INSTRUCTIONS Identification of Bacteria From a Mixed Culture (100 point lab project; BIOL260) I. Introduction. i. The identification of unknown bacteria is a time-honored part of most microbiology courses. 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. II. Rationale. i. Upon completion of this project, the successful student will demonstrate: a. The ability to apply basic scientific principles in solving real and hypothetical microbiological problems using the scientific method; b. An ability to effectively communicate complex scientific information; c. Effective personal time management, organization, and study skills. ii. The specific learning objectives involve mastery of techniques and concepts presented throughout the course. They include a demonstration of the ability to: a. Streak for isolation from a mixed broth culture by obtaining and identifying different colony types; b. Perform aseptic technique by maintaining working and reserve stock cultures without contamination; c. Make Gram stains and correctly obtain and interpret gram reactions; d. Effectively use all-purpose, selective, differential, and selective-differential media. e. Select only the most significant media and reagents for characterization of the biochemical activities of the unknown bacterial species. f. Collect and evaluate data in a logical manner. g. Present work in detailed daily lab notes, demonstrating skilled observation with information presented logically, orderly, and neatly. III. Project Format. i. Phase I: Isolation. a. Pairs of students will receive a broth culture containing a mixture of two bacterial species. b. As a team, you will isolate each species into pure culture. c. The process for this isolation is shown in Figure A below. ii. 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. 1. Descriptions of the available tests/media will be provided. a. Lab textbooks give additional information. b. Information about a species characteristics can be found in BERGEY'S MANUAL and in lab manuals. In cases where the lab manuals and 1
Bergey's Manual provide conflicting information, Bergey's Manual should be assumed to be correct. 2. The sequence of tests should display DICHOTOMOUS logic. a. Dichotomous logic involves a series of yes/no or true/false questions. b. SHOTGUN logic should not be used, and will result in the loss of a substantial number of points b. You should save examples of all of your cultures. 1. These examples will verify the information contained in your daily lab notes (see IV. Below). IV. Daily Lab Notes i. You are required to write detailed notes of your activities, observations, thoughts, etc for every lab class. You will keep these in your lab drawer where you can access them. Notepaper will be provided. The completed daily lab notes must be handed in to the instructor at the end of the project. Further details will be provided in the lab. ii. NOTE: it is important to write down all lab 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. V. Unknowns Report 100 points. i. You must submit a typewritten, hard copy report that details your thoughts and activities for each day of unknowns work. a. With the exception of the microorganism paragraph (see f. below), the report should be written using bullet points, and NOT in an essay style. b. Please use 12 point font and do NOT double space. c. Your 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. d. You should not omit data that makes your project look better. e. Drawings, photos, and tables of information are very helpful. 1. You may photograph your test results using the ipads provided in the lab, and include these in your report; however, this is not required. 2. Drawings, photos, tables, etc., must be completely labeled and discussed within the body of your report. f. Each report entry must begin with the date and time. g. 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; instead, just say you did a Gram stain. h. 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. i. It is MANDATORY that your report include either: (i) a dichotomous tree (flowchart style) or (ii) a table outlining the process you used to identify your unknown organism. An example of a suitable Table is provided on the next page. j. 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 2
determined the identity of your microorganism. 1. This part of the report only needs to be a paragraph or two in length; do not exceed one typed page for this section. 2. 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. a. Use the APA citation style for citing references and for the LITERATURE CITED page. For e.g., see The Cornell University APA Citation Guide http://www.library.cornell.edu/newhelp/res_strategy/citing/apa.html EXAMPLE TABLE 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. ** Plagiarism (continued) I wish to remind you that I take plagiarism very seriously, as does the college. The following definition of plagiarism is from the section entitled "Academic Dishonesty" at http://www.clark.edu/about_clark/policies/student_code.php "Plagiarism, which includes using another person's ideas, words, or other work in an instructional course without properly crediting that person." If plagiarism is identified, I will subtract 5% at a minimum. Be aware that repeated instances of plagiarism by a student can be punished more severely, including loss of all points in an assignment up to (the most severe) expulsion from the college. If you are unsure about what constitutes plagiarism, please go through the online tutorial found at: http://www.clark.edu/library/iris/use/use_home.shtml 3
VI. Grading Criteria: i. 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 on the bulletin board in the lab (and posted on Moodle). ii. Logic of tests performed only the most relevant tests, using a dichotomous approach, should be performed. A shotgun approach will score poorly. a. Running tests to confirm the results of earlier tests is not necessarily a waste of media or effort. The important thing to describe is your rationale for each test that you run. iii. Completeness of report each entry should include as much detail as possible. a. What tests were run? Why? What were the possible conclusions? What were the results of the tests? What conclusions did you draw? 1. All results should always be recorded, regardless of whether or not the tests were successful. b. Diagrams, photographs, and tables of information are very helpful, as long as the information is also explained in the text of your report. c. Completeness and accuracy of species description. iv. Organization, clarity, and neatness of the daily lab notes are important. a. All information (except in diagrams) must be typed. b. Each entry should be dated. c. Effectiveness of written communication. 1. Your report should be free of grammatical and spelling errors. 2. Your report should be concise, easy to read, and easy to understand. d. Successful identification of your unknowns is worth a small percentage of the grade. 1. It is much more important to show logical thinking than it is to actually correctly identify your bacteria. e. Lack of proper laboratory technique may be costly. 1. Points will be deducted if your daily lab notes entries do not correspond to events witnessed by the instructor (remember, the instructor will have plenty of opportunity to monitor your work BE HONEST!!!). 2. Lack of adherence to safety rules may not only cost you points, but may get you dismissed from class. f. Five points will be deducted from the score of each team member who identify the same bacterial species. v. Each student must submit a report, written in your own words. a. YOU MAY NOT PERFORM THE ANALYSES AS A TEAM (although you must consult with one another) AND YOU MAY NOT SUBMIT REPORTS THAT ARE WRITTEN AS THOUGH THE PROJECT WAS PERFORMED AS A TEAM. b. The only teamwork will occur at the beginning of the project during the isolation of the unknown species. 4
Student 1 Student 2 TSA (for purity check) TSA (for purity check) TSA slants 5