Ex. 17: Nitrate Respiration (Nitrate Reduction Test)

Objectives?

Vocabulary• Oxidation – reduction reactions• Reductase • Nitrate vs. Nitrite• Denitrification

Nitrate Reduction and Denitrification

Some bacteria only produce nitrate reductase, other bacteria also produce nitrite reductase

nitrate reductase

nitrite reductase

Day 1:Materials needed per table: Nitrate broth tubes (4) 1 sterile Q-tip

Three pure cultures needed per table: Alcaligenes faecalis

(negative) Proteus vulgaris

(positive for nitrite) Pseudomonas aeruginosa

(denitrifier)Each student takes responsibility to inoculate one of the

nitrate broth tubes

Reagents and Steps1. Test for accumulation of nitrite:

Add 5 drops of nitrate reagents A (dimethyl-naphthylamine) and B (sulfanilic acid) red color means nitrite present (+)

2. If step one is negative: Add small pinch of Zinc powder red means

nitrate was present. Why? And what is the meaning?

3. No color development in step 1 or 2 Why? And what is the meaning?

Possible Results and Interpretation

Table 17-1: Possible Nitrate Test Results

Result Interpretation Symbol

Red color after addition of reagents A and B

Nitrate reduction to nitrite (NO3 → NO2)

+No color after the

addition of reagents A and B

Incomplete test; addition of zinc dust required

No color change after addition of zinc dust

Nitrate reduction past nitrite to gaseous or non-gaseous nitrogen compounds

+

Red color after addition of zinc dust No nitrate reduction

Nitrate Reduction TestTest tubes after 24 hour incubation and before adding reagents A and B

A. faecalisNegative Control P. vulgaris

P. aeru- ginosa

. . . after adding reagents A and B

What do you do next?

A. faecalisNegative Control P. vulgaris

P. aeru- ginosa

. . . after adding Zinc to tubes 1, 2 and 4

A. faecalis P. vulgarisP. aeru- ginosaNegative

Control

Table 17-2: Nitrate Utilization PatternsPattern Nitrate Nitrite Gas Interpretation

NO3 NOT used

present absent absent No nitrate reduction

NO2

producedabsent present absent Nitrate reduction to nitrite

Gas produced absent absent Produced

and escapedDenitrification (Nitrate reduction all the way to gas)

Step 1 Step 2

???Step 1

Step 2