Exercise 9

What is the main purpose of fermentation and respiration in cells?

conversion of organic molecules into energy (ATP)

What is the first step in both fermentation and respiration?

glycolysis

• glucose → pyruvate

What determines what happens to pyruvate after glycolysis?

the catabolic system the organism uses

• fermentation or respiration

What is the final electron acceptor in respiration?

an inorganic molecule

What is the final electron acceptor in aerobic respiration?

oxygen (O2)

What is the general pathway for respiration?

glycolysis → krebs cycle → electron transport chain (ETC)

What is the electron transport chain?

a series of electron transfers to molecules with progressively more positive reduction potentials

What are examples of final electron acceptors in anaerobic respiration?

nitrate (NO3-), sulfate (SO4^2-)

How do we indirectly test for the presence of an ETC in bacteria?

by testing for electron transfer activity, often using indicators that detect oxidative reactions

What harmful byproducts can be produced during aerobic metabolism?

reactive oxygen species (ROS)

• superoxide radicals (O2-)

• hydrogen peroxide (H2O2)

Which bacteria typically have enzymes to break down ROS?

aerobic and facultative anaerobes

What enzymes help detoxify ROS in bacteria?

• superoxide dismutase (SOD): converts superoxide radicals

• catalase: breaks down hydrogen peroxide (H2O2) into water and oxygen

What enzyme does the catalase test detect?

catalase, which converts hydrogen peroxide (H2O2) → water + oxygen

How is the catalase test performed?

add 3% hydrogen peroxide to the edge of the bacterial colony

How do you interpret a positive catalase test?

bubbling from oxygen release indicates catalase is present

How do you interpret a negative catalase test?

no bubbling, indicating catalase is absent

What is the main function of cytochrome c oxidase?

to remove electrons from cytochrome c (oxidize it) and transfer electrons to oxygen (reduce O2)

How can cytochrome c be detected in the lab?

by using a chromogenic reducing agent that changes color when oxidized

What is an example of a chromogenic reducing agent used in the oxidase test?

dimethyl-p-phenylenediamine

What enzyme does the oxidase test detect?

cytochrome c oxidase, which transfers electrons to oxygen

What indicator/reagent is used in the oxidase test?

Dimethyl-p-phenylenediamine hydrochloride (an aromatic amine)

What happens when cytochrome c oxidase is present?

the reagent is oxidized, producing a dark blue to black color

What indicates a negative oxidase test?

no color change; reagent remains colorless

What does nitrate respiration utilize as the final electron acceptor?

Nitrate (NO₃⁻), an inorganic molecule

What enzyme is required to convert nitrate to nitrite?

nitrate reductase

What is denitrification?

some bacteria can reduce nitrate all the way to N₂ gas using other enzymes

What is assimilatory nitrate reduction?

reduction of nitrate to ammonium (NH₄⁺) for incorporation into cellular molecules

What enzyme does the nitrate broth test detect?

nitrate reductase, which reduces nitrate (NO3-) → nitrite (NO2-)

What reagents are added to detect nitrite in Nitrate Broth?

Nitrate I (sulfanilic acid) and Nitrate II (dimethyl-alpha-naphthylamine)

What indicates a positive result for nitrate reductase?

formation of a brick red color after adding the reagents

What is the purpose of adding zinc powder?

To catalyze the reaction if nitrate is still present and not reduced by the organism

How do you interpret a red color after zinc addition?

Negative result – nitrate was not used; the organism does not have nitrate reductase

How do you interpret no color after zinc addition?

Nitrate was reduced beyond nitrite (e.g., denitrification) – positive for nitrate reduction

What enzyme does the urea broth test detect?

urease, which degrades urea → 2 ammonia (NH3) + CO2

How does urease activity affect the pH of the medium?

increases pH, making the medium more alkaline

What pH indicator is used in urea broth?

phenol red

How is the color interpreted in urea broth?

