Enterobacteriacea and Pseudomonas:

In the previous lab, 4 Gram-negative bacilli were streaked on the selective and differential MacConkey agar. MacConkey agar 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.

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.

The other medium inoculated was the SIM tube that tests for 3 different physiological properties, 1) H 2S production, 2) tryptophanase activity (indole production), and 3) motility. H 2S 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.

Expected Results

Escherichia coli

MacConkey: Ferments lactose (striking pink colony).

Glucose tube: Ferments glucose with gas production (yellow with a bubble in the Durham Tube).

SIM: Does not produce H 2S gas, indole positive (rose ring), and may or may not be motile.

Proteus mirabilis

MacConkey: Does not ferment lactose (colorless colony).

Glucose tube: Ferments glucose with gas production (yellow with a bubble in Durham Tube).

SIM: Produces H 2S, does not produce indole (no rose ring), and motile (variable with some species/strains).

Shigella dysenteriae

MacConkey: Does not ferment lactose (colorless colony).

Glucose tube: Ferments glucose without producing gas (yellow broth, no bubble in Durham Tube).

SIM: Does not produce H 2S, indole negative, and non-motile.

Pseudomonas aeruginosa

MacConkey: Does not ferment lactose (colorless colony).

Glucose tube: Does not ferment glucose (red broth).

SIM: Does not produce H 2S, indole negative, and motile.

In today's lab we will continue the characterization of Gram-negative bacilli. The nitrate test, which is important for the identification of both Gram-positive and Gram-negative bacteria, will be performed.

Rachel Watson, M.S.
AG 5010
766-3524
Cell: 307-760-2942
rwatson@uwyo.edu