Gelatin Hydrolysis Test

 

What is the purpose of the test? 

 

The purpose is to see if the microbe can use the protein gelatin as a source of carbon and energy for growth. Use of gelatin is accomplished by the enzyme gelatinase.

 

 

How is gelatinase activity determined? 

 

A medium containing gelatin is used. We have all seen how gelatin forms a semisolid substance. In the medium it serves as the solidifying agent (substituting in place of agar) in addition to its role as a source for carbon and energy. When gelatin is used, the medium changes from semisolid to liquid and cannot be resolidified.

 

 

What medium is used?  

 

The medium used is nutrient gelatin. The medium is a nutrient broth to which 12% gelatin is added, converting it into a semisolid medium.

 

 

How is the test performed?  

 

An inoculum from a pure culture is transferred aseptically to a sterile tube of nutrient gelatin. The inoculated tube is incubated at 35-37 C for 24 hours and the results are determined. Because even undigested gelatin becomes liquid at temperatures modestly above the incubation temperature, it is important to determine whether the gelatin has actually been digested by gelatinase. This is accomplished by placing the tube into a refrigerator for a few minutes, exposing it to temperatures that cause undigested gelatin to resolidify. If the gelatin has been digested, the medium in the tube will fail to solidify after refrigeration. If gelatinase is present, the liquid medium will fail to solidify upon refrigeration

 

 

What reagents are added?

 

None.

 

 

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

 

Inoculation of Medium

 

1. Select the nutrient gelatin medium.  

2. Start your Bunsen burner.  

3. Select the inoculating wire tool.  

4. Flame your inoculating wire to sterilize it.  

5. Remove the caps from your test tubes.

6. Flame the mouths of your test tubes.  

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.  

9. Flame the mouths of your tubes once again.  

10. Replace the caps on the test tubes.  

11. Re-flame the inoculating tool.  

 

Incubation of the Inoculated Medium

 

12. Place the inoculated tube 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. Drag and drop the tube in the refrigerator.  

16. Remove the tube from the refrigerator.  This will initiate a video that will demonstrate whether the cooled culture was able to resolidify or not and thus will provide the information needed to determine the result of the test.  If it has solidified, it will not flow when the tube is tilted.  This is an indication of a negative test.  If the medium flows when the tube is tilted, the gelatin has been digested.  This is a positive result for the presence of the enzyme gelatinase.  

17. Record test result.  

18. Dispose of the culture.