Optical Density

Last Wednesday (February 6, 2013) I worked with Eun Ji. We didn't do much because she was very busy and had already done her experiment before hands.

At 2 pm, we attended a mandatory safety education at one of the largest laboratory because a few weeks ago an undergraduate was in a lab alone and for some reason hurt himself / herself quite severely. They demonstrated the emergency eye wash and shower. They also invited some people to try pulling the "pole" (I don't know how to describe it) so we can get familiar with the force. It's actually really fun because it was my first time to see the shower on! In the end, they really emphasizes that safety is more important than flooding the laboratory.

Later, Eun Ji showed me how to measure optical density of bacteria. Optical density (OD) is the measure of the amount of light absorbed by a suspension of bacterial cells or a solution of an organic molecule with the use of a spectrophotometer. The more bacteria in a solution, the higher the OD. Eun Ji started to grow her bacteria the day before and measure its OD every 2hrs. Each type of bacteria has the greatest protein production efficiency at a certain OD, in this case at OD = 0.5. Thus, when Eun Ji's bacteria reaches OD = 0.5, she will inject a reagent, and the bacteria will start to make the protein she wants. The last time she checked, the OD was still 0.4, so she hypothesize  it would reach 0.5 at 2 pm.

However, when we checked it, the OD has already exceeded 0.8!! Eung Ji explained that it's because the bacteria had now reached the exponential phase (2nd), in which they multiply very rapidly. I thought we had to start the whole experiment all over again, but luckily all we need to do is to dilute it:)

So we went back to the original lab to dilute the solution. However, doubling the volume does not necessary means lowering OD by half, so we can only do some trial-and error. We really need to well-mix the solution when adding new medium, so she keeps pouring back and forth the solutions (though she said ideally we would use pipettes, but for the sake of time...).

The middle two have bacteria (milky), and the outer two are the new medium we want to add to the original solution to dilute them.

The second time we got 0.65, still too high. We ending up adding about 200ml of medium, and the OD was successfully reduced to 0.53!

OD measured by spectrophotometer; OD = 0.532

Eun Ji then added some reagents to the solution and took them down to a cell growth room where the cell incubator shaker was set in 30C. Then, she will have to check it 6 hrs later. Eventually, she will break the bacteria and collect the desired protein.

It was interesting to see how much process one has to do just to get a desired OD although I didn't get to touch anything because Eun Ji said that type of E.coli. can, but rarely, cause diarrhea, which is also why she used alcohol to wipe the flask every time she opened it. We ended early because she has a group meeting at 3:30, so I spent the last half hour doing my work. Next week I will be working with Namita, and I am looking forward what we are going to do together:)