Microbiology Laboratory Tests: Media, Biochemical Assays, and Bacterial Identification

Selective medium

Medium that allows only certain microorganisms to grow while inhibiting others

Differential medium

Medium that contains substances causing some bacteria to appear different from others

EMB agar purpose

Selective for Gram negative bacteria and differential for lactose fermentation

EMB vigorous lactose fermenter

Metallic green sheen or dark purple colonies

EMB moderate lactose fermenter

Purple colonies

EMB non-lactose fermenter

Pink, clear, or colorless colonies

Phenol red carbohydrate test positive result

Yellow color indicating acid production

O/F glucose test purpose

Determines whether carbohydrate metabolism is oxidative or fermentative

O/F oxidative organism result

Open tube yellow, oil-covered tube green

O/F fermentative organism result

Both open and oil-covered tubes yellow

O/F negative result

Both tubes remain green

Methyl Red (MR) test purpose

Detects mixed acid fermentation from glucose

MR positive result

Immediate red color

MR negative result

Yellow or no color change

Voges-Proskauer (VP) test purpose

Detects 2,3-butanediol fermentation (acetoin production)

VP positive result

Red color within 30 minutes

VP negative result

No color change or yellow/brown color

Citrate test purpose

Determines ability to use citrate as sole carbon source and ammonium salts as sole nitrogen source

Citrate positive result

Growth with intense blue color

Citrate negative result

No growth and remains green

IMViC tests

Indole, Methyl Red, Voges-Proskauer, Citrate

E. coli IMViC results

Indole positive, MR positive, VP negative, Citrate negative

Klebsiella aerogenes IMViC results

Indole negative, MR negative, VP positive, Citrate positive

Catalase test purpose

Detects enzyme catalase which breaks hydrogen peroxide into water and oxygen

Catalase positive result

Bubbles

Catalase negative result

No bubbles

KOH test purpose

Confirms Gram reaction by detecting cell wall lysis

KOH positive result

String formation indicating Gram negative

KOH negative result

No strings indicating Gram positive

Nitrate reduction test purpose

Determines ability to reduce nitrate

Nitrate positive result

Red after reagents A & B OR no color after zinc

Nitrate negative result

Red after zinc added

Oxidase test purpose

Detects cytochrome C oxidase enzyme

Oxidase positive result

Blue or purple within 30 seconds

Oxidase negative result

No color change within 30 seconds

SIM medium purpose

Tests for hydrogen sulfide production, indole production, and motility

SIM H2S positive result

Black precipitate in tube

SIM H2S negative result

No blackening

SIM indole positive result

Red color at top after Kovac's reagent

SIM indole negative result

No color change

SIM motility positive result

Diffuse growth away from stab line

SIM motility negative result

Growth only along stab line

Flagella function

Allow bacterial motility through rotation

Brownian motion

Random vibration of cells that is not true motility

Swarming motility

Concentric rings of growth across agar plate

Organism known for swarming

Proteus mirabilis

Phenylalanine test purpose

Detects phenylalanine deaminase enzyme

Phenylalanine positive result

Green color after ferric chloride

Phenylalanine negative result

Yellow color

Urea test purpose

Detects urease enzyme which hydrolyzes urea into ammonia and carbon dioxide

Urea positive result

Bright pink or cerise color

Urea negative result

No color change

Starch hydrolysis test purpose

Determines ability to hydrolyze starch

Starch positive result

Clear zone around streak after iodine

Starch negative result

Dark agar up to streak line

Hydrogen sulfide production mechanism

Sulfur reduced to H2S which reacts with iron salts to form black precipitate

Mixed acid fermentation organisms

Escherichia, Salmonella, Proteus

Butanediol fermentation organisms

Klebsiella, Serratia

Mineral oil in O/F test purpose

Creates anaerobic environment for fermentation tube