Enterobacter aerogenes and other species of Enterobacteriaceae are easy to identify using the API-20E test kit. This kit, manufactured by bioMeriuex Inc., includes 20 mini-tubes or wells for the test. The test consists of inoculating a pure bacterial suspension into the test wells and reading the color reactions that occur. Identification relies on converting the results of these color reactions into a seven-digit code known as the Analytical Profile Index. Matching this code with the manufacturer’s database provides an identification of the organism.
- Enterobacter aerogenes pure culture
- Ampule containing 5 ml of sodium chloride 0.85 percent medium
- Incubation box
- Demineralized or distilled water
- Mineral oil
- Touch-tone telephone
Isolate the organism you wish to identify on a culture medium specific for Enterobacteriaceae. Use the API 20E test kit only with pure cultures and not directly on mixed-culture specimens.
Open an ampule containing 5 ml of sodium chloride 0.85 percent medium. Remove a single colony from an 18- to 24-hour-old isolation plate with a pipette, and add it into the ampule. Mix to obtain a homogenous suspension. Use this suspension as soon as you finish preparing it.
Set up an incubation box that consists of honeycombed wells in a tray fitted with a lid. Open the box, and add 5 ml of demineralized or distilled water into the tray wells. Unpack the strip provided in the API 20E test kit, and place it into the incubation box.
Fill the tube and the cupule of the wells marked "GEL," "VP" and "CIT" with the suspension you have prepared. In the remaining wells, fill the bacterial suspension into the tube but not the cupule. Add mineral oil into the wells marked "ADH," "ODC," "LDC," "URE" and "H2S" to create an anaerobic environment.
Cover the incubation box with the lid, and incubate at a temperature between 34 and 38 degrees Celsius for 18 to 24 hours.
Observe the wells, and note the reactions on the results sheet. Provided a minimum of three tests are positive, proceed to the color reactions using the chemical test solutions, or reagents, that came with the kit. Add one drop of TDA reagent into the well marked "TDA" and a drop each of VP1 and VP2 reagents into well marked "VP." If fewer than three tests came out positive, incubate the strips for another 24 hours before adding these reagents.
Observe the color changes produced in the wells. A reddish brown color in TDA indicates a positive reaction. Wait for 10 minutes after adding reagents from the kit into VP1 and VP2. Consider a red or pink color as positive and a pale pink color appearing after 10 minutes as negative.
Add a single drop of JAMES reagent from the test kit to the well marked "IND." Consider a pinkish color in the entire cupule as a positive reaction.
Perform the oxidase test as specified by the kit manufacturer, and record its results along with the results of the previous 20 tests.
Use the reading table provided with the test kit to allocate values for the reactions observed in each of the test wells. Add these values as prescribed to obtain the seven-digit profile number.
Feed this seven-digit profile number into the kit manufacturer’s database through a touch-tone telephone. Wait to receive the identification provided by the database. If the profile number does not provide specific results, perform the supplementary tests specified by the kit manufacturer.