Antibiotic Resistance

Antibiotic Resistance A study in natural selection Connections ● The microbiology lab techniques you learn in this lab will be important for future coursework and possibly for working in research labs on campus. ● You have likely heard about medical problems caused by the evolution of antibiotic resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). The same evolutionary forces at work in this experiment are responsible for the development of antibiotic resistance in medical contexts. ● This activity relates to the Microbial Evolution research area in our foundations lab courses. Goals/Skills After completing this lab activity you will: ● Understand general sterile technique and common sources of contamination in a lab setting. ● Be able to count bacterial colonies on an agar plate and estimate cell population size by dilution plating. ● Be able to streak a plate to obtain single bacterial colonies. ● Be able to describe how natural selection acts on existing variation in a population. Make your own copy of this document Remember to make a copy of this document (File>Make a copy), or download a copy (File>Download) to use as your working document. 1

Introduction Natural selection occurs when there is a heritable phenotypic variation within a population, and when having a certain phenotype confers a fitness advantage. Since organisms with the selected phenotype will go on to produce more offspring, over many generations the frequency of the genetic variant responsible for the phenotype will increase in the population. In this lab, we will see if a selective environment will cause a change in the frequency of a phenotype in the bacterium Escherichia coli ( E. coli ). The selective environment will be bacterial media with antibiotics in it. The phenotype in question is antibiotic resistance (i.e., the ability to survive and reproduce when there is antibiotic present in the media). E. coli is a bacterium that naturally occurs in the intestinal tract of mammals. You will notice, in the experiment details below, that we incubate E. coli at 37°C when we work with it in the lab. 37°C is 98.6°F (human body temperature). This is the optimal temperature for E. coli , and the temperature at which they reproduce the fastest. The antibiotic rifampicin is toxic to E. coli because it inhibits RNA synthesis by binding to RNA polymerase and preventing the production of new proteins required for metabolism and growth. Although rifampicin is toxic to E. coli , mutations can occur that enable E. coli to overcome or avoid the toxic effects of the antibiotic. Microbiology techniques In addition to studying natural selection with our rifampicin experiment, you will also learn some important microbiology lab techniques. In this lab activity you will learn how to keep your bench space, lab equipment, and tools sterile in order to prevent contamination of your bacterial cultures while you work (and to prevent your cultures from contaminating surfaces in the lab!). You will also learn some common techniques we use to inoculate bacterial cultures, determine whether a culture is diverse or homogenous, isolate individual colonies of bacteria to work with, and figure out the population density in a liquid culture (how many cells/ml in a culture). These microbiology techniques will be important for future coursework, and are common ones you will come across in many research labs at the university. Overview of the experiment In the first part of this experiment, we will determine if there is existing variability in our E. coli culture, upon which natural selection can act. In other words, we want to find out whether or not there are rifampicin-resistant mutants already present in the population of mostly antibiotic- sensitive E. coli . You will also prepare experimental subcultures of your E. coli culture and see 2

Day 1: Microbiology Skills & Starting the Experiment Sterile technique Microorganisms are everywhere. They are in the air, water, and virtually all surfaces around you. They are also all over you! To study a particular sample of a microorganism, we need to be sure that we have a culture containing only the intended microorganism. In order to do this, we need to use sterile equipment and techniques that prevent unwanted organisms from entering our cultures. Note that sterile technique also prevents the accidental release of the microorganisms you are working with. You won’t work with pathogenic microorganisms in the foundations labs, but it’s possible that in the future you will work with something that poses a health risk. Good sterile technique prevents contamination from going both ways (i.e., from contaminating your lab work and preventing people in the lab from getting exposed to the organism you’re working with). The basic principles of sterile technique are to work quickly and to avoid contamination of your sample from the air or any surfaces/instruments that your sample might come in contact with. Never touch anything sterile with your hands, and avoid breathing directly onto sterile items. Items such as inoculating loops, petri dishes, pipette tips, microfuge tubes, and any glassware used should all be sterilized prior to use. Your lab instructor can show you the autoclave tape that indicates whether or not an item has been sterilized in the autoclave. Also, remember that any item exposed to the air is at risk of contamination. Anything with a lid (test tubes, pipette tip boxes, petri dishes, etc.) should be kept closed except for the brief moment when you need to remove the lid to do your work. Streaking for single colonies When working with bacteria cultures, it is often desirable to obtain single, isolated colonies on a media plate. This can help you separate species from a mixed culture, and is also a way to start a culture of genetically identical cells. Each individual colony comes from one individual cell; therefore a colony is made up of individuals that are genetically identical to each other. In this lab, you will practice the skill of streaking a plate for single colonies (often referred to as “streak plating”). This streak plate will not be part of the antibiotic resistance experiment, but it will help you practice an important microbiology lab skill. Before you get a media plate and begin streaking the E. coli , follow the steps below to practice your streaking plan. 4

