any individual colonies or the confluent "lawn" of bacteria? If so, your culture is growing and you will be able to do your experiments. You can make a streak plate at any point to verify you have a pure culture. Does the lawn have a uniform color and texture? If so, then it's hopefully a pure culture.

What is your deadline for your science board? You are just exploring your topic and you will have lots of ideas for projects, probably 1000 more projects than you will have time for. If your project is due within a month, then I recommend deciding on your project as soon as possible. If it's not due until March, then you can plan to decide within a week or two. You do want to have a definite experiment with quantitative results for the science board, and you need to allow a few days after the end of the experiment to allow time for writing up the board.

Yes, you can officially start after the bacteria have grown up overnight on the agar slant. Why don't you define your question exactly and plan the experiment that will answer that question before you do too much work, however.

Donna Hardy

[Trader]

B. subtilis is growing...I moved it to the refrigerator (I hope I did the right thing by not keeping it in the incubator?) -- e. coli isn't growing! )=. There is a very small colony in some of the plates, but now I'm thinking that's a contaminant. I left it in the incubator nevertheless...to be left over the weekend o=.

The project registration forms are due in March, and the regional fair is in March 24. I really want to contribute something useful

Hopefully that gives me time?

My current research question is --

What is the optimum temperature for e. coli and b. subtilis which yields the highest amount of viable organisms and is there a difference with the optimum temperature which yields the shortest doubling time?

I really want to contribute something very interesting to the science fair (I think I'll have no chances of qualifying, but I just want to increase my chances. Good news is that I am going to continue this project waaaay after the science fair!

I ... guess I am one of those "I love science but... I'm afraid my research question isn't much" kind of a person -- thanks for your patience [as I realize that after 70 posts now, I have done nothing...much. Please realize that I definitely want to get something original in and I truly appreciate your help throughout the way.

[donnahardy2]

Hi Trader,

Good job in growing the B. subtilis! The spores formed by this organism allows it to survive for a very long time; the E. coli culture was probably too old and had lost viability. The stray colony or two are most likely a contaminant, but it's good to continue the incubation. Do you have any Gram stain reagents? You could check to see if the colonies are Gram-negative rods, and this would help confirm that you have the right organism. If all of the colonies that grow appear to be identical, then it would be worthwhile to check further. If you see different types of colonies, then you have contaminants.

Your project questions are good, and will make a good basic project. You can include additional information in your background section to explain the reason you are doing your project. What is your hypothesis, or guess about the maximum number of viable organisms and the minimum generation time? This should be based on literature references.

It looks like you will be doing your project on B. subtilis to start with. Do you have any local contacts yet that can help you? My friend from Shanghai has not heard back from any of her contacts yet.

Donna Hardy

[Trader]

I thought of doing a Gram stain, though excuse my lack of knowledge in the area, but

Wouldn't using a streak plate to have individual colonies of the bacteria be more effective in making sure it is a pure culture than a Gram stain, which only shows if the bacteria is a Gram positive or negative bacteria?

Perhaps Gram stain is more efficient though -- I'll look into the materials .

I asked my science teacher about my hypothesis and apparently we don't necessarily need a number in our hypothesis? I could instead describe what to expect when the bacteria is actually grown at optimum temperature, but I'm still not sure about the scientific method part of the hypothesis...

Would it be okay to have a hypothesis like "When grown at the optimum temperature, b. subtilis and e. coli will result in the highest amount of viable organisms at the end of the obtained stationary phase which is the same temperature as the temperature which causes the lowest doubling time during the log phase"?

I've tried searching for references, but all other literature are way beyond mine (which makes me feel really bad, because it seems like everyone's project is fancier <_<) and though I have seen some references of "e. coli enters the stationary phase 18-24 h after incubation". I've searched for "optimum temperature for b. subtilis and e. coli" and there were results ... that makes my project very very unoriginal. Blah... But I don't know if that's in LB/nutrient agar. Either way, my project is so unoriginal )=. But it's important to set foundations...

