Wednesday, March 30, 2011

microbiology indentification of unknown organisms

Micro 240 Lab Unknown Guidelines Spring 2011 1. Each tube contains 2 organisms, 1 tube for G+ and 1 tube for G-. Tubes should be thoroughly mixed before transferring bacteria. ALL PLATES MUST BE STREAKED FOR ISOLATION!!!!! 2. Each student will have two large TSA plate for the initial isolation streak from the tubes to start Unknown Identification. Record your unknown number on the plate and on your Unknown Report form. Return your Unknown tubes to your Instructor. All original tubes are disposed of after lab as certain organisms will produce metabolites that inhibit other organisms in the tube and certain organisms eat the other bacteria.
3. You will need to return at 24 hours and 48 hours after you have streaked your unknowns. Look for different colony types on your plate. Certain bacteria will produce pinpoint colonies. Transfer 1 colony of each different colony type from the original isolation plate onto regular sized NA plates (subculture). Keep your large plate until you have isolated all possible organisms from it. You must transfer colonies within the first 2-3 days. After 3 days of incubation, the large plates
must be placed in the cold room tub. Longer incubation will result in overgrowth and inhibition of the organisms, which will impact your ability to identify the organisms. If you do not see different colony types, streak a NA plate from the thickest part of your initial streak on the large plate. ALL PLATES MUST BE STREAKED FOR ISOLATION!!!!! 4. A pure culture
5. All results
must be confirmed by colony morphology of a separated colony and a Gram stain. Isolated colonies should be transferred to a new NA, incubated, checked for purity and kept in the cold room to serve as a source of inoculum for biochemical tests. Pure cultures MUST be turned in with the Unknown Identification Report form. MUST be recorded on the Unknown Identification Report form. An Instructor must perform or supervise all coagulase, oxidase and Lancefield tests. You will need to request blood agar and urea slants when needed as these media cannot be stored at room temperatures for extended periods of time. Bring report forms, keys, biochemical tables, instructions with you to all labs and Open Labs. ALL PLATES MUST BE STREAKED FOR ISOLATION!!!!! 6. Biochemical tests on your dichotomous key may be started once a pure culture of the bacteria has been obtained. You will want to take isolated colonies of pure cultures from your NA subculture plate to start all biochemical tests.
7. Each student must perform their own work. You will lose points if another student helps you with the Unknown ID.
8. All media and tests used
9. Students are responsible for keeping their cultures properly labeled and stored. Students are responsible for keeping the work area clean and decontaminated. Tape all plate bags shut after you have removed the plates that you need. The plates will dry out if left open for long periods of time. Please be courteous to your classmates, Open Lab Technicians, and Instructors as you work on unknowns. You must behave professionally at all times.
Each student must identify at least 2 organisms, 1 Gram + bacterium and 1 Gram – bacterium.
You will need to fill out an Unknown Identification Report for all organisms tested, even if a final identification is not determined. This means 4 total reports. Unknown Identification
must be recorded on the Media Log at the side of the room. Do NOT use excessive media, know what you are doing and why. Set up only the test indicated as the next step on your key. You must set up tests sequentially as results for the previous tests are noted. Do NOT set up all tests at the same time. This is NOT following your key. You will lose points if you do not follow your key. 100 points total Dichotomous key for Gram + organisms 5 points Dichotomous key for Gram – organisms 5 points Followed keys for identification of organisms-10 points Logging all media and tests, excess media NOT used-
35 points
Colony characteristics, cell shape and arrangement, Gram reaction -
10 points for each of the 2 Unknowns Identified 10 points Biochemical Test information- complete and correct- 15 points Genus and species –spelled completely and correctly - 10 points Correct identification of additional organisms with all steps correctly performed, is worth 10 points extra credit. You may turn in results early if you have completed testing. Signed dichotomous keys must be turned in with the worksheets and plates with isolated pure cultures.

Bacterial Unknowns and Dichotomous Keys Micro 240 Lab Spring 2011 The idea of a Dichotomous key is to select a test that will allow you to tell 2 different organisms apart. To identify a microorganism you will need more than 1 test, you will be making a type of flow chart for your dichotomous keys. You will actually be using the dichotomous keys you create to identify the unknown microorganisms in this lab. The first step for your Unknown ID is to isolate single colonies and Gram Stain. When you have Gram Stain information for your pure culture you can start to identify the bacterium. You will need to make 1 dichotomous key for Gram positive bacteria and 1 dichotomous key for Gram negative bacteria. You will use the biochemical tests listed in the tables posted on Blackboard. The tables of biochemical tests include certain characteristics, growth media, and reagent based tests available for use in the identification in Micro 240 Lab at UAA. Print the tables and bring to lab. You must key out and conclusively identify each of the 17 organisms listed.
To start your keys select tests that will place the organisms in groups, such as shape for the Gram positive organisms. Now you can work on a series of tests to differentiate the Gram positive rod shaped bacteria from each other as shown in the example below that shows the identification of
You need to key out ALL 17 organisms on the Biochemical Tests tables for Gram Positive and Gram Negative Bacteria. You must have
We will discuss dichotomous keys in lab February 28 to March 3, 2011. Your final dichotomous key for Gram Positive organisms and your key for Gram Negative organisms must be filled in with ink or typed and are due March 14 to17, 2011. Your Instructor must sign and date the acceptance of this key. You must follow this key for identification of your G+ and G- Unknowns without variation unless the variation is discussed, signed, and dated by your Instructor in ink.
Lactobacillus plantarum. Genus names and abbreviations are always capitalized, species names are in lower case. Underline or italicize all genus and species names. at least 2 tests that differentiate the bacteria from other bacteria. When you have the organism differentiated from all others, you need 1 confirmatory test for this organism. At this point you can predict the result of the test based on which organism the previous tests indicated. Select a test that will have a positive result for this organism. Negative results can be false, due to insufficient inoculation with live organism. You can not repeat any tests that you had performed when testing for this specific organism. Due to cost; coagulase, Lancefield Groups, and oxidase tests can ONLY be used as confirmatory tests for the specific organisms that would produce a positive result or a grouping indication with this test and only by an Instructor.
Share |

No comments: