PreLab Activity: Read through the entire lab! Come prepared to explain to your group what this lab is about

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1 BIO101Lab weeks 4 and 5 PreLab Activity: Read through the entire lab! Come prepared to explain to your group what this lab is about The Evolution of the bacterium, Pseudomonas fluorescens Introduction Evolution is defined as the change over time. Biological evolution is defined as the heritable genetic change in a population over multiple generations (time) in the presence of selective pressures. These changes can be small such as the development of bacterial resistance to an antibiotic or the improved eyesight of mice living in dark sewers. Some changes, however, can be large. These changes can eventually lead to distinct species that are unable to breed with one another. But which changes cause organisms to evolve? Certain traits are selected for naturally via their conferring success or fitness to an organism in a given environment. These traits are maintained in the gene pool of a population of organisms, and are heritably passed on to subsequent generations during reproduction. You may have heard this process called natural selection. The ability to pass on traits to the next generation is what Darwin meant by the fitness of an organism. He was not describing strength, but rather the ability of organisms to survive and reproduce, thus maintaining their traits in the next generation. The environment in which this population lives provides the selective pressures that determine which set of genes confer success in that environment. This clarification is important to note; it is not the organism, but rather the environment that selects for change. As you may have already guessed, the traits we are describing are genes made up of DNA. At the heart of evolution, and unbeknownst to Darwin, is the genetic code. Random and spontaneous mutations arise in the DNA of all living organisms. Some of the mutations are harmful to the organisms, such as those that cause cancers in humans, whereas others are helpful for surviving in a particular environment. For example, malaria is a serious parasitic disease (primarily in Africa) caused by four different Plasmodium species transmitted to humans through the bite of an infected female Anopheles mosquito. In humans, the malaria parasite reproduces in the liver and red blood cells, causing sickness (i.e. vomiting, fever, anemia). If malaria goes untreated, then the parasite can eventually cause death. Moreover, chronic malarial infections are associated with a childhood cancer known as Burkitt s lymphoma. Current drug therapy works to help clear the infection, but some malaria have now become resistant the traditional therapies (yet another example of evolution). However, there are some individuals who are resistant to malarial infections altogether because they contain a genetic mutation in the HbS gene (encodes for hemoglobin) that prevents the replication of the parasite in the red blood cells. These individuals are more fit for survival in the African countryside where malarial infections are common. Unfortunately, these individuals develop a disease known as sickle cell trait, which is caused by the same mutation that protects them from malarial infections (this is an example of an

2 evolutionary trade-off). Whereas a mutation in HbS provides an advantage to these individuals to survive so they may pass on their genetic material to the next generation (Darwin s definition of biological fitness), individuals with sickle cell trait have a shorter overall survival than African individuals who do not have the mutation. Moreover, some mutations have no effect whatsoever. The random mutations that provide success within an environment will be passed to the next generation to ensure their success, so long as they survive long enough to reproduce. Today, we will examine evolution in a controlled, laboratory setting. You will work with a bacterium known as Pseudomonas fluorescens. Pseudomonas is a large phylum of bacteria that all have at least three similar traits: they are motile (have flagella to help them move in culture), they are strict-aerobes (require oxygen to grow), and they form biofilms. P. fluorescens is a non-pathogenic (does not cause human disease) species that grow in the soil, plants, and water surfaces. You will work with a particular strain of P. fluorescens called SBW25, which grows in the soil, and is commonly associated with plant roots. Today you will use the SBW25 strain to study evolution in a static culture and observe the heritability of adaptive traits that are at the heart of evolution.

3 Materials used in this Laboratory Exercise: - King s B broth - King s B agar plates - SBW25 wild-type bacteria streaked on a King s B media agar plate - Sterile pipettes, 5 and 10 ml - Pipette bulb or pipetaid - P200, P1000 pipettes - P200, P1000 pipette tips - Sterile test tube or culture tube, 13 mm diameter or larger - Tubes for growing P. fluorescens in static culture, each with 6.5 ml of King s B broth - Flint or lighter - Bunsen burner - Spreading rod - Ethanol jar - Inoculating loop - Sharpie marker - Sterile water - Sterile epitubes, 1.5 ml, each with 500 µl of King s B broth - Bleach - Vortex - 28 C incubator - Test tube rack

