Lab 24: Gram Negative Rods
Biochemical tests
• identification of gram negative rods
• IMViC series of tests
– Indole
– MR
– VP
– Citrate
Indole Test
• Determine if the
organism can
produce the enzyme
tryptophanase which
cleaves tryptophan,
generating indole
• Add 4-5 drops of
Kovacs reagent
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MR test
• Determine if the
organsim can
produce acid end
products from
glucose
fermentation
• Add 4-5 drops of
methyl red (pH
indicator)
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VP test
• Determine if the
organism can
produce alkaline and
non-acid end
products from
fermentation of
glucose.
• Add __ drops of VPA, __ drops of VP-B
and incubate at 37oC
for 30 minutes.
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Citrate test
• Determine if the
organism can
metabolize citrate as
the sole carbon
source
• pH indicator is brom
thymol blue
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Sugar Fermentation Broths
• Determine if the organism can ferment
the sugar (glucose, lactose, or sucrose)
• pH indicator is phenol red
– Turns yellow in acidic conditions
– Turns hot pink in alkaline conditions
• Look for gas production in the durham
tube
Phenol red fermetation broths
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Urea Test
• Determine if the
organism can
produce the
enzyme urease
which cleaves
urea generating
ammonia
• pH indictor is
phenol red
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Phenylalanine
Slants
• Tests for the presence of the enzyme
phenylalanine deaminase
• If produce enzyme, organism will break
down phenylalanine to phenylpyruvic
acid
• When you add ferric chloride, slant
turns green
Motility Deeps
• Used to determine if the organism
produces flagella
• Look at the tube in all dimensions
• If you see a “fanning” of growth, or lack
of a stab line, then it is motile
Catalase Test
• Use the TSA plate, find an isolated
colony to test
• Determine if the organsim produces the
enzyme catalase which breaks down
hydrogen peroxide generating oxygen
and water
• Add few drops of hydrogen peroxide
and look for bubbles
Oxidase
• Test for the
presence of enzyme
cytochrome oxidase
• Use sterile swab to
pick up a colony and
add a few drops of
“oxidase reagent” to
swab
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MacConkey Agar
• Selective and
differential agar
– Selects for gram
negative rods
– Differentiates
lactose
fermenters from
non-fermenters
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XLD plates
• Selective and differential agar (selects for gram
negative rods, also helps differentiate Salmonella
from Shigella)
• Organisms which ferment lactose, sucrose, or xylose
will generate yellow colonies on agar
• Organisms which decarboxylate lysine will raise the
pH and colony will appear red
• Salmonella will appear as red colonies with black
precipitate (produce hydrogen sulfide)
• Shigella will not ferment or produce hydrogen sulfide
XLD plates
• Salmonella are black
colonies
• Other gram negatives
are yellow to orange
colonies
Hektoen Enteric Plates
• Selective and differential agar (selects for gram
negative rods, differentiates between those that
ferment lactose and/or sucrose)
• Contains a high concentration of bile salts to inhibit
gram positive
• Organisms that ferment produce yellow colonies,
those that don’t ferment generate blue-green colonies
HE plates
• Organisms that ferment produce yellow
to orange colonies, those that don’t
ferment generate blue-green colonies