• cerise → pH > 8.1 (strongly alkaline, positive urease)

• red → neutral pH

• yellow → acidic pH

What enzyme does the gelatin test detect?

gelatinase, which hydrolyzes gelatin → amino acids

Why is gelatin used in the gelatin test?

because gelatin solidifies at low temperatures, allowing easy detection of hydrolysis

How is the gelatin test read?

after chilling the tube in an ice bath; if the gelatin remains liquid, the test is positive

What indicates a negative gelatin test?

gelatin remains solid after chilling, indicating no gelatinase activity

What enzyme does the phenylalanine slant detect?

phenylalanine deaminase, which converts phenylalanine → phenylpyruvic acid (PPA) + ammonia (NH3)

What reagant is added to detect PPA?

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

How is a positive result indicated in phenylalanine slant?

deep green color forms in the presence of PPA

How is a negative result indicated in phenylalanine slant?

no color change after adding ferric chloride

What does the S in SIM medium stand for, and what does it test?

sulfide; tests for H2S production

• H2S reacts with iron in the medium → black precipitate (FeS)

What does the I in SIM medium stand for, and how is it tested?

indole; tests for tryptophanase activity

• add kovacs reagent to detect indole

What does the M in SIM medium stand for, and what does it test?

motility; detects whether bacteria can move away from the stab line in soft agar

What serves as the final electron acceptor in fermentation?

an organic molecule

How does the energy yield of fermentation compare to respiration?

fermentation produces less energy than respiration

What happens to pyruvate during fermentation?

its converted into organic acids and gases

Name common fermentation pathways

• Mixed acid fermentation

• 2,3-butanediol fermentation

How is the term “fermentation” used in relation to polysaccharides?

it refers to the breakdown of polysaccharides into monomers that can be fermented

• ex: lactose → glucose + galactose → pyruvate

What do MR and VP tests detect?

two different fermentation pathways — mixed acid (MR) and 2,3-butanediol (VP)

Can the MR and VP tests be performed on the same culture tube?

no, they must be done separately

What does the Methyl Red (MR) test detect?

Mixed acid fermentation, which produces stable, strong acids

What reagent is used in the MR test?

methyl red reagent

What is a positive MR result?

red color - indicates high acid production (pH ≤ 4.4)

What is a negative MR result?

yellow/orange color - indicates low acid concentration (pH > 4.4)

Vogues-Proskauer (VP) tests for

2,3-butanediol fermenters

• glucose → acetoin → 2,3-butanediol

2,3-butanediol fermenters produce

the acetoin intermediate

Acetoin can interact with

VP I and VP II (Barritt’s reagents)

What enzyme does the tryptone broth test detect?

tryptophanase

What reaction does tryptophanase catalyze?

tryptophan → Indole + Pyruvate + Ammonia (NH₃)

What reagent is added to Tryptone Broth to detect indole?

Kovac’s reagent (contains DMABA and HCl in amyl or butyl alcohol)

What is the function of Kovac’s reagent?

It is nonpolar and extracts indole to the top layer of the tube

What indicates a positive indole test?

a cerise color at the top layer

hat indicates a negative indole test?

no color change

• top layer remains yellow/unchanged

What does the Simmons Citrate Slant test for?

the ability of an organism to use citrate as its sole carbon source for growth

What is the indicator in the Simmons Citrate Slant?

bromothylmol blue

What causes a color change in the medium?

alkaline byproducts from citrate utilization increase pH, turning the medium blue

What is the primary indicator of a positive test for Simmons Citrate test?

growth on the slant

• color change to blue may accompany growth but not required

What is the primary indicator of a negative test for Simmons Citrate test?

no growth and the medium remains green

The four tests of IMViC can be used to differentiate

E.coli from Enterobacter aerogenes

The four tests of IMViC

• indole

• methyl-red

• voges-proskauer

• citrate

E.coli in IMViC is positive for

indole and MR, but not VP or citrate

Enterobacter aerogenes in IMViC is positive for

VP and Citrate, but not indole or MR