2. General Microbiology Laboratory Biochemical Tests

3. Amylase Production (Starch Hydrolysis Test) Amylase Production (Starch Hydrolysis Test)  Starch agar is a differential medium that tests the ability of an organism to produce certain exoenzymes, including α-amylase and oligo-1,6-glucosidase, that hydrolyze starch.  Starch molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade starch into subunits that can then be utilized by the organism.  Starch agar is a simple nutritive medium with starch added. Since no color change occurs in the medium when organisms hydrolyze starch, we add iodine to the plate after incubation. Iodine turns blue, purple, or black (depending on the concentration of iodine) in the presence of starch.  A clearing around the bacterial growth indicates that the organism has hydrolyzed starch.

4. Procedures  Streak the test organism across a small portion of the agar surface.  Incubate at 37 oC for 48 hours.  Cover the surface with iodine. Rotate to distribute the iodine into a thin layer. Do not flood the plate.  Iodine will turn blue when it reacts with starch. A clear zone will be seen where starch has been digested.

5. Starch Hydrolysis Test Starch agar before inoculation

6. Starch Hydrolysis Test

7. Bacillus subtilis Bacillus subtilis colony on a culture medium containing starch. The culture plate has been flooded with a weak iodine solution, which reveals a zone of clearing around the colony (arrow). This zone represents the area where the starch has been hydrolyzed so that it is no longer available to react with the iodine solution.

8. Gelatin Liquefaction  Determine an organism's ability to produce proteolytic-like enzymes and liquefy gelatin.  Differentiate between species in the genera Staphylococcus and Clostridium as well as aid in the identification of other species and genera.  This test is used to differentiate Gram-negative species. Serratia, Pseudomonas, and Vibrio are positive for this test. The practicality of this test was not appreciated until, the development of the rapid procedures.

9. Gelatin Liquefaction  Gelatin is a simple protein. When in solution, will be a solid at a temperature of 25 o C or lower.  Gelatin will be a liquid at a temperature of 26 o C or higher.  Some microorganisms can liquefy gelatin by producing the extracellular enzyme called “Gelatinase”.  When bacteria that produce the enzyme gelatinase are grown in a gelatin medium, the enzyme breaks up the gelatin molecule and the medium cannot solidify even at cold temperatures.

10. Procedures  Inoculate gelatin deeps and incubate for up 30 days.  To determine whether liquefaction has occurred place the tube in the refrigerator for 30 minutes  Remove and check the tube for liquefaction. if negative, continue incubation until liquefaction occur.

11. Result

12. Gelatin Liquefaction  Positive strong: liquefaction occurs within 3days.  Positive week: liquefaction occurs in 4-30 days.  Negative :No liquefaction after 30 days.

13. X-ray film  An alternative method for detecting gelatinase production is the use of X-ray film that is coated with a green gelatin emulsion.  Organisms that produce gelatinase remove the emulsion from the strip.  Inoculate each of the two cultures into a separate tube of 0.5 ml saline. The suspension should be very turbid.  Insert a strip of the X-ray or gelatin film into each saline suspension.  Incubate the tubes at 35°C. Observe at 1, 2, 3, 4, and 24 hours for removal of the gelatin emulsion from the strip with subsequent appearance of the transparent strip support

14. Result Gelatin strip test. The organism on the left does not hydrolyze gelatin and, therefore, no clearing of the gelatin film is seen. On the right, the portion of the strip submersed in the organism suspension has cleared, indicating gelatin hydrolysis.

15. End of lecture