Decarboxylase Test- Principle, Procedure and Result Interpretation

Decarboxylase Test- Principle, Procedure and Result Interpretation

Decarboxylase Test- Principle, Procedure and Result Interpretation

Objective of Decarboxylase Test

This test is used to differentiate decarboxylase producing Enterobacteriaceae from other gram-negative rods.

Principle of Decarboxylase Test

This test measures the enzymatic ability (decarboxylase) of an organism to decarboxylate (or hydrolyze) an amino acid to form an amine. Decarboxylation, or hydrolysis, of the amino acid results in an alkaline pH and a color change
from orange to purple.

Media: Peptic digest of animal tissue (5 g), beef extract (5 g), bromcresol purple (0.1 g), cresol red (0.005 g), dextrose (0.5 g), pyridoxal (0.005 g), amino acid (10 g), pH 6.0.

Procedure of Decarboxylase Test (Moeller’s Method)

A. Glucose-Nonfermenting Organisms

  1. Prepare a suspension (≥McFarland No. 5 turbidity standard) in brain-heart infusion broth from an overnight culture (18 to 24 hours old) growing on 5% sheep blood agar.
  2. Inoculate each of the three decarboxylase broths (arginine, lysine, and ornithine) and the control broth (no amino acid) with 4 drops of broth.
  3. Add a 4-mm layer of sterile mineral oil to each tube.
  4. Incubate the cultures at 35°-37°C in ambient air. Examine the tubes at 24, 48, 72, and 96 hours.

B. Glucose-Fermenting Organisms

  1. Inoculate tubes with 1 drop of an 18- to 24-hour brain-heart infusion broth culture.
  2. Add a 4-mm layer of sterile mineral oil to each tube.
  3. Incubate the cultures for 4 days at 35°-37°C in ambient air. Examine the tubes at 24, 48, 72, and 96 hours.

Result Interpretation of Decarboxylase Test

Positive: Alkaline (purple) color change compared with the control tube.
Negative: No color change or acid (yellow) color in test and control tube. Growth in the control tube.

Limitations of Decarboxylase Test

The fermentation of dextrose in the medium causes the acid color change. However, it would not mask the alkaline color change brought about by a positive decarboxylation reaction.

Quality Control

Positive:
Lysine—Klebsiella pneumoniae (ATCC33495)
Ornithine—Enterobacter aerogenes (ATCC13048)
Arginine—Enterobacter cloacae (ATCC13047)
Negative:
Lysine—Enterobacter cloacae (ATCC13047)
Ornithine—Klebsiella pneumoniae (ATCC13883)
Arginine—Klebsiella pneumoniae (ATCC33495)

Decarboxylase Tests- Principle, Procedure and Result Interpretation

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