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General Microbiology Laboratory
Biochemical Tests
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.
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.
Starch Hydrolysis Test
Starch agar before inoculation
Starch Hydrolysis Test
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.
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.
Gelatin Liquefaction
Gelatin is a simple protein. When in solution, will be a solid
at a temperature of 25oC or lower.
Gelatin will be a liquid at a temperature of 26oC or
 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.
Inoculate gelatin deeps and incubate for up 30
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
Gelatin Liquefaction
Positive strong: liquefaction occurs within
Positive week: liquefaction occurs in 4-30
Negative :No liquefaction after 30 days.
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
 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
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.
End of lecture