Biochemical Test Flashcards
biochemical reactions bacteria use to break down organic compounds and reactions they use to synthesize new bacterial parts from the resulting carbon skeleton.
Bacterial Metabolism
a. Diagnostic schemes analyze each unknown
microorganism for:
- utilization of various substrates as a carbon
source - production of specific end products from
various substrates - production of an acid or alkaline pH in the
test medium
● Breakdown of chemical substrate through the degradative process of catabolism coupled with oxidation-reduction reactions
Energy Production
Bacteria use biochemical pathways to catabolize (breakdown) carbohydrates and produce energy by two mechanisms:
Fermentation
Respiration
● anaerobic process carried out by both obligate and facultative anaerobes
● electron acceptor is an organic compound
Fermentation
● less efficient in energy generation — beginning substrate is not completely reduced
● a mixture of end products (lactate, butyrate, ethanol, and acetoin) accumulates in the medium — identification of anaerobic bacteria
Fermentation
Application of Fermentation
- Voges-Proskauer
- Methyl Red Tests
● efficient energy-generating process
● molecular oxygen is the final electron acceptor
● obligate aerobes and facultative anaerobes
Respiration
NOTE!!!!!!!!!!!!!!!!!
● Certain anaerobes can carry out anaerobic respiration, in which inorganic forms of oxygen, such as ____ and ____, act as the final electron acceptors
nitrate and sulfate
● ability to use acetamide as the sole source of carbon
● produce acylamidase, which deaminates acetamide to release ammonia resulting in an alkaline pH
Acetamide Utilization
Result of Acetamide Ulitization
✓Positive: BLUE COLOR
✓Negative: No color change
QC for Acetamide Utilization
✓Positive: Pseudomonas aeruginosa
✓Negative: Escherichia coli
● ability to use acetate (sodium acetate) as the sole
● source of carbon differentiate Shigella sp. from Escherichia coli
Acetate Utilization
Result for ACETATE UTILIZATION
✓Positive: Medium becomes alkalinized = BLUE
✓Negative: No growth or no indicator change to blue
QC for Acetate Utilization
✓Positive: Escherichia coli
✓Negative: Shigella sonnei
● presumptive identification and differentiation of Streptococcus pyogenes
● distinguish staphylococci species (resistant) from micrococci (susceptible)
Bacitracin (TAXOA) Susceptibility
Result for Bacitracin (TAXOA) Susceptibility
✓Positive: ZOI>10 mm
✓Negative: No zone of inhibition
QC for Bacitracin (TAXOA) Susceptibility
✓Positive: Streptococcus pyogenes
Micrococcus luteus
✓Negative: Streptococcus agalactiae
Staphylococcus aureus
● 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
Bile Esculin Test
Result for Bile Esculin Test
✓ Positive: GROWTH and BLACKENING of the
agar slant
✓ Negative: Growth and no blackening of medium
QC for Bile Esculin Test
✓ Positive: Enterococcus faecalis
✓ Negative: Escherichia coli
● differentiates Streptococcus pneumonia
(positive–soluble) from alpha-hemolytic
streptococci (negative–insoluble)
● Bile/Bile salt (sodium desoxycholate) rapidly lyses pneumococcal colonies
● Lysis depends on amidase → autolytic enzyme
Bile Solubility Test
Result for Bile Solubility Test
✓ Positive: Colony disintegrate
✓ Negative: Intact colonies
QC for Bile Solubility Test
✓ Positive: Streptococcus pneumonia
✓ Negative: Enterococcus faecalis
● detect BUTYRATE ESTERASE
● identification of Moraxella (Branhamella) catarrhalis
● bromochlorindolyl butyrate → indoxyl + O2 → Indigo
Butyrate Disk
Result for Butyrate Disk
✓ Positive: Development of a BLUE COLOR
(5-minute incubation)
✓ Negative: No color change
QC for Butyrate Disk
✓ Positive: Moraxella catarrhalis
✓ Negative: Neisseria gonorrhoeae
● differentiate Streptococcus agalactiae (positive)
from other streptococcal species
● CAMP factor acts synergistically with the beta-lysin of S. aureus to cause enhanced lysis of RBC
CHRISTIE, ATKINS, AND MUNCH-PETERSON
(CAMP) TEST
Result for CAMP Test
✓Positive: Arrowhead-shaped zone of
beta-hemolysis
✓Negative: No enhancement of hemolysis
QC for CAMP Test
✓Positive: Streptococcus agalactiae
✓Negative: Streptococcus pyogenes
CAMP Positive Organisms
- Streptococcus agalactiae
- Listeria monocytogenes
- Proprionibacterium acnes
Revere CAMP Positive Organisms
- Clostridium perfringens
- Corynebacterium pseudotuberculosis
- Corynebacterium ulcerans
- Corynebacterium urealyticum
● differentiates catalase-positive MICROCOCCAL and STAPHYLOCOCCAL spp. from catalase-negative
STREPTOCOCCAL spp.