Interesting references for support -- http://aem.asm.org/cgi/reprint/71/12/7920.pdf for e. coli growth and I'm not sure but does this http://jb.asm.org/cgi/reprint/119/2/560.pdf describe b. subtilis growth? It says growth of cell surface.

It gives a doubling time, which is good too. I know this is somewhat last-minute, but is there any way to make this project "original" while focusing on the basics of it?

I was thinking about finding out a difference between the temperatures that give lowest doubling rate, and temperatures that give highest amount of viable organisms, and a possible difference? That would actually be quite interesting.

Also, I recently came across this http://www.phys.chuo-u.ac.jp/labs/matus ... 8-3875.pdf and was wondering if this would account for a possible error in identifying the strain through looking at its morphology?

As for "good basic projects" -- they won't happen to have some chances of 'doing well' in science fairs do they? (Sorry, it's in my personality to be a bit competitive, though I realize the need of a good foundation)

I've tried the phone numbers for the Shanghai Society of Microbiolgy several times, and nobody has picked up...perhaps they are out of office? I'll try again until someone picks up -- though would it help if I contact the other societies that are also of sciences, but not of the same subfield such as the Shanghai Society of Biotech?

Thanks for your patience and help

[donnahardy2]

Hi Trader,

You are correct; the streak plate is used to confirm that you are working with a pure culture. The Gram stain would confirm that the bacterium matches the characteristics of E. coli. Don't worry about doing this, however, if you don't have the reagents readily available. Did the E. coli culture grow?

For your hypothesis, 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. Please don't worry that your project is not original. I assure you that science fair judges much prefer a basic, well-done project to a fancy one that is done poorly. You are doing this particular project because it will give you invaluable experience that you need for future projects. And, in doing it and making careful observations, you may discover something new.

Your idea to find out if there is a different temperature for highest number of viable organisms and shortest doubling time is an interesting idea. You could add total cell volume to your measurements if you had a centrifuge available to measure the volume of the cell pellet after growth. 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

The reference you found on the fractal growth patterns of B. subtilis is an interesting paper, perhaps you could analyze the growth characteristics of your strain. The authors found a difference with nutrients and surface moisture on the agar plates. Maybe you could expand on this idea. Please don't add more than one more variable to your experiment. Ithink you are finding that it takes a lot of work just to do a basic experiment.

Otherwise, I can’t think of a way to make this project completely original. You are probably right that this project will not be a top prize-winning project, but it is still worthwhile doing. And, you never know what the competition will be doing; it’s best to do the best possible job, and you will win if yours is the best project that is entered into the fair.

The Shanghai Society for Microbiology doesn’t sound like it is very active. The phone number must be staffed by volunteers. Was there an address that you could write to?

Donna Hardy

[Trader]

Thanks for the boost and I know it's not about winning -- people often tell me that that often gets in the way.

The e. coli is not growing...I've made an order for more e. coli and hopefully it'll come before the end of this week.

I would want to know if the optimum temperature that makes the highest amount of viable organisms would be the optimum temperature which yields the shortest doubling time -- thanks for the method!

Yes the fractal growth seemed interesting but I think it would be more on topic (because I eventually want to check out my "timing" sensor strain method) if I find out whether the highest amount of viable organism temperature = the lowest doubling rate.

That's going to be my research question -- it wouldn't happen to be "original" would it? (Sorry,... it's part of me to ask that question ><!)

It looks like I won't have access to the slant tubes so I did make 25 nutrient plates and hopefully can begin my culture tomorrow, and run through my first "recording" of b. subtilis at least. And see what to do when e. coli comes here. However,

Just to confirm -- after overnight incubation and moving the plates into the refrigerator, we can expect them to last about one month right?

(This may seem a bit weird, but I LOVE MAKING PLATES! xD but if I continue at this rate, it'll be all I do =P)

Thank you for the encouragement -- I do need it . I think of this entire thing -- science fair projects, as a marathon and not a sprint, you know what I mean?

The SSM -- I doubt they would reply because it does seem like they are inactive. But I do know the address and I'll drop by sometime to see what's going on there =P.

