Bacteriology Handouts PA

BACTERIOLOGY HANDOUTS
Objectives
1. Isolate, identify and analyze the bacteria that cause disease in humans.
2. Prediction and interpretation of antimicrobial susceptibility patterns
1a. Bacterial Structure

A. Cytoplasmic Structures
1. Nuclear area - single circular chromosome
2. Plasmid - small circular extra chromosomal dsDNA that confers antibiotic resistance
3. Ribosomes - consists of RNA and protein that serves as the site of protein synthesis
4. Metachromatic granules - reserves of polyphosphates
5. Spores – thick walled, highly durable refractile resting cells
B. Cell Envelope Structures
A. Plasma Membrane - phospholipid bilayer (embedded with proteins) that envelopes the cytoplasm
B. Periplasmic Space - gel like matrix between cell membrane and cell wall that degrades and detoxifies macromolecules
C. Peptidoglycan Layer - repeating disaccharide attached by polypeptides that maintains the shape of the cell.
D. Outer Membrane - phospholipid bilayer with LPS, lipoproteins, porins (control the passage of solutes)
C. Surface Polymers or Appendages
1. Capsule - gelatinous polymer of polysaccharide and/or polypeptide that surround cells
2. Flagella - long protein filaments which rotate and cause bacteria to be motile.
Arrangements: a. monotrichous; b. amphitrichous; c. lophotrichous; d. peritrichous
3. Fimbriae and Pili - hair like appendages that is shorter, straighter and thinner than flagella.
a. Fimbriae (common pili) - evenly distributed, from few to several hundred that facilitates adherence of cells
b. Pili (sex or conjugation pili) - protein tubes, longer than fimbriae that join bacterial cell for DNA transfer
4. Axial filaments - bundles of fibrils anchored at one end of spirochete and spirals around the cell. Its rotation of
filaments propels the spirochete in a spiral motion
2b. Host-Microorganisms Interactions

A. Characteristics: Found in body sites of healthy persons. Either resident or transient
B. Usual Flora at Body Sites
1. Skin - Armpit, groin (diptheroids), hair follicles, sweat glands & sebaceous glands (S. epidermidis & P. acnes)
2. Upper respiratory tract - Mouth (viridans strep, G− anaerobes), nose & pharynx (diplococci, diptheroids)
3. GI tract - Esophagus, stomach, SI, colon (90% obligate anaerobes, Staphylococcus, Enterococcus, Enterobacteriaceae)
4. Lower genitourinary tract -Urethra & vagina (Lactobacillus, anaerobic sporeformers, G+ cocci, Diptheroids)
C. Role of the Usual Microbial Flora
1. In host defense - Activates the immune system and blocks colonization of extraneous pathogens
2. In infectious disease –Opportunists when its natural habitat is damaged, disturbed by trauma or if the host’s i u e
system is compromised
D. Microbial Factors in Pathogenesis of Infection
1. Pathogenicity - The ability to produce disease in an individual.
a. True Pathogen - Organisms that cause disease in healthy individuals (B. anthracis and Y. pestis)
b. Opportunistic Pathogen - Organisms that cause opportunistic/iatrogenic infections (H. influenzae, S. epidermidis)
2. Virulence - Is the relative ability of the organisms to cause disease. Depends on virulence factor which allows the
organisms to (a) resist phagocytosis, (b) adhere to surface structures, (c) survive intracellularly and (d) produce
enzyme and toxins ( substances that disrupt cell metabolism)
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i. Exotoxins: Secreted by the organism into the environment. The organism must possess the gene ii.
Endotoxins : Lipid A of the outer membrane. Released upon lysis of the organism
Characteristic Exotoxins Endotoxins
Organism Type G(+) / G(-) G(-)
Chemical Nature Simple protein Lipid A
Stability at 100°C Labile Stable
Ab neutralization Detoxified Not detoxified
Biologic Activity Individual to toxin Same for all toxins
1c. Control of microorganism
A. Sterilization Versus Disinfection
1. Disinfectants - chemical agents applied to inanimate objects
2. Antiseptic - a substance applied to the skin for reducing the number of bacteria
B. Methods of Disinfection and Sterilization
1. Physical Methods
a. Heat (°C) Time Required Applications
Boiling Water 100 15 mins.
Disinfects
Pasteurization 63 (72) 30 mins. (15 secs)
Oven (Dry Heat) 160-180 1.5 – 3 hrs.
Autoclave (Moist Heat) 121 15 min. at 15 psi Sterilizes
132 30-60 min. at 15 psi
b. Filtration Applications
Plastic polymers or cellulose esters (0.22 μm) parenteral and antibiotic solutions & vaccines.
HEPA filters (0.3 μm) biological safety cabinets
c. Radiation
X rays, gamma rays and UV disposables: syringes, catheters or gloves
2. Chemical Methods
a. Antiseptics b. Disinfectant (Sterilizers)
Type Agents Type Agents

