To use Excel in graphing and calculating doubling time. Subculture a slant culture of Bacillus subtilus into TSB. Subculture a broth culture of Staph epidermidis onto a TSA slant. Up to a 3-log difference was observed between counts on SMAC and tryptone soy agar (TSA). By continuing to use our website, you are agreeing to, An Evolution Unit That Develops Students’ Capacity to Construct Arguments from Evidence. Run the tops of the tubes through the heat to create an updraft (taking air contaminants AWAY from the tube entrance). Look at the section below in INTERPRETION to read your tube results. (Fig. A film of broth culture can be seen across the loop as you remove it from the tube. Examples of Results from Spectrophotometry, Example of Results from Standard Plate Method, https://mckernanmicrobiology.wikispaces.com, https://mckernan microbiology.wikispaces.com, http://visionandchange.org/files/2013/11/aaas-VISchange-web1113.pdf, http://www.ucpressjournals.com/reprintinfo.asp, Using Unplugged Computational Thinking to Scaffold Natural Selection Learning, The Misuse of Pedigree Analysis in the Eugenics Movement, Developing & Testing Curricula for Teaching Evolutionary Concepts at the Elementary School Level, What John Scopes Told His Family & Friends about His Trial, Building Argumentation Skills in the Biology Classroom. Media is the nutrient-rich material that provides food to the microbes.. Pick up a loopful of your inoculum from either a broth or an agar culture. Until you become well-acquainted with this procedure, you might want to draw the 3 sections that you will streak inside of (Fig. 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. But happy to evaluate. 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. Streak out cultures for isolation and identify colonies. Then tubes are returned to their appropriate water baths. 2), Go into the tube to take your inoculum and QUICKLY place the inoculum into the new medium tube. The students inoculate their trypticase soy broth (TSB) tubes and learn how to use the spectrophotometer to measure optical density, which includes setting the wavelength, zeroing the instrument with an uninoculated broth tube, and then measuring the optical density of their freshly inoculated TSB tube. 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. Comparison to literature and related studies, Design improvements and future work are included. Subculture TSA plate culture of E. coli into TSB, a TSA slant, and a TSA plate. An agar medium contains agar (1.5-2%) as a solidifying agent---isolation and purification. Incubated aerobically for 48 hours at 35°C. Size – The size of Escherichia coli is about 1–3 µm × 0.4–0.7 µm (micrometer).. Generally it is best to keep E. coli well away from the other cultures because it may overgrow them. Why would we need to know that? Then subtract x2 from x1 to obtain the doubling time of E. coli at 25°C. I walk the class through the steps of scientific inquiry. Trypticase soy agar (TSA) plates, 16 per group. So the challenge is helping students determine where sources of error could have occurred. (2011). Part 2: 1. 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. 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. Incubate aerobically for 24 hours at 35°C. Then the results are evaluated and the class brainstorms on whether the results confirm their hypothesis, and why or why not; whether the two methods gave comparable doubling times, and why or why not; the advantages and disadvantages of the two methods; and finally, how the procedures might be improved and what further study might include. Agar plates for disposal go into the red biohazard autoclave bag on the floor below the tubes. It has a garbage like oder. Identify different ways by which bacteria grow in culture—in agar deeps, on agar slants, on agar plates, in broths. Sterilize the tops of the tubes again (to eliminate potential air contamination again) and replace the caps. You will also have a mixed culture with 2 species in order to learn how to best separate and isolate bacterial species. 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. Sterilize the loop again. (A) Growth curve of E. coli grown at 25°C and 37°C. The biology department at Chestnut Hill College has endeavored to incorporate the research process into all of its undergraduate biology courses, beginning long before the recommendations in Vision and Change in Undergraduate Biology Education (AAAS, 2011). 1), and be sure that the ENTIRE wire is sterilized. In the second session, “Parts of a Scientific Paper” are presented in a PowerPoint (https://mckernanmicrobiology.wikispaces.com). MORPHOLOGY OF ESCHERICHIA COLI (E. COLI) Shape – Escherichia coli is a straight, rod shape (bacillus) bacterium.. 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. The inoculum is obtained by first shaking the culture a bit, and then going into the broth with the loop. Dilution bottles with 99 mL sterile distilled water, 12 per group. Why streak from the bottom of the agar slant medium up to the top in a straight line, rather than a back and forth wavy inoculation from side-to-side on the slant? Now Excel will calculate the x value for a given y by programing with this equation: x = LN(y/0.0441)/0.028, obtained from the equation for the line at 37°C. 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. Mannitol Salt Agar (MSA) Plate . Students have often said that they like the lab because of its hands-on aspects: taking readings, seeing the growth curve emerge, pipetting, serial dilutions and plating, and the quantitative analysis of raw data to calculate doubling times.
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