Experiment 2: Bacterial Quantification PDF

Experiment 2: Bacterial Quantification by Spectrophotometry and CFU Counting After this lab, students should be able to: Demonstrate the use of serial dilutions [something about the math] Set up a growth curve experiment and calculate growth rate Compare the results from two di?...

Experiment 2: Bacterial Quantification by Spectrophotometry and CFU Counting After this lab, students should be able to: Demonstrate the use of serial dilutions [something about the math] Set up a growth curve experiment and calculate growth rate Compare the results from two di?erent methods Purpose: To calculate the doubling time of an E. coli culture through two methods and compare the two methods. Background Growth Curves Bacteria grow by binary fission, experiencing exponential growth as the number of cells doubles every generation over a constant time interval. The amount of time that it takes for a bacterial cell to divide (or for a culture to double in density) is known as the doubling time. The rate of doubling is dependent on many things, including environmental conditions such as temperature and availability of nutrients, as well as characteristics of the bacteria being studied. A typical bacterial growth curve exhibits distinct regions corresponding to the di?erent stages of bacterial growth. During the Lag Phase, cells adapt to the medium and there is limited cell division while the culture responds to the change in environment. During the Exponential Phase, cells divide rapidly through binary fission, where one cell becomes two. This phase is where cells are growing at their maximum rate. During the Stationary Phase, division slows as nutrients decline and waste products build up. Eventually, growth will be minimal, and the bacterial density will level o? at a steady maximum. Some growth curves will additionally include a Death Phase, where cells die o? due to lack of nutrients, resulting in a decline in cell density. Our experiment will not last long enough to reach this point, but you may see it depicted in some resources. Lag Exponential or Number of Bacteria (log scale) Stationary Phase Death Phase Phas Log Phase Time (minutes) Figure 2.1: Example Bacterial Growth Curve Quantifying Bacterial Growth To quantify bacterial growth, the change in cell density over time is measured. Two common ways of measuring cell density will be used today to monitor the growth curve of your bacteria: spectrophotometric measurements of turbidity (OD600), and viable cell counts, measured in Colony Forming Units per milliliter of media (CFU/mL). Turbidimetric (OD600) Measurements The basis of turbidimetric measurements is the fact that bacteria are large enough to scatter light when grown in a liquid culture. Cellular components scatter light passing through the liquid suspension, and the density of these components increase as the culture grows. Thus, the turbidity of the solution is proportional to the cell density in the solution. We will use spectrophotometers to measure this turbidity over the course of the experiment. A spectrophotometer works by passing light of a specific wavelength through a sample and measuring what proportion of light reaches a detector on the other side. By comparing to a “blank” of liquid media containing no bacteria, the machine will report the absorbance of the solution – a representation of how much light is blocked by the contents of the solution. Denser solutions with more cells will have higher absorbance readings because less light is transmitted through the turbid solution. OD600 = 0.000 Light Source Sterile LB Broth Spec detects No bacteria = light @600nm no absorbance OD600 = 0.630 Light Source LB Broth with Some light is blocked or scattered by growing bacteria bacteria, increasing the absorbance Figure 2.2: Example of Spectrophotometric measurement of cell density For this experiment, we will be measuring the absorbance at 600 nanometers. Viable Cell Counts The other method we will be using to quantify bacterial growth is through a viable cell count. One downside of OD600 measurements is that a dead cell scatters light the same way as a living cell, even though it is not part of the growth at that point in time. In a viable cell count, a small volume of media is plated on LB agar at each time point and incubated overnight. The number of colonies on each plate are counted after incubation, with the assumption that each colony on the plate