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Biochemical Tests for Gram Negative Organisms

MacConkey Plates

MacConkey agar is a selective media that supports the growth of gram-negative bacteria and inhibits the growth gram-positive bacteria. It also differentiates on the basis of lactose fermentation. Colonies of lactose fermenting bacteria appear dark pink/red and non-lactose fermenting bacteria will appear colorless.


MacConkey plate with E. coliMacConkey plate with Shigella and Pseudomonas

Glucose Broth Tubes

Glucose fermentation broth tubes test for the ability of the organism to ferment glucose. Organic acids are the products of fermentation. These acids lower the pH of the medium causing the pH indicator, phenol red, to change from red to yellow. Fermentation can occur with or without the production of gas. If gas is produced, it is trapped in the inverted Durham tube and can be seen as a bubble. Gas cannot be produced without fermentation of the sugar. Other sugars are also utilized in this manor to test for various fermentation capabilities.



Glucose broth tube results

SIM Tubes (Sulfur, Indole, Motility)

The SIM tube tests for 3 different physiological properties, 1) H2S production, 2) tryptophanase activity (indole production), and 3) motility. H2S production is indicated by the presence of a black precipitate. Tryptophanase is an enzyme that cleaves the amino acid tryptophan into pyruvic acid, ammonia and indole. This reaction is determined by adding Kovac’s reagent to the SIM tube following incubation. Kovac’s reagent combines with indole to produce a rose/red color. Motility is determined by observing growth/turbidity away from the line of inoculation.



SIM tube results

Kligler's Iron Agar (KIA)

Kligler's Iron Agar (KIA) is commonly used to distinguish members of Enterobacteriaceae from other Gram-negative bacilli and to identify lactose negative enterics, which are often more serious pathogens of the Gastrointestinal tract. KIA medium measures an organism’s ability to ferment glucose and lactose, to utilize protein, and to produce H2S. Although interpretation of the medium is somewhat complicated, the ability to determine these three properties all in one test makes KIA media particularly useful.



KIA tube results

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