● H2O2 → H2O + O = EFFERVESCENCE
CATALASE TEST
Result for CATALASE TEST
✓ Positive: Copious bubbles are produced
✓ Negative: No or few bubbles are produced
QC for CATALASE TESTCATALASE TEST
✓ Positive: Staphylococcus aureus
✓ Negative: Streptococcus pyogenes
● isolate and purify Pseudomonas aeruginosa from contaminated specimens
● ability of an organism to grow in the presence of cetrimide
Cetrimide Agar
Result for Cetrimide Agar
✓ Positive: Growth, variation in color of colonies
✓ Negative: No growth
QC for Cetrimide Agar
✓ Positive: Pseudomonas aeruginosa
✓ Negative: Escherichia coli
● 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
Citrate Utilization
Result for Citrate Utilization
✓ Positive: Growth on the medium = BLUE
✓ Negative: Absence of growth
QC for Citrate Utilization
✓ Positive: Enterobacter aerogenes
✓ Negative: Escherichia coli
● differentiate S. aureus (+) from coagulase-negative staphylococci
Coagulase Test
Two forms of Coagulase
- Bound Coagulase→ clumping factor
- Free Coagulase→ extracellular protein enzyme
Result for Coagulase-Slide Test
✓ Positive: Clumping in 10 seconds or less
✓ Negative: No clumping
Result for Coagulase-Tube Test
✓ Positive: Clot of any size
✓ Negative: No clot
QC for Coagulase-Test
✓ Positive: Staphylococcus aureus
✓ Negative: Staphylococcus epidermidis
● differentiate decarboxylase producing
Enterobacteriaceae from other gram-negative
rods
● Amino acid → Amine = alkaline pH →from orange to purple
● pH indicator: BROMCRESOL PURPLE
Decarboxylase Tests (Moeller’s Method)
Result for Decarboxylase Tests (Moeller’s Method)
✓ Positive: Alkaline (PURPLE) color change
✓ Negative: No color change or acid (yellow)
QC for Decarboxylase Tests (Moeller’s Method)
✓ Positive:
▪ Lysine—Klebsiella pneumoniae
▪ Ornithine—Enterobacter aerogenes
▪ Arginine—Enterobacter cloacae
✓ Negative:
▪ Lysine—Enterobacter cloacae
▪ Ornithine—Klebsiella pneumoniae
▪ Arginine—Klebsiella pneumoniae
● distinguish Serratia sp. (+) from Enterobacter sp., Staphylococcus aureus (+) from other species, and Moraxella catarrhalis (+) from Neisseria sp.
● ability to hydrolyze DNA (DNAase)
● DNA–methyl green complex→ from green to colorless zone
DNA Hydrolysis (DNAse Test Agar)
Result for DNA Hydrolysis (DNAse Test Agar)
✓ Positive: COLORLESS around the test organism
✓ Negative: Medium remains green
QC for DNA Hydrolysis (DNAse Test Agar)
✓ Positive: Staphylococcus aureus
✓ Negative: Escherichia coli
● presumptive identification and differentiation of Enterobacteriaceae
● Esculin is hydrolyzed to esculetin, which reacts with Fe3+ and forms a dark brown to black precipitate
Esculin Hydrolysis
Result for Esculin Hydrolysis
✓ Positive: BLACKENED MEDIUM
✓ Negative: No blackening
QC for Esculin Hydrolysis
✓ Positive: Enterococcus faecalis
✓ Negative: Escherichia coli
● ability to ferment carbohydrates
Fermentation Media
differentiate enteric bacteria from
coryneforms
ANDRADE’S FORMULA
Result for ANDRADE’S FORMULA
✓ Positive: Indicator change to PINK
✓ Negative: Growth, but no change in color
QC for ANDRADE’S FORMULA
✓ Positive, with gas: Escherichia coli
✓ Positive, no gas: Shigella flexneri
distinguish enterococci from streptococci
Bromocresol Purple
Result for Bromocresol Purple
✓ Positive: Indicator change to YELLOW
✓ Negative: Growth, but no change in color
QC for Bromocresol Purple
✓ Positive, with gas: Escherichia coli
✓ Negative, no gas: Moraxella osloensis
● presence and arrangement of flagella
Flagellar Stain (Wet Mount Technique)
QC for Flagellar Stain (Wet Mount Technique)
✓ Peritrichous: Escherichia coli
✓ Polar: Pseudomonas aeruginosa
✓ Negative: Klebsiella pneumonia
MOTILITY of diff. Bacteria in Flagellar Stain
● Rapid darting/shooting star = Vibrio (monotrichous)
● Darting = Campylobacter
● Twitching = Kingella, Bartonella
● Tumbling (end over end) = Listeria
● Gliding = Capnocytophaga, Mycoplasma
pneumoniae
● Corkscrew (axial filaments) = Spirochetes
● Spinning = Leptospira
➢ gelatinases hydrolyze gelatin
➢ presumptive identification of Staphylococcus sp., Enterobacteriaceae, and some gram-positive bacilli
Gelatin Hydrolysis
Result for Gelatin Hydrolysis
✓ Positive: Partial or total liquefaction at 4°C within 14 days
✓ Negative: Complete solidification
QC for Gelatin Hydrolysis
✓ Positive: Bacillus subtilis
✓ Negative: Escherichia coli
● differentiate a pyocyanogenic pseudomonads from other Pseudomonas spp.
