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.
Procedure
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.
A Bacillus subtilis is on the left. Escherichia coli is on the right.
B After iodine has been added, the clearing in the medium around B.
subtilis demonstrates a positive result for starch hydrolysis. E. coli, with no
clearing, is negative.
Gelatin Liquefaction
Determine an organism's ability to produce proteolytic-like
enzymes and liquefy gelatin.
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.
Staphylococcus aureus, which is gelatinase positive can be
differentiated from S. epidermidis(gelatinase-negative).
Bacillus anthracis, B. cereus, and several other members of
the genus are Gelatinase positive, as are Clostridium tetani
and C. perfringens.
Gelatin is a protein derived from collagen a
component of vertebrate connective tissue.
Gelatinase comprise a family of extracellular
enzymes produced and secreted by some
microorganisms to hydrolyze gelatin
Gelatin is a simple protein.
 will be a solid at a temperature of 25oC or lower.
 will be a liquid at a temperature of 26oC or higher.
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.
Two methods used to determine gelatinase production
1) The Gelatin stab method.
2) The Gelatin strip method.
The Gelatin Stab Method
1) Inoculate nutrient gelatin deep tubes (contain 12% gelatin)
and incubate for up 30 days.
2) To determine whether liquefaction has occurred place the
tube in the refrigerator for 30 minutes
3) Remove and check the tube for liquefaction. if negative,
continue incubation until liquefaction occur.
If the gelatin is still intact (the bacteria did not produce
gelatinase), the media will solidify in the refrigerator and a
negative test result is recorded.
If the organism has produced sufficient gelatinase, the tube will
remain liquid (at least partially) and not solidify in the
refrigerator. A positive test result is recorded.
Some organisms may produce gelatinase in rather small
quantities. Thus, a tube with a negative gelatinase result should
be reincubated for 30dys. Whenever desired, the tube may be
refrigerated and results observed. If the tube is still negative
after 30days of incubation (completely solidifies when
refrigerated), it can be reasonably concluded that this organism
does not produce gelatinase.
The 'Serratia marcescens' on the left is positive for gelatinase
production, as evidenced by the liquidation of the media.
The 'Salmonella typhimurium' on the right is negative, as
evidenced by the solidity of the media.
Results
Positive strong:
liquefaction occurs with 3days
Positive weak :
liquefaction occurs in 3-30 days
Negative :
No liquefaction after 30 days
The Gelatin Strip Method
An alternative method for detecting gelatinase production is
the use of X-ray film(Gelatin Strip) that is coated with a green
gelatin emulsion.
Organisms that produce gelatinase remove the emulsion from
the strip.
1) Inoculate each of the two cultures into a separate tube of 0.5
ml saline. The suspension should be very turbid.
2) Insert a strip of the X-ray or gelatin film into each saline
suspension.
3) 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
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.