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PREPARED BY: DR. MA. CRISTINA LIWANAG

METHODS of BACTERIAL IDENTIFICATION

➔ Manual method
➔ Semi-automated – API – Analytical Profile Index
➔ Automated – VITEK System
➔ Gram Staining – it provides preliminary information regarding the likely presence or absence of a pathogen.
➔ In identification of organisms, it can be phenotypic, genotypic or immunologic
 Genotypic – identifying the organism using molecular methods; involves characterization
 Phenotypic – macroscopic and microscopic characteristics are considered.
▪ In colony morphology, odor is considered because there are some organisms that produces
odor
▪ Pigment production is also noted
▪ Resistance and susceptibility of organisms in antibiotic agents
IMPORTANT MICROSCOPIC & COLONY MORPHOLOGY
S. aureus ➔ Medium sized butyrous (oily looking, buttery looking) colonies, seen in grape-like
clusters
o Pathogenic staphylococci
o Coagulase +
o Can be observed in 5% SBA
o Beta-hemolytic on BAP
o BAP & MSA & CNA – used to initially isolate S. aureus; produces yellow halo on MSA
CoNS (S. epidermidis & S. ➔ White colonies & coagulase (-)
saprophyticus)
o Gamma-hemolytic
o Light pink or colorless on MSA
S. pneumoniae ➔ DOME shaped (umbonate, raised, elevated) colonies after 24 hrs. and nail head
colonies (crater like colonies/colonies with checker appearance) after 48 hrs.
➔ Microscopically lancet shaped or bullet shaped diplococci
o A.k.a. diplococcus pneumoniae
o Alpha-hemolytic on BAP
Micrococci ➔ Strictly aerobic cocci in tetrads
o Gamma-hemolytic
S. pneumoniae ➔ Lysed colonies ff. addition of 10% sodium desoxycholate in bile solubility testing
o Incubated for 30 mins.
o Viridans strep. – bile solubility negative
Genus Neisseria ➔ Bean shaped diplococci
o N. gonorrhea – always pathogenic
o N. meningitidis – can be a normal flora
Moraxella catarrhalis ➔ Hockey puck colonies – colonies remaining intact when pushed across the plate
using inoculating loop
C. perfringens ➔ Double/target hemolysis colonies surrounded by inner beta and outer alpha
o Spore former; classified as anaerobe
o 3 procedures to identify C. perfringens: double target hemolysis, reverse CAMP test, litmus milk test
(producing stormy fermentation of milk)
o Beta hemolysis is due to theta toxin and beta hemolysin
o Alpha hemolysis is due to alpha toxin and lecithinase
C. difficile ➔ Colonies with horse stable or barn yard odor
➔ (+) fluorescence chartreuse (yellow fluorescence) on BAP
o Causes pseudomembranous colitis or Antibiotic associated diarrhea
o Produces toxins A & B
 Toxin A is an enterotoxin
 Toxin B is cytotoxin
o Usually detected through cytotoxin detection through immunoassays
Bacillus anthracis ➔ Microscopically: disjointed bamboo fishing rod
➔ On BAP it will develop colonies with swirling projection; Medusa head/lion head
colonies
➔ Colonies with beaten egg white consistency
➔ On Gelatin media: Inverted fir tree on tube media
➔ On microscope: String of pearl pattern because of its susceptibility to penicillin
o Spore former, aerobic
o Non-motile and gamma hemolytic
Clostridium tetani ➔ Terminally located spores and swollen
Listeria monocytogenes ➔ Tumbling motility at room temp “end over end” motility
➔ Umbrella like growth on agar surface
o Gram + non-spore forming bacilli

