RHICabral | 2014

STAINING TECHNIQUES
stain

Gram

acid fast

capsule

capsule 2

primary stain

crystal violet

carbol fuchsin

crystal violet

Mordant

Gram's iodine
95% ethanol

tap water

secondary
stain

safranine

20% copper
sulfate
20% copper
sulfate

decolorizer

3% HCl in 95%
ethanol
methylene
blue

spore
malachite
green
-

safranin

purple cells
with white
halos
purple cells
only
B. subtilis,
some E. coli, P.
aeruginosa,
Salmonella, S.
marcescens,
some S. aureus

green
endospore
inside red cell

positive result

purple

red

light blue halo

around blue ell

negative result

pink

blue

blue cell only

Mycobacteriu
m, Nocardia

B. subtilis,
some E. coli, P.
aeruginosa,
Salmonella, S.
marcescens,
some S. aureus

positive
organisms

source: Penuliar | 2014

B. subtilis, S.
aureus

red cells only

B. subtilis

RHICabral | 2014
BIOCHEMICAL TESTS
test

starch
hydrolysis

citrate
utilization

lactose
fermentation

gelatin
hydrolysis

indole test

substrate

enzyme

product

indicator

positive
organisms

starch

-amylase
(exo), oligo1,6glucosidase
(exo)

disaccharides,
glucose

Lugol's iodine

B. subtilis

citrate

citrate
permease
(endo), citrase

pyruvic acid,
oxaloacetic
acid, carbon
dioxide

bromothymol
blue

E. coli, P.
aeruginosa,
some
Salmonella

lactose

lactose
permease
(endo), galactosidase
(endo)

glucose,
galactose

phenol red

E. coli (acid +
gas), S. aureus
(acid only)

gelatin

gelatinase
(exo)

amino acids

10% mercuric
chloride

B. subtilis, P.
vulgaris, P.
aeruginosa, S.
marcescens, S.
aureus

tryptophan

tryptophanase
(endo)

indole,
pyruvate,
ammonia

Ehrlich reagent
(dimethylamin
obenzaldehyd
e)

E. coli, P.
vulgaris

fructose 6phosphate

phenol red

B. subtilis, E.
coli,
Salmonella, S.
aureus

amino acids

10% mercuric
chloride

B. subtilis

ferric
ammonium
citrate, phenol
red

B. subtilis (A/K)
E. coli (A/AG)
P. vulgaris
(K/AG+)
P. aeruginosa
(K/K)
S. typhimurium
(K/AG+)
S. marcescens
(K/A)
S. aureus (A/A)

mannitol
hydrolysis

mannitol

casein
hydrolysis

casein

triple sugar
iron test

dextrose
(0.1%), sucrose
(1%), lactose
(1%), sodium
thiosulfate,
peptones

source: Penuliar | 2014

mannitol
permease,
mannitol-1phosphate-5dehydrogenas
e
caseinase
(exo)
sucrase, galactosidase,
glycolytic
enzumes,
thiosulfate
reductase,
cysteine
desulfurase

pyruvic acid,
lactic acid,
carbon
dioxide,
hydrogen gas,
hydrogen
sulfide, ferrous
sulfide

RHICabral | 2014
TRIPLE SUGAR IRON TEST INTERPRETATION
results

symbol

interpretation

red/yellow

K/A

yellow/yellow

A/A

red/red
red/no color change

K/K
K/NC

yellow/yellow with bubbles

A/AG

red/yellow with bubbles

red/yellow with bubbbles
and black precipitate
red/yellow with black
precipitate
yellow/yellow with black
precipitate

K/AG

glucose fermentation, peptone catabolized

glucose and lactose and/or sucrose
fermentation
no fermentation, peptone catabolized
no fermentation, peptone used aerobically
glucose and lactose and/or sucrose
fermentation, gas produced
glucose fermentation only, gas produced
glucose fermentation only, gas poduced,
hydrogen sulfide produced
glucose fermentation only, hydrogen sulfide
produced
glucose and lactose and/or sucrose
produced, hydrogen sulfide produced

source: Penuliar | 2014

K/AG+
K/A+
A/A+

positive
organisms
S. marcescens
S. aureus
P. aeruginosa
E. coli
P. vulgaris, S.
typhimurium
-

RHICabral | 2014

test/organism

B. subtilis

E.coli

P. vulgaris

P. aeruginosa

S.
typhimurium

S.
marcescens

S. aureus

SUMMARY OF BIOCHEMICAL TESTS FOR EACH TEST ORGANISM

starch hydrolysis
citrate utilization
lactose fermentation
gelatin hydrolysis
indole test
mannitol hydrolysis
casein hydrolysis
triple sugar iron test

(+)
(+)
(-)
(+)
(-)
(+)
(+)
A/K

(-)
(-)
(+)
(-)
(+)
(+)
(-)
A/AG

(-)
(+)
(-)
(+)
(+)
(-)
(-)
K/AG+

(-)
(-)
(-)
(+)
(-)
(-)
(-)
K/K

(-)
(+)/(-)
(-)
(-)
(-)
(+)
(-)
K/AG+

(-)
(-)
(-)
(+)
(-)
(-)
(-)
K/A

(-)
(-)
(+)
(+)
(-)
(+)
(-)
A/A

source: Penuliar | 2014