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Author Topic:   Application of the Scientific Method: Antibiotic Resistance
Taq
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Posts: 9973
Joined: 03-06-2009
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Message 16 of 20 (692165)
02-28-2013 11:37 AM


Basic Methodologies
Moving on from our hypotheses, we need to get a better feel for the tools that we can use in our experiments. The advantages of using bacteria like E. coli is that they are clonal organisms, they have a very short generation time, and they are easily cultivated. So let's put these characteristics to work.
First, we need to go back the opening post. As I stressed, the entire experiment started with a single bacterium that formed a single colony. What we do next is transfer this single colony to some liquid culture and let them multiply overnight. This will give us a ton of bacteria to work with, and they will still all share a common ancestor. However, these bacteria have been accumulating mutations as they replicate so we will have a genetically diverse population over these many generations that have occurred since we founded that original colony with a single bacterium.
As noted in the post above, E. coli don't have legs. When a founding bacterium is placed on a plate the descendants stay right next to the founder. Think of it like the children of farmers using the land adjacent to their parent's farm. As long as you keep the founders far enough apart you will get nice separate colonies like those in the picture above. However, if you put the founders close together you will get a "lawn" of bacteria where the borders of one colony butt up against the colony next to it. What you get is a continuous layer of bacteria much like your lawn of grass at home, and this plate of bacteria will now be referred to as the master plate. The master plate will contain colonies founded by genetically diverse descendants of a single bacterium.
Now we will introduce replica plating. This process can be described as bacteria stamping where our bacterial lawn serves as our ink pad. What you do is take your round pad that is just a little smaller than the diameter of the plate and press it down on the bacterial lawn.
Once you have "copied" the master plate you stamp it onto the replica plates making sure to record the orientation of the stamp so you can keep track of where the bacteria came from on the original master plate.
For this experiment, we create a master plate on media without antibiotic. We then replica plate onto media containing antibiotics.
What predictions do each of these hypotheses make?
1. Random mutations: If resistance is due to mutations that occur prior to antibiotic exposure then they will already exist on the master plate, and they will be clonal. Therefore, we should find resistant colonies on the replica plates at the same position on each replica plate since they came from the same position on the master plate.
2 and 3. Non-random mutations and/or resistance induction: Since each bacteria has the same chance or same mechanism of producing resistance we should NOT see colonies appear at the same position on each of the replica plates.
What are the results? We see colonies at the same position on each of the replica plates. This is consistent with random mutations, and inconsistent with non-random mutations or induced resistance.
Any questions?

Replies to this message:
 Message 17 by herebedragons, posted 03-01-2013 9:03 AM Taq has replied
 Message 18 by herebedragons, posted 03-01-2013 9:21 AM Taq has replied

  
herebedragons
Member (Idle past 858 days)
Posts: 1517
From: Michigan
Joined: 11-22-2009


Message 17 of 20 (692212)
03-01-2013 9:03 AM
Reply to: Message 16 by Taq
02-28-2013 11:37 AM


Re: Basic Methodologies
Any questions?
First a comment then a question.
You did a good job of describing the methodologies of this experiment, but since you titled this thread "Application of the Scientific Method" I had hoped you would have spent a bit more time explaining how the scientific method was applied. I think it is easy for those that work with this stuff on a regular basis to take it for granted, but for those "non-science types" it is not quite as intuitive. I have seen a lot of confusion as to how to actually use the scientific method to come to conclusions.
For example, I like how you tested against competing hypotheses, but I don't think that issue would be clear to someone unfamiliar with the concept. It is critical that you test not only your hypothesis but the competing or null hypothesis. How often have you heard that a particular result is compatible with an ID hypothesis, which it may indeed be, but if the results are also compatible with the null hypothesis, then you cannot draw a conclusion based on those results. These are the types of things that maybe could be better addressed in this thread.
I'm going to put my question in a separate post.
HBD

Whoever calls me ignorant shares my own opinion. Sorrowfully and tacitly I recognize my ignorance, when I consider how much I lack of what my mind in its craving for knowledge is sighing for. But until the end of the present exile has come and terminated this our imperfection by which "we know in part," I console myself with the consideration that this belongs to our common nature. - Francesco Petrarca
"Nothing is easier than to persuade people who want to be persuaded and already believe." - another Petrarca gem.

