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 VUMIE Online© Online Lab , complete the following steps:
Inoculation of Medium
1. Select the starch agar medium.
2. Using an inoculating loop, complete the process of a tube-to-plate aseptic transfer to inoculate the medium. Forgotten how to do these things? Watch the "Show Me How To" videos.
Incubation of the Inoculated Medium
3. Place the inoculated tube into the 35-37 C incubator.
4. Press the New Day button to move forward 24 hours. Forgotten how to do these things? Watch the "Show Me How To" videos.
Addition of Reagents
5. Incubate this test for only 24 hours.
6. Retrieve the incubated culture from the incubator.
7. This is a test that requires addition of reagents to bring about the change in appearance of a positive test.
Select the dropper tool and the appropriate reagent needed from the chemical shelf. For this test, select Iodine. Remove the plate lid and add the reagent.
Determination of Test Results
8. 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.
9. Record test result.