BY 206 Lab Tope Week 7 (Ex 2-7; 5-4, 7, 9, 10, 13, 16, 30)

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FLUID THIOGLYCOLLATE BROTH

anaerobic, microaerophilic

FLUID THIOGLYCOLLATE BROTH

This liquid medium promotes the growth of a variety of fastidious to nonfastidious microorganisms. It is generally associated with the cultivation of ____ and ____ bacteria.

yeast extract and pancreatic digest of casein

FLUID THIOGLYCOLLATE BROTH

Key components of this medium are yeast extract, pancreatic digest of casein, dextrose, sodium thioglycollate, L-cysteine, and resazurin.

Which two provide nutrients to the medium?

sodium thioglycollate and L-cysteine

FLUID THIOGLYCOLLATE BROTH

Key components of this medium are yeast extract, pancreatic digest of casein, dextrose, sodium thioglycollate, L-cysteine, and resazurin.

Which two reduce oxygen to water?

resazurin

*pink when oxidized, colorless when reduced

FLUID THIOGLYCOLLATE BROTH

Key components of this medium are yeast extract, pancreatic digest of casein, dextrose, sodium thioglycollate, L-cysteine, and resazurin.

Which one acts as an indicator of O₂?

aerotolerant anaerobe

*doesn't need oxygen for growth but can tolerate its presence; growth all throughout the medium

FLUID THIOGLYCOLLATE BROTH

What type of organism is this based on their aerotolerance?

facultative anaerobe

*doesn't need oxygen for metabolism but uses it when it's present; growth all throughout but heavier at the top

FLUID THIOGLYCOLLATE BROTH

What type of organism is this based on their aerotolerance?

strict anaerobe

*lack the metabolic enzyme system for using oxygen, so it's poisoned by it; growth is heavier at the bottom

FLUID THIOGLYCOLLATE BROTH

What type of organism is this based on their aerotolerance?

strict aerobe

*need oxygen to grow; heavy growth at the top where oxygen is most plentiful

FLUID THIOGLYCOLLATE BROTH

What type of organism is this based on their aerotolerance?

microaerophile

*requires only small amounts of oxygen; grows near the top

FLUID THIOGLYCOLLATE BROTH

What type of organism is this based on their aerotolerance?

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METHYL RED & VOGES-PROSKAUER TESTS (MR-VP)

IMViC (Indole, Methyl red, Voges-Proskauer, Citrate)

MR-VP TESTS

The methyl red and Voges-Proskauer tests are components of what battery of tests?

Enterobacteriaceae

MR-VP TESTS

The methyl red and Voges-Proskauer tests are used to distinguish between members of the family ____ and differentiate them from the other G(-) rods.

peptone

MR-VP TESTS

Methyl red and Voges-Proskauer broth contains peptone, glucose, and a phosphate buffer.

Which is used to provide protein?

glucose

MR-VP TESTS

Methyl red and Voges-Proskauer broth contains peptone, glucose, and a phosphate buffer.

Which is used to provide fermentable carbohydrate?

phosphate buffer (potassium phosphate)

MR-VP TESTS

Methyl red and Voges-Proskauer broth contains peptone, glucose, and a phosphate buffer (potassium phosphate).

Which is used to resist pH changes in the medium?

mixed acid fermentation

*overcomes the phosphate buffer and lowers the pH (produces stable acid end products)

MR-VP TESTS (methyl red test)

The Methyl red test detects organisms capable of performing . . .

MR-VP TESTS (methyl red test)

MR (+) organisms lower the broth's pH permanently.

methyl red indicator dye

MR-VP TESTS (methyl red test)

Following incubation, mixed acid fermentation is verified by the addition of what?

-positive test

-mixed acid fermentation

MR-VP TESTS (methyl red test)

A red color indicates . . .

4.4

MR-VP TESTS (methyl red test)

Methyl red is red at pH ___.

-negative test

-no mixed acid fermentation

MR-VP TESTS (methyl red test)

A yellow color indicates . . .

6.2

MR-VP TESTS (methyl red test)

Methyl red is yellow at pH ___.

a negative or inconclusive test

MR-VP TESTS (methyl red test)

An orange color indicates . . .

organisms that are capable to ferment glucose, but quickly convert their acid products to acetoin and 2,3-butanediol

MR-VP TESTS (VP test)

The Voges-Proskauer (VP) test was designed for . . .

MR-VP TESTS (VP test)

VP(+) organisms initially may produce acid and temporarily lower the pH, but because the 2,3-butanediol fermentation end products are neutral, the pH at completion of the test is near neutral.

diacetyl

MR-VP TESTS (VP test)

Adding VP reagents oxidizes acetoin to . . .

guanidine nuclei

MR-VP TESTS (VP test)

Diacetyl reacts with ____ from peptone to produce a red color.

-positive test

-2,3 butanediol fermentation (acetoin produced)

MR-VP TESTS (VP test)

A red color indicates . . .

-negative test

-NO 2,3 butanediol fermentation (acetoin is not produced)

MR-VP TESTS (VP test)

No color change (or development of copper color) indicates . . .

. . . . .

OXIDASE TEST

cytochrome c oxidase

OXIDASE TEST

The oxidase test is designed to identify bacteria containing the respiratory enzyme . . .

Neisseria, Moraxella

OXIDASE TEST

It is used for the presumptive identification of the oxidase (+) ____ and ____.

