Theory of Metabolic Tests and Reactions Used to Identify Gram Neg. Enteric Bacteria
[Don’t use this sheet as instructions on how to set up or process your rest results!]
Test Name
Carbohydrate
Fermentation Tubes
with Phenol Red
Substance(s) or
ability being tested
for
Chemistry
Fermentation of a
particular
monosaccharide
Phenol red turns yellow when bacteria ferment
sugars and produce acids (with or without gas) in
the first 24 hrs. Gas is captured in the inner
Durham tubes if it is produced during
fermentation. After about 48 hours, when the
sugar is exhausted, bacteria utilize the peptone in
the medium, producing a hot pink (alkaline)
product. Bacteria which cannot ferment the sugar
will grow immediately on the peptone and may
stay red or turn hot pink after only 24 hrs.
(Note this is different
from our Kleyn and
Bicknell lab manual
that uses methyl red
and different are
produced)
Methyl Red/VogesProskauer (MRVP)
Indole Production
from Tryptone
Broth
Fermentation of
glucose (if at all) to
acetoin and onward
to butanediol (uses
the butanediol
fermentation
pathway)
MR and VP tubes are interpreted separately.
Adding methyl red to a grown culture turns red if
acid was produced. The VP reagent is added to a
grown culture in the presence of O2 and makes a
red ring at the broth surface if acetoin is present
(typical of Enterobacter bacteria). MRVP must
be read within 24-48 hours as nearly all cultures
become alkaline after 72 hours due to catabolism
of the peptone into ammonia.
Presence of the
enzyme
tryptophanase
The amino acid tryptophan can be hydrolyzed
into indole, ammonia, and pyruvate if the enzyme
tryptophanase is present. Addition of Kovacs
Reagent produces a red layer at the top of
tryptone broth if indole is present.
Negative/Control
Positive Result
Red
(above pH 6.8)
Yellow
(below pH 6.8)
Phenol red turns hot
pink/fuschia if the
medium becomes
alkaline or > pH 8.2
Yellow MR test (no
change)
Red MR test
Yellow VP test (no
change)
Red ring at surface
for VP test
No change in broth
compared to control
Red layer at the top
of the broth
Metabolic Tests and Reactions Used to Identify Gram-negative Enteric Bacteria pg. 1
Test Name
Oxidase
production
Substance(s) or
ability being tested
for
Presence of enzyme
cytochrome oxidase
in electron transport
chain
Citrate permease
enzyme
Citrate
utilization
Urease enzyme
Urease Test on
Urea Agar
MacConkey
Agar for gram
negatives and
lactose
utilization
Motility Test
Fermentation of
lactose and selection
for gram negatives
Flagellum/flagella
present
Chemistry
Negative/Control
Positive Result
Oxidase reagent turns purple-gray within 1 minute
when dripped onto plated bacterial colonies
No change in colony
color
Colony or streak
turns pinkish-purple
Green citrate medium with bromthymol blue
indicator turns blue as citrate-utilizing bacteria
convert citrate to alkaline products. Even though
citrate is broken down into acidic produces, citrateutilizers make an overwhelming amount of alkaline
products.
The urea in urea agar slants is deaminated when
NH2 groups are removed, producing alakine
ammonium ions (NH4+). Phenol red in the medium
turns hot pink/fuschia in alkaline conditions.
Only gram negative bacteria should grow (except
Enterococcus and some Staphylococcus) as the bile
salts and crystal violet in the medium inhibit gram
positives. Neutral red, a colorless pH indicator in
the agar, turns red when the lactose is fermented to
produce acid. Lactose-fermenting gram neg.
enterics are called coliforms
Agar slant remains
green throughout
(pH < 6.9)
Agar slant turns
blue in some
portion
(pH > 7.6)
Medium remains a
peach color, or even
turn yellow if bacteria
produce acid
Medium remains clear
to faintly pink and
colonies are white or
crème colored
Medium turns
fuschia/hot pink
Organisms that are motile “swim” away from
inoculation tunnel, often creating an “inverted
Christmas tree” effect in an agar deep.
Medium turns
reddish and
colonies themselves
become pink or
tinged with pink
when lactose is
fermented (pH <
6.8)
Medium remains dull Wisps of and
yellow with whitish- opaque growth
creamy opaque growth extend away from
only within the
the inoculation
inoculation tunnel
tunnel; “inverted
Christmas tree”
[Don’t use this sheet as instructions on how to set up or process your rest results!]
Metabolic Tests and Reactions Used to Identify Gram-negative Enteric Bacteria pg. 2