TECHNICAL DATA SHEET #585 REV.

OXIDATIVE-FERMENTATIVE MEDIA
(OF BASAL MEDIA)
PRODUCT:
Tube Media:a
OF Basal Medium, Control, Hugh-Leifson, item no. T7140
OF Basal Medium With 1% Specific Carbohydrate, Hugh-Leifson, item no.’s T7142 (dextrose), T7143 (fructose),
T7146 (lactose), T7148 (maltose), T7150 (mannitol), T7162 (sucrose), T7156 (xylose)
OF Medium, Control, King, item no. T7178
OF Medium With 1% Specific Carbohydrate, King, item no.’s T7180 (dextrose), T7186 (lactose), T7188 (maltose),
T7191 (mannitol), T7192 (sucrose), T7196 (xylose)

a
see catalog for ordering options

PURPOSE:
OF basal medium is a low peptone-containing medium that detects the acid produced by microorganisms that require oxygen for
the degradation of carbohydrates. The utilization of OF Glucose Medium allows for the categorization of microorganisms as
fermenters, oxidizers, or nonoxidizers. In addition, the motility of the microbe can be determined with this semisolid medium.

PRINCIPLE:
Hugh and Leifson1,2 first realized that microorganisms utilizing carbohydrates via an aerobic metabolic pathway produced only a
minimal amount of acid. In media containing large amounts of peptones, these same microbes would utilize the peptones,
producing enough alkaline by-products to obscure the acid produced. Hugh and Leifson decreased the ratio of peptone to
carbohydrate in the medium to a 0.2:1 ratio which minimized the formation of alkaline by-products; the subsequent increased
carbohydrate concentration enhanced the acid production by the microbes.3 The semisolid consistency of the agar prevents
dilution of any acid produced, as well as keeping the acid concentrated at the site of production. Bromthymol blue or phenol red
acts as an indicator and changes to yellow when acid is formed.

The Centers for Disease Control prefers the phenol red indicator (King OF formulation); others use bromthymol blue indicator,
which may be more sensitive, but may also be somewhat inhibitory.4 Studies at the CDC revealed that the correlation between
Hugh-Leifson and King OF media are excellent and either can be used when interpreting results from Weaver’s tables (CDC
approach).3 The sodium chloride and potassium phosphate in the Hugh-Leifson formulation act as buffers and enhance the
detection of acid production.

FORMULAS:
Approximate, per liter of deionized filtered water.

(1) Hugh-Leifson OF Basal Medium, Control:

Pancreatic Digest of Casein .......................... 2.00 g
Sodium Chloride ............................................ 5.00
Dipotassium Phosphate ................................. 0.30
Bromthymol Blue ........................................... 0.08
Agar ............................................................... 3.00
Final pH 6.8 ± 0.2 at 25°C

(2) King OF Basal Medium, Control:

Pancreatic Digest of Casein .......................... 2.00 g
Agar ............................................................... 3.00
Phenol Red .................................................... 0.03
Glycerol ......................................................... 10.00 ml
Final pH 7.3 ± 0.2 at 25°C

(3) Hugh-Leifson OF Basal Medium With Carbohydrate:

Same as (1) above except it contains 10.0 g of a specific carbohydrate.

27120 SW 95th Avenue • Wilsonville, OR 97070 • 800.547.0659

Page 1
 TECHNICAL DATA SHEET #585 REV. 2
(4) King OF Basal Medium With Carbohydrate:
Same as (2) above except it contains 10.0 g of a specific carbohydrate.

PRECAUTIONS:*
For in vitro diagnostic use. Observe approved biohazard precautions.

Storage: Upon receipt store at 2-8°C away from direct light. Media should not be used if there are signs of contamination,
deterioration (discoloration or evaporation), or if the expiration date has passed.

Limitations: Some microorganisms are unable to grow in OF Basal Media and require repeat testing using 2% serum or 0.1%
yeast extract.4

Slow-growing microorganisms may not produce color changes for several days.

Species that are especially active on amino acids may cause weak acid reactions to reverse, making interpretation confusing.

If the oxidation reaction is delayed or weak, an alkalinity may be observed on the surface, but with further incubation the alkaline
reaction reverts to acid. Microorganisms may attack carbohydrates at different rates. For example, Pseudomonas maltophilia
produces an early alkaline reaction on glucose OF media, becoming weakly acid upon further incubation; however, on maltose
OF media, acidity is rapidly produced.

