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Bacterial Growth Question


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#1 Brand New

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Posted 01 May 2014 - 10:58 AM

I'm in college, currently studying Biomedical Science. As part of my course I have to undergo 6 weeks of experiments. I have chosen to investigate the sensitivity of E.coli k12 to UV light.

 

I have a very general question as to why some of my plates have a confluent growth of individual colonies and others have a confluent mat growth? (they were all inoculated using lawn spread technique).

 

I have attached a couple of pictures (I don't know how well they will turn out).

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#2 bob1

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Posted 01 May 2014 - 12:44 PM

You need to tell us what you did exactly to reach this point...



#3 Brand New

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Posted 01 May 2014 - 12:59 PM

I had inoculated nutrient broth with E.coli k12 the day before from my E.coli k12 stock slope and placed it in a shaking incubator at 37 degrees centigrade overnight. The day after I used a P200 Gilson and transferred 100 microlitres of the E.coli k12 broth culture to the nutrient agar. I then used a glass spread to evenly spread the E.coli over the agar. I did the exact same for E.coli strain B. My experiment was basically comparing the sensitivity of both bacteria to UV light, so I exposed them to UV light for different amounts of time under a UV fish tank light. After that I wrapped my k12 in tin foil and placed both plates in an incubator at 37 degrees centigrade and observed my results the next day. For 5 seconds exposure, my k12 had a confluent growth of individual colonies. However, with my strain B I had a confluent mat growth.

 

I hope this helps.



#4 bob1

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Posted 01 May 2014 - 01:02 PM

The images you posted don't show confluent colonies for the Doc4 image- they are discrete but probably too many to count easily.  How did your no UV exposure controls look?  As you have used two different strains - what is the difference between the two?  Could these differences account for the pattern?



#5 Brand New

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Posted 01 May 2014 - 01:13 PM

I have attached photos of my controls. I was just wondering as to why sometimes I get a mat growth with no sort of, distinct individual colonies and sometimes I get a plate covered in a lot of small individual colonies. Is there a reason to why bacteria grows this way? I really don't know or understand.

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#6 bob1

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Posted 01 May 2014 - 01:30 PM

So your strain B and K12 plates look like they don't have colonies - more of a lawn (it's usually referred to as this, rather than "mat") - so did your treatment cause this?  Or do some of your control plates also show the discrete colonies?

 

If the controls show discrete colonies - its probably a technical error...



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Posted 01 May 2014 - 01:56 PM

My controls had no individual colonies, they were just lawns. My controls were not exposed to any UV light, they were just placed straight in the incubator.

 

I will attach other photos to show the difference, the best one to show is the 5 second exposure to UV light. You can see from the k12 plate that it show lots of small individual colonies covering the plate. Whereas with the strain B it is a lawn.

 

I have also attached photos of my 2 minute exposure to the UV light. With the K12 is shows 2 very small individual colonies. Whereas with the strain B there are a lot more and larger colonies present.

 

I just can't understand why sometimes I get individual colonies and sometimes I get a lawn. I don't know if it is to do with the UV light only killing some of the bacteria and the bacteria present has either survived or repaired it's DNA? Do you know if there is a reason as to why bacteria may grow sometimes in individual colonies and at other times as a lawn?

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#8 bob1

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Posted 01 May 2014 - 04:03 PM

The fact that your controls are always the same indicates that they are working as they should... therefore what conclusions can you draw (I'm not giving you the full answer because this is your assignment!).  You are on the right track with your ideas above.

 

Hint - compare to selection with an antibiotic.



#9 Brand New

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Posted 02 May 2014 - 01:48 AM

When you say compare to a selection with an antibiotic, do you mean how an antibiotic would create zones of clearing due to the bacteria being killed off and that the UV light has had this sort of effect on the E.coli. It has killed certain areas on the dish and some bacteria has either survive or it has undergone photoreactivation due to the formation of pyrimidine dimers caused by the UV radiation? Because if not, then I'm at a complete loss...



#10 bob1

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Posted 03 May 2014 - 01:50 AM

More or less correct - if you coat a plate with antibiotic (not using disks, antibiotic in the medium) and then plate a lawn of bacteria  only resistant colonies will grow - this is equivalent what you are seeing.

 

As your untreated controls cover the plates in both cases you can assume that under normal conditions the bacteria grow fine, but when UV treated one of the strains shows colonies, and the other a lawn - this would indicate that one of the strains is resistant (lawn) and one at least partially susceptible to UV (colonies).  The exact mechanism of how they are resistant or how some survive would be something that could be studied further, but the (known) differences between the strains may tell you something



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Posted 03 May 2014 - 02:47 PM

ah I see! When conducting the experiment I did find that when both strains were exposed to UV for 5+ mins there was no growth at all. However, in the case of the k12, exposure for 5 seconds showed only 2 individual colonies, where as with the strain B, even at 2 minutes exposure there was still around 80~ odd colonies. So, I mean, from that it does show that the k12 is more sensitive to UV than the strain B. Or maybe I should say, more sensitive at those particular wavelengths.

But thank you very much for your help, it is very much appreciated.



#12 bob1

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Posted 03 May 2014 - 03:09 PM

You should specify the type of UV used (A, B, C) and if possible the wavelength range.



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Posted 04 May 2014 - 01:25 AM

It's UVC, however, with the fish tank I couldn't find out which particular wavelength it emits, so I don't even know if it may be more than one wavelength. During my 6 weeks though I did use a UV spectrophotometer and I was getting results showing a wavelength of 251nm was the optimum and I repeated my experiment and still found it was the best, but when I looked it up, other people were saying it was around 260nm so who knows. Maybe it was an anomaly or I messed up the experiment twice! 



#14 phage434

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Posted 04 May 2014 - 07:17 AM

Most UVC lamps emit at 254 nm nominally. I don't know how sharp that peak is, but likely quite sharp, since it is a mercury emission line.



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Posted 05 May 2014 - 07:02 AM

Oh really? thank you, that's helps a lot to know that since that is close to the 251nm which I was using on the spec. and it will give a good comparison.






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