Nitrate Reduction and Denitrification Some bacteria only produce nitrate reductase, other bacteria also produce nitrite reductase nitrate reductase 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 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?
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
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
... after adding reagents A and B What do you do next? A. faecalis Negative Control P. vulgaris P. aeru- ginosa
... after adding Zinc to tubes 1, 2 and 4 A. faecalisP. vulgaris P. aeru- ginosa Negative Control
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
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