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o Used as presumptive test is hanging drop
o It can tolerate cold enrichment at 4°C
Corynebacterium ➔ Palisade (side by side arrangement), picket fence, Chinese letter, XYV formation
diphtheriae ➔ Gray to black colonies on CTBA
➔ Black colonies with brown halo on Tinsdale media
Erysipelothrix ➔ In Gelatin media: Bottle brush like/test tube brush like or pipe cleaner pattern of
rhusiopathiae growth
o Occupational hazard in those handling meat, poultry and fish
o Known to cause erysipeloid (skin infection)
o H2S + on TSI
M. tuberculosis ➔ Cauli flower like growth
o Mycobacterium – slow grower – takes weeks to grow on media
o Needs whole egg for growth
 Lowenstein Jensen, Petragnani’s & ATS
M. xenopi ➔ Colonies appearing like bird’s nest on corn meal agar
M. bovis ➔ Water droplet colonies
o Causes tb in cattles
o Causes intestinal tb in man
o Bacille Calmette-Guerrin (BCG) is prepared using M. bovis
M. leprae ➔ Microscopically cigar packet arrangement
➔ Mice foot pads or foot pads of armadillo
o A.k.a. Hansen’s bacillus; causes leprosy
 Leprosy – a chronic disease affecting the skin, mucus membranes and peripheral nerves
o Non-culturable
o Gram + bacilli
M. kansasii ➔ Cross barred bacillus
➔ Photochromogen
o Mycobacteria may be classified into 3 categories
 MTB complex – M. tuberculosis and bovis
 Runion’s classification: MOPP – mycobacterium other than tuberculosis; NTM – non tuberculous
mycobacteria (photochromogens, scotochromogen, non-photo chromogens and rapid growers)
o M. kansasii - #2 NTM – TB in AIDS px
o M. avium - #1 NTM – TB in AIDS px
Klebsiella pneumoniae ➔ On EMB – pink-purple mucoid colonies
➔ + on string test
E. coli ➔ On EMB – pink to purple colonies with greenish metallic sheen
Lactose Fermenter ➔ Enteric bacilli – pink to purple colonies on Mac Conkey Agar
Enterobacter spp. ➔ On EMB – pink to purple colonies with dark center
➔ Fish eye appearance
Salmonella ➔ SSA – colorless colonies with black center
Shigella ➔ SSA – colorless colonies without black center
o S. sonnei – is classified as late lactose fermenters
o Salmonella & Shigella – non-lactose fermenters
o H2S detection – differential test to differentiate Salmonella & Shigella
 Salmonella – H2S + (with black center); causes bloody diarrhea
 Shigella – H2S – (w/o black center)
Salmonella ➔ Black colonies on BSA
o BSA – selective media for Salmonella
o BSA is selective because of brilliant green inhibitory agent
C. perfringens ➔ Box car morphology – due to the square ends
Calymmatobacterium ➔ Safety pin appearance due to accumulation of polar granules
granulomatis
o Y. pestis – assumes safety appearance due to bipolar staining
 Uses Wayson Stain or methylene blue
 Wayson stain – most accurate
Genus PROTEUS ➔ P. vulgaris/P. mirabilis – colonies with burnt chocolate/burnt gun powder
o Shows swarming on BAP
Prodigiosin ➔ Red pigment produced by Serratia marcescens
o S. marcescens – lipase, gelatinase and DNAse +
Y. pestis ➔ Stalactite growth/flocculent (clumps adhering on the sides of the tube) growth on
broth
➔ Hammered copper colonies on BAP
➔ Turbidity on broth
Y. enterocolitica ➔ Bull’s eye colonies on CIN
➔ Will produce pink colonies with red center

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o CIN – selective media for Y. enterocolitica; contains mannitol as fermentable and neutral red as indicator
P. aeruginosa ➔ PYOCYANIN (+) and PYOVERDIN (+)
➔ Colonies with corn tortilla like/corn taco odor/odor similar to overripe grapes
o Considered as #1 ICU isolate
o Causes nosocomial infection – common in hospital environment
o Pyocyanin – water soluble blue pigment
o Beta-hemolytic, non-fermenter and strict aerobe
o Cetrimide agar is used to initially isolate
Pyoverdine ➔ P. stutzeri produces wrinkled colonies
Genus Haemophilus ➔ Bacterial genera that require X & V factors
H. influenzae ➔ Satellitism – luxuriant growth neat the source of incorporated V factor
H. haemolyticus ➔ Haemophilus spp. Requiring both X & V factors
H. aegypticus,
H. influenzae
H. parahaemolyticus ➔ Haemophilus spp. Requiringonly V factors
H. parainfluenzae
H. paraphrophilus
H. ducreyi ➔ Microscopic: school of fish; rail road track or finger print appearance
o Causes STD – soft chancre
H. influenzae ➔ With colonies appearing like “dew drops”
CAPNOPHILIC ➔ Genus Neisseria
➔ Genus Streptococci
Campylobacter ➔ Microaerophilic & capnophilic
V. mimicus & V. cholerae ➔ Species in genus Vibrio considered not HALOPHILIC
Genus Vibrio – 8-10% salt ➔ Species in genus Vibrio considered HALOPHILIC
Glucose ➔ All members of the genus Vibrio ferment
V. vulnificus ➔ The only lactose fermenting vibrio
Eikenella corrodens ➔ PITS and corrodes the agar
➔ Colonies with bleach like odor
➔ Normal flora
Prevotella spp. & ➔ Brick read fluorescens on UV
Porphyromonas ➔ Produces black pigment on plated media
o Gram (-) anaerobic bacilli
Genus Veilonella ➔ Red fluorescence
o Gram (-) anaerobic cocci
Fusobacterium nucleatum ➔ Microscopically appears like toothpick because of tapered ends
➔ Colonies may appear like breadcrumbs
➔ Produces speckled colonies
Bifidobacterium spp. ➔ Anaerobic bacilli with BIFID or bifurcated ends
➔ Appear like dog biscuit under the microscope?
Actinomyces israelii ➔ Anaerobe
➔ Produces colonies with molar tooth appearance
Aggregatibacter ➔ Part of HACEK – star shaped colonies
actinomycetemcomitans
o HACEK – causes endocarditis
Alcaligenes faecalis ➔ Apple fruity odor
Streptobacillus ➔ Puff balls/fluff balls growth on broth
monoliformis ➔ String of beads
o Causes rat bite fever (may be acquired through animal bite or scratch) and Haverhill fever – through ingestion
of contaminated milk
Bordetella pertussis ➔ Colonies appearing like mercury droplets
Staphylococcus aureus ➔ On MSA produces colonies with odor similar to an OLD SOCK
Borrelia spp. ➔ Loosely coiled spirochetes
o Blood spirochetes
o Needs vector (arthropod transmitted)
Leptospira ➔ Tightly coiled spirochetes
Treponema pallidum ➔ Non-culturable spirochete
➔ Can be maintained alive using testicular chancre of rabbits
Mycobacterium & ➔ Not gram stained – cell wall less
Ureaplasma
Gardnerella vaginalis ➔ Produces clue cells on pap-stained smears
Mycoplasma spp. ➔ Fried egg appearing colonies