Alcohol (50%-70%) ethanol, isopropanol Aldehydes formaldeyde (1-8%), glutaraldehyde (2%)
Halogens iodophors Halogen chlorine and chlorine compounds (Bleach)
Heavy Metals AgNO3 & HgCl2 Detergents quaternary ammonium compounds
Phenolics chlorhexidine, triclosan Phenolics hexachlorophene
1d. Clinical Laboratory Safety

A. Routes of Infection
1. Mucous Membrane contact - rubbing the eyes (conjunctiva) or nose with contaminated hands
2. Airborne - inhalation of aerosols produced during centrifugation of unstoppered tubes (M. tuberculosis, Brucella)
3. Ingestion - failure to wash hands after work, eating, drinking and mouth pipetting (Salmonella and Shigella)
4. Direct Inoculation - puncture by contaminated needles and broken glass (Hepatitis virus)
B. Safety from Infectious Agents
1. Safety Program
a. Standard Precaution: Blood and body fluids from all patients should be considered infectious b. Work and
Environmental Practice Controls
• No mouth pipetting, eating, drinking, smoking or applying cosmetics
• No recapping of needle, dispose needles to sharps container
• Disinfect workstations, wash hands frequently and minimize generation of aerosols
• Wear personal protective equipment (PPE) and work in BSC
2. Biologic Safety Cabinets (BSCs)
• Containment barrier that protects the worker from aerosolized organism. Air is sterilized by UV and HEPA filter
• Cabinet Classification:
a. Class I - Room air pass into the cabinet sterilizing only the air to be exhausted
b. Class II - Sterilize air that flows over the work surface and the air to be exhausted
c. Class III - Self contained ventilated system. Closed front contain attached gloves
3. Biosafety Levels
a. Biosafety Level-1 - For handling organisms not known to consistently cause disease in healthy adults. Work done
in open bench tops with adherence to standard precautions. Limited access, presence of hazard warning signs,
decontamination of infectious waste (autoclave).
b. Biosafety Level-2 - For handling common or likely encountered pathogens in a routine clinical laboratory. Use BSC
I or II. Trained personnel, presence of biosafety manual and sharps containers.
c. Biosafety Level-3 - For handling organisms that can be transmitted by aerosols. Controlled access. Ducted air
ventilation, special lab clothing and personal respirator.
d. Biosafety Level-4. Research facilities handling exotic viruses and potential bioterrorist agents. Personnel and all
materials are decontaminated before leaving the facility. Non Circulating ventilation system. Maximum
containment and use of class II or III BSCs.
1e. Specimen Collection & Processing

