Biochemical Testing Reference Chart

A set of 100 practice flashcards reviewing bacterial biochemical tests, including media, indicators, reagents, positive and negative results, and biochemical reactions for Urease, TSI, IMViC, PAD, Catalase, and Oxidase.

Urease Test

Medium for Urease Test

$$\text{H}_2\text{O} + \text{urea}$$

Indicator for Urease Test

Phenol red.

Reagent for Urease Test

None (—); no reagent is added to the medium.

Positive Result for Urease Test

Pink, indicating an alkaline pH shift caused by ammonia accumulation.

Negative Result for Urease Test

Yellow, indicating acidic conditions and no urea hydrolysis.

Biochemical Reaction for Urease Test

$\text{urea} \xrightarrow{\text{urease}} \text{CO}_2 + \text{H}_2\text{O} + \text{ammonia}$.

Products of the Urease Reaction

$\text{CO}_2 + \text{H}_2\text{O} + \text{ammonia}$.

TSI

Triple Sugar Iron agar, a multi-test medium used to evaluate carbohydrate fermentation, peptone utilization, and hydrogen sulfide production.

Indicator in TSI Agar

Phenol red.

Lactose/Sucrose Fermentation (TSI)

A test within TSI agar evaluating an organism's capacity to ferment lactose and/or sucrose into acid.

Medium for Lactose/Sucrose Fermentation

TSI Triple Sugar Iron agar.

Indicator for Lactose/Sucrose Fermentation

Phenol red.

Reagent for Lactose/Sucrose Fermentation

None (—).

Positive Result for Lactose/Sucrose Fermentation

Yellow throughout the slant and butt, recorded as A/A.

Negative Result for Lactose/Sucrose Fermentation

No change, recorded as K/K (red).

Biochemical Reaction for Lactose/Sucrose Fermentation

$\text{lactose/sucrose} \rightarrow \text{acid}$.

A/A Designation (TSI)

Acid slant / Acid butt (yellow/yellow), indicating fermentation of lactose and/or sucrose in TSI agar.

K/K Designation (TSI)

Alkaline slant / Alkaline butt (red/red), indicating that carbohydrates were not fermented (no change).

Glucose Fermentation (TSI)

A test within TSI agar evaluating whether an organism ferments glucose into acid.

Medium for Glucose Fermentation

TSI (Triple Sugar Iron) agar.

Indicator for Glucose Fermentation in TSI

Phenol red.

Positive Result for Glucose Fermentation (TSI)

Yellow butt with a red slant, recorded as K/A.

Negative Result for Glucose Fermentation (TSI)

No change, recorded as K/K (red).

Biochemical Reaction for Glucose Fermentation

$\text{glucose} \rightarrow \text{acid}$.

K/A Designation (TSI)

Alkaline slant / Acid butt (red slant/yellow butt), indicating glucose fermentation only.

Peptones (TSI Test)

The metabolic catabolism of peptones in TSI agar when carbohydrates are not fermented or become depleted.

Medium for Peptones Reaction

TSI (Triple Sugar Iron) agar.

Positive Result for Peptones in TSI

Red/pink, indicating an alkaline shift from ammonia production.

Negative Result for Peptones in TSI

No change.

Biochemical Reaction for Peptones Utilization

$\text{peptones} \rightarrow \text{ammonia (alkaline)}$.

$\text{H}_2\text{S}$ Test (TSI)

A test assessing hydrogen sulfide ($\text{H}_2\text{S}$) production in TSI agar.

Medium for $\text{H}_2\text{S}$ Test

TSI (Triple Sugar Iron) agar.

Indicator for $\text{H}_2\text{S}$ in TSI

Ferrous sulfate.

Positive Result for $\text{H}_2\text{S}$ Production

Black precipitate formation in the medium.

Biochemical Reaction for $\text{H}_2\text{S}$ Production

$\text{Sodium thiosulfate} \rightarrow \text{H}_2\text{S} + \text{Ferrous sulfate (black)}$.

IMViC

A group of four diagnostic tests: Indole, Methyl Red, Voges-Proskauer, and Citrate.

Indole Test

A biochemical test determining whether an organism hydrolyzes tryptophan into indole, pyruvic acid, and ammonia.

Medium for Indole Test

Sulfide-Indole-Motility (SIM) medium.

Indicator for Indole Test

None (—); detection requires the addition of an external reagent.

Reagent for Indole Test

Kovac's reagent.

Kovac's Reagent

The chemical reagent added to Sulfide-Indole-Motility medium to detect indole production.

Positive Result for Indole Test

A red layer forming at the surface of the medium after adding Kovac's reagent.

Negative Result for Indole Test

A yellow layer remaining at the surface of the medium after adding Kovac's reagent.

Biochemical Reaction for Indole Test

$\text{Tryptophan} \xrightarrow{\text{tryptophanase}} \text{indole} + \text{Pyruvic acid} + \text{ammonia}$.

Tryptophan

The amino acid substrate in Sulfide-Indole-Motility medium degraded in the indole test.

