Kaori Marie Sembrano

BIOCHEMICAL TESTS

REACTION/EXPECTED INCUBATION Medium

TEST PURPOSE Example of Organisms tested INDICATOR
REQUIREMENT
RESULT Used
Green (-)
Ability to use
Royal Blue (+)
Acetamide Acetamide as source (substrate) Acetamide – Escherichia coli Bromthymol 35-37*C for up
Utilization of Carbon Acylamidase (ep) Ammonia Blue Indicator to 4 days
Pseudomonas aeruginosa
*Deamination rxn Streptophomonas
maltophila
Ability to use Acetate
Green (-)
as source of Carbon Growth on a medium with Royal Blue (+)
Bromthymol 35-37*C for up
Acetate Utilization Sodium Acetate, in w/c the
Shigella sonnei Blue Indicator to 7 days
Diff. Shigella spp. organisms utilizes Acetate Escherichia coli
From Escherichia coli
Shigella flexneri
Presumptive Id. And
diff. of β-hemolytic
18-24 hrs at 35-
GROUP A 0.04 U of
(+) (-) 37*C:
Streptococci (Strep. bacitracin in
- in ambient air
Bacitracin pyogenes) from other Bacitracin inhibits the synthesis
Streptococcus pyogenes Streptococcus agalactiae
the FIRST
for staphylococci Blood Agar
Susceptibility groups of the spp. of bacterial cell walls QUADRANT
- in 5-10% CO2
Micrococcus luteus Staphylococcus aureus for streptococci
Distingish >10 mm ZOI
diff.
Staphylococci spp.
From Micrococci
Sodium desoxycholate (bile
salt) lowers the surface tension
Intact colonies (-)
between the bacterial cell
membrane and the medium; Colony disintegration
Diff. Streptococcus Enterococcus faecalis
helps accelerate the organisms (+) 10% Sodium 35-37*C for 30 5% Sheep blood
Bile Solubility Test pneumoniae from α-
natural autolytic process. desoxy- minutes agar
hemolytic streptococci Streptococcus agalactiae
Streptococcus pneumoniae cholate
Presence of Amidase= autolytic
Streptococcus mitis
enzyme in pneumococcal
colonies
Streptococci and Enterococci
Presumptive Id. Of are not inhibited by Bile salts.
Enterococci from the (the rest of the gram (+) are
Streptococcus Bovis inhibited only) Growth but no color
Group Growth and presence of change (-)
Growth in the presence of 4% Ferric
dark brown/black ppt (+) 35-37*C for 48
Bile Esculin Test Diff. Enterococci and bile and hydrolysis of Escherichia coli
ammonium
hrs
Beef extract
(substrate) Esculin to (ep) citrate
Group D Enterococcus faecalis
Streptococci from the Esculetin. Streptococcus pyogenes
rest of the Group-D
3+
viridans Streptococci Esculetin reacts with Fe to
form a dark brown/black ppt
Detect Butyrate
esterase to aid the
Blue color (+)
identification of (substrate) Bromochlorindolyl indigo (-)
Butyrate Disk Moraxella butyrate – butyrate esterase  RT for 5 minutes
Moraxella catarrhalis
(Branhamella) (ep) Indoxyl Neisseria gonorrhoeae
catarrhalis
Kaori Marie Sembrano
REACTION/EXPECTED INCUBATION Medium
TEST PURPOSE Example of Organisms tested INDICATOR
REQUIREMENT
RESULT Used

“ Christie-Atkins- Group B-streptococci produces Arrow-head zone (+) No arrowhead/

Munch-Peterson” Test CAMP factor (haemolytic enhancement of
Arrowhead
protein) that acts with β-lysin Streptococcus agalactiae hemolysis (-) 35-37*C in Sheep blood
CAMP Test Diff. Group B- Staphylococcus aureus to
enhanced
ambient air agar
hemolysis
streptococci from cause an enhanced lysis of the Listeria monocytogenes Streptococcus pyogenes
other streptococci spp. rbc

2 toxins produced by aeorobic

Diff. catalase (+) and facultative anaerobes:
micrococcal and -2 bubbles (+) no bubble (-)
H2O2 & superoxide (O )
Catalase Test staphylococcal spp. 30% H2O2
From catalase (–_ Staphylococcus aureus Streptococcus pyogenes
streptococcal spp. (substrate) H2O2 – catalase 
H2O & oxygen

Isolate and purify

Cetrimide inhibits the growth of Growth and color change
Pseudomonas Cetrimide
bacteria by releasing (ep) (+) No growth and no
auruginosa (cetyl-
Nitrogen and Phosphorus change (-) 35-37*C for up
Cetrimide Agar Pseudomonas aeruginosa
trimethyl-
to 7 days
Determine the org. ammonium
P. auruginosa is resistant to (yellow-green to blue-green Escherichia coli
ability to grow in the bromide)
cetrimide colonies)
presence of Cetrimide

ID of org. using
Sodium citrate for
(substrate) Citrate – citrate
carbon source, and Blue color (+)
permease  (ep) pyruvate
Ammonium salts for
Green (-)
nitrogen source Enterobacter arogenes Bromthymol 35-37*C for up Simmons‟ Citrate
Citrate Utilization (substrate) Ammonium
blue to 7 days agar
Phosphate – citrate Escherichia coli
Part of IMViC to diff. Kliebsiella pneumoniae
(permease) (ep) ammonia
Enterobacteraceae
and ammonium OH
from other gram (–)
rods

2 forms of coagulase: Bound &

Free coagulase

Bound: Slide test:: “clumping

factor” is bound to the bacterial
cell wall and reacts directly with
fibrinogen ; precipitation of
fibrinogen on the staph. Cell=
Diff. Staphylococcus Macroscopic clumping / No clumping / no clot (-)
clumping
aureus (+) from clot (+)
Coagulase Test coagulase (-) Staphylococcus
Free: Tube test:: “an
staphylococci Staphylococcus aureus epidermidis
extracellular CHON enzyme”
that forms a clot when S.
aureus is incubated with plasma
; clotting mech. Is due to
activation of CRF (coagulase-
reacting factor) that reacts with
fibrinogen to form a fibrin clot
Kaori Marie Sembrano
REACTION/EXPECTED INCUBATION Medium
TEST PURPOSE Example of Organisms tested INDICATOR
REQUIREMENT
RESULT Used
(acid) yellow color (-)
“Moeller‟s method” Amino acid – decarboxylase  (alk) Purple color (+)
amine
Lysine- Enterobacter
Diff. decarboxylase- Lysine- Klebsiella 35-37*C for up
cloacae Bromcresol *dextrose= sugar
Decarboxylase Test producing Decarboxylation pneumoniae
Ornithine- Klebisella purple
to 4 days in
base
Enterobacteriaceae - 3 CHONs used: Ornithine- Enterobacter ambient air
pneumoniae
from other gram (-) Lysine, Ornithine, Arginine aerogenes
Arginine- Klebsiella
rods (LOA) Arginine- Enterobacter
pneumoniae
cloacae

diff. through the

prodxn of
Deoxyribonuclease
No color (+)
Medium remains green
distinguishes:
(-)
Serratia spp. from (substrate) DNA – Staphylococcus aureus Aerobically at
DNA Hydrolysis Enterobacter sp. deoxyribonuclease  (ep) Methyl green 35-37*C for 13- DNase agar
Escherichia coli
DNA-methyl green complex Serratia spp. 24 hrs
Staphylococcus
Neisseria spp.
aureus from other spp. Moraxella catarrhalis

Moraxella catarrhalis
from Neisseria spp.

Hydrolysis of (substrate) presence of dark No ppt and presence of

Esculin to (ep) Esculetin. brown/black ppt and loss fluorescence under
Ferric
Diff. and Id of of fluorescence under wood’s lamp (-) 35-37*C for up
Esculin Hydrolysis Enterobacteriaceae 3+ wood’s lamp (+)
ammonium
to 7 days
Esculetin reacts with Fe to citrate
Escherichia coli
form a dark brown/black ppt Enterococcus faecalis
ANDRADE’S FORMULA BROMCRESOL PURPLE
Diff. base on the ability
to ferment CHO
*Andrade’s
With durham‟s tube to
formula: 35-
capture gase during 3 basal CHO: glucose, Purple (-) *Andrade‟s
Pink (+) 37*C for up to 7
metabolism lactose, sucrose Yellow formula :
Yellow (+) *Andrade‟s days in ambient
* Andrade’s formula /straw (-) Strepto- peptone
Escherichia formula air
Fermentation Media (uses dextrose): Andrade’s formula: yellow (-) coccus medium
coli (w/ gas) Escherichia *Bromcresol *Bromcresol
Diff. Enterics form to pink (+)= acid is produced Pseudo- mitis *Bromcresol
coli (w/ gas) purple purple : 35-
Coryneforms Bromcresol purple: purple (-) monas purple : BHI
Shigella 37*C for up to 4
*Bromcresol purple to yellow (+) = acid is produced aeruginosa Moraxella broth
flexneri days in ambient
(uses sorbitol): osloenis
air
Diff. enterococci from
streptococci

