Biology 240 Clark College Kibota 1IDENTIFICATION OF BACTERIA FROM A MIXED CULTURE (100 POINTS TOTAL)I. IntroductionThe identification of unknown bacteria is a time-honored part of microbiology courses. Itwill challenge your knowledge and skill in performing laboratory techniques, your ability tocritically evaluate the information obtained from these techniques, and your ability toeffectively communicate this information. II. RationaleA. Upon completion of this project, the successful student will demonstrate:1. the ability to apply basic scientific principles in solving real and hypotheticalmicrobiological 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 presentedthroughout the course. They include a demonstration of the ability to:1. Streak for Isolation from a mixed broth culture by obtaining and identifyingdifferent colony types;2. Perform Aseptic Technique by maintaining working and reserve stock cultureswithout 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 thebiochemical activities of the unknown bacteria.6. Collect and evaluate data in a logical manner.7. Present work in a detailed journal demonstrating skilled observation withinformation presented logically, orderly, and neatly.III. Project FormatA. Phase I: Isolation1. Pairs of students will receive a broth culture containing a mixture of two bacterialspecies.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 slantsBiology 240 Clark College Kibota 2XMixed Broth (contains two different species)TSA37°CAzide37°CMacConkey37°CTSA30°CAzide30°CMacConkey30°CStreak for IsolationDay1Day2Store in refrigerator. Use stock culture to run metabolic and morphological tests.Use reserve culture if stock becomes contaminated.Days3 onFIGURE A: Ideal Isolation ProceduresStock 1Reserve1Stock 2Reserve2From among these sixplates, identify bothcolony types thatrepresent differentspecies. Pick cellsfrom each colony typeto start a stock cultureand a duplicate reserveculture.Grow at appropriatetemperature (ascertainedfrom Day 1 plates).These are TSA slants.Biology 240 Clark College Kibota 3B. Phase II: Species Identification1. 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'SMANUAL and in the lab textbook. In cases where the lab textbookand Bergey's Manual provide conflicting information, Bergey's Manualshould 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 pointsA. Unknowns Report1. You must submit a typewritten (or word processed) report that details yourthoughts and activities for each day of unknowns work.a) These descriptions should include the tests you run, what information youhoped to gain from these tests, the observations you made from testsinoculated 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 advicefrom other sources (e.g., the instructor, other students), but all work should beYOUR OWN.5. A description of your microorganism including diseases, modes of transmission,common sources/places it is found, treatments, and any other interestinginformation.6. All background literature/sources you use should be cited. You must give creditto the source of any information that is not yours.a) Use a standard format (such as MLA) for citing references and for theLITERATURE CITED section.Biology 240 Clark College Kibota 4 B. The following are the grading criteria, starting with the most important and ending withthe 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 dichotomousapproach, should be performed (a shotgun approach will score poorly).a) Running tests to confirm the results of earlier tests is not necessarily wastedmedia or effort. The important thing to describe is your rationale for eachtest 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? Whatwere the results of the tests? What conclusions did you draw?(1) ALL RESULTS SHOULD BE RECORDED (whether or not the tests weresuccessful).b) Diagrams, photographs, and tables of information are very helpful (as longas 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 thegrade.a) It is much more important to show logical thinking than it is to actuallycorrectly 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 toevents witnessed by the instructor