Starch Hydrolysis

 

What is the purpose of the test? 

 

The purpose is to see if the microbe can use starch, a complex carbohydrate made from glucose, as a source of carbon and energy for growth. Use of starch is accomplished by an enzyme called alpha-amylase.

 

 

How is alpha-amylase activity determined? 

 

A medium containing starch is used. After inoculation and overnight incubation, iodine reagent is added to detect the presence of starch. Iodine reagent complexes with starch to form a blue-black color in the culture medium. Clear halos surrounding colonies is indicative of their ability to digest the starch in the medium due to the presence of alpha-amylase.

 

 

What medium is used?  

 

The medium used is starch agar. The medium is a nutrient agar to which starch is added.

 

 

How is the test performed?  

 

An inoculum from a pure culture is streaked on a sterile plate of starch agar The inoculated plate is incubated at 35-37 C for 24 hours. Iodine reagent is then added to flood the growth. Presence of clear halos surrounding colonies is positive for their ability to digest the starch and thus indicates presence of alpha-amylase.

 

 

What reagents are added?

 

Iodine reagent is added after incubation to flood the surface of the plate.

 

 

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

 

Inoculation of Medium

 

1. Select the starch agar medium.  

2. Start your Bunsen burner.  

3. Select the inoculating loop tool.  

4. Flame your inoculating loop to sterilize it.  

5. Remove the cap from your inoculum and your lid from your sample plate.  

6. Flame the mouth of your inoculum tube.  

7. Use the sterile inoculating tool to pick up an inoculum from the culture tube of the unknown bacterium.  

8. Immediately transfer the inoculum into the fresh, sterile medium. NOTE: you must streak the inoculum back and forth across the plate for several seconds to inoculate the plate. You will know it has been successfully done when streak marks appear on the agar surface.  

9. Flame the mouth of your inoculum tube once again.

10. Replace the cap on the inoculum tube and the plate lid.  

11. Re-flame the inoculating tool.  

 

Incubation of the Inoculated Medium

 

12. Place the inoculated plate into the 35-37 C incubator.  

13. Press the New Day button to move forward 24 hours.  

 

Determination of Test Results

 

14. Incubate for the appropriate length of time. For this test, 24 hours is sufficient for determining the result.  

15. Retrieve desired incubated culture from the incubator.  

 

Addition of Reagents

 

16. Select the dropper tool and the appropriate reagent needed from the chemical shelf. For this test, select Iodine Reagent  

17. Remove the lid from the plate  

18. Place the dropper above the plate and add the reagent to the culture. The starch in the plate is changed to blue-brown by the iodine reagent. Areas where starch has been digested by bacterial growth exhibit clear halos in the midst of the dark plate, indicating a positive alpha-amylase, or starch hydrolysis test. Plates containing bacteria without alpha-amylase are uniformly dark, a negative result.  

19. Record test result.  

20. Dispose of the culture.