Tryptophanase

The enzyme that degrades tryptophan into indole, pyruvic acid, and ammonia.

Pyruvic Acid (Indole Test)

One of the three metabolic products formed alongside indole and ammonia during tryptophan hydrolysis.

Methyl Red Test

A biochemical test detecting mixed acid fermentation from glucose.

Medium for Methyl Red Test

MRVP broth.

Reagent/Indicator for Methyl Red Test

Methyl red.

Positive Result for Methyl Red Test

Red color following the addition of methyl red reagent, indicating low pH.

Negative Result for Methyl Red Test

Yellow color following the addition of methyl red reagent.

Biochemical Reaction for Methyl Red Test

$\text{glucose} \rightarrow \text{acid products}$.

Voges-Proskauer Test (Voges-Proskaur)

A biochemical test determining if an organism ferments glucose into neutral end products.

Medium for Voges-Proskauer Test

MRVP broth.

Indicator for Voges-Proskauer Test

None (—).

Reagent for Voges-Proskauer Test

Barritt's reagent.

Barritt's Reagent

The chemical reagent added to MRVP broth in the Voges-Proskauer test.

Positive Result for Voges-Proskauer Test

Rose/pink color development after adding Barritt's reagent.

Negative Result for Voges-Proskauer Test

Yellow or no change after adding Barritt's reagent.

Biochemical Reaction for Voges-Proskauer Test

$\text{glucose} \rightarrow \text{neutral products}$.

MRVP Broth

The shared liquid medium utilized to carry out both the Methyl Red and Voges-Proskauer tests.

Citrate Test

A biochemical test determining whether an organism can utilize citrate as its sole source of carbon.

Medium for Citrate Test

Citrate agar.

Indicator for Citrate Test

Bromthymol blue.

Reagent for Citrate Test

None (—); bromthymol blue is already present in the medium.

Positive Result for Citrate Test

Blue color change, indicating an alkaline shift.

Negative Result for Citrate Test

Green (no change), indicating no citrate utilization.

Bromthymol Blue

The pH indicator in citrate agar that is green at neutral pH and turns blue under alkaline conditions.

PAD

Phenylalanine deaminase test.

Medium for PAD Test

Phenylalanine deaminase agar.

Indicator for PAD Test

None (—); detection requires the addition of an external chemical reagent.

Reagent for PAD Test

Ferric chloride.

Ferric Chloride

The reagent added to phenylalanine deaminase agar to react with phenylpyruvic acid.

Positive Result for PAD Test

Green color development following the addition of ferric chloride.

Negative Result for PAD Test

Yellow color following the addition of ferric chloride.

Biochemical Reaction for PAD Test

$\text{Phenylalanine} \xrightarrow{\text{phenylalanine deaminase}} \text{phenylpyruvic acid} + \text{ammonia}$.

Phenylpyruvic Acid

The deamination product that complexes with ferric chloride to yield a green color in a positive PAD test.

Phenylalanine Deaminase (Enzyme)

The enzyme that removes the amino group from phenylalanine, generating phenylpyruvic acid and ammonia.

Catalase Test

A biochemical test used to detect the presence of catalase, which breaks down toxic hydrogen peroxide.

Medium for Catalase Test

None (—); the test is typically performed directly on a glass slide or culture.

Indicator for Catalase Test

None (—); bubble formation serves as the direct observable indicator.

Reagent for Catalase Test

Hydrogen peroxide ($\text{H}_2\text{O}_2$).

Hydrogen Peroxide ($\text{H}_2\text{O}_2$)

The reagent substrate added in the catalase test to observe effervescence.

Positive Result for Catalase Test

Bubbles, demonstrating the production and release of oxygen gas.

Negative Result for Catalase Test

No bubbles, indicating an absence of catalase activity.

Biochemical Reaction for Catalase Test

$\text{H}_2\text{O}_2 \xrightarrow{\text{catalase}} \text{O}_2 + \text{H}_2\text{O}$.

Catalase (Enzyme)

The enzyme that catalyzes the decomposition of hydrogen peroxide into oxygen and water.

Oxygen Gas in Catalase Test

$\text{O}_2$ gas formed during hydrogen peroxide degradation that appears as visible bubbles.

Oxidase Test

A biochemical test determining whether an organism produces cytochrome c oxidase.

Medium for Oxidase Test

None (—).

Indicator for Oxidase Test

None (—).

Reagent for Oxidase Test

P-aminodimethylaniline oxalate (Pg. 208).

P-aminodimethylaniline Oxalate

The redox reagent used in the oxidase test that changes color when oxidized.

Page 208 Reference

The reference cited on the chart detailing the reagent P-aminodimethylaniline oxalate.

Positive Result for Oxidase Test

Dark blue/purple color change.

Negative Result for Oxidase Test

No change (no color development).

Reading Window for Oxidase Test

Read results within $30$ seconds.

Significance of the 30-Second Oxidase Window

Results must be read within $30$ seconds to avoid false-positive results caused by the non-enzymatic auto-oxidation of the reagent in ambient air.