TABLE OF CONTENTS:

§ Staphylococcus .......... 3

§ Streptococcus .......... 4

§ Gram negative cocci .......... 5

§ Mycobacteria .......... 6

§ Nocardia .......... 8

§ Corynebacteria .......... 8

§ Spore-forming bacteria .......... 9

§ Anaerobic bacteria .......... 11

§ Enterobacteriaceae .......... 12

§ Vibrionaceae .......... 16

§ Non fermentative organisms .......... 17

§ Parvobacteria .......... 19

§ HACEK .......... 21

§ Unusual bacteria .......... 21

§ Miscellaneous bacteria .......... 24

§ Water Bacteriology .......... 26

§ Milk Bacteriology .......... 26

 BACTERIOLOGY

GRAM STAIN
Purpose Reagents Gram (-) Gram (+)
Primary Crystal violet Purple Purple
Mordant (↑ affinity of dye to Iodine Purple Purple
the organism)
Decolorizer Acetone alcohol Colorless Purple
Counterstain Saftanin Red Purple

General rule:
1. ALL COCCI ARE GRAM (+) except Neisseria, Veilonella, Moraxella
2. ALL BACILLI ARE GRAM (-) except Mycobacteria, Corynebacteria, Clostridia, Bacillus,
Lactobacillus, Listeria, Erysipelothrix, Nocardia, Actinomyces
3. All spiral organisms are reported as Gram (-)
4. Yeasts are Gram (+)

Cell wall: basis for Gram staining

§ Gram (+)
o Impermeable to alcohol
o Thick peptidoglycan (murein)
o Teichoic acid
o Mycolic acid (Mycobacteria)
o Exotoxin
§ Gram (-)
o Permeable to alcohol
o Thin peptidoglycan
o Lipopolysaccharide (loose layer)
o Outer membrane
o Periplasm
o Lipid A
o Exotoxin and endotoxin
o Somatic (O) Ag - serotyping

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 BACTERIOLOGY

STAPHYLOCOCCUS Coagulase negative

Under the microscope: [Gram positive in grape-like clusters] § S. hominis: novobiocin (R), urease (+)
§ S. simulans: novobiocin (S), urease (+), PYR (+)
§ S. sciuri: novobiocin (R), oxidase (+), ALP (+)
§ S. schleiferi: lactose, BG (+), endocarditis
§ S. cohnii, S. xylosus

Coagulase negative Staphylococci

Staphylococcus epidermidis
- Skin flora
- Blood culture contaminant
- Bacteremia
- Prosthetic valve endocarditis
Micrococcus Staphylococcus Rothia - Novobiocin (S)
Catalase + + v.
O/F test Oxidative Fermentative Fermentative Staphylococcus saprophyticus
Mod. Oxidase + - - - Novobiocin (R)
Bacitracin S R R - PYR (-)
Furazolidone R S R - Glucosidase (+)
Lysostaphin R S R - UTI in young women
Staphylococcus aureus
- Yellow orange colony Clinically important Staphylococci:
- CATALASE (+) S. aureus S. epidermidis S. saprophyticus
- COAGULASE (+) Colony Yellow White White
- Nitrate and VP (+) Catalase + + +
- Gelatin (+) Coagulase + - -
- PYR (-) Mannitol + - -/+
Novobiocin S S R
Virulence factor: DNAse + - -
§ Protein A – cell wall, anti-phagocytic, virulence
Phosphatase + + -
§ Enterotoxin – food poisoning
Gelatinase + + +
§ Beta hemolysin
§ Leukocidin – Panton-Valentine – lyse WBC Mannitol positive (S. aureus)
§ TSST-1 – toxic shock syndrome
§ Exfoliatin (epidermolysin) – skin scalded syndrome
(Ritter’s)
§ Beta lactamase, DNAse
§ Staphylokinase – dissolve clot (fibrinolysin)
§ Hyaluronidase – spreading factor
§ Gelatinase – liquefy gel
§ Lipase – fat splitting enzyme
*Note: Catalase is not a virulence factor

Diseases caused:
Skin infections, ocular pathogen, carbuncles,
furuncles, folliculitis, cellulitis, wound infections,
impetigo, bacteremia, endocarditis, osteomyelitis,
septic arthritis Staphylococcus aureus vs. Staphylococcus epidermidis
on blood agar
Toxigenic diseases:
§ Skin scalded syndrome Staphylococcus aureus
§ Food poisoning - Yellow, cream, beta-hemolytic
§ Toxic shock syndrome
§ Nosocomial infection Staphylococcus epidermidis
- White, non-hemolytic
Like S. aureus:
1. Staphylococcus intermedius: slide coagulase (+), Methicillin Resistant S. aureus
VP/acetoin (-), PYR (+) - Penicillin (R)
2. Staphylococcus lugdunensis: slide coagulase (+), - Oxacillin (R)
PYR (+), endocarditis - Cefoxitin screen (+)
3. Staphylococcus haemolyticus: beta-hemolytic,
coagulase (-), endocarditis Methicillin Sensitive S. aureus
- Penicillin (R)
Coagulase positive: - Oxacillin (S)
§ S. aureus - Cefoxitin screen (-)
§ S. pseudiintermedius
§ S. intermedius
§ S. delphini
§ S. lugdunensis

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 BACTERIOLOGY

STREPTOCOCCUS Gamma streptococcus

- Gram-positive cocci in chain, pairs - No hemolysis (no zone)
- CATALASE NEGATIVE - E. faecalis, E. faecium, S. bovis (Group D)
- Pinpoint colonies Lancefield Classification
- Facultative anaerobes Based on carbohydrate on the cell wall
- Capnophilic (5-10% CO2) Added with antisera → (+) agglutination
- Medium of choice: sheep’s blood agar
- Selective medium: PEA, CNA The following are the bacteria under Lancefield classification:
Streptococcus pyogenes (Group A Streptococci)

- Flesh-eating bacteria
- Drug of choice: penicillin
- Gram (+) cocci in chain
- Beta hemolytic on blood agar
- Catalase negative
- Bacitracin (S), SXT (R), PYR (+)
- M protein – cell wall, anti phagocytic, virulence →
react with Factor H
- Streptolysin O – O2 labile, Ag, anaerobic, lytic, sub surface
Staphylococci vs. Streptococci hemolysis
Staphylococci Streptococci - Streptolysin S – O2 stable, non Ag, surface hemolysis
Catalase + - - Erythrogenic toxin – scarlet fever
Hemolysis +/- +
- Streptokinase – thrombolytic drug (AMI)
CO2 - + - Hyaluronidase AKA spreading factor (Duran Reynal Factor)
Colony Pinhead Pinpoint
Diseases:
Beta-hemolytic Gamma-hemolytic Alpha-hemolytic
Strep Strep Strep - MAJOR THROAT PATHOGEN
Group A Strep Group D Strep S. pneumoniae - Pharyngitis
-Bacitracin (S) -Bile esculin HOH -Optochin (S) - Complications: acute glomerulonephritis,
-PYR (+) (+) -Bile solubility (+) rheumatic fever/rheumatic heart disease
-SXT (R) - Scarlet fever
S. viridans Dick’s test (red) – skin test for scarlet fever
Group B Strep -Optochin (R) Schultz-Charlton (rash face) – immunity test
-CAMP (+) -Bile solubility (-) - Erysipelas, impetigo, wound burn
-Hippurate HOH (+) - Toxic shock syndrome, puerperal sepsis, childbed
-SXT (R)
fever, pyoderma, necrotizing fasciitis
Group C, F, G Streptococcus agalactiae (Group B Streptococci)
-SXT (S)
Hemolysis on blood agar - Beta-hemolytic, vaginal flora
- #1 Neonatal meningitis, septicaemia
- CAMP TEST (+) – arrowhead zone of β-hemolysis
- Hippurate hydrolysis (+)
- Granada medium for S. agalactiae
Group C, F, G Streptococci

- Beta-hemolytic
- Bacitracin (R), SXT (S)

Group C:
- Streptococcus equisimilis
- Streptococcus equi
- Streptococcus dysagalactiae
Beta-hemolytic Streptococci
Bacitracin SXT (Cotrimoxazole) Organism Diseases:
S R Group A Strep - Pharyngitis, bacteremia, pneumonia, cellulitis,
R R Group B Strep abscess
R S Group C, F, G Strep
Group D Streptococci
Smith’s and Brown’s Classification I. Enterococcus
Based on hemolysis on blood agar Enterococcus faecalis (index of fecal contam of
sea/marine water)
Alpha streptococcus Enterococcus faecium
- Incomplete (greenish zone) hemolysis Enterococcus durans
- S. pneumoniae, Viridans streptococcus
- Drug resistant (vancomycin resistant enterococci)
- UTI, wound, nosocomial pathogen
*Alpha prime – zone of alpha hemolysis surrounded by zone
of beta hemolysis after refrigeration
II. Non-enterococcus
Streptococcus bovis (causes colon cancer)
Beta streptococcus Streptococcus equines
- Complete (colorless/yellowish zone) hemolysis
- S. pyogenes, S. agalactiae, Groups C, F, G

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 BACTERIOLOGY

*Bile hydrolysis test Vancomycin Resistant Streptococci – Streptococcus

- (+) blackening on bile esculin medium Associated with Bacterial Endocarditis (SBE)
- Differentiates group D from other strep Pediococcus Leuconotoc
- Presumptive test for group D Vancomycin R R
Bile esculin + +
Enterococcus Non-enterococcus 6.5% NaCl + +
Bile esculin + + PYR - -
6.5% NaCl + - LAP + -
PYR & Hippurate + - MRS broth - +
Penicillin R S Other info:
Growth 45°C + + 1. Aerococcus spp.: PYR (-), LAP (+), environment,
Growth 10°C + - UTI
The following are not anymore classified under Lancefield: 2. Gemella spp.: oral flora, Gram (+) cocci in pair,
Streptococcus pneumoniae cluster, chain, tetrad
3. Alloiococcus spp.: PYR & LAP (+), otitis media
- Gram (+) diplococcic, lancet-shaped 4. Streptococcus urinalis: PYR & LAP (+), 6.5% NaCl
- Alpha-hemolytic (+), growth at 10°C (-), UTI
- Virulence: capsule (mucoid colonies) 5. Enterococcus spp.: PYR & LAP (+), 6.5% NaCl (+),
- Optochin sensitive > 16 mm novobiocin, growth at 10°C (+)
- Grows on blood agar, chocolate agar, gentamicin Abiotrophia spp and Granulicatella
blood agar but not on MacConkey agar - Nutritionally variant streptococci
- On blood agar: round, brownish, alpha-hemolytic, - Streptococcus associated with bacterial endocarditis
mucoid - Requires Vit. B6 (pyridoxine)
- Neufeld Quellung (+) capsular swelling – - Staph streak test (+)
confirmatory test - (-) BAP, CAP
- Francis test – skin test for pneumococci - (+) Pyridoxal medium

