Nitrate Respiration (Nitrate Reduction Test)

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Presentation transcript:

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 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?

Possible Results and Interpretation 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 Test Test tubes after 24 hour incubation and before adding reagents A and B Negative Control P. aeruginosa A. faecalis P. vulgaris

. . . after adding reagents A and B Negative Control P. aeruginosa A. faecalis P. vulgaris What do you do next?

. . . after adding Zinc to tubes 1, 2 and 4 P. aeruginosa Negative Control A. faecalis P. vulgaris

Nitrate Utilization Patterns Step 1 Step 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)

Step 1 ??? Step 2 the end