UNMC Clinical Laboratory Science Program
Page 1
Enterobacteriaceae Biochemical Tests and Media
Mahon, 2nd edition, Chapter 16, pages 463-514
Biochemical Test
or Media
Cytochrome
oxidase
Test Principle
Same as Micrococcaceae worksheet
Expected Results
Positive/ Negative/
Sensitive Resistant
Same
Same
Test/media result for the Enterobacteriaceae
Enterobacteriaceae =
(p 185, 465)
Reagent:
Media containing nitrate, alpha-naphthylamine,
sulfanilic acid, zinc dust
Before
zinc:
Before
zinc:
After
zinc:
After zinc:
Same
Same
Enterobacteriaceae =
Principle:
Nitrate
reduction
(p. 465, 506, 11321133)
OF glucose
utilization
The capability of an organism to reduce nitrates to
nitrites. Organisms demonstrating nitrate reduction
have the capability of extracting oxygen from nitrates
to form nitrites and other reduction products. The
presence of nitrites in the test medium is detected by
the addition of alpha-naphthylamine and sulfanilic
acid, with the formation of a red diazonium dye. The
addition of zinc dust is to confirm a negative reaction
or to see if organism reduced nitrates all the way to
N2O gas.
Same as Micrococcaceae worksheet
Enterobacteriaceae =
(p 465, 1133)
Reagent:
Used to speciate the Enterobacteriaceae.
Media contains glucose, peptone, pH indicator,
bromcresol purple and cresol red, and amino acid
ornithine
Principle:
Ornithine
Bacteria possessing ornithine decarboxylase
Decarboxylase enzyme is capable of decarboxylating ornithine.
(p 501, 504-505)
The
enzyme removes a molecule of CO2 from the amino
acid to form alkaline-reacting amines. This results in
a color change of a pH indicator.
Methods include: Moeller decarboxylase broth,
Falkow lysine broth, and MIO media
Reagent:
Proteus species =
Phenylalanine, ferric chloride
Phenylalanine
Deaminase
(p 501, 504, 1134)
Principle:
Bacteria possessing phenylalanine deaminase
enzyme is capable of deaminating phenylalanine to
phenylpyruvic acid. Addition of ferric chloride results
in a green color if phenylpyruvic acid is present.
Providencia species =
Morganella species =
Most other Enterobacteriaceae =
UNMC Clinical Laboratory Science Program
Page 2
Enterobacteriaceae Biochemical Tests and Media
Mahon, 2nd edition, Chapter 16, pages 463-514
Biochemical Test
or Media
Indole
production
(p 185, 467, 501-502,
505)
Citrate
utilization
(p 467, 501-502, 1122)
Gelatin
liquefaction
(p: 492-495, 1126)
Test Principle
Used to speciate the Enterobacteriaceae.
Principle:
Escherichia coli =
Bacteria that possess the enzyme tryptophanase
are capable of cleaving tryptophan, producing
indole, pyruvic acid and ammonia. Indole can be
detected by adding para-dimethylaminobenzaldehyde (Ehrlich’s or Kovac’s reagent) and observing
a red color.
Reagent: Sodium citrate, pH indicator
This test is determines the ability of an organism
to utilize sodium citrate as its only carbon source
for metabolism. Bacteria that can grow on this
medium turn the bromthymol blue indicator from
green to blue.
Reagent: Gelatin
Escherichia coli =
Klebsiella species =
Proteus species =
Principle:
This test is used to determine the ability of an
organism to produce proteolytic enzymes
(gelatinases) that liquefy gelatin.
Motility
Principle:
(p 465, 501, 505, 11311132)
Used to determine if an organism is motile. An
organism must possess flagella to be motile. If
organism is motile, it will spread out into the
medium from the site of inoculation.
Reagent: o-nitrophenyl-β-D-galactopyranoside
(ONPG)
Principle:
(p 185, 498-499)
Used to speciate the Enterobacteriaceae.
