Hi Barbara,
On a different thread you posted ...
I would like it fully explained the exact detailed process in determining bacterial resistant to antibiotics. The explanations so far on other threads are open to interpretation because the conclusions are not shared by many viewers.
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*10
11 or 100*2
32 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,
WK
Edited by Wounded King, : No reason given.