● ability of an organism to grow at 42°C
GROWTH @ 42°C TEST
Result for Growth @ 42°C Test
✓ Positive: Good growth at both 35°and 42°C
✓ Negative: No growth at 42°C but good growth at 35°C
QC for Growth @ 42°C Test
✓ Positive: Pseudomonas aeruginosa
✓ Negative: Pseudomonas fluorescens
● 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
Hippurate Hydrolysis
Result for Hippurate Hydrolysis
✓ Positive: Deep purple color
✓ Negative: Colorless or slightly yellow pink color
QC for Hippurate Hydrolysis
✓ Positive: Streptococcus agalactiae
✓ Negative: Streptococcus pyogenes
Bacteria in Hippurate Hydrolysis
- Streptococcus agalactiae
- Listeria monocytogenes
- Campylobacter jejuni subsp. jejuni
- Gardnerella vaginalis
Tryptophan → INDOLE + KOVAC’S REAGENT
(dimethylamine-benzaldehyde and hydrochloride) or EHRLICH’S REAGENT = RED COLOR
Indole Production
Result for Indole Production
✓ Positive: PINK- TO WINE-COLORED RING
✓ Negative: No color change
QC for Kovac’s Method (Indole)
✓ Positive: Escherichia coli
✓ Negative: Klebsiella pneumoniae
QC for Ehrlich’s Method (Indole)
✓ Positive: Haemophilus influenzae
✓ Negative: Haemophilus parainfluenzae
QC for Kovac’s Method - Anaerobic (Indole)
✓ Positive: Porphyromonas asaccharolytica
✓ Negative: Bacteroides fragilis
presumptive identification of CATALASE-NEGATIVE GRAM- POSITIVE COCCI
● Hydrolysis of Leucine beta-naphthylamide by
leucine aminopeptidase → beta-naphthylamine + cinnamaldehyde reagent = RED COLOR
Leucine Aminopeptidase (LAP) Test
Result for Leucine Aminopeptidase (LAP) Test
✓ Positive: Development of a red color within 1
minute
✓ Negative: No color change or development of a slight yellow color
QC for Leucine Aminopeptidase (LAP) Test
✓ Positive: Enterococcus faecalis
✓ Negative: Aerococcus viridans
● ability to metabolize litmus milk: fermentation, reduction, clot formation, digestion, and the formation
of gas
Litmus Milk Medium
Result for Litmus Milk Medium
➢ 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 for Litmus Milk Medium
➢ Fermentation: Clostridium perfringens
➢ Acid: Lactobacillus acidophilus
➢ Peptonization: Pseudomonas aeruginosa
● LIA: lysine, peptones, a small amount of glucose, ferric ammonium citrate, and sodium thiosulfate
● GLUCOSE FERMENTED→ BUTT BECOMES
ACIDIC (YELLOW)
● LYSINE DECARBOXYLASE PRESENT
→CADAVERINE → ALKALINE STATE (PURPLE)
● OXIDATIVE DEAMINATION of lysine and in the presence of FERRIC AMMONIUM CITRATE and FLAVIN MONONUCLEOTIDE, forms BURGUNDY COLOR on the slant
Lysine Iron Agar
● pH indicator: Bromocresol Purple
▪ yellow at or below pH 5.2
▪ purple at or above pH 6.8
Lysine Iron Agar
Result for Lysine Iron Agar
✓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 for Lysine Iron Agar
✓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
LIA reaction for Salmonella
K/K
LIA reaction for Shigella and Citrobacter
K/A
LIA reaction for Proteus, Providencia, Morganella (PPM)
R/A
● ability to produce and maintain stable acid end products from glucose fermentation, overcome the buffering capacity of the system, and to determine the ability to produce 2,3-BUTANEDIOL or ACETOIN from glucose fermentation
Methyl Red/Voges-Proskauer (MR-VP) Tests
o detects mixed acid fermentation that lowers the pH
o red at pH 4.4 and yellow at pH 6.2
Methyl Red
o detects the ability to convert the acid products to acetoin and 2,3- butanediol → alpha-naphthol is added, followed by potassium hydroxide (KOH) → RED
Voges-Proskauer Test
QC for MR-VP Tests
✓ MR positive/VP negative: Escherichia coli
✓ MR negative:/VP positive: Enterobacter aerogenes
Medium for MR-VP Tests
MR-VP medium or Clurk and Lubs Dextrose
Broth
