Lab 7 Notebook
BS07 Secondary Assays for Bacterial Characterization
Objective: To profile bacterial populations based on additional biochemical approaches to
better determine growth/metabolic characteristics.
Notes:
Indole test: Biochemical test to determine a bacterial species ability to concert (breakdown)
tryptophan into indole.
Procedure: Test #1
1. Using a sterile loop pick an isolated colony from BAP (blood agar
plate).
2. Smear directly onto the reaction area of the slide.
o One sample per quadrant.
3. Examine test area for color change within 20 seconds.
o Indole negative = yellow color (tryptophan is not being
broken down)
o Indole positive = red/pink color and pink/orange color (tryptophan is
being broken down)
4. Indole test is important because indole is actually responsible for regulating
various aspects of bacterial physiology.
o Ex: spore formation, plasmid stability, drug resistance
o If a bacterium is indole positive: more resistant to drugs and
medications and will survive in harsher environments if they go into
spore formation.
Procedure: Test #2
1. Using a sterile loop pick an isolated colony from BAP (blood agar
plate).
2. Inoculate bacteria in media broth. (Contains either a tryptophan or a
peptone base broth)
3. Grow at 37°C overnight.
4. Add ~5 drops of Kovac’s reagent to culture.
o Indole negative = yellow color
o Ex: pseudomonas is indole negative
o Salmonella is indole negative
o Indole positive = red/pink-orange color
o Ex: gram-negative E.coli are indole positive
o If nothing happens (clear yellow), tells you that the bacteria lack the
ability to break down tryptophan.
TSI Test: (Triple Sugar Iron) agar is used to assess the ability of bacteria to ferment sugars and/or
produce hydrogen sulfide gas.
o Commonly used to test for enteric bacteria (gram-negative bacteria in your
intestines or gut)
o Examples: salmonella and shigella
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, o If bacteria are able to ferment in any of the sugars, it would be glucose,
lactose, or sucrose. Upon fermentation, it produces acid which changes the
pH of the indicator in the agar and you get a color change.
Procedure:
o Use a sterile loop to inoculate slant from liquid culture.
o Press loop into the agar slant.
o Slant contains a pH indicator (red).
o While removing loop, streak slanted side of agar with loop.
o Grow at 37°C overnight.
o (Next day) examine samples.
o First tube: color change, went from red to yellow
o Indicates fermentation of sugars.
o Has empty space at bottom, gas has been produced, pushing
agar up into the tube.
o Has not turned black, gas has been produced but it is not
hydrogen sulfide gas
o Second tube: color gradient, pink color at top and migrates down into
the yellowish orange color.
o Indicates that we only have glucose fermentation, and glucose
only.
o Has not turned black or changes in the agar for the gas pocket,
no gas is being produced.
o Third tube: producing hydrogen sulfide gas.
o Fourth tube: negative control, untreated or uninoculated sample.
API Test: (Analytical profile index) Broad, multi-panel test used for rapid characterization of
bacterial species.
o One stop for multiple assays and answers.
Procedure:
1. Fill all tubes with bacteria culture:
o Fill CIT, VP and GEL entirely (RED ARROWS!)
o Add mineral water to ADH, LDC, ODC, H2S and URE = anaerobic (BLUE
ARROWS!)
o H2S= hydrogen sulfide gas
o GLU= glucose
o SOR = sorbitol
2. Add 5mL distilled water into tray, add API strip and cover with lid
3. Incubate at 37°C overnight.
4. (Next day) examine samples.
o (2nd photo)
o 20 wells
This study source was downloaded by 100000829684747 from CourseHero.com on 03-18-2022 22:09:20 GMT -05:00
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BS07 Secondary Assays for Bacterial Characterization
Objective: To profile bacterial populations based on additional biochemical approaches to
better determine growth/metabolic characteristics.
Notes:
Indole test: Biochemical test to determine a bacterial species ability to concert (breakdown)
tryptophan into indole.
Procedure: Test #1
1. Using a sterile loop pick an isolated colony from BAP (blood agar
plate).
2. Smear directly onto the reaction area of the slide.
o One sample per quadrant.
3. Examine test area for color change within 20 seconds.
o Indole negative = yellow color (tryptophan is not being
broken down)
o Indole positive = red/pink color and pink/orange color (tryptophan is
being broken down)
4. Indole test is important because indole is actually responsible for regulating
various aspects of bacterial physiology.
o Ex: spore formation, plasmid stability, drug resistance
o If a bacterium is indole positive: more resistant to drugs and
medications and will survive in harsher environments if they go into
spore formation.
Procedure: Test #2
1. Using a sterile loop pick an isolated colony from BAP (blood agar
plate).
2. Inoculate bacteria in media broth. (Contains either a tryptophan or a
peptone base broth)
3. Grow at 37°C overnight.
4. Add ~5 drops of Kovac’s reagent to culture.
o Indole negative = yellow color
o Ex: pseudomonas is indole negative
o Salmonella is indole negative
o Indole positive = red/pink-orange color
o Ex: gram-negative E.coli are indole positive
o If nothing happens (clear yellow), tells you that the bacteria lack the
ability to break down tryptophan.
TSI Test: (Triple Sugar Iron) agar is used to assess the ability of bacteria to ferment sugars and/or
produce hydrogen sulfide gas.
o Commonly used to test for enteric bacteria (gram-negative bacteria in your
intestines or gut)
o Examples: salmonella and shigella
This study source was downloaded by 100000829684747 from CourseHero.com on 03-18-2022 22:09:20 GMT -05:00
https://www.coursehero.com/file/102330523/Lab-7-Notebookdocx/
, o If bacteria are able to ferment in any of the sugars, it would be glucose,
lactose, or sucrose. Upon fermentation, it produces acid which changes the
pH of the indicator in the agar and you get a color change.
Procedure:
o Use a sterile loop to inoculate slant from liquid culture.
o Press loop into the agar slant.
o Slant contains a pH indicator (red).
o While removing loop, streak slanted side of agar with loop.
o Grow at 37°C overnight.
o (Next day) examine samples.
o First tube: color change, went from red to yellow
o Indicates fermentation of sugars.
o Has empty space at bottom, gas has been produced, pushing
agar up into the tube.
o Has not turned black, gas has been produced but it is not
hydrogen sulfide gas
o Second tube: color gradient, pink color at top and migrates down into
the yellowish orange color.
o Indicates that we only have glucose fermentation, and glucose
only.
o Has not turned black or changes in the agar for the gas pocket,
no gas is being produced.
o Third tube: producing hydrogen sulfide gas.
o Fourth tube: negative control, untreated or uninoculated sample.
API Test: (Analytical profile index) Broad, multi-panel test used for rapid characterization of
bacterial species.
o One stop for multiple assays and answers.
Procedure:
1. Fill all tubes with bacteria culture:
o Fill CIT, VP and GEL entirely (RED ARROWS!)
o Add mineral water to ADH, LDC, ODC, H2S and URE = anaerobic (BLUE
ARROWS!)
o H2S= hydrogen sulfide gas
o GLU= glucose
o SOR = sorbitol
2. Add 5mL distilled water into tray, add API strip and cover with lid
3. Incubate at 37°C overnight.
4. (Next day) examine samples.
o (2nd photo)
o 20 wells
This study source was downloaded by 100000829684747 from CourseHero.com on 03-18-2022 22:09:20 GMT -05:00
https://www.coursehero.com/file/102330523/Lab-7-Notebookdocx/