A. Basic Principles of Specimen Collection
1. Fundamentals
a. Collect the specimen in the acute phase of the infection
b. Select the correct anatomic site
c. Pa kage the spe i e that ill ai tai the orga is ’s ia ility
2. Collection Procedures
a. Blood Culture - highest concentration occurs before the fever spikes. Draw 2 sets from right and left arms, respe ti
ely hr apart. l per set is collected in adults and 5-10 ml per set in children
b. CSF & Body Fluids - collect L by needle aspiration and place in sterile, screw-cap tube or anaerobic
transporter c. Gastrointestinal Tract
i. Gastric Biopsy - rapid urease test or culture for H. pylori
ii. Rectal Swab or Feces - for isolation of E. coli & Vibrio spp. Plated on enrichment or selective enteric
media d. Genital Tract
i. Female - cervix, urethra, vagina; Male - prostate, urethra. For isolation of N. gonorrhoeae
ii. Collected using swab moistened with transport medium. Plated in Thayer Martin Medium e.
Lesion/wound/abscess
i. Superficial - swab along outer edge using swab moistened with transport medium
ii. Deep - aspirate with needle and syringe and place in an anaerobic transport system f.
Lower Respiratory Tract
i. Sputum - gargle with water, cough deeply into sterile, screw cap container (AFB, Legionella and Nocardia) g.
Upper Respiratory Tract
i. Collected using swab moistened with transport medium
ii. Nasopharynx: Whooping cough - B. pertussis
iii. Pharynx (Throat): Strep Throat - S. pyogenes; Epiglotitis - H. influenzae; Oral gonorrhea – N.
gonorrhoeae h. Urine
i. Clean Catch Mid Stream, Catheter, Suprapubic aspirate
ii. Placed directly in a sterile, screw-cap container
iii. For diagnosis of lower UTI (cystitis, urethritis) and upper UTI (glomerulonephritis)
3. Labeling and Requisitions
a. Specimen Label: Patients name, age and gender; identification nu er; patie t’s roo u er or location;
requesting physician; culture site; date and time of collection
b. Requisition Form: Patie t’s a e, age a d ge der; patie t’s roo u er or lo atio ; physi ia ’s a e;
address;
culture site; date and time of collection; clinical diagnosis or patient history; name of individual transcribing orders
B. Preservation, Storage & Transport
1. Specimen Storage
Refrigerator Temp. (4°C) Room Temp. (22°C) Body Temp. (37°C)
Foreign Devices Abscess, lesion, wounds CSF
Feces Body Fluids
Urine Genital Samples
Sputum Nasopharynx, Throat
2. Anticoagulant: 0.025% Sodium polyanethol sulfonate (SPS) and Heparin
3. Holding or Transport Medium: Stuarts & A ie’s (Dacron, Rayon or Calcium Alginate swabs) or JEMBEC System
C. Specimen Receipt and Processing

1. Criteria for Rejection
a. Unmatched requisition and specimen label
b. Specimen transported at improper temperature, fixative and media or has exceeded 2 hrs. c.
Specimen collected in improper areas.
d. Quantity not sufficient (QNS), leaking or dried specimen.
e. “putu ith < WBC’s a d > EC/lpf.
2. Macroscopy: Swab or Aspirate; Stool (consistency); presence of blood, clot and mucus; volume and turbidity
3. Specimen Preparation: Homogenization; Concentration (centrifugation or filtration); Decontamination
1f. Microscopic Examination of Infected Materials
A. Introduction
1. Confirm that the specimen is representative
2. Identify agents using direct visual detection using stains
3. To guide the workup of specimen for culture.
B. Preparation of Samples
Smear Specimen Fixation

Wet Preps Fluids or semisolids a. Slide warmer at
Drop Clear, pus, swab rinse, tissue homogenate 60 C for i s.
b. Flooding with 95%
Pellet Blood culture, Dilute
methanol for 1 min.
Rolled Swab material (Pus)
Pull-apart Thick, granular, mucoid
C. Stains
1. Simple stain: colors the forms and shapes. E.g. Methylene Blue
2. Differential stain: colors specific components. E.g. Gram Stain, Acid Fast Stain
3. Antibody or Probe-mediated stain: directed specifically at identification of an organism
General Morphology
Wright- Sample with cellular
Giemsa background
Selected Morphology Genus (Species)-Specific Stains

Methylene Blue Metachromatic granules Intracellular organism
Antibody or DNA
Acid Fast Stains Mycolic Acid Lacks cell wall
probe stains
Not resolved by light microscope
Gram Stain Cell Wall
India Ink Capsules
Schaefer Fulton Endospores
Leifson Flagella
D. Microscopes
Microscope Magnification Application
Bright Field 10-1000 Stained cells
Dark Field 10-400 For cells not readily stained
Phase-contrast 10-400 Living or unstained cells
Fluorescence 10-400 Cells stained w/ fluorochromes
Review Questions
1. All of the following sites contain normal flora, EXCEPT:

