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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
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