BIOCHEMICAL

TEST
RAM AGLUGUB, RMT
ACETAMIDE UTILIZATION
➢ability to use acetamide as the sole source of
carbon
➢produce acylamidase, which deaminates
acetamide to release ammonia resulting in an
alkaline pH
Result
✓Positive: Blue color
✓Negative: No color change
QC
✓Positive: Pseudomonas aeruginosa
✓Negative: Escherichia coli
ACETATE UTILIZATION
➢ability to use acetate (sodium acetate) as the
sole source of carbon
➢differentiate Shigella sp. from Escherichia coli
Result
✓Positive: Medium becomes alkalinized (blue)
✓Negative: No growth or no indicator change to blue
QC
✓Positive: Escherichia coli
✓Negative: Shigella sonnei
BACITRACIN (TaxoA) SUSCEPTIBILITY
➢presumptive identification and differentiation of
Streptococcus pyogenes
➢distinguish staphylococci species (resistant) from
micrococci (susceptible)
Result
✓Positive: zone of inhibition >10 mm
✓Negative: No zone of inhibition
QC
✓Positive: Streptococcus pyogenes
Micrococcus luteus
✓Negative: Streptococcus agalactiae
Staphylococcus aureus
BILE ESCULIN TEST
➢differentiates Enterococci and group D streptococci from
non–group D viridans streptococci
➢growth in the presence of 4% bile and to hydrolyze esculin
to esculetin which reacts with Fe3+ to form dark brown to
black precipitate
Result
✓Positive: Growth and blackening of the agar slant
✓Negative: Growth and no blackening of medium
QC
✓Positive: Enterococcus faecalis
✓Negative: Escherichia coli
BILE SOLUBILITY TEST
➢differentiates Streptococcus pneumonia (positive–soluble)
from alpha-hemolytic streptococci (negative–insoluble)
➢Bile or bile salt (sodium desoxycholate) rapidly lyses
pneumococcal colonies
➢Lysis depends on amidase→ autolytic enzyme
Result
✓Positive: Colony disintegrate
✓Negative: Intact colonies
QC
✓Positive: Streptococcus pneumonia
✓Negative: Enterococcus faecalis
BUTYRATE DISK
➢detect BUTYRATE ESTERASE
➢identification of Moraxella (Branhamella) catarrhalis
➢butyrate esterase hydrolyze bromochlorindolyl butyrate
that releases indoxyl, in the presence of oxygen
spontaneously forms indigo
Result
✓Positive: Development of a blue color (5-minute incubation)
✓Negative: No color change
QC
✓Positive: Moraxella catarrhalis
✓Negative: Neisseria gonorrhoeae
CHRISTIE, ATKINS, AND MUNCH-PETERSON
(CAMP) TEST
➢differentiate Streptococcus agalactiae (positive) from
other streptococcal species
➢Listeria monocytogenes (+)
➢CAMP factor acts synergistically with the beta-lysin of S.
aureus to cause enhanced lysis of RBC
Result
✓Positive: Arrowhead-shaped zone of beta-hemolysis
✓Negative: No enhancement of hemolysis
QC
✓Positive: Streptococcus agalactiae
✓Negative: Streptococcus pyogenes
 CAMP TEST
CAMP Positive Organisms:
1. Streptococcus agalactiae
2. Listeria monocytogenes
3. Proprionibacterium acnes
Reverse CAMP Positive Organisms:
1. Clostridium perfringens
2. Corynebacterium pseudotuberculosis
3. Corynebacterium ulcerans
4. Corynebacterium urealyticum
CATALASE TEST
✓differentiates catalase-positive MICROCOCCAL and
STAPHYLOCOCCAL spp. from catalase-negative
STREPTOCOCCAL spp.
