1. Ex. 16: Nitrate Respiration (Nitrate Reduction Test)
Objectives?

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

3. Nitrate Reduction and Denitrification
nitrite reductase
nitrate reductase
Some bacteria only produce nitrate reductase, other bacteria also produce nitrite reductase

4. 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

5. Reagents and Steps Test for accumulation of nitrite:
Add 5 drops of nitrate reagents A (dimethyl-naphthylamine) and B (sulfanilic acid)  red color means nitrite present (+)
If step one is negative:
Add small pinch of Zinc powder  red means nitrate was present. Why? And what is the meaning?
No color development in step 1 or 2  Why? And what is the meaning?

6. Possible Results and Interpretation
Table 16-1: 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 

7. Nitrate Reduction Test
Test tubes after 24 hour incubation and before adding reagents A and B
Negative
Control
P. aeru- ginosa
A. faecalis
P. vulgaris

8. . . . after adding reagents A and B
Negative
Control
P. aeru- ginosa
A. faecalis
P. vulgaris
What do you do next?

9. . . . after adding Zinc to tubes 1, 2 and 4
P. aeru- ginosa
Negative
Control
A. faecalis
P. vulgaris

10. Table 16-2: Nitrate Utilization Patterns
Step 1
Step 2
Table 16-2: Nitrate Utilization Patterns
Pattern
Nitrate
Nitrite
Gas
Interpretation
NO3 NOT used
present
absent
No nitrate reduction
NO2
produced
Nitrate reduction to nitrite
Gas produced
Produced and escaped
Denitrification (Nitrate reduction all the way to gas)

11. Step 1
???
Step 2
the end