biochemical tests part one differentiation of organisms based on their ability to break down complex...
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BIOCHEMICAL TESTS
PART ONE
Differentiation of organisms based on their ability to break down complexMacromolecules in to simpler nutritional constituents
Macromolecules are polymers of monomeric subsunits.
Hydrolysis reactions cleave polymers into monomers by adding water.
Protein(Amino Acids)
Starch (Sugars)Fat (Triglycerides And other lipids)
Macromolecules and their (Monomeric Subunits)
Starch: a polymer of sugars
The hydrolysis reaction is the reverse of the dehydration reaction
Muscles store sugar as glycogen.
STARCH TEST 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
B.cereus
P.aeruginosa
Results:
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
Procedure:
Divide one tributyrin agar plate into thirds and inoculate with E.coli, Bacillus cereus, and P.aeruginosa E.coli
B.cereusP.aeruginosa
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
B.cereusP.aeruginosa
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
Carbohydrate Fermentation(Phenol Red Carbohydrate Broth)
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 Tubes4 Phenol Red Sucrose Broth Tubes4 Phenol Red Lactose Broth Tubes
Inoculate the above tubes with the following organisms:
E.coli, P.vulgaris, A.faecalis, S.aureus
TRIPLE SUGAR IRON TEST (TSI) Used to differentiate among the different
groups of Enterobacteriaceaebased 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 Red1% 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: 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
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