General Microbiology
Laboratory
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Among the many enzymes that bacteria may
produce are exoenzymes (those that are
excreted) used to degrade large polymers
into smaller compounds.
For example, starch digestion results from
the action of amylase released into the
surrounding medium.
The starch is a polysaccharide that cannot
pass across the cell membrane.
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Amylase breaks starch into smaller sugar
residues that can enter the cell and be
processed by respiration or fermentation.
Gelatinase is another exoenzyme. It can
cause the liquefaction of media solidified by
gelatin (rather than agar).
Caseinase is an enzyme that hydrolyzes
casein, the major protein component in milk.
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Lipase production is common to bacteria that
grow in foods rich in fats such as butter and
mayonnaise.
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This enzyme breaks fats into its components
glycerol and fatty acids.
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Agar which contains lipids prepared from egg
yolks is used in identifying lipolytic activity.
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Most enzymes are endoenzymes.
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They are produced in the cell and catalyze
intracellular reactions.
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Among the kinds of reactions that are used as
evidence in identification of unknown
bacteria are:
a) The breakdown of toxic wastes such as
hydrogen peroxide or urea.
b) The reduction of nitrate or oxygen.
c) The degradation of specific amino acids.
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Amylase activity is demonstrated using
starch agar, a medium containing starch as
the carbohydrate source.
Amylase hydrolyzes starch. Starch is a
polysaccharide--a long chain of glucose
molecules linked by glycosidic bonds.
Amylase breaks the glycosidic bonds,
producing small oligosaccharides and free
glucose.
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Amylase production is tested by growing
organisms on starch agar.
After incubation, the starch agar is flooded
with Gram's iodine.
The iodine reacts with starch to produce a
dark purple or brown color.
If amylase is present, clear zones will appear
in the starch agar where hydrolysis has
occurred.
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POSITIVE CONTROL:
Bacillus subtilits
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NEGATIVE CONTROL:
E.coli
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Streak each 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.
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Gelatin Liquefaction
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Gelatin is liquefied by the virtue of the
production of an enzyme called gelatinase.
Gelatin is an incomplete protein; it lacks
tryptophan.
However, the ability to hydrolyze gelatin is a
well established bacterial classification
characteristic.
Gelatin is produced by the hydrolysis of
collagen, a large protein found in the
connective tissues of animals.
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Normally, gelatin produces a gel in water
below 25°C.
When gelatin is hydrolyzed, it loses its ability
to form a gel.
In this experiment, you will use nutrient
gelatin in place of nutrient agar.
If a given microbe produces gelatinase, the
nutrient gelatin will liquefy.
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Gelatin
polypeptide
Amino acid “Liquid”
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This test is used to differntiate Gram- egative
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.
The gelatin stab method employs nutrient
gelatin deep tubes that contain 12% gelatin.
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1Inoculate
gelatin
deeps
using
bacteriological needle for up to 30 days.
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2- 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.
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Positive: Strong: Liquefaction occurs within 3
days.
Positive: weak: Liquefaction occurs in 4-30
days.
Negative: No liquefaction after 30 days.
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Figure (1): Result of
Gelatin Liquefaction:
The tubes to the right
depict a gelatinase
negative (A) and
elatinase positive (B
and C) reactions.
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Good
Luck
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