Fermentation of Simple Carbohydrates
When a microorganism is able to ferment glucose, as determined by an OF-glucose test, additional testing can be done to determine the extent of the organism's metabolic flexibility. Many microbes can ferment a wide variety of sugars and other microorganisms are more limited in how they can acquire energy from sugars. Common sugars that some bacteria can ferment and others cannot include: adonitol, dulcitol, glucose, lactose, mannitol, and sucrose.
The General Recipe for Carbohydrate Fermentation Media is:
Purple broth base (1% peptone, 0.1% beef extract, 0.5% NaCl, 20 µg/ml bromcresol purple)
plus 1% of the desired carbohydrate that you would like to test.
In BISC209, we will be able to provide you with some, but not all, of those sugars (listed in the first paragraph) in fermentation tubes. Please check with your instructor to see if your plan for carbohydrate fermentation testing is feasible.
PROCEDURE FOR INOCULATING CARBOHYDRATE FERMENTATION TUBES
Inoculate one tube of available carbohydrate media for each organism:
Use your inoculation loop to aseptically transfer a tiny amount of growth from a stock slant or rrom an isolated pure colony.
Mix gently, being careful not to strike the inverted Durham tube (the small glass tube that is sitting upside down in the purple broth base tube).
Incubate for 24 to 72 hrs (or longer, if appropriate).
After incubation is complete, compare an inoculated tube to an uninoculated tube.
A color change to yellow in the fermentation media indicates a positive test (+). The basis of this change is that fermentation of carbohydrates 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 below 6.8.
In positive tests only, gas production, as part of the fermentation process, is detected by gas bubbles trapped in the smaller inner tube (Durham tube). If gas is present the result is recorded as +/gas.
A negative test carbohydrate fermentation test (-) is determined by a purple to gray color in the media despite obvious microbial growth in the tube. Gas production is not assessed in negatives.
When a microbe is Negative for acid production from sugars (as tested here), you might want to test for non-acid products, such as acetoin. In that case, you would use the methyl red and Voges-Proskauer tests (MRVP) in Enzyme tests .
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