PLEASE HELP ME WITH MY EXPERIMENT! It involves viruses.

[bhh1988]

I want to do an experiment on the Effect of pH, temperature, and UV light on the frequency of prophage induction in bacteria. I'm thinking of using E.Coli or Lactococcus lactis, but most importantly, I need a procedure for my experiment! I've never worked iwth viruses before so I don't know where to get them, exactly which ones are lysogenic, or exactly how to prepare them. I'm sure it'd involve lots of precise measurements and timing, which I really have no way of knowing except through an expert's help & experience. PLEASE HELP ME OUT OR GIVE ME ANY TIPS OR IDEAS YOU HAVE OR REFER ME TO SOMEONE WHO CAN!!!!! THIS EXPERIMENT REALLLLLLY INTERESTS ME AND I DON'T WANT TO GIVE IT UP! THANKS!!!!!! Thanks so much for your time guys.

[jessicahua]

Hi!

This sounds like a very interesting project. Here is a site that might be useful:

1. http://www.freesciencefairproject.com/b ... teria.html (pH effect on bacteria)

I'll post more information later.

[hiramuzammal]

I found a few articles that might help

1. "Thermal Enhancement of Ultraviolet Mutability in a tif-1 uvrA Derivative of Escherichia coli B/r: Evidence That Ultraviolet Mutagenesis Depends upon an Inducible Function"
http://www.pubmedcentral.nih.gov/articl ... &tools=bot

2. "Effects of Diverse Environmental Conditions on LC3 Prophage Stability in Lactococcus lactis"
http://aem.asm.org/cgi/content/full/71/2/721

3. "Lysogenic"
http://en.wikipedia.org/wiki/Lysogenic

4. "Viruses"
http://www.cat.cc.md.us/courses/bio141/ ... ysolc.html

Good Luck.

[jessicahua]

Hi, I'm back again!

Here are some sites that I thought might be helpful or interesting. They are not exactly similar to your project, but they do have aspects that might help :

E. Coli Preparation:

1. http://www.newton.dep.anl.gov/askasci/m ... e00148.htm (preparing e. coli)
2. http://www.icampus.ucl.ac.be/ANIBIOL/do ... age01.html (more on the preparation of e.coli)
3. http://www.accessexcellence.org/AE/AEPC ... .coli.html (this looks like a good source to show how to prepare e. coli because I did a lab like this last year which was very similar)

UV Light

1. http://www.usc.edu/CSSF/History/2004/Projects/S1307.pdf (not sure about this one )

Interesting:

1. http://www.ncbi.nlm.nih.gov/entrez/quer ... t=Abstract (interesting)

I think you should be able to prepare all of your samples in the same way, and for the different experiments just alter your variable. I hope these sites are helpful. If you need more help feel free to ask!

[jessicahua]

Well...I'm back again!

I hope those sites were useful. Here are some more sites:

Purchase E. Coli or other bacteria:

1. http://www.picultures.com/index.vnss
2. http://www.atcc.org/
3. http://sargentwelch.com/category.asp?c= ... eid=GL143A

Purchase Petri Dishes ans Agar:

1. http://www.physlink.com/estore/cart/Pet ... thAgar.cfm

[bhh1988]

thank you guys sooooo much. Everything really helped but especially jessica's website: http://www.icampus.ucl.ac.be/ANIBIOL/do ... chor888687

Wow did that help. Except I have one little question with that website as of right now. I looked at hte procedure like 3 times and one thing that confuses me is on part 3: Infect bacteria with recombinant phages.

It says:
Make serial dilutions as following:
in all pink tubes pipette 270 µl of SM buffer


add 30 µl of the phage suspension into the first tube
mix carefully
remove 30 µl from this mix, and add it to the second tube
mix carefully
repeat this operation with the next tubes and so on.

Infect E. coli with the phage dilutions
transfer 100 µl of each pink tube into the corresponding white tube.
add 100 µl of competent bacteria to each tube of the 2nd row.

