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Micrococcus species Staphylococcus aureus

• Gram Stain Gram stain
• Gram Stain
– Gram positive cocci – Gram positive cocci in clusters
– Characteristically in tetrads • Colony morphology
– Usually larger than – Round, smooth, white, creamy
Staphylococcus species colonies with beta-hemolysis
– May have yellow pigment
• Colony
C l morphology
h l with extended incubation
– Micrococcus luteus = yellow pigment • Biochemical tests
– Micrococcus roseus = pink pigment – Catalase positive
• Biochemical tests – Coagulase positive
– Catalase positive • Pathology
– Modified oxidase positive – Wound & skin infections, toxic shock syndrome, food
BAP poisoning, pneumonia, osteomyelitis, bacteremia, etc.

Staphylococcus epidermidis Staphylococcus saprophyticus

• Gram Stain • Gram Stain
– Gram positive cocci in clusters – Gram positive cocci in clusters
• Colony morphology • Colony morphology
– Round, smooth, white, creamy
– Round, smooth, white, creamy
colonies, no hemolysis
colonies, no hemolysis
– May have yellow pigment
• Biochemical tests • Biochemical tests
– Catalase positive – Catalase positive
– Coagulase negative – Coagulase negative
– Novobiocin “S” – Novobiocin “R”
• Pathology • Pathology
– Normal skin flora, nosocomial infections, – UTI’s in young, sexually active females
prosthetic valve endocarditis, catheters, septicemia

Microdase (Modified Oxidase) Test Bacitracin (“A”, “BC”) Disk

• Rapid test • Overnight disk diffusion test
– Two minutes for reaction • Concentration of bacitracin is 0.04 UI
– Detects enzyme – cytochrome oxidase • Used to differentiate between
– Based on oxidase reaction but contains DMSO to – Micrococcus species = “S” (zone of no growth)
release cytochrome oxidase
– Staphylococcus
p y species
p = “R” (g
(growth right
g upp to disk))
• Used
U d tto diff
ti t bbetween
t Staphylococcus
Micrococcus Staphylococcus
– Staphylococcus species
• No color change = negative for
cytochrome C
– Micrococcus species Micrococcus

• Blue color = positive for cytochrome C


Coagulase Tube Test Mannitol Salt Agar

• Tests for free coagulase – an extracellular • Media classified as selective and differential
enzyme that causes a clot to form when – Selective for Staphylococcus species (↑ NaCl)
bacterial cells are incubated in rabbit plasma – Differential for the fermentation of mannitol, the sole
carbohydrate in the media
• Used to differentiate between • Mannitol fermented  ↓ pH  indicator turns pink to yellow
– Staphylococcus aureus = positive (clot at 4 hours) • Staphylococcus
p y aureus = p
positive,, Staphylococcus
• Some strains of Staph. aureus may
lyse the clot (due to staphylokinase)
yellow colonies surrounded by aureus

so it is important to check reaction yellow zone

every ½ hour for the first 4 hours) – Note: some coag neg Staph can be +
– Other Staphylococcus species = Coag neg • Other Staphylococcus species
negative (no clot at 24 hours) Staph sp.
& Micrococcus species = negative,
• Report as “Coagulase reddish colonies – no color
negative Staphylococcus species”
change of media Staphylococcus
species, not

DNase Agar Novobiocin Disk

• Media classified as differential • Disk diffusion test using novobiocin (antibiotic) at
• Inoculum is a dime-sized area of growth a concentration of 5 ųg for the presumptive
• Used to detect the production of identification of Staphylococcus saprophyticus in
an active DNase exoenzyme by urine cultures
aerobic bacteria • Staphylococcus saprophyticus = “R”
– Organism
O i grows up to
t the
th disk
di k
• Used to differentiate Staphylococcus
or a zone <16 mm
– Staphylococcus aureus = Staphylococcus
positive, pink color around growth
• Other Staphylococcus species = “S”
– Other Staphylococcus species =
– Organism’s growth is inhibited
negative, no color change
by antibiotic with a zone > 16 mm
species, not
aureus Staphylococcus species,
not saprophyticus