GRAM NEGATIVE COCCI

Genera included:
§ Neisseria – aerobic
§ Moraxella – aerobic
§ Veilonella – anaerobic

- Gram negative diplococcic

- Oxidase and catalase (+)
- Grow well on chocolate agar

Neisseria gonorrhoeae: (+) CAP, (-) BAP

Neisseria meningitides: (+) CAP, (+) BAP
Normal flora/Saprophytic:
- N. subflava
- N. flavescens (yellow)
- N. mucosa
- N. sicca
Diseases:
Pelvic Inflammatory Disease (PID)
- #1 adult bacterial meningitis
1. Chlamydia trachomatis
- Pneumococcal pneumonia
2. Neisseria gonorrhoeae
- Otitis media (most common cause)
3. Mycoplasma hominis
- Lobar pneumonia (rusty sputum)
- Bacteremia, community acquired pneumonia
Antibiotics inhibit:
Viridans Streptococci
§ Vancomycin – Gram (+) bacteria
§ Colistin – Gram (-) bacteria
- Optochin resistant
§ Nystatin – Yeast
- Bile insoluble
§ Trimethoprim lactate – swarming Proteus
- #1 oral flora
§ Anisomycin – Yeast
Species:
§ Amphoterecin – Yeast
Streptococcus mitis
Streptococcus sanguis – subacute endocarditis due
to dental procedure * OXIDASE [TAXO N] (+) “PPAM ViC HeN”
§ Pseudomonas
Streptococcus mutans – dental plaques/caries
§ Plesiomonas
Gram positive cocci, catalase negative, beta hemolytic strep
Test Group A Group B Grp C, F, G § Aeromonas
Bacitracin S R R § Moraxella
SXT R R S § Vibrio
CAMP - + - § Campylobacter
PYR + - - § Helicobacter
Hippurate - + - § Neisseria

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 BACTERIOLOGY

Neisseria gonorrhoeae
- Kidney (coffee) bean shaped inside
polymorphonuclear neutrophils
- OXIDASAE POSITIVE and ferments glucose
- SUPEROXOL CATALASE TEST (+) – uses 30% H2O2
- Virulence: “pili”, endotoxin
- Diseases: gonorrhea (clap), ophthalmia neonatorium,
salphingitis, epididymitis, arthritis, PID, leading cause
of STD, Fitz-Hugh Curtis syndrome
- Penicillinase-producing N. gonorrhoeae – resistant to
penicillin

OTITIS MEDIA
1. Streptococcus pneumoniae
2. Haemophilus influenzae
3. Moraxella catarrhalis

MYCOBACTERIA
Penicillinase (+) → (R) to penicillin → do beta-lactamase test - ACID FAST BACILLI
Beta lactamase test is held on primary culture because - Mycolic acid
plasmid is lost on subculture; (+) color change - Slow growers except: M. fortuitum, M. chelonei
Neisseria meningitides - “Much granules”; aerobic non sporeformer, non-
- Gram (-) kidney shaped diplococi motile
- CAP: smooth, gray-brown, mucoid - In the cell wall: N-glycolylmuramic acid, high lipid
- OXIDASE POSITIVE
- CHO ferment: glucose & maltose Three groups:
- Carrier: nasopharynx A. M. tuberculosis complex (causes TB)
- Virulence: capsule, endotoxin, pili, IgA protease 1. M. tuberculosis – pulmonary TB
- Serotypes A, B, C, Y, W135 – capsule Ag 2. M. bovis – intestinal TB (BCG vaccine source)
- Fatal meningitis, meningococcemia, DIC 3. M. africanum – pulmonary TB (Africa)
- Waterhouse-Freiderichsen syndrome –
hemorrhage in adrenal gland B. MOTT/NTM – NON TB MYCOBACTERIA

Moraxella catarrhalis C. M. leprae: agar (-)

- URT commensal, Gram (-) diplococcic
M. tuberculosis M. bovis
- OXIDASE POSITIVE, reduce NO3 to NO2
Niacin + -
- DNAse POSITIVE (best to differentiate from other
Nitrate + -
Moraxella spp.) TCH R S
- ASACCHAROLYTIC, beta lactamase (+) Catalase - v.
- 3rd cause of otitis media AFB Grading National Standard (DSSM)
- Bronchopulmonary (LRT); Sinusitis (URT)
0 : No AFB/300 fields
+n : 1-9 AFB/100 fields
Oxidase CHO DNAse TMA 1+ : 10-99 AFB/100 fields
N. gonorrhoeae + Glucose - +
2+ : 1-10 AFB/field in at least 50 fields
N. meningitides Glucose,
+ - + 3+ : >10 AFB/field in at least 20 fields
maltose
M. catarrhalis + None + +
Butyrate Disk (Tributyrin HOH) Test Mycobacterium tuberculosis (Koch bacillus)
(+) blue color (M. catarrhalis)
(-) no color change (N. gonorrhoeae)
CHO Fermentation Test
Glucose Maltose Lactose Sucrose
Neisseria
+ - - -
gonorrhoeae
Neisseria
+ + - -
meningitides
Neisseria
sicca
+ + - +
(wrinkled
colony)
Neisseria
lactamica + + + -
(ONPG +)
Moraxella
catarrhalis - - - -
(hockey puck
colony) - Obligate aerobe; require 5% CO2 for growth

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 BACTERIOLOGY

- Virulence: cord factor (clumping/aggregation of acid Mycobacteria Other Than Tuberculosis (MOTT) or Non TB
fast organisms seen on smear) and sulfatides Mycobacteria (NTM)
*based on photoreactivity, pigment production, rate of growth,
*Mantoux – skin test for tuberculosis biochemical test

For culture: A. Photochromogens – ROUNYON’S Group I

A. Agar based medium - Growth in the presence of light
§ Duboi’s Oleic Acid Albumin medium M. kansasii: NO3 (+), yellow, pulmonary disease
§ Mitchison’s medium M. marinum: NO3 (-), swimming pool granuloma
§ Middlebrook 7H10-7H11: used in AST M. asiaticum: pulmonary disease
M. simiae: niacin (+), NO3 (-)
B. Egg-based medium M. intermedium: pulmonary disease
Malachite green – inhibits normal flora M. novocastrense: cutaneous disease
§ Lowenstein-Jensen medium (best)
§ American Thoracic Society medium B. Scotochromogens – ROUNYON’S Group II
§ Petragnani medium - Growth in the presence/absence of light
§ Dorset Egg medium M. scrofulaceum: scrofula (cervical adenitis), Tween 80 (-),
Urease (+)
C. Liquid media (rapid culture) M. gordonae: tap water bacillus, Tween 80 (+), Urease (-)
§ Bactec 12B, Septi-Chek AFB, Middlebrook 7H9/7H12 M. szulgai: pulmonary disease, cervical adenitis
M. tuberculosis on Lowenstein-Jensen agar M. interjectum: lymphadenitis, pulmonary disease
M. heckeshomense: pulmonary disease
M. kubicae: pulmonary disease
M. tusciae: tap water, cervical lymphadenitis
M. cookie, M. hiberniae: non-pathogens

C. Non photochromogens – ROUNYON’S Group III

- Growth in the absence of light
M. avium complex (MAC): pulmonary dse in AIDS, Tellurite
(+), Catalase (+); MAC=M. avium & M. intracellulare
M. intracellulare: Battey bacillus
M. ulcerans: Buruli ulcer
Gene Expert – rapid, sensitive, definitive test for TB M. xenopi: pulmonary disease
M. malmoense: pulmonary disease
Biochemical Test for Mycobacteria M. genovense: disseminated disease in AIDS
M. haemophilum: disseminated disease, beta-hemolytic
1. NIACIN TEST M. paratuberculosis: inflammatory bowel disease (Johne’s);
- Definitive test Crohn’s disease
- Principle: niacin + niacin ribonucleotide + aniline dye M. celatum, M. conspicuum, M. heidelbergense, M. terrae,
+ cyanogens bromide = yellow (+) M. trivial, M. gastri: non pathogens
- M. tuberculosis (+); M. bovis (-)
D. Rapid growers – ROUNYON’S Group IV
2. CATALASE TEST AT 68°C (Heat stable) M. fortuitum: NO3 (+)
- Medium: Tween 80; Reagent: 30% H2O2 M. chelonei: NO3 (-)
- Result: >45 mm height of gas bubbles M. phlei: CO2 (+), mycobactin
- M. kansasii (+); M. tuberculosis (-) M. smegmatis: urine AFB (+)
M. abscessus: disseminated disease
3. NITRATE REDUCTION TEST
- (+) result: pink/red M. fortuitum M. chelonei
Arylsulfatase + +
4. TWEEN 80 HOH TEST Mac without + +
crystal violet
- (+) Red
Nitrate + -
- M. kansasii (+); M. avium (-)
5% NaCl and
+ -
Iron Uptake
5. TELLURITE REDUCTION TEST
Mycobacterium leprae (Hansen bacilli)
- Tellurite → black metallic tellurium
- AFB, cigarette-packet/picket fence
- Used to ID M. avium - Not cultivable in agar; specimen: tissue
- Hydrolyze 3,4-dihydroxy-phenylalanine
6. ARYLSULFATASE TEST (Rapid growers)
- Tropism to peripheral nerves (lost of sensation)
- Tripotassium phenolphthalein disulfide/sulfate acted - Causes: Leprosy (Hansen’s disease)
upon by arylsulfatase to produce free phenolphthalein
- Lepromatous/Multibacillary – Lepromine (-); many
- (+) pink/red: M. fortuitum-chelonei
AFB, CMI (-), Leonine face
- Tuberculoid/Paucibacillary – Lepromine (+), few AFB,
7. TCH SUSCEPTIBILITY TEST
CMI (+), Nodules
- M. bovis (S); M. tuberculosis (R)
- Culture: foot pads of armadillo
- Fite Faraco stain
*IGRAS (Immunology)
- Interferon Gamma Release Assay
- Patient with BCG vaccine; T-cell based test