● differentiate STAPHYLOCOCCUS from MICROCOCCUS SPP. by detection of the enzyme oxidase
● oxidase reacts with the oxidase reagent and cytochrome C to form the indophenol
Microdase Test (Modified Oxidase)
Result for Microdase Test (Modified Oxidase)
✓ Positive: Development of BLUE TO
PURPLE-BLUE COLOR
✓ Negative: No color change
QC for Microdase Test (Modified Oxidase)
✓ Positive: Micrococcus luteus
✓ Negative: Staphylococcus aureus
● diffuse zone of growth extending out from the line of inoculation
Motility Testing
Result for Motility Testing
✓ Positive: Spread out from the site of inoculation
✓ Negative: Remain at the site of inoculation
QC for Motility Testing
✓ Positive: Escherichia coli
✓ Negative: Staphylococcus aureus
● determine gas formation during glucose fermentation
● Lactobacillus spp. and Leuconostoc sp. produce gas
● Selective medium: SODIUM ACETATE and AMMONIUM CITRATE
MRS Broth
Result for MRS Broth
✓ 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 for MRS Broth
✓ Positive: Lactobacillus lactis
✓ Negative: Escherichia coli
● presumptively identify various genera of
Enterobacteriaceae and verotoxin-producing E. coli (neg)
● B-d-glucuronidase hydrolyzes
4-methylumbelliferyl-ẞ-d-glucuronide to
4-methylumbelliferyl which fluoresces blue under long wavelength UV
4-METHYLUMBELLIFERYL-SS-D-GLUCURONIDE
(MUG) TEST
Result for MUG test
✓ Positive: Electric blue fluorescence
✓ Negative: Lack of fluorescence
QC for MUG Test
✓ Positive: Escherichia coli
✓ Negative: Klebsiella pneumoniae
● 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 alpha naphthylamine to produce a red,
water-soluble azo dye
● Zinc is added to validate colorless result for the presence of untreated nitrate
Nitrate Reduction
Result for Nitrate Reduction
✓ Positive: RED
✓ Negative: No color change
QC for Nitrate Reduction
✓ Positive: NO3+, no gas: Escherichia coli
✓ Positive: NO3+, gas: Pseudomonas aeruginosa
✓ Negative: Acinetobacter baumannii
● reducing nitrite to nitrogen do not turn color and do produce gas in the nitrate reduction test
Nitrite Reduction
Result for Nitrite Reduction
✓ Positive: No color change to red 2 minutes; gas production in Durham tube
✓ Negative: Broth becomes red; no gas production is observed
QC for Nitrite Reduction
✓ Positive: Proteus mirabilis
✓Negative: Acinetobacter baumannii
● ability to produce B-GALACTOSIDASE which hydrolyzes substrate ONPG to form orthonitrophenol (yellow product)
● distinguishes late LLF from NLF of Enterobacteriaceae
● Indicates presence of B-galactosidase
O-NITROPHENYL-B-D-GALACTOPYRANOSIDE
(ONPG) TEST
Result for ONPG Test
✓ Positive: YELLOW (presence of B-galactosidase)
✓ Negative: Colorless
QC for ONPG Test
✓ Positive: Shigella sonnei
✓ Negative: Salmonella typhimurium
● 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
OPTOCHIN (TAXO P) SUSCEPTIBILITY TEST
Result for OPTOCHIN (TAXO P) SUSCEPTIBILITY TEST
✓ Positive: ZOI ≥ 14 mm in diameter, with 6-mm disk
✓ Negative: No zone of inhibition
QC for OPTOCHIN (TAXO P) SUSCEPTIBILITY TEST
✓ Positive: Streptococcus pneumoniae
✓ Negative: Streptococcus pyogenes
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
OXIDASE TEST (KOVAC’S METHOD)
Result for OXIDASE TEST (KOVAC’S METHOD)
✓ Positive: Development of a dark purple color within 10
✓ Negative: Absence of color
QC for OXIDASE TEST (KOVAC’S METHOD)
✓ Positive: Pseudomonas aeruginosa
✓Negative: Escherichia coli
Reagent for the Oxidase Test for Neisseria
Reagent: 1% dimethyl-p-phenylenediamine hydrochloride
(+): colonies become purple black
Reagent for the Indophenol Method
Reagent: p-aminodimethylaniline hydrochloride (or Oxalate) and alpha-naphthol
(+): Intense Blue color within 2 minutes
