Enterobacteralles Lecture Objectives

What is the purpose/principle of the Indole (Ehrlich’s and Kovac’s) test?

To detect whether an organism can split indole from the amino acid tryptophan.

Blue= positive; Pink = negative

What is the purpose/principle of the Triple Sugar Iron Agar/ Kligler Iron Agar test?

To identify an organism’s ability to ferment lactose, sucrose, and/ or glucose, with or without the production of gas. And the production of hydrogen sulfide is also determined.

SLANT/BUTT

K/A (Red/yellow) = Glucose only

A/A (yellow/yellow) = Glucose + lactose and or sucrose

K/K(red/red) OR no change = neither glucose nor lactose/sucrose (nonfermenter)

Bubbles = CO2 gas production (NOT H2S)

Black ppt. = H2S production (occurs most often in the butt)

What is the purpose/principle of the motility test?

To determine if an organism is motile (flagellated) or non motile (nonflagellated)

Concentrated turbidity around stab = non motile

Distributed turbidity from stab (ex “fuzzy catepillar”) = motile

What is the purpose/principle of the Urease test?

determine an organism’s ability to split urea by the enzyme urease

Pink = positive (fast reactions (within hours) are known as strong urease producers)

No change / nickelodeon orange = non-urease producer

What is the purpose/principle of the Oxidase test?

to determine the presence of oxidized cytochrome C (cytochrome oxidase enzyme).

Filter paper/ colony turning a deep blue = presence confirmed

no color = presence not confirmed

What is the purpose/principle of the MUG test?

MUG = 4-methylumbelliferyl-ß-D-glucuronidase
Testing organism for production of ß-glucuronidase
Tube or disk test
Positive test = fluorescence with UV light
Helpful screening test for E. coli
Rapid test

What are the substrate(s), product(s), and reagent(s) used in the Indole (Ehrlich’s and Kovac’s) test?

Media: Peptone or tryptone broth (with little or no glucose so the bacteria will try to utilize tryptophan!

Substrate: tryptophan

Ehrlich’s procedure:

• extract with chloroform, ether or xylene

• Reagent: P-dimethylaminobenzaldehyde-HCl in ethyl alcohol

• Use Ehrlich’s for nonfermenting GNRs and anaerobes

• More sensitive test

Kovac’s procedure:

• Reagent: P-dimethylaminobenzaldehyde-HCl in alcholol

• Used for enterobacterales

• less sensitive

• Spot indole (rapid test) avalible

What are the substrate(s), product(s), and reagent(s) used in the Triple Sugar Iron Agar/ Kligler Iron Agar test?

Media: 0.1% glucose, 1.0% lactose, 1.0% sucrose, and peptones.

Reagents: Phenol red indicator (yellow = acidic, red = basic), Ferrous sulfate, Sodium thiosulfate.

For the detection of H2S production:

Sodium thiosulfate reacts with the acid environment H2S, which then reacts with ferrous ions (from ferrous sulfate), to form the black pigment (ferrous sulfide)

What are the substrate(s), product(s), and reagent(s) used in the motility test?

No substrates or products produced - just growth

Medium: semi-solid medium (0.4%) - agar stabbed

Reagent: Tetrazolium salts are somtimes incoporated. Reduced to formazan (red) when incorporated into cells (red mans movement), but not always used because the salts are inhibitory to some organisms.

What are the substrate(s), product(s), and reagent(s) used in the Urease test?

Media: Christensen’s media (buffer, urea, glucose, peptone, phenol red)

Substrate: Urea

Reagent: none

What are the substrate(s), product(s), and reagent(s) used in the Oxidase test?

Reagents:
1% tetramethyl-p-phenylenediamine dihydrochloride

OR
Dimethyl-p-phenylenediamine + alpha naphthol

can be done on filter paper, plate, tube, or impregnated packaged strips. Do NOT use nichrome loops ( use wooden applicator sticks), and do NOT use selective media!

What are the substrate(s), product(s), and reagent(s) used in the MUG test?

MUG = 4-methylumbelliferyl-ß-D-glucuronidase