In, © 2015 by National Association of Biology Teachers. Discussion (20–30 minutes) of the activity’s importance; forming a hypothesis; and explanation of procedures and their purpose. Detail of colonies of E.coli and Pseudomonas aeruginosa on tryptic soy agar (TSA). Wash your hands with soap both BEFORE and AFTER lab, and, in addition, when you have a spill. I walk the class through the steps of scientific inquiry. Can we repeat the results observed by others? But changing the y-axis to a logarithmic scale, adding an exponential line, and using the equation for the line to calculate doubling time is new to many students. Plate spinners. 4), on the back (bottom of plate containing agar medium) with a sharpie pen (Fig. Then, in sequence: 1 mL from bottle A is transferred to bottle B, resulting in a 1:104 dilution, and the bottle is shaken as before; 1 mL from bottle B is added to bottle C, resulting in a 1:106 dilution, and this bottle is shaken as before; 1 mL from bottle B is pipetted on the plate labeled “104”; 0.1 mL from bottle B is pipetted on the plate labeled “105”; 1 mL from bottle C is pipetted on the plate labeled “106”; and 0.1 mL is pipetted onto the plate labeled “107”. It reduces bacterial contamination since the bacteria, even if they get into the plate in between the lid and bottom, would have to go UP to get to the agar. or TSA. Replace the lid and invert the plate. To calculate the doubling time, set up a ratio and solve for X: X = 34.78 minutes = the time it takes for the culture to double at 25°C. This measurement is their T = 0 minutes reading, and the experiment has begun. Lift the top film and with a 3M™ Electronic Pipettor or equivalent held perpendicular to plate, dispense 1 mL of sample suspension onto center of bottom film. The combination of soy and casein peptones supply organic nitrogen in the form of amino acids and polypeptides, making the medium highly nutritious. An explanation of how doubling time is calculated from each method is covered (https://mckernan microbiology.wikispaces.com). Doubling time in minutes of E. coli grown at 25°C and 37°C. E. coli overnight culture. Be sure that the new medium is already labeled so you do not confuse the various cultures. The procedure used here was adapted from Benson (2013). You will also have a mixed culture with 2 species in order to learn how to best separate and isolate bacterial species. Enterobacter aerogenes growing on MacConkey agar. You can do this by 1) sticking the hot loop in the agar at the edge of the agar away from the bacteria, or 2) just holding the loop for a few seconds while it cools. The classes are usually composed of biology majors in their junior year, with an occasional sophomore or a few seniors. 3). In the culture at that point in our experiment, 42 × 105 = 4.2 × 106 CFU/mL. Once the two time points have been calculated, we can use these numbers to calculate doubling time. 3. Serial dilutions prepared and plates inoculated. Therefore, in a 3-hour laboratory session, when an overnight culture of E. coli is heavily inoculated into media already at the appropriate temperatures, it is possible to obtain a complete growth curve. Click here to let us know! (My PowerPoint presentation for this lab is available at https://mckernanmicrobiology.wikispaces.com.). Then subtract x2 from x1 to obtain the doubling time of E. coli at 25°C. It teaches the nature of the research process, from asking questions and developing a testable hypothesis to writing a scientific paper, as well as the concepts of bacterial growth and two classic techniques for measuring bacterial growth, spectrophotometry, and standard plate method. These procedures also give students another opportunity to practice aseptic technique, handling of bacterial cultures, serial dilutions, pipetting, and micropipetting – and the feeling that they are really becoming scientists. The presentation of data in tables and graphs and the use of computers to help with data analysis and interpretation are integral to the research process. Staphylococcus aureus (Gram positive) - growth with yellow halo Dissolved balls were spread evenly across the surface of the agar with an L‐shaped spreader and incubated at 37°C for 48 h ( Clesceri et al. Eliminate potential contamination of bacterial cultures by using. Tubes for disposal go inn test tube racks at disposal area in corner of the lab. The equation for an exponential line is y = bemx, where b is the y intercept and m is the slope. The parts of a scientific paper are covered in detail (see https://mckernanmicrobiology.wikispaces.com); descriptions of what should be included in each section are posted for students to use as a guide in writing the paper. Dip a sterile cotton swab in water and swab a large area of the surface being tested (i.e. Enterobacter Aerogenes and Esterichia Coli Meme Culture: A selective culture plate with a niche joke from a viral internet campaign. (Fig. Number of colonies on each of the dilution plates for both times and incubation temperatures. Repeat this procedure for the 25°C growth curve. Typically, 100 mm round plates are for growth/selection, 150 mm round plates are for screening, and rectangular plates can be used with automated systems. One common source of error is the students’ micropipetting skills – which is often obvious when examining the CFU results. epidermidis. The gas should be turned all of the way on, so that the level is parallel with the rubber tubing. Subject: Using a Simple Escherichia coli Growth Curve Model to Teach the Scientific Method, (Optional message may have a maximum of 1000 characters.). In the second lab session, students count the number of colonies that have grown on TSA plates (see Figure 3 for an example of the plates) that were inoculated as described above. The objectives of the laboratory exercise are as follows: To review and practice scientific inquiry. (Tables 2, 3, and 4 and Figure 2 were taken from actual results obtained by students.) In addition to nutrients and growth factors, and perhaps agar, there are additives such as NaCl salt, pH buffers, and pH indicators that allow biochemical reactions to be identified. However, there is a large difference in doubling time obtained from the two methods for the 25°C data. Then program Excel to calculate the x1 value [x = LN(y/0.1858)/0.0078], which is the time in the growth curve where OD = 0.5. After supplementation with blood (e.g. E.coli is a lactose-fermentor on MacConkeys agar. All rights reserved. Using two methods gives the students an opportunity to discuss how different methods can be used to measure the same parameter – in this case, doubling time. I emphasize that an understanding of how this number was arrived at is important. For example (from Table 3), at 40 minutes and a temperature of 25°C, the 105 plate is best with 42 colonies. Petri dish with E.coli on triptic soy agar (TSA). The samples are then spread over the surface of the agar by placing the plate onto the plate spinner, alcohol-flaming the bent glass rod, and using the rod to spread the bacteria while spinning the plate. In (B), a closer look at the plate shows isolated colonies of E. coli (white colony; white arrow), S. aureus (yellow colony; yellow arrow), and C. violaceum (purple colony; blue arrow). I ask the students to choose a y value that is on the line, so that this is an optical density that occurred during the exponential phase of growth. Use an inoculating loop for the agar plate and the broth media. E.coli is normal flora of the intestines of all animals and humans. Label all test tubes and petri plates with your name (initials), your table #, date, exercise #, and name of organism. Sterilize the tops of the tubes again (to eliminate potential air contamination again) and replace the caps. Different species of bacteria will be used so that you can become familiar with different growth patterns. In the lecture portion of the course, the concepts of binary fission, four phases of bacterial growth, factors affecting bacterial growth, and techniques for measuring bacterial growth are covered. Isolation plate of E.coli E.coli E.coli is a Gram-negative rod. It appears as a off white, round colonie on TSA media. Figure 2B shows just the data points during the exponential phase of growth at each temperature and the resulting straight-line relation and equations that resulted. To write a science research paper. Then tubes are returned to their appropriate water baths. THE LAST PERSON ON THE TABLE CHECKS TO MAKE SURE THAT ALL 4 GAS JETS ARE OFF! 12. What improvements of methods and further steps are needed in research on the problem? ALWAYS check agar plates carefully to make sure that there are no mold or bacterial contaminants on the plate: if so, discard the plate in the autoclave bag. Copyright © 2021 National Association of Biology Teachers. Using their data, I demonstrate how to calculate the CFU/mL for one time point at one temperature. The effect of incubation temperature on generation time. In most undergraduate microbiology courses, the laboratory portion is very technique oriented (e.g., Gram staining, identification of bacteria, testing for antimicrobial resistances). Escherichia coli, Staphylococcus aureus, and Chromobacterium violaceum was streaked onto a TSA plate and incubated for 24 hours at 37°C (A). Lift the lid of the plate just a bit, in order to get your inoculating loop into it: DO NOT TAKE THE ENTIRE LID OFF. Look at the section below in INTERPRETION to read your tube results. Why or why not? Two members of the group work on generating the data for E. coli grown at 25°C, and two work on generating the data for E. coli grown at 37°C. Sterile 1-mL pipets, micropipettes, and micropipette tips. If your goal is to identify the type of growth pattern, then just bring the loop straight up the slant. From this data, the doubling for E. coli at 25°C is 88.87 minutes (see https://mckernanmicrobiology.wikispaces.com). Sterile bent glass rods for spreading bacterial samples on TSA plates. Streak each of the following bacterial strains on separate TSA plates: • the laboratory E. coli [ATCC 25922] stock culture (negative genotype control strain); • the E. coli astA [Strain ECTOIL] culture (positive genotype [toxin astA] control strain); • the E. coli isolate (ECENV; unknown genotype) isolated from the prior experiment; and 2), Go into the tube to take your inoculum and QUICKLY place the inoculum into the new medium tube. Leavitt et al. Comparison to literature and related studies, Design improvements