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Topic 18: Biochemical Tests Review

  • How the Biochemical Tests Work/How Substrates are Broken Down
    • Simple sugar (glucose & fructose) are broken down by endoenzymes into acids
    • Complex sugar (lactose & sucrose) are broken down by exoenzymes into simple sugars which are then broken down by endoenzymes into acids
    • Amino acids (ornithine & tryptophan) are broken down by endozymes into ammonia
    • Proteins (peptone & gelatin) are broken down by exoenzymes into amino acids which are then broken down by endoenzymes into ammonia
  • Eosin Methylene Blue (EMB)
    • Selective for gram neg & differential for lactose fermentation
    • Results
    • Thick strong growth: gram neg organism
    • No growth: no gram neg organism
    • Colorful (pink, black, purple, metallic green) growth : lactose fermentation
    • No colorful growh: no lactose fermentation
    • Gram pos organims may grow but growth is stunted
    • EMB is selective due to methylene blue and eosin
  • Phenylethyl Alcohol (PEA)
    • Selective for gram pos
    • Results
    • Opaque growth : gram pos
    • Transparent/Weak/No growth: not gram pos
    • Phenylethyl alcohol inhibits growth of gram neg
  • OF Glucose
    • Differential for oxidation and fermentation of glucose
    • Results
    • Aerobic tube yellow & anaerobic tube green : pos for glucose oxidation only
    • Aerobic tube & anaerobic tube yellow: pos for glucose oxidation & fermentation
    • Aerobic tube green & anaerobic tube green : neg for glucose oxidation and fermentation
    • Yellow : acid produced; green/blue: ammonia produced
    • pH indicator is bromthymol blue
    • can also test organism’s motility and oxygen requirements
  • Starch Hydrolysis
    • Differential for production of amylase
    • Results
    • Clearing after addition of iodine : pos for amylase and starch hydrolysis
    • No clearing after addition of iodine: neg for amylase and starch hydrolysis
    • Amylase is exoenzyme that breaks down starch
  • Fermentation Tubes (glucose, lactose, sucrose, xylose, trehalose, mannitol)
    • Differential for fermentation of specified carbohydrate
    • Results
    • Yellow color : pos for fermentation
    • Red/Pink color : neg for fermentation and pos for pepone
    • Bubbles in Durham tube : gas production
    • No bubbles in Durham tube : no gas production
    • True fermentation : acid and gas production
    • Fermentation : acid production
    • No fermentation : no acid or gas production
    • Incubate less than 24 hrs
    • pH indicator is phenol red
  • Gelatin Hydrolysis (Gelatinase Test)
    • Differential for production of exoenzyme gelatinase
    • Results
    • Liquid medium : pos for gelatinase
    • Solid medium : neg for gelatinase
    • Incubate 4-7 days at room temperature
  • Urease Test
    • Differential for production of urease
    • Results
    • Fuchsia : pos for urease
    • Orange/yellow/no color: neg for urease
    • incubate 24-48 hrs
    • pH indicator is phenol red
  • Peptone Iron Deep
    • Differential for liberation of hydrogen sulfide from amino acid
    • Ferrous iron reacts w/ H2S and produces black precipitation
    • Results
    • Black color : pos for H2S
    • No color change : neg for H2S
    • Tests motility
  • MIO Deep
    • Differental
    • Tests for production of indole, ornithine decarboxylase, and motility
    • Results
    • purple : pos for ornithine decarboxylase
    • yellow : neg for ornithine decarboxylase
    • red after kovac’s reagent : pos for indole
    • no colar change after kovac’s reagent : neg for indole
  • Phenylalanine Deaminase Test
    • Differential
    • Tests for production of phenylalanine deaminase
    • Results
    • Forrest green color : pos
    • No color change : neg
    • Testing reagent is 10% ferric chloride (after incubation)
  • Nitrate Reduction Test
    • Differential
    • Tests for reduction of nitrate
    • Nitrate reduction is an anaerobic process
    • Results
    • Red/pink color after adding reagents A & B : pos (reduced to nitrate)
    • No color change after A, B, & Zinc : pos (reduced to nitrous oxide or nitrogen gas)
    • Red after A, B, & Zinc : neg (nitrate never reduced)
    • Zinc is a reducing agent and will confirm whether or not nitrate has been reduced
  • Oxidase Test
    • Differential
    • Tests for presence of cytochrome c
    • Results
    • strip turns purple : pos
    • no color change : neg
    • only strict aerobes will yield pos result
  • Catalase Test
    • Differential
    • Tests for production of catalase (breaks down into water and O2)
    • H2O2 is byproduct of aerobic respiration
    • Results
    • Bubble : pos
    • No bubbles : neg
  • IMViC Tests
    • Identifying enterics (faculative anaerobes, gram neg, non-endospore forming, bacilli, live in large intesting
    • Indole Test
    • Differential
    • Tests for production of endoenzyme tryptophanase and production of indole
    • Results
      • red/purple after kovac’s : pos for indole and tryptophanase
      • no color change : neg for indole and tryptophanse
    • Methyl Red Test
    • Differential
    • Tests for production of stable acid from glucose
    • Results
      • Red/pink after methyl red : pos for stable acid production
      • Yellow/orange after methyl red : neg for stable acid production
    • Voges-Proskauer Test
    • Differential
    • Tests for production of acetoin (unstable acid)
    • Add VP 1 & VP 2 reagents and leave cap off
    • Results
      • Red: post for acetoin production
      • gray/brown : neg for acetoin production
    • Citrate Utilization Test
    • Differential
    • Tests if organism can use citrate as sole carbon source and production of citrase
    • Results
      • Blue : pos
      • Green: neg
    • pH indicator is bromthymol blue