O-F GLUCOSE MEDIA
Determination of oxidative and fermentative carbohydrate metabolism
O-F glucose is a semisolid medium incorporating a pH indicator, 1 % glucose, and
peptones. The type of metabolism a bacterium has---aerobic use of a sugar vs.
anaerobic use of a sugar---determines the color results in the 2 tubes used. The
overlay of oil or vaspar wax prevents oxygen from getting into the medium of 1
tube: the other tube has air inside. Aerobic use of sugar is oxidative metabolism,
anaerobic use is fermentative. Bacteria that oxidize carbs are usually obligate aerobes,
whereas those that ferment are usually facultative anaerobes. The fermentation will
produce far more acid than the aerobic oxidation, but the O/F glucose test is very
sensitive to the small amounts of acid produced via aerobic means.
As the organism grows and uses the sugar, the resulting acid will change the pH, and
the pH indicator will change colors. The indicator is brom thymol blue, starting as a
forest green color at a neutral pH, and ending as a yellow with low pH and a blue with
high pH. Using 2 tubes allows the identification of whether the organism is oxidative
(only open tube acidic) or fermentative (both tubes acidic). If the organism uses the
peptones rather than the glucose, the pH will become basic
OBJECTIVES:
Determine whether a bacterium is oxidatively or fermentatively using glucose.
MATERIALS NEEDED: per table
2 O-F glucose tubes per organism being run
sterile mineral oil (or sterile petrolatum or Vaspar)
INSTRUCTOR cultures: an oxidizing bacterium, a fermenting bacterium, and an
inert/nonuser (probably Pseudomonas, E. coli, and Alkaligenes fecalis .
THE PROCEDURE:
1. Inoculate the medium by taking an inoculum with a NEEDLE, and stabbing
through the medium to about 1/4 inch of the bottom of each of 2 tubes. Repeat
this with the other tube.
2. Overlay 1 tube with a half-inch of sterile mineral oil
3. Incubate at 25 or 37 C for a couple of days (might want to keep questionable +
reactions or negative reactions for 3-4 days)..
YOUR INSTRUCTOR WILL INOCULATE 3 demo bacteria for the entire class:
Pseudomonas, E. coli, and Alkaligenes fecalis .
INTERPRETATION:
If you get a NEGATIVE reaction with no acid in either tube, it may be a good idea to
confirm it by running another glucose test using sugar discs or phenol red
glucose. However, this is a more sensitive glucose test than phenol red since there is
less peptone in the medium (can counteract the acidity of the glucose use) and the bro
thymold indicator is more sensitive to small amounts of acidity than phenol red indicator.
Those bacteria which use the peptides rather than the sugar will produce basic end
products, causing the brom thymol blue to become a blue-green or blue. Although this
is a biochemical reaction, it is of no consequence in the reading of this test.
REACTION
TUBE W/O OIL
WITH OIL OVERLAY
oxidation
Yellow - top tube only
No yellow
blue or green
fermentation
entire tube yellow
entire tube yellow
green
inert
entire tube
entire tube
blue or green
NEGATIVE
QUESTIONS:
1. The pH indicator in this medium is:
2. Why might you get a blue color rather than a yellow color reaction?
3. You are identifying the ability of the microbe to utilize the sugar _____.
Fall 2011 - Jackie Reynolds, Richland College, BIOL 2421