arose from a single isolated bacterium. By taking into account the volume of liquid plated, one can calculate the number of colony forming units per milliliter of solution (or CFU/mL) at each time point and plot a growth curve in this manner. Serial Dilutions Viable cell counts require that it is possible for a researcher to count the number of cells on each plate. However, bacteria are incredibly miniscule, and it is possible for there to be thousands of bacteria present in a single drop of liquid culture. Scientists use a process called serial dilutions to methodically dilute a liquid culture until the concentration of bacteria is low enough that it will be possible to count the number on a given plate. In our serial dilution setup, an initial stock solution is diluted 10-fold into a volume of fresh media – by adding 20µL of bacterial culture to 180µL of sterile LB, we have made a sample that is 1/10 the concentration (10-1) of the original. 20µL of this sample is then added to another 180µL of sterile LB, creating a sample with 1/100 (10-2) of the original. This process is repeated several more times, resulting in a series of samples that are all 10 times more dilute than the previous. We can then add a small volume from specific dilutions to LB plates and count the growth later. Helpful Hints for Serial Dilutions: Careful pipetting is essential for this process to succeed! Make sure your equipment is set to the right volume and that you are pipetting properly. Set up your arrangement with your lab partner and make sure you are on the same page about what sample is which – you will have a lot of LB by the end of this process, and small miscommunications can harm your results. When diluting the bacteria, make sure that you are changing pipette tips after each dilution! It is also important to thoroughly mix the sample at each dilution by slowly pipetting up and down. Materials (for each pair of students): Flask of sterile LB broth, 25mL Tube of sterile LB broth, 15mL 96-well plate for making serial dilutions LB agar plates Cuvettes for OD600 measurements Sterile glass beads for plating Procedure: 1. Prior to lab, your instructors inoculated 25mL of LB broth with a sample of E. coli and have been incubating it at 37°C. Your TA will tell you which culture is assigned to your group - retrieve it from the shaking incubator and bring it to your lab bench. Examine the culture – is it cloudy? 2. Pipet 2mL of this culture into an empty cuvette, and 200µL into well A1 of your 96-well plate. These will be your samples for time point 0. 3. Label your flask with your group info and return it to the shaking incubator. Set a timer for the next time point (either 30 or 20 minutes – your TA will specify). 4. Perform the measurements on the time 0 samples according to the following protocols. Between time points, you can prepare for the next time points and plan with your partner. 5. At each time point, take another 2mL of your culture for the OD600 readings and add 200µL to the next column in your 96-well plate for your serial dilutions. Repeat until the end of class. 6. Clearly label all your plates and tape them together for your TA to incubate. Place all liquid cultures in the waste receptacles in the fume hood, and solid waste into the biohazard bins as directed by your TA. Taking OD600 Measurements 1. Add 2mL of sterile liquid LB to an empty cuvette to serve as a blank for the spectrophotometer. o Save this tube between measurements! 2. At each time point, take 2mL of bacterial culture from the flask and add it to a fresh cuvette. 3. Confirm the spectrophotometer is set to 600nm. Place the tube of sterile LB in the spectrophotometer, close the lid, and press the “blank” button. Wait for the machine to read 0.000. 4. Remove the blank and replace with the bacterial culture. Close the lid, wait for the spectrophotometer to settle, and record the OD600. o It’s okay if it doesn’t settle completely, but the value shouldn’t be changing by more than a couple of thousandths. Serial Dilutions and CFU Plating 1. For convenience and ease of organization, we will be making serial dilutions in a 96-well plate. Each column will contain dilutions for a given timepoint, with descending rows containing serial 1:10 dilutions (see Fig 2.3). 2. At the first time point, pipet 200µL of your culture into well A1. 3. Pipet 180µL of sterile LB broth into wells B1 through H1 in the first column. 