Originally Posted by Alvin719
Thanks, but I have a question. Why do I have to put it in ice? I understood that he said that putting it in the water bath with make the cell membrane permeable, but what is the point of putting it in ice, water bath, back to ice, and then let it stand in room temperature for 10 minutes? Thanks
Keeping the E.coli in the ice bath decreases the membrane channel activity. For your experiment, you have to get each of the variables (LB, AMP and ARA) inside the cell, so one way is to create small holes in the membrane using chemicals. I'm sure you're going to do this or your teacher will explain it but one way is to use CaCl2. When you rupture the membrane, the contents inside the cell may leak so the ice bath prevents this.
When you put it into the water bath, the membrane undergoes heat shock so the previously made holes will seal.
When you return the cells to the ice bath, you decrease the activity of the channels and prevent the heat shock from causing too much damage. Remeber that the membrane is 3-D so the water will affect all of the membrane, meaning some parts can get denatured so the ice bath serves as a buffer in this sense.
Returning the E.coli to room temperature allows for it to be at around 37C, which is somewhere around 100F. E.coli lives inside you and this temperature is the optimal temperature for it to grow, so when you introduce each sample to each platelet of LB, AMP and ARA, you want the E.coli to be at maximal effeciency for the effects to take place.
I guess an analogy of it would be someone whose heart has stopped beating (the ice bath). When you try to use the paddles, you attempt to bring the person back (the water bath) but don't want to keep using the paddles when the person's heart is beating (the ice bath acting as a buffer). Then, you bring them to normal body temperature, stabalize them and give any necessary medications (room temperature). That's the way I think of it and at each step of the way, I think of how the microbiology will lead to the analogical goal.