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 BACTERIOLOGY

- Phenolase test – separates M. leprae from other CORYNEBACTERIA

Mycobacteria with the use of 3,4-dihydroxyphenyl- - Pleomorphic Gram positive rods
alanine - Club shape, chinese letters, palisade
- Wade-Fite technique – used to detect AFB in - Characteristic: Babes-Ernst metachromatic
paraffinized tissues granules
- Lepra cell – macrophage containing acid-fast bacilli - Non motile, no spore, no capsule
- Treatment: Dapsone - In BAP: raised, translucent, gray colonies
- CATALASE POSITIVE

Listeria Corynebacterium
Motility + at 22°C -
Esculin HOH + -
NOCARDIA Salicin + -
- Partially acid fast CAMP + -
- Gram positive branching rod (Actinomycete) Corynebacterium diphtheria (Kleb Loeffler’s Bacillus)
- Lysozyme resistant, catalase (+), urease (+) - Virulence: exotoxin heat labile A, B
- Cause pneumonia - Disease: Diphtheria – grayish pseudomembrane on
- Fungus-like bacteria tonsils, pharynx, larynx
- Has extensive aerial hyphae - “Bull neck appearance”
- Specimen: OPS, NPS, skin swab
*anaerobic for Actinomyces
Sulfur granules for Actinomyces Biotype of C. diphtheria
Variant:
1. gravis – gray, large, non hemolytic, starch/glycogen
fermentation (+)

2. intermedius – black, small, non hemolytic

3. mitis – medium-sized, black, beta hemolytic,

starch/glycogen fermentation (-)

Lab ID
1. Culture on blood agar, Loeffler’s serum agar, Pai coagulated
egg, Clauberg, MacLeod’s
* Tinsdale (black colony with brown halo)
* Potassium tellurite – inhibits normal flora; gray to black col
* Cystine tellurite BAP – gun metal gray colonies
2. G/S: Gram (+) bacilli in V, X or Chinese letters=
3. LAMB Stain – metachromatic granules
Nocardia Actinomyces 4: Catalase and DNAse
Oxygen Aerobic Anaerobic (+); Urease (-)
Acid fast Acid fast organism Non acid fast 5. CHO Fermentation test:
Catalase + - glucose and maltose
Urease + - 6. Elek test –
Sulfur granules +/- + confirmatory; (+)
precipitation line diphtheria
toxin in vitro test
7. Schick test – skin test
for diphtheria

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 BACTERIOLOGY

Pathogens (Toxigenic) Urease Nitrate Starch HOH

C. diphtheriae - + +/-
C. ulcerans + - +
C. pseudotuberculosis + +/- -
Diphtehroids
- Normal flora of skin, oral, conjunctive, GUT
- Non toxigenic
- Does not produce gray-black colonies on tellurite
media
- Endocarditis due to bacteremia
Diphtheroids Feature Disease
Ferment dextrose,
C. xerosis saccharose & Conjunctivitis
maltose
Hoffman’s
C. pseudodiphtheriticum Endocarditis
bacillus – oral
JK bacillus, drug Prosthetic valve
C. jeikeium resistant, skin flora endocarditis (HIV)
Skin, conjunctiva,
C. amycolatum Endocarditis
oral flora
C. auris Human flora Otiits media
Leifsonia aquatic Fresh water Bacteremia
Kurthia Environment Bacteremia
Coral red
C. minutissimum Erythrasma
fluorescence
Reverse CAMP (+)
Arcanobacterium
w/ S. aureus Bacteremia
haemolyticum inverted triangle
Skin flora,
C. urealyticum UTI
Urease (+)
Rhodococcus (Corynebacterium) equi
- Pleomorphic (rod-cocci); partial AFB, Gram (+)
- Common in HIV
- Mucoid, salmon pink colonies
- Urease, catalase & nitrate (+)
- CAMP test (+) with S. aureus = arrowhead zone

Bacillus cereus (Fried rice bacillus)

- Beta hemolytic, MOTILE, Penicillin (R), Lecithinase (+)
SPORE-FORMING - Fried rice bacillus – spores on rice grain (food
poisoning)
BACILLUS & CLOSTRIDIA - Virulence: enterotoxin, pyogenic toxin
Bacillus Clostridium
- Food poisoining, eye endophthamlmitis
Oxygen Aerobic Anaerobic
Catalase + - - Blood bank contaminant (RT)
Gas - + - Selective: mannitol egg yolk polymyxin B agar,
PEMBA, Bacillus cereus medium
Bacillus anthracis (Anthrax bacillus)
- Gram (+) rods, chain – bamboo, square end *Note: heat shock method – growth & enhance endospores
(Also note: Least severe of food poisoning – S. aureus)
- Non motile, spore forming, zoonotic
- Largest pathogenic bacteria to human
Bacillus subtilis (Hay bacillus)
- Virulence: exotoxin (edema toxin, lethal toxin,
- Gram (+) rod in chain; central spore
protective Ag) and capsule (poly-d-glutamic acid)
- Common laboratory contaminant, eye infection in
- MacFadyean’s – capsular stain
heroin addicts, pneumonia, bacteremia, wounds
Disease:
Bacillus stearothermophilus – flat sour spoilage, no gas
- Malignant pustule – black eschar [cutaneous
anthrax]
Bacillus thuringiensis – pesticides and insecticides exposure,
- Woolsorter’s – [pulmonary anthrax], Ragpicker’s dse
wound, burn, pulmonary disease
- Gastroenteritis – [intestinal anthrax]
Differences on BAP:
Laboratory diagnosis: B. anthracis – non hemolytic, irregular, serrated, swirling
- Selective: PLET (Polymyxin Lysozyme EDTA P. aeruginosa – beta hemolytic, serrated, moist colony, ground glass
Thallous Acetate) B. subtilis – beta hemolytic, ground glass, dry colony
- Medusa head (ground glass) colony, inverted pine
tree
- Catalase (+)
- String of pearl test on blood agar
- Ascoli test – serologic precipitation test: detect
anthrax antigen
- Penicillin susceptibility test (10 units) – presumptive
- PCR – definitive test

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 BACTERIOLOGY

B. anthracis versus B. cereus Clostridium botulinum (Canned good bacillus)

B. anthracis B. cereus - Virulence: botulinum toxin – block release of
Motility - + acetycholine (flaccid paralysis) – most potent toxin
Capsule + - - Botulinum (neurotoxin) – most potent exotoxin
Hemolysis - + (BH)
Growth @ 45°C - + Disease:
Salicin ferment - + - Food botulism – most severe type of food poisoning
Penicillin G S R - Wound botulism – spore on wound
- Infant botulism – floppy baby syndrome; sudden
infant death syndrome (SIDS) crib death
CLOSTRIDIUM
- Obligate anaerobe Gram (+) endospore Clostridium tetani (Tack head bacillus)
- Habitat: human and animal - TERMINAL oval spore (tennis racket, drumstick) –
- Saccharolytic except C. tetani and C. septicum has iron affinity
- Ethanol shock or heat shock spore test (+) - Asaccharolytic
- Virulence: exotoxin (tetanosapsmin) – binds to
Three types of Clostridium: ganglioside receptors and inhibit neurons in CNS
1. Neurotoxic: C. tetani, C. botulinum (spastic paralysis)
2. Histotoxic: C. perfringes, C. septicum
3. Enteric: C. difficile

Clostridium perfringens / Clostridium welchii

- Encapsulated, non motile, double hemolysis
- Box car shaped bacillus
- Virulence: alpha toxin, enterotoxin, beta toxin
- Source: wound contact with soil

Diseases:
- Gas gangrene (myonecrosis)
- Food poisoning (enterotoxins)
- Necrotic enteritis (pig bel)
- Neutropenic enterocolitis

Lab ID:
- BAP – target or double zone of hemolysis Diseases:
- Nagler test – detect C. perfringens toxin (α- - Lockjaw
toxin/lecithinase); (+) opalescence on agar w/o anti- - Risus sardonicus
toxin - Opisthotonus
- Reverse CAMP test – S. agalactiae and C.
perfringens (+) arrow head zone of beta hemolysis Clostridium ramosum
- Stormy fermentation of milk – (+) coagulate casein + - Round terminal spore; ferment glucose
gas
- Chopped meat (anaerobic broth) – growth + gas Clostridium difficile
- Colon flora
- Antibiotic (clindamycin) associated
pseudomembranous enterocolitis – major cause of
diarrhea in hospitals

Lab ID:
- Direct detection of cytotoxin from the stool – toxin
detection by EIA
- Tissue culture – gold standard for toxin identification
of C. difficile
- Medium: Cycloserine Cefoxitin Fructose Agar
(yellow horse manure odor); red to yellow =
fermentation of fructose

