Oxidation/Fermentation (OF) Medium (CDC Method)
A visual guide to the OF medium test, used to classify bacteria as fermenters, oxidizers, or non-utilizers based on their carbohydrate metabolism under aerobic and anaerobic conditions.
OF Dextrose Tubes A-C
Compare the open (aerobic) and oil-overlaid (anaerobic) tube pairs. Phenol red turns yellow under acid conditions.
Open tube turns completely yellow due to acid production from glucose fermentation and oxidation.
Oil-overlaid anaerobic tube turns completely yellow, demonstrating fermentative metabolism in the absence of oxygen.
Open tube shows acid production (yellow color) restricted to the oxygen-exposed top surface layer only.
Overlaid tube remains completely red. The organism cannot ferment carbohydrate anaerobically.
Open tube remains completely red; no acid is produced aerobically.
Oil-overlaid tube remains completely red; no acid is produced anaerobically.
Open tube turns completely yellow due to acid production from glucose fermentation and oxidation.
Oil-overlaid anaerobic tube turns completely yellow, demonstrating fermentative metabolism in the absence of oxygen.
Open tube shows acid production (yellow color) restricted to the oxygen-exposed top surface layer only.
Overlaid tube remains completely red. The organism cannot ferment carbohydrate anaerobically.
Open tube remains completely red; no acid is produced aerobically.
Oil-overlaid tube remains completely red; no acid is produced anaerobically.
What to look for
| Visual clue | Meaning | Student shorthand |
|---|---|---|
| Yellow color (Acid) | Carbohydrate is metabolized to acid byproducts, lowering pH | Positive reaction (A) |
| Red or Alkaline color | No carbohydrate metabolism; peptone utilization may turn medium deep red | Negative or Neutral (K) |
| Mineral Oil Overlay | Creates an anaerobic environment inside the overlaid tube | Differentiates fermentation from oxidation |
| Yellow top, red bottom | Acid produced exclusively at the aerobic surface through oxidative pathways | Oxidation pattern |
OF Dextrose Interpretive Matrix
| Pattern | What to call it | How to think about it |
|---|---|---|
| Open: Yellow / Overlaid: Yellow | Fermentation (F) | Organism can ferment glucose anaerobically (e.g., E. coli). |
| Open: Yellow (top) / Overlaid: Red | Oxidation (O) | Organism uses glucose oxidatively only (e.g., P. aeruginosa). |
| Open: Red / Overlaid: Red | Non-utilizer (Asaccharolytic) | Organism does not metabolize glucose (e.g., Moraxella). |
- OF medium uses Hugh and Leifson's formulation: high carbohydrate-to-peptone ratio to prevent alkaline peptone amines from masking weak acid.
- Fermenters turn both tubes completely yellow (e.g., Escherichia coli).
- Oxidizers turn only the open tube yellow at the top, leaving the overlaid tube red (e.g., Pseudomonas aeruginosa).
F is for Fermentation = both yellow. O is for Oxidation = open top yellow only. Non-utilizer = both red.