and future work are included. ; E. coli is the normal flora of the human body. FIG. What I have done is combine them into an exercise that focuses on scientific inquiry, research design, data analysis, and scientific writing. Size – The size of Escherichia coli is about 1–3 µm × 0.4–0.7 µm (micrometer).. To disrupt clumps of bacterial cells, the bottle is shaken in a long arc motion 25 times. The results presented above are actual results from a group of students. All rights reserved. Optical density (OD) readings taken for 140–180 minutes. Therefore, if y (optical density) is known, as in this case, the time (x) can be solved as x = ln(y/m)/b. -TSA plate-Glucose mineral salt plate-EMB plate-MSA plate-MAC plate. The procedure used here was adapted from Kleyn et al. Put the loop part into the incinerator until it glows red. Sterilize the loop in the flame and let it cool before continuing to spread the bacteria. Of what use is it to know what kind of growth pattern on an agar slant or a broth medium an organism has? Approximately 40 minutes into the experiment, 1 mL of the culture is removed for serial dilutions and plating as shown in Figure 1. Also, science needs to be repeatable. Review the parts of a scientific paper and the rubric for grading. ; The primary habitat of E. coli is in the gastrointestinal (GI) tract of humans and many other warm-blooded animals. It is critical in the risk assessment of certain foods to be able to enumerate stresse … 5). Using a sterile agar medium plate (lift the lid just enough to insert the loop), streak a vertical line straight down. Heat the inoculating wire of the loop until red-hot (Fig. ], Importance or relevance of research project, Equipment and material How data were collected and procedures. So the challenge is helping students determine where sources of error could have occurred. Move the loop in a zig-zag pattern across the agar until 1/3 of the plate is covered, finishing the first section. Candida albicans (ATCC ® 10231) colonies growing on TSA (Cat. Your instructor will show you how to perform these procedures, but here is a brief description of the methods you will use. 4.2 The presence of some lateral diffusion of blue color away from the target Count colonies on plates prepared in session 1. Why use a streak plate to grow a bacterium rather than an agar medium slant or a broth medium. Typical results of colony counts are shown in Table 3. Plates are incubated at 37°C for 24–48 hours, and the colonies are counted. demonstrated the versatility of TSA by cultivating both aerobic and anaerobic microbes using TSA. There are 2 ways to inoculate the slant: Use the needle to inoculate the deeps or semi-solid agars. G62). Mannitol Salt Agar (MSA) Plate . This exercise is usually run in the fourth week of the semester, so that students already have some practice with handling of bacterial cultures and aseptic technique. Recipient(s) will receive an email with a link to 'Using a Simple Escherichia coli Growth Curve Model to Teach the Scientific Method' and will not need an account to access the content. Next, the procedures are reviewed. Most students at this level have used Excel to make tables and graph data. Tryptic Soy Agar (TSA) Hand Plate is a general growth medium for the isolation and cultivation of microorganisms. Unless otherwise noted, LibreTexts content is licensed by CC BY-NC-SA 3.0. From these two data points, the magnitude of increase in culture can be calculated by dividing: So now the culture has increased 3.45-fold in 60 minutes (the time differs between collection of two samples), but we need to determine how many minutes it took the culture to double. Now double the y value to 0.16 and again solve for x2. Tubes are vortexed, and the OD readings are recorded every 20 minutes over the next 2.5 to 3 hours. c. Escherichia coli: colonies and confluent growth appear bright pink to red and surrounded by a pink precipitate (cloudiness) in the agar surrounding the growth . Then, wipe cotton swab all over a Tryptic Soy Agar (TSA) plate and cover. aerogenes BioBall TM were each aseptically dropped onto the surface of separate TSA (TSA‐S) and PCA (PCA‐S) plates and 1 ml of autoclaved tap water was added. 2. The growth of E. coli was inhibited by the high salt concentration. E. coli was discovered by Theodor Escherich in 1885 after isolating it from the feces of newborns. By continuing to use our website, you are agreeing to, An Evolution Unit That Develops Students’ Capacity to Construct Arguments from Evidence. Because we are interested in determining the doubling time during the exponential phase of growth only, the data points for Figure 2B were chosen by determining the beginning and end of the exponential phase from Figure 2A. (B) Exponential phase of growth for E. coli at 25°C and 37°C. Cultivation 24 hours, 37°C in an aerobic atmosphere. From this data, the doubling for E. coli at 37°C is 24.76 minutes (see https://mckernanmicrobiology.wikispaces.com). Cultivation 24 hours, 37°C in an aerobic atmosphere. What are optimal conditions? The standard plate method invites review of serial dilutions, the concept of colony-forming units, and how to determine the concentration of microorganisms.
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