✓Catalase converts hydrogen peroxide to water and
oxygen
Result
✓Positive: Copious bubbles are produced
✓Negative: No or few bubbles are produced
QC
✓Positive: Staphylococcus aureus
✓Negative: Streptococcus pyogenes
CETRIMIDE AGAR
➢isolate and purify Pseudomonas aeruginosa from
contaminated specimens
➢ability of an organism to grow in the presence of
cetrimide
Result
✓Positive: Growth, variation in color of colonies
✓Negative: No growth
QC
✓Positive: Pseudomonas aeruginosa
✓Negative: Escherichia coli
CITRATE UTILIZATION
➢ability to use sodium citrate (sole carbon source) and
inorganic ammonium salts (sole nitrogen source)
➢citrate-permease converts citrate to pyruvate
➢ammonium phosphate → ammonia and ammonium
hydroxide→ alkaline pH → bromthymol blue indicator from
green to blue
Result
✓Positive: Growth on the medium (Blue)
✓Negative: Absence of growth
QC
✓Positive: Enterobacter aerogenes
✓Negative: Escherichia coli
COAGULASE TEST
✓differentiate S. aureus (positive) from coagulase-negative
staphylococci (negative)
Two Forms of Coagulase
1. Bound Coagulase→ clumping factor
2. Free Coagulase→ extracellular protein enzyme
Result
1. Slide Test
✓ Positive: Clumping in 10 seconds or less
✓ Negative: No clumping
2. Tube Test
✓ Positive: Clot of any size
✓ Negative: No clot
QC
✓Positive: Staphylococcus aureus
✓Negative: Staphylococcus epidermidis
DECARBOXYLASE TESTS (MOELLER’S METHOD)
➢differentiate decarboxylase producing
Enterobacteriaceae from other gram-negative rods
➢ability to decarboxylate amino acid to form an amine
resulting in an alkaline pH →from orange to purple
Result pH indicator: BROMCRESOL PURPLE
✓Positive: Alkaline (purple) color change
✓Negative: No color change or acid (yellow)
QC
✓Positive:
▪ Lysine—Klebsiella pneumoniae
▪ Ornithine—Enterobacter aerogenes
▪ Arginine—Enterobacter cloacae
✓Negative:
▪ Lysine—Enterobacter cloacae
▪ Ornithine—Klebsiella pneumoniae
▪ Arginine—Klebsiella pneumoniae
DNA HYDROLYSIS (DNASE TEST AGAR)
➢distinguish Serratia sp. (positive) from Enterobacter sp.,
Staphylococcus aureus (positive) from other species, and
Moraxella catarrhalis (positive) from Neisseria sp.
➢ability to hydrolyze DNA (DNAase)
➢DNA–methyl green complex→ from green to colorless zone
Result
✓Positive: Colorless around the test organism
✓Negative: Medium remains green
QC
✓Positive: Staphylococcus aureus
✓Negative: Escherichia coli
ESCULIN HYDROLYSIS
➢presumptive identification and differentiation of
Enterobacteriaceae
➢Esculin is hydrolyzed to esculetin, which reacts with Fe3+
and forms a dark brown to black precipitate
Result
✓Positive: Blackened medium
✓Negative: No blackening
QC
✓Positive: Enterococcus faecalis
✓Negative: Escherichia coli
FERMENTATION MEDIA
➢ability to ferment carbohydrates
Andrade’s formula → differentiate enteric bacteria from
coryneforms
Result
✓ Positive: Indicator change to pink
✓ Negative: Growth, but no change in color
QC
✓ Positive, with gas: Escherichia coli
✓ Positive, no gas: Shigella flexneri
Bromocresol purple → distinguish enterococci from streptococci
Result
✓ Positive: Indicator change to yellow
✓ Negative: Growth, but no change in color
QC
✓ Positive, with gas: Escherichia coli
✓ Negative, no gas: Moraxella osloensis
FLAGELLA STAIN (WET MOUNT TECHNIQUE)
➢Presence and arrangement of flagella
QC
✓Peritrichous: Escherichia coli
✓Polar: Pseudomonas aeruginosa
✓Negative: Klebsiella pneumonia
GELATIN HYDROLYSIS
➢gelatinases hydrolyze gelatin
➢presumptive identification of Staphylococcus sp.,
Enterobacteriaceae, and some gram-positive bacilli
Result
✓Positive: Partial or total liquefaction at 4°C within 14 days
✓Negative: Complete solidification
QC
✓Positive: Bacillus subtilis
✓Negative: Escherichia coli
GROWTH at 42°C
➢differentiate a pyocyanogenic pseudomonads from other
Pseudomonas sp.
