Phenylalanine Deaminase Test


What is the purpose of the test? 


This test determines whether the microbe produces the enzyme phenylalanine deaminase, which is needed for it to use the amino acid phenylalanine as a carbon and energy source for growth.



How is presence of phenylalanine deaminase determined? 


If phenylalanine deaminase is present, a degradation product is produced from phenylalanine. The product combines with iron compounds in an acidic environment to produce a green color.



What medium is used? 


The medium used is phenylalanine agar, a nutrient medium enriched with 0.2% phenylalanine. It is allowed to solidify at a slant.



How is the test performed? 


An inoculum from a pure culture is transferred aseptically to a sterile tube of phenylalanine agar to streak the slant. The butt of the tube need not be stabbed. The inoculated tube is incubated at 35-37 C for 24 hours. Then, reagents must be added to allow results to be determined.



What reagents are added? 


After incubation, five drops of 10% ferric chloride and five drops of 0.1N HCl are added and the tube is gently shaken. A positive result is indicated if a green color develops within five minutes.


To perform this test in VirtualUnknown™ Microbiology, complete the following steps:


Inoculation of Medium


1. Select the phenylalanine agar slant medium.  

2. Start your Bunsen burner.  

3. Select the inoculating loop tool.  Flame your inoculating loop to sterilize it.  

4. Remove the caps from your test tubes.  Flame the mouths of your test tubes.  

5. Use the sterile inoculating tool to pick up an inoculum from the culture tube of the unknown bacterium.  Immediately transfer the inoculum into the fresh, sterile medium.  

6. Flame the mouths of your tubes once again.

7. Replace the caps on the test tubes.  

8. Re-flame the inoculating tool.  


Incubation of the Inoculated Medium


9. Place the inoculated tube into the 35-37 C incubator.  

10. Press the New Day button to move forward 24 hours. Incubate for the appropriate length of time. This test is properly interpreted after 24 hours.  

11. Retrieve desired incubated culture from the incubator.  


Addition of Reagents


12. Select the dropper tool and the appropriate reagent needed from the chemical shelf. For this test, you will use two reagents: 10% ferric chloride solution and 0.1N HCl.  Select 10% ferric chloride reagent from the reagent list.

13. Remove the cap from the tube. Place the end of the dropper into the tube and add the 10% ferric chloride reagent to the sample.

14. Select the 0.1N HCl reagent from the reagent list.   Place the end of the dropper into the tube and add the 0.1N HCl reagent to the sample.  


Determination of Test Results


15. Observe test result. For this test, the reagent turns the liquid below the slant green when giving positive results. If negative, no color change is observed.  

16. Record test result. Dispose of the culture.