IMVP + simmon's citrate, nitrate reduction (lab 8)

what is the purpose of the indole test?

• to see if certain enteric bacteria are able to hydrolyze tryptophan 

  • Indole = product of breaking down tryptophan 

• Useful in differentiating e. Coli from other enteric bacteria 

what is used to determine the indole test? what does a positive test look like?

• Determined using Kovac’s Reagent 

  • Red color → + indole test 

  • Bacteria = + for tryptophan hydrolysis

    • Means that enteric bacteria has the ability to hydrolyze tryptophan 

what is the MR-VP test

• Used to identify certain enteric bacteria based on which pathway they use to ferment glucose. 

• Methyl-Red and Vogues Proskauer (2-3 Butanediol fermentation) = both are IMVC tests 

• Bacteria are commonly either one OR the other, rarely do they test positive for both  MR and VC tests 

what does the MRVP test identify?

identifies glucose-fermenting Enterobacteriaceae family members 

what does a positive methyl red test (part of MR-VP) look like

• When bacteria ferment glucose, they produce acid which decrease the pH & makes it acidic

  If MR broth turns red, bacteria = mixed acid fermenter 

what does a positive VP test look like

• Alternate pathway for glucose fermentation 

• Tests to see presence of acetoin 

Gradual formation of Red color in broth tube → bacteria uses 2-3 butanediol pathway fermentation instead; acetoin is present

Simmons Citrate Agar 

• Some bacteria can use citrate as a carbon source

  • Helps distinguish gram negative bacterial species 

• BTB = used as pH indicator 

what does a positive citrate test look like

• If bacteria is able to use citrate, then they will make an enzyme called citrate permease, which converts citrate → pyruvate 

• If bacteria is able to produce enzyme citrate permease to break down citrate, BTB will turn from green to blue, 

  • pH changes from neutral to alkaline 

Nitrate Reduction 

• All members of enteric bacteria = able to reduce nitrate into nitrite via enzyme called nitrate reductase 

• Gram negative bacteria use nitrate as final electron acceptor in anaerobic respiration 

how do you determine nitrate reduction?

• Examine for gas in Durhams tube 

  • Gas = nitrate was reduced a gas, which is a + test 

• If no gas was found, reagent A & B will be added to test for presence of nitrite 

  • Red color → nitrite is present 

• Gas production OR red color development as the result of adding in the A & B reagent + indicates + test for nitrate reduction