Lab 7 Introduction to Secondary Characterization Assays
Objective: To profile bacterial population based on additional biochemical approaches
to better determine growth/ metabolic characteristics
Indole Test: Biochemical test to determine a bacterial species ability to convert
(breakdown) tryptophan into indole.
Procedure: Test #1
1. Using a sterile loop pick an isolated colony from BAP
2. Smear directly onto the reaction area of the slide
3. Examine test area for color change within 20 seconds
Procedure: Test #2
1. Using a sterile loop pick an isolated colony from BAP
2. Inoculate bacteria in media broth
3. Grow at 37C overnight
4. Add ~5 drops of Kovac's reagent to culture.
TSI test: [Triple Sugar Iron] agar is used to assess the ability of bacteria to ferment
sugars and/or produce hydrogen sulfide gas.
Procedure:
, 1. Using a sterile loop inoculate slant from liquid culture
2. Press loop into the agar slant
3. While removing loop, streak slanted side of agar with loop
4. Grow at 37 degrees overnight
5. Examine samples the next day
API test: Broad, multi-panel test used for rapid characterization of bacterial species.
Procedure:
1. Fill all tubes with bacteria culture
- Fill CIT, VP and GEL entirely
- Add mineral oil to ADH, LDC, ODC, H2S AND URE = anaerobic
2. Add 5 mL distilled water into tray, add API strip and cover with lid
3. Incubate at 37 degrees overnight
4. (Next day ) examine samples
Notes:
Prepare the materials and safety materials equipment.
Indole Test
● Indole negative
o Yellow
▪ Tryptophan is not being broken down
● Indole positive
o Red or pinkish/Orange color
▪ Tryptophan is being broken down
*Remember you don’t always get a perfect shade of color.
Objective: To profile bacterial population based on additional biochemical approaches
to better determine growth/ metabolic characteristics
Indole Test: Biochemical test to determine a bacterial species ability to convert
(breakdown) tryptophan into indole.
Procedure: Test #1
1. Using a sterile loop pick an isolated colony from BAP
2. Smear directly onto the reaction area of the slide
3. Examine test area for color change within 20 seconds
Procedure: Test #2
1. Using a sterile loop pick an isolated colony from BAP
2. Inoculate bacteria in media broth
3. Grow at 37C overnight
4. Add ~5 drops of Kovac's reagent to culture.
TSI test: [Triple Sugar Iron] agar is used to assess the ability of bacteria to ferment
sugars and/or produce hydrogen sulfide gas.
Procedure:
, 1. Using a sterile loop inoculate slant from liquid culture
2. Press loop into the agar slant
3. While removing loop, streak slanted side of agar with loop
4. Grow at 37 degrees overnight
5. Examine samples the next day
API test: Broad, multi-panel test used for rapid characterization of bacterial species.
Procedure:
1. Fill all tubes with bacteria culture
- Fill CIT, VP and GEL entirely
- Add mineral oil to ADH, LDC, ODC, H2S AND URE = anaerobic
2. Add 5 mL distilled water into tray, add API strip and cover with lid
3. Incubate at 37 degrees overnight
4. (Next day ) examine samples
Notes:
Prepare the materials and safety materials equipment.
Indole Test
● Indole negative
o Yellow
▪ Tryptophan is not being broken down
● Indole positive
o Red or pinkish/Orange color
▪ Tryptophan is being broken down
*Remember you don’t always get a perfect shade of color.
