Microbiology Exam 2

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Catalase test purpose
Produce catalase as detected by O2 gas produced (bubbles).
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Catalase procedure
-Spread loop of organism on slide -Add drop of H2O2
Catalase test results
catalase positive: bubbles catalase negative: no bubbles
Oxidase test purpose
- if a bacterium possesses the enzymes (Cytochrome c oxidase)
oxidase test procedure
-take a colony of bacteria -scrape it onto a small piece of filter paper -add a drop of oxidase reagent
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oxidase test results
-Positive Oxidase Test: blue within 20-30 seconds -Negative Oxidase Test: No color change within thirty seconds
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Reagent used in Oxidase test
oxidase reagent
Triple Sugar Iron
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TSI test
- catabolic abilities of bacterium when given glucose, sucrose, & lactose -produces hydrogen sulfide gas (H2S)
TSI test results
-low pH: yellow -pH 7: Orange -alkaline: red
Verbalizing/reading data
- K/A= utilize glucose - K/K and NC/NC= no utilize glucose, lactose, sucrose -K/A+H2S= Utilize glucose and hydrogen sulfide production -A/A + gas= glucose, lactose, sucrose
Citrate (Simmon's Citrate)
- Determine if bacterium utilizes citrate as a source of carbon.
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Carbon source for citrate
Sodium Citrate
pH indicator for citrate
bromthymol blue
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Citrate utilization
-enzymes: citrate permease & citrase
citrate permease
imports citrate into the cell
breaks down citrate
Citrate medium colors
-Blue: at alkaline pH -Green: at natural pH
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Citrate test results
citrate positive: blue Citrate negative: green
Urease Broth
- determine if bacterium possesses enzyme urease
What is urease?
-Acts on urea to produce ammonia -ammonia drive pH up -Phenol red turns orange to hot pink
Urease results
-urease positive: hot pink -urease negative: no change
MRVP broth (Methyl Red Voges Poskauer)
-determines if the bacterium follows a mixed acid fermentative pathway -or Butylene Glycol Fermentative pathway
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MRVP reaction
- bacteria can metabolize carbon under anaerobic conditions
methyl red
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MRVP reaction
- positive: Red tube -negative: orange/yellow
What carbohydrate(s) does a bacterium use if it presents as an A/A in a TSI tube?
fermentation of glucose, sucrose, and lactose.
If you inoculated a pure culture of bacteria into an MIO tube and the tube is turbid after 24 h of incubation, is it motile or nonmotile?
What is the substate in urease broth that yield the produce ammonia?
Urease acts on urea to produce ammonia.
. If hydrogen sulfide gas is colorless, how do you determine it's presence in a TSI tube?
-by observing the chemical reaction -the black color at the butt of the tube formed by gas and iron
Why do you have to wait 3-5 days to determine a VP reaction?
if it is read too soon the results may be a false-positive.
If you have an A/A TSI tube and pink colonies on a MAC plate, what carbohydrate(s) does your bacterium definitely utilize?
bacterium utilizes lactose fermentation.
Why do you have to see a yellow MIO tube 24 after inoculation and incubation turn to purple after 48 h incubation to determine the presence of ODC?
- determine the presence of ODC (Ornithine Decarboxylase) -it allows the microbe to use the ornithine.
What does catalase do?
Catalase is an enzyme that decomposes hydrogen peroxide to the components water and oxygen.
What cytochrome are you looking for when you perform an oxidase test?
Cytochrome c oxidase.
Why is MAC both selective and differential?
-it selectively allows only Gram-negative bacteria to grow -whilst differentiating which bacteria can ferment lactose.
How does Kovac's reagent help you determine indole production?
Without Kovac's reagent we would not be able to visualize the reaction because Indole is a colorless compound.
What enzymes must a bacterium possess to be "citrate positive"
citrate, citrase, and citrase permease.
how do you know if a bacterium is "citrate positive"?
look for a blue color, if it lacks these enzymes then it remains green
What are the three sugars and their concentrations in a TSI tube
-Lactose: 1.0% -Sucrose: 1.0% -Glucose: 0.1%
What are the two fermentation pathways being screened for in MRVP broth?
-Mixed Acid Fermentation -Butylene Glycol Fermentation
utilize glucose only
No utilize glucose, lactose, sucrose
utilize glucose only with hydrogen sulfide gas
A/A + gas
utilize glucose, lactose, sucrose
What is the substrate and the enzyme and product when working with urease broth?
Substrate: urea, Enzyme: urease, Product: Ammonia
What is the substrate and the enzyme and the product(s) when working with MIO medium?
Substrate: Motility, Indole, ODC, Enzyme: Ornithine decarboxylase, Product: Enterobacteria, Indole
pH indicator in TSI medium
Phenol red
pH indicator for Urease Medium
Phenol red or Phenolphthalein
pH indicator for MIO medium
Bromcresol purple
How do you determine if Mixed Acid fermentation has occured?
Add 2-3 drops of methyl red into culture. Red, = positive results. orange/yellow = negative result.
What is the intermediate compund that will eventually lead to butylene glycol?
Acetoin - acetoin react with alpha-napthol and you will see a red color - red color after adding alpha-napthol = positive for butylene glycol fermentation