FP (CL, SIM, Nitrate Reduction, Starch, Citrate)

• SIM (Sulfur-Indole-Motility) medium differential or selective?

  differential
• How many tests are performed in this one medium?

  3 (SIM)

  Sulfur Reduction

  Indole production

  Motility
• Sulfur Reduction to H2S occurs in two different ways depending on enzymes

  1. Enzyme cysteine desulfurase catalyzing cysteine to pyruvate conversion. This conversion leads to reduction of sulfur to H2S
  2. Enzyme thiosulfate reductase catalyzing sulfur (in the form of sulfate) reduction to H2S at the end of an anaerobic respiratory electron transport chain.
• Black in medium = Sulfur reduction +

  Examples: Salmonella, Drancisella, Proteus
• No black in medium = sulfur not reduced -

  Examples: morganella morganii & Providencia rettigeri
• Iron (Fe) from FeSO4 in the medium combines with H2S produced by bacteria to form iron sulfide (Fe2S3), which gives a black precipitate (SIM)
• Indole production: Tryptophanase (if produced by bacterium) hydrolyzes tryptophan in the medium into pyruvate, ammonia, and indole
• Koavac’s Reagent (drops on top of medium) turns from clear to pink/red when indole is present (+)
• Reagent color is unchanged (SIM)


  Tryptophan is not broken down into indole and pyruvate (-) = clear
• Clark & Lubs Broth aka MR-VP Broth is used to distinguish between the metabolic pathway that an organism uses to break down pyruvate.
• Two major pathways: Mixed Acid (MR test) & 2,3 Butanediol pathways (VP test)
• Nitrate reducing bacteria (enterics) contain enzyme nitrate reductase and perform single step reduction of nitrate (NO3) to nitrite (NO2)
• Other bacteria in process known as denitrification are believed to contain several enzymes capable of reducing nitrate and products to N2
• Other products of nitrate reduction

  NH3 (ammonia), NO (nitric oxide), N2O (nitrous oxide)
• Nitrate Reduction Test components 


  nitrate broth: beef extract, peptone, potassium nitrate (KNO3)
• Nitrate reduction test differential or selective?


  Differential
• In contrast to many differential media nitrate broth has no color indicators
• Suifanilic acid and alapha-naphthylamine (reagents A & B) added to nitrate broth
•  If broth in nitrate reduction test turns red, nitrate was reduced to nitrite [+]
• If there is no color change in nitrate reduction broth it is inconclusive meaning nitrate was not reduced or it was reduced to other nitrogenous compounds (NO3 > N2 or NH4)
• Powdered Zinc is added to tubes with no color change.
• Red color after reagents are added

  nitrate reduction to nitrite (NO3 > NO2) +
• No color (after addition of reagents)

  incomplete test; requires addition of zinc powder
• No color change (after addition of zinc powder)

  nitrate reduction to nongaseous nitrogenous compounds (NH3, NO, N2O) +
• Red color (after addition of zinc powder)


  No nitrate reduction (-)
• Starch Utilization / Hydrolysis 


  Starch agar is used to test for the breakdown of starch by amylase
• Starch agar contains: beef extract and peptone to support growth, soluble starch and agar
• Steps: Inoculate plate w sterile loop > plate flooded with Grams iodine
• Grams iodine reacts with starch to give a purple blue color medium
• Clearing in medium (+) = organism hydrolyzed starch in media
• No clearing in medium (-) = none of starch is hydrolyzed
• Starch is a polysaccharide with repeating alpha-D-glucose subunits. If bacteria has amylase it breaks it down into single subunits
• Starch agar selective or differential?

  differential
• Citrate Slant Agar selective or differential?

  Both