Biochemical Identification of Gram (-)
Bacteria
I. Carbohydrate Utilization
 Lactose utilization-most important carbohydrate
determination test
 Lactose- glucose-galactoside bond-galactose
 B-galactoside permease-transport enzyme
 B-galactosidase-enzyme hydrolyzes lactose into
glucose and galactose.
 LFs-both enzymes; NLFs-neither; LLFs/DLFs-lack B-
gp but possess B-galactosidase
 CHO utilization: Oxidation(Aerobic); Fermentative
(Anaerobic)
 Asaccharolytic-do not use CHO; use organic
molecules for energy & carbon sources
A. Oxidation-Fermentation Tests
 Differentiating glucose fermenters
(Enterobacteriaceae) from non-glucose fermenters
(Pseudomonas)
 Hugh-Leifson O/F Basal Medium (OFBM)
 Contains 1% Carbohydrates and 0.2% peptone
original color: yellow
Fermenter: Change in color in both tubes.
Enterobacteriaceae
Oxidizer: Change in color in
tubes without mineral oil.
Pseudomonas
Nonoxidizer: No change in
color in both tubes. Alcaligenes faecalis
B. Triple Sugar Iron Agar
 Test whether a gram-negative rod utilizes glucose,
and lactose/sucrose with phenol red as indicator
Kligler Iron Agar(KIA)-glucose & lactose only
 Contents:
a. 1% lactose, 1% sucrose and 0.1% glucose
b. Sodium thiosulfate and Ferrous sulfate
o Bacterium (acid envi) + Sodium thiosulfate
→ H2S gas
o H2S + Ferric ions → Ferrous sulfide (black
precipitate)
c. Phenol red (pH indicator)
slant butt agar
slant – aerobic or anaerobic
inoculating needle: stab and streak method
K/K – alkaline/alkaline: did not change color
H2S--no color black
K/A – change in color (yellow: acid)
H2S+ - automatic acidic environment
gas – represented by a space or crack
late fermenters – konting red
C. ONPG (o-nitrophenyl galactopyranoside)
 Test the ability of organism to produce β-
galactosidase and hydrolyzes ONPG into galactose &
о-nitrophenol
 (yellow). (delayed/late lactose fermenters)
Positive:
Yellow (Presence of β-galactosidase)
Lactose and dLFs, Shigella sonnei
Negative:
Colorless (absence of enzyme)
Non-lactose fermenters
II. Glucose Metabolic Ends Products
A. Methyl Red Test MR-VP (Clark and Lubs
medium)
 Determines the end products of glucose fermentation
 First pathway produces mixed acid (MR - red)
 Second pathway produces acetoin (VP - pink-red)
 Add 5 or 6 drops of methyl red per 5 ml broth
 Glucose → Pyruvate → Mixed acid + Methyl Red →
Red
Positive: Bright red color
indicative of mixed acid
fermentation
(Escherichia coli)
Negative: Yellow Color
(Klebsiella and
Enterobacter)
B. Voges-Proskauer Test – detection of the
production of acetylmethylcarbinol (acetoin) ->
oxidized to diacetyl
 Add 6 drops of α-naphthol and 2 drops of 40% KOH to
1 ml broth, expose to O2 and stand for 10-15 mins.
Positive: Red (pink-red) color at
the surface of the medium
(Klebsiella and Enterobacter)
Negative: Yellow Color (copper
like) at the surface of the medium
(Escherichia coli)
III. Amino Acid Utilization IV. Miscellaneous Test
A. Decarboxylase and Dihydrolase Test A. Citrate, Malonate, or Acetate Utilization
 Determines whether an organism is capable of  Determine the ability of an organism to use sodium
decarboxylating an amino acid to form an citrate, malonate or acetate as the sole source of
amine/diamine carbon
 Contents: Glucose, peptones, bromcresol purple and  Indicator: Bromthymol blue
cresol red (pH indicators), amino acid at 1%  N2 souce: NH4 salts
concencentration
1. Lysine: Lysine decarboxylase (enzyme) →
Cadaverine (amine) [product] + CO2
2. Ornithine: Ornithine decarboxylase →
Putrescine
3. Arginine: Arginine dihydrolase → Citrulline →
Ornithine
lysine and ornithine: decarboxylation
arginine: hydrolase original color: green
Positive: Blue
Only slant no butt

