Nitrate Respiration Nitrate Reduction Test Day 2 Materials

Nitrate Respiration (Nitrate Reduction Test) Day 2: Materials needed per table: The four incubated Nitrate broth tubes from the last session § Nitrate Reagents A and B § Zinc powder and spatula Each student takes responsibility to test one of the nitrate broth cultures

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 Possible Nitrate Test Results Result Interpretation Red color after addition of reagents A and B Nitrate reduction to nitrite (NO 3 → NO 2) 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 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 Nitrate Utilization Patterns Pattern Nitrate Nitrite Gas Interpretation NO 3 NOT used present absent No nitrate reduction NO 2 produced absent present absent Nitrate reduction to nitrite absent Produced and escaped Denitrification (Nitrate reduction all the way to gas) Gas produced absent

Step 1 ? ? ? Step 2