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A large proportion of disease causing Gram negative bacterial genera belong to the oxidase negative family Enterobacteriaceae (the “enterics”). Many microbiological media/methods exist for the characterization and identification of enteric organisms. The first that we will cover is the differential utilization of citrate (citric acid). Citrate is a carbohydrate which can be utilized as a carbon and energy source by certain enteric genera but not by others. Although the name "enteric" is derived from the fact that these organisms inhabit animal intestines, some enteric genera (see the “aerogenese-group” in the discussion of EMB agar in exercise #10) are found in non-clinical environments as well. These genera are capable of citrate utilization, and include Citrobacter, Enterobacter and Klebsiella. Salmonella is also citrate positive eventhough it is an enteric genus not belonging to the aerogenese-group. Three of our Gram negative non-fermentors (Pseudomonas, Alcaligenes and Acinetobacter) are also citrate positive. Those enteric species found only in animal feces are not capable of citrate utilization. These are referred to as the "coli group" and include the following genera: Escherichia, Shigella, Proteus and our 4th Gram negative non-fermentor, Aeromonas.

Simmons citrate agar media contains the pH indicator bromthymol blue, a chemical that is green at neutral pH, and blue at alkaline pH. Organisms capable of citrate utilization will cause the pH of Simmons citrate agar to increase by virtue of the consumption of citric acid (the base NH4OH is generated from NH4H2PO4 during the metabolic process). Any color change from green to blue is considered positive for citrate utilization. To conduct the test, inoculate a Simmons citrate agar slant using your loop. Incubate the slant at 37oC for 24-48hrs. and examine for any color change. SEE IMAGE: Simmons citrate: left negative, right positive.

NOTES:

* We will only use this test to differentiate Gram negative rods.

* Most carbohydrate utilization media will become acidic as the organism produced acid from sugar

utilization. Simmons Citrate agar is different. Consumption of the citrate, which is an acid, will result

on the media becoming alkaline (basic) resulting in the color change from green to blue.

* Any blue color anywhere on the slant constitutes a positive reaction here.

O/F Glucose deeps

O/F glucose is a semisolid medium incorporating bromthymol blue as a pH indicator, 1 % glucose, and the protein peptone. Here O stands for “oxidative” metabolism of glucose, and F stands for “fermentative” metabolism of glucose. Despite the intended meaning of these terms, the O/F glucose test is actually an oxygen requirement test as it relates to glucose metabolism. That being said, we could take O to mean obligately aerobic, and F to mean facultative. These are the only 2 options as none of our Gram negative rods are aerotolerant anaerobes.

As discussed in the citrate lab above, bromthymol blue has a green color at neutral pH. Bromthymol blue turns yellow at an acidic pH. Two O/F glucose tubes are inoculated. One of the tubes is then overlayed with sterile mineral oil which prevents oxygen from diffusing into the medium, and renders this tube anaerobic.  Microbial activity in this tube (the F tube) must be in the form of fermentation. Fermentation carried out by a facultative organism will result in the production of a large amount of acid, causing the F tube to turn yellow. With no oil covering the other tube (the O tube), oxygen will diffuse into the top half of the media allowing aerobic respiration to occur. Carbon dioxide produced from respiration will dissolve in water in the media resulting in the formation of carbonic acid, which will cause the O tube to turn yellow. Facultatives will cause both tubes to turn yellow, which is interpreted as an F result (facultative glucose catabolism). Obligately aerobic organisms will cause the O tube to turn yellow, with no reaction in the F tube. This is interpreted as an O result (aerobic glucose catabolism).

We may also see blue coloration, usually at the top of the O tube, due to utilization of the peptone resulting in accumulation of a basic ammonia by-product. The other possible result is “no reaction,” which is no color change in either tube.

Results: SEE IMAGES: O/F glucose: F reaction O reaction no reaction

 MATERIALS NEEDED: 

2 O/F glucose tubes per organism being run

sterile mineral oil

1. Inoculate both tubes of media with a NEEDLE, stabbing through the medium to the bottom as you did for motility tests.

2. Overly 1 tube with ¼ to ½ inch of sterile mineral oil.

3. Incubate both tubes at 37 degrees C for 24-48hrs.

4. Examine tubes for color changes.

Expected results:

NOTES:

* We will only use this test to differentiate Gram negative rods.

* A common mistake on this medium is to apply oil incorrectly. Remember that you should place oil on top of only 1 deep. Place no less than ¼” of oil on the deep.