B. DNase
 Test the ability of the organism to hydrolyze DNA
 Oligonucleotides formed will be detected by 1 N HCL
Moeller
Decarboxylase Positive: Hydrolysis of the
Base Medium surrounding medium (Clear
Zone) or halo (S. aureus and S.
Positive: marcescens)
Alkaline
(purple) color Negative: No clearing observed
(S. epidermidis and S.
Negative: acid saprophyticus)
(yellow) color Methyl green
metachromatic dye
Decarboxylase Reactions
C. Gelatin Liquefaction
LDC ODC ADH
K. subs.
 Used to determine the ability of an organism to
+ - -
pneumoniae produce gelatinases that liquefy gelatin. Gelatinase
K. subs. + - - breaks up proteins into peptides & amino acids.
oxytoca Positive: partial or total
E. + + - liquefaction of inoculated tube. Ex:
aerogenes S. aureus, Corynebacteria
E. cloacae - + +
P. vulgaris - - - Negative: Complete solidification
P. mirabilis - + - of tube. Ex: S. epidermidis,
Listeria
B. Deaminase Test
 Test the ability of an organism to oxidatively D. Indole Production
deaminatephenylalanine to phenylpyruvic acid  Test for the ability of an organism to split tryptophan to
form the compound indole.
Positive: Green color develops on slant
after FeCl3 is added (Proteus spp.
Providencia and Morganella or PPM)
Tryptophan broth Kovac’s reagent PDAB (gives red color)
Negative: slant remains original color Positive:
after the addition of FeCl3. Pink to wine colored ring after
addition of Kovac’s reagent
(Escherichia coli, P. vulgaris)

Negative:
No color change
(Klebsiella, Enterobacter, P.
mirabilis)
 F. Lysine Iron Agar Slant (LIA)
 Nitrate and Nitrite Reduction Test- differentiate  Determines the ability of the microorganism to
between bacteria based on their ability or inability to decarboxylate or deaminate lysine and form H2S
reduce nitrate (NO3−) to nitrite (NO2−) using anaerobic  Contents: Lysine (amino acid), Glucose (carbon
respiration source), H2S Indicator (ferric ammonium citrate &
zinc, alpha-naphthol nitrate and sulfuric acid - reagent sodium thiosulfate) and bromcresol
 no change with alpha- purple (pH indicator)
naphthol & sulfuric  Principle:
acid: nitrite absent o When glucose is fermented, the
 turns red with alpha- butt of the medium becomes
naphthol & sulfuric acidic (yellow)
acid: nitrite present, o If the organism decarboxylates
positive nitrate lysine, a purple butt / slant
reduction test forms; deaminates lysine, a
 no change with zinc: burgundy, plum, reddish-purple
positive nitrate color slant forms.
reduction test deamination only occurs
 turns red with zinc: anaerobically so it’s on slant
negative nitrate
reduction test

 Inoculate nitrate broth with an isolate and incubate for

48 hours.
 Add 10-15 drops each of sulfanilic acid and
N,Ndimethyl-1-naphthylamine. If the bacterium
produces nitrate reductase, the broth will turn a deep
red within 5 minutes at this step.
 If no color change is observed, then the result is
inconclusive. Add a small amount of zinc to the broth.
If the solution remains colorless, then both nitrate
reductase and nitrite reductase are present. If the
K/R: numerator
solution turns red, nitrate reductase is not present.
o K – decarboxylase
o R – deaminase
Organisms Nitrate reduction reading: alkaline/alkaline decarboxylase (+) /
Acinetobacter (-) Negative deaminase (+) H2S (-)
calcoaceticus
Enterobacter aerogenes + Positive G. Motility Indole-Ornithine (MIO)
Escherichia coli + Positive  Semisolid medium that detects motility, indole and
Salmonella typhimurium + Positive ODC
 Contents: Ornithine (amino acid), Glucose (carbon
E. Urease (Christensen’s agar [usual] or Stuart’s broth) source), and bromcresol purple (pH indicator)
 Test the ability of an organism to produce the enzyme
urease which hydrolyzes urea.
 Indicator is phenol red (Urea →ammonia + CO2)

Positive: Red (magenta)

throughout medium
Proteus, Providencia and
Morganella: rapid urease
producers

Positive: Red (magenta) slant

or pink to orange through
medium slow or weak urease
producer

Negative: no color change /

yellow

(w) (s) – weak or strong

H. Sulfide-Indole-Motility Agar (SIM)
 Determines the ability of the microorganism to form
H2S, indole, and observe for motility

Indole Production: Pink to wine

colored ring after addition of
Kovac’s reagent (tube 2)

H2S Positive: Blackening of the

medium (tube 4)

Motile: Diffuse growth extending

laterally from line of inoculation (tube 1-4)

Miscellaneous Test
 IMVC
o Indole
 Kovac’s
 Ehrlich’s
o MRVP
 Methyl Red
 Voges-Proskauer
o Citrate
 Biochemical Test – usually used to identify bacteria
o TSI
o LIA
o SIM
o SCA
o Urease

V. Rapid Biochemical Tests

A. Spot Indole
B. ONPG
C. Oxidase
D. Catalase
E. Bile Solubility
F. PYR
G. Rapid Urease
H. Rapid hippurate hydrolysis
I. MUG
J. LAP

 Oxidase Test (tetramethyl-p-phehylenediamine

dihydrochloride) - determines presence of cytochrome
oxidase.

Positive: (violet color)

Psedomonas, Neisseria,
Micrococcus, Plesiomonas.

Negative: (colorless)
Enterobacteriaceae,
Staphylococcus