Motility Lecithinase Lipase Lactose Glucose

C. perfringens - + - + +
C. botulinum + - + - +
Reverse CAMP test C. tetani + - - - -
C. difficile + - - - +

*Lipase (+) – C. botulinum, C. novyii, C. sporogenes

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 BACTERIOLOGY

ANAEROBIC BACTERIOLOGY 5. Fusobacterium necrophorum – Vincent’s angina

- Collection: needle aspiration; Specimen: aspirate & (synergistic infection caused by 2 organism: F.
tissue; Transport & storage: room/ambient temp necrophorum & B. vincentii), Lemierre’s disease,
colony: opalescent with speckles on stereoscope and
Media: (+) chartreuse fluorescence
- Schaedler, Lombard Dowell agar 6. Bacteroides ureolyticus – pitting of agar
- Wilkins-Chalgren broth & agar – antimicrobial 7. Lepotrichia spp. – pointed and blunt ended cell
susceptibility test medium for anaerobes fusiform rods
- Anaerobic PEA – gram positive anaerobes
- THIO – enriched broth with hemin, Vit K *Note: B. vincentii – causes trench mouth & Vincent’s angina
- BBE – B. fragilis (blackening due to bile and esculin)
- Anaerobic KVBE – gram negative anaerobes Gram (+) Anaerobic cocci
1. Peptostreptococcus anaerobius – SPS sensitive,
ID of anaerobic bacteria: indole (-)
Direct gram stain → Streak (BBE, LKV, PEA, BAP, CAP, THIO, 2. Parvimonas (Micromonas) micra – SPS sensitive,
EYA) → colony check, gram stain → aerotolerance test – SC milky halo around colony
to CAP in CO2 → SC on BAP, CAP incubate anaerobically at 3. Peptostreptococcus asaccharolyticus
37°C for 24-48 hrs → perform KVC, SPS, NO3 test 4. Peptococcus niger (Staphylococcus like) - catalase
positive
Methylene blue Resazurin 5. Finegoldia, Gallicola, Peptoniphilus,
Aerobic Blue Pink Murdochinella, S. anaerobius, Anaerococcus
Anaerobic Colorless colorless
Gram (-) Anaerobic cocci
Gas Liquid Chromatography – definitive test for anaerobes 1. Veilonella parvula – fluoresce red UVL, oral flora
2. Megasphera, Acidaaminococcus, Anaeroglobus
Characteristics of Anaerobes and Negativicoccus
Brick red fluorescence Prevotella, Porphyromonas
Red fluorescence Veillonella
Pitting of agar B. ureolyticus
Double zone of hemolysis C. perfringens
Swarming C. tetani, C. septicum GRAM NEGATIVE BACILLI
Molar tooth col., sulfur granules Actinomyces israelii
Breadcrumb colony F. nucleatum
MacConkey agar
Horse odor (CCFA) C. difficile
ID (Kanamycin-Vancomycin-Colistin)
1. RRR – B. fragilis
2. RSR – Porphyromonas
3. RRS – Prevotella
4. SRS – B. urealyticus, Fusobacterium (+) (-)
5. SSR – Clostridium Enterobacteriaceae Haemophilus,
Pseudomonas HACEK,
Gram (+) Anaerobic bacilli ↓ Pasturella,
1. Actinomyces – fungus like bacteria (ray fungus) OXIDASE Legionella,
- Actinomyces bovis – lumpy jaw ↓ Moraxella,
(+) Pseudomonas Brucella,
- Actinomyces israelii – draining sinus tract with
(-) Enterobacteriaceae B. pertussis,
sulfur granules Francisella
2. Bifidobacterium dentium – GIT, oral flora
3. Eubacterium lentum – GIT, oral flora
4. Propionebacterium acne – acne, skin flora, blood
culture contaminant, RBC contam, anaerobic
diphtheroid, catalase and indole (+), propionic acid
5. Lactobacillus – pregnancy, inhibits G. vaginalis,
promote C. albicans, Doderlein bacillus, catalase (-),
aerotolerant anaerobe
6. Mobiluncus – bacterial vaginosis (G. vaginalis),
motile

Gram (-) Anaerobic bacilli

§ GIT flora

1. Bacteroides fragilis – needs 20% bile, colon flora,

black on BBE, capsule (virulence factor), catalase (+),
bile resistant
2. Porphyromonas asaccharolytica – black pigment,
red fluorescence on UVL, vancomycin (S) H2S (+) “PECS”
3. Prevotella melaninogenica – black pigment, red § Proteus
fluorescence on UVL, vancomycin (R) § Edwardsiella
4. Fusobacterium nucleatum – breadcrumb colonies, § Citrobacter
fusiform rod, spindle shape § Salmonella

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 BACTERIOLOGY

Lysine decarboxylase [LDC] (+) “KEEESSA” ENTEROBACTERIACEAE

§ Klebsiella - Glucose fermenters
§ E. coli - Motile (petrichous) except Klebsiella & Shigella
§ Enterobacter - OXIDASE NEGATIVE except Plesiomonas
§ Edwardsiella - Catalase positive, nitrate reducer
§ Serratia - Selective: Mac, EMB, DCA, SSA, BSA
§ Salmonella - Antigenic: capsule (K), cell wall (O), flagella (H)
§ Arizona Lactose fermenters Non lactose fermenters
E. coli Proteus-Morganella-
Indole (+) “P-M-PEECSS” Klebsiella Providencia
§ Proteus-Morganella-Providencia Arizona Salmonella
§ E. coli Citrobacter Shigella
Enterobacter Serratia (LLF)
§ Edwardsiella
Hafnia Yersinia
§ Citrobacter diversius Plesiomonas
§ Shigella flexneri/boydii Edwardsiella
§ Serratia Media Colonies Organisms
Voges-Proskauer (+), Methyl red (-) “KESH” Colorless Shigella
§ Klebsiella SSA
Colorless w/ black Salmonella
§ Enterobacter Mac Red/pink E. coli
§ Serratia GMS, Red, LF: K. Pneumoniae
EMB
§ Hafnia Colorless NLF: Shigella, Salmonella
Yellow Vibrio cholera
TCBS
Citrate (+) “PPECKAS” Bluish-green V. parahemolyticus
SMac Colorless EHEC (E. coli O157-H7)
§ Proteus
§ Providencia Other Enterics:
§ Enterobacter Uropathogenic E. coli (UPEC) UTI, cystitis, pyelonephritis
Meningitis/sepsis associated
§ Citrobacter Neonatal meningitis, K1 Antigen
E. coli (MNEC)
§ Klebsiella
Non-motile, NLF, anaerogenic
§ Arizona Alkalescens dispar E. coli
§ Salmonella enteritidis Biochemically inert, blood and
Ewingella Americana wound infections
Urease (+) “KPMPS” NLF, malignancy (colon
Hafnia alvei
§ Klebsiella cancer), GIT infections
§ Proteus
§ Morganella RAPID LACTOSE FERMENTERS (Coliforms)
§ Providencia
§ Serratia Escherichia coli (Colon bacillus)
- # 1 cause of UTI, gram negative sepsis
PAD, Urease, LD (+) “P-M-P” - #2 neonatal meningitis (K1 antigen)
§ Proteus - Nosocomial, wound, bacteremia, pneumonia
§ Morganella
§ Providencia TSI: A/AG IMVC: +, +, -, - LOA: +, +, -
Greenish metallic sheen on EMB
Non motile at 35°C “SKY” MUG (+) except E. coli O157-H7
§ Shigella, Klebsiella, Yersinia

KCN → differentiate Salmonella (-) from Proteus (+)

Malonate → differentiate Citrobacter (+) from Salmonella (-)

Enteric Media
pH
Medium Inhibitor CHO LF NLF
Indicator
Eosin Y Eosin Y
Red/pink
EMB Methylene Lactose Methylene Colorless
GMS
blue blue
Crystal
Mac violet Lactose Neutral red Red/pink Colorless
Bile salt
Xylose
Red/
XLD Bile salt Lactose Phenol red Yellow
Colorless
Sucrose
Salicin Brom-
Green/
HEA Bile salt Lactose thymol Yellow
Sucrose blue
colorless Biotypes:
DCA Bile salt Lactose Neutral red Red/pink Colorless 1. Enterotoxigenic E. coli (ETEC)
Bile salt - Traveller’s diarrhea
SSA Brilliant Lactose Neutral red Red Colorless - Cholera like toxin or heat labile enterotoxin
green
Brilliant Bismuth
- Watery diarrhea without mucus
BSA Glucose Black col. - Salmonella - O6; O8; O25
green sulphite
Brom-thymol
TCBS Bile salt Sucrose blue Yellow Green

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 BACTERIOLOGY

2. Enteropathogenic E. coli (EPEC)

- Infantile diarrhea (pathogenicity island)
- Watery diarrhea with mucus
- O111, O114

3. Enteroinvasive E. coli (EIEC)

- Dysentery (shigella) like diarrhea (invasion)
- Stool with RBC, WBC, mucus
- O124, 143, 164
- Sereny test – virulence test

4. Enterohemorrhagic E. coli (EHEC)

- Verotoxin E. coli (VTEC)
- Hemolytic Uremic Syndrome
- Hemorrhagic colitis
- Bloody diarrhea without WBC
- Shigella-like toxin – verotoxin K. pneumoniae on MacConkey agar
- E. coli O157-H7 : Sorbitol MacConkey (-); MUG (-)
LDC VP & Urease Indole
K. pneumoniae + + -
K. oxytoca + + +
K. ozaenae + - -
K. rhinoscleromatis - - -

Biochemical Test Result of Rapid Lactose Fermenters

TSI LIA
E. coli A/A + gas K/K
K. pneumoniae A/A + gas K/K
E. aerogenes A/A + gas K/K
E. cloacae A/A + gas K/A

I M V C U
E. coli + + - - -
K. pneumoniae - - + + +
E. aerogenes - - + + -
5. Enteroaggregative E. coli E. cloacae - - + + -
- Acute and chronic diarrhea
- Aggregative adhesion fimbriae LATE LACTOSE FERMENTERS
- Stacked brick pattern of cells
Arizona sp
Enterobacter - Related to Salmonella (Salmonella arizona – the
- UTI, wound, septicaemia only lactose fermenting Salmonella)
- Virulence: capsule
TSI: A/AG H2S ONPG (+) LIA: K/K
TSI: A/AG IMVC: -, -, +, +
Urease (-) except E. gergoviae
Citrobacter
Lysine Ornithine Arginine - Citrate (+)
E. aerogenes + + - - LDC negative
E. gergoviae (urease +) + + - - C. koser (diversus) – neonatal meningitis
Hafnia (Enterobacter) alvei + + - - C. freundii – UTI, pneumonia, endocarditis
E. cloacae (-) LDC control - + +
Cronobacter (Enterobacter) TSI: A/AG H2S ± LIA: K/A ONPG (+)
- + +
sakazakii (yellow)
Pantoea (Enterobacter) - - -
agglomerans (yellow)

Klebsiella pneumoniae (Friedlander’s bacillus)

- Capsule, non motile
- Pneumonia, wound, meningitis, UTI
- Pyogenic liver abscess

TSI: A/AG LIA: K/K LOA: +, - , - IMVC: -, -, +, +

Urease & malonate (+)
Mac- mucoid, lactose fermenter
(+) string test due to mucoid colony

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 BACTERIOLOGY

TSI Indole Malonate Salmonella

C. freundii A/AG H2S - + - Black colony on SSA, BSA (S. typhi)
C. diversus/ - (+) Selenite F, tetrathionate broth (enrichment)
A/AG + +
koseri - TSI: K/AG H2S; LDC (+)
C.
A/AG + - - Aerogenic except S. typhi, S. gallinarum
amalonaticus
- Motile except S. gallinarum, S. pullorum
Biochemical Test Result of Late Lactose Fermenters - Kauffman-White scheme: serotype of Salmonella
TSI LIA - Vi, O, H Ags (S. typhi); Vi (heat)
Arizona sp A/A + gas + H2S K/K + H2S
- S. paratyphi A: H2S & LDC (-)
C. freundii A/A + gas + H2S K/A
C. diversus A/A + gas K/A - Related to Citrobacter
- Pea soup stool (Salmonellosis)
I M V C U
Arizona sp - + - + - Diseases:
C. freundii - + - + - 1. S. typhi: typhoid fever, meningitis, osteomyelitis
C. diversus + + - + - 2. S. paratyphi A & B: paratyphoid fever
3. S. paratyphi C (S. cholera suis): septicaemia, most
NON-LACTOSE FERMENTERS severe
4. S. enteritidis: gastroenteritis (poultry); #1 on the
Proteus-Morganella-Providence (Proteeae) group Philippines
- Phenylalanine deaminase [PAD] (+) 5. S. typhimurium: food poisoning
- Lysine deamination (+)
- Urease (+) except Providencia alcalifacien
- LOA -, -, - except Morganella and P. mirabilis
(ornithine positive)
- Indole (+) except P. mirabilis