➢ability of an organism to grow at 42°C
Result
✓Positive: Good growth at both 35°and 42°C
✓Negative: No growth at 42°C but good growth at 35°C
QC
✓Positive: Pseudomonas aeruginosa
✓Negative: Pseudomonas fluorescens
HIPPURATE HYDROLYSIS
➢Hippuricase hydrolyzes hippuric acid→ GLYCINE AND
BENZOIC ACID
➢Glycine is deaminated by ninhydrin, which is reduced
during the process
➢end products of the ninhydrin oxidation react to form a
PURPLE-COLORED PRODUCT
Result
✓Positive: Deep purple color
✓Negative: Colorless or slightly yellow pink color
QC
✓Positive: Streptococcus agalactiae
✓Negative: Streptococcus pyogenes
 HIPPURATE HYDROLYSIS
1. Streptococcus agalactiae
2. Listeria monocytogenes
3. Campylobacter jejuni subsp. jejuni
4. Gardnerella vaginalis
INDOLE PRODUCTION
➢Tryptophanase hydrolyze tryptophan to form indole which then
reacts with Kovac’s reagent (dimethylamine-benzaldehyde
and hydrochloride) or Ehrlich’s reagent producing a red color
Result
Positive: Pink- to wine-colored ring
Negative: No color change
QC
A. Kovac’s Method
✓ Positive: Escherichia coli
✓ Negative: Klebsiella pneumoniae
B. Ehrlich’s Method
✓ Positive: Haemophilus influenzae
✓ Negative: Haemophilus parainfluenzae
C. Ehrlich’s Method (Anaerobic)
✓ Positive: Porphyromonas asaccharolytica
✓ Negative: Bacteroides fragilis
LEUCINE AMINOPEPTIDASE (LAP) TEST
➢presumptive identification of catalase-negative gram-
positive cocci
➢Hydolysis of Leucinebeta-naphthylamide by leucine
aminopeptidase
➢resulting beta-naphthylamine produces a red color upon
addition of cinnamaldehyde reagent
Result
✓Positive: Development of a red color within 1 minute
✓Negative: No color change or development of a slight yellow
color
QC
✓Positive: Enterococcus faecalis
✓Negative: Aerococcus viridans
LITMUS MILK MEDIUM
➢ability to metabolize litmus milk: fermentation, reduction,
clot formation, digestion, and the formation of gas
➢Fermentation of lactose → litmus turns pink
➢Sufficient acid: casein is coagulated→ solidifying the milk
➢hydrolyze casein→ straw colored
➢reduce litmus→ colorless in the bottom of the tube
QC
Fermentation: Clostridium perfringens
Acid: Lactobacillus acidophilus
Peptonization: Pseudomonas aeruginosa
LYSINE IRON AGAR (LIA)
➢Lysine Iron Aagar: lysine, peptones, a small amount of
glucose, ferric ammonium citrate, and sodium thiosulfate
➢glucose fermented→ butt becomes acidic (yellow)
➢lysine decarboxylase present, cadaverine is produced→
alkaline state (purple)
➢Oxidative deamination of lysine and in the presence of
ferric ammonium citrate and flavin mononucleotide, forms
burgundy color on the slant
pH indicator: Bromocresol Purple
▪ yellow at or below pH 5.2
▪ purple at or above pH 6.8
Result
✓Alkaline slant/alkaline butt (K/K)—lysine decarboxylation
and no fermentation of glucose
✓Alkaline slant/acid butt (K/A)—glucose fermentation
✓Red slant/acid butt (R/A)—lysine deamination and
glucose
QC
✓Alkaline slant and butt: H2S positive: Citrobacter freundii
✓Alkaline slant and butt: Escherichia coli
✓Alkaline slant and butt: H2S positive: Salmonella
typhimurium
✓Red slant, acid butt: Proteus mirabilis
METHYL RED/VOGES-PROSKAUER (MR-VP) TESTS