Prodxn of Gelatinase

Presumptive Id for Partial or complete

Solid (-)
Staphylococcus spp., liquefaction (+)
Gelatin Hydrolysis Enterobacteriaceae,
Gelatinase liquefies gelatin
Escherichia coli
and some gram (+) Bacillus subtilis
bacilli
Kaori Marie Sembrano
REACTION/EXPECTED INCUBATION Medium
TEST PURPOSE Example of Organisms tested INDICATOR
REQUIREMENT
RESULT Used
(substrate) Hippuric acid –
hippuricase  (ep) glycine &
benzoic acid Deep purple color (+) yellow/colorless (-) 0.2 ml
Hippurate Presence of
ninhydrin 35* for 2 hrs
Hydrolysis Hippuricase
Glycine- ninhydrin  purple Streptococcus agalactiae Streptococcus pyogenes reagent
colored product

KOVAC’S RGNT EHRLICH’S RGNT

Presence of Yellow
Pink/wine
Tryptophanase (substrate) Tryptophan – color (-)
color (+)
tryptophanase  Indole,
* Kovac’s reagent – pyruvate, ammonia Haemo-
Pink/wine Yellow Haemo-
dimethylamine- philus
color (+) color (-) philus Kovac‟s rgnt Tryptophane
Kovac’s rgnt- for para-
Indole Production benzaldehyde & influenzae
influenzae Ehrlich‟s rgnt broth
hydrochloride Enterobacteriaceae
Escherichia Klebsiella (an-
* Ehrlich’s reagent – (anaerobic)
coli pneumoniae aerobic)
same with kovac‟s + Ehrlich’s rngt- for other gram Porphy-
ethyl alchohol ; more (-) bacilli romonas
Bacteroides
sensitive asaccharo-
fragilis
lytica

(substrate) Leucine β-
Presumptive ID of
naphtylamide – leucine
Leucine catalase (-) gram (+)
aminopeptidase  β- red color (+) Yellow color (-) Cinna-
cocci
Aminopeptidase naphthylamine maldehyde RT for 5 mins
(LAP) test Enterococcus faecalis Aerococcus viridans rgnt
Detection of Leucine
β-naphthylamine reacts with
aminopeptidase
cinnamaldehyde = red color (+)
Components of LIA: Lysine,
peptones, Glucose, Ferric
ammonium citrate, and Sodium
thiosulfate

Butt: (substrate) Lysine – lysine (K/K)

decarboxylase  Cadaverine {purple/purple) :
(purple) ; (neut. The organic lysine
acids formed by glucose decarboxylation
Diff gram (-) bacilli
fermentation) and the butt but no glucose (R/A)
based on
reverts to alkalinity fermentation (K/A) {red/yellow) :
decarboxylation
(anaerobic butt) and - if decarboxylase is not {purple/yellow) : Lysine 35-37 *C in tight
Lysine Iron Agar deamination (aerobic produced, the butt remains Escherichia coli glucose deamination & Bromcresol cap tube and in
(LIA) slant) of lysine and the acidic (yellow) fermentation only glucose purple ambient air for
(w/ H2S) fermentation 18-24 hr
formation of H2S Slant: (substrate) Lysine –lysine Shigella flexneri
deaminase + ferric ammonium Citrobacter Proteus mirabilis
citrate & flavin mononucleotide freundii
 burgundy/red color
-if deamination does not occur, Salmonella
slant remains purple (-) typhirium

Alk slant/Alk butt (K/K)