This message is a reply to:
 Message 16 by Taq, posted 02-28-2013 11:37 AM Taq has replied

Replies to this message:
 Message 19 by Taq, posted 03-01-2013 7:24 PM herebedragons has not replied

  
herebedragons
Member (Idle past 858 days)
Posts: 1517
From: Michigan
Joined: 11-22-2009


Message 18 of 20 (692215)
03-01-2013 9:21 AM
Reply to: Message 16 by Taq
02-28-2013 11:37 AM


Re: Basic Methodologies
What are the results? We see colonies at the same position on each of the replica plates. This is consistent with random mutations, and inconsistent with non-random mutations or induced resistance
Based on this experiment we can conclude that the mutations for resistance to this antibiotic are consistent with random mutations. But is it reasonable to infer that all mutations operate under the same principals? Can the mechanisms of antibiotic resistance in prokaryotic cells be extrapolated to eukaryotic cells or metazoans? I don't think that is a reasonable leap (from this experiment alone). * which I guess is a part of the discussion about the application of the scientific method as well - recognizing the limitations of a study and proposing new directions for research. (This is probably the thing that fascinates me most about science is that finding an answer to a hypothesis doesn't resolve an issue it provides more opportunity for questions!)
So what follow up experiment would you propose to further strengthen the connection between this study and mutations in eukaryotes and/or metazoans?
HBD

Whoever calls me ignorant shares my own opinion. Sorrowfully and tacitly I recognize my ignorance, when I consider how much I lack of what my mind in its craving for knowledge is sighing for. But until the end of the present exile has come and terminated this our imperfection by which "we know in part," I console myself with the consideration that this belongs to our common nature. - Francesco Petrarca
"Nothing is easier than to persuade people who want to be persuaded and already believe." - another Petrarca gem.

This message is a reply to:
 Message 16 by Taq, posted 02-28-2013 11:37 AM Taq has replied

Replies to this message:
 Message 20 by Taq, posted 03-01-2013 7:30 PM herebedragons has not replied

  
Taq
Member
Posts: 9973
Joined: 03-06-2009
Member Rating: 5.7


Message 19 of 20 (692313)
03-01-2013 7:24 PM
Reply to: Message 17 by herebedragons
03-01-2013 9:03 AM


Re: Basic Methodologies
You did a good job of describing the methodologies of this experiment, but since you titled this thread "Application of the Scientific Method" I had hoped you would have spent a bit more time explaining how the scientific method was applied.
I did fail to spell it out in a concise manner. The steps are there in the posts, but they are spread out and not put together in an obvious way.
Observation: Bacteria become resistant to antibiotics
Hypothesis: Random mutations produce a new phenotype that is resistant to antibiotics.
Null hypothesis: The new phenotype is not due to random mutations.
Experiment: Produce antibiotic replica plates from drug free master plate.
Predictions: The hypothesis predicts that resistance will be clonal and the mutation will arise prior to exposure to antibiotics. If the colonies on the replica plates come from the same position on the master plate then the hypothesis is supported. If the resistant colonies do not come from the same position on the master plate then the null hypothesis is supported.
Experimental results: Resistant colonies on the antibiotic plates come from the same area of the master plate.
Conclusion: The results of the experiment are consistent with random mutations producing a resistant phenotype in the absence of antibiotics.
Further experiments: . . . coming soon

This message is a reply to:
 Message 17 by herebedragons, posted 03-01-2013 9:03 AM herebedragons has not replied

  
Taq
Member
Posts: 9973
Joined: 03-06-2009
Member Rating: 5.7


Message 20 of 20 (692316)
03-01-2013 7:30 PM
Reply to: Message 18 by herebedragons
03-01-2013 9:21 AM


Re: Basic Methodologies
Based on this experiment we can conclude that the mutations for resistance to this antibiotic are consistent with random mutations. But is it reasonable to infer that all mutations operate under the same principals?
It is not reasonable without further experimentation. If, however, disparate phenotypes arise in a similar manner as seen with antibiotic resistance then you can start to hypothesize that there is a basic mechanism that ties all of the together. For example, what if bacteriophage resistance can also be shown to come about in a similar fashion, or mutations that revert a negative mutation in a lactase gene? Even more, what if it is shown that deleterious mutations arise in a similar manner, such as those that knockout an important amino acid synthesis pathway?
I think it is also important to point out that the simple experiment discussed in the previous posts was first done before DNA had even been discovered. Since then we have a much better understanding of the molecular basis of genetics. However, I think it is important to start with these foundational experiments to show how we got to the theory we have now. I think it is also important to show that we do not just assume that mutations are random. We have reasons why we conclude that they are random.

This message is a reply to:
 Message 18 by herebedragons, posted 03-01-2013 9:21 AM herebedragons has not replied

  
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