1. This diagram shows an agar media plate divided into 3 sections. Draw a similar diagram on a piece of paper or on the whiteboard in the lab. 2. On the diagram, draw a small dot or circle in sector 1, to indicate where you will initially spread the bacteria onto the plate with your inoculating loop. 3. Draw your streak pattern in the first sector by making a tight zigzag line that begins at the dot from the previous step and fills most of the sector. The lines in your zigzag should not touch each other. 4. Start your zigzag streak for the second section by crossing your zigzag in the first section and then streaking into the second section. Continue your zigzag in the second section. 5. Finally, cross your zigzag in section 2 and continue it into section 3, completing the zigzag in the third section. Procedure for streaking a plate for single colonies You will work with a lab partner on the antibiotic resistance experiment. However, it is important for every person to demonstrate that they can get single colonies on a plate. 1. Swab your bench with 70% ethanol, and allow it to dry. 70% ethanol is flammable! Do not spray ethanol on your bench in the presence of an open flame. Be aware of what students next to you and on the other side of the bench are doing (they may already have their Bunsen burner lit). 2. Obtain an LB media plate. On the bottom of the plate, not the lid, write your name or initials, the date, and your lab section number. 3. Get an E. coli stock plate from your lab instructor. 4. It is common practice to also label a plate with the bacteria’s strain number. Look at the stock plate - if the strain number is there you can add that information to the label on your own plate. 5. It is recommended that you draw lines on the bottom of the plate to designate the three sectors, as shown in the diagram above. 6. Check your Bunsen burner: Make sure the needle valve on the bottom is not closed all the way, and make sure the collar is not completely open. Light your Bunsen burner, and adjust the flame so it is low and blue. 7. Flame your wire loop, starting at the end closest to the handle and working your way to the loop end. Each section should turn orange before you move to 5

air up and away from the bench space. This helps prevent bacteria, mold spores, and other microbes in the environment from landing on your work. 5. Once you’re confident about the amount of antibiotic to add to the LB+rif tube, add that and briefly vortex mix the tube. 6. Obtain a tube of E. coli stock culture from your lab instructor. This will be the inoculum for your test tube cultures. 7. Using proper sterile technique, add 100 μl of the stock culture to each of your tubes. 8. If you’re going to move onto dilution plating fairly soon, you can leave your Bunsen burner lit. If you’re going to take a break you should turn off the gas valve now. 9. Place your tubes in the 37°C shaking incubator. These tubes will incubate for ~48 hours, until the next lab period. Dilution plating, round 1 This set of plates will show you what is present in your initial cell population at the beginning of the experiment. Is there diversity in this culture, with respect to the ability to withstand rifampicin? When plating samples for counting, the goal is to obtain plates with a reasonable number of colonies to count accurately, and with enough colonies to give meaningful data. At the low end of this range, we aim to get at least 30 colonies. At the high end of this range, we want plates that have a number of colonies that can be reasonably counted (i.e., the colonies aren’t overlapping each other so much that they cannot be accurately counted, which can happen when you have several hundred colonies on a plate). Because cultures can vary substantially in density, the optimal dilution for obtaining a good number of colonies won’t be the same for all cultures. This is why you will plate out a few different dilutions of the E. coli stock culture in order to cover the range of dilutions that are most likely to yield a countable and useful number of colonies. 1. Light your Bunsen burner, if it’s not already lit from inoculating your subculture tubes. 2. Using good sterile technique, do a serial dilution from the E. coli stock culture (not from the subcultures you just prepared, but from the original tube of E. coli culture you were given today). Use the diagram below as a reference for which dilutions to make. ○ Use liquid LB media as the diluent. ○ Note that most steps are 1:10 dilutions here, but one step is a 1:100 dilution. ○ Be sure to vortex each tube as you go! 7