4 Procedure: Day 1 (10/6) Today s Laboratory Objectives: You and your partners will observe the shaken and static cultures of Pseudomonas fluorescens and wild-type colonies of P. fluorescens on an agar plate. Next, you will perform a serial dilution from the shaken and static cultures so you will be able to calculate the number of bacteria in your culture and identify the morphologies of the bacteria in the respective cultures. The SBW25 strain of Pseudomonas fluorescens was grown in a shaken culture of King s B medium (which is selective for the fluorescent Pseudomonads) prior to today. Typically, cultures of bacteria are shaken if the organism is an aerobe (requires oxygen to live) because the shaking ensures that all the bacteria in the culture are supplied with equal concentrations of nutrients and oxygen. Therefore, all bacteria in the culture have an equal opportunity for success. The environment of a shaking culture is not stressful to P. fluorescens. Prepared for you is a sample of the P. fluorescens from the shaken culture on an agar plate of King s B medium. Each colony started as a single cell and all cells in that colony are genetically identical. Observations of the wild-type colony of P. fluorescens and the shaken culture 1. At your bench there should be both a shaken culture of P. fluorescens and an agar plate with colonies of P. fluorescens from this shaken culture. Observe the shaken culture of P. fluorescens. Keep this image in mind as we will compare it to static culture in a few minutes. 2. Observe the agar plate of the P. fluorescens. This plate contains colonies of bacteria from a shaken culture. Describe the colony (i.e. what is its color, morphology or shape, and luster). We will consider these colonies to be wild-type throughout the lab. Observations of the static culture What happens if we change the environment of the P. fluorescens? For example, what if the culture isn t shaken, but rather is static? 3. Get a static culture of P. fluorescens for your group from the back bench. Be careful handling the static culture. It is a fragile environment that will fall apart if you are to shake it around too much. Walk carefully to your bench so that the members of your group can see the culture in its static form. Prior to today, a single colony of the wild-type P. fluorescens was inoculated into a static culture. The lid was put on loosely to allow air-flow in and out of the tube, but to prevent evaporation. The culture was incubated at room temperature for 10 days. 4. Observe your static culture. Draw/Sketch what you observe and then write out what you observe while trying to be as descriptive as possible. While viewing the culture, ask yourself, What are the environmental differences of a shaken and static culture? Work with your partners to explain what you think has happened. What information about P. fluorescens supports your hypothesis? Compare the static culture to the shaken culture. Are there similarities or differences between what you see in one and the other?

5 5. After observing both the static and shaken cultures, separately, mix the contents of each culture completely by either pipetting the cells up and down with a sterile pipette, or vortexing the tube (make sure the lid is on tight before vortexing). 6. Using a sterile pipette, add 4.5 ml of sterile water to each of 8 sterile test tubes. Label the tubes using a sharpie with numbers 1 through 8. Serial Dilution of the static and shaken cultures Next, you will determine how many bacteria are in your cultures (per ml) and the morphologies of the colonies (which actually correlate with genetic changes of P. fluorescens). One way to accurately count the number of bacteria you have in a culture is to perform a serial dilution or a dilution series (Figure 1). In brief, from a culture one makes successive dilutions of that culture to a concentration that will allow for counting colonies of bacteria on an agar plate (called a colony forming unit, or CFU). Remember that each colony represents a single bacterium that multiplied. Therefore, each colony represents one bacterium. One can then calculate backwards to determine the number of bacteria in the original culture by calculating the number of bacteria on a plate and knowing the total dilution that was made from the original culture. There is an additional benefit to serial dilutions: It allows one to observe individual colonies of bacteria and their morphologies. We will determine the number of bacteria in the static and shaken cultures as well as observe the morphology of the bacterial colonies from those cultures. 7. Perform a dilution series of your bacteria from the static culture. Review the next few steps of the lab before beginning the procedure. Fill out the blanks in Figure 1 with how much water you add to each tube prior to diluting, how much volume of each tube you transfer to the next, and the volume that you plate. This will help you determine the dilution of the original static or shaken culture that each plate represents. 8. Add 500 µl (0.5 ml) of the homogenized static culture to the first tube labeled 1. This is a 1 to 10 dilution of your static culture (500 µl of culture in 5.0 ml of total volume, or 500 µl /4.5 ml). Mix thoroughly. 9. Take a new sterile pipette, remove 500 µl from the tube labeled 1 and transfer to the test tube labeled 2. This is a 1 to 10 dilution of the tube labeled 1, and a 1 to 100 dilution from the original culture (1/10 x 1/10 = 1/100). 10. Continue to serial dilute your samples from one tube to the next until you have diluted your bacteria through all eight tubes. Remember to use sterile techniques to ensure you are not contaminating your cultures. Use a new, sterile pipette for each dilution. 11. Obtain three King s B medium plates. Label the bottom of the plates with your partners and your initials, the date, and either 6, 7 or Pipette up 100 µl of the microcentrifuge tube labeled 6 and transfer this to your King s B medium plate labeled 6. How much is this tube labeled 6 diluted from your original culture? What fraction of 1 ml is 100 µl? These numbers will be important to calculate how many bacteria were in your original culture.