Alk slant/Acid butt (K/A)
Red slant/Acid butt (R/A)
Kaori Marie Sembrano
REACTION/EXPECTED INCUBATION Medium
TEST PURPOSE Example of Organisms tested INDICATOR
REQUIREMENT
RESULT Used
MR detects mixed acid
Diff. members of the fermentation that lowers the pH
Enterobacteriaceae of the broth (red- acidic) ;
family (yellow/orange- negative result)
Methyl Red
MR (-) / VP (+)
Maintenance of stable Voges-Proskauer detects the (MR) = pH 4.4
Methyl-Red/ Voges- {yellow/red)
acid end products from organisms ability to convert acid MR (+) / VP (-) {red/yellow) MR should not
Sugar base:
Proskauer Test glucose fermentation products to acetoin & 2,3-
Enterobacter aerogenes
Secondary be read before
Dextrose
(MRVP) butanediol Escherichia coli rgnt for VP: α- 48 hrs
Detect other naphthol &
Enterobacter cloacae
organisms that 2 types of VP: 40% KOH
produce neutral end VP test (Barritt‟s method) for
prods: 2,3-butanediol gram (-) rods
& acetoin VP test (Coblentz method) for
streptococci
(substrate) β- d-glucopyranosid
uronic acid -- β-d- Lack of fluorescence (-)
4-Methyl- Presumptively identify
glucuronidase  (ep) aglycons Electric blue fluorescence
various genera 35-37 C in a
umbelliferyl-β-D- & d-glucuronic acid (+) Kliebsiella pneumoniae
Enterobacteriaceae & closed container
Glucuronide (MUG) verotoxin-prod. for 2 hrs
*hydrolysis of substrate turns to Escherichia coli Verotoxin-prod.
test Escherichia coli
4-methylumbelliferyl (fluoresces Escherichia coli
blue under UV light)
Does not require addxn of Zinc
No color change= Yellow red color w/o gas (-):
Determine the org. can dust Sol. A=
color with gas (+) :
reduce NITRITE TO sulfanilic acid 35-37 C for 48
Nitrite Reduction GASEOUS No color change after addxn of
Acinetobacter baumanii
Sol. B= α- hrs
BHI broth
Proteus mirabilis
NITROGEN reagents/ during reduction of naphthylamine
Alcaligenes piechardii
nitrite to gaseous nitrogen
Alcaligenes faecalis
Determine the ability
(substrate) Nitrate – nitrate
to reduce NITRATE to NO3+, no gas:: red color
reductase  (ep) Nitrite +
NITRITE (+) : Escherichia coli Sol. A=
sulfanilic acid  diazonium salt No color (-)
sulfanilic acid 35-37 C for 48
Nitrate Reduction + α-naphthylamine = red, water Nitrate broth
All Enterobacteriaceae NO3+, with gas:: red color Sol. B= α- hrs
soluble azo dye Acinobacter baumannii
family reduce Nitrate (+) : Pseudomonas naphthylamine
aeruginosa
Requires addxn of Zinc dust
Supportive test only
(substrate) ONPG -- β-
Determine the ability galactosidase 
to produce β- Orthonitrophenol (O-
galactosidase nitrophenol) : yellow cmpd Yellow color (+)
O-Nitrophenyl-β-D- No color (-)
37*C in ambient
Galactopyranoside The test distinguishes Lactose fermenters must able Proteus vulgaris
Salmonella typhimurium
ONPG disk
air for 4 hrs
(ONPG) Test late lactose fermenters to transport (β-galactoside
from non-lactose permease) and hydrolyse (β- Shigella sonnei
fermenters of galactosidase ) the
Enterobacteriaceae lactose  glucose and
galactose
Determine the effect of Optochin interferes ATPase and (-)
(+) ZOI: 14-16
Optochin ( Ethyl the prodxn of ATP.
Optochin (P Disk) Hydrocupreine
mm is 5% Sheep blood
Streptococcus pyogenes
Susceptibility Test Streptococcus pneumoniae considered agar plate
Hydrochloride) to the Optochin lyses Pneumococci .
susceptible
organism α-Streptococci are resistant Streptococcus mitis
Kaori Marie Sembrano
REACTION/EXPECTED INCUBATION Medium
TEST PURPOSE Example of Organisms tested INDICATOR
REQUIREMENT
RESULT Used
„Kovac‟s Method”

