Genetic variability in a bacteria species

Well it doesn't but then that site isn't the original paper, it is a simplified precis. The actual original replication experiment paper can be found here (PDF). There will be more than one bacterium in the culture plated out, but it is unlikely that there will be more than 1 resistant bacterium so close together as to appear as only 1 colony unless the resistance strain already has a high frequency. Not there, but this is standard practice. To make sure that there is one originating bacterium the sample to be plated out will usually be grown from a colony from a streaked plate, a technique specifically developed to isolate colonies originating from a single bacterium.In the original paper they say 'In a typical experiment, a dense broth culture was grown from a single colony on plain agar'.When discussing a streptomycin resistance based experiment (Page 7 of the PDF) they mention having to grow up massive amounts of bacteria because the mutation was so rare. 20 or 30 plates with 3 X 10⁹ bacteria plated out on them and only 3 resistants. Can you explain to us how this could be the case if the mutant was pre-existing (even as just 1 bacterium) in the initial starting colony as you posit?TTFN,WK

OK, now I'm confused. How does the Lederberg's 1952 paper documenting their experiments not relate to the the Berkley site offering a precis of the basic methodology of the Lederberg's experiments published in 1952?Did you actually read and understand either of them? No, it wasn't, it showed the evolution of resistance. Except you didn't reference anything other than the Berkley page which refers to the Lederberg's 1952 work, the only applicable paper is the one I linke to, the fact that you are incapable of recognising that the two are the same is your own problem. The antibiotic in the Lederberg's paper is Streptomycin rather than penicillin, but otherwise there is no difference. I don't know how it is taught at Berkley I only know what that page shows. Like I already said, it is a very simplified explanation of the experiment. The only bit they leave out that is relevant to our discussion is that before the initial plating out the bacterial cuture came from a clonal isolate and was susceptible to the phage/antibiotic. A vastly huge number, essentially incalculable. If you have a more specific question I might be able to be a bit more specific. Well why would you assume that was the survival rate of a mutation? And why would you assume only one mutation occurred in the experiment? With a relatively small bacterial genome and a sufficient period of incubation, not neccessarily a massively long time, there is a very good probability that every single possible point mutation that the genome could undergo has occurred at least once. There is little to no reason to suppose that there is only one possible mutation which gives rise to resistanceIn a selection experiment the reason it survives is because in the presence of antibiotics resistance is a very beneficial trait. Unless it is highly detrimental otherwise I don't know why you think it should necessarily be lost from the population once it has arisen. They do, why you think they don't is beyond me. The Lederberg's paper shows that resistance to bacteriophages and streptomycin can also arise, and the chances are that in some cases you could simply use the replicate plate method to screen for as many selective conditions as you like, that was one of the entire points of the Lederberg's methodology. Because there is no way that makes sense. If the DNA was there why wasn't the original bacteria resistant? If it is turned on in response to the antibiotic why aren't the majority of the colonies on the replicate plates resistant. Well this is vague enough only to be very unhelpfull, care to provide an actual specific example? One point I would make is that people are diploid and bacteria aren't. Bacteria don't have recessive traits like sexual organisms, there is essentially no skipping of generations for bacterial traits.I think you need to take more time to understand these experiments rather than just trying to shotgun nonsensical questions in hopes that one of your wild shots will find some target. If you understood these experiments at all you would understand why many of your questions make no sense.TTFN,WK

Hi Barbara,On a different thread you posted ... I think this is probably a more suitable venue for such a discussion. I'd also like to point out that your argument about explanations being open to question seems vague and unclear.Can you think of anything that isn't open to question? No matter how ridiculous a proposition there is often someone willing to argue it, there is just coming up a conference which questions the idea that the Earth is not the centre of the universe. The fact that someone disagrees, especially with the conclusions, doesn't really make an explanation any better or worse.Be that as it may, I'll try and take you step by step through the process.1. An initial clonal population grown from one cell is isolated on non-selective media, i.e. media which provides sufficient nutrients for the bacteria to grow freely. This is usually done using the plate streak method which has been discussed earlier and indeed was discussed on the page you yourself linked to in Message 34.2. One colony is picked off the plate and transferred to a non-selective liquid culture medium such as Luria broth.a) If at this stage you want to check that your chosen colony is susceptible to the antibiotic you want to test you can allow the colony to regrow and then place it into a liquid culture medium containing your selective antibiotic, if there is no growth observed then you can be sure that the original bacteria did not have resistance. Alternatively the plate replicated method could be used to copy the entire original plate to a selective solid medium and see if anything grew from our initial colony.3. This is then allowed to culture overnight at 37^oC, allowing for the lag phase and several rounds of replication (which we can call generations) during the log phase. During this period many common strains of E. coli will have a doubling time ~20-30 minutes which means that over ~14-16 hours if we had 100 cells from our original isolated colony we should now have ~4.3*10¹¹ or 100*2³² cells. Bear in mind that 100 cells would be an incredibly small number for a picked colony.4. If we follow the Lederberg's protocol, which was being discussed previously, the next step is to plate out the cells again onto non-selective media. To some extent the concentration of the cells we want to plate out is dependent on how frequent our mutation is.If we have some idea of the frequency then we will have an idea what concentration to use. If we don't then we may want to make a series of dilutions or even to concentrate the cells from our culture and then make a series of dilutions. In the Lederberg's experiments they plated out a fairly substantial number of cells sufficient to produce a lawn of bacteria.5. The plates are allowed to grow up for ~4-6 hours, the replica plating method, stamping with a velvet cloth and transferring it to a new plate, is then used to make a copy of each plate onto a corresponding selective plate containing antibiotic.a) With a very dense lawn multiple copies may be desirable as you will probably not get a transfer of every discrete clonal population. Such replicates can be compared to identify the regions containing the resistanct bacteria.6. The replica plates are cultured overnight and any bacteria which have already gained resistance will be able to grow on the selective plates.7. If a confluent lawn of bacteria was the source then resistant clonal areas can be identified on the corresponding non-selective plate and used to isolate single resistant bacteria. If the initial plating was at a low enough density then single resistant colonies may already be isolated enough.This method allows resistant bacteria to be isolated from previously non-resistant bacteria without the principal replicating line ever being exposed to the antibiotic. If we keep picking our colonies from the non-selective plates only guided by our replicate plates.Do you think you understand the approach? We can know that our original strain was not resistant and we can identify and select for a resistant strain from an ongoing population which is never exposed to the antibiotic.Can you explain what you think is open to interpretation? I put it to you that all of ICANT's objections were due to his failure to read and understand the experimental procedure from the original paper which he was directed to multiple times. As a reminder that paper is 'REPLICA PLATING AND INDIRECT SELECTION OF BACTERIAL MUTANTS', J Lederberg and E Lederberg, Journal Lof Bacteriology, 1952 (PDF).TTFN,WKEdited by Wounded King, : No reason given.