How To Test Oxidation Fermentation – Procedure, Uses And Interpretation

Carbohydrates contain carbon, hydrogen and oxygen in the ratios of CH20, it is an organic compound that is found in many different kinds of food and grain.

Different organisms use carbohydrates differently depending on the enzymes that are present in this situation.

How To Test Oxidation Fermentation - Procedure, Uses And Interpretation

Considering the pattern of fermentation is different per species, genera or by group of organisms, this property has been extensively utilized within microbiology labs as a method for biochemical differentiation of microbes.

Put simply, an oxidation fermentation test determines if certain bacteria metabolize carbohydrate oxidatively, through fermentation, or are non-saccharolytic leading to lack of ability to use carbohydrates.

We have provided the basic procedure and uses of these tests as well as how to interpret their data.


We can determine whether an organism is fermentative or oxidative thanks to a test pioneered by Hugh and Leifson, known as Hugh and Leifson’s medium, or commonly as the ‘OF Medium’.

A sugar such as glucose, xyclose, mannitol, lactose, sucrose, or maltose can be added to the medium as a fermentable carbohydrate, to see what happens.

This is Hugh and Leifson’s Medium or the OF 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

Read our method below


The succinct method below should be followed in order to get the best results for interpretation.

  1. Obtain a pure isolated colony from an 18-24 hour culture.
  2. Inoculate each tube by stabbing the agar with a sterilized utensil to approximately ¼ inch from the bottom of the tube. Tubes should be inoculated in duplicate for each test organism.
  3. Either sterile mineral oil, sterile melted paraffin, sterile melted petrolatum should be sourced and applied to each of the duplicate tubes.
  4. Tighten the cap of the overlaid tube you have just applied the oil, paraffin, or petrolatum to and then loosen the cap of the non overlapped tube
  5. Both tubes should ideally be incubated aerobically at 35 degrees celsius or 95 degrees Fahrenheit for potentially up to 14 days or two weeks.
  6. You should examine each tube each day to monitor color changes, check out the results section to see what the colors reference.

Expected Results And Their Interpretation

These are the results we expect from these results as well as what these color indicators mean and how we can interpret them in order to get the most from these tests.

Yellow Color

A yellow color on the indicator means that there is a positive result for carbohydrate utilization, and this is indicated by the color yellow in the medium.

But these results do still require some interpretation for this specific test.

To know that the results show an oxidative bacteria there must be a development of a yellow coloration in the open tube only.

However, if there is a development of coloration in both the open tube and the closed tube this is clear evidence that the bacteria is fermentative rather than oxidative.

Medium Remains Green Or Turns Blue

Medium Remains Green Or Turns Blue

If the medium remains the same color, green, or turns a slightly blue color, this is unfortunately the indicator of a negative result.

This means that the bacteria you are studying is actually a non-oxidizer and non-fermenter which you should use in your review of the test and what it means in the wider terms of your experiment.

Uses Of This Test

So, you have your results, how can we use these results to make wider assumptions about said bacteria? Ultimately, your results will affect the hypothesis you are testing greatly.

If you are seeking to identify gram-negative bacterias, the ability of said bacteria to ferment or oxidize a specific carbohydrate can be helpful in this identification.

Moreover, this test can be used to determine if an organism can use carbohydrate substrates to produce acid byproducts.

If you find your bacteria is a fermentative bacteria then they are routinely tested for their ability to produce acid from six other carbohydrates (The ones mentioned: glucose, xyclose, mannitol, lactose, sucrose and maltose.

The test is regularly used by those who are interested in fermentation. Another random use is to identify different substrates of rust caused by bacteria oxidizing.

What Limitations Are There To The Test?

Well, in order to undertake a complete identification of the bacteria, we should consider that biochemical, immunological, molecular, or mass spectrometry should be undertaken on the colonies from the pure culture in order for complete identification.

Moreover, a slow growing organism may take much longer to produce results than we expect, more than several days.

In terms of the medium used, not all microorganisms can grow within the medium, it may be necessary to use a basal medium containing dextrose to confirm a negative reaction in this case.

Similarly, certain mineral oils could be too acidic and lead to an erroneous result that needs verification.

Further Reading

Undertaking further reading can be necessary to form real interpretation of these results and form assumptions that can be verified by the best.

Your results and how they affect your hypothesis can require some further reading to truly understand what your results mean. More so, this can require spectrometry rather than the OF test to get the real results worth testing.

If you want to learn more about these tests from the experts, consider consulting these texts listed below:

  • Cappuccino J.G. and Sherman N.  2008.  Microbiology: A Laboratory Manual, 8th ed. Pearson Benjamin Cummings, San Francisco, CA, USA
  • Murray, P.R., E.J. Baron, J.H. Jorgensen, M.L. Landry, and M.A. Pfaller. 2007. Manual of Clinical Microbiology. 9th ed. ASM Press, Washington, D.C.

Final Thoughts

By following these directions you should be able to find out if a bacteria is oxidative or if the bacteria is fermentative.

These results are still subject to interpretation, but following the interpretations listed you should be able to identify at least a negative or positive result. 

Although, how you use these results will completely depend upon your own use of these bacteria and the reasons why you want to know if the oxidative or fermentative.

Many fermenters use this test often to find new bacteria to ferment. The bacteria that is not fermentative can be tested for many other things as well and can lead to discovery

We hope this guide to the OF test has helped you find the results you need.

Jennifer Dawkins

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