Oxidation-Fermentation (OF) Test (or Hugh-Leifson Test)
Oxidation-Fermentation (OF) Test (or Hugh-Leifson Test)
The Oxidation-Fermentation (OF) Test tests the metabolism of sugar by prokaryotic cells. Cells may metabolize sugar in a variety of pathways, and the OF Test is studying whether sugar is metabolized by aerobic respiration or by an anaerobic pathway including fermentation. The OF medium has a low agar and peptone content, and a high sugar content, making it a semi-solid medium that is unlikely to go alkaline from protein utilization if the sugars are metabolized. Sugar fermentation produces fairly strong acids while aerobic respiration produces only weak acids, either of which will turn the indicator, Bromthymol Blue, yellow. Bromthymol Blue is green around a neutral pH and blue at an alkaline pH (>7.5).
Two tubes are stab inoculated with an organism, then one tube is covered with mineral oil, creating an anaerobic environment to study fermentation. The other tube is left uncovered with oil, creating an aerobic environment to study aerobic respiration. Organisms that do not utilize the sugar in both tubes will leave the media blue or green (non-saccharolytic, non-sugar user). Organisms that only engage in aerobic respiration will turn the media yellow in the top of the uncovered (aerobic) tube while the covered tube remains blue or green.
Organisms that only slowly ferment the sugars will be slightly yellow at the top of both tubes. Organisms that slowly ferment the sugars and aerobically respire the sugars will also be slightly yellow at the top of both tubes. Organisms that strongly ferment the sugars will be yellow throughout both tubes. Organisms that both stronly ferment and aerobically respire the sugars will also be yellow throughout both tubes. Obviously, this test can only differentiate organisms into three or four groups:
- Non-sugar metabolisers
- Aerobically respiring organisms
- Organisms that either aerobically respire and ferment or ferment only
- And organisms that either aerobically respire and slowly ferment or slow fermenters only
Aim of Test: To differentiate bacteria species on their ability of aerobically respire or ferment sugar and to determine motility. This test allows for the differentiation of the fermenting Enterobacteriaceae from the aerobically respiring Pseudomonas and Bordetella and from the non-sugar using Alcaligenes and Moraxella
Procedure:
- Obtain two tubes for each strain being tested (your instructor will inform you of which strains to test).
- Label the tubes, including your name, the date, and the test name.
- Do NOT use your Wire loop, as this will soon ruin it. Use your inoculating needle for this test.
- If assaying for motility, care must be taken with the stab. Be certain that your needle is straight and that your stab is straight down and then pulled up exactly the way it went down.
- Aseptically transfer some bacteria to the inoculating needle. This is done by flaming your needle, COOLING IT WELL, picking up bacteria on the needle by rolling the tip in a colony of bacteria (oe scaping it). Sometimes students get no growth in this test from just briefly cooling the tip of the needle: the heat from above the tip moves down the needle and kills the bacteria. So coool it well.
- Stab the needle straight down into the tube going about 2/3 of the way down and then pulling the needle straight out the way you came in.
- A poor stab will make the motility reading difficult, so repeat the test if needed.
- Repeat this procedure for the second tube of the set.
- Overlay one tube of each set with a thin layer of sterile mineral oil (about 4 mm).
- Remind your instructor to set up a control tube (an uninoculated control). Gently place your tubes in the rack where instructed. Rough handling may distort the test.
- Incubate the tubes at room temperature for about 5 days (or at 35-37 C for 2 days).
Results:
- Examine the tubes for color changes, noting where the color has changed. If tubes have no visible growth and have not changed color or the organism is slow growing, additional incubation time may be required.
- Look for motility as in the motility test.
- Check your results against the table below.
Note to the Instructor:
Although at first glance this test looks pretty interesting, it actually adds little but confirmation to the knowledge obtained from the Utilization of Glucose Test and the Thioglycollate Test. If given a choice between the two, the latter tests are recommended instead of the OF Test.
Although at first glance this test looks pretty interesting, it actually adds little but confirmation to the knowledge obtained from the Utilization of Glucose Test and the Thioglycollate Test. If given a choice between the two, the latter tests are recommended instead of the OF Test.
Post a Comment