1. Ex. 17: Nitrate Respiration (Nitrate Reduction Test) Objectives?

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

3. Nitrate Reduction and Denitrification  Some bacteria only produce nitrate reductase, other bacteria also produce nitrite reductase nitrate reductase 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 1.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?

6. Possible Results and Interpretation Table 17-1: Possible Nitrate Test Results ResultInterpretation Symbol Red color after addition of reagents A and B Nitrate reduction to nitrite (NO 3 → NO 2 ) + 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 A. faecalis Negative Control P. vulgaris P. aeru- ginosa

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

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

10. Table 17-2: Nitrate Utilization Patterns PatternNitrateNitriteGasInterpretation NO 3 NOT used presentabsent No nitrate reduction NO 2 produced absentpresentabsentNitrate reduction to nitrite Gas produced absent Produced and escaped Denitrification (Nitrate reduction all the way to gas) Step 1Step 2

11. ??? Step 1 Step 2