Incubate for 20 min at 37°C in an incubator

First of all, I'm assuming that the first tube will end up being just a junk tube. because I think they're saying that I have to take 30 microliters from the first tube's mix and put that in each of the other tubes, meaning that all the other tubes will end up with 300 microliters and the first one with 180 microliters. But it gets even more confusing later when it says add 100 microliters of competent bacteria to each tube of the 2nd row. What is the 2nd row? In the picture, the second row is the pink row of tubes, but what are the white row of tubes for hten? Just to throw away 100 microliters of the phage dilutions? I'm scared that I might be making too many assumptions here because of this slight confusion, and I know that I don't want to make any mistakes or else everything will go wrong. Anyone have anything to say to make that part clearer for me? Thanks so much guys.

Also, where do I get viruses? lol

[jessicahua]

That part was unclear and confusing. I believe you add 30 microliters to the first tube. After you mix it, then take 30 microliters out of the first and add it to the second tube. After that, mix the suspension in the second tube and then take out 30 from the second tube and add it to the third. That way each tube would have the same amount of the mixture. I don't think you keep taking out the mixture from the first tube. (This information pertains to what you do with the PINK tubes)

Actually, you mixed up the picture. If you look under the part "Organize your bench" there is a list of what you need. It says that the first row contains the PINK tubes and the second row has the WHITE tubes. So, you take 100 microliters from a PINK tube and add it to the corresponding WHITE tube. Then you add 100 microliters of competent bacteria to the WHITE tubes. To sum it all up, you will be adding 200 microliters to the WHITE tubes. You will NOT add anything to the PINK tubes in this part.

I hope that helped!

[jessicahua]

One more thing...

Why do you need viruses? Also, what virus do you need?

[bhh1988]

about that 30 microliters thing...

The reason why I didn't think you keep transferring from 2 to 3 then from 3 to 4 etc. is because the amounts don't end up being the same. We put 30 microliters of virus in the first, which already had 270 microliters of the buffer. So that means 300 microliters in all, meaning a tenth of that is virus and 90% is buffer. So transferring 30 microliters of that into the second tube, 90% of that 30 microliters is buffer and 3 of those microliters is virus. So now the second tube has 3 microliters of virus out of a total of 300 microliters. That's a hundredth of the solution that is viruses. If you keep doing that, then by the time you're at the 5th one, you've got barely any viruses in there compared to the first one. Therefore, that would mean that all the pink tubes have a different concentration of viruses, and that's why I thought that way couldn't be.

That second row thing makes sense to me now. I totally missed that.

And the viruses and bacteria: I'll get back to you on that. I actually didn't see the atcc website that you gave me at the time so I'll check that out and ask any questions if i still have any. Thank you so much! Let me know what you think about that whole transferring solution thing with the pink tubes. THANKS!

[jessicahua]

Hi!

hmmm...I never thought about how little of the phage susepnsion you would have in the last tube. I reread part 3, and I still get the same thing. However, I will ask my teacher about this tomorrow, and I sent an email to the person who created that website. Hopefully, they will be able to prvide me with an answer. I'll post again tomorrow with the correct procedure.

[phamlinh]

Hi there,

I wanted to post a clarification about serial dilutions. It's a very common technique that is particularly relevant to microbiology. Jessica explained it correctly the first time. Remember that as long as you mix the solutions thoroughly, you're dealing with a concentration, and not just a volume.

The protocol described on the webpage is called a serial 1:10 dilution. You're absolutely correct that you're transferring things serially, and the volumes are going to change. In other words, you start with 300ul in tube 1, take 30 and add to 270ul in tube 2. Mix well. Take 30 from tube 2 and add to 270ul in tube 3....etc. You do not go back to the original tube. As you make the dilutions, only the last tube will have 300ul and all of the other tubes will have 270. This is okay because you're working with a concentration, not just a strict volume.

The point of serial dilutions is to find a range in which you will be able to detect bacterial lysis by phage (i.e. viruses that infect bacteria). For example, if you added 1 billion phage particles to a solution of 100 bacteria, you would lyse all of the bacteria. If you added 1 phage particle to 100 bacteria, you may not be able to detect that one lysed bacteria. For this reason, microbiologists do serial dilutions.

Let's go back to that 1 billion number. In scientific notation, that's 1X10^9. What happens if you dilute that 10fold? You would now have a dilution that is 1X10^8. How many phage particles do you have left if you've performed a 1:10 dilution 5 times in a row? Please post the response so we know that you're thinking along the correct lines. In any case, you'll find that it's more than you might expect.

