TEST

MEDIA

Gram Stain

Growth in
Selective and
Differential
Media

PURPOSE

COMPONENTS

-to determine the Gram

reaction of the bacterium

Mannitol Salt Agar

(MSA)
- red colored medium
-selective for gram
positive bacteria
- mannitol (C6H14O6)crystalline alcohol used
as a diuretic and in
testing kidney function
Eosin- Methylene
Blue Agar
- red orange medium
- selective for gram
negative bacteria +
family
Enterobacteriaceae
- eosin (C20H8Br4O5)dye used as biological
stain for cytoplasmic
structures
MacConkey Agar
(MCA/ MAC)
-red colored medium
- selective for isolation
of gram-negative bacilli

- differentiate mannitol
fermenters from nonfermenters, & select
bacteria that can highly
survive the high NaCl
concentration
- fermentation
(enzymatically controlled
breakdown of an energy
rich compound)
- used for isolation,
identification,
differentiation of lactosefermenting, gram
negatives and non- lactose
fermenting, gram
negatives

Phenol red (pH

indicator)

- differentiate lactosefermenting, gram

negatives and non- lactose
fermenting, gram
negatives

- peptone
(nitrogen source)
-lactose (carbon
source)
- neutral red (pH
dye)

REAGENTS
-Crystal Violet
-Gram Iodine
-Ethanol
-Safranin
7.5% Nacl
(inhibiting
reagent for
other bacteria)

- Eosin Y (pH
indicator)
- Methylene blue
(pH indicator)
*indicators are
toxic to gram
positives e.g. S.a
(no growth)

-bile salts
(reduces
surface tension)
-crystal violet
(inhibits cell
wall synthesis)
* inhibit growth
of gram
positives

EXPECTED
RESULT
- Purple (gram
positive)
- Pink/ red (gram
negative)
-Yellow colonies w/
yellow coloration
(acidic pH)mannitol
fermenters (+)
- Colorless colonies
w/ pink coloration
(basic pH)- nonmannitol
fermenters (-)
- Colonies have
greenish metallic
sheen with blue
black center-lactose
fermenters (+)
- colonies that have
no metallic sheen,
colorless -nonlactose fermenters
(-)
- Red or intense
pink colonies
-lactose fermenters
(+)
- Colorless/ creamcolored colonies non-lactose
fermenters (-)

QUALITY
CONTROL

Staphylococc
us aureus (+)
Staphylococc
us epidermis
(-)

- E. coli &
Enterobacter
aerogenes(+)
Pseudomona
s
aeruginosa(-)

- E. coli (+)
Enterobacter
aerogenes (-)

Catalase Test

NA (Nutrient Agar)

-detects ability of microbes

to produce catalase,
which decomposes H2O2
(hydrogen peroxide) to
H2O and O2

Starch
Hydrolysis

Starch Agar
- colorless medium
- amylase acts on
starch and is secreted
out of the cell and
diffuses into
surrounding medium
-disaccharides and
monosaccharaides
enter the cell and are
acted upon
endoenzymes
Glucose
fermentation broth

-to determine which

bacteria possesses
amylase, exoenzyme for
breaking down starch

- starch

-used to indicate acid and

gas production
- to test ability of organism
to ferment glucose

- Phenol red/
Bromcresol
purple (pH
indicator)

Nutrient broth +
0.5% glucose
-light yellow colored
medium

-determines whether the

microbe performs mixed
acids fermentation when
supplied with glucose

Sugar
fermentation
on Durham
rube

Methyl red
Test

-3%

H 2 O2

- gram iodine

- Methyl red

-Formation of
bubbles
(catalase present,
+)
- no bubble
formation (no
catalase, -)

- medium turns
blue black after
addition of gram
iodine (starch was
not degraded, -)
- no blue black
complex after gram
iodine addition;
Colorless zone
surrounding the
colonies (+)
-medium coloration
turns from red to
yellow as sign of
acid production (+)
- air bubbles are
trapped in the
inverted tube as
sign of gas
production (+)
- medium coloration
turns red (+)- acid
production
-medium coloration
turns yellow (-)

Pseudomona
s a., E. coli,
S.a. (+)
Streptococcu
s,
Enterococcus
(-)
- Bacillus s.
(+)

- E. coli (+)

VogesProskauer

Nutrient broth +
0.5% glucose
-light yellow colored
medium

Citrate
Utilization

SCA ( Simmons
Citrate Agar)

Sugar
Utilization

TSIA (Triple Sugar

Iron Agar)/ Kliglers
iron agar
- Glucose is
catabolized to yield
pyruvate, which is
further oxidized to
CO2, H2O and energy

- Confirms whether the

organism has metabolized
pyruvate further to neutral
compounds

- to determine the ability

of gram negative bacteria
to catabolize glucose,
lactose, sucrose

- 5% VP reagent
A (alphanaphtol)
-VP reagent B
(40% KOH)
- citrate (carbon
source)
- ammonium
dihydrogen
phosphate
(nitrogen source)
- bromthymol
blue (pH
indicator)- turns
yellow when
acidic
- glucose
-lactose
-sucrose
-phenol red (pH
indi)

-pink to red color

(+)
- there is no change
in medium
coloration (-)
-medium coloration
changes from green
to blue
- no change in
color; medium turns
yellow/ acidic (-)

K/A- red slant &

yellow butt; alkaline
slant, acid butt
(glucose, nonlactose fermenters)
A/A- yellow slant &
yellow butt; acid
slant, acid butt
(glucose, lactose/
sucrose fermenters)
K/K- red slant & red
butt; alkaline slant,
alkaline butt
(degrade peptone
both in the
presence& absence
of O2)
K/NC- red slant &
no change in butt
color (nonfermenters; only
utilize peptone