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Chlamydia pneumoniae ➔ With pear shaped elementary bodies
o Chlamydia trachomatis & Chlamydia psittaci – produces round elementary bodies
Campylobacter spp. ➔ S-shaped, wings of seagulls
➔ Darting motility
Pasteurella multocida ➔ Musty odor colonies/smells like mushroom
Cardiobacterium hominis ➔ Part of “HACEK” group that forms rosette arrangement

➔ SUBACTUE BACTERIAL ENDOCARDITIS (SBE) – caused by Viridans streptococci

➔ Gram (-) coccobacilli that causes SBE – HACEK
 Haemophilus parainfluenzae
 Aggregatibacter spp.
 Cardiobacterium hominis
 Eikenella corrodens
 Kingella kingae

PATHOLOGY-DISEASES & VIRULENCE FACTOR

Staphylococcus aureus
➔ Toxin Mediated diseases
 Toxic shock syndrome –TSST1 or enterotoxin F
 Food poisoning – enterotoxins A, B, and D
 Scalded skin syndrome/ritter’s disease –exfoliatin
➔ Non-Toxin mediated diseases
 Bullous impetigo – skin infection
 Skin infections (boils, carbuncles, furuncles, cellulitis)
 Stye – eye infection
IMPORTANT VIRULENCE FACTORS
Coagulase ➔ Causes bacterial cell to agglutinate in plasma; converts fibrinogen into
fibrin
➔ Major virulence factor
Beta hemolysin ➔ Causes beta-hemolysis
PVL-Panton valentin leucocidin ➔ Causes destruction of WBCs
Beta Lactamase or Penicillinase ➔ Causes resistance to penicillin
DNAse or Thermonuclease ➔ Decreases viscosity of exudates allowing more mobility
Enterotoxins A & B ➔ Food poisoning
Exfoliatin ➔ Associated with scalded skin syndrome
Lipase ➔ Initiates skin infections
TSST1 ➔ Causes toxic shock syndrome
Staphylokinase ➔ Promotes fibrinolysis; dissolution of clot
Protein A ➔ Prevents phagocytosis
Hyaluronidase/Duran Raynal Factor ➔ Enhances ability of an organism to invade tissues
CoNS (S. epidermidis & S. saprophyticus)
➔ Staphylococcus epidermidis – prosthetic heart valve infection – biofilm production
➔ Staphylococcus saprophyticus – UTI in young female
➔ S. pyogenes – group A beta-hemolytic streptococci
 Major throat pathogen
 Causes pharyngitis
 Necrotizing fasciitis – rapidly progressing skin infection
 Scarlet fever – symptom: strawberry tongue
 Erysipelas
➔ Post-streptococcal sequelae – acute glomerulonephritis and rheumatic fever
 Unwanted outcome of pharyngitis
➔ Dick’s Test – detects susceptibility to scarlet fever
IMPORTANT VIRULENCE FACTORS
M Protein ➔ Major virulence factor
➔ Prevents phagocytosis
Protein F ➔ Provides adherence to ET cells
Hyaluronidase ➔ Spreading factor
Streptokinase ➔ Promotes fibrinolysis; dissolution of clot
Erythrogenic Toxin/Pyrogenic ➔ Causes rashes in scarlet fever
Toxin ➔ Schultz Charlton Test – to determine if rashes are due to scarlet fever or not
Streptolysin O ➔ Oxygen labile; antigenic; causes sub-surface hemolysis on BAP
➔ Will cause hemolysis only when incubated anaerobically
Streptolysin S ➔ Oxygen stable; non-antigenic; causes surface hemolysis on BAP

