Ex. 17: Nitrate Respiration
(Nitrate Reduction Test)
Objectives?
Vocabulary
• Oxidation – reduction reactions
• Reductase
• Nitrate vs. Nitrite
• Denitrification
Nitrate Reduction and
Denitrification
nitrite reductase
nitrate reductase
Some bacteria only produce nitrate reductase, other
bacteria also produce nitrite reductase
Day 1:
Materials needed per table: Three pure cultures needed
 Nitrate broth tubes (4)
per table:
 Alcaligenes faecalis
 1 sterile Q-tip
(negative)
 Proteus vulgaris
(positive for nitrite)
 Pseudomonas aeruginosa
(denitrifier)
Each student takes responsibility to inoculate one of the
nitrate broth tubes
Reagents and Steps
1. Test for accumulation of nitrite:
Add 5 drops of nitrate reagents A (dimethylnaphthylamine) 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
Red color after addition
of reagents A and B
Nitrate reduction to nitrite (NO3 →
NO2)
No color after the
Incomplete test; addition of zinc dust
addition of reagents A
required
and B
Nitrate reduction past nitrite to
No color change after
gaseous or non-gaseous nitrogen
addition of zinc dust
compounds
Red color after addition
of zinc dust
No nitrate reduction
Symbol
+
+

Nitrate Reduction Test
Test tubes after
24 hour
incubation and
before adding
reagents A and B
Negative
Control
A. faecalis
P. vulgaris
P. aeruginosa
. . . after adding reagents A and B
Negative
Control
A. faecalis
P. vulgaris
P. aeruginosa
What do you
do next?
. . . after adding Zinc to tubes 1, 2 and 4
Negative
Control
A. faecalis
P. vulgaris
P. aeruginosa
Step 1
Step 2
Table 17-2: Nitrate Utilization Patterns
Pattern
Nitrate
Nitrite
Gas
Interpretation
NO3 NOT
used
present
absent
absent
No nitrate reduction
NO2
produced
absent
present
absent
Nitrate reduction to nitrite
Gas
produced
absent
absent
Produced Denitrification (Nitrate
and escaped reduction all the way to gas)
Step 1
???
Step 2