Microbiology Lab Quiz 4

IMViC

• Indole Production test

• Methyl Red test

• Voges Proskauer test

• Citric Utilization test

Indole Production Test Purpose

ID bacteria that produce tryptophanase

Tryptophanase

Enzyme that breaks down tryptophan

Indole Production Test Media

Tryptophan broth (contains tryptophan broth)

Indole Production Test Substrate

Tryptophan

Indole Production Test Enzyme

Tryptophanse

Indole Production Test Products

Pyruvic acid + ammonium + indole (colorless)

Kovac’s reagent

Reacts with indole —> Rosindole (pink)

Positive Indole Production Test

Pink ring at top of tube

Negative Result for Indole Production Test

Yellow ring at top of tube

Purpose of Methyl Red Test

To distinguish between bacteria that produce used acids from glucose

Purpose of Voges Proskauer Test

Distinguish between bacteria that produce neutral product acetoin from glucose

MRVP media

Glucose-supplemented nutrient broth

VP1

Alpha-naphthol

VP2

Guanine in KOH

Alpha-naphthol binds to

Acetoin

Guanidine in KOH binds to

Diacetyl

Positive MRVP

Red

Negative MRVP

Yellow

Citrate Utilization Test Purpose

Test if bacteria can utilize sodium citrate as the only carbon source

Citrate Utilization Test Media

Simmon’s Citrate Agar Slant

Sodium Citrate

Only carbon source

Ammonium

Only nitrogen source

pH indicator in Citrate Utilization Test

Bromthyol blue

Citrate Utilization substrate

Citrate

Citrate Utilization Enzyme

Citrate permease

Citrate Utilization Test products

Ammonia + ammonium hydroxide

Positive Citrate Utilization Test

Blue

Negative Citrate Utilization Test

Green

Catalase Test purpose

To ID bacteria that produce catalase

Commonly used to differentiate between Staphylococcus and Streptococcus (both gram positive)

Catalase and Oxidase Test Media

Nutrient agar slant

Catalase Test Substrate

H2O2

Catalase Test products

H2O + O2 (bubbles)

Oxidase Test purpose

ID bacteria that produce cytochrome C oxidase

Positive Oxidase Test Result

blue/purple

Nitrate reduction test purpose

Test if bacteria can reduce nitrate

Nitrate reductase

Enzyme that reduces nitrate → nitrite

Product of denitrification

N2

KNO3

Source of NO3

No gas in Durham Tube

Organism is incapable of denitrification → continue to step 2

gas in Durham tube

Organism may be capable of denitrification → continue to step 2

sulfanilic acid and alpha-napthylamine

Used to test for the reduction of nitrite

Organism reduces nitrate into nitrite only

Red after adding sulfanilic acid and alpha-napthylamine

Organism is completely incapable of reducing nitrate

Red after adding zinc powder

Organism reduces nitrate into some other nitrogenous compound

Red after adding zinc powder

Selective media

Selects for growth of specific bacteria; prevents unwanted bacteria from growing

Enriched media

Contains chemicals that enhance the growth of desired bacteria

Differential media

Differentiates between different types of bacteria

PEA

Phenylethyl alcohol agar

Phenylethyl alcohol

Selective agent in PEA

Gram-positive bacteria

PEA selects for

MSA

Mannitol salt agar

7.5% NaCl

Selective agent in MSA

salt-tolerant bacteria

What does MSA select for

Mannitol and phenol red

Differential agents in MSA

MSA differentiates between

Mannitol fermenters and non-mannitol fermenters

Mannitol fermenters

Yellow around colonies

Non-mannitol fermenters

Red/bright pink around colonies

EMB

Eosin Methylene Blue

Eosin and Methylene Blue

Selective agents in EMB

Gram-negative bacteria

What does EMB select for

Lactose, eosin, and methylene blue

Differential agents in EMB

EMB differentiates between

Lactose fermenters and non-lactose fermenters

Lactose fermenters

Pink, purple, or green metallic sheen

Non-lactose fermenters

Pale/colorless

BAP

Blood agar plate

Components of BAP

Tryptose agar enriched with 5% sterile sheep blood

Fastidious

Hard to grow

What does BAP differentiate

Hemolysis

Hemolysis

Break apart RBC

Beta hemolysis

Complete hemolysis (clear)

Alpha hemolysis

Partial hemolysis (cloudy grey/green)

Gamma hemolysis

No hemolysis (red)

Staphylococcus General Characteristics

• Gram positive cocci

• Catalase positive

• Salt tolerant

• Sometimes confused with Micrococcus

Staphylococcus Bacitracin Susceptibility Test purpose

Differentiate between Staphylococcus and Micrococcus

Staphylococcus Bacitracin susceptibility test media

Tryptose agar plate

All Staphylococcus are

Bacitracin resistant

All Micrococcus are

Bacitracin sensitive

Bacitracin sensitive — Micrococcus

Zone of inhibition present

Bacitracin resistant — Staphylococcus

No zone of inhibition

Purpose of Coagulase Test

ID bacteria that produce coagulase (Staphylococcus aureus)

Coagulase Test Media

Rabbit Plasma — contains fibrinogen

Coagulate Test Substrate

Fibrinogen

Coagulate Test Enzyme

Coagulase

Coagulate Test product

Fibrin (clots)

General Streptococcus Characteristics

• Gram-positive cocci

• Catalase negative

• Fastidious → grow on BAP

• sometimes confused with Enterococcus

BAGG Test purpose

ID Enterococcus faecalis

BAGG test media

BAGG Broth

BAGG

Buffered Azide Glycerol Glucose

Sodium azide

Inhibits growth of Gram-negatives

Glucose

Fermentable sugar

Bromocresol Purple

pH indicator

Acidic: yellow

neutral/basic: purple

Enterococcus faecalis

Ferments glucose → acid

Positive BAGG test

Yellow

Negative BAGG test

Purple

Streptococcus bacitracin Susceptibility test purpose

Differentiate between Group A Streptococcus (GAS) and Group B Streptococcus (GBS)

Streptococcus Bacitracin Susceptibility Test and CAMP Test media

BAP

GAS

Bacitracin resistant Streptococcus

No zone of inhibition

GBS

Bacitracin sensitive Streptococcus

Zone of inhibition present

CAMP Test purpose

identify Streptococcus agalactiae (GBS)