Principle:
<0.4% agar concentration in media
to allow free spread of organism
(o-nitrophenyl
galactosidase)
Test/media result for the Enterobacteriaceae
Reagent: Amino acid tryptophan in broth,
para-dimethylaminobenzaldehyde
Reagent:
ONPG test
Expected Results
Positive/
Negative/
Sensitive Resistant
This test is used to determine the ability of an
organism to produce β -galactosidase, an enzyme
that hydrolyzes the substrate ONPG to form a
visible (yellow) product, orthonitrophenol. This
determines lactose fermentation in slow lactose
fermenters. This test is not a substitute for the
determination of lactose fermentation because
only the enzyme β-galactosidase is measured.
Serratia species =
Most other Enterobacteriaceae =
Klebsiella =
Shigella =
Yersinia =
Most other Enterobacteriaceae =
Used to speciate the Enterobacteriaceae.
UNMC Clinical Laboratory Science Program
Page 3
Enterobacteriaceae Biochemical Tests and Media
Mahon, 2nd edition, Chapter 16, pages 463-514
Biochemical Test
or Media
VogesProskauer
(Acetylmethylcarbinol)
(p 198, 467, 498, 500501, 1131)
Test Principle
Reagent:
Principle:
Expected Results
Positive/
Negative/
Sensitive Resistant
Test/media result for the Enterobacteriaceae
Used to speciate the Enterobacteriaceae.
Potassium hydroxide, alpha-naphthol
This test is used to determine the ability of an
organism to metabolize glucose to pyruvic acid,
then to acetyl-methyl-carbinol (acetoin). In the
presence of atmospheric oxygen and 40% KOH,
acetoin is converted to diacetyl, and then alphanaphthol is added to catalyze or bring out a red
complex.
Reagent: Urea
Used to speciate the Enterobacteriaceae.
Principle:
Urease
production
(p 185, 467, 501, 503,
1139)
Urease is an enzyme possessed by many species
of microorganisms that can hydrolyze urea to
ammonia, CO2 and H2O. The ammonia reacts in
solution to form ammonium carbonate, resulting in
alkalinization and an increase in the pH of the
medium. The pH change will cause the phenol
red indicator to change to red.
Ingredients:
A/A =
Medium in tube as a slant contains agar base,
dextrose, lactose (10x concentration of dextrose),
phenol red and ferrous sulfate. Medium is red
uninoculated.
K/K =
Examples of Enterobacteriaceae that are lactose
fermenters:
Principle:
KIA
(p. 497-499, 11271128)
This test is used to determine the ability of an
organism to ferment glucose and lactose and
produce hydrogen sulfide. Fermentation of
glucose but not lactose, changes the color from
red to yellow (butt and slant) initially. With
continued incubation, slant changes back to red
due to reversion of reaction under aerobic
conditions. Butt remains yellow due to anaerobic
conditions. Fermentation of both glucose and
lactose changes color from red to yellow, and
both butt and slant stay yellow due to high levels
of lactose being fermented. Black precipitate
forms when organism is able to produce H2S from
ferrous sulfate.
K/A =
Examples of Enterobacteriaceae that are non-lactose
fermenters:
Black precipitate =
Gas =
UNMC Clinical Laboratory Science Program
Page 4
Enterobacteriaceae Biochemical Tests and Media
Mahon, 2nd edition, Chapter 16, pages 463-514
Ingredients:
Expected Results
Positive/
Negative/
Sensitive Resistant
A/A =
Principle:
K/K =
Biochemical Test
or Media
Test Principle
Medium in tube as a slant contains
agar base, dextrose, lactose and sucrose (both 10x
concentration of dextrose), phenol red and ferrous
sulfate. Medium is red uninoculated.
TSI
(p 497-499, 1138)
This test is used to determine the ability of an
organism to ferment glucose, lactose, and/or
sucrose, and produce hydrogen sulfide.