Hemolysis Bacitracin (“A”, “BC) Disk

• Beta (β) hemolysis • Overnight disk diffusion test
– Complete lysis of RBC’s surrounding colony causing • Concentration of bacitracin Group A Strep

a clear zone or clearing of blood from the agar is 0.04 UI

• Alpha (α) hemolysis • Used to differentiate between
– Partial lysis of RBC’s surrounding colony causing a – Streptococcus pyogenes (Group A
i h di
l ti iin media
di Strep) = “S” (any zone of no growth
• Non-hemolytic around the disk)
– Also known as Gamma • There are a small % of other
beta-hemolytic Strep that are “S”
hemolysis (δ)
– No lysing of RBC’s and no – Most other beta-hemolytic Strep = “R”
color change of the medium (growth up to edge of disk) Beta Strep
not Group A

surrounding colony


Hippurate Hydrolysis Test CAMP Test

• A rapid test that detects the enzyme hippuricase • An overnight test that detects the extracellular
– Na hippurate  benzoic acid + glycine  blue/purple color change
Grp B Strep Ninhydrin
CAMP factor
hydrolyzes Reagent
2 hr, 35°C 15 min. – A streptococcal protein that acts synergistically with
Staphylococcal beta-hemolysis on sheep RBC’s
• Used to differentiate between
• Used to differentiate between
– Streptococcus
p agalactiae
(Group B Strep) = positive, – Streptococcus agalactiae Group B Strep
blue/purple color (Group B Strep) = positive,
“arrow” formation
– Other beta-hemolytic
Strep = negative, no color Positive Negative
change – Other beta-hemolytic aureus
Strep = negative, no “arrow”
Beta Strep not Group B

Optochin (“P”) Disk PYR Hydrolysis Test

• Overnight disk diffusion test • Rapid test
• Disk contains 5 ųg ethyl-hydrocupreine – Enzyme L-pyrroglutamyl-aminopeptidase hydrolyzes the
substrate L-pyrrolidonyl-beta-naphthylamide to produce a beta-
hydrochloride naphthylamine
• Used to differentiate between – When the reagent cinnamaldehyde combines with the beta-
naphthylamine, a bright red color is produced
– Streptococcus Positive
pneumoniae = “S” Strep
• Used
U d tto diff
ti t
(>14 mm zone of no – Streptococcus pyogenes = positive
growth around the disk) – Enterococcus species = positive
– Other Streptococcus
species = “R” – Other Streptococcus species =
(growth up to edge of negative (no color change)
disk) Strep

6.5% Sodium Chloride Test Bile Esculin Hydrolysis

• Certain GPC are able to grow in 6.5% NaCl • Certain GPC are able to grow in the presence of
whereas others are inhibited by this salt bile and hydrolyze esculin to form a blackening
concentration of media, whereas others are inhibited by the
• Used to differentiate between bile
– Enterococcus species = • Used to differentiate between
i i ((growth,
h turbid)
bid) Growth – Enterococcus species = Neg-
– Group D Streptococcus = No positive (black) itive
negative (no growth, clear) – Group D Streptococcus =
– Streptococcus species positive (black)
viridans group = negative – Other Streptococcus species
= negative (no color change)


Streptococcus pyogenes (Group A) Streptococcus agalactiae (Group B)

• Gram Stain • Gram Stain
– Gram positive cocci in pairs/chains – Gram positive cocci in pairs/chains
• Colony morphology • Colony morphology
– Small, transparent, smooth colony – Small, grayish-white mucoid colony
with well-defined zone beta-hemolysis with small zone beta-hemolysis
• Biochemical tests • Biochemical tests
– Catalase negative – Catalase negative
– Bacitracin (A) “S” – Bacitracin (A) “R”
– PYR positive – Hippurate positive
• Pathology – CAMP positive
– Pharyngitis, skin infections, • Pathology
necrotizing fasciitis, toxic shock – Neonate meningitis,
syndrome, post-streptococcal sequelae endometritis, wound infections

Streptococcus pneumoniae Streptococcus species viridans group

• Gram Stain • Gram Stain
– Gram positive cocci in pairs – Gram positive cocci in pairs
(lancet-shaped) (lancet-shaped)

