Nitrate Respiration (Nitrate Reduction Test)
Ex. 17: Nitrate Respiration (Nitrate Reduction Test)Objectives?VocabularyOxidation reduction reactionsReductase Nitrate vs. NitriteDenitrification Nitrate Reduction and DenitrificationSome bacteria only produce nitrate reductase, other bacteria also produce nitrite reductase
nitrate reductasenitrite reductaseDay 1:Materials needed per table: Nitrate broth tubes (4)1 sterile Q-tipThree 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 tubesReagents and StepsTest 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 InterpretationTable 17-1: Possible Nitrate Test ResultsResultInterpretationSymbolRed color after addition of reagents A and BNitrate reduction to nitrite (NO3 NO2)+No color after the addition of reagents A and BIncomplete test; addition of zinc dust requiredNo color change after addition of zinc dustNitrate reduction past nitrite to gaseous or non-gaseous nitrogen compounds +Red color after addition of zinc dustNo nitrate reduction
Nitrate Reduction TestTest tubes after 24 hour incubation and before adding reagents A and BA. faecalisNegative ControlP. vulgarisP. aeru- ginosa. . . after adding reagents A and B
What do you do next?A. faecalisNegative ControlP. vulgarisP. aeru- ginosa. . . after adding Zinc to tubes 1, 2 and 4
A. faecalisP. vulgarisP. aeru- ginosaNegative ControlTable 17-2: Nitrate Utilization PatternsPatternNitrateNitriteGas InterpretationNO3 NOT usedpresentabsent absentNo nitrate reductionNO2producedabsentpresentabsentNitrate reduction to nitriteGas producedabsentabsentProduced and escapedDenitrification (Nitrate reduction all the way to gas)
Step 1Step 2???
Step 2the end