Biochemical Tests
Fermentation
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Glucose acts as an electron donor to one
or more of its organic products
End products include alcohols, organic
acids, and CO2
Today we use two tests
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Phenol red broth
Triple sugar iron (TSI)
Phenol Red Broth
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Contains:
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Carbohydrate
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peptone
Phenol red (pH indicator)
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Dextrose/glucose
Lactose
Sucrose
Below 6.8 = yellow
6.8-7.4 = red
Above 7.4 = pink
Durham tube – inverted to catch gas bubbles
TSI
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Similar to phenol red broth, but has a 3 sugars
in one slant
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Also tells if organism reduces sulfur
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Only contains a small amount of glucose (0.1%), so if
the organism only ferments glucose, it will quickly run
out and will resort to breaking down protein, which
will increase pH and cause red coloration
Black precipitate
Streak the surface and stab it (get to oxygen
poor area)
Starch Hydrolysis
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Starch can be broken down into glucose if the
proper enzymes are produced by bacteria
Iodine turns from yellow to black in the presence
of starch
So if the bacteria produces starch hydrolyzing
enzymes, there will be no starch present and
iodine will not produce black color
Casein Hydrolysis
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Casein = milk protein
Casease (enzyme) breaks down casein
into amino acids to be taken up by
bacteria
Casease-positive bacteria will produce a
clear zone
Gelatin Hydrolysis
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Gelatin is another protein
Gelatinase (enzyme) breaks down gelatin
into amino acids to be taken up by
bacteria
Gelatin liquifies when it’s broken down
Lipid Hydrolysis
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Enzyme lipase breaks down lipids
Spirit blue dye gets pulled toward bacteria
when lipids are taken up
Indole/H2S
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SIM determines
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Sulfur reduction (black precipitate)
Indole production from trypophan (red color
with Kovac’s reagent)
Motility (radiating growth)
Methyl Red-Vogues Proskauer (MRVP)
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Methyl red test determines mixed-acid
fermentation
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Vogues Proskauer test determines if fermenting
bacteria utilize the butylene glycol pathway
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Addition of methyl red reagent gives red color for
positive bacteria
Addition of vogues proskauer reagent gives red color
for positive bacteria
Because both result in red color, next week we
need to pour ½ of each broth into a fresh
culture tube
Citrate Utilization
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Determines if bacteria use citrate as
carbon source
Contains bromthymol blue dye
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Green at pH 6.9
Blue at pH 7.6
Produces alkaline products when citrate is
used
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So it turns BLUE