Ex_5-7_Nitrate_Reduction_T..pptx

MBIO 2815 Ex 5-7 Nitrate Reduction Test Anaerobic respiration does NOT use O2 as a final electron acceptor but instead uses NONOXYGEN electron acceptors such as NITRATE (NO3-) CATABOLIC PATHWAYS GLUCOSE enters the cell AEROBIC RESPIRATION ANAEROBIC RESPIRATION FERMENTATION if nitrate (NO3-) is used as the final electron acceptor than the bacteria can perform either DENITRIFICATION to produce nitrogen gas or AMMONIFICATION to produce ammonia MBIO 2815 Ex 5-7 Nitrate Reduction Test Glycolysis Krebs cycle ETC NO3- e- MBIO 2815 Ex 5-7 Nitrate Reduction Test Denitrification = a multi-step process performed by a group of enzymes that leads to the production of nitrogen gas from NO3 NO3 nitrate NO2 nitrite NO nitric oxide N2O nitrous oxide N2 nitrogen gas MBIO 2815 Ex 5-7 Nitrate Reduction Test Ammonification = a multi-step process performed by a group of enzymes that leads to the production of ammonia from NO3 NO3 nitrate NO2 nitrite NH4 ammonia MBIO 2815 Ex 5-7 Nitrate Reduction Test NO3 nitrate NO2 nitrite nitrate reductase The 1st step in either denitrification OR ammonification is the reduction of NITRATE (NO3) to NITRITE (NO2) by the enzyme NITRATE REDUCTASE Purpose of the nitrate reduction test: to determine if an organism can use nitrate for either denitrification or ammonification MBIO 2815 Ex 5-7 Nitrate Reduction Test NO2 nitrite NO nitric oxide N2O nitrous oxide N2 nitrogen gas NO3 nitrate NH4 ammonia Nitrate Broth Key components: POTASSIUM NITRATE (KNO3) the nitrate source A Durham tube to monitor gas production MBIO 2815 Ex 5-7 Nitrate Reduction Test Nitrate broth does NOT contain any color indicators so prior to inoculation the broth is clear MBIO 2815 Ex 5-7 Nitrate Reduction Test Possible results from the nitrate reduction test: After overnight growth gas production is seen (i.e., an air pocket or bubbles in the Durham tube) If present the source of the gas must be determined (i.e., gas through denitrification or fermentation?) MBIO 2815 Ex 5-7 Nitrate Reduction Test Production of gas in the nitrate reduction test alcohols, acids & gases NO3 nitrate NO2 nitrite nitrate reductase N2 nitrogen gas Fermentation Denitrification MBIO 2815 Ex 5-7 Nitrate Reduction Test Production of gas in the nitrate reduction test If the organism used in a nitrate test is a NON-FERMENTOR as determined by a fermentation test (e.g. OF test or phenol red broth) then the gas must be produced through denitrification and must be nitrogen gas alcohols, acids & gases NO3 nitrate NO2 nitrite N2 nitrogen gas Fermentation Denitrification MBIO 2815 Ex 5-7 Nitrate Reduction Test Production of gas in the nitrate reduction test organism is a non-fermentor source of gas: denitrification type of gas: nitrogen gas NO3 nitrate NO2 nitrite N2 nitrogen gas Other nitrogen compounds NITRATE REDUCTASE Organism MUST have nitrate reductase for denitrification Organism is nitrate reductase POSITIVE (test is finished) MBIO 2815 Ex 5-7 Nitrate Reduction Test Production of gas in the nitrate reduction test If the organism used in a nitrate test is a FERMENTOR as determined by a fermentation test (e.g. OF test or phenol red broth) then the type of gas produced is not known and the test must be continued alcohols, acids & gases NO3 nitrate NO2 nitrite N2 nitrogen gas Fermentation Denitrification ? MBIO 2815 Ex 5-7 Nitrate Reduction Test Possible results from the nitrate reduction test: Growth overnight but NO gas production OR gas production but organism is a fermentor 2 reagents must be added to the culture: Reagent A = sulfanilic acid Reagent B = naphthylamine