Proteus
- Swarm on BAP & CAP but not on Mac
- TSI: K/AG H2S
- PAD (+)
- # 2 UTI, renal stone (urease – virulence)
- Burnt chocolate cake odor
Species:
- P. vulgaris: indole positive (Ox2, Ox19, Oxk)
- P. mirabilis: indole negative (Oxk)
Proteus ↔ Rickettsia

TSI Urease Ornithine

Morganella
K/AG + +
morganii
Providencia K/AG ± -
stuartii
Providencia K/AG + -
rettgeri
Providencia K/AG - -
alcalifaciens
Proteus Salmonella Shigella
K/AG H2S + + Motility + -
mirabilis
H 2S + -
Morganella Providencia LOA +, +, + -, -, -
Ornithine + - Indole - +/-
Citrate - + Invasive - +
Blood culture + -
Biochemical Test Result of P-M-P group
Related Citrobacter E. coli
TSI LIA
P. vulgaris K/A + gas + H2S R/A
P. mirabilis K/A + gas + H2S R/A Salmonella Citrobacter
P. rettgeri K/A + gas R/A LDC + -
M. morganii K/A + gas R/A Malonate - +
MacConkey NLF LF
I M V C U
P. vulgaris + + - + + Proteus Salmonella
P. mirabilis - + - + + KCN + -
P. rettgeri + + - + + Urease + -
M. morganii + + - - + PAD + -
LDC - +

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 BACTERIOLOGY

Shigella Yersinia
§ Non motile, colorless on SSA, Acetate (-)
§ TSI: K/A; LIA: K/A; LDC (-) Yersinia pestis (Plague bacillus)
§ LOA: -, -, - except S. sonnei (ornithine +) - Bipolar bodies (Wayson)
§ Related to E. coli (acetate +) - Safety pin, stalactite (+); V and W Ags
§ Biochemically inert - Non motile, urease and ornithine (-)
§ Dysentery bloody stool with mucus & pus cells - Rat flea bite
§ Culture – fresh stool with mucous flecks and rectal - Bubonic, pneumonic, septicemic PLAGUE
swab of ulcer (best) - Black death (bioterrorism)
§ Shigellosis (4F): food, fever, finger, feces

Ornithine
O Ag Mannitol
ONPG
S. dysenteriae (shiga) A - -
S. flexneri (strong) B + -
S. boydii (boyd’s) C + -
S. sonnei (duval) D + +

*S. sonnei – cross react w/ Plesiomonas shigelloides

Serratia marcescens
- NLF/LLF
- Red (prodigiosin)
- LOA: +, +, -
- Arabinose (-) Safety pin appearance of Y. pestis
- DNAse, Lipase, Gelatinase, Citrate (+)
- Drug resistant Yersinia enterocolitica
- Nosocomial, UTI, bacteremia, pneumonia - Oxidase (-), motile at 22°C but not at 35°C
- S. rubidae – red pigment; arabinose (+) - Cold enrichment at 4°C (same as Listeria)
- S. liquifacien – blood bag contaminant - Bull’s eye colony on CIN
CIN (+): Y. enterocolitica [oxidase (-)];
Biochemical Test Result of Salmonella-Shigella-Serratia Aeromonas [oxidase (+)]
TSI LIA - Zoonotic, unpasteurized milk, enterocolitis
S. typhi K/A + small H2S K/K - Arthritis and erythema nodosum
S. enteritidis K/A + gas + H2S K/K - Appendicitis and blood bag contaminant
S. dysenteriae K/A K/A
S. sonnei K/A K/A
S. flexneri K/A K/A
S. boydii K/A K/A
S. marcescens K/A or A/A K/K

I M V C U
S. typhi - + - - -
S. enteritidis - + - + -
S. dysenteriae - + - - -
S. sonnei - + - - -
S. flexneri + + - - -
S. boydii + + - - -
S. marcescens + - + - +

Edwardsiella tarda
- Indole (+), NLF
- TSI: K/AG H2S (like Salmonella except Salmonella is Bull’s eye colonies of Y. enterocolitica
Indole [-])
- IMVC: +, +, -, - (like E. coli except E. coli is LF) Yersinia pseudotuberculosis
- Lysine decarboxylase (+) - LOA: -, -, -
- Extra-intestinal pathogen (wound, bacteremia) - Urease (+)
- Diarrhea, fish & turtles - Acute mesenteric lymphadenitis, septicaemia
- Animal pathogen (pseudotubercles)

Y. Y. Y.
pestis enterocolitica pseudotuberculosis
Motility - + +
Urease - + +
Ornithine - + -
Sucrose - + -
TSI K/A A/A K/A

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 BACTERIOLOGY

VIBRIONACEAE
- Vibrio, Aeromonas, Plesiomonas Serotype Ogawa Inaba Hikojima
- Stool pathogen Anti-Ogawa + - +
- All are oxidase (+), catalase (+), indole (+) Anti-Inaba - + +
- All ferment glucose; polar flagella
*VCO1 El Tor – more common; pandemic cholera agent
Vibrio
- Comma shape, motile (monotrichous) Lab ID
- Oxidase (+) except V. mitschnikovii - Darkfield microscopy: darting, shooting star
- O129 susceptible - Oxidase (+)
- Halophilic except V. cholerae, V. mimicus - O129 Sensitivity test (S)
- Alkaliphilic - Polymyxin B susceptibility test: (R) for El Tor
- LOA: +, +, - - Cholera red test: NO3, Indole (+)
- Nitrate reduction (+)
- MacConkey: colorless colony except V. vulnificus Vibrio parahemolyticus
- Transport medium: Cary-Blair - Halophilic (8% NaCl), Indole (+)
- Buffered Glycerol Saline: transport medium for stool - Non sucrose fermenter (green on TCBS)
but is not used when isolating Vibrio because glycerol - Kanagawa positive (BH on Wagatsuma agar)
destroys Vibrio - LOA: +, +, -
- Gastroenteritis (seafoods – Japan)
- TSI: K/A

6%
Disease TCBS Oxidase
NaCl
V. cholerae Cholera - Yellow +
V. alginolyticus Gastroenteritis + Yellow +
Green
V. parahemolyticus Gastroenteritis + +
Arabinose
Green
V. vulnificus Sepsis, wound + +
Lactose
V. fluvialis Gastroenteritis + Yellow +
V. furnissi Rare + Yellow +
V. mimicus Gastroenteritis - Green +
V. metschnikovii Gastroenteritis -/+ Yellow -
Grimonti hollisae Gastroenteritis + Green +
Photobacterium
Wound + Green +
damsel

Vibrio cholerae Aeromonas spp

- Non halophilic, 1-3% NaCl, Indole (+) - Non halophilic
- Rice watery stool (cholera) - O129 resistant
- String test positive (0.5% Na desoxycholate): - TSI: A/AG (glucose & sucrose)
differentiate V. cholerae from others - (+) oxidase, catalase, DNAse, esculin HOH
- Sucrose fermenter (yellow on TCBS) - BAPA: beta hemolysis
- TSI: A/A - LOA: +, -, +
- Cholera toxin (choleragen) – increases cAMP - Opportunistic (freshwater)
leading to loss of H2O, Na, K (dehydration) - Red leg disease, diarrhea, wound, septicaemia
- Pfeippers phenomenon – lysis of V. cholerae
- Causes Thrombotic thrombocytopenina purpura Plesiomonas spp
- Non halophilic
- O129 sensitive/resistant
- TSI: K/A or A/A (glucose) – inositol
- Oxidase, catalase and inositol positive
- LOA: +, +, +
- Opportunistic (freshwater), diarrhea, wound,
septicaemia

Vibrio Aeromonas Plesiomonas

NaCl + - -
Motility + + +
Oxidase + + +
O129 S R S/R
susceptibility
LOA +, +, - +, -, + +, +, +
Classification of V. cholerae O1 (VCO1) DNAse,
Biotype Classical El Tor hemolysis, - + -
Polymyxin Susceptibility S R esculin HOH
Lysis by bacteriophage + -
Chicken RBC agglutination - +
Hemolysis of sheep RBC - +
Voges Proskauer test - +

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 BACTERIOLOGY

Campylobacter
- Spiral or curved rods, motile darting sea gull wing C. jejuni H. pylori
- Oxidase and catalase positive Oxidase + +
- Microaerophilic Catalase + +
Microaerophilic + +
- Growth at 37-42°C
Urease - +
- Zoonotic, indoxyl acetate (+) except C. lari
Growth at 42°C + -
- Selective media: Skirrows, Butzler, CAMPY-BAP, Colony Gray-white Translucent
CAMPY-CVA, Cefoperazone Charcoal Desoxycholate
Agar (CCDA)

NON FERMENTATIVE ORGANISMS

§ Pseudomonas aeruginosa
§ Acinetobacter
§ Stenotrophomonas maltophilia

DIAGNOSTIC TESTS
1. O-F tests
- Hugh & Leifson medium
- 1% glucose, 1% agar, peptone
- High CHO, low peptone
- Indicator: bromthymol blue
- Results: (+) yellow [acid]; (-) green [no acid]

Open (tube w/o Close (tube w/

mineral oil) mineral oil)
Green
OXIDIZER Yellow
(Pseudomonas)
Yellow
FERMENTER Yellow
(E. coli)
Green
NON UTILIZER Green
(Alkaligenes)

2. Growth at 42°C = (+) growth at 35°C and 42°C

Diseases: § (+) P. aeruginosa
1. C. jejuni: #1 gastroenteritis (US, world) from poultry, § (-) P. fluorescens
Guillain-Barre syndrome (post infection complication,
paralysis) 3. Cetrimide test
2. C. fetus: animal abortion § (+) growth – P. aeruginosa
3. C. coli: gastroenteritis (diarrhea) from swine § (-) no growth – E. coli

