Gram Negative Rods: Biochemical and Media Identification

Vocabulary practice flashcards covering selective and differential media, carbohydrate metabolism, and key biochemical diagnostic tests for Gram-negative rods.

Selective Medium

A culture medium designed to allow only certain microorganisms to grow while inhibiting the growth of others, such as EMB agar using eosin and methylene blue dyes to inhibit Gram-positive bacteria.

Differential Medium

A culture medium containing specific substances that cause particular bacteria to take on an appearance that distinguishes them from other bacteria.

EMB (Eosin Methylene Blue) Agar

A selective and differential medium containing eosin Y and methylene blue that inhibits Gram-positive bacteria and differentiates Gram-negative lactose fermenters: vigorous fermenters form metallic green sheen/purple colonies, moderate fermenters form purple colonies, and non-fermenters form pink or colorless colonies.

Arrangement of Flagella

The structural distribution patterns of bacterial motility organelles, categorized as polar (a single flagellum at one pole), lophotrichous (a tuft of flagella at one pole), or peritrichous (flagella distributed all around the cell perimeter).

Oxidative-Fermentation (O/F) Carbohydrate Test

A differential test using two semisolid agar tubes per organism—an unsealed oxidative (O) tube and an anaerobic fermentation (F) tube overlaid with sterile mineral oil—to identify whether carbohydrate breakdown occurs fermentatively (both yellow), oxidatively only (open O tube yellow), or neither (both remain green).

Methyl Red (MR) Test

A test differentiating bacteria based on mixed-acid fermentation of glucose; production of organic acids lowering broth pH to $5.0$ or less turns the added methyl red indicator red.

Voges-Proskauer (VP) Test

A test detecting $2,3\text{-butanediol}$ fermentation of glucose; the neutral end product is oxidized to acetoin, which reacts with reagents VP1 (alpha-naphthol) and VP2 (KOH) to develop a pink-to-red color within $30\,\text{minutes}$.

Citrate Utilization Test

A biochemical test determining if an organism can use citrate as a sole carbon source and ammonium salts as a sole nitrogen source; bacterial utilization produces alkaline ammonia, causing bromothymol blue indicator to turn from green to deep Prussian blue.

SIM: Hydrogen Sulfide ($H_2S$) Production

The reduction of elemental sulfur by bacteria coupled to organic oxidation; produced $H_2S$ gas reacts with ferrous ammonium sulfate and sodium thiosulfate to form ferrous sulfide, visible as a black precipitate.

SIM: Indole Production

The enzymatic cleavage of tryptophan into indole, ammonia, and pyruvic acid by tryptophanase; detected by adding Kovac's reagent, which reacts with indole to form a deep red ring at the top of the medium.

SIM: Motility Test

The evaluation of bacterial swimming in a $0.4\%$ semisoft agar medium stab-inoculated with a needle; motile bacteria produce diffuse turbidity away from the stab line, whereas nonmotile bacteria grow strictly along the line of inoculation.

Brownian Motion

The non-vectorial shaking or wiggling movement of cells caused by the random molecular bombardment of surrounding fluid particles, distinct from true directional swimming motility.

Swarming Motility

A rapid, coordinated outward movement across an agar plate chasing nutrients (characteristic of Proteus mirabilis), leading to concentric rings of growth over the entire surface rather than isolated colonies.

Phenylalanine Deaminase Test

A test identifying the oxidative deamination of phenylalanine to phenylpyruvic acid and ammonia by phenylalanine deaminase; addition of $10\%$ ferric chloride reacts with phenylpyruvic acid to yield a dark green complex.

Urea Hydrolysis Test

A test detecting urease, which hydrolyzes urea to carbon dioxide and ammonia; the resulting alkalinity shifts the phenol red indicator from orange (pH $6.8$) to an intense cerise pink-red color (pH $8.1$ or greater).

IMViC Tests

A panel of four biochemical tests—Indole, Methyl Red, Voges-Proskauer, and Citrate—used to differentiate enteric Gram-negative rods, such as distinguishing Escherichia coli ($+$ / $+$ / $-$ / $-$) from Klebsiella aerogenes ($-$ / $-$ / $+$ / $+$).

Catalase Test

A slide test differentiating organisms by their ability to produce catalase, which hydrolyzes hydrogen peroxide ($H_2O_2$) into water ($H_2O$) and oxygen ($O_2$), confirmed by immediate bubbling.

KOH Test

A rapid test confirming Gram status by mixing cells with $3\%$ KOH; Gram-negative cell walls lyse and release DNA strings that stick to an inoculating loop, while Gram-positive walls remain intact.

Nitrate Reduction Test

A differential test evaluating nitrate reduction; turning red with reagents A and B indicates reduction to nitrite, turning red only after zinc indicates unreduced nitrate (negative), and remaining colorless after zinc indicates reduction to ammonia or nitrogenous gas.

Cytochrome C Oxidase Test

A test identifying the respiratory enzyme cytochrome C oxidase using a swab inoculated with bacteria and oxidase reagent ($N,N,N',N'\text{-tetramethyl-p-phenylenediamine}$), producing a dark blue to purple color within $30\,\text{seconds}$.

Starch Hydrolysis Test

A differential plate assay detecting starch breakdown; following incubation, flooding the plate with Gram's iodine reveals starch hydrolysis as a clear zone surrounding bacterial growth against a dark background.