6 13. Sterilize your spreading rod with some ethanol and a flame. To do this, dip the rod into the beaker containing ethanol and then simply put it over the flame. Be careful with this step as you are working with an open flame!!! After the flame dies out, your spreader is sterile. 14. Now GENTLY spread the cells around the whole King s B medium agar plate so the bacteria is evenly spread onto the plate. Place your spreading rod back into the ethanol to kill any bacteria that may be on the rod. 15. Repeat steps 9-11 for both the test tubes labeled 7 and Have each member of the group inoculate bacteria on a 6, 7, and 8 plate from the same dilution series. In total, you will have 3 plates of each dilution, 6, 7, and After the water containing your bacteria has dried, place the plate upside down in and incubate the plates at 28 C. 18. Repeat steps 7-16 for the shaken culture. 19. Place your used materials in the proper location. Dispose of used pipette tips properly. Add bleach to all test tubes and the tube that contains the remains of the static culture. 20. Prior to observing your plate, discuss with your partners what you expect to see on the agar plates of bacteria from both the shaken and static cultures. What changes do you expect to see in the bacteria from the two different environments?

7 Day 2 (10/7) Today s Laboratory Objectives: You will observe the King s B medium plate you streaked with bacteria from the static and shaken cultures. Observation of agar plate with bacteria from static and shaken cultures 21. Observe the plates from the shaken cultures. You will see that each of your dilutions has a different number of colonies. Pick one set of plates (i.e. the 6, 7 or 8 ) that has between colonies (if your group has such a set of plates). Each member of the group should have their own plate to count. Average the number of colonies from the three plates in that set. Next, take a look at your other sets of plates (i.e. those you did not count). Approximately how many colonies are on these plates? What is the relationship between the number of colonies in each set of plates? Hint: Think about the procedure for the serial dilution and what tube was used to plate each sample. 22. Now, the numbers you get represent how many cells were in 100 µl of that dilution. For example, you might have counted the set of 6 plates, so you are counting the number of colonies from 100 µl of the 6 th dilution. What we want to know is how many were in 1 ml of the original culture. To calculate the number of colonies (also known as colony forming units, CFUs) per 1 ml of the original culture you need to know what your dilutions were (i.e. were you doing 1:2, 1:5, 1:10, 1:50 dilutions?), how many colonies there are on your plate, what dilution plate you counted from, and what fraction of 1 ml is 100 µl. Try calculating this on your own and we will go over it together when we all meet together. 23. Next, you will likely find that you have colonies that look different from one another from the static culture. In total, you may have up to 3 different colony morphologies (one of which will be the wild-type colony that you observed). Draw a picture of the colony morphologies and write a description of each (as you did on Day 1). What distinguishes the colonies from each other? Give a single or twoword name to each colony type to describe what you see (be creative). Include a brief rationale for the name. We will talk about these colonies at the next lab. Extra Notes: Count a plate with colonies from both the shaken and static cultures. Make sure to note which dilution plate (either 6, 7, or 8) you have counted. Hopefully, you see only (or predominantly) wild-type colonies on the plate with bacteria from the shaken culture. 24. Using the set of plates you counted to determine the CFUs (above), count the number of colonies for all morphologies on plates from both the static and shaken cultures and calculate the percentage of the total number of colonies each represents. Each member of the group should count their own plate. Average these percentages from the plates you counted. 25. Perform steps 5-8 for the plates from the static culture. Extra Notes: If you have 3 observable morphologies, count the number of colonies for each of those three morphologies. 26. You should now know the following: the total number of colonies from one set of plates for both the shaken and static cultures, the dilution number of the set of

