Oxidation Fermentation Test Procedure

In this oxidation fermentation test procedure post we have briefly explained about oxidation fermentation test principle, objectives, requirements, oxidation fermentation test procedure, uses and limitations of oxidation fermentation test.

Oxidation Fermentation Test

Oxidative fermentative test determines if bacteria metabolise carbohydrates oxidatively or by fermentation, or if they are non-saccharolytic and so unable to utilise the carbohydrate in the media. Only aerobic circumstances allow oxidative organisms to metabolise glucose or other carbs, implying that oxygen is the ultimate hydrogen acceptor. Other species digest glucose and use another material, such as sulphur, as a hydrogen acceptor.

This fermentative process is oxygen-free, and organism cultures can be aerobic or anaerobic. An acid is formed when a carbohydrate is broken down. The Hugh and Leifson test, as it is sometimes called, uses a semi-solid medium in tubes containing the carbohydrate under test (typically glucose) and a pH indicator.

To create anaerobic conditions, two tubes are inoculated and one is immediately sealed. In both tubes, the Enterobacteriaceae cause an acid reaction throughout the medium. Organisms such as Alcaligenes faecalis, which cannot break down the carbohydrate aerobically or anaerobically, create an alkaline reaction in the open tube but no change in the covered tube.

Gas generation and motility can also be recorded using Hugh and Leifson’s medium. The Baird-Parker medium modification is used to test staphylococci and micrococci.


Oxidation fermentation test reveals whether specific gram-negative rods use fermentation or aerobic respiration to consume glucose (oxidatively). Pyruvate is transformed to a range of mixed acids during anaerobic fermentation, depending on the kind of fermentation.

In the presence or absence of oxygen, the high concentration of acid produced during fermentation will convert the bromthymol blue indicator in oxidative fermentative test medium from green to yellow. Because some non-fermenting gram-negative bacteria use aerobic respiration to consume glucose, they only create a modest quantity of weak acids during glycolysis and the Krebs cycle.

The detection of weak acids generated is made easier by the drop in peptone and increase in glucose. To aid acid detection, a dipotassium phosphate buffer is used.


1. Fresh cultures

2. Inoculating loop

3. Oxidative Fermentative medium

4. Two tubes


Hugh and Leifson’s medium: Peptone 2.0gm/L,  Sodium chloride 5.0gm/L,  Dipotassium phosphate 0.30gm/L,  Glucose (Dextrose) 10.0gm/L,  Bromothymol blue 0.030gm/L, Agar 3.0gm/L,  Final pH ( at 25°C) 7.1±0.2.

Oxidation Fermentation Test Procedure

1. Remove the oxygen from 2 tubes of medium by boiling them for 10 minutes and allowing them to cool before using.

2. Insert a straight wire vertically to roughly 0.5cm from the bottom of both tubes to stab-inoculate them.

3. To establish anaerobic conditions, incubate one tube aerobically while the other is either incubated anaerobically or sealed with a layer of melted soft paraffin to a depth of about 3cm above the medium. Place the controls next to the test organism.

4. Incubate for 48 hours or longer at 35°C. Slow-growing species may require more time incubation; check tubes daily for colour change.


OF test

Gram negative rod

Positive result: Oxidation- Acid in aerobic tube only (yellow colour in aerobic tube, green in anaerobic tube). Fermentation: Acid in both tubes (yellow colour) example: Pseudomonas aeruginosa, Escherichia coli.

Negative result: Neither fermentation nor oxidation; No acid production (blue or green colour in aerobic tube, green in anaerobic tube) example: Acinetobacter Iwoffii.

Gram positive cocci

Positive result: Oxidation – Acid in aerobic tube only (yellow colour in aerobic tube, purple in anaerobic tube). Fermentation: Acid in both tubes (yellow colour) example: Micrococcus luteus, Staphylococcus aureus.

Negative result: (Neither fermentation nor oxidation) No acid production /No colour change (purple colour in both tubes).


1. Oxidation fermentation test allows gram-negative bacteria to be identified based on their capacity to oxidise or ferment a specific carbohydrate. Oxidation fermentation test used to see if an organism produces acid by products from carbohydrate substrates.

2. The ability of non-fermentative bacteria to create acid from six carbohydrates is widely studied: glucose, xylose, mannitol, lactose, sucrose, and maltose.


1. For complete identification, biochemical, immunological, molecular, or mass spectrometry testing on colonies from pure culture is indicated. Slow growing organisms may take several days to yield effects.

2. Oxidation fermentation test medium does not support the growth of several bacteria. To confirm the negative reaction, another dextrose-containing basal medium may be required. Some mineral oils are acidic, which might lead to inaccurate readings.

Further Readings