Oxidative-Fermentative Test (Hugh and Leifson)- Principle, Test Procedure Results and Interpretation

 

Objective

To detect the oxidation or fermentation of carbohydrates by bacteria.

Principle

Saccharolytic microorganisms degrade glucose either fermentatively or oxidatively.

The end products of fermentation are relatively strong mixed acids that can be detected in a conventional fermentation test medium. However, the acids formed in oxidative degradation of glucose are extremely weak, and the more sensitive oxidation-fermentation medium of Hugh and Leifson (OF medium) is required for their detection.

The OF medium of Hugh and Leifson differs from carbohydrate fermentation media as follows:

The concentration of peptone is decreased from 1% to 0.2%.

The concentration of carbohydrate is increased from 0.5% to 1.0%.

The concentration of agar is decreased from 1.5% to 0.3%, making it semisolid.

The lower protein/carbohydrate ratio reduces the formation of alkaline amines that can neutralize the small quantities of weak acids that may form from oxidative metabolism. The relatively larger amount of carbohydrate serves to increase the amount of acid that can potentially be formed. The semisolid consistency of the agar permits acids that form on the surface of the agar to permeate throughout the medium, making interpretation of the pH shift of the indicator easier to visualize. Motility can also be observed in this medium.

Media and Reagents

For comparison, the formulas for a conventional carbohydrate fermentation medium and OF medium are as follows:

Carbohydrate Fermentation Medium

Peptone	10 g
Sodium chloride	5 g
d-glucose	5 g
Bromcresol purple	0.02 g
*Agar	15 g
Distilled water	1 L
pH	7.0

*Agar is omitted from broth medium.

 

OF Medium of Hugh and Leifson

Peptone	2 g
Sodium chloride	5 g
d-glucose	10 g
Bromthymol blue	0.03 g
Agar	3 g
Dipotassium phosphate	030
Distilled water	1 L
pH	7.1

OF medium should be poured without a slant into tubes with an inner diameter of 15–20 mm to increase surface area.

Quality Control

Glucose fermenter: Escherichia coli

Glucose oxidizer: Pseudomonas aeruginosa

Nonsaccharolytic: Moraxella species

Test Procedure

Two tubes are required for the OF test, each inoculated with the unknown organism, using a straight needle, stabbing the medium three to four times halfway to the bottom of the tube. One tube of each pair is covered with a 1-cm layer of sterile mineral oil or melted paraffin, leaving the other tube open to the air. Incubate both tubes at 35°C and examine daily for several days.

Results

Interpretation

Acid production is detected in the medium by the appearance of a yellow color. In the case of oxidative organisms, color production may be first noted near the surface of the medium. Following are the reaction patterns:

Open Tube	Covered Tubes	Metabolism
Acid (yellow)	Alkaline (green)	Oxidative
Acid (yellow)	Acid (yellow)	Fermentative
Alkaline (green or blue)	Alkaline (green or blue)	Nonsaccharolytic

These color reactions are shown in Color Plate 7-1D. For slower-growing species, incubation for 3 days or longer may be required to detect positive reactions.

Oxidative-Fermentative Test (Hugh and Leifson)

Reference

1. BBL Manual of Products and Laboratory Procedures. 5th Ed. Cockeysville, MD: BioQuest, 1973:129–130.
2. Hugh R, Leifson E. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram-negative bacilli. J Bacteriol 1953;66:24–26.
3. MacFaddin JF. Biochemical Tests for Identification of Medical Bacteria. 2nd Ed. Baltimore, MD: Williams & Wilkins, 1980:260–268.