This sounds like a very interesting project, and I wish you a lot of luck. Please make sure to talk to your teacher before purchasing bacteria and phage. You want to make sure you have approval before doing these experiments. Note that bacteria like E. coli and B. subtilis are very safe, and don't require much paperwork to get approval. They also both have well characterized phage that infects them.

If you're anywhere near a university, I would highly recommend that you contact a microbiology lab. They may be able to give you bacteria cultures and phage for free.

Please post if this doesn't make sense or if you have any more questions.

Best of luck,
Linh

[bhh1988]

first off, i really wanna thank you guys for helpign me out with this. I am seriously at least a hundred times better off right now then I was two days ago. Thanks a lot Linh for enlightening me with the serial dilutions thing. And to answer your question, the 5th tube I think should have 1X10^5 viruses. But from the sound of it, it seems like the whole purpose of that serial dilutions thing is simply to see what a good concentration is for the experiment. Therefore, my experiment wouldn't really begin until AFTER I do that fairly lengthy procedure and figure out an appropriate concentration. But now I wonder if I would have to do something similar with my other factors like pH and temperature. Just like I want to find a good concentration so I don't end up with a bunch of completely lysed plates, wouldn't I want to know how much to raise or lower the temperature or pH to see good results? Let's say I had three levels of pH, that is, 2, 7 and 11. It might be hard to compare them if the ones with pH 2 and 11 are both completely lysed. I don't want them so lysed that I can't compare them, but at hte same time I want there to be significant enough difference as well. Therefore, I want to find a good, sensible pH level and temperature. I'll have to think about how I'd find out what levels I want to test my factors at.

[jessicahua]

I know you already have your question answered, but this is the email that I got back from the person who desgined the website:

You should prepare several tubes containing 270 microlitres of SM medium
In the first tube, you add 30 microliters of your phage suspension. You mix.

You phage are now diluted 10 times.
You take 30 microliter of this mix. You add them to the second tube containing 270 microliters of SM. You mix and you have now a 100 times dilution of you phages.
You take 30 microliter of this last mix. You add them to the third tube containing 270 microliters of SM. You mix and you have now a 1000 times dilution of you phages
Yu go on this way...
Maybe you should see on:
http://www.icampus.ucl.ac.be/SBIM2520/d ... plate.html

(make sure to look at that site it might help you to make sure that you are doing everything correctly)

[phamlinh]

Greetings,

I'm glad to hear that you understand serial dilutions now. It also looks like you're thinking about your project very carefully, which is good to hear. Are you going to be able to contact a microbiology lab at a local university? It's important for us to know as we help you with your experimental design.

Just to let you know, the serial dilutions are not a big deal to perform. In fact, you would want to do them every time you conduct an experiment. But it's great to see you thinking ahead of time. It's clear that you understand why serial dilutions are performed, but perhaps I should provide a little context.

Typically, your phage is going to be in solution. That solution will be kept in the fridge, or frozen down as a stock for use each time. But cold temperature could potentially inactivate the phage. Let's say that one time you thaw the virus, 100% are active. The second time you perform the experiment and thaw the virus (or get it out of the fridge) 70% are active. And maybe the third time you replicate the experiment, only 10% are active. For this reason, it may be important to perform a serial dilution each time you perform the experiment. But remember, it only takes about 10 minutes to do this part of the experiment. And it's mostly as a control to ensure that you're in the right dilution range.

Along these same lines, you're exactly correct that you would want to vary the temperature and the pH along a range. But in this case, these are your experimental conditions, so you do want to test a whole range. You say that you want a good sensible range, but this is the purpose of your experiments.

Here's an example of an experimental setup.

You would first do your serial 1:10 dilutions. Let's say you make the final volume of each dilution 900microliters. Then you could divide that 900microliters and put each of the tubes at different temperatures. The same is true of pH.

If this doesn't make sense, try drawing out a list of all the tubes you would need for the various conditions. This is always a good idea to get a handle of your experimental design. And if it still gets too confusing, let us know and we'll help you out.

By the way, I would strongly suggest that you only test one variable at a time. For example, change the pH, but keep the temperature constant and vice versa. If you change both variables at the same time, you won't know which one is contributing to your results.

Best of luck,
Linh