E.coli (A/A)
Ps.a. (K/NC)

aerobically)

H2 S
Production

Gelatin
Hydrolysis
Test

TSIA (Triple Sugar

Iron Agar)
-bacteria reacts w/
Na2S2O3 in an acid
envi thru reduction rxn
to yield SO3 and S2
-H2S reacts w/
NH4Fe(SO4)2 to
produce FeS
Nutrient Broth

- to determine the ability

of gram neg bacteria to
liberate sulfides from
ferrous ammonium sulfate
or sodium thiosulfate

-determines the ability of

an org to produce
gelatinases, which can
degrade gelatin to
peptides and amino acids
-to determine ability to
break down tryptophan

Indole
Production

Tryptone Broth

Motility Indole
Ornithine test

MIO ( Motility Indole

Ornithine)

-determine the motility of

bacteria

Reactions in
Litmus Milk

Litmus Milk

-used to determine the

action of bacteria on milk

-ferrous sulfate
(H2S formation
indi)

Tryptophan

- dehydrated skin
-litmus

-black ppt (FeS)

covers the butt
(acidic, +)
-no black ppt (-)

-Kovacs
reagent

- medium was
liquefied (+)
- medium remained
solid (-)

B.s. (+)

- A pink to red ring

develops on the
surface of the broth
(+, indole presen

E.coli (+)
E. a. (-)

- dense to no
growth (-)
- thick growth along
the area of stab
spreading to edge
of tube (+)
pink color= acidic
- purplish- blue
color= alkaline
-white color=
reduction; litmus
serves as an e
acceptor
- hard curd w/ clear

E.coli (pink;
curd & gas
prod.)
Ps.a. (purple
blue)

Nitrate
reduction test

Nitrate broth
-nitrate reacts w/
sulfanilic acid and
naphthylamine to
produce sulfobenzeneazo-naphtylamine

-used to determine if an
org is capable of reducing
nitrate(NO3) to nitrite
(NO2) or to other
nitrogenous compounds
via nitrate reductase

KNO3 (source of
nitrate)

Lysine
Decarboxylati
on and
Deamination
test

LIA (Lysine iron

agar)

-test for ability of org to

deaminate lysine (aerobic)
or decarboxylate lysine
(anaerobic)

-lysine, glucose,
peptone
-bromcresol
purple
- sodium

-Nitrate reagent
A (sulfanilic
acid)
-Nitrate reagent
B
(naphthylamine)

supernatant= acid
curd/ clot due to
pption of casein by
acid produced from
lactose
- soft curd followed
by peptonization=
rennet curd
Dissolution of clot=
peptonization due
to digestion of curd/
milk proteins
- Bubbles in
coagulated milk=
gas production
- Distinct red color
which may turn
brown rapidly is
observed after
adding the two
reagents (+, nitrate
present)
-there is change in
color to red after
adding a pinch of
zinc powder (-,
unreduced NO3 was
present and
reduced to NO2)
- Colorless after
add. Of Zn powder
(+, no NO3 to
reduce)
- Purple slant,
purple butt;
alkaline, alkaline
(Lysine
decarboxylation;

thiosulfate and
ferric ammonium
citrate

K/K)
- red slant, yellow
butt; rxn bet
ammonia & ferric
ammonium citrate,
acid (Lysine
deamination, +)
- purple slant,
yellow butt
(negative reaction)
- blackening (

H2 S
Urease Test

Stuarts Urea Broth

- Differential medium

OxidativeFermentation
test

Oxidative
Fermentation tubes

- test the ability of enzyme

to produce an exoenzyme
called urease that
hydrolyzes ammonia and
CO2; differentiates
Protaceae and Entero
- Used to determine those
org that utilize carbo
aerobically (oxidation)
from those that utilize
anaerobically
(fermentation)
-determines the presence
of bac. Enzyme,
cytochrome oxidase

Production)

Phenol red (pH

indicator)
Urea

Change in color
from bright pink to
reddish color (+)urease prod.

- Proteus
mirabilis (+)
-E. coli. (-)

-Carbohydrate
(1%)
-Peptone (0.2%)/
tryptone
containing
glucose
-bromthymol
blue (pH indi)

-no rxn- no color

change
- (+) oxidationtube w/o oil turns
yellow (top); tube
w/ oil has no
change
- (+) fermentationboth tubes turns
yellow
-Alkaline rxn- blue
color dev.=only
peptone and not
carbohydrate was
utilized

-P.
aeruginosa
(+)
- E. coli. (-)

Casein
hydrolysis
test

Skim Milk Agar

- casein- responsible
for the white color of
milk

-test to detect prod of

proteases and caseases
that digest casein to
soluble peptides which
results to clear zone

Coagulase
Test

Citrated rabbit
plasma
- coagulase- enzyme
produced by several
mircobes that enables
conversion of
fibrinogen to fibrin
Tributyrin Agar
-differential medium

-use to distinguish bet diff

types of Staphylococcus
isoletes
- to determine whether an
org coagulates or not

Tributyrin
Agar (Lipid
Utilization)

-only lipase test

- to determine lipase,
enzyme that breaks down
lipids

- White agar turns

clear and colorless
around green
colony (when
digested by
exoenzymes)lipase activity and
breakdown of
triglyceride
- Rabbit plsama

-halo surrounds the

areas (+)

-P.
aeuriginosa
(+)
- S.
marcescens
clear zone
with red
pigment
- E. coli. (-)
no hydrolysis
-S.a (+)
- S.e (-)