A) Trachea B) Urethra C) Small intestine D) Groin area
2. The biosafety level practice for handling blood samples suspected of containing HIV and HBV:
A) BSL I B) BSL II C) BSL III D) BSL IV
3. Which of the following specimen preparation procedure is commonly applied to pus discharge? A) Homogenization
B) Concentration C) Decontamination D) None of these
E. Terminology for Direct Examinations
Gram Positive Bacilli

Small Listeria, Corynebacterium
Large Clostridium, Bacillus
Diptheroid Corynebacterium
Beaded Mycobacterium, Corynebacterium
Branched Nocardia, Actinomyces
Bifid/V forms Bifidobacterium
Gram Positive Cocci Gram Negative Coccobacilli

Chains Streptococcus Singly Fastidious G (-) Bacilli
Cluster Staphylococcus, Viridans Strep.,
Chains, Masses Gram (-) anaerobes
Diplococci Streptococcus pneumoniae
Encapsulated Gram Negative Bacilli
S. pneumoniae, S. pyogenes
Small Haemophilus, Legionella, Bordetella,
Gram Negative Cocci Brucella, Francisella, Pasteurella
Neisseria spp., Medium Enterics, Pseudomonads
Diplococci
Moraxella catarrhalis Curved/Spiral Vibrio, Campylobacter, Helicobacter
Spiral Borrelia, Leptospira, Treponema
F. Examination of Prepared Material
Cells & structures Associations
Amorphous debris Necrosis, heavy protein fluid
Epithelial cells Cell surface in collection site
Mononuclear cells Chronic inflammation
Mucus Irritation of glandular surfaces
G. Examples of Sample Observations Purulence Acute inflammation, exudation
Red blood Cells Trauma, hemorrhage
1g. Traditional Cultivation
A. Introduction
1. To grow (cultivate) and isolate all bacteria in the specimen
2. To determine which bacteria that grow are most likely causing the infection
3. To obtain sufficient growth of clinically important bacteria and allow identification
B. Nutritional Requirements
1. Types of Bacteria by Nutrient Requirements
a. Fastidious - Complex nutritional requirement b. Non fastidious - Basic nutritional requirement
2. Media Classifications
a. Supportive (general isolation) media - support growth of most non fastidious organisms
b. Enriched (nonselective) media - supplement added to supportive media for growth of fastidious
microbes
i. Sheeps Blood Agar - trypticase soy agar w/ 5% sheep's blood
• Enriched & differential. Determines hemolytic patterns
• Beta- complete clearing of RBC, Alpha - greenish discoloration around the colony, Gama - no effect ii.
Chocolate Agar - blood are lysed when added to molten base releasing hemin & NAD
• Can support for N. gonorrhoeae & Haemophilus spp.
c. Enrichment Broth - permits growth of certain bacteria while inhibitory to others
d. Selective Media - permits growth of certain bacteria while inhibitory to others.
e. Differential Media - provides a distinct cultural appearance of microorganisms.
f. Antibiotic Media - Selective for a certain group of bacteria through addition of antibiotics
g. Back-up Broth - Broth w/ agar (0.075% ) & thioglycolic acid (reducing agent) creating anaerobiosis C.
Environmental Requirements
1. O2 and CO2 Availability
a. Obligate aerobe - requires oxygen for growth
b. Facultative anaerobe - can grow either with or without oxygen
c. Obligate anaerobe - cannot grow in the presence of oxygen
d. Aerotolerant anaerobe - can survive in the presence of oxygen but do not use oxygen for metabolism
e. Microaerophile - requires a reduced level of oxygen for growth
f. Capnophilic - requires extra carbon dioxide (5% to 10%)
2. Temperature, pH & Moisture
a. Temperature: 35-37 C (42 C for C. jejuni, 0 C for L. monocytogenes & Y. enterocolitica)
b. pH: neutral (6.5-7.5)
c. Moisture: humidified incubators and sealing of agar plates
D. Use of Colonial Morphology

