"Cell Culture Cell Cycle Lab"
Cell Culture/ Cell Cycle Lab Introduction During the next two weeks, we will be exploring the world of cell growth and the cell cycle. As future biomedical engineers, you will almost undoubtedly have to work with cells of many types. Although there are many differences between different cell types, there are a few commonalities most cells share. Two of these similarities are 1) all cells grow and 2) most cells divide regularly. In order to study cell behavior, a scientist (you!) needs to understand how cells grow in order to keep them healthy in an in vitro (outside the body) environment. When cells are incubated in media containing nutrients for them to live off of, the cells experience a particular growth pattern, known as the growth curve. There are four major phases of this curve (see Figure 1): 1. Lag Phase. Immediately after inoculation of the cells into fresh medium, the cell population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity. The length of the lag phase can be influenced by the chemical composition of the media and initial number of cells. (Fig. 1, A) 2. Exponential (log) Phase. The exponential phase of growth is a pattern of regular growth where all the cells are dividing in an exponential manner (total cell number=No*2n, where No=initial number of cells and n=number of generations). The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. (Fig 1, B) 3. Stationary Phase. Exponential growth cannot continue forever in a closed system. Over time, the nutrients in the media decrease (because the cells consume it all), and the amount of waste produced by cells increase. For these reasons, cell population growth levels off. During this time the rate of cell division equals the rate of cell death. (Fig 1, C) 4. Death Phase. Detrimental environmental changes like nutrient deprivation and toxic buildup cause an exponential death phase. If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. (Fig 1, D) Log10 number of cells C D B A time Figure 1: graph of typical growth curve It is important to note that all cells have varying lengths of time that they are in a particular phases. All cells, both prokaryotic and eukaryotic, grow and divide in a time and environment dependent manner. Only by measuring the number of cells over time that one can give a good prediction of cell growth phase as a function of time. Once the cell growth graph is obtained, one can estimate the growth rate constant () and mean generation time (g). The growth rate constant is a measure of the number of doublings that occur per unit of time in an exponentially growing culture. The mean generation time indicates how long it takes to double the cell population. Equations to describe this information is found below: µ = ( (log10 Nt - log10 N0) 2.303) / (t - t0) g = (log10 Nt - log10 N0) / log102 Nt=number of cells at time t N0=number of cells at initial time point t= time when number of cells are Nt to= time when number of cells are No (note: growth rate constant and generation time are related to the exponential phase and the points should be selected from this phase) Overview of Lab In this lab, you will be given a solution of E. Coli bacteria cells in the middle of the log phase of their growth curve. You are to monitor their growth directly and indirectly: 1) counting the number bacterial colonies and 2) using the turbidity of the cells in the flask, you will measure optical density (percent absorbance). You will then analyze those results to obtain the growth rate constant and doubling time. Proctocol Before you begin, do the following Clean your area with ethanol very well. Strike up the bunsen burner label 4 sets of tubes 10-1, 10-2, 10-3, … 10-6. Put .9mL of sterile dH20 into each tube using sterile technique. Every 30minutes, do the following: 1. Remove 100uL from the flask of cells and put into test tube labeled 10-1. Mix well. Take a fresh tip and put 100uL from the 10-1 sample into the test tube labeled 10-2. Continue these serial dilutions until you have gotten down to 10-6. 2. You will use the 10-6, 10-5, and 10-4 samples to plate onto the LB/Amp plates. Remove 100uL from the 10-6 test tube and put it onto the LB/Amp plate. Spread the cells all over the plate with a flamed cell spreader. Let your plates stay on the bench for a few minutes before you put them in the incubator. When you put them in the incubator, do so with the lid facing down to prevent moisture from getting onto the plates. 3. Remove another 3mL from the flask and put into a test tube to be read in the spectrophotometer for percent absorbance (optical density). After you have taken the OD, make sure you write down the time and the reading. 4. Return the next day and count your plates to see how many colonies you have. If there you have plates where there are too many colonies to count, then count only the plates with higher dilutions. To calculate how convert number of colonies to cells/mL: (number of colonies)*(inverse of dilution concentration)(10) if you count 30 colonies in the 10-4 solution, and plate 0.1mL onto the plate, (30*104*10) = 3*106 cells/mL **** Do at least 4 time points. NOTE: The more timepoints you do, the better your graph will look!!!**** 5. Calculate the log of the number of cells/mL, and draw a graph of your growth curve as log10 number of cells as a function of time, ie: Time No. cells/mL 1og10(x) OD600 counted (x) reading 0 50 1.69 2.1 Graph the bolded boxes 6. Calculate the growth rate constant and generation time from the log phase values you recorded and equations above. Additonal Informtion Because you received the culture in the middle of the growth curve, you will be provided with the timepoints, cell number/mL, and optical densities in the lag phase: Time (h): Viable cells/ml: O.D. 600: 0.5 3.1x101 0.64 1.0 3.2x101 0.65 1.5 8.4x101 0.85 2.0 2.58x102 1.10 2.5 8.57x102 1.36 3.0 2.11x103 1.55 3.5 9.80x103 1.89 4.0 2.04x104 2.05 4.5 5.92x104 2.28 5 5.0 1.90x10 2.53