Update:

Just did a gram stain on b. subtilis but it turned out red. I wonder why? (I did it twice), and roughly I did

Took a slide, drops of sterilized water on it, inoculated w/ sterilized loop (tapped three times to make sure it went in), then made the plate. Then left it to cool... and then I added violet crystal stain, 1 min, washed off w/ tap water, added gram iodine, 30 s, washed off w/ decolorizer (50% acetone, 50% ethanol or ethyl alcohol (same thing?), then immediately added on safranine, 1 min, washed off w/ tap water, and let it try out in the open.

I think it has something to do with the decolorizer? Because...maybe it shouldn't be "washed off"? But only have a few drops of it until it trickles down?

Did I do anything wrong? o= Because I wonder why after two times, it still turned out red as opposed to the ... purple that is expected.

On the instructions it said that after the decolorizer, it is supposed to have retain the primary stain (purple), but I had blue. I wonder why?

I hope what I had is b. subtilis, or else I would have no bacteria left <_</

[donnahardy2]

Hi Trader,

I agree that the growth curve experiments are more related to your ultimate goal for this project, even though it's not a completely original project. The fractal growth study is interesting, but unrelated.

I hope your new culture of E. coli will come soon. I'm glad you were able to reorder it.

It's disappointing that you can't get slant tubes. However, you can keep your culture growing on the agar plates and the culture will probably stay viable in the refrigerator if the plates don't dry out. Any type of small glass bottle with a screw capped tube could be adapted for a slant culture, and you may want to keep looking around for something like this that could be used to maintain your cultures for a longer period of time.

It's good that you like to make agar plates. If you are careful not to let the plates dry out in the refrigerator, they should last for a month. It might helpful to repackage them in the plastic sleeves that the original plates come in, but it's very important not to let them accidentally get exposed to air and get contaminated. You should be storing the plates upside down; sorry if I had mentioned this before.

The Bacillus subtilis should be Gram-positive purple, not red. Did you look at the cells under the microscope? If so, did they look anything like this:

http://www.microbelibrary.org/asmonly/D ... asp?id=369

Did you heat-fix the smear for a second or two over an open flame? I would try leaving the iodine on for a longer period of time, and try using 95% ethanol for the decolorizing step. I have never used ethanol/acetone, and don’t know if it would make a difference. The acetone is more non-polar than ethanol, and might wash the crystal violet from the Gram-positive cell wall. If the cells have the right shape (rods), then try making fresh Gram stain reagents. Was the crystal violet blue or purple? It should be dark purple. If the cells are not long rods, then you may not have B. subtilis. What do the colonies look like? B. subtilis is white and colonies get wrinkled over time.

Donna Hardy

[Trader]

I... added a drop of sterilized water on an unsterilized plate (which now leads me to ask, sterilized water is not required for the entire process right?, inoculated one loop of what is hopefully b. subtilis, and dabbed that end two times in the water to make sure there's some of it in the water.

Then I waved the glass slide over a flame until the water eventually dries out...I think that's heat-fixing it? Though it certainly took more than 2 seconds... I wonder if I am doing this wrong?

One question regarding storage -- I've stored them up side down but there are a lot of water droplets that are on the lid that I find myself actually having to quickly open the lid, shake the water away, then close it -- which makes it very possible for contamination.

I haven't been storing them in plastic bags yet because I thought there was in fact too much moisture -- is this true?

Ok, I'll see what happens when I try the new decolorizing method.

I tried with 95% ethanol and longer iodine...but it still remained red. It was a darker red though,... almost violet... but definitely "dark red" more.

On my "Gram stain protocol", it said that even as early as the "decolorizer" stage, after I washed the slide with the decolorizer, it should retain the "primary stain", that is, violet crystal.

As soon as I poured on the "decolorizer" however, for all three trials the bacteria turned blue -- would this be a "primary" stain? But somehow it seems like as soon as I put on safranin, it just always turn red.