37°C 42°C Nalidixic Cephalothin Hippurate

C.jejuni + + S R +
C. coli + + S R -
C. fetus + - R S -
Pseudomonas spp
C. jejuni C. lari C. coli - Oxidase (+) except S. maltophilia
Catalase + + + - Motile except B. mallei
Oxidase + + + - MacConkey (+) = colorless colony
Hippurate + - - - TSI: K/K or neutral reaction
Indoxyl acetate + - + - O-F = yellow (O), green (F)
- Opportunistic infection (environment)
- Brevundimonas vesicularis, Acidovorax facilis: Mac (-)
- Ralstonia pickettii: glucose non oxidizer
- Burkholderia gladioli: oxidase (-), plant pathogen,
cystic fibrosis
Helicobacter pylori
- Formerly Campylobacter
Pseudomonas aeruginosa
- Oxidase and catalase (+)
- B. pyocyanus
- Microaerophilic
- Blue pus agent
- Urease (+)
- Oxidase & catalase (+)
- Peptic ulcer, gastritis, cancer
- Motile (monotrichous)
- Selective media: Skirrow’s, Mod. TMA, CAMPY-CVA
- LDC (-)
- Urea breath test: rapid test for H. pylori
13 C-Urea &&&&&&&&&&&&&&&⃗
𝑈𝑟𝑒𝑎𝑠𝑒 13 CO2 (exhale) - Fruity grape-like or corn tortillas odor
- Pyocyanin (blue green), fluorescein (yellow green),
or pyoverdin
- Fluorescein: P. putida, P. fluorescens
- Growth at 42°C

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 BACTERIOLOGY

- Acetamide = carbon source Burkolderia pseudomallei (Whitmore’s bacillus)

- O/F test: +/- (oxidize glucose) - Melioidosis or Glanders like (sepsis, pneumonia)
- Virulence: exotoxin A (inhibits protein synthesis), - Wrinkled colony on ashdown medium
endotoxin, alginate (biofilms), pili - O-F: +/- (lactose oxidizer); arginine (+)
- Growth at 42°C; motile (lophotrichous)
Diseases: - Safety pin appearance
- #1 cystic fibrosis, pneumonia, meningitis - Vietnamese time bomb (bioterrorism)
- #2 burn, wound (ecthyma gangrenosum)
- Swimmer’s ear (otitis externa), endocarditis, UTI
- Contact lens infection, conjunctivitis
- Dermatitis – “jacuzzi” hot tub syndrome
- (+) iodine preparation
- Ventilator associated pneumonia
- #1 ICU isolate, #1 NFO, #1 Opportunistic

Lab ID of P. aeruginosa
- Cetrimide (+): selective media
- BAP: gray, spreading, serrated, metallic sheen,
mucoid, beta hemolytic
- MacConkey: colorless with green pigment
- MHA: bluish-green colonies (pyocyanin)
- (+) NO3, Urease, Gelatin HOH
- LOA: -, -, + B. pseudomallei showing bipolar staining (safety pin appearance)
- Glucose oxidizer

Wrinkled colonies on Ashdown medium

Burkholderia mallei
P. aeruginosa on cetrimide agar
- Glander’s disease
37°C 42°C Pyocyanin Fluorescein - O-F: +/- (glucose, maltose, lactose)
P. aeruginosa + + + + - Only non motile pseudomonad
P. fluorescens + - - + - No disk diffusion test
P. putida + - - +

Gelatin HOH & Proteolysis Pseudomonas stutzeri

P. fluorescens + - Brown (buff colored) wrinkled colony
P. putida - - 6.5% NaCl (+), NO2-N2 gas, non lactose oxidizer,
arginine (-)

Burkholderia cepacia
- Oxidase & LDC (+) Stenotrophomonas maltophilia
- Motile (lophotrichous) - Xanthomonas/Pseudomonas
- Green colony on CAP - Oxidase (-), DNAse (+), LDC (+)
- Yellow on Oxidative Fermentative Polymyxin B - O-F: +/- (glucose, maltose)
Bacitracin Lactose - Motile (lophotrichous)
- Yellow to pink on PC agar (violet, bile salts, polymyxin - Large, smooth, glistening, lavender green colony on
B, ticarcillin & phenol red) SBAP
- Earthy or dirt like odor - Yellow pigment (TSA); ammonia-like odor
- Pink colony on Mac (lactose oxidizer) - Brown pigment on heart infusion agar with tyrosine
- Yellow = B. cepacia & P. stutzeri (wrinkled colony) - Nosocomial – catheter related infection, bacteremia,
- #2 cystic fibrosis, pneumonia, sepsis wound, UTI, pneumonia
- Cause onion bulb rot in plants and foot rot in - 4th common Gram (-) rod isolate
humans
- ID by commercial system based on phenotype and
genotype to confirm Shewanella putrefaciens
- TSI: K/K H2S
- Oxidase (+)

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 BACTERIOLOGY

Other NFO: Moraxella lacunata (Morax Axenfield)

Acinetobacter - Blepharoconjunctivitis
- #2 NFO - Nitrate, oxidase, catalase (+)
- Oxidase (-), catalase (+) - Mistaken as Neisseria
- Non-motile, not reduce nitrate - Asaccharolytic (CHO -)
- Gram negative coccobacilli - MacConkey (-)

Moraxella spp.
M. nonliquifaciens Nitrate (+), spread & pit agar
M. osloensis Acetate (+) semi opaque colonies, GUT flora
M. lacunata Liquefy serum, digest Loeffler slant
M. lincolnii Not pit the agar

Chryseobacterium (Flavobacterium) meningosepticum

- (+) oxidase, DNAse, gelatine HOH, indole
- New name: Elizabethkingia
- Neonatal meningitis, sepsis
- Yellow (flavin), non motile, Mac (-)
- Nebulizers, beta-lactamase

Methylobacterium – pink colony, Mac (-), grow at 25°C

- BAP: round, opaque, mucoid colony Weeksiella verosa – brown, mucoid colony, urease (-)
- Mac: purple colony Bergeyella zoohelcum – yellow sticky colony, urease (+)
- Mistaken as Neisseria
- Drug resistant, UTI, wound, diarrhea Eikenella corrodens
- Nosocomial pathogen - One of HACEK
- Risk: ICU, burn units, multiple antibiotics - Human bite wound “clenched fist”
- 3rd common gram (-) rod isolate - Corrode (pitting of) agar, bleach like odor, SBE
- Acinetobacter transformation test – differentiate agent
asaccharolytic, nitrate-reducing strains of - MacConkey (-)
Acinetobacter from CDC group no. 1
- Acinetobacter CDC group no. 1: animal bite wound, Kingella spp.
coccoid to medium colony, NO3 (+), Mac (-) - One of HACEK
- Cause SBE
A. baumanii/ A. lwoffi - Ferment glucose
A. anitratus
Growth at 42°C + - - K. Denitrificans: non hemolytic
OF Glucose + - - K. Kingae: beta hemolytic
Old name Herella vaginocola Mima polymorpha - Pits the agar

TSI: K/K Acinetobacter Pseudomonas Oxidase Catalase Mac

Motility - + Acinetobacter - + +
Oxidase - + Alkaligenes + + +
NO3 - + Flavobacterium + + -/+
Moraxella + + -
Kingella + - -
Eikenella + - -
Alkaligenes (Pseudomonas) faecalis
- Stool flora
- Asaccharolytic = O/F test: -/-
- Oxidase & catalase (+) PARVOBACTERIA
- Feather edge colonies with green zone (Gram negative bacilli or coccobacilli, fastidious, aerobic, CO2, Mac negative)
- Apple like fruity odor Diagnostic tests for Parvobacteria
- UTI, wound, diarrhea - Oxidase +/-
- Motile (peritrichous) - Require X (hemin) and V (NAD)
- Satellitism (H. influenzae)
A. faecalis Acinetobacter - Medium: chocolate agar (horse blood) + 5% CO2
Oxidase + - - No growth on MacConkey agar
Motility + - - Require X factor for in vitro growth
Nitrate + -
Haemophilus
X V Porphyrin
Non-oxidizer, Oxidase (+) H. influenzae + + -
Roseomonas Mucoid, pink colonies, Mac (+), Urease (+) H. aegypticus + + -
Delfia acidovorans Orange, indole (+), polar flagella H. haemolyticus + + -
Psychrobacter Grows at 25°C than 35°C H. parainfluenzae - + +
Comamonas Polar flagella H. parahaemolyticus - + +
Achromobacter NO3 (+), bacteremia, pneumonia H. paraphrophilus - + +
Myroides Spreading colony, yellow, fruity odor H. ducreyi + - -
H. aphrophilus - - +
If Porphyrin (+), does not require X factor

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 BACTERIOLOGY

Growth Factor Tests for Haemophilus Culture Media:

1. Porphyrin test = X factor (ALA) test; (+) = red 1. Mercury drop (pearl like) colonies on potato blood
fluorescence glycerol agar or Bordet Gengou – common medium,
2. X and V strip test = MHA non selective
A. XV growth = H. influenzae
B. V and XV growth = H. parainfluenzae
C. X and XV growth = H. ducreyi
3. Satellitism (Staph streak test) = BAP with S. aureus
(other sources: C. albicans, S. pneumoniae) as V
factor
4. Beta hemolysis on horse BAP: H. haemolyticus / H.
parahemolyticus

Haemophilus influenzae (Pfeiffer’s bacillus)

- Virulence: capsule (type b), IgA protease,
lipopolysaccharide (LPS), pili
- Satellite around S. aureus (in BAP)
- #3 cause of bacterial meningitis
1. S. pneuomoniae
2. N. meningitides
3. H. influenzae (children)
- Major cause of acute epiglotitis
- Otitis media, conjunctivitis, pneumonia, sepsis,
cellulitis, cystic fibrosis 2. Regan Lowe (charcoal horse blood with cephalexin &
- GS: Gram (-) coccobacilli amphotericin B) – best
- CAP: grayish, dew drop mousy odor 3. Jones Kendrich (charcoal, yeast extract)
- Oxidase positive 4. Charcoal cephalexin blood agar, Stainer and Scholte
- Porphyrin test negative
- Beta lactamase test (+) Transport: half strength Regan Lowe, Cold casein hydrolysate,
- Satellitism test: growth near S. aureus casamino acid broth, fluid medium

Anti-PT ELISA: reliable serologic test for B. pertussis

Mac, Regan
Species Motile Urease Oxidase Lowe
BAP
B. pertussis 3-6
- - + -
(human) days
B. parapertussis 2-3
- + - +
(pertussis-like; animal) days
B. bronchiseptica 1-2
+ + + +
(kennel cough) days