3. You will make 2 sets of dilution plates, both from the serial dilution you just made. One set will be on plates with LB media, and the other set will be on LB+rifampicin plates. Obtain the plates you’ll need, according to the list below. Label each plate with the type of media (LB or LB+rif), your name/initials, today’s date, and the dilution you will plate. It is important to note that you are only going to plate 1/10 of the volume in each of your serial dilution tubes (that’s 100 μl out of the total 1000 μl in each tube). This is an additional 1:10 dilution. We’ll therefore refer to the final dilution on each plate as the “total effective dilution”. This total effective dilution is the number you’ll need when you do your calculations later. When you label your plates, make sure it’s clear whether you’re writing down the dilution you plated or the total effective dilution (or write both down). ○ Dilutions to plate on LB plates: ■ 10 -5 (10 -6 total effective dilution) ■ 10 -6 (10 -7 total effective dilution) ■ 10 -7 (10 -8 total effective dilution) ○ Dilutions to plate on LB+rifampicin plates (50 μg/ml rifampicin) : ■ Undiluted E. coli culture (10 -1 total effective dilution) ■ 10 -1 (10 -2 total effective dilution) ■ 10 -2 (10 -3 total effective dilution ■ 10 -3 (10 -4 total effective dilution) 4. Make sure you have a glass media spreader, a jar of 95% alcohol, and a lid for the jar. Set the glass media spreader in the jar of alcohol and keep the lid nearby in case you accidentally light the alcohol in the jar on fire (if that happens, put the lid on the jar to extinguish the flame). 8

information. You can borrow a streak plate from someone else in the lab who got single colonies (once they’re done looking at it). This plate can serve as your stock plate to pick a colony off of. ` Results of dilution plating, round 1 1. Count the number of colonies on each of your LB plates, and enter those numbers in Table 1. If a plate has several hundred colonies, you may want to divide the plate into sections to help you keep track. You can count each section and add them up to find the total, or you could count one section and multiply to get an estimate of the total number of colonies on the plate (consider the fact that you now have an estimate rather than an actual colony count, though). 2. Count the colonies on each of your LB+rifampicin plates, and enter those numbers in Table 2. Table 1. Results of first round of dilution plating, LB media plates. Dilution you plated Total effective dilution Number of colonies 10 -5 10 -6 10 -6 10 -7 10 -7 10 -8 Table 2. Results of first round of dilution plating, LB+rifampicin media plates. Dilution you plated Total effective dilution Number of colonies Undiluted culture 10 -1 10 -1 10 -2 10 -2 10 -3 10 -3 10 -4 10

Liquid cultures - making observations Bring your two test tube cultures (LB and LB+rifampicin) to your bench to make observations. Use the space below to write down your observations. Is the media relatively cloudy, or is it clear? Do the cultures in the two tubes look similar to each other, or different? Dilution plating, round 2 You will now make sets of dilution plates from each of your test tube cultures. The protocol here is the same as the one you followed on day 1, so look at the day 1 instructions for more specific instructions if you’re unsure about a certain step. 1. Light your Bunsen burner and adjust to a blue flame. 2. From each of your 2 test tube cultures (LB and LB+rifampicin), make the same serial dilution that you did on day 1 (refer to the serial dilution diagram in the day 1 protocol). ○ Be sure to label your serial dilution sets in a way that you know which one is which. ○ Remember to vortex each tube, including your glass test tube culture, as you work on the serial dilution. 3. Get the LB and LB+rifampicin media plates you need in order to make the dilution plate sets listed below. Remember that the goal is to get plates with more than 30 colonies, but not so many that it’s impossible to count accurately. It’s reasonable to predict that the colony grown in regular LB will have results similar to your first round of dilution plates, so you’ll plate the same concentrations as last time from your LB culture. For the culture grown in LB+rifampicin, we don’t know how many colonies might grow on the antibiotic plates. We will therefore plate a wider range of dilutions from that test tube culture. ○ From the LB test tube culture: ■ Dilutions to plate on LB plates: ● 10 -5 (10 -6 total effective dilution) ● 10 -6 (10 -7 total effective dilution) ● 10 -7 (10 -8 total effective dilution) ■ Dilutions to plate on LB+rifampicin plates (50 μg/ml rifampicin) : ● Undiluted E. coli culture (10 -1 total effective dilution) ● 10 -1 (10 -2 total effective dilution) ● 10 -2 (10 -3 total effective dilution ● 10 -3 (10 -4 total effective dilution) ○ From the LB+rifampicin test tube culture: ■ Dilutions to plate on LB plates: ● 10 -5 (10 -6 total effective dilution) ● 10 -6 (10 -7 total effective dilution) 11