➢ability to produce and maintain stable acid end products from
glucose fermentation, to overcome the buffering capacity of the
system, and to determine the ability to produce 2,3-butanediol
or acetoin from glucose fermentation
METHYL RED
→detects mixed acid fermentation that lowers the pH
→red at pH 4.4 and yellow at pH 6.2
VP
→detects the ability to convert the acid products to acetoin and
2,3-butanediol→ alpha-naphthol is added, followed by potassium
hydroxide (KOH)→ red
QC
MR positive/VP negative: Escherichia coli
MR negative:/VP positive: Enterobacter aerogenes
MICRODASE TEST (MODIFIED OXIDASE)
➢differentiate Staphylococcus from Micrococcus spp. by
detection of the enzyme oxidase
➢oxidase reacts with the oxidase reagent and cytochrome
C to form the indophenol
Result
✓Positive: Development of blue to purple-blue color
✓Negative: No color change
QC
✓Positive: Micrococcus luteus
✓Negative: Staphylococcus aureus
MOTILITY TESTING
➢diffuse zone of growth extending out from the line of
inoculation
Result
✓Positive: Spread out from the site of inoculation
✓Negative: Remain at the site of inoculation
QC
✓Positive: Escherichia coli
✓Negative: Staphylococcus aureus
MRS Broth
➢determine gas formation during glucose fermentation
➢Lactobacillus spp. and Leuconostoc sp. produce gas
➢Selective medium: sodium acetate and ammonium
citrate
Result
✓Positive: Leuconostoc sp.—Growth, gas production indicated by
a bubble in the Durham tube
✓Positive: Lactobacillus spp.—Growth, no gas production
✓Negative: No growth
QC
✓Positive: Lactobacillus lactis
✓Negative: Escherichia coli
4-Methylumbelliferyl-β-D-Glucuronide
(MUG) Test
➢presumptively identify various genera of
Enterobacteriaceae and verotoxin-producing E. coli (neg)
➢β-d-glucuronidase hydrolyzes 4-methylumbelliferyl-β-d-
glucuronide to 4-methylumbelliferyl which fluoresces blue
under long wavelength UV
Result
✓Positive: Electric blue fluorescence
✓Negative: Lack of fluorescence
QC
✓Positive: Escherichia coli
✓Negative: Klebsiella pneumoniae
NITRATE REDUCTION
➢nitrate reductase converts the nitrate (NO3) to nitrite (NO2)
➢reduction is determined by adding sulfanilic acid and alpha-
naphthylamine
➢sulfanilic acid and nitrite react to form a diazonium salt→
couples with the alphanaphthylamine to produce a red,
water-soluble azo dye
➢Zinc is added to validate colorless result for the presence of
untreated nitrate
Result
✓Positive: Red
✓Negative: No color change
QC
✓Positive: NO3+, no gas: Escherichia coli
✓Positive: NO3+, gas: Pseudomonas aeruginosa
✓Negative: Acinetobacter baumannii
NITRITE REDUCTION
➢reducing nitrite to nitrogen do not turn color and do
produce gas in the nitrate reduction test
Result
✓Positive: No color change to red 2 minutes; gas production in
Durham tube
✓Negative: Broth becomes red ; no gas production is observed
QC
✓Positive: Proteus mirabilis
✓Negative: Acinetobacter baumannii
o-Nitrophenyl-β-D-Galactopyranoside (ONPG) Test
➢ability to produce β-galactosidase which hydrolyzes
substrate ONPG to form orthonitrophenol (yellow product)
➢distinguishes late LLF from NLF of Enterobacteriaceae
➢Indicates presence of β-galactosidase
Result
✓Positive: Yellow (presence of β-galactosidase)
