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Ex. 17: Nitrate Respiration (Nitrate Reduction Test). Objectives?. Vocabulary. Oxidation – reduction reactions Reductase Nitrate vs. Nitrite Denitrification. . Some bacteria only produce nitr a te reductase, other bacteria also produce nitr i te reductase. - PowerPoint PPT Presentation
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Ex. 17: Nitrate Respiration (Nitrate Reduction Test)
Objectives?
Vocabulary• Oxidation – reduction reactions• Reductase • Nitrate vs. Nitrite• Denitrification
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 Steps1. 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
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 TestTest tubes after 24 hour incubation and before adding reagents A and B
A. faecalisNegative Control P. vulgaris
P. aeru- ginosa
. . . after adding reagents A and B
What do you do next?
A. faecalisNegative Control P. vulgaris
P. aeru- ginosa
. . . after adding Zinc to tubes 1, 2 and 4
A. faecalis P. vulgarisP. aeru- ginosaNegative
Control
Table 17-2: Nitrate Utilization PatternsPattern Nitrate Nitrite Gas Interpretation
NO3 NOT used
present absent absent No nitrate reduction
NO2
producedabsent present absent Nitrate reduction to nitrite
Gas produced absent absent Produced
and escapedDenitrification (Nitrate reduction all the way to gas)
Step 1 Step 2
???Step 1
Step 2
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