Sterile paraffin and sterile liquid mineral oil seals are not as effective as a sterile petrolatum seal. Some types of mineral oil are
acidic and can contribute to false-positive results.

PROCEDURE:*
Specimen Collection: Not applicable since these media are not for primary isolation but, rather, are used in characterizing pure
cultures. Isolated organisms, established isolation techniques, and tests for purity are necessary before inoculating these
media. Direct inoculation of specimens will produce erroneous results. Information on specimen collection may be found in
standard reference texts.

Method of Use, Tubes: Prior to inoculation, the medium should be brought to room temperature. Using sterile water or saline,
prepare a heavy suspension of the microorganism from a pure culture growing for 18-24 hours; growth from a TSI is a good
source, if pure growth has been inoculated. Inoculate a pair of OF tubes with the liquid solution; using a straight needle, stab two
tubes to approximately 1/4 inch from the bottom of the tube. Overlay one tube with 1-2 milliliters of sterile melted petrolatum;
loosen the screw cap of the other tube. Incubate aerobically at 35°C and examine daily for 3-4 days; incubation of up to 14 days
may be necessary.

Microorganisms that oxidize only glucose will not ferment any other carbohydrate. In determining other carbohydrate utilization
of such microbes, the sealed tube may be omitted.

Interpretation:
Carbohydrate Utilization** Open Tube, Plate: Sealed Tube:
Fermenter Acid (Yellow) Acid (Yellow)

Oxidizer Acid Alkaline

(Yellow near the surface) (Hugh-Leifson: Green)
(King: Orange)

Nonoxidizer Alkaline Alkaline

(Hugh-Leifson: Blue/Green) (Hugh-Leifson: Green)
(King: Red/Orange) (King: Orange)

** Use an inoculated OF Basal Medium Control as a comparison.

Motility: Motile microorganisms migrate from the stab line, diffusing into the media.

Nonmotile microbes grow around the stab line; the surrounding medium remains clear. Some microbes are nonmotile at 35?C
and motile between 15-25°C. Such microbes need to be tested at room temperature.

27120 SW 95th Avenue • Wilsonville, OR 97070 • 800.547.0659

Page 2
 TECHNICAL DATA SHEET #585 REV. 2
Material Required but Not Provided: Standard microbiological supplies and equipment such as loops, needles, incubators,
and incinerators, as well as petrolatum, are not provided.

QUALITY CONTROL:*
Carbohydrate Used: Microorganisms Used (ATCC#): Expected Results:

Control Pseudomonas aeruginosa (27853) no change

Klebsiella pneumoniae (13883) no change
Morganella morganii (25830) no change

Dextrose, Mannitol Pseudomonas aeruginosa (27853) oxidation

Klebsiella pneumoniae (13883) oxidation/fermentation
Moraxella osloensis (10973) no change

Fructose, Maltose, Stenotrophomones maltophilia (13637) oxidation

Klebsiella pneumoniae (13883) oxidation/fermentation
Moraxella osloensis (10973) no change

Lactose, Sucrose Bulkholderia cepacia (25416) oxidation

Klebsiella pneumoniae (13883) oxidation/fermentation
Moraxella osloensis (10973) no change

Xylose Pseudomonas fluorescens oxidation

Klebsiella pneumoniae (13883) oxidation/fermentation
Moraxella osloensis (10973) no change
Key: See “Interpretation”

User Quality Control: Check for signs of contamination and deterioration.

BIBLIOGRAPHY:
1. Finegold, S. M., and E. J. Baron, Bailey and Scott’s Diagnostic Microbiology, 7th ed., C. V. Mosby, St. Louis, 1986.
2. Hugh, R., and E. Leifson, J. Bacteriol., 66:24-26, 1953.
3. Koneman, E. W., et al., Color Atlas and Textbook of Diagnostic Microbiology, 3rd ed., J. B. Lippincott, Philadelphia, 1988.
4. Lennette, E. H., et al., Manual of Clinical Microbiology, 4th ed., American Society for Microbiology, Washington, D. C., 1985.
5. MacFaddin, J. F., Biochemical Tests for Identification of Medical Bacteria, 1st ed., Williams and Wilkins, Baltimore, 1976.

*For more detailed information, consult appropriate references and/or details in the preface of the PML Technical Manual.

PML MICROBIOLOGICALS, INC.

Data #585
Copyright 1989 by PML Microbiologicals, Inc.
Revision Date: January 2001

27120 SW 95th Avenue • Wilsonville, OR 97070 • 800.547.0659

Page 3