Proteins, amino acids, enzymes lab 24, sulfide, indole, motility agar

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Purpose of sulfur reduction test

Used to differentiate members of enterobacteriacae
ESP sulfur reducers from non reducers


What medium used in indole production, motility, and sulfur reduction test

SIM medium


What is SIM medium

Semi solid medium with casein and animal tissues as sources of amino acids
Iron containing compound
Sulfur in form of sodium thiosulfate


What is sulfur reduced to

Anaerobic activity, accomplished 2 ways depending in enzymes present


What are the two enzymes that reduce sulfur

1. Cysteine desulfurase
2. Thiosulfate reductase


Cysteine desulfurase

Catalyzes putrefaction of amino acid cysteine to pyruvate


Putrefaction of cysteine

Cysteine + h2o + h (cysteine desulfurase) pyruvate (for fermentation or respiration)
Byproduct of is h2s and nh3


Thiosulfate reductase

Enzyme that catalysts the reduction of sulfur (in form of sulfate) at the end of the anaerobic respiratory etc


What do both systems produce

H2s(hydrogen sulfide gas)
When either reaction occurs h2s produced combines with iron (ferrous ammonium sulfate in medium) to form ferric sulfide (feS) a black precipitate


What is blackening of media indicating

Positive test
H2S produced combines with ferris ammonium sulfate in media and forms ferric sulfide a back precipitate


Putrefaction of cystein

Reaction is mechanism for getting energy ut of amino acid cysteine


Indole test

Identifies bacteria capable of producing indole using enzyme tryptophanase
Uses SIM medium


How is Nicole production made possible in SIM medium

Presence of tryptophan (in casein and animal protien)


What does tryptophanase do?

Hydrolyses tryptophan to pyruvate, ammonia (by deamination) and indole


How is tryptophanase detected

Addition if kovacs reagent after incubation


Kovacs reagent

Contains DMABA AND HCL dissolved in amyl alcohol


What happens when kovacs reagent added to SIM

Forms liquid layer over solid media
DMABA then reacts with any ndole present and produces a quinoa dal compound that turns the reagent layer red


Formation of red layer

Positive reaction and presence of tryptophanase p 75


Motility test agar

Semisolid medium with reduced agar concentration to .4% just enough to maintain form but also a,low for motility


How is motility agar inoculated

Stabbing with needle


How is motility detected

As diffuse growth radiating from central stab line p 84

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