Biology 240 Clark College Kibota 1

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1 Biology 240 Clark College Kibota 1 IDENTIFICATION OF BACTERIA FROM A MIXED CULTURE (100 POINTS TOTAL) I. Introduction The identification of unknown bacteria is a time-honored part of microbiology courses. It will challenge your knowledge and skill 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 A. Upon completion of this project, the successful student will demonstrate: 1. the ability to apply basic scientific principles in solving real and hypothetical microbiological problems using the scientific method; 2. an ability to effectively communicate complex scientific information; 3. effective personal time management, organization, and study skills. B. The specific learning objectives involve mastery of techniques and concepts presented throughout the course. They include a demonstration of the ability to: 1. Streak for Isolation from a mixed broth culture by obtaining and identifying different colony types; 2. Perform Aseptic Technique by maintaining working and reserve stock cultures without contamination; 3. Gram Staining by consistently obtaining and interpreting correct gram reactions; 4. Effectively use all-purpose, selective, differential, and selective-differential media. 5. Select only the most significant media and reagents for characterization of the biochemical activities of the unknown bacteria. 6. Collect and evaluate data in a logical manner. 7. Present work in a detailed journal demonstrating skilled observation with information presented logically, orderly, and neatly. III. Project Format A. Phase I: Isolation 1. Pairs of students will receive a broth culture containing a mixture of two bacterial species. 2. As a team, you will isolate each species into pure culture. 3. The process for this isolation is shown in Figure A (on the next page). 4. Grading Criteria (see Unknowns Grading Sheet) a) Number of different colonies identified. b) Efficient use of resources (1) Minimum isolation utilization (a) 2 each: TSA plates, Azide plates, MacConkey plates (b) 4 TSA slants

2 Biology 240 Clark College Kibota 2 FIGURE A: Ideal Isolation Procedures X Mixed Broth (contains two different species) Streak for Isolation Day 1 TSA Azide MacConkey TSA Azide MacConkey From among these six plates, identify both colony types that represent different species. Pick cells from each colony type to start a stock culture and a duplicate reserve culture. Day 2 Stock 1 Reserve 1 Stock 2 Reserve 2 These are TSA slants. Days 3 on Grow at appropriate temperature (ascertained from Day 1 plates). Store in refrigerator. Use stock culture to run metabolic and morphological tests. Use reserve culture if stock becomes contaminated.

3 Biology 240 Clark College Kibota 3 B. Phase II: Species Identification 1. Select ONE of your stock cultures. 2. Run a series of tests to determine the species identity of this culture. a) Descriptions of the available tests/media are given on the course website. (1) Lab textbooks give additional information. (2) Information about species characteristics can be found in BERGEY'S MANUAL and in the lab textbook. In cases where the lab textbook and Bergey's Manual provide conflicting information, Bergey's Manual should be assumed to be correct. b) The sequence of tests should display DICHOTOMOUS logic. (1) Dichotomous logic involves a series of yes/no or true/false questions (see Figure B). 3. You should save examples of all of your cultures, slides, etc. a) These examples will verify the information contained in your lab journal. IV. Unknowns Report (Due TUESDAY, MAY 31) 100 points A. Unknowns Report 1. You must submit a typewritten (or word processed) report that details your thoughts and activities for each day of unknowns work. a) These descriptions should include the tests you run, what information you hoped to gain from these tests, the observations you made from tests inoculated on the previous day. b) Drawings/photos and tables of information are very helpful. 2. Each report entry must begin with the date and time. 3. Each page should be numbered. 4. These thoughts and words should be YOUR OWN. You may receive advice from other sources (e.g., the instructor, other students), but all work should be YOUR OWN. 5. A description of your microorganism including diseases, modes of transmission, common sources/places it is found, treatments, and any other interesting information. 6. All background literature/sources you use should be cited. You must give credit to the source of any information that is not yours. a) Use a standard format (such as MLA) for citing references and for the LITERATURE CITED section.

4 Biology 240 Clark College Kibota 4 B. The following are the grading criteria, starting with the most important and ending with the least important (An example of the Grading Sheet is attached in this packet): 1. 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 wasted media or effort. The important thing to describe is your rationale for each test that you run. 2. 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 BE RECORDED (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 text). c) Completeness and accuracy of species description. 3. Organization, clarity, and neatness of the lab journal are important. a) All information (except in diagrams) must be typed. b) Each entry should have the date and each page should be numbered. 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. 4. Successful identification of your unknowns is worth a small percentage of the grade. a) It is much more important to show logical thinking than it is to actually correctly identify your bacteria (guessing is not worth much). 5. Lack of proper laboratory technique may be costly. a) Points will be deducted if your lab journal entries do not correspond to events witnessed by the instructor (remember, the instructor will have plenty of opportunity to monitor your work BE HONEST!!!). b) Lack of adherence to safety rules may not only cost you points, but may get you dismissed from class. 6. Five points will be deducted from the score of each team member who identify the same bacteria. C. Each student must submit a report (written in your own words). 1. 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. 2. The only teamwork will occur at the beginning of the project during the isolation of the unknown species.

5 Biology 240 Clark College Kibota 5 FIGURE B. Example of a Dichotomous Process Species A B C D E F G Cell Morphology Glucose Fermentation Lactose Fermentation Sucrose Fermentation Bacilli Cocci A, C, E, G B, D, F