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