• Colony morphology • Colony morphology

– Small, round, glistening, mucoid,
– Small, round, glistening, mucoid, dome-shaped colony with alpha-hemolysis
dome-shaped colony with alpha-hemolysis
– Autolytic changes (center collapses) when older
– Autolytic changes (center collapses) when older
• Biochemical tests
• Biochemical tests – Catalase negative
– Catalase negative – Optochin (P) “R”
– Optochin (P) “S” – Bile esculin negative
– Bile solubility positive • Pathology
• Pathology – Normal flora, opportunistic, subacute bacterial endocarditis
– Pneumonia, sinusitis, otitis media, bacteremia, meningitis

Enterococcus species MacConkey Agar

• Classified as a selective and differential media
– Selective for Gram negative rods (Gram positive
organisms are inhibited by bile salts)
– Differential for lactose fermentation
• Media contains lactose as its only carbohydrate
• Indicator is neutral red (agar is pink)
• Lactose Fermenter Non”F” ”F”
– Pink colonies
• Non-lactose Fermenter
– Clear colonies
– Note: Proteus will not
swarm on this media


Hektoen-Enteric (HE) Agar Eosin Methylene Blue (EMB) Agar

• Classified as a selective and differential media
– Selective for Gram negative rods
• Classified as a selective and differential media
(Gram positive organisms are inhibited by bile salts) – Selective for Gram negative rods (Gram positive
– Differential for lactose & sucrose fermentation
• Media contains lactose and sucrose organisms are inhibited by eosin dye)
• Indicator is bromthymol blue (agar is green)
• Lactose and/or Sucrose “F” (Yellow colonies)
– Differential for lactose fermentation
• Lactose & Sucrose Non-“F” (Bluish-green colonies) • Media contains lactose as its only carbohydrate
• H2S production = black precipitate • Indicators is eosin & methylene blue
• Lactose Fermenter
– Dark-pink-centered colonies
• Lowers pH which precipitates dye
Lactose & – Note: E. coli = metallic green
Lactose &/or Non-”F” sheen
Sucrose ”F” Lactose & with H2S +
Sucrose • Non-lactose Fermenter
– Clear colonies Escherichia coli

Xylose Lysine Desoxycholate (XLD) Agar Salmonella-Shigella (SS) Agar

• Classified as a selective and differential media • Classified as a selective and differential media
– Selective for Gram negative rods (Gram positive – Selective for pathogenic Enterics
organisms are inhibited by bile salts) (Gram positive organisms are inhibited by bile salts)
– Differential for lactose, sucrose and xylose fermentation – Differential for lactose fermentation
• Media contains lactose, sucrose, and xylose • Media contains lactose as its only carbohydrate
• Indicator is phenol red (turns yellow when pH decreases) • Indicator is neutral red (agar is pink)
– Differential for lysine decarboxylation (LDC) • Lactose “F” (Red/Pink colonies)
• Media contains lysine
• Lactose Non-“F” (Colorless colonies)
• Yellow colonies = Fermenter of carbohydrates
• H2S production = black precipitate
• Yellow colonies with black centers = “F” & H2S +
• Colorless/red colonies = Non “F”
• Red colonies with black centers = LDC + & H2S +

Oxidase Test Glucose OF Media

• Enzyme cytochrome oxidase in the presence of • Fermentative (“F”)
the reagent tetra-methyl- (or dimethyl-) para- organism will produce an
phenylene-diamine-dihydrochloride to form a acid reaction (yellow)
throughout tube
blue color within 30 seconds
• Oxidative (“O”) organism
– Positive = blue color development within 30 seconds will produce an acid Non-“F” Uninoculated
– Negative
g = no color development
p reaction in the upper
pp 1/3 Non-“O” “O” “F”

• Must use a platinum loop or sterile wooden stick, of media

because nichrome loops can cause a false • Non-fermenter & Non-
positive oxidizer (Non-“F” & Non-
Positive Negative
“O”) will produce an
• Tetra-methyl reagent is alkaline reaction (blue) in
more sensitive than upper portion of tube due
dimethyl reagent to peptone metabolism. gas


Kligler’s Iron Agar (KIA) Lysine Iron Agar (LIA)