Fermentation of dextrose but no lactose or sucrose,
changes the color from red to yellow (butt and slant)
initially. With continued incubation, slant changes
back to red due to reversion of reaction under
aerobic conditions. Butt remains yellow due to
anaerobic conditions.
Fermentation of dextrose plus lactose and/or
sucrose changes color from red to yellow, and both
butt and slant stay yellow due to high levels of
lactose being fermented. Black precipitate forms
when organism is able to produce H2S from ferrous
sulfate.
Ingredients: Medium in tube as a slant contains
agar base, lysine, small amount of glucose, ferric
ammonium citrate, sodium thiosulfate and
bromcresol purple (pH indicator). Medium is purple
uninoculated.
LIA
(p 505, 1129)
Used to speciate the Enterobacteriaceae.
K/A =
Black precipitate =
Gas =
LDA =
LDA =
Organism
Proteus species
Principle:
This test is used to determine whether a GNR
decarboxylates or deaminates lysine and forms
hydrogen sulfide. Butt: Glucose fermentation
(acidic) will turn butt yellow. If the organism
produces lysine decarboxylase, cadaverine is
formed. Cadaverine neutralizes the organism acids
formed by glucose fermentation, and the butt reverts
to alkaline state (purple). If decarboxylase is not
produced, butt remains acidic (yellow). Slant: If the
oxidative deamination of lysine occurs, a compound
is formed that, in the presence of ferric ammonium
citrate and a coenzyme, flavin mononucleotide,
forms a burgundy (wine-red) color on the slant. If
deamination does not occur, the LIA slant remains
purple. Black precipitate = H2S production from
ferric ammonium citrate.
Test/media result for the Enterobacteriaceae
Providencia species
LDC =
LDC =
Salmonella species
Citrobacter species
H2S =
H2S =
LDA
LDC
H2S
UNMC Clinical Laboratory Science Program
Page 5
Enterobacteriaceae Biochemical Tests and Media
Mahon, 2nd edition, Chapter 16, pages 463-514
Biochemical Test
or Media
Test Principle
Media classification:
Selective and differential for GNR
Ingredients:
MacConkey
agar
(p 314-316, 490-491,
964, 1129-1130)
Peptone base with lactose, gram positive organisms
inhibited by crystal violet and bile salts. Neutral red
as indicator.
Principle:
pH indicator changes to dark pink/red when
organism ferments lactose. Non-lactose fermenters
remain clear and colorless.
Media classification:
Selective and differential for GNR – esp. Salmonella
and Shigella
Hektoen
enteric agar
(HE)
(p 490-491, 964, 11261127)
Ingredients:
Peptone base agar with bile salts, lactose, sucrose,
salicin, and ferric ammonium citrate. Indicators
include bromthymol blue and acid fuchsin.
Expected Results
Positive/
Negative/
Sensitive Resistant
Test/media result for the Enterobacteriaceae
Examples of Enterobacteriaceae that are lactose
fermenters:
Examples of Enterobacteriaceae that are non-lactose
fermenters:
Colony morphology:
E. coli =
Salmonella =
Principle:
Gram positive organisms and some coliforms are
inhibited by high bile salt concentration. GNR that
will grow can be differentiated as to lactose/sucrose
fermenters / non-fermenters. H2S production can
also be determined.
Shigella =
Media classification:
Colony morphology:
Differential, selective medium for the isolation and
differentiation of Salmonella and Shigella spp. from
other gram negative enteric bacilli.
E. coli =
Ingredients:
Xylose lysine
desoxycholate
agar (XLD)
(p 490-491, 964, 11391140)
Yeast extract agar with lysine, xylose, lactose,
sucrose, and ferric ammonium citrate. Sodium
desoxycholate inhibits gram positive organisms;
phenol red as indicator.
Principle:
Sucrose, lactose and xylose can be fermented.
Lysine can be decarboxylated. Sodium thiosulfate
can be used for production of H2S.
Salmonella =
Shigella =
Citrobacter =