Microbiology Exam 2

Catalase test purpose

Produce catalase as detected by O2 gas produced (bubbles).

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

oxidase test results

-Positive Oxidase Test: blue within 20-30 seconds -Negative Oxidase Test: No color change within thirty seconds

Reagent used in Oxidase test

oxidase reagent

TSI

Triple Sugar Iron

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.

Carbon source for citrate

Sodium Citrate

pH indicator for citrate

bromthymol blue

Citrate utilization

-enzymes: citrate permease & citrase

citrate permease

imports citrate into the cell

citrase

breaks down citrate

Citrate medium colors

-Blue: at alkaline pH -Green: at natural pH

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

MRVP reaction

- bacteria can metabolize carbon under anaerobic conditions

MR

methyl red

VP

Voges-Proskauer

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?

Motile

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

K/A

utilize glucose only

K/K

No utilize glucose, lactose, sucrose

K/A+H2S

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