Brucella species
- Facultative intracellular
- No capsule, obligate aerobe, non motile
- Category B bioterrorism
- Zoonotic – Erythritol (animal placenta) enhance
growth
- Trypticase soy broth, Wisconsin, Castaneda broth
X factor (hemin) = blood agar; V factor (NAD) = S. aureus - Brucellosis, undulant fever, malta fever,
Mediterranean fever, Gibraltar fever, Cyprus fever
Haemophilus ducreyi - Bang’s disease; animal abortion
- X factor - Endocarditis, laboratory acquired infection
- Chancroid; sexually transmitted - Unpasteurized milk: common means of brucellosis
- Soft chancre (T. pallidum: hard chancre) - H2S (+): B. abortus, B. melitensis, B. suis
- Growth on CAP + vancomycin (selective) - Particle agglutination – rapid test for Brucella
- Lateral flow dipstick – screening of Brucella outbreak
Haemophilus aegypticus - B. canis is not a bioterrorism agent
- Koch Week’s bacillus - 1:160 titer – significant titer for brucellosis in SAT
- X and V factor - Brucella cross reacts with Francisella
- Pink eye conjunctivitis; Brazilian purpuric fever

Bordetella pertussis
- Capsule, obligate aerobe
- Whooping cough bacillus
- Catarrhal, paroxysmal, convalescence
- Require cysteine & methionine for growth
- MacConkey (+) except B. pertussis

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 BACTERIOLOGY

UNUSUAL BACTERIA
Dye Inhibition Test Animal § Spirochetes (only serology)
Urease CO2 Thionine Fuchsin abortion § Mycoplasma (no cell wall)
B. abortus Not § Chlamydia (intracellular)
+ + Inhibited Cattle
(Bang’s) inhibited
§ Rickettsia (intracellular)
Not Not Goat &
B. melitensis + -
inhibited inhibited sheep
Not SPIROCHETES
B. suis + - Inhibited Swine Diagnosis Disease
inhibited
Not Syphilis, yaws,
B. canis + - Inhibited Dog Treponema Serology
inhibited pinta, bejel
Culture
Cell wall: Weil’s disease or
Leptospira
diaminopimelate infectious jaundice
Francisella tularensis (Bacterium tulareri) acid (DAP)
- Require cystine & cysteine (also Bordetella, Legionella) Borrelia Giemsa Lyme, relapsing
- Catalase (+), oxidase (-), beta lactamase (+) (blood spirochete) Serology fever
- Urease (-), Mac (-), non motile, capsule, aerobe
- Tularaemia – Ohara, market men’s disease
- Deerfly, lemming, rabbit fever, water-trapper’s Treponema pallidum
- Laboratory acquired infection (most common); - Non cultivable on agar medium
inhalation, ticks, deerfly, rabbit - Obligate intracellular (rabbit’s testicle)
- Forshay – skin test for tularaemia, bioterrorism, PCR
test, poorly staining Gram (-) rod, mistaken as H. Disease: Syphilis
influenzae 1. Primary – hard chancre
- Major biohazard in the lab 2. Secondary – condylomata lata, skin rash; highly
- Beta-lactamase (-) to differentiate from HACEK infectious, best time to do serology
- F. philomiragia: oxidase and gelatine HOH (+) 3. Latent – asymptomatic (serology)
4. Tertiary – gummas (granuloma) – cardiovascular
Pasturella multocida syphilis, neurosyphilis, Tabes dorsalis (paralysis of
- Gram negative coccobacilli spinal cord)
- Capsule, non motile, bipolar stain, safety pin 5. Congenital syphilis – stillbirth, abortion
- (+) oxidase, catalase, indole, nitrate
- Penicillin (S) - Blood transmitted, sexually transmitted, congenital
- Glucose fermenter infection (transplacental)
- Grow on BAP, CAP but not on MacConkey - Serologic test: TORCH test (Toxoplasma: cerebral
- Animal bite wound, pneumonia calcification; Cytomegalovirus: #2 mental retardation)
- Endocarditis, meningitis, arthritis, liver cirrhosis - Jarisch-Herxheimer reaction: phenomenon where
- Cat bite infection, shipping fever in cattles large quantities of toxin are released as bacteria dies
- Musty or mushroom-like odor during treatment, post (arsenic) treatment reaction
- TREATMENT: PENICILLIN
Note:
§ Haemophilus: X & V test Lab ID
§ Bordetella: Urease & Oxidase 1. Dark field microscopy – corkscrew motility
§ Brucella: Thionine & Fuchsin 2. Levaditi silver impregnation
§ Francisella: Oxidase, Beta-lactamase, & Urease 3. Serology:
§ Pasturella: Mac (-) a. VDRL, RPR, TRUST (Reagin test) – screening
b. FTA-ABS, TPHA, MHA-TP, HATTS – confirmatory
if: (+) RPR, (-) FTA-ABS = biologic false positive

Other Treponemes
HACEK (Skin diseases, not sexually transmitted, can cause biologic false [+])
Haemophilus aphrophilus; Actinobacillus § T. pertenue – yaws/framboise
actinomycetemcomitans; Cardiobacterium hominis; Eikenella § T. carateum – pinta
corrodens; Kingella kingae
(Gram negative coccobacilli, fastidious, CO2, Mac [-], SBE) § T. endemicum – bejel
Oxidase Catalase
H. aphrophilus + - Leptospira interrogans icterohemorrhagica
A. actinomycetemcomitans - + - Hook ends spiral, obligate aerobes, zoonoti
C. hominis + - - Causes Weil disease / Leptospirosis (animal urine)
E. corrodens + -
K. kingae + -
- Specimen:
1st week – blood, CSF (acute)
2nd week – urine (chronic)
Features Disease
H No X & V Endocarditis
- Culture media:
Star like colony, bipolar stain, dots and Fletcher’s – rabbit serum (enrichment/selective)
A Periodontitis, meningitis
dashes of Morse code (+) fatty acid (30°C, 6-8 weeks)
Endocarditis, Hemolytic Noguchi
C Teardrop, rosette, indole (+)
uremic syndrome
Ellinghaussen McCullough Johnson Harris
E Asaccharolytic, bleachlike, pits agar Human bite wound
K Pitting of agar, twitching motile Osteoarthritis
Note: L-biflexa-non pathogenic, PCR- blood w/ EDTA,
5-fluorouracil (Fletcher) – inhibit contaminant

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 BACTERIOLOGY

Lab ID of Leptospirosis - Elementary body: infectious

1. Dark field microscopy: corkscrew motility - Reticulate body: reproductive
2. Culture: confirmatory/definitive test
3. Serology
a. Macroscopic Agglutination Test (MAT) – screen
serum + Ag (killed Leptospira) = (+) agglutination
b. Microscopic Agglutination Test (MIT) – confirm
serum + Ag (live Leptospira) = (+) agglutination w/
darkfield microscope

Borrelia (Blood spirochete)

A. Relapsing fever due to:
- Borrelia recurrentis (epidemic) louse bite
- Borrelia anserina, toricatae, parkeri – tick bite
- Diagnosis: Wright/Giemsa blood/Bone marrow
- Best to diagnose relapsing fever: Giemsa stained
peripheral blood smear

B. Lyme disease
- Borrelia burgdorferi
- # 1 tick borne disease in US
- Tick bite of Ixodes dammini
- Primary stage – erythema chronicum migrans (bull’s
eye rash)
- Secondary stage – meningitis, cardiac
- Tertiary stage – arthritis
- Culture on Barber Stoenner Kelly at 33°C for 6-12 Chlamydia trachomatis
weeks - TRIC (Trachoma and Inclusion Conjunctivitis) agent
- Best to diagnose Lyme disease: serology - Lymphogranuloma venerium (LGV) – buboes
Screening: ELISA, IFAT (Ab test) - Frei test – skin test for LGV
Confirmatory: Western blot (protein), p39 (late - #1 non gonococcal urethritis (NGU), pelvic
marker), flagellar 41 KD (acute marker) inflammatory disease (PID)
- Sensitive to sulfonamide
- Reiter’s syndrome associated

Non gonococcal urethritis

1. Chlamydia trachomatis
2. Ureaplasma urealyticum
3. Mycoplasma genitalium

Lab ID of C. trachomatis
- Iodine or Giemsa stain of glycogen containing
inclusion bodies( Halberstadter prowazeik)
- McCoy (best medium) – gold standard
Brachyspira spp - DFA – Chlamydia Ag
- Forms “false brush border” on intestine - PCR/NAAT – definitive test
- Transport at 4°C
Chlamydia - Swab: Dacro/Rayon swab on sucrose phosphate
- Gram (-) bacilli buffer
- Obligate intracellular parasite
- Energy “ATP” parasite
- Inclusion body (Giemsa stain) – diagnostic
- Bedsonia (old name); Chlamydophila (new name)

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 BACTERIOLOGY

Chlamydia psittaci Trophyrema whipplei

- Parrot fever or psittacosis (ornithosis) – cyst of the - Whipple’s disease
birds (diarrhea) - Gram positive actinomycete
- Man: pneumonia (inhalation of bird droppings) - PAS stained macrophages
- Non glycogen inclusion body (Giemsa) - Non cultivable
- Resistant to sulphonamide - PCR – definitive test
- 1:32 = significant titer (CFT, IFAT) - Stool disease; specimen: tissue, stool, gastric
washing
Chlamydia pneumoniae (TWAR)
- Human to human transmission – pneumonia Mycoplasma
- (+) human lines and Hep-2 cell, IFT - No cell wall (pleomorphic), non motile, smallest
- Guillain Barre syndrome with Campylobacter - Equal to the size of Poxvirus (300 nm)
- Has DNA & RNA unlike a (Pox)virus which only has
RNA
*Shell Vial Culture - Fried egg/mulberry colony (Dienes stain) –
- Rapid culture for virus and Chlamydia methylene blue
- 1-2 days (+) - Require sterol for growth except Acholeplasma
- Penicillin (R), SPS (S), CAP (+)
- Transport: 4°C on 0.5% albumin – TSB, 2SP, SP-4,
Rickettsiae – Rickettsia, Orientia, Ehrlichia Trypticase phosphate broth, 10% bovine serum
albumin
Rickettsia
- Obligate intracellular except Coxiella (extracellular via
inhalation) Mycoplasma pneumoniae (Eaton agent)
- Arthropod borne - Pleuropneumonia like organism (PPLO)
- Cross react with Proteus (Weil-Felix) - Primary atypical pneumonia – walking pneumonia
- Mulberry (fried egg) colony (aerobic with CO2
Lab ID - Selective: PPLO agar, Edward Hayflick’s, biphasic
1. Special stain: Gimenez, Macchiavelo, Castaneda, SP4, Mod. NYCA (4 weeks – report as negative)
Giemsa - Confirm: hemadsorption test – attachment to RBC
2. Culture: embryonated egg (best), cell culture - Cold agglutination test/DFA
3. Weil-Felix test – Rickettsial Ab - Best: inhibition of growth by specific antisera