Enterobacteriaceae, Pseudomonadaceae

OXIDASE TEST

It is also used to differentiate the oxidase (-) ____ from the oxidase (+) ____.

tetramethyl-p-phenylenediamine

OXIDASE TEST

Cytochrome c oxidase oxidizes and catalyzes the reduction of cytochrome c by a chromogenic reducing agent called . . .

flavoproteins, iron-sulfur proteins, and cytochromes

OXIDASE TEST

What are three important ETC molecules unable to donate protons?

20 seconds

OXIDASE TEST

How much time is allotted for a color change to occur after adding the reagent?

-positive test

-cytochrome c oxidase is present

OXIDASE TEST

Dark blue/purple within 20 seconds indicates . . .

-negative test

-cytochrome c oxidase NOT present

OXIDASE TEST

NO color change to dark blue/purple within 20 seconds indicates . . .

. . . . .

CITRATE UTILIZATION TEST

Enterobacteriaceae

CITRATE UTILIZATION TEST

This test was designed to differentiate members of the ____, all of which are facultative anaerobes.

IMViC (Indole, Methyl red, Voges-Proskauer, Citrate)

CITRATE UTILIZATION TEST

This test is part of a test series known as the . . .

sodium citrate

CITRATE UTILIZATION TEST

The citrate utilization test is used to determine the ability to use ____ as its sole source of carbon.

Simmons citrate agar

*defined medium

CITRATE UTILIZATION TEST

What agar is used?

citrate permease

CITRATE UTILIZATION TEST

What enzyme do bacteria possess to transport citrate into the cell and perform citrate fermentation?

ammonium (in the form of ammonium phosphate)

CITRATE UTILIZATION TEST

These organisms must also be able to survive with ____ as their sole nitrogen source.

citrate lyase

CITRATE UTILIZATION TEST

What enzyme hydrolyzes citrate into oxaloacetate and acetate?

bromothymol blue dye

*green at pH 6.9

*blue at pH 7.6

CITRATE UTILIZATION TEST

What is used as a color indicator?

-positive test

-citrate is utilized

CITRATE UTILIZATION TEST

A blue color indicates . . .

-positive test

-citrate is utilized

CITRATE UTILIZATION TEST

No color change, but growth on the slant indicates . . .

-negative test

-citrate is NOT utilized

CITRATE UTILIZATION TEST

No color change, and no growth on the slant indicates . . .

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MALONATE UTILIZATION TEST

Enterobacteriaceae

MALONATE UTILIZATION TEST

The malonate test differentiate between Escherichia and Enterobacter, but now includes ____.

succinate dehydrogenase

MALONATE UTILIZATION TEST

What enzyme does the citric acid cycle require to convert succinate into fumarate?

malonate (malonic acid)

MALONATE UTILIZATION TEST

What is added to the medium to replace succinate as the substrate in the reaction?

glucose

MALONATE UTILIZATION TEST

What is added to the medium to promote growth of organisms that are slow to respond?

buffers

MALONATE UTILIZATION TEST

What is added to the medium to stabilize it at pH 6.7?

-positive test

-malonate is utilized

MALONATE UTILIZATION TEST

A dark blue color indicates . . .

-negative test

-malonate is NOT utilized

MALONATE UTILIZATION TEST

NO color change or a slightly yellow color indicates . . .

. . . . .

STARCH HYDROLYSIS (AMYLASE TEST)

Gardnerella vaginalis

STARCH HYDROLYSIS (AMYLASE TEST)

Starch agar was originally designed for cultivating Neisseria. But it is now used to isolate and presumptively identify . . .

STARCH HYDROLYSIS (AMYLASE TEST)

Starch agar also aids in differentiating members of the genera Corynebacterium, Clostridium, Bacillus, Bacteroides, Fusobacterium, and Enterococcus.

α-amylase and oligo-1,6-glucosidase

STARCH HYDROLYSIS (AMYLASE TEST)

Organisms that produce and secrete the extracellular enzymes ____ and ____ are able to hydrolyze starch.

iodine

STARCH HYDROLYSIS (AMYLASE TEST)

What reagent is added to detect the presence or absence of starch in the vicinity around the bacterial growth?

-positive test

-α-amylase/oligo-1,6-glucosidase are present

STARCH HYDROLYSIS (AMYLASE TEST)

Clearing around growth indicates . . .

-negative test

-α-amylase/oligo-1,6-glucosidase is NOT present

STARCH HYDROLYSIS (AMYLASE TEST)

NO clearing around growth indicates . . .

. . . . .

CASEIN HYDROLYSIS (CASEASE TEST)

casease

CASEIN HYDROLYSIS (CASEASE TEST)

The casein hydrolysis test is used for the cultivation and differentiation of bacteria that produce the enzyme ____.

casein

*what gives milk its white color

CASEIN HYDROLYSIS (CASEASE TEST)

Casease hydrolyzes the milk protein . . .

undefined

CASEIN HYDROLYSIS (CASEASE TEST)

What type of medium is milk agar?

-positive test

-casease is present

CASEIN HYDROLYSIS (CASEASE TEST)

Clearing in agar indicates . . .

-negative test

-casease is absent

CASEIN HYDROLYSIS (CASEASE TEST)

NO clearing in agar indicates . . .

ur doing great hun

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ENTEROPLURI-TEST (ENTEROTUBE II)

Enterobacteriaceae

ENTEROTUBE II

The Enterotube II is a multiple test system used for rapid identification of bacteria from the family ____.

enteric

ENTEROTUBE II

The Enterotube II is used to identify ____ bacteria based on glucose, adonitol, lactose, arabinose, sorbitol, dulticotl fermentation, lysine and ornithine decarboxylation, sulfur reduction, indole production, acetoin production from glucose fermentation, phenylalanine deamination, urea hydrolysis, and citrate utilization.