Changes

How did Lenski obtain Cit- clones from later populations after Cit+ had become the dominant phenotype? It's pretty standard procedure. First, keep in mind that these populations contained a minority of Cit- cells. Now, a small liquid growth is made from the frozen glycerol stock of cells from a given generation, say 32,000. This is grown to an appropriate OD and then plated on a standard rich media agar plate. Provided that the plated sample isn't too dense, this plating will deposit somewhere between 15-100 individual cells on the agar, with plenty of spacing between them. The plate is then incubated at 37 C for a given time, during which the individual cells replicate to form small, visible colonies. These colonies will consist solely of cells that are genetically identical to the original cell deposited on the agar. This is all well and good, but how do you find which of the colonies are Cit-? Here you use a technique called replica plating. The agar plate is gently inverted onto a sterile swatch of velvet so that some of the cells from each colony are deposited onto the fabric. Next, a citrate-only agar plate is pressed against the velvet, transferring cells from the fabric to the new plate in exactly the same spacial orientation as the first plate. The cells are then allowed to grow on the citrate-only plate. When we then compare the two plates, we look for colonies on the rich plate that did not grown on the citrate-only plate. These colonies will be Cit-, and can be used for the replay experiments. [[User:Gerlach|Gerlach]] 13:45, 14 July 2008 (EDT)

How did Lenski obtain Cit- clones from later populations after Cit+ had become the dominant phenotype? It's pretty standard procedure. First, keep in mind that these populations contained a minority of Cit- cells. Now, a small liquid growth is made from the frozen glycerol stock of cells from a given generation, say 32,000. This is grown to an appropriate OD and then plated on a standard rich media agar plate. Provided that the plated sample isn't too dense, this plating will deposit somewhere between 15-100 individual cells on the agar, with plenty of spacing between them. The plate is then incubated at 37 C for a given time, during which the individual cells replicate to form small, visible colonies. These colonies will consist solely of cells that are genetically identical to the original cell deposited on the agar. This is all well and good, but how do you find which of the colonies are Cit-? Here you use a technique called replica plating. The agar plate is gently inverted onto a sterile swatch of velvet so that some of the cells from each colony are deposited onto the fabric. Next, a citrate-only agar plate is pressed against the velvet, transferring cells from the fabric to the new plate in exactly the same spacial orientation as the first plate. The cells are then allowed to grow on the citrate-only plate. When we then compare the two plates, we look for colonies on the rich plate that did not grown on the citrate-only plate. These colonies will be Cit-, and can be used for the replay experiments. [[User:Gerlach|Gerlach]] 13:45, 14 July 2008 (EDT)

:Just a correction: The Cit+ cells didn't dominate the cultures at generations 32,000 & 32,500. Cit+ mutants represented 12% and 19% of the population at these times points, respectively. Replica plating is definitely one means of identifying Cit- and Cit+ clones but because the Cit+ cells represented a fraction of the total, it is simple enough to streak the samples to individual colonies (founded by single cells) and test each colony individually. Otherwise, a nice description of replica plating (a technique developed by Joshua Lederberg that allowed him do the research for which he was awarded one of the 1958 Nobel Prizes in Medicine)

:Just a correction: The Cit+ cells didn't dominate the cultures at generations 32,000 & 32,500. Cit+ mutants represented 12% and 19% of the population at these times points, respectively. Replica plating is definitely one means of identifying Cit- and Cit+ clones but because the Cit+ cells represented a fraction of the total, it is simple enough to streak the samples to individual colonies (founded by single cells) and test each colony individually. Otherwise, a nice description of replica plating (a technique developed by Joshua Lederberg that allowed him do the research for which he was awarded one of the 1958 Nobel Prizes in Medicine)--[[User:Argon|Argon]] 18:38, 14 July 2008 (EDT)

== 1. Lenski's "historical contingency" hypothesis, as specifically depicted in Figure 3, is contradicted by the data presented... ==

== 1. Lenski's "historical contingency" hypothesis, as specifically depicted in Figure 3, is contradicted by the data presented... ==