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

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