I was also wondering about the sterile technique part of this -- I cleaned the slide w/ ethanol, then made the bacteria smear (using method mentioned above), then it was completely left in the open air. Wouldn't there be contamination between when I dropped drops of sterile water on the slide to when the bacteria is inoculated in there? I'm looking for possible cases of contamination (if applicable here?) to explain why three trials resulted in a gram-negative reading...

I've also tried bacteria from two different petri plates. ... I'll analyze the morphology tomorrow (despite the G- reading) and see if hopefully it was just the staining process that I got wrong -- that it is still b. subtilis.

Regarding streak plates, I was wondering

Why would it be necessary to have individual colonies [obtained from streak plates] to create a stock culture?

I'll also look into the agar slants. The concept is really cool! It can last longer due to its larger surface area right?

Thanks for all your help and patience!

[deleted-42343]

Hi!

I am the TLC "Expert" that Terik mentioned. So sorry I have not commented sooner. I've read through some of your comments, but not all. It sounds like you know much more about the topic of using biology and TLC than I do! I made a new technique for TLC, but it was more of a general technique, and to test it I used different concentrations of chemicals, so it was not related to biology. If you are still thinking about using TLC for your experiment, I think I could be most helpful with the actual technique part of it. I might be able to help with giving you some tips on how to do TLC properly, and some cool ways on how to analyze the results.

The technique I worked with was to help analyze TLC plates to determine the original concentration that was used. The main equipment you would need are TLC plates and a digital camera with manual exposure. You can easily make your own TLC chamber out of some kitchen glassware.

If you want to read the paper I wrote on this technique, it might be helpful in analyzing your results. It would help you determine the amount of each compound present in the bacteria you are studying. The link has information about the (free) software I wrote to analyze the images of the TLC plates and there is a copy of the research paper, too.

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

I am very impressed with your drive and I'm sure your project with turn out great because you have so much enthusiasm for the topic.

If you have any questions, please let me know. TLC can appear very easy to do but it often takes a bit of practice to get the technique down.

[donnahardy2]

Amber,

Welcome! It's always great to have a TLC expert to provide advice on this project. I'm sure Trader would like to be doing the TLC experiments, but he does not have an AHL sensor strain to work with yet. Do you happen to know any microbiologists who are doing research in this area?

Trader,

Your questions are always very perceptive. Sterile water is used because bacteria can grow to very high levels of 100,000 or higher per ml in pure water that has been sitting around, and could interfere with the Gram stain. But this usually is not a critical point and using freshly prepared water will work well.

Your procedure for preparing the Gram stain is OK. You might want to let the culture smear air dry and then heat-fix the sample for about 2 seconds.

Your description of the results is correct, except for the final step when the crystal violet was washed out of the cells. Gram-positive cell walls should retain the crystal violet. I don't know why the B. subtilis is turning red on the Gram stain. If the morphology is right, then maybe you will need new Gram stain reagents. If you don't see long rods under the microscope, then you may need to new culture of B. subtilis. However, maybe the culture has turn to spores, but I wouldn't expect that from a culture that is less than a week old. You may not be able to resolve this until you have another culture of a known Gram-positive bacterium to test. Some Bacillus species decolorize easily, and I don’t know if B. subtilis is one of those organisms.

Do all of the colonies on your Petri dishes look identical? They should be if this is a pure culture. Look on the oldest Petri dish you have and see if the colonies are getting wrinkles. Bacillus species usually have colonies that are kind of wrinkled; it's very distinctive.

You do a streak plate to isolate the stock culture to make sure that you have a pure culture. If you take the sample for the stock culture from a lawn, you may pick up a contaminant. Keeping pure cultures is a constant concern of microbiologists, and using a contaminated culture will invalidate results.

I’m worried about the drops of water on the Petri dish lids. Many bacteria are motile, including E. coli and B. subtilis, and if one motile bacterium gets into a drop of water, it will swim through and contaminate all of your plates. When you do your plate counts with this batch of Petri dishes, keep one plate to incubate as a sterile control to make sure the plates are not contaminated. The next time that you pour plates, leave the lid tilted slightly while the agar is solidifying and cooling so the surface of the plates can air dry. This will take just a few minutes extra. You should be decontaminating the work surface with 70% ethanol or equivalent before you pour plates, and hopefully you have a quiet place to prepare the plates to avoid contamination at this step.

