BIOCHEMICAL TESTS
PART ONE
Differentiation of organisms based on their ability to break down complex
Macromolecules in to simpler nutritional constituents
Protein
Starch
Fat
Macromolecules are polymers of monomeric subsunits.
Hydrolysis reactions cleave polymers into monomers by adding water.
Macromolecules and their (Monomeric Subunits)
Protein
Starch (Sugars)
(Amino Acids)
Fat (Triglycerides
And other lipids)
Muscles store sugar as glycogen.
Starch: a polymer of sugars
The hydrolysis reaction is the reverse of the dehydration reaction
STARCH TEST

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Differentiates bacteria based on their
ability to hydrolyze starch with the extra
cellular enzyme amylase
Starch (a polysaccharide) is to large to
pass through the bacterial cell wall; to
be of metabolic value to the bacteria,
starch must be split into smaller
fragments or individual glucose
molecules
Procedure:

Divide one starch plate into thirds and
inoculate with E.coli, Bacillus cereus,
and P.aeruginosa
E.coli
P.aeruginosa
B.cereus
Results:
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
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Starch and its sugar subunits are clear
in the medium.
Iodine is used to detect the presence
or absence of starch in the vicinity
around the bacteria
Iodine reacts with starch and
produces a blue-black color.
After addition of iodine to the plate
media, any microbial starch hydrolysis
by the exoenzyme amylase reveals a
clear zone around the bacterial growth
Lipid Hydrolysis Test


Used to identify bacteria capable of
producing the exoenzyme lipase
Triglycerides (a possible bacterial
carbon and energy source) are too large
to enter the bacterial cell; bacteria that
produce and secrete lipase hydrolyze
triglycerides into glycerol and 3 fatty
acid chains
Triglyceride Hydrolysis by Lipase
Procedure:

Divide one tributyrin agar plate into
thirds and inoculate with E.coli, Bacillus
cereus, and P.aeruginosa
E.coli
P.aeruginosa
B.cereus
Results:


Tributyrin agar plates
contain the triglyceride
tributyrin and are
initially opaque;
lipase-positive
organisms will exhibit a
clear zone around their
growth—tributyrin has
been hydrolyzed
Casein Hydrolysis Test



Identifies bacteria capable of
hydrolyzing casein (protein) with the
enzyme casease
Casein is the protein that gives milk it’s
color.
To be utilized by certain bacteria, casein
must be broken down in to its smaller
subunits, amino acids
Procedure:

Divide one skim milk agar plate into
thirds and inoculate with E.coli, Bacillus
cereus, and P.aeruginosa
E.coli
P.aeruginosa
B.cereus
Results:

Bacteria that secrete
the proteolytic
exoenzyme casease
hydrolyze milk
protein thus creating
a zone of clearing
around the bacterial
growth
Gelatin Hydrolysis



Test for the ability of an organism to produce
the exoenzyme gelatinase which digests and
liquefies gelatin
Gelatin is a protein derived from collagen, a
connective tissue found in vertebrates
Gelatin is too large to enter the bacterial cell;
however its amino acids my be used as an
energy source or built back up into bacterial
protein
Procedure:

3 Nutrient gelatin tubes are stab
inoculated with the following organisms
then incubated:
E. coli
B.cereus
P.aeruginosa
Results:


Since nutrient gelatin melts at 28°C, care
must be taken to distinguish between
organisms capable of producing gelatinase
and gelatin tubes that are affected by
incubator temperature
After incubation, tubes should be
refrigerated; tubes inoculated with gelatinase
positive organisms remain liquid after
refrigeration

After refrigeration, E.coli is
gelatinase negative and
P.aeruginosa is gleatinase
positive
Carbohydrate Fermentation
(Phenol Red Carbohydrate Broth)


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Differential test to detect the ability of
an organism to ferment various
carbohydrates
Phenol Red is the pH indicator (yellow
below 6.8 and red above 7.4)
A Durham tube (inverted small tube) is
used to trap any gas produced from the
fermentation of various sugars
Procedure:


Obtain and label the following tubes:
4 Phenol Red Glucose Broth Tubes
4 Phenol Red Sucrose Broth Tubes
4 Phenol Red Lactose Broth Tubes
Inoculate the above tubes with the
following organisms:
E.coli, P.vulgaris, A.faecalis, S.aureus
Results of Carbohydrate
Fermentation Tests:
TRIPLE SUGAR IRON TEST
(TSI)


Used to differentiate among the different
groups of Enterobacteriaceae
based on their ability to ferment glucose,
lactose and/or sucrose
Also differentiates between groups capable of
reducing sulfur to hydrogen sulfide gas (H2S)
Procedure:


Medium contains:
1% Lactose
Phenol Red
1% Sucrose
Sodium thiosulfate
0.1% Glucose
Obtain 6 TSI agar slants; inoculate with the
following organisms: P.aeruginosa, E.coli,
P.vulgaris, C.freundii, P.mirabilis, A.faecalis

Medium is inoculated by stab and streak
Results:


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Red slant/Red butt = no fermentation
Red slant/Yellow butt = only glucose
fermentation
Yellow slant/yellow butt = lactose
and/or
sucrose
fermentation
Dark color: Hydrogen Sulfide produced