Summary of Biochemical Tests
in Microbiology
“Better living through chemistry”
Catalase Test
Summary
Tests for presence of catalase.
Background
Catalase is an enzyme that
breaks down hydrogen
peroxide into water and
oxygen gas. Evolution of
oxygen gas bubbles is
positive.
All + except EF –
Methods
Loop of bacteria added to a
drop of Hydrogen Peroxide
H2O2
Blood Agar Test
Summary
Tests for presence of
hemolysin.
Background
Hemolysin is an enzyme that
destroys red blood cells.
Methods
Bacteria streak and loop stabs
to blood agar.
3 Types of Blood
Agar Results
Summary
Alpha – Partial hemolysis
Beta – Complete hemolysis
Gamma – No hemolysis
Background
Alpha hemolysis partially
oxidizes hemoglobin changing it
to green.
All B+ except SE and EF
Beta hemolysis lyses RBC
completely making a clear zone
Gamma hemolysis – no activity
Mannitol Salts
Agar (MSA) Test
Summary
Selective for gram +
Differential for fermentation
of mannitol sugar.
Background
Gram + bacteria tolerate
sodium chloride (NaCL) salt.
SA + EF + SE - and others –
Methods
Streak surface of MSA Agar.
Watch for change from pink
to yellow as a +
McConkeys Test
Summary
Tests for fermentation of
lactose.
Background
Selects for gram - , Crystal
violet inhibits gram +
Lactose and neutral red
produce a dark pink
differentiating lactose +
EC, CLO, KP are +
Methods
Loop of bacteria streaked on
Mac agar
EMB Agar Test
Summary
Tests for fermentation of
lactose.
Background
Selects for gram - , Eosin and
methylene blue inhibit gram +
Lactose and dyes produce a
dark pink differentiating
lactose + in CLO and a green
sheen in EC
EC, CLO, KP are +
Methods
Loop of bacteria streaked on
EMB agar
Nitrate Reduction
Test
Summary
Tests for nitrate reductase and
nitrite reductase.
Background
Potassium Nitrate KNO3 as
substrate. Nitrate  Nitrite
 N2
Methods
. Add Nitrate A & B and
cherry red means + for nitrate
reductase
All +
Follow up with Zinc = grey = +
for nitrite reductase
PA +
Triple Sugar Iron (TSI) Test
Summary
Differentiates gram – enterics
by fermentation patterns.
Background
Lactose and glucose are
substrates. Phenol red is the
indicator. pH of 6.8 or lower
turns yellow.
Methods
Stab and streak TSI slant.
Red slant & yellow but =
glucose fermentation. ST SF PA
Yellow slant and yellow butt =
lactose and glucose
fermentation. SA SE EF BS EC
CLO KP PV(false)
Red slant & red butt = no
fermentation.
Black butt = Hydrogen sulfide
production
Urease Test
Summary
Differentiates organisms using
urease to break down urea
into ammonia
Background
Substrate is urea. pH
indicator is phenol red (pH 6=
yellow, 6.8 topaz, 8 = fuchsia)
Methods
Loop inoculation of urea
broth.
SA SE KP PV are +
IMViC Indole Test
Summary and Background
Differentiates enteric
organisms such as EC and PV
which are able to metabolize
the amino acid tryptophan
into indole and pyruvic acid.
This test also detects motility
of bacteria and H2S2
production.
Methods
Needle inoculation of SufideIndole-Motility (SIM) media.
Substrate is tryptophan. Add
Kovac’s indole reagent. A red
color is + for indole
production.
Black = + for H2S2
Motility is observed as
turbidity radiating out from
the inoculation line.
IMViC Methyl Red
Summary and Background
Differentiates enteric
organisms such as EC from
CLO using the timing of
glucose fermentation
products. Both produce acids
but CLO further converts to
non acidic ethanol and then
elevates the pH.
Methods
Loop inoculation of MR VP
broth. Add methyl red
indicator.
Red = + for acid conditions.
SA SE BS EC KP PV ST SF
Yellow is – indicating more
basic conditions. EF CLO PA
Citrate Test
Summary
Differentiates organisms using
citrate as a carbon source.
Background
Organisms using citrate must
also have nitrogen found in
the medium as ammonium
dihydrogen phosphate.
Citrate fermentation produces
ammonia causing the
indicator (bromothymol blue)
to turn deep blue.
Methods
Loop inoculation of citrate
slant.
Prussian blue = + for citrate
fermentation and alkaline
conditions.
Green or yellow = - (low pH)