My coworker who was originally from Shanghai heard from her microbiologist friend, who, unfortunately is too busy writing grant proposals to help. We are now looking for a contact in a local college or university who could provide local assistance. Did any of your teachers graduate from a local university? If so, perhaps they could refer you to someone.

Donna Hardy

[Trader]

Dear Amber,

I've read so much about you!! I'm a big fan. -- I love science and though my project isn't too original, I know I'll have the best time yet out of it. Especially at the fair. Getting to see what other people do what be one of the best parts.

The TLC experiment would be great. But then right now (I live in China) and we have a Chinese New Year holiday...so I'm afraid I won't be able to get to my equipment until ... Feb. 5 (which does puts a further strain on the little time I have left to do a science fair project).

Originally I was hoping that I could detect AHLs in v. fischeri through TLC, but it seems like I could neither get v. fischeri nor a sensor strain within 2 months' time, so I decided to test whether the "substitute sensor strain" method would work. Before that happens, I'll need to find out the optimum temperature, making my current research question

Is there a difference between the optimum temperature that produces the highest amount of viable organisms and the optimum temperature that produces the lowest doubling time in b. subtilis?

The lack of response from the one local microbiology society in Shanghai, as well as the microbiologists to be found here )=, as well as the distance/time/price restraints I have has been troublesome, but I'll hope to solve that all next year .

I think TLC might not work unless I have a sensor strain, like Donna said, so I'm not sure if it would be most efficient if I test to see if the "substitute sensor strain" method would actually work...I would be able to know in about 3 months time.

But for now, I really want to try TLC but I can't -- Sorry for not being able to continue with TLC after the introduction of its use -- ... in 3 months I might be able to continue?

-----

Dear Donna,

I love the concept part of each question -- thank you for your patience in answering them!

As you may have read above...we have a Chinese New Year holiday ... which means that I am pretty much unable to do much (but of course, type out a research plan, finalize my hypothesis with proof, review of pertinent literature to get an actual project going, outline my plan, as well as getting down all the concepts, getting down all the why's, how's, hopefully a day to day plan [but from experience already I'll know that it'd be great if I can finish up to half of what I plan ]) until Feb. 5...

I think I might stick with the research question above, (with it being original would be a bonus, but I certainly want to find out) ...

Once again I find myself with a lot of articles in my hand -- time to read again!

Sorry for this additional delay ^^. I haven't been able to examine the morphology of the bacteria ... it would have been very useful to see if it is actually b. subtilis.

I did get some e. coli, so I would be able to fit in "e. coli" in the research question above.

One more question though -- when I am to "heat fix" the smear, does that mean [previously] letting the slide (that has just had the sterile water on it and the b. subtilis in it, dry out due to the fire) cool, and then wave it over the flame for 2 seconds then continuing?

It remains Gram negative in the gram stain results . Just to confirm, we are supposed to wash with tap water, each stain after its use (with the exception of the decolorizer) right? ... I even tried with a different plate, and it seemed like it didn't work.

Based on my understanding of bacteria ... (which is very limited )

Would it be possible/likely that the lawn of bacteria has b. subtilis but also a contaminant, and that I should take the individual colonies I see in the streak plates and attempt to do a gram stain on those?

But I'm thinking, if I made the streak plate from the lawn of bacteria...wouldn't the contaminant also be in the streak plate?

Sorry...my understanding regarding having a pure culture which I know is b. subtilis is really poor ...

Thank you for your patience and understanding once again!

--> On a somewhat unrelated note, I've been browsing for summer programs and the science summer programs (especially internships like RSI) were particularly interesting! Of course I can't get into RSI and it's deadline is over, but do you by any chance happen to be familiar with any similar internships that can help with science research (particularly with biology, perhaps microbiology) -- it would be a GREAT opportunity to check out -- I can come to the states every summer ^^!!