Microbiology FINAL lab

MR (Methyl Red) Test

Substrate:

• GLUCOSE

What enzyme breaks that down? Enzyme:

• GLYCOLITIC ENZYME

What product gets made? Product:

• Lactate, Pyruvate, formate, acetate

Reagent: METHYL RED

What color means positive? : RED

VP (Voges- Proskauer) Test

Substrate:

• Glucose

Enzyme: (i dont think we need to know this)

• Butanediol fermentation pathway

What product gets made? Product:

• Acetoin

Reagents: ALPHA Naphthol & KOH

What color means positive? : RED

What is the microbe eating? What ______ started the reaction?

SUBSTRATE

Difference between MR & VP?

BOTH start with glucose

MR: detects acids VP: detects acetoin

CATALASE TEST

Substrate:

• Hydrogen peroxide (H2O2)

What enzyme breaks that down? Enzyme:

• CATALASE

What products are made? Products:

• O2

• H2

Reagent: HYDROGEN PEROXIDE

What means positive: BUBBLES

OXIDASE TEST (Kovacs Oxidase Test)

Substrate:

• Electrons moving through ETC

What enzyme breaks that down? Enzyme:

• Cytochrome c OXIDASE

Reagent: crazy name that we probably dont need to know

What color when positive? : PURPLE

Nitrate Broth

Substrate:

• Nitrate

What enzyme break this down? Enzyme:

• Nitrate reductase

What product is made? Product:

• Nitrite (bc it was reduced)

Reagents: SULFANILIC ACID + ALPHA NAPHTHYLAMINE

Color when positive: RED

Simmons Citrate Test

Substrate:

• Citrate

what breaks that down? Enzyme:

• Citrase

What products are made? Product:

• Acetate + Formate

What color means positive: BLUE

TRICK: Sky is Blue, Sky contains citrate

Phenylalanine Agar (PPA Slant)

Substrate:

• Phenylalanine

What enzyme breaks this down? Enzyme:

• Phenylalanine Deaminase (takes away Amino Acids)

What product is made? Product:

• Phenylpyruvic Acid

Reagent: FERRIC CHLORIDE

Positive color: GREEN

Starch Agar

Substrate:

• Starch

What enzyme breaks this down?:

• Amylase

What products are made?

• Glucose

Reagent: Iodine

Positive creates: CLEAR ZONE

Urea Broth

Substrate:

• Urea

What enzyme breaks Urea down?

• Urease

Product = Ammonia

Positive color: HOT PINK

Skim Milk Agar

Substrate:

• Casein (milk protein)

What enzyme breaks down Casein? Enzyme:

• Casease

What product is made?

• Amino Acids

Positive?: CLEAR ZONE

Nutrient Gelatin

Substrate:

• Gelatin

What breaks down gelatin? Enzyme:

• Gelatinase

What product is made?

• Amino acids

Positive?: LIQUID AFTER refrigeration

if it resolidifies, its negative

Spirit Blue Agar (tributyrin agar)

Substrate:

• Fat

Enzyme:

• Lipase

Product: FATTY ACIDS + GLYCEROL

Positive? HALO around growth

SIM test

S= Sulfur I= indole M= Motility

For Sulfur: DOES BACTERIA PRODUCE H2S (hydrogen sulfide gas)?

• (+) color = BLACK

For Indole: Does Tryptophanase break down Tryptophan?

• Add Reagent to find out: KOVACS regent

• (+) result = BRIGHT RED LAYER @ top

For Motility:

• (+) result: CLOUDINESS outward of stab line

• (-) result: growth restricted to stab line

TSIA (Triple Iron Agar)

Red Slant/ Yellow Buutt (K/A):

• ONLY glucose fermented

Yellow Slant/Yellow Buttt (A/A):

• Glucose, lactose, & Sucrose Fermented

Red Slant/ Red Buttt (K/K):

• NO SUGARS FERMENTED

Gas production?

• (+) result: Cracks/Lifting in agar

H2S gas production?

• (+) result: BLACK precipitate

PEA (Phenylethyl Alcohol Agar)

• Selective

• Gram (+)

  • bacterial growth indicates

MAC (MacConkey Agar)

• Selective AND Differential

  • selects for Gram (-)

  • differential for LACTOSE= PINK/RED

    • otherwise colorless

EMB (Eosin Methylene Blue)

• Selective AND Differential

  • selects for Gram (-)

  • differential for LACTOSE

    • STRONG lactose fermenter= GREEN

    • WEAK lactose fermenter= purple/pink

    • NO FERMENTER = colorless

MSA (Mannitol Salt Agar)

• Selective AND Differential

  • Selective for: SALT TOLFERANT ORGANISMS

  • Differential for: Mannitol fermentation

    • Fermenter= YELLOW

    • Non-fermenter= RED/PINK

Thioglycolate Broth

Purpose: O2 Tolerance

Bacteria on TOP ONLY:

• Obligate Aerobe= needs O2

Bacteria on BOTTOM ONLY:

• Obligate Anaerobe= dies in O2

Throughout but Mostly on Top::

• facultative anaerobe

EVEN THROUGHOUT:

• Aerotolerant

(not sure if we learned this) Thin band below top:

• Mircoaerophile

Litmus milk

Ferment lactose? → Acid forms = PINK

Digest casein? → Proteolysis= CLEARIN

Reduce litmus?= WHITE

DNAse Test

Substrate:

DNA

Enzyme:

DNAse

Product:

DNA Fragments

(+) result= clear zone around growth bc DNA was destroyed

(-) result= NO clear zone

Litmus Milk