MBIO 2815 Ex 5-7 Nitrate Reduction Test Possible results from the nitrate reduction test: NO3 nitrate NO2 nitrite HNO2 nitrous acid H2O (in media) OH- HNO2 nitrous acid reagents A & B RED COLOR MBIO 2815 Ex 5-7 Nitrate Reduction Test Possible results from the nitrate reduction test: NO3 nitrate NO2 nitrite HNO2 nitrous acid Reagents A & B + RED COLOR Nitrate reductase red color after addition of reagents A & B nitrous acid available for reagents A & B to react with nitrite available to react with H2O to produce nitrous acid nitrate in broth converted to nitrite Since nitrite produced organism is nitrate reductase POSITIVE MBIO 2815 Ex 5-7 Nitrate Reduction Test Possible results from the nitrate reduction test: NO3 nitrate NO2 nitrite HNO2 nitrous acid Reagents A & B + RED COLOR Nitrate reductase NO red color after addition of reagents A & B NO nitrous acid available for reagents A & B to react with NO nitrite available to react with H2O to produce nitrous acid no nitrite present but test MUST CONTINUE ? MBIO 2815 Ex 5-7 Nitrate Reduction Test Possible results from the nitrate reduction test: NO3 nitrate NO2 nitrite HNO2 nitrous acid Reagents A & B + RED COLOR Nitrate reductase No red color after addition of reagents A and B DOES NOT indicate that the organism is nitrate reductase negative Possibility #1: organism does NOT have nitrate reductase and cannot convert nitrate to nitrite MBIO 2815 Ex 5-7 Nitrate Reduction Test Possible results from the nitrate reduction test: No red color after addition of reagents A and B DOES NOT indicate that the organism is nitrate reductase negative NO2 nitrite other nitrogen compounds NO3 nitrate NH4 ammonia Possibility #2: organism has reduced all nitrate to nitrite and all nitrite has been converted to other nitrogen compounds Nitrate reductase MBIO 2815 Ex 5-7 Nitrate Reduction Test Possible results from the nitrate reduction test: No red color after addition of reagents A and B NO2 nitrite NO3 nitrate Addition of zinc powder required zinc powder Zinc powder catalyzes the reduction of any nitrate source in the broth to nitrite MBIO 2815 Ex 5-7 Nitrate Reduction Test Possible results from the nitrate reduction test: After reagents A & B added no color change so zinc powder added zinc powder reduces nitrate to nitrite nitrite reacts with H2O to form nitrous acid nitrous acid reacts with reagents A & B already in the media red color is produced zinc powder NO2 nitrite NO3 nitrate reduction by zinc powder MBIO 2815 Ex 5-7 Nitrate Reduction Test Possible results from the nitrate reduction test: NO2 nitrite NO3 nitrate reduction by zinc powder red color after addition of zinc indicates that nitrate was still present in the broth after bacterial growth since nitrate remained in media there was NO reduction of it to nitrite Organism is nitrate reductase NEGATIVE MBIO 2815 Ex 5-7 Nitrate Reduction Test Possible results from the nitrate reduction test: After reagents A & B added no color change so zinc powder added NO nitrate for zinc powder to reduce to nitrite NO nitrite to react with H2O to form nitrous acid NO nitrous acid to react with reagents A & B already in the media NO red color NO2 nitrite NO3 nitrate reduction by zinc powder zinc powder MBIO 2815 Ex 5-7 Nitrate Reduction Test Possible results from the nitrate reduction test: NO2 nitrite NO3 nitrate reduction by zinc powder no color after addition of zinc indicates that no nitrate was present in the broth after bacterial growth nitrate must have been rapidly reduced to nitrite and then nitrite converted to other nitrogen compounds Organism is nitrate reductase POSITIVE