8 plates (i.e. 6, 7, or 8 ), the types of colony morphologies in the shaken and static cultures, and the percentages of each of those colony morphologies make up in the shaken or static cultures. Extra Notes: Take 15 minutes out of your weekend to search the internet to determine how to calculate the number of bacteria per ml in the original culture based on the following: the number of colonies you counted and which dilution plate was counted. Additionally, you need to know what fraction of 1 ml is 100 ul. This is important because the number of bacteria you have counted on each plate represents the number of bacteria per 100 ul (the volume you pipetted onto the agar plate). Starting static cultures from 3 colony morphotypes 27. Next, you will test the heritability of the changes observed in the morphology of the mutant bacteria. You will grow both wild-type and mutant colonies of P. fluorescens in separate static cultures. a. Obtain an inoculating loop, 3 epitubes with King s B broth, and 3 flat-bottom culture tubes with 6 ml of King s B broth. The inoculating loop is by the Bunsen burner and the tubes are on the back table. b. Turn on the Bunsen burner c. Using aseptic techniques pick a colony of one of the morphology types you see on your plate and transfer the colony to the eptibube using an inoculating loop. To do this, place the inoculating loop into the flame of the Bunsen burner until it glows red-orange. Pull the loop out of the flame and let it cool in the air for 10 seconds. Pick up a colony off the agar plate with the loop. Open your epitube and swirl the inoculating loop in the broth to dislodged the bacteria from the loop. Close the epitube and vortex it to suspend the bacteria in the broth. Flame the inoculating loop to kill any bacteria remaining on it. d. Take the full amount of broth from the epitube and transfer it to the flatbottom tube using sterile technique. Make sure to run the mouth of the flat-bottom tube over the flame after you have removed the cap. After inoculating the flat-bottom tube with the bacteria, flame the mouth of the tube, again, screw the cap onto the tube, and then unscrew it ¼ to ½ turns so that air can get in and out of the tube (remember, Pseudomonads need oxygen to live). e. Label each tube with initials and an indicator to denote with which colony you inoculated the static culture. f. Put the inoculating loop into the flame until it becomes red-hot to sterilize it and then repeat the process for the other colonies morphs you have seen on your plates. g. Let the tubes incubate at 28 C (in the same incubator as your plates incubated) for 6 days. 28. Put the plate of the colonies from the static culture with which you inoculated the three cultures into the fridge until Thursday (Day 8).

9 Day 8 (10/13) Today s Laboratory Objectives: You will observe the static culture you started from the different morphotypes. This will provide evidence that the morphology and biofilm formation are associated, and that both indicate a heritable (genetic) change. Observation of static cultures 29. Observe your test tubes. What do the culture look like? In what ways do the two cultures look alike? In which ways do they differ? In a few sentences, compare and contrast the different static culture. If you were to plate the bacteria from the static culture on King s B medium plates, what would you expect to see? Why?

10 Post-lab Questions: 1. Explain how the static culture represents at least 3 different environments that could select for different populations. 2. Which colony type(s) gave rise to the biofilm? What could account for the differences between the appearance of the static cultures from the different morphotypes? 3. Explain how the different colony morphologies indicate that evolution has occurred. 4. How do the growth patterns of the different colonies in the static culture show heritability? 5. Describe how the lab is a model of evolution and natural selection? Address: (1) the various environments (shaken and static culture, and the agar plate), (2) natural selection created by those environments, (3) heritable change, and (4) time. 6. How does environment influence adaptation? 7. Think of an example of evolution (it can t be P. fluorescens or the HbS and malaria example given in the introduction of this lab). In a few sentences, describe how this example represents evolution using the terms we used to describe the evolution of P. fluorescens at the end of the lab.

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