Day 3: Collecting & Analyzing Results Checking on your streak plates (if applicable) If you repeated the streak-plating protocol during the last lab, check on your plate to see if you’ve obtained single colonies or not. Consult with your lab instructor. Results of dilution plating, round 2 Like your first round of dilution plates, count the number of colonies on each plate. Enter the data into the appropriate rows on Tables 3-6. Table 3. Results of second round of dilution plating, culture grown in LB, colonies on LB plates. Dilution you plated Total effective dilution Number of colonies 10 -5 10 -6 10 -6 10 -7 10 -7 10 -8 Table 4. Results of second round of dilution plating, culture grown in LB, colonies on LB+rifampicin plates. Dilution you plated Total effective dilution Number of colonies Undiluted culture 10 -1 10 -1 10 -2 10 -2 10 -3 10 -3 10 -4 Table 5. Results of second round of dilution plating, culture grown in LB+rifampicin, colonies on LB plates. Dilution you plated Total effective dilution Number of colonies 10 -5 10 -6 10 -6 10 -7 10 -7 10 -8 13

Table 6. Results of second round of dilution plating, culture grown in LB+rifampicin, colonies on LB+rifampicin plates. Dilution you plated Total effective dilution Number of colonies Calculating CFU/ml How to calculate CFU/ml Now that you have the colony counts from your dilution plates, you can use this information to calculate the size of the bacteria population in each of your cultures. With bacterial populations, we talk about population sizes in terms of the number of colony forming units (CFUs) per ml of media. Colony forming units are assumed to represent single cells. However, if cells adhere to one another, colonies may arise from small groups of cells rather than single cells. This is why it would not be completely accurate to call the measure “cells/ml”. To calculate CFU/ml, you need to take into account both the number of colonies you counted and the total effective dilution on the plate. Remember that each tube in your serial dilution had a total volume of 1 ml (1000 μl). Because you only put 100 μl from each tube on the plate, you effectively carried out an additional 1:10 dilution. Therefore, you want to use the total effective dilution when you calculate CFU/ml. Here is a sample CFU/ml calculation: Plated: 10 -6 total effective dilution Colony count: 64 colonies ( colony count ) × ( dilution factor ) = CFU / ml The “dilution factor” in the above equation is the inverse of the total effective dilution. In this example, the dilution factor is 10 6 . So, the calculation would be 64 × 10 6 = 6.4 × 10 7 CFU / ml 14

( ( rifampicinresistant CFU / ml ) ( total populationCFU / ml ) ) × 100 = Percentage of populationthat' sresistant ¿ rifampicin Calculate the percent of the population that was resistant to rifampicin in the original E. coli stock culture, and in each of your two subcultures. Record those numbers in the right-most column of Table 7. Thinking about the relationship between sample size and variation Before you leave lab today, you are going to add your CFU/ml and percent resistance data to a class data sheet. Why are we compiling data from multiple replicates of this experiment, rather than just having you draw conclusions from your own experiment? Let’s spend some time thinking about sample size and its relationship to variation. We’ll think back to the data you collected in the zebrafish experiment, since that is a simple type of data that you are already familiar with. Then you can apply this thinking to your CFU/ml and percent resistance data set from this lab. Specifically, let’s examine the relationship between sample size and the standard deviation (spread) of the data. Q1 Working individually, describe the relationship between sample size and the standard deviation of data. Write your answer below and wait for your lab instructor before moving on to the next section. The data table below shows heart rate measurements from untreated (control) zebrafish taken by students in a previous semester. The mean and standard deviation were calculated for various sample sizes (samples were chosen randomly, and heart rates with a value of “0” were discarded). 16

Q2 Based on this table, how would you describe the relationship between sample size and the standard deviation of the data? Support your answer with data from the table. When you are done writing, wait for your lab instructor before moving on. Compiling class data & making figures Add your data from Table 7 to the class data sheet, where your lab instructor will compile all of the data for your lab section. Once you have the data sheet with everyone’s data, you’ll be able to calculate summary statistics (mean, standard deviation, etc.). Your lab instructor will lead a discussion about what sort of figure would be appropriate to make with these data. 17