Exercise 9 studyguide

Biochemical tests that yield within minutes

catalase

oxidase

Tube media tests for other metabolic processes

nitrate broth

gelatin

urea broth

phenylalanine

SIM

IMViC

Fermentation vs Respiration

Conversion of organic molecules to an energy source (ATP)

glucose→pyruvate

Respiration 

Final electron acceptor is an inorganic molecule

-oxygen → aerobic respiration

-Nitrate/Sulfate/etc → anaerobic respiration

RESPIRATION=

Glycolysis →Kreb’s Cycle → ETC

-transfer of electrons to molecules with a more positive reduction potential

-WE ARE INDIRECTLY TESTING FOR THE PRESENCE OF AN ETC

Catalase Test Science

-during aerobic metabolism, reactive oxygen species (ROS) may be produced:

• Superoxide radicals

• Hydrogen Peroxide (H2O2)

Some bacteria have enzymes capable of breaking down these toxins

-Superoxide dismutase (SOD)- superoxide radicals to hydrogen peroxide

-Catalase- hydrogen peroxide to water and oxygen

Catalase Test

-Detects Catalase

-convert hydrogen peroxide to water and oxygen

H2O2 → 2H2O + O2

Add 3% hydrogen peroxide to the edge of the colony

POSITIVE for catalase = BUBBLIING

Cytochrome C oxidase test Science

-main function is to remove electrons from cytochrome c (oxidase) and transfer them to oxygen.

-can also reduce cytochrome c by oxidizing chromogenic reducing agent, dimethyl-p-phenyleneduamine

Oxidase test

-detects cytochrome c oxidase

(transfers electrons to final electron acceptor-oxygen)

-INDICATOR- dimethyl-p-phenylenediamine hydrochloride

-in the presence of cytochrome c oxidase, regent is oxidized and turns BLUE TO BLACK

Nitrate Respiration science

-utilize nitrate (NO3) as final inorganic electron acceptor

-requires enzyme nitrate reductase to convert nitrate to nitrate

-some bacteria have other enzymes to reduce nitrate all the way down to N2 (denitrification)

-others can reduce nitrate to ammonium for incorporation (assimilatory nitrate reduction)

Nitrate Broth

-detects nitrate reductase- which reduces nitrate to nitrite (NO3 → NO2)

-Nitrate 1 (sulfanilic acid) and Nitrate 2 (dimethyl-alpha-naphthylamine) reacts with NO2 → RED BRICK COLOR

-if nitrate is not used and is residual, then zinc powder will catalyze the reaction

-a red result after Zn addition indicates that nitrate was not used- organism did not have enzyme

Urea Broth

-detects urease

-degrades urea → 2 ammonia (NH3) and carbon dioxide (CO2)

-increases pH (ammonia) - more alkaline

-CONTAINS; urea and pH indicator phenol red

-pH increases = positive for urease → CERISE COLOR

Gelatin

-detects the presence of gelatinase (breaks down gelatin)

-Catalyzes the hydrolysis of gelatin → amino acid

-gelatin solidifies in cold temperature, so put into ice bath

-if after ice bath it is liquidy and not solidified then it contains gelatinase and broke down gelatin

Phenylalanine slant

-medium detects production of phenylalanine deaminase (breaks down phenylalanine to PPA)

Phenylalanine → phenyl pyruvic acid (PPA) + ammonia (NH3)

ADD 5-10 drops of 10% ferric chloride (FeCl3) regant

-in the presence of PPA FeCl3 appears a deep green color

PP IN THE POOL TURNS GREEN

SIM broth

S-Sulfide

• H2S produced by bacterium reacts with Fe in medium to produce FeS

• reduced sulfur with iron is black precipitate

I-Indole

• Add 3-5 drops of Kovac’s regent

• POSITIVE FOR INDOLE IS PINK

• if posses tryptophanase it can break down tryptophan, indole is product

M-Motility

• POSITIVE FOR MOTILITY IS CLOUDY

Fermentation

-electrons get dumped back to pyruvate

-final electron acceptor is an organic molecule

-Pyruvate → general end products are organic acids and gases

-general term for the breakdown of polysaccharides into monomers that can be fermented

Example of Mixed Acid Fermenter

E. coli

Example of 2,3-butabediol fermentation

Enterobacter aerogenes

MR-VP broth

-one medium

-but Methyl Red (MR) and Vogues-Proskauer (VP) tests must be done separately

Methyl Red test

-tests for mixed acid fermenters

-add 3-4 drops of methyl red reagent

-detects high acid production, lowering pH to 4.4 or less

-if it remains red color then it is positive for mixed acid fermenters

(low acid does not allow retention of red)

Voges-Proskauer (VP) test

-tests for 2,3-butanediol fermenters

glucose →acetoin→2,3-butanediol

-2,3-butanediol fermenters produce the acetoin intermediate

-acetoin reacts with Barritt’s reagents 

• VP 1 (Alpha Naphthol) -10 drops to intensify red color

• VP 2 (KOH)- produces alkaline condition condition that favors acetoin oxidation.

When acetoin oxidation → diacetyl and O2 is made. PRESENCE OF O2 PRODUCES RED COLOR. On top.

Tryptone Broth

-detects tryptophanase

-Hydrolyzes tryptophan → indole, pyruvate, ammonia

INDOLE- reacts with Kovac’s reagent

• Para-dimethylaminobenzaldehyde 

• (DMABA)

Indole + Kovac’s reagent = CERISE ring on top

Simmons Citrate Slant

Medium detects utilization of citrate as sole carbon source for growth

INDICATOR- bromothymol blue

-blue reaction may accompany growth, but the PRIMARY INDICATOR of Citrate utilization is GROWTH

-growth is almost always accompanies by a blue color change

-BLUE IT GREW

IMViC is

-set of 4 tests that can be used to differentiate E. coli from Enterobacter aerogenes

4 tests:

• Indole (SIM or tryptone broth)

• MR

• VP

• Citrate

E.coli= POS indole, POS MR, NEG VP, NEG Citrate

E. aerogenes= NEG indole, NEG MR, POS VP, POS Citrate