● ability to oxidize or ferment specific carbohydrates
● glucose, xylose, mannitol, lactose, sucrose, and maltose
OXIDATION/FERMENTATION (OF) MEDIUM (CDC METHOD)
● 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
Hugh and Leifson’s Formula
Carbohydrate Fermentation Medium that is Base Medium
a. Peptone water
b. Trypticase
c. Tryptone B
Carbohydrate Fermentation Medium that is 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 for Fermentation
- Rusell’s Double Sugar (RDS)
● contains glucose and lactose with Andrade’ Acid Fuchsin - Smith Fermentation Tube
Result for OF Medium
✓ 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 for OF Medium
✔ Fermenter (Glucose): Escherichia coli
✓ Oxidizer (Glucose): Pseudomonas aeruginosa
● 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
Phenylalanine Deaminase Agar
Result for Phenylalanine Deaminase Agar
✓ Positive: GREEN COLOR on slant
✓ Negative: Slant remains original color
QC for Phenylalanine Deaminase Agar
✓ Positive: Proteus mirabilis
✓ Negative: Escherichia coli
● presumptive identification of Streptococcus pyogenes and enterococci by the presence of L-pyrrolidonyl arylamidase
● L-pyrrolidonyl- B-naphthylamide → B-naphthylamine + N,N- methylaminocinnamaldehyde reagent → BRIGHT RED PRECIPITATE
L-PYRROLIDONYL ARYLAMIDASE (PYR) TEST
Result for L-PYRROLIDONYL ARYLAMIDASE (PYR) TEST
✓ Positive: Bright red color within 5 minutes
✓ Negative: No color change or an orange color
QC for L-PYRROLIDONYL ARYLAMIDASE (PYR) TEST
✓ Positive: Enterococcus faecalis
Streptococcus pyogenes
✓ Negative: Streptococcus agalactiae
● ability of an organism to utilize pyruvate
● differentiation between Enterococcus faecalis (+) Enterococcus faecium (-)
● Pyruvic acid → added to the broth to determine whether the microorganism is able to use pyruvate
● Bromothymol blue indicator changes from blue to yellow
Pyruvate Broth
Result for Pyruvate Broth
✓ Positive: Indicator changes from green to yellow
✓ Negative: No color change
QC for Pyruvate Broth
✓ Positive: Enterococcus faecalis
✓ Negative: Streptococcus bovis
● 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: Bromocresol Purple
Salt Tolerance Test
Result for Salt Tolerance Test
✓ Positive: Visible turbidity in the broth, with or
without a color change from purple to yellow
✔ Negative: No turbidity and no color change
QC for Salt Tolerance Test
✓ Positive: Enterococcus faecalis
✓ Negative: Streptococcus bovis
● 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
Triple Sugar Iron Agar (TSI)
● 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
Triple Sugar Iron Agar (TSI)
Result for TSI
✓ 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 for TSI
✓ A gas production: Escherichia coli
✓ K/A, +/- gas production, H2S+: Salmonella
typhimurium
✓ K/K: Pseudomonas aeruginosa
✓ K/A, H2S+: Proteus mirabilis
✓ K/A: Shigella flexner
● 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
● Medium: CHRISTENSEN’S UREA AGAR or BROTH
Urease Test (Christensen’s Method)
Result for Urease Test (Christensen’s Method)
✓ Positive: Change in color of slant from light orange to magenta
✓ Negative: No color change
QC for Urease Test (Christensen’s Method)
✓ Positive: Proteus vulgaris
✓ Weak positive: Klebsiella pneumonia
✓ Negative: Escherichia coli
● differentiation Haemophilus species
X and V Factor Test
Result for X and V Factor Test
✓ 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 for X and V Factor Test
✓ 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
heat stable substance known as HEMIN or HEMATIN
X factor
heat labile substance known as NAD or COENZYME I
supplied by yeast, potato extract and bacteria
(Staphylococci, Pneumococci and Neisseria)
V factor
