The nitrate test, which is important for the identification of both Gram-positive and Gram-negative bacteria, will be performed.
In today's lab we will continue the characterization of Gram-negative bacilli. SIM: Does not produce H 2S, indole negative, and motile. Glucose tube: Does not ferment glucose (red broth). SIM: Does not produce H 2S, indole negative, and non-motile. Glucose tube: Ferments glucose without producing gas (yellow broth, no bubble in Durham Tube). SIM: Produces H 2S, does not produce indole (no rose ring), and motile (variable with some species/strains). Shigella is a non-lactose fermenter on MacConkey agar and will give a K/N reaction with no H 2 S production on TSI slant. Commonly encountered lactose-fermenting Gram-negative bacilli include K lebsiella spp., E. Glucose tube: Ferments glucose with gas production (yellow with a bubble in Durham Tube). From the time Roux first recognized curved gram negative rods in the stools of patients with cholera. MacConkey: Does not ferment lactose (colorless colony). It has also become common to use the media to differentiate bacteria by their abilities to ferment sugars other than lactose. SIM: Does not produce H 2S gas, indole positive (rose ring), and may or may not be motile. MacConkey agar is used for the isolation of gram-negative enteric bacteria and the differentiation of lactose fermenting from lactose non-fermenting gram-negative bacteria. Glucose tube: Ferments glucose with gas production (yellow with a bubble in the Durham Tube). MacConkey: Ferments lactose (striking pink colony). Motility is determined by observing growth/turbidity away from the line of inoculation. Some media contain lactose sugar (macconkey). Kovac’s reagent combines with indole to produce a rose/red color. CLED stop growth of gram positive and allows growth of gram negative bacteria and macconkey medium as well. This reaction is determined by adding Kovac’s reagent to the SIM tube following incubation. Tryptophanase is an enzyme that cleaves the amino acid tryptophan into pyruvic acid, ammonia and indole. H 2S production is indicated by the presence of a black precipitate. 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. Gas cannot be produced without fermentation of the sugar. If gas is produced, it is trapped in the inverted Durham tube and can be seen as a bubble. Fermentation can occur with or without the production of gas. These acids lower the pH of the medium causing the pH indicator, phenol red, to change from red to yellow. Organic acids are the products of fermentation. Glucose fermentation broth tubes test for the ability of the organism to ferment glucose. Colonies of lactose fermenting bacteria appear dark pink/red and non-lactose fermenting bacteria will appear colorless. It also differentiates on the basis of lactose fermentation.
MacConkey agar supports the growth of Gram-negative bacteria, and inhibits the growth Gram-positive bacteria. In the previous lab, 4 Gram-negative bacilli were streaked on the selective and differential MacConkey agar. THE VIRTUAL EDGE: Lab 16 Enzyme-Mediated Biochemical Activities of Bacteria: Physiological Testing I
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