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➔ Will case hemolysis only when incubated aerobically
➔ Streptococcus Agalactiae
 #1 cause of neonatal meningitis
 Capsule – major virulence factor
➔ Streptococcus pneumoniae
 #1 cause of adult bacterial meningitis
 Causes pneumonia (rusty brown sputum)
 Otitis media – ear infection
 Capsule
➔ Viridans Streptococci
 Major throat flora – causes SBE
➔ S. mutans - #1 contributor of dental carries
HACEK Group
➔ Agents of endocarditis
Neisseria gonorrhea
➔ Gonorrhea – STD
➔ Ophthalmia neonatorum – gonorrhea form of conjunctivitis acquired by newborns when the discharge from infected
mother accumulates in their eye conjunctiva
 Can be prevented using silver nitrate
Neisseria meningitidis
➔ Normal flora of oro & nasopharynx
➔ Meningitis in those between 5-29 y/o
➔ Meningococcemia – meningococci in blood
➔ Waterhouse Friedrichsen Syndrome – severe form of meningococcemia characterized by bleeding of adrenal
glands; disseminated intravascular coagulation DIC
Moraxella catarrhalis
➔ Known to cause otitis media
Bacillus anthracis
➔ Gram +, aerobic, spore former
➔ Non-motile and gamma hemolytic
➔ Selective media: polymyxin lysozyme EDTA thallous acetate
➔ Colonies with swirling projections – medusa head, lion head
➔ Colonies with beaten egg white consistency – tenacious
➔ On gelatin media – inverted fir tree/pine tree pattern
➔ Forms sting of pearl appearance – susceptibility to penicillin
➔ Anthrax bacillus because it is the known causative agent of the disease anthrax
➔ Cutaneous anthrax – most common type but lease severe
 May be acquired through contact with infected spores – black eschar
➔ Intestinal – ingestion of spores – most severe; least common
➔ Pulmonary – woolsorter’s disease; rag picker’s disease or hide porter’s disease
 Inhalation of spores
➔ Virulence factors
 Poly-D-glutamate capsule
 Exotoxin with 3 components – edema factor, lethal factor and protective antigen
Genus Clostridium
➔ Clostridium perfingens – myonecrosis/gas gangrene
 Histotoxic
 Reverse CAMP test +; double/target hemolysis
 Stormy fermentation of milk (litmus milk test)
➔ Clostridium difficile – pseudomembranous colitis; antibiotic associated diarrhea
 Enterotoxin
 Toxin A (enterotoxin) and toxin B (cytotoxin)
 Detection of cytotoxin through immunoassays
 + fluoresce chartreuse on BAP
➔ Clostridium tetani – causes tetanus – spastic paralysis due to tetanospasmin
 Tetanospasmin – major virulence factor; an exotoxin which will block release of neurotransmitters → spastic
paralysis → locked jaw → sardonic smile
 Diagnosis of tetani is based on symptoms – rhesus sardonicus
➔ Clostridium botulinum – causes botulism – home canned good intoxication
 Neurotoxin – botulinum toxin, a very potent toxin
 Causes flaccid paralysis – muscles are totally unresponsive
 Infant botulism – may develop as a result of spore ingestion via breast feeding
 Symptom: floppy baby syndrome
 SID/sudden infant death syndrome/crib death

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 Laboratory confirmation is done by demonstrating the presence of toxin in serum, stool, or food, or by
culturing C. botulinum from stool, a wound or food.
 Not cultured; Von ermengen’s bacillus
 On BAP – alpha-hemolytic, + lipase reaction
Corynebacterium diphtheriae
➔ A.k.a. Kleb Leoffler’s bacillus
➔ Diphtheria due to release of diphtheria toxin “pseudo membrane formation/swollen neck appearance” (characteristic
symptom)
 Characterized by low grade fever, mild soar throat and body weakness
➔ Modified Elek’s Test – in vitro toxigenicity test
➔ Animal inoculation/Guinea pig lethal test
Listeria monocytogenes
➔ Primarily an animal pathogen
➔ Can tolerate cold enrichment
➔ Agent of food poisoning due to consumption of contaminated soft cheese and coleslaw
➔ Can cause septicemia; still birth, meningitis in elderly & immunocompromised

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