Determines the
presence of * avoid using
(substrate) Tetramethyl-p-
CYTOCHROME Dark purple color (+) 1% nickel-base alloy
phenylenediamine
OXIDASE activity for No color (-) tetramethyl-p- wires containing
Oxidase Test dihydrochloride – Cytochrome 10 seconds
the identification of Pseudomonas aeruginosa phenylene- chromium and
oxidase  (end product)
oxidase (-) Escherichia coli diamine Iron = false
Indophenol
Enterobacteriaceae, Neiseria gonorrhoeae positive results
differentiating them
from other gram (-)
bacilli
Deamination
Determine the ability
End products: Ammonia and
to oxidatively Green color-complex (+) Yellow color (-) 35-37*C in
Phenylalanine deaminate
Phenylpyruvic acid 10% ferric
ambient air with
Deaminase Test chloride
Phenylalanine to Proteus mirabilis Escherichia coli loose cap
Phenylpyruvic acid is detected
phenylpyruvic acid
with 10% ferric chloride
(substrate) L-pyrrolidonyl-β-
Presumptive
naphthylamide -- L-pyrrolidonyl
identification of
arylamidase (end product) β- Bright red ppt (+)
L-Pyrrolidonyl GROUP A N,N- methyl-
Naphthylamine Orange color (-)
STREPTOCOCCI and amino- @ RT for 2
Arylamidase Test ENTEROCOCCI by
Enterococcus faecalis
cinnamaldehy minutes
disk
(PYR) β-naphthylamine is reactive with Streptococcus agalactiae
the presence Of L- de
cinnamaldehyde Streptococcus pyogenes
Pyrrolidobyl
Arylamidase
(substrate) Tryptophan –
Tryptophanase (end product)
None/Pink color (-)
INDOLE 1% para-di-
Determine the Blue green (+)
methyl-amino-
Spot Indole Test presence of
Indole reacts with
Klebsiella pneumoniae
cinnamaldehy
30 seconds x
TRYPTOPHANASE Escherichia coli
Cinnamaldehyde de
Enterobacter cloacae
color change within 20 seconds

Alk slant/ no change Butt

(K/K) : Glucose, Lactose and
Sucrose Non-Utilizer
Determine gram (-)
rods ferment
GLUCOSE and Alk Slant/ Acid Butt (K/A):
K/A (red/yellow):
LACTOSE or Glucose Fermentation Only
K/K (Red/Red): A/A (yellow all) 10 parts lactose:
SUCROSE and forms Phenol Red 35-37*C in
Triple Sugar Iron H2S Acid Slant/Acid butt (A/A):
with gas: Shigella flexneri
and Ferrous ambient air for
10 parts sucrose:
Test (TSI) Pseudomonas 1 part glucose
Glucose, Sucrose, and/or Sulfate 18-24 hr
aeruginosa Escherichia coli *with H2S: and peptone
Diff. Lactose Fermenter
Proteus vulgaris
Enterobacteriaceae
from the rest of gram *Black ppt indicates H2S gas
(-) rods and ferrous sulphide prodxn

*Bubbles or cracks indicate

prodxn of H2 or CO2
Kaori Marie Sembrano
REACTION/EXPECTED INCUBATION Medium
TEST PURPOSE Example of Organisms tested INDICATOR
REQUIREMENT
RESULT Used

“Christensen‟s Orange (+)

Hydrolysis of Urea via Urease Red (-) 35-37*C in
Method” *sugar base-
Urease Test Proteus vulgaris
Phenol Red ambient air for
dextrose
End product: Ammonia and CO2 Escherichia Coli 48 hr-7 days
Determines the ability Klebsiella pneumoniae
to produce UREASE
Impregnated Disks with X,V, or
Differentiation of Growth around XV disk only- Haemophilus influenzae
both are placed directly on to BHI Agar
Haemophilus spp
the confluent inoculation Tryptic Soy Agar
Growth around XV and V disks – Haemophilus 35-37*C in
X and V Factor Test X factor- Hemin. parainfluenzae
XV,X, V Disks
ambient air
Haemophilus
The organism will grow only Agar
V factor- Nicotinamide
around the disk that provides Nutrient Agar
Adenine Dinucleotide Growth around XV and X disks – Haemophilus ducreyi
the appropriate factor for growth