✓Negative: Colorless
QC
✓Positive: Shigella sonnei
✓Negative: Salmonella typhimurium
OPTOCHIN (Taxo P) SUSCEPTIBILITY TEST
➢aka ETHYL HYDROCUPREINE HYDROCHLORIDE
➢lyses Streptococcus pneumoniae (positive test), but
alpha-streptococci are resistant (negative test)
➢zone of 14 to 16 mm is considered susceptible
➢Presumptive identification of Streptococcus pneumoniae
Result
✓Positive: ZOI ≥ 14 mm in diameter, with 6-mm disk
✓Negative: No zone of inhibition
QC
✓Positive: Streptococcus pneumoniae
✓Negative: Streptococcus pyogenes
OXIDASE TEST (KOVAC’S METHOD)
➢presence of cytochrome oxidase which oxidize
tetramethyl-p-phenylenediamine dihydrochloride to
indophenol (dark purple)
➢identification of oxidase-negative Enterobacteriaceae,
differentiating them from other Gram (-) bacilli
Result
✓Positive: Development of a dark purple color within 10
✓Negative: Absence of color
QC
✓Positive: Pseudomonas aeruginosa
✓Negative: Escherichia coli
Oxidation/Fermentation (of) Medium (CDC
Method)
➢ability to oxidize or ferment specific carbohydrates
➢glucose,xylose, mannitol, lactose, sucrose, and maltose
Hugh and Leifson’s formula
→low peptone-to-carbohydrate ratio and a limiting amount of
carbohydrate.
→reduced peptone limits the formation of alkaline amines that may mask
acid production resulting from oxidative metabolism
Carbohydrate Fermentation Medium
A. Base Medium→ sugar-free
a. Peptone water
b. Trypticase
c. Tryptone Broth
B. pH Indicators
a. Bromcresol Purple → Purple (alk) to Yellow(acid) at pH 6.3
b. Andrade’s Acid Fuchsin→ Pale Yellow (alk) to Reddish Pink
(acid) at pH 5.5
c. Phenol Red → Red (alk) to Yellow at pH 7.9
Other Methods
1. Rusell’s Double Sugar (RDS)
→conatins glucose and lactose with Andrade’ Acid Fuchsin
2. Smith Fermentation Tube
Result
✓Positive: Acid production (A) indicated by the color
indicator changing to yellow
✓Weak-positive (Aw): Weak acid formation
✓Negative: Red or alkaline (K) color in the deep
✓No change (NC) or neutral (N)
QC
✓Fermenter (Glucose): Escherichia coli
✓Oxidizer (Glucose): Pseudomonas aeruginosa
PHENYLALANINE DEAMINASE AGAR
➢ability to oxidatively deaminate phenylalanine to
phenylpyruvic acid
➢Morganella, Proteus, and Providencia can be
differentiated from other members of the
Enterobacteriaceae family
➢Phenylpyruvic acid → detected by adding a few drops of
10% FERRIC CHLORIDE→ green colored complex
Result
✓Positive: Green color on slant
✓Negative: Slant remains original color
QC
✓Positive: Proteus mirabilis
✓Negative: Escherichia coli
L-PYRROLIDONYL ARYLAMIDASE (PYR) TEST
➢presumptive identification of Streptococcus pyogenes and
enterococci by the presence of L-pyrrolidonyl arylamidase
➢L-pyrrolidonyl arylamidase hydrolyzes L-pyrrolidonyl- β-
naphthylamide to produce a β-naphthylamine which can be
detected in the presence of N,N-
methylaminocinnamaldehyde reagent → bright red
precipitate
Result
Positive: Bright red color within 5 minutes
Negative: No color change or an orange color
QC
Positive: Enterococcus faecalis
Streptococcus pyogenes
Negative: Streptococcus agalactiae
PYRUVATE BROTH
➢ability of an organism to utilize pyruvate
➢differentiation between Enterococcus faecalis (+) and