• Lactose Fermenter, Glucose • Detects deamination (LDA)
Fermenter and decarboxylation (LDC) of
– Acid (yellow) slant & butt (A/A) the amino acid lysine
• Lactose Non-Fermenter, • Slant
– LDA positive = wine-red
Glucose Fermenter
– LDA negative = purple (alkaline)
– Alkaline (red) slant & acid butt
(K/A) • Butt
– LDC positive = purple (alkaline)
• Glucose Non-Fermenter – LDC negative = yellow (acid)
– Alkaline (red) slant & butt (K/K)
• H2 S
• H2 S – Positive = Black precipitate
– Positive = Black precipitate GF GF GF – Negative = No precipitate
– Negative = No precipitate H2S neg H2S pos
• Note: This test is invalid if the Unino- K/A K/K Red/A
organism is unable to ferment LDC – LDC + LDC – LDC +
glucose! H2 S + H2 S – H2 S –

Ornithine Decarboxylase Media Indole Test

• Detects decarboxylation • Detects the enzyme Tryptophanase
(ODC) of the amino acid Tryptophane ---------- Indole ----------------------- Cherry Red color
ornithine tryptophanase
Kovac’s Reagent

• Top 1/3 of media always • Indole positive

purple ODC – ODC +
– Cherry red color
• Butt after
ft adding
ddi Kovac’s
K ’
– ODC positive = purple Reagent
– ODC negative = yellow • Indole negative
(lower 2/3 or tube) Negative Positive

– No color reaction
• Note: This test is invalid if
after adding Kovac’s
the organism is unable to Reagent
ferment glucose!

Motility S Media Nitrate Reduction Test

• Motile • Detects organism’s ability to reduce nitrate (NO3) to
– Diffuse clouding nitrite (NO2) or nitrogen gas (N2↑)
away from the line • Add 2 reagents to the Motility S tube
of inoculation – Alpha-naphthylamine & sulfanilic acid

• Non-motile • NO3  NO2

– Add reagents
g = Red color
Growthth only
l along
the line of • NO3
inoculation – Add reagents = No color reaction
Non-motile Pos Neg
Motile – Add zinc dust = Red color
• NO3  N2↑
– Add reagents = No color reaction
– Add zinc dust = No color reaction


Simmon’s Citrate Media Christiansen’s Urea Media

• Detects organism’s ability • Detects the enzyme urease
to use citrate as its sole Urea + H2O -------- ammonium carbonate
source of carbon urease

• Positive
– Blue color – alkaline end
• If the organism can hydrolyze
products,, bromothymole
p y Negative
g urea, there will be an increase
blue indicator turns blue in pH due to the alkaline end
when pH increases product. The indicator, phenol
– Growth on slant – with or
without color change
red, will turn a bright pink color.
• Negative • Positive = pink color
– No color change and no • Negative = no color change
growth on slant
Positive Strong Negative

Gelatin Klebsiella pneumonia

• Detects proteolytic enzymes which hydrolyze • Growth on sheep blood agar is very mucoid
large protein molecules into smaller peptides
and peptones
• Reagent = acidic mercuric solution
• Positive
– Clear zone adjacent to line of
growth after addition of reagent
• Negative
– A milky color in area adjacent to
growth after addition of reagent

Escherichia coli Proteus mirabilis

• Growth on sheep blood agar is very often beta- • Growth on sheep blood agar swarms across the
hemolytic entire plate

One colony swarms to edge of plate


Oxidase Test CTA Sugars for Neisseria ID

• Enzyme cytochrome oxidase in the presence of • Detects oxidation of carbohydrates (CHO)
the reagent tetra-methyl- (or dimethyl-) para- • Indicator is phenol red
phenylene-diamine-dihydrochloride to form a • CHO oxidized
blue color within 30 seconds – Decrease in pH, indicator turns yellow
– Positive = blue color development within 30 seconds • CHO not utilized Positive
– Negative
g = no color development
p – No pH change,
change indicator stays red
• Must use a platinum loop or sterile wooden stick,
because nichrome loops can cause a false
positive Positive Negative

• Tetra-methyl reagent is Organism Glucose Maltose Lactose Sucrose DNase Nitrate

more sensitive than N. gonorrhoeae

N. meningitidis
+ +
- -
dimethyl reagent N. lactamica + + + - - -
M. catarrhalis - - - - + +

Thayer-Martin (TM) Media Moeller’s Decarboxylase Tests

• A selective agar for Neisseria gonorrheae, N. • Detects enzymatic ability of organism to decarboxylate
meningitidis, and N. lactamica (normal flora) or hydrolyze an amino acid to form an amine
• pH increases (alkaline) and changes indicator to purple
• Agar contains antibiotics
• Amino acids tested include:
– Colistin – inhibits Gram negative rods
– Arginine, Lysine and Ornithine
– Vancomycin – inhibits Gram positive organisms
• Positive
– Nystatin – inhibits yeast – Purple color
• Modified TM agar • Negative
– Contains antibiotic – Yellow/purple or green/purple
trimethoprim/ • Note: this media works with
sulfamethoxazole to glucose “F”, glucose “O”, and Positive Negative
inhibit Proteus species glucose Non-“F” Non-“O”

Pigment Production for Pseudomonas Pseudomonas aeruginosa

• Gram negative rod
• Use Trypticase Soy Agar (TSA slant) to view
pigmentation – Beta-hemolytic
– Flat, spreading colony
• Pigments – Fruity, grape-like smell
– Pyoverdin – yellow-green / • Oxidase positive
yellow-brown • Glucose “O”
– Pyocyanin – blue • A i i dih
Arginine dihydrolase
d l positive
• Lysine decarboxylase negative
– Combination of pyoverdin
• Ornithine decarboxylase negative
& pyocyanin – green • Nitrate variable
– Pyorubin – red • Esculin hydrolysis negative
– Pyomelanin – brown / black • Gelatin variable “O” Pigment ADH+ ODC- LDC-
• DNase negative
• Growth at 42°C
• Sometimes produces green or red pigment


Stenotrophomonas maltophilia X & V Quad Plate

• Gram negative rod • Used for the speciation of
• Oxidase negative Haemophilus
• Glucose “O” • Determines requirement of X
• Arginine dihydrolase negative and V factors and hemolysis
• Lysine decarboxylase positive on horse or rabbit blood
• Ornithine decarboxylase negative • X Factor
• Nitrate variable – Hemin,
Hemin heat stable
• Esculin hydrolysis variable • V Factor
• Gelatin positive – NAD, heat labile IV
• DNase positive • Quadrants I
• No growth at 42°C – I contains X factor II

• Sometimes can develop a – II contains V factor

lavender-green to light purple – III contains X & V factors
pigment – IV contains horse blood

Haemophilus species Pasteurella multocida

• Gram negative coccobacilli • Gram negative rod
• Fastidious – Small (can be coccoid), BAP

– Will not grow on sheep blood agar can exhibit bipolar staining
– Must use Chocolate agar • No growth on MAC
– Requires CO2
• Oxidase positive OF
• Porphyrin test Glucose
– Delta
aminolevulinic acid (ALA) is substrate
• Glucose “F” weak
– Determines if organism has enzyme required to produce hemin (apple green color)
(X factor) • Nitrate positive MAC

– If positive, organism does not require X factor

• ODC positive
Requires Requires Hemolysis ALA-Porphyrin
X V on horse blood test • Indole negative
H. Influenzae Positive Positive None Negative
• Urease negative
H. Parainfluenzae Negative Positive None Positive
H. Hemolyticus Positive Positive Beta Negative • “S” to 2 units of Penicillin
H. parahemolyticus Negative Positive beta Positive

Campylobacter jejuni Aeromonas hydrophila

• Curved Gram negative Gram Stain • Gram negative rods BAP

rod • Beta-hemolytic on BAP

• Grows at both 37°C & • Grows on MAC
– Lactose variable
42°C in 10% CO2
(microaerophilic) • Oxidase positive
• Glucose “F”
• Oxidase positive
• LDC variable
• Darting motility on wet • ODC negative MAC
prep • Indole positive
• Hippurate hydrolysis • Gelatin positive
positive • VP positive
• Nalidixic Acid “S” • Esculin positive
• Cephalothin “R” • DNase positive
Campy BAP


Acinetobacter species Acid Fast Stain / Ziehl-Neelsen Stain

• Gram negative coccobacilli BAP
• Mycobacteria are readily stained with 1° stain,
• Oxidase negative which binds to mycolic acid in their lipid-rich cell
• Acinetobacter baumannii wall. This stain cannot be removed (decolorized)
and organism is referred to as being acid-fast.
– Glucose “O”
– Lactose “F” • Primary Stain is
– Grows at 42°C
Acinetobacter baumannii carbo-fuschin
• Acinetobacter lwoffii • Decolorizer is
– Glucose Non-“F”, Non-“O”
acid alcohol
– Lactose Non-“F” • Counterstain is
– No growth at 42°C methylene blue
• Nitrate negative • Acid Fast Bacilli (AFB)
– Red branching rods
• Non-motile against a blue background

Kirby Bauer Susceptibility E-Test Susceptibility

MIC Bacteroides fragilis group

• Most common anaerobic organism isolated in
the clinical laboratory
• Pleomorphic Gram negative Gram stain
• Grows in 20% bile
• Hydrolyzes esculin
• Penicillin “R”
• Beta-lactamase positive AnaBAP


Clostridium perfringens Gram stain Bacillus species

• Anaerobic organism • Large Gram positive rods Gram
• Large Gram positive rods – With age can appear Gram
– Blunt, rounded ends variable or Gram negative
(“boxcar” shaped) – Can see spores (if organism
– May see spores is stressed)
Double zone
• BAP – double zone of beta-hemolysis
• Blood Agar
beta-hemolysis – Large colonies BAP
• Catalase negative – Rough edges
• Lecithinase positive – Beta-hemolytic
on egg yolk agar
• Motile
• Causes
– Gas gangrene
• Considered a contaminant
Gas in
Thio broth
– Food poisoning
Egg yolk agar

Bacillus anthrasis Listeria monocytogenes

• Large Gram positive rods • Small Gram positive rods BAP

– Subterminal spores (not at end or in the middle) (can be coccobacilli)

• Blood agar
• Blood Agar
– Grayish white colony
– Non-hemolytic BAP – Small zone beta-hemolysis
• Non-motile • Catalase p
• Causes anthrax – Demonstrated on T-soy agar
slant – tube catalase
• Esculin hydrolysis positive
Hippurate Esculin
• Hippurate hydrolysis positive
• Motility
– Wet prep = tumbling motility
– 25°C semisolid media = inverted
umbrella Motility
Gram stain

CTA Sugars for Corynebacterium ID Corynebacterium diphtheriae

• Detects oxidation of carbohydrates (CHO) • Pleomorphic Gram positive rods
• Indicator is phenol red – Often palisading
– Can be club-shaped Gram stain
• CHO oxidized
– Decrease in pH, indicator turns yellow • BAP
– May have a very small
• CHO not utilized
zone beta-hemolysis
– No pH change,
change indicator stays red Positive
• Catalase positive
Organism Glucose Maltose Sucrose Urease Nitrate
• Nitrate positive
C. diphtheriae + + - - + • Glucose positive
+ +/- - - -
C. jeikeium
• Maltose positive
Diphtheroids +/- +/- +/- +/- +/-
• Sucrose negative
• Urease negative
• Note: must test for the diphtheria toxin


Corynebacterium jeikeium Diphtheroids

• Pleomorphic Gram positive rods • Pleomorphic Gram positive rods
– Often palisading – Often palisading Gram stain
Gram stain
– Can be club-shaped – Can be club-shaped
– Non-hemolytic – Non-hemolytic
– Opaque colonies – Opaque colonies
• Catalase positive • Catalase positive
• Nitrate negative • Nitrate variable
• Glucose positive • Glucose variable
• Maltose variable • Maltose variable
• Sucrose negative • Sucrose variable
• Urease negative • Urease variable
• Key to identification – resistant to most antibiotics used • Considered normal skin flora
to treat Gram-positive infections. It is “S” to vancomycin or contaminant
• Causes infections in immunocompromised persons

Albert’s Stain Cystine-Tellurite Blood Agar (CTBA)

• Methylene blue stain • Classified as a selective and differential media
• Selective for Corynebacterium
• Stains Corynebacterium species – Potassium tellurite inhibit
irregularly, giving a beaded appearance many non-coryneform bacteria
• Differential for tellurite
• Metachromatic areas of cell stain more
– Corynebacterium species
intensely (dark purple/black) than rest of (also Staph & Strep)
cell and are referred to as Babès-Ernst • Colonies appear gray or black
granules (or metachromatic granules) – Corynebacterium diphtheriae,
Corynebacterium ulcerans, &
Corynebacterium pseudotuberculosis
• Brown halo surrounding colony due to cystinase activity