Module 9 Prelab 2: Oxidase & Nitrate Reduction Tests

what does the oxidase test for?

bacterium that has the oxidase enzyme (used in aerobic respiration)

oxidase

enzyme that helps in the process of aerobic respiration

what is oxidase present in?

non-enteric gram negative bacteria

purpose of the oxidase test

determine if a bacterium has the oxidase enzyme, which helps in the process of aerobic respiration

procedure of oxidase test

1. Transfer bacteria to filter paper or a cotton swab.

2. Add a drop of oxidase reagent to the bacteria.

3. Wait 1-2 mins

appearance of positive oxidase test

the reagent turns purple within 1-2 minutes

meaning of positive oxidase test

the bacteria have the oxidase enzyme and it performs aerobic respiration

appearance of negative oxidase test

no color change

meaning of negative oxidase test

the bacteria do not have the oxidase enzyme and it doesn’t perform aerobic respiration

enteric bacteria such as Escherichia coli are oxidase _____

negative

Why should reactions for the oxidase test be recorded within two minutes?

if you wait too long, the oxygen in the air will react and you will get a false positive

How is the oxidase test helpful in distinguishing Gram-negative Enterobacteriaceae from other Gram-negative bacilli such as Pseudomonas or Alcaligenes?

it shows which bacteria is enteric

purpose of nitrate reduction test

to determine whether a bacterium can reduce nitrate (NO₃⁻) to nitrite (NO₂⁻) or further reduce it to nitrogen gas (N₂) as part of its metabolic processes

what does the nitrate reduction test test for?

nitrate reductase — whether bacteria can use nitrate instead of oxygen for respiration

what does the nitrate reduction test tell us?

if the bacterium can switch to anaerobic respiration using nitrate instead of oxygen

• tells us if they are faculatative anaerobes

what kind of respirators are Pseudomonas?

obligate aerobes

do obligate aerobes like Pseudomonas use cytochrome c oxidase?

yes, obligate aerobes use oxidase

Pseudomonas result for oxidase test

positive test

how is the positive test for Pseudomonas on the nitrate test detected?

a gas bubble in the durham tube

• detected on the first try

what kind of respirators are enterobacteriaceae?

facultative anaerobes — they can switch between using oxygen and nitrate

do faculatative anaerobes like enterobacteriaceae use cytochrome c oxidase?

no, they use a different enzyme so they test negative for the oxidase test

E. coli result for oxidase test

negative

what is added to help bacteria reduce nitrate to nitrite if it is able?

sulfanilic acid (A) and alpha-naphthylamine (B)

how does Enterobacteriaceae test on nitrate test?

1. nitrate is still present at first

2. reduces to nitrate when A&B are added

how does a positive reduction from nitrate to nitrite look like after adding A&B?

dark pink

• detected on the second try

how does a negative reduction from nitrate to nitrite look like after adding A&B?

super pale pink/no change

how does a positive test for use of any nitrate look like after adding zinc powder look?

no change

how does a negative test for use of any nitrate look like after adding zinc powder look?

dark pink

• detected on the third try

what kind of respirators are Alcaligenes?

obligate aerobes

alcaligenes result for oxidase test

positive

does Alcaligenes have nitrate reductase? can they use nitrate for respiration?

no, they don’t use nitrite at all

how does Alcaligenes test on nitrate test?

totally negative—it never uses nitrate

what do you do if you see that the bacteria does not reduce nitrate to nitrite?

keep testing to see if it even reacts to nitrate by adding zinc

what is used to detect nitrate in nitrate test after discovering that the bacteria doesnt reduce at all?

zinc powder

positive result after you add zinc powder

no color change

negative result after you add zinc powder

red color

which bacteria is this?

Pseudomonas

which bacteria is this?

e. coli

which bacteria is this?

alcaligenes