Carbohydrate Fermentation Media
Most bacteria can catabolize carbohydrates using either an oxidative or fementative metabolic pathway or both. The oxidative breakdown of carbohydrates requires oxygen to complete the catabolism while fermentative pathways does not require oxygen and can occur in some bacteria only in the absence of oxygen and in other both in the presence and absence of oxygen.
OF Glucose medium is useful to screen for these two basic abilities: oxidative only catabolism or the presence of a fermentative pathway. (The original paper: Rudolph Hugh and Einar Leifson. 1953. "The Taxonomic Significance of Fermentative versus Oxidative Metabolism of Carbohydrates by Various Gram Negative Bacteria." J. Bacteriol. 66 (1): 24-26.)
OF Glucose contains a high concentration of carbohydrate and a small amount of peptone to support the growth of non oxidative, nonfermentative bacteria.
Observe and record the color and texture of an uninoculated OF Glucose deep.
Inoculate 2 tubes (one with a cotton plug or loose cap and one with a screw cap)for each organism you are testing.
Aseptically transfer your isolate to each of the tubes and "stab" the agar with your loop.
Incubate the tubes for the appropriate time.
Observe the OF Glucose medium. The bromthymol blue indicator in the medium will turn yellow if acids are produced indicating the carbohydtrates have been digested. Alkaline by products from the metabolism of the peptone turn the media blue. If both tubes are yellow the organism can ferment carbohydrates.
Sugar Fermentation Media General Recipe for Carbohydrate fermentation media: Purple broth base (1% peptone, 0.1% beef extract, 0.5% NaCl, 20 µg/ml bromcresol purple) plus 1% of the desired carbohydrate.
Commonly Used Fermentation Media
Inoculate one tube for each organism: adonitol, dulcitol, glucose, inositol, lactose, mannitol, and sucrose. Incubate for 24 to 72 hours at 35C. The fermentation of carbohydrates by Gram negative organisms gives rise to the production of pyruvic acid causing a change in color of the media from purple to yellow due to the reduction in pH.
A color change to yellow in the fermentation media indicates a positive test (+).
In positive tests only, gas production is evidenced by gas bubbles trapped in the smaller inner tube (Durham tube). If gas is present the result is recorded as +/gas.
A negative test (-) is evidenced by a purple to gray color and obvious growth* in the tube.
Example, the lactose fermentation test contains 1% lactose dissolved in the purple broth base.
Phenol Red Agar with sugars mannitol or sucrose: 1% mannitol(or sucrose) is added to phenol red agar base (1% peptone, 0.1% beef extract, 0.5% NaCl, 1.5% agar and 25µl/ml phenol red) to test for the fermentation of mannitol by the bacteria.
A positive test is a yellow color due to the formation of acidic products of mannitol fermentation. The indicator, phenol red, turns yellow when the pH falls below 6.8. Staphylococcus aureus usually ferments mannitol within 24 to 48 hours.
An inoculum should be spread on the slant and then stabbed to the bottom of the butt, a region that remains fairly anaerobic. The inoculated slants should then be incubated at 37°C for 24-48 hours. Yellow color indicates a positive (+) test.
Links to Labs in the Soil Microbes Project