*Note: Mycoplasma hominis

Excluded from Rickettsiae: - Large fried egg colony
- Coxiella (extracellular) - Post abortal fever
- Bartonella (Rochalimeae) – agar (+) - Post partum fever, PID
- Arthritis associated with agammaglobulinemia
Ticks = vector - Media: A7/A8, NYCA, SP4, argining
Endothelial cells – site of multiplication (rash)
Ureaplasma urealyticum
Rickettsia spp Vector Disease - T strain (tiny fried egg)
Rocky mountain spotted - Urease positive (brown)
R. rickettsi Tick
fever - Non gonococcal urethritis
R. akari Mite Rickettsial pox - Media: A7/A8, NYCA, SP4 – urea
Murine (endemic) typhus
R. typhi Rat flea - No haze in broth because of its small size
fever
- Crosses placenta
Epidemic typhus fever
R. prowazekii Louse Brill-Zinsser disease –
recrudescence typhus
Orientia tsutsugamushi Chigger Scrub typhus Mycoplasma and Ureaplasma
Bartonella quintana Louse Trench fever § Biochemical profile test: glucose, arginine, urease
Ehrlichia chaffensis Tick Monocytic Ehrlichoisis § Susceptibility test: broth or agar dilution
Ehrlichia equi (morulae) Tick Granulocytic Ehrlichoisis
Coxiella burnetti (via inhalation) Q fever

Ehrlichia
- Morulae, tick transmitted, destroy leukocyte
- Sennetsu fever
- WBC Morulae – intraphagosomal aggregates of
replicating Ehrlichia
- Anaplasma phagocytophilum – human granulocytic
anaplasmosis
- PCR, serology, immunohistology – diagnosis of
rickettsia; transport temperature = 4°C

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 BACTERIOLOGY

*Note: Spirillum minus/minor – rat bite fever, spiral

MISCELLANEOUS BACTERIA (Sodoku fever)
Gardnerella (Corynebacterium/Haemophilus) vaginalis
- Oxidase and catalase (-), SPS (S)
- Hippurate & starch HOH (+)
- Bacterial vaginosis, grayish, foul smelling
- CLUE CELLS (squamous EC with Gram [-]
bacilli/coccobacilli) on cytologic exam
- GS, PAP, WET MOUNT – best for clue cells
- Whiff/Sniff test: (+) fishy amine like odor (10% KOH)
- Treatment: METRONIDAZOLE
- Note: Nugent Scoring System – used for diagnosis of
vaginosis
- Selective media: beta hemolytic on Human blood
tween 80 agar, V agar, Columbia CNA

Chromobacterium violaceum
- Violet colored (violacein)
- NH4 cyanide; Mac – non lactose fermenter
- Opportunistic – environment, abscess, cellulitis
- Quorum sensing control

Clue cell

Calymmatobacterium (Klebsiella) granulomatis

- Safety pin, capsule, non motile
- Granuloma inguinale (donovanosis) – STD genital
ulcer
- Donovan bodies (Giemsa) – blue rods found inside
mononuclear phagocytes or histiocytes
- Media: human monocytes
Capnocytophaga sp
- Gliding motility (spreading colony)
- Periodontal disease (oral flora), fusiform rod
- On sheep’s blood agar: yellow (2-7 days)
- SPS (S)

Donovan bodies

Streptobacillus monoliformis
- Produce “L” forms; defective cell walls
- String of beads, fluff balls (broth)
- Fried egg (heart infusion)

Disease: Bartonella spp. (former Rickettsia family)

- Rat bite fever – animal bite - Gram (-) rod, Oxidase (-), Mac (-), BAP (-), CAP (+)
- Haverhill disease – ingestion - Bartonella cross react w/ Chlamydia, Coxiella
- SPS (S) - Zoonotic (cats), insect bite (flea)
- Medium: whole blood, serum, ascetic fluid (cotton ball, - Detection: Warthin starry silver stain,
bread crumbs) immunofluorescent stain, PCR
- Cultured on CAP & co-cultivation in cell culture
- Blood w/ EDTA, Biopsy: Columbia/Heart Infusion Agar with
5% sheep/horse blood

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 BACTERIOLOGY

Bartonella bacilliformis Erysipelothrix rhusiopathiae

- Destroy RBC - Gram (+) rod, non motile, H2S producer, catalase (-)
- Vector: sandfly - The only Gram (+) bacteria that produces H2S
- Carrion’s disease - Test tube brush, pipe cleaner, bottle brush
- Verruga peruana – skin eruption - Erysipeloid (Butcher’s cut/Diamond cut)
- Oroya fever – anemia

BARTONELLA Vector Disease

B. bacilliformis Sandfly Carrion’s disease
B. quintana Body louse Trench fever, endocarditis
Cat Scratch Disease – major
Cat endocarditis, bacillary
B. henselae scratch angiomatosis, Peliosis
hepatitis, neuroretinitis
Cat Minor Cat Scratch Disease
B. clarridgeiae
scratch No endocarditis
B. elizabethae Fleas Endocarditis
Legionella pneumophila
- Philadelphia strain
- Short Gram (-) rods, aerobic, motile
- Gram stain: 0.1% fuchsin instead of safranin
- Require L-cystine and iron for growth
- Air conditioning, water cooling system
- Broadstreet pneumonia & Pontiac fever
- Transport: 4°C; Storage: -70°C; pH: 6.9
- Specimen of choice: bronchoalveolar lavage
- Monoclonal immunofluorescent stain: definitive dx
- Intracellular infection (virulence): heat shock protein,
outer membrane protein (OMP), definitive organelle
trafficking (DOT), intracellular multiplication (ICM)

Lab ID
1. DFA – Legionella Ag Note:
2. Cultured best on BCYE – blue green colony/cut glass Erysipelas = Streptococcus
colony (4 days); Feeley-Gorman – brown colony Erythrasma = Corynebacterium
3. Stain: Dieterle silver stain – black Erysipeloid = Erysipelothrix

L. micdadei – acid fact organism, Pittsburgh pneumonia Listeria Erysipelothrix

monocytogenes rhusiopathiae
L. bozemanii – WIGA agent, pneumonia
Catalase + -
Motile (25°C) + -
Specime: BAL, urine, sputum, blood, stool Hemolysis Beta Alpha
Voges Proskauer + -
Listeria monocytogenes H2S produce - +
- Gram (+) rod, motile at RT Bile esculin &
+ -
- Tumbling (broth); umbrella like motility (semi solid) Hippurate
- Inverted Christmas tree Gluconate + -
- Beta hemolysis on SBAP McBride, Cold
Media BAP
- Cold enrichment at 4°C (+) & McBride (+) enrichment
- Listerolysin O – O2 labile hemolysin Non Branching Catalase (-), Gram (+) rod:
Anton test : ocular virulence test H2S (+) on TSI,
Erysipeloid,
Neonatal meningitis, sepsis Erysipelothrix large & smooth
bacteremia
colony, α-hemolytic
Infantiseptica granulomatous
Clue cells, SPS (S),
- Food poisoning (coleslaw, cheese) small gray colony,
- Fetal abortion, still birth Gardnerella Vaginosis
β-hemolytic in
- CAMP test w/ S. aureus = (+) block (rectangular) HBTA
hemolysis Multiple colonial
Lactobacillus
- Oxidase negative (aerotolerant morphologies & Bacteremia
- Ferment glucose, salicin, trehalose alpha-hemolytic,
anaerobe)
resemble strep
Reverse CAMP (+),
small to large, white,
Arcanobacterium gray colony, beta Pharyngitis, cellulitis
hemolytic, resemble
strep

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 BACTERIOLOGY

WATER BACTERIOLOGY
- E. coli – index of fecal contamination
- Sodium thiosulfate – neutralize chlorine

Stages of water bacteriology (conventional)

1. Presumptive test
Lactose broth/Lauryl Tryptose broth + water → incubate at
35°C for 24 hours → gas in Durham tubes (+); 48 hrs (-)

2. Confirmatory test
EMB/Endo agar + inoculums (24 hrs incubation) → colony (+)

3. Completed test
Lactose broth fermentation tube with phenol red → inoculums
incubate at 35°C for 24-48 hours → acid & gas (+) → Gram
negative rods

Multiple Tube Fermentation Test

- Gold standard test (5 test tubes)
- Reported in MPN (most probable number)
- Positive: >1.1 MPN/100 mL (not potable)
- Negative: <1.1 MPN/100 mL

Stages of MTFT
1. Presumptive test
Triple Strength Lactose Tube broth + water → (+) gas in
Durham tube; (-) no gas after 48 hours

2. Confirmatory test for total coliforms

Brilliant green lactose broth → (+) gas [non fecal origin]

3. Confirmatory test for fecal coliforms

EC broth at 44°C → (+) gas [fecal origin]

Most probable number

Number of positive tubes per 100 mL
0 < 1.1
1 1.1
2 2.6
3 4.6
4 8.0
5 > 8.0

MILK BACTERIOLOGY
- Pasteurization: sterilization of milk
- Pathogens: Salmonella, V. cholerae, B. abortus, C.
diphtheria, M. bovis, B. anthracis, Coxiella, FMDV,
cowpox virus, S. pyogenes
- Normal flora:
P. syncyanea – blue milk
F. synxanthum – yellow
P. aeruginosa – blue green
S. marcescens – red; S. lactis – souring of milk
B. subtilis – hay bacteria; proteolytic action on
coagulated milk
Alcaligenes viscosus – slimy or ropy milk

Bacteriologic Analysis of Milk

1. Methylene Blue Reduction Test – dye is decolorized
(reduced) by the oxygen containing bacteria; (+) colorless,
(-) blue

2. Standard Agar Plate Count - # of colonies x dilution factor

3. Microsonic or Breed Count

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Original content by A. Aldave (ACTS Review Center) Modified by: Ma. Christyl Vie R. Felias, RMT