Ex 17 Nitrate Respiration Nitrate Reduction Test Objectives

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

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

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

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

Nitrate Reduction and Denitrification nitrite reductase nitrate reductase Some bacteria only produce nitrate reductase,

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: Three pure cultures needed Nitrate broth tubes (4)

Day 1: Materials needed per table: Three pure cultures needed Nitrate broth tubes (4) per table: 1 sterile Q-tip 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 1. Test for accumulation of nitrite: Add 5 drops of nitrate

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 Table 17 -1: Possible Nitrate Test Results Result Interpretation Red

Possible Results and Interpretation Table 17 -1: Possible Nitrate Test Results Result Interpretation Red color after addition of reagents A and B Nitrate reduction to nitrite (NO 3 → NO 2) Symbol + No color after the Incomplete test; addition of zinc dust addition of reagents A required and B 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 tubes after 24 hour incubation and before adding reagents A and

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.

. . . 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

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

Step 1 Step 2 Table 17 -2: Nitrate Utilization Patterns Pattern Nitrate Nitrite Gas

Step 1 Step 2 Table 17 -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 Gas produced absent Produced Denitrification (Nitrate and escaped reduction all the way to gas)

Step 1 ? ? ? Step 2

Step 1 ? ? ? Step 2