4. With a fresh pipette tip, add 20µL of culture from well A1 to well B1. Pipet up and down slowly to mix. Well B1 now contains a 1:10 dilution of the culture at the first time point. 5. With a new, fresh pipette tip, add 20µL of B1 into C1 and mix. Repeat until you have made dilutions down the entire column, mixing and changing tips after each dilution. a. The 8th row now contains a 10-7 dilution of your original culture. 6. Plate the dilutions from rows E, F, and G (10-4, 10-5, and 10-6) for the time point. Your TA will tell you which of the following plating methods to use.  7. At the next time point, repeat steps 2 – 6 in the next column until you have completed all time points. Figure 2.3: Potential schematic of 96-well plate for serial dilutions Drop Plate Method 1. Take an LB agar plate and divide into three equal sections. Label the plate with your initials and time point and label each section for one of the three dilutions. 2. Carefully pipette 10µL of a dilution onto its section. Repeat twice more so that you have three drops of the dilution in the same section – be careful not to let them touch! 3. Repeat the process with the other two dilutions in the other two quadrants. Glass Bead Spreading 1. Obtain three LB agar plates. Label them with your initials, the time point, and the dilution. 2. Carefully pipette 100µL of the appropriate dilution onto the plate. 3. Add 6-8 glass beads to the plate and return the lid. Shake gently for 30 seconds to spread the liquid across the plate. Let sit for 2 minutes, then dispose of the glass beads in the burn-up bin. a. Don’t use more glass beads than are necessary! b. Spreading may also be done with a disposable plastic “hockey stick” depending on supplies. Data Table Time of Absorbance # of colonies Dilution Volume plated Calculated incubation (min) @600nm on plate (in mL) CFUs/mL 0 10-4 10-5 10-6 Data Analysis: Count the number of colonies on your plates. If there are too many colonies to the point where it is not possible to count individual colonies, you may write “TMTC” for “too many to count.” If you used the drop plate method, write down all three values for a given dilution, and average them if they are similar. Self-check: if your serial dilutions were done perfectly, each dilution would be a factor of 10 more concentrated than the next lowest. How close are your values to this ideal? Calculate the density of the original culture in CFUs/mL for each dilution at each time point. This can be done with the following formula: # 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑜𝑛 𝑝𝑙𝑎𝑡𝑒 𝐶𝐹𝑈/𝑚𝐿 = 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 ∗ 𝑣𝑜𝑙𝑢𝑚𝑒 𝑝𝑙𝑎𝑡𝑒𝑑 (𝑖𝑛 𝑚𝐿) For example, if you counted 137 colonies on your 10-5 plate using the glass bead method: 137 𝐶𝐹𝑈 = 1.37 ∗ 10# 𝐶𝐹𝑈𝑠/𝑚𝐿 10!" ∗ 0.1 𝑚𝐿 Once you have calculated the CFUs/mL for each dilution at a given timepoint, compare them to each other. They should be relatively close! Average them if they are close together. If they are not close, look at your plates and see if you can find any potential causes of error in the way you plated, such as a clumping of colonies that might indicate you did not spread the liquid evenly. Questions for Homework: 1. Organize your data in a table, including the absorbance and CFUs/mL at each timepoint. Don’t forget units! 2. Create two graphs: OD600 vs. time and CFU/mL vs. time. Do your graphs follow the same trend? How similar do they appear? 3. After constructing the previous graphs, construct a plot of OD600 vs CFU/mL. What trend do you expect to see? What trend do you actually see? Suggest an explanation for any di?erences. 4. Identify the portion of time when your samples are in the exponential phase of growth. How can you tell they are in this phase? What stage of growth were your bacteria in at timepoint 0? 5. Using the following equation, calculate the generation time (also called doubling time) for three successive pairs of time points during exponential growth (e.g. 20 – 40, 40 – 60, and 60 – 80). Average and report the generation time for your culture. For clarity, t is the time at which the measurement was taken, and N is the bacterial concentration at that time. You may use either CFU/mL or OD600, but you must use the same metric for all three comparisons. 0.3 ∗ (𝑡$ − 𝑡% ) 𝑔= 𝑙𝑜𝑔$% 𝑁$ − 𝑙𝑜𝑔$% 𝑁% 6. How does your generation time compare to the known generation time of ~20 minutes for E. coli? If these are di?erent, what explanations can you provide to explain this discrepancy?