1. Isolation of bacteria from specimens
a. Isolation Streaking (Three Sector T Streak) - standard pattern for inoculating specimen
b. Cross streaking- for quantization of CFU in urine specimens
2. Evaluation of colony morphologies
a. Size: pinpoint, small, medium, large
b. Form/Margin: punctiform, circular, filamentous, irregular, rhizoid
c. Elevation: flat, raised, convex, umbilicate, umbonate
d. Density: transparent, translucent, opaque
e. Color: white, gray yellow or buff
f. Consistency: Brittle or crumbly, creamy or butyrous , dry or waxy, sticky
g. Pigment: P. aeruginosa - green; S. marcescens - brick-red; Prevotella melaninogenica - brown-black
h. Odor: S. aureus - old sock : P. aeruginosa - grape like; P. mirabilis - pudrid;
Haemophilus - mousy or mouse nest; Nocardia - freshly plowed field
1h. Phenotyping Scheme
1. Microscopic Morphology & Gram Reaction 5. Nutritional Requirements and Metabolic Capabilities
2. Macroscopic Morphology (Colony appearance) a. Enzyme capabilities (Enzyme based test)
3. Environmental Requirements b. Presence of Metabolic Pathways
4. Susceptibility to Antimicrobial Agents
2. Gram (+) Cocci

I. Catalase Test
• Test the ability to convert H2O2 into O2 & H2O (3% H2O2 –Catalase→ O2 + H2O)  Positive: Copious
bubble Staphylococcus spp. & Micrococcus spp.
 Negative: No or few bubbles Streptococcus spp. & Enteroccus spp. II.
Oxidase Test
• Test for the position of Cytochrome C
 Positive: Development of purple-blue color Micrococcus spp. 
Negative: No color change Staphylococcus spp.
2a. Gram (+) Staphylococci

I. General Characteristics
• G(+) cocci in clusters, Catalase (+), Oxidase (-), facultative anaerobes, 7.5-10% NaCl (+)
II. Clinically Significant Species
A. Staphylococcus aureus: Most clinically significant Staphylococcus sp., present in skin surfaces, nosocomial infections
1. Virulence Factor
a. Enterotoxins: A, B & D: food poisoning; B: pseudomembrane colitis
b. Toxic Shock Syndrome Toxin-1 (Enterotoxin F): Menstruating-associated toxic shock syndrome c. Exfoliative
Toxin: Scalded skin syndrome
d. Cytolytic Toxins: Hemolysins(α, β, , Panton-Valentine Leucocidin (ɣ-Hemolysin)
e. Enzymes: i. Staphylocoagulase (Coagulase) - fibrinogen  fibrin ; ii. Staphylokinase (Fibrinolysin) -
dissolve fibrin clots; iii. Protease, Hyaluronidase, Lipase f. Protein A: Binds the Fc portion of IgG
g. Beta lactamase(Penicillinase): cleaves the β-lactam ring of penicillin
2. Clinical Infections
a. Skin and wound infections – i. folliculitis & furuncles; ii. boils & carbuncles; iii. bullous impetigo
b. Scalded skin syndrome; c. Toxic shock syndrome; d. Food poisoning
B. Staphylococcus epidermidis
1. Virulence Factor: Exopolysaccharide sli e or biofilms
2. Infections: Hospital acquired UTI, prosthetic valve endocarditis
C. Staphylococcus saprophyticus
1. Virulence Factor: Adherent to Urogenital tract epithelium
2. Infections: UTI in sexually active young females and in older women with indwelling catheters
D. Staphylococcus lugdunensis; E. Staphylococcus haemolyticus
III. Laboratory Diagnosis
A. Isolation
1. Specimen: Aspirate or Swabs
2. Culture Media:
a. Sheeps Blood Agar (SBA): Enriched and Differential (5% Sheeps blood)
• S. aureus: Medium to large. Pigmented yellow. β-hemolytic
• S. epidermidis: Small to medium. Gray-white. ɣ-hemolytic
• S. saprophyticus: Small to medium. White-yellow or orange. ɣ-hemolytic
b. Mannitol Salt Agar (MSA): Selective (7.5% NaCl) and Differential (D-mannitol, phenol red)
• S. aureus: Growth w/ fermentation (yellow halos)
• S. epidermidis: Growth w/o fermentation (remains pink ) c. Laboratory
Diagnosis
Organism Slide Coagulase Tube Coagulase DNase MSA Fermentation Novobiocin (5μg)
S. aureus + + + +
S. epidermidis ̶ ̶ ̶ ̶ S
S. saprophyticus ̶ ̶ ̶ ̶ R
a. Coagulase Test- Test for the ability to convert fibrinogen into fibrin. Differentiate S. aureus from Coagulase (-)
Staphylococci. 2 types: Bound and Free coagulase a1. Coagulase
Slide Test- Dete ts ou d oagulase lu pi g fa tor .
Positive (+): Macroscopic clumping
Negative (-): No clumping; a2. Coagulase
Tube Test - Detects free coagulase.
Positive (+): Clot of any size
Negative (-): No clot
b. DNase Test - Test for DNA hydrolysis.
Positive (+): clear zone
Negative (-): No clearing
c. Novobiocin Susceptibility - Test for susceptibility to 5 μg Novobiocin. Susceptible(S): zone diameter >16 mm
Resistant (R): zo e dia eter
2b. Gram (+) Streptococci