Enterococcus faecium (-)
➢Pyruvic acid→ added to the broth to determine whether
the microorganism is able to use pyruvate
➢Bromthymol blue indicator changes from blue to yellow
Result
✓Positive: Indicator changes from green to yellow
✓Negative: No color change
QC
✓Positive: Enterococcus faecalis
✓Negative: Streptococcus bovis
SALT TOLERANCE TEST
➢ability of an organism to grow in high concentrations of
salt
➢differentiate enterococci (+) from nonenterococci (-)
➢Medium: Brain-Heart Infusion Broth containing 6.5% NaCl
➢Indicator: Bromcresol Purple
Result
✓Positive: Visible turbidity in the broth, with or without a color
change from purple to yellow
✓Negative: No turbidity and no color change
QC
✓Positive: Enterococcus faecalis
✓Negative: Streptococcus bovis
SPOT INDOLE TEST
➢determine the presence of tryptophanase
➢Tryptophanase breaks down tryptophan → indole,
detected by its combination with certain aldehydes to
form a colored compound
➢Indole + Cinnamaldehyde = blue-green compound
Result
✓Positive: Development of a blue color within 20 seconds
✓Negative: No color development or slightly pink color
QC
✓Positive: Escherichia coli
✓Negative: Klebsiella pneumoniae
TRIPLE SUGAR IRON AGAR (TSI)
➢determines whether Gram (-) rod ferments glucose and
lactose or sucrose and forms hydrogen sulfide (H2S)
➢differentiate members of the Enterobacteriaceae family
from other gram (-) rods
➢Composition: 10 parts Lactose, 10 parts sucrose, 1 part
glucose and peptone
➢Phenol Red→ pH indicator
➢Ferrous Ammonium Sulfate→ H2S Indicator
➢CO2 and hydrogen gas (H2) → presence of bubbles or
cracks or by separation of the agar
Result
✓Alkaline slant/ Alkaline butt (K/K)→ glucose, lactose, and
sucrose nonutilizer;
✓Alkaline slant/Acid butt (K/A)→ glucose fermentation only
✓Acid slant/Acid butt (A/A)→ glucose, sucrose, and/or lactose
fermenter
✓Black Precipitate→ production of ferrous sulfide and H2S gas
(H2S+)
✓Bubbles or cracks→ gas production
QC
✓ gas production: Escherichia coli
✓K/A, +/− gas production, H2S+: Salmonella typhimurium
✓K/K: Pseudomonas aeruginosa
✓K/A, H2S+: Proteus mirabilis
✓K/A: Shigella flexner
UREASE TEST (CHRISTENSEN’S METHOD)
➢ability to produce urease, which hydrolyzes urea to ammonia
and CO2
➢Proteus sp. → rapidly hydrolyze urea
➢ammonia alkalinizes the medium
➢pH Indicator: PHENOL RED → from light orange at pH 6.8 to
magenta (pink) at pH 8.1
Result
✓Positive: Change in color of slant from light orange to magenta
✓Negative: No color change
QC
✓Positive: Proteus vulgaris
✓Weak positive: Klebsiella pneumonia
✓Negative: Escherichia coli
X and V FACTOR TEST
➢differentiation Haemophilus species
Result
✓Positive:
▪ Growth around the XV disk → requirement for both factors
▪ Growth around the V disk, no growth around the X disk, and light
growth around the XV disk shows a V factor requirement
✓Negative: Growth over the entire surface of the agar indicates
no requirement for either X or V factor
QC
✓Haemophilus influenza→ halo of growth around the XV disk, no
growth on the rest of the agar surface
✓Haemophilus parainfluenzae→ halo of growth around the XV
and V disks
✓Haemophilus ducreyi → halo of growth around the XV and X
disks