Ex. 16: 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: 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

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 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 

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

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

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

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)

Step 1 ??? Step 2 the end