• Introduction: G(+) cocci in pairs / chains, Catalase (-), aerotolerant anaerobesa
and some are capnophilic
• Cell Wall (Polysaccharide) Structure Pattern of hemolysis

Species B. Lancefield C. Browns Common Terms (Group)
S. pyogenes A β A
S. agalactiae B β B
S. dysagalactiae, S. equi C β C
S. bovis group D α,γ D Non Enterococcus
E. faecalis, E. facium D α,β,γ D Enterococcus
S. pneumoniae – α Pneumococcus
S. anginosus, mutans, mitis A, C, F, G, N α, β, Viridans streptococcus

A. Streptococcus pyogenes
1. Virulence Factor
a. Protein M & F, Lipoteichoic acid: Adherence to mucosal/epithelial cell
b. Hyaluronic acid capsule; c. streptodornase (nuclease); d. hyaluronidase
e. Streptolysin O: Subsurface hemolysin (O2 labile) and induces anti-streptolysin O
f. Streptolysin S: Surface hemolysin (O2 stable) and non-immunogenic
g. Streptokinase: Fibrinolysin
h. Streptococcal pyrogenic exotoxin A: Scarlet fever and toxic shock-like syndrome
a. Bacterial pharyngitis & tonsilitis
b. Pyodermal Infections: Impetigo, erysipelas, cellulitis, scarlet fever
c. Necrotizing fasciitis (hospital gangrene); d. Toxic shock-like syndrome (TSLS)
e. Post-streptococcal sequelae: rheumatic heart fever and acute glomerulonephritis
B. Streptococcus agalactiae
1. Virulence Factor: Capsule
2. Infections: Obstetric complications, neonatal sepsis (pneumonia, meningitis), endometritis
C. Groups C and G Streptococci
1. S. dysagalactiae subsp. equisimilis (Large-colony forming β-hemolytic isolates)
2. S. anginosus group (Small-colony forming β-hemolytic isolates)
D. Streptococcus pneumoniae
1. Virulence Factor: Capsule
2. Infections: Pneumonia, meningitis
3. Others: Diplococci and capnophilic
E. Viridans Streptococci
1. Virulence Factor: Capsule, dextran and adhesins
2. Infections: Subacute bacterial endocarditis, septicemia and cavities
F. Enterococcus
1. Virulence Factor: Adhesins, cytolysins
2. Infections: Endocarditis, bacteremia and UTI
C. Colony Characteristics
Species Description
Group A pinpoint, large zone of β hemolysis
Group B larger, narrow zone of β hemolysis

Viridans small; α, β,ɣ he olyti
Group D small; α, β,ɣ hemolytic
Pneumococci glistening, dome-shaped, mucoid, umbilicated; α hemolytic
D. Biochemical Identification
1a. Bacitracin (Taxo A) -Test for susceptibility to 0.04 U Bacitracin. Positive – Group A
1b. Sulfamethoxazole-Trimethoprim - Test for susceptibility to 1.25 μg of SXT disk. Positive – Not Group A or B
2. PYR Hydrolysis - Test for the ability to hydrolyze the substrate L-pyrrolidonyl-β-napththylamide
• Positive - Pink to cherry-red color: Group A or D Enterococcus ; Negative - No color change or orange color
3. CAMP Reaction - Test for the synergistic hemolysis between group B Streptococcus & S. aureus.
• Positive - Enhanced hemolysis in arrowhead pattern: Group B
4. Hippurate Hydrolysis - Test for ability to hydrolyze hippuric acid to benzoic acid & glycine (+ Ninhydrin) Positive -
Deep blue (purple): Group B ; Negative: Colorless
5. Bile Esculin Hydrolysis - Tests for ability to grow in 40% bile & hydrolyze esculin; Positive - blackening: Group D
6. Salt Tolerance Test - Test the ability to grow in 6.5% NaCl; Positive -turbidity/color change (purpleyellow): D Entero
7. Optochin (Taxo P) Susceptibility - Test for susceptibility to Taxo P; Positive - zone of inhibition: S. pneumoniae
8. Bile Solubility Test - Test for solubility to bile salt (2% Na desoxycholate)
Positive - Colonies disintegrates: S. pneumoniae , Negative - Colonies remains intact
1. β he olyti Bacitracin PYR SXT CAMP/Hippurate
A S + R -
B R - R +
Not A/B R - S -
2. α he olyti Optochin BEA Hydrolysis 6.5% NaCl PYR

S. pneumoniae S - - -
D Enterococcus R + + +
D Non-enterococcus R + - -
Viridans Strep. R S - -
3. Β, α, ɣ he olyti BEA Hydrolysis 6.5% NaCl PYR

D Enterococcus + + +
D Non-enterococcus + - -
Viridans Strep. S - -
2a. Aerobic Gram Positive Bacilli (Catalase Positive)
I. Clinically Significant Species

Corynebacterium diptheriae
1. Virulence Factor: Diptheria Toxin (Encoded by the tox gene) - block protein synthesis
2. Clinical Infections: a. Respiratory (pharyngitis characterized by the development of an exudate

membrane pseudo e ra e a d b. cutaneous diphtheria (non-healing ulcers and necrotic
lesions)
3. Laboratory Diagnosis
a. Microscopy: Club shape (Coryneform); Pleomorphic (Palisades, V & L forms, chinese letters,
picket fences); Irregularly staining (metachromatic areas)
b. Colony: i. SBA (Narrow zone of β-hemolysis); ii. Cystine-tellurite blood agar & Tinsdale's agar
(black colonies w/ brown halo); iii. Loeffler Serum / Pai Agar (produces metachromatic granules)
4. Toxigenicity Test – Elek Test
5. Identification
Characteristic C. diptheriae C. ulcerans C. pseudotuberculosis C. jeikeium
Ti sdale’s Halo + + + -
Urease - + + -
Gelatin Hydrolysis - + - -
a. Urease Test: Test the ability to hydrolyzes urea (Urea –Urease ammonia)
Positive: red/magenta (rapid urease) or orange (weak urease producer); Negative: no change / yellow
b. Gelatin hydrolysis: Test the ability to produce proteolytic enzymes and liquefy gelatin
• Positive: partial or total liquefaction; Negative: complete solidification
B. Corynebacterium jeikeium
1. Virulence Factor: Multiple antibiotic resistance
2. Clinical Infections: Iatrogenic infections (prosthetic heart valves)
3. Other Characteristics: Nonhemolytic and lipophilic (5% SBA w/ 1% tween 80)
C. Listeria monocytogenes
1. Virulence Factor: a. Protein p60 (adhesion and penetration to phagocytes) and; b. Listeriolysin O (cytