Unknown bacteria #47
Microsoft Word - Unknowns.doc INSTRUCTIONS Identification of Unknown Bacteria Introduction. The identification of unknown bacteria is a time-honored part of most microbiology courses. Although you will not actually be doing this project, you will be doing it virtually, it is hoped that it will challenge your knowledge and skills in performing laboratory techniques, your ability to critically evaluate the information obtained from these techniques, and your ability to effectively communicate this information. Rationale. Upon completion of this project, the successful student will demonstrate: The ability to apply basic scientific principles in solving real and hypothetical microbiological problems using the scientific method; An ability to effectively communicate complex scientific information; Effective personal time management, organization, and study skills. The specific learning objectives involve mastery of techniques and concepts presented throughout the course. They include a demonstration of the ability to: Virtually streak for isolation from a broth culture; Virtually perform aseptic technique by maintaining working and reserve stock cultures without contamination; Virtually make Gram stains and correctly obtain and interpret Gram reactions using a microscope; Virtually effectively use all-purpose, selective, differential, and selective-differential media. Virtually select only the most significant media and reagents for characterization of the biochemical activities of the unknown bacterial species. Virtually collect and evaluate data in a logical manner. Present work in detailed daily lab notes, demonstrating skilled observation with information presented logically, orderly, and neatly. Project Format. Phase I: Isolation. Students will virtually receive a broth culture containing a bacterial species. Some species will be G+ and some species will be G-. Virtually, you will isolate each species into pure culture. The process for this isolation is shown in Figure A below. Phase II: Species Identification. a. Using your (ONE) pure culture from Phase I, run a series of tests to determine the identity of the species in the culture. Descriptions of the available tests/media will be provided. Lab textbooks give additional information. Information about a species’ characteristics can be found in BERGEY'S MANUAL and in lab manuals. In cases where the lab manuals and Bergey's Manual provide conflicting information, Bergey's Manual should be assumed to be correct. The sequence of tests should display DICHOTOMOUS logic. Dichotomous logic involves a series of yes/no or true/false questions. SHOTGUN logic should not be used, and will result in the loss of a substantial number of points. Daily Lab Notes. You must write detailed notes of your activities, observations, thoughts, etc. for every lab class. You will use these as the basis for the Unknowns Report you will turn in. NOTE: it is important to document all of your activities, whether they give good or poor results. This is critical for when you write your Unknowns Report, as you cannot trust your memory to try and recall what you did during the project. Unknowns Report—75 points. You must submit online a report that details your thoughts and activities for each day of unknowns work. With the exception of the microorganism paragraph (see f. below), the report should be written using bullet points, and NOT in an essay style. Please use 12 point font and do NOT double space. Your daily descriptions should include the tests you run, the information you hoped to gain from these tests, the observations you made from tests inoculated on the previous day. You should not omit data that makes your project “look better.” Drawings, photos, and tables of information are very helpful. You may photograph your test results and include these in your report; however, this is not required. Drawings, photos, tables, etc., must be completely labeled and discussed within the body of your report. Each report entry must begin with the date and time. You do NOT have to describe the step-by-step details of each test you run. For example, if you run a Gram stain it is unnecessary to describe each step of the process; instead, just say that you made a Gram stain of your culture. The thoughts and words in your report should be YOUR OWN. You may receive advice from other sources (e.g., the instructor, other students), but all work should be YOUR OWN. It is MANDATORY that your report include a table outlining the process you used to identify your unknown organism. An example of a suitable table is provided on the next page. Microorganism paragraph. A description of your microorganism including diseases, modes of transmission, common sources/places it is found, treatments, and any other interesting information will be given at the end of your report after you have determined the identity of your microorganism. This part of the report only needs to be a paragraph or two in length; do not exceed one typed page for this section. All background literature/sources you use should be cited. You must give credit to the source of any piece of information that is not your own original thought. Use the CSE (numeric) or APA citation style for citing references and for the LITERATURE CITED page. EXAMPLE TABLE (** NOTE: this table is NOT the same thing as the table(s) shown in the Sample Report by Dr Kibota **) TEST: Gram Catalase PR-mannitol PR-glucose Citrate RESULT: G+ coccus positive negative Acid positive SPECIES: E. faecalis M. luteus M. luteus M. luteus M. varians M. varians M. varians M. varians M. varians S. aureus S. aureus S. epidermidis S. epidermidis S. epidermidis S. epidermidis S. agalactiae S. bovis S. mitis S. pneumoniae S. pyogenes ** NOTE: for each species, keep it in the same row throughout the table. ** Grading Criteria. Listed below are the grading criteria, starting with the most important and ending with the least important. Further details can be found in the Grading Sheet in the lab. Logic of tests performed—only the most relevant tests, using no more than one dichotomous approach, should be performed. A shotgun approach will score poorly. You may only perform one test/lab period or receive a penalty. Running tests to confirm the results of earlier tests should only be performed under certain circumstances, for example, if there was a procedural error. If you DO repeat a test, you must be able to adequately describe your rationale for repeating the test in your Final Report. If not, points will be deducted. Completeness of report—each entry should include as much detail as possible. What tests were run? Why? What were the possible conclusions? What were the results of the tests? What conclusions did you draw? All results should always be recorded, regardless of whether or not the tests were successful. Diagrams, photographs, and tables of information are very helpful, as long as the information is also explained in the text of your report. Completeness and accuracy of species description. Organization, clarity, and neatness of the daily lab entries are important. All information (except in diagrams) must be typed. Each entry should be dated. Effectiveness of written communication. Your report should be free of grammatical and spelling errors. Your report should be concise, easy to read, and easy to understand. Successful identification of your unknowns is worth a percentage of the grade. It is much more important to show logical thinking than it is to actually correctly identify your bacteria. Lack of proper laboratory technique may be costly. Points will be deducted if your daily lab entries do not correspond to actual events. Lack of adherence to safety rules may not only cost you points, but may get you dismissed from class. Each student must submit a report, written in your own words. YOU MAY NOT PERFORM THE ANALYSES AS A TEAM AND YOU MAY NOT SUBMIT REPORTS THAT ARE WRITTEN AS THOUGH THE PROJECT WAS PERFORMED AS A TEAM. Day 1 MacConkey 30C Azide 30C TSA 30C MacConkey 37C Azide 37C TSA 37C X Streak for Isolation Figure A: Ideal Isolation Procedures. Day 2 Unknown Broth (contains either species; G+ or G-) Azide selects against G- species. MacConkey selects against G+ species. From among these six plates, identify the colony types that represent the two different species. Pick cells from each type of colony to start stock and duplicate reserve cultures on TSA slants, and a TSA purity check plate on Day 2. TSA slants& TSA purity check plates: Grow at appropriate temperature (ascertained from Day 1 plates). Stock 1 Reserve 1 Stock 2 Reserve 2 TSA Purity Check Plate 1 TSA Purity Check Plate 2 Day 3 and on Store in the refrigerator. Use your stock culture to run metabolic and morphological tests. Use your reserve culture only if your stock culture becomes contaminated. Figure B. Example of a Dichotomous Process. A B C D E F G Species Cell Morphology PR Sucrose + - + - - - - PR Lactose + - - - + + - PR Glucose + + + - + + + Refer to Table B (above) to answer the following hypothetical questions by using dichotomous logic. Day 3 1) If your organism is a bacillus shaped bacterium, would the PR Glucose test be a logical test to run? Why or why not? 2) If your organism is a coccus shaped bacterium, would the PR Glucose test be a logical test to run? Why or why not? 3) If your organism is a coccus shaped bacterium that is negative for lactose fermentation, what test should you run to determine the identity of your organism? If you do not understand this process (dichotomous logic) discuss it with your lab instructor as soon as possible! Hello students, This email is to tell you what your unknown bacteria number is. Please post it in your Unknown Notes page in canvas (I will need it to send you your test results. If there is no number, you will not get results.) This number represents the unknown bacteria that I am sending to you. You will need to streak it out and grow it virtually (meaning, you need to do nothing, but we are virtually doing what you would do in the real lab). Then you will set up the gelatin test today, 10/20, it takes at least one week to run (see the gelatin test in Canvas under Week 5 Lab) before you get results. You will do the Gram stain on Sunday, 10/25 but not look at the cells until 10/27. And you will set up the PR-Lactose test on Monday, 10/26. That way, you will be following dichotomous logic