Microbiology lab 7 secondary characteristic assays_v2.docx...

  • Portage Learning
  • BIOD 152
  • Lab Report
  • lboettcher
  • 10
  • 86% (29) 25 out of 29 people found this document helpful

This preview shows page 1 - 5 out of 10 pages.

Microbiology lab (19:15) Lab 7: Introduction to secondary characterization Assays AP Reference Chart
API strip and template Lab Notebook LB07 Secondary Assays for Bacterial Characteristics Objective: To profile bacterial populations based on additional biochemical approaches to better determine growth/metabolic characteristics Notes: 3 main tests 1. Indole Test 2. TSI Test 3. API Test Indole Test : Biochemical test to determine a bacterial species ability to convert or break down tryptophan into indole. Looks like an oxidase test; has four quadrants where you can place your bacterial sample Procedure: Test #1 1. Using a sterile loop pick an isolated colony from BAP 2. Smear directly onto the reaction area of the slide; dispose of sterile loop into bioharzard waste 3. Examine test area for color change within 20 seconds Notes Indole negative=yellow color Indole positive=red/pink orange color (pink-top right quadrant); tryptophan is being broken down
Indole best is important because indole is responsible for regulating various aspects of bacterial physiology; this include pore formation, plasma and stability as well as drug resistance. If bacteria is able to break down tryptophan into indole it will be able to be more resistance to drugs/medications as well as surviving harsh environments. Procedure: Test #2 (use it in a liquid culture) 1. Using a sterile loop pick an isolated colony from BAP 2. Inculcate bacteria into media broth (contains tryptophan or peptone base broth) 3. Grow at 37 degrees Celsius overnight (Go from clear transparent media to cloudiness indicating hat the culture has grown) 4. Add around 5 drops of Kovac’s reagent to culture Findings/Results: See on right hand side of handout there is a strong + reaction because you see bright pink band at the top which means indole is being produced. In this particular bacteria whereas you can also have a weaker tryptophan isn’t being broken down as efficiently but you still get the slight pink color sample. Both considered indole positive for this test. Whereas if no color change and remained a yellow color broth that would be indole negative.
TSI Test: [Triple Sugar Iron] agar is used to assess the ability of bacteria to ferment sugars and/or produce hydrogen sulfide gas; uses agar slant shown in picture in right. In test tubes we have agar which we’ve seen in sterile petri dishes but now done tube to produce nice gradient slant effect Procedure: 1. Use a sterile loop to inoculate slant from liquid culture 2. Press loop into the agar slant 3. While removing loop from the agar at the bottom of the tube, lightly streak it along gradient of slant (slanted side of agar with loop) as you pull out; dispose of sterile loop in biohazard bin 4. Grow at 37 degrees Celsius over night for overnight growth 5. (Next day) examine samples Right hand side multiple colors and variants that came out as result of rest

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture