Medically Important Bacteria

GRAM POSITIVE BACTERIA

Gram Positive Cocci
STAPHYLOCOCCI
MICROCOCCI
STREPTOCOCCI
Family Micrococcaceae
• 3 genera:
• Staphylococci
• Micrococci
• Planococci
• Stomatococci
Laboratory Diagnosis
• 1. Gram Stain

• Staphylococcus: Gram (+) cocci in clusters (Grape like cocci).

• Micrococci: Gram (+) cocci in tetrads (packets of 4) or sarcinae

(packets of 6).
Gram Stain
• Staphylococci • Micrococci
2. Growth on BAP
• Staphylococci
= Creamy white pinhead colonies with characteristic hemolytic pattern.

4 Types of Hemolytic Pattern:

• Alpha hemolysis
• Beta hemolysis
• Gamma hemolysis
• Alpha-prime hemolysis
4 Types of Hemolytic Pattern

ALPHA HEMOLYSIS BETA HEMOLYSIS

• Incomplete hemolysis • Complete hemolysis
• The colony is surrounded by a colorless or
• The colony is surrounded by a yellow zone.
green or brown zone. • Staphylococcus aureus
4 Types of Hemolytic Pattern

GAMMA HEMOLYSIS ALPHA-PRIME HEMOLYSIS

• Non-hemolytic • Colony is surrounded with an
alpha-zone of hemolysis and
an OUTER beta-zone of
hemolysis.
3. Growth on Loeffler’s Serum Slant (LSS)
• Enhances the pigmentation of
Staphylococcus

• Staphylococcus aureus: Gold

and Yellow colonies
• Staphylococcus citreus:
Lemon-yellow colonies
• Staphylococcus albus
(Staphylococcus epidermidis):
Porcelain white colonies
4. Growth on Mannitol Salt Agar (MSA)
• Selective/differential for
Staphylococcus spp.
• Inhibitor: 7.5% NaCl
• CHO: Mannitol
• pH indicator: Phenol red
• Staphylococcus aureus: yellow
colonies
• Staphylococcus epidermidis: pink
colonies
• Staphylococcus saprophyticus:
pink colonies
5. Catalase test
• Differentiate Staphylococci
from Streptococci
• Reagent: 3% H2O2
• Positive result: Effervescence
or bubbles
• Staphylococci: Catalase (+)
• Streptococci: Catalse (-)
6. Test to differentiate Staphylococcus from
Micrococcus
Test Staphylococcus Micrococcus
Aerobic growth (+) (+)
Anaerobic growth (+) (-)
Lysostaphin Sensitive Resistant
Bacitracin Resistant Sensitive
Modified oxidase Negative Positive
Glucose utilization Fermenter Oxidizer

Modified oxidase (Microdase):

Reagent: tetramethylparaphenylene diamine dihydrochloride with DMSO
(dimethylsulfoxide)
Positive result: BLUE
6. Test to differentiate Staphylococcus from
Micrococcus
• CHO Oxidation Fermentation (OF) test
• CHO: Glucose
• pH indicator: Bromthymol Blue

Genera Open tube Closed tube

Staphylococcus Yellow Yellow

Micrococcus Yellow Green

6. Test to differentiate Staphylococcus from
Micrococcus

MODIFIED OXIDASE OF TEST

7. Coagulase test
• A. Slide Coagulase
• Detects: cell-bound coagulase
or CLUMPING FACTOR
• Reagent: Rabbit’s plasma or
EDTA plasma
• Positive: Clumping or clot
7. Coagulase test
• B. Tube Coagulase
• Detects: Free Coagulase
• Reagent: 0.5 mL Rabbit’s
plasma + colony incubated for
4 hours at 37 ˚C
• Positive: formation of a gel-like
fibrin clot
8. DNAse Test (DNA Hydrolytic test)
• Medium: DNA medium with
METHYL GREEN
• Positive result: Clearing of the
medium (Staphylococcus
aureus)
• DNAse (+):
• Staphylococcus aureus
• Moraxella
• Serratia
9. Novobiocin test
• Staphylococcus epidermidis:
associated with bacterial
endocarditis following the
insertion of artificial heart valves
• Novobiocin: SENSITIVE

• Staphylococcus saprophyticus:
important cause of UTI in young
women
• Novobiocin: RESISTANT
Family Streptococcaceae
• Streptococci
• Gram-positive cocci occurring in pairs or in chains
• Nonmotile
• Non-spore forming
• Facultative anaerobes
• Catalase negative
Classification
• Lancefield Classification
• By antigenic character of a group-specific cell wall polysaccharide
• Almost 20 Lancefield groups are identified
• Brown’s Classification
• Classified according to the pattern of hemolysis
Hemolysis Species
Alpha Streptococci pyogenes,
Viridans strep. And some
Group D.
Beta Streptococci pyogenes and
Lancefield Classification
Group Species
A Group A Streptococci
(Streptococci pyogenes)
B Group B Streptococci
(Streptococci agalactiae)
C Streptococci equisimilis
Streptococci zooepidemicus
Streptococci equi
Streptococci dysagalactiae
D Entrococci
Entrococci faecalis
Entrococci faecium
Entrococci durans
Entrococci avium

Non-Entrococci
Streptococcus bovis
Streptococcus equinus
Brown’s Classification

Hemolysis Species
Alpha Streptococci pyogenes, Viridans strep.
And some Group D.

Beta Streptococci pyogenes and

Streptococci agalactiae, Group C and D

Gamma Most members of Group D

Laboratory Diagnosis
• 1. Gram stain: Gram (+) cocci in chains or in pairs
• Streptococcus pneumoniae: Lancet/bullet or Flame-shaped
diplococci
2. Growth on BAP
• White pinpoint colonies with hemolytic pattern
• Same as Staphylococci
3. Catalase test
• Catalase negative
4. Bile esculin hydrolysis
• Determine the ability to grow
in 40% bile and esculin
hydrolysis
• Positive result: esculetin reacts
with ferric chloride to form
brown-black ppt
• Positive for: Group D
Streptococci
 Test to differentiate Group D Enterococcus from Group
D non-Enterococcus

GROUP D Growth in 6.5% NaCl PYR

Enterococci Positive Positive

Non-Enterococci Negative Negative

PYRase Test
• Test for: Pyrrolidylarylamidase
• Substrate: PYR-L-pyrrolidyl-
beta-napthylamide
• Color developer: P-DAB (para-
dimethylaminobenzaldehyde)
• Positive color: RED
• Positive for: Group D
Enterococcus and Group A
Streptococcus
6. CAMP Reaction – Christie, Atkins, Munch, Peterson
• Positive result: arrow-head
hemolysis
• Positive for: Group B
streptococci (Streptococci
agalactiae)
7. Hippurate hydrolysis test
• Determine the hydrolysis of
sodium hippurate to benzoic
acid and glycine + ninhydrine
• Positive for: Group B
streptococci (Streptococcus
agalactiae)
• Positive result: VIOLET COLOR
 8. Bacitracin and Sulfamethoxazole-trimethoprim
Susceptibility test
GROUP BACITRACIN SXT Notes
(TAXO A)
A (S. pyogenes) Sensitive Resistant PYRase (+)
B (S. agalactiae) Resistant Resistant CAMP & Hippurate
(+)
C Resistant Sensitive
D Enterococcus Resistant Resistant Bile esculin
hydrolysis, Growth
in 6.5% NaCl and
PYRase
D Non-Enterococcus Resistant Resistant Bile esculin
Hydrolysis
9. Immunology Test
• Dick’s Test
• Susceptibility test for Scarlet
fever
• Arm + Erytrogenic toxin = (+)
REDNESS or ERYTHEMA
• Schultz-Charlton Test
• Diagnostic Test for Scarlet fever
• Arm + Anti-toxin = (+)
NEUTRALIZATION or
BLANCHING PHENOMENON
 10. Test to Differentiate Streptococcus pneumoniae
from Viridans Streptococci

TEST Streptococcus pneumoniae Viridans Streptococci

Mouse virulence Positive Negative
Inulin fermentation Positive Negative
Bile solubility Positive Negative
Optochin Sensitive Resistant
Taxo P
Chemical name: Zone of inhibition
Ethylhydrocuprein ≥ 14mm
hydrochloride
Neufeld Quellung Positive (Capsular swelling) Negative
10. Test to Differentiate Streptococcus pneumoniae
from Viridans Streptococci

MOUSE VIRULENCE INULIN FERMENTATION

10. Test to Differentiate Streptococcus pneumoniae
from Viridans Streptococci

BILE SOLUBILITY OPTHOCIN TEST

10. Test to Differentiate Streptococcus pneumoniae
from Viridans Streptococci

NEUFELD QUELLUNG
11. Francis test
• Detection of pneumococcal antibodies
Gram Positive Bacilli
BACILLUS
CLOSTRIDIUM
CORYNEBACTERIUM
LISTERIA
ERYSIPELOTHRIX
LACTOBACILLUS
KURTHIA AND ROTHIA
Bacillus
• Gram positive bacilli that form spores AEROBICALLY.
• Catalase POSITIVE
Bacillus anthracis
• is the most virulent, and the agent of ANTHRAX
• * Cutaneous anthrax = malignant pustule (black eschar)
• * Woolsorter’s disease or pulmonary anthrax
• * Gastrointestinal anthrax
• Bacillus anthracis is the largest bacteria, largest pathologic bacilli
and it is NON-MOTILE.
Laboratory Diganosis
COLONIES LIFTED BY LOOP OR
NEEDLE
MEDUSA HEAD COLONIES “BEATEN EGG CONSISTENCY”
Laboratory Diagnosis
FROM CULTURE BAMBOO
FROM PATIENT CAPSULATED POLE ARRANGEMENT
Laboratory Diagnosis
STRING OF PEARLS
(MHA WITH PENICILLIN)
Bacillus cereus
• Is associated with food poisoning isolated from several foods: rice,
other cereals, vegetables and milk.
• “FRIED RICE POISONING” = vomiting and diarrhea
• MOTILE
Bacillus anthracis vs Bacillus cereus

Test Bacillus anthracis Bacillus cereus

Catalase Positive Positive

Lecithinase Positive Positive

Motility NON-MOTILE MOTILE

Hemolysis on BAP GAMMA HEMOLYSIS BETA HEMOLYSIS

Clostridia
• Gram positive bacilli that form spore ANAEROBICALLY.
• Catalase NEGATIVE.
Clostridium perfringes
• Cause of myonecrosis (GAS GANGRENE) and food poisoning
• In solid media and thioglycollate broth, spores are USUALLY ABSENT
• DOUBLE ZONE of HEMOLYSIS
• LECITHINASE POSITIVE
• REVERSE CAMP
• NAGLER REACTION
• Sulfhemoglobinemia
• Bacillus aerogenes or Clostridium welchii
LABORATORY DIAGNOSIS
LECITHINASE POSITIVE
NAGLER REACTION REVERSE CAMP
Laboratory diagnosis
DOUBLE ZONE HEMOLYSIS
INNER: COMPLETE (THETA TOXIN)
OUTER: INCOMPLETE (ALPHA TOXIN AND
LECITHINASE)
Clostridium botulinum
• Agent of food and wound botulism, as well as infant botulism.
• HONEY, FLOPPY BABY SYNDROME.
• Botulism results from the liberation of botulism toxin, a powerful
neurotoxin associated with FLACCID PARALYSIS.
• Spores are OVAL and SUBTERMINAL.
• LIPASE POSITIVE
Laboratory Diagnosis

FLACCID PARALYSIS LIPASE TEST

Clostridium tetani
• Devil’s grin/ Risus sardonicus
• Etiologic agent of TETANUS, which result from entry of the organism
or spores into the puncture wound.
• Produces TETANOSPASMIN (neurotoxin), associated with SPASTIC
CONTACTIONS/ LOCK JAW
• Spores are ROUND and TERMINAL (Tack head/drum stick/lollipop
bacilli).
Laboratory Diagnosis

LOCK JAW MORPHOLOGY

Clostridium difficile
• Difficult to identify
• Important cause of antibiotic-associated pseudomembranous colitis
and diarrhea
• Spores are OVAL and SUBTERMINAL
• Cultured on CCFA = Cycloserine, Cefoxitin, Fructose Agar (Yellow
colonies)
• Clindamycin = 1st ANTIBIOTIC
Laboratory Diagnosis

CCFA MORPHOLGY
 Biochemical test for CLOSTRIDIA SPP.

MOTILITY LECITHINASE LIPASE LACTOSE GLUCOSE

Clostridium Negative Positive Negative Positive Positive
perfinges
Clostridium Positive Negative Positive Negative Positive
botulinum
Clostridium Positive Negative Negative Negative Negative
tetani
Clostridium positive Negative Negative Negative Positive
difficile
Corynebacterium diphtheriae
• The organism produces powerful exotoxin that first attacks the mucous
membrane of the respiratory tract; this results from inflammation and
pseudomembrane formation of the oropharynx leading to respiratory
obstructions.
• Klebs-Loeffler bacillus, agent of diphtheria
• Diphtheria toxin = BULL’S NECK APPERANCE
• BABES-ERNST GRANULES = metachromatic granules/volutin granules
• Club shape, Barbed/dumbell, palisade/picket fence, snapping
formation/Chinese character arrangement
• NON-MOTILE
Laboratory diagnosis
NITRATE REDUCTION
POSITIVE UREASE NEGATIVE
Laboratory diagnosis
SUGAR TEST
= GLUCOSE MALTOSE FERMENTED
CATALASE POSITIVE = SUCROSE NOT FERMENTED
Culture
LSS
(LOEFFLERS SERUM SLANT) PAI’S COAGULATED EGG
Culture
MODIFIED TINSDALE’S
AGAR TELLURITE AGAR
COLONIAL TYPES OF
CORYNEBACTERIUM
DIPHTHERIAE
Gravis type
* 1 to 2 mm colonies on blood agar,
LARGEST COLONIAL TYPE
COLONIAL TYPES OF
CORYNEBACTERIUM
DIPHTHERIAE
Mitis type
Fried egg appearance on blood agar
(clear colonies with white centers);
bleachlike odor on tellurite medium
COLONIAL TYPES OF
CORYNEBACTERIUM
DIPHTHERIAE
Intermedius type
Small colonies (0.5 mm) on blood
agar; black colonies with gray
borders on tellurite agar
Toxigenecity test
IN VIVO (ANIMAL
INOCULATION = GUINEA PIG) IN VITRO (ELEK’S TEST)
Diptheriods
• Corynebacterium jeikieum (formerly group JK)
• Multi-drug resistant
• Resistant to a number of antibiotics
• Associated with endocarditis, pneumonia and peritonitis

• Corynebacterium pseudodiphthericum – normal flora, throat

• Corynebacterium xenosis – conjunctiva
• Corynebacterium acnes – skin
• Corynebacterium minitissimum - erythemia
Listeria monocytogenes
• Found in the environment in soil, water, sewage, and decaying
vegetation and in the feces of humans, swine and poultry
• Major source of infection is CONTAMINATED FOOD (cabbage, fruit,
dairy ptoducts)
• Meningitis, pneumonia, abortion, stillbirth, endocarditis,
conjunctivitis, and urethritis.
• Perinatal human listeriosis (granulomatosis infanseptica)
• MOTILE
Motility Testing
HANGING DROP SEMISOLID MEDIUM
(UMBRELLA-LIKE/ INVERTED
(TUMBLING MOTILITY) CHRISTMAS TREE -LIKE GROWTH)
Listeria vs Erysipelothrix

Test Listeria monocytogenes Erysipeltohrix

rushiopathiae
Catalase Positive Negative

H2S Negative Positive

Motility Motile Non-motile

Culture
• McBride medium
CAMP test
• Positive result
• Streptococcus agalactiae = Catalase negative
• Listeria monocytogenes = Catalase positive
Virulence test
• Anton’s test (Ocular test of Anton’s)
• Inoculated to the conjuctival sac of rabbit
• (+) Purulent conjunctivitis
 Listeria vs Corynebacterium
Test Listeria Corynebacterium
Motility MOTILE NON-MOTILE
Fermentation of Salicin Positive Negative
Erysipelothrix rushiopathiae
• Causative agent of ERYSIPELOID, cutaneous inflammation of hand
or fingers, SEAL FINGER or WHALE FINGER.
• Veterinary infection and occupational hazard for those handling
meat, poultry and fish.
• Gelatin test: TEST TUBE BRUSH or BOTTLE BRUSH GROWTH
Lactobacillus acidophilus
• Normal flora of the mouth, gastrointestinal tract and vaginal canal
(DODERLEIN BACILLI, BOAS-DOPPLER’S BACILLUS)
• Nonpathogenic and has little clinical significance
• Cultured on TOMATO JUICE AGAR
• Lactobacillus casein
• Shirota strain
• Yakult
Other Gram positive bacilli

KURTHIA BESSONNII ROTHIA

• Infection among
immunosuppressed patients.
• Found in soil, opportunistic
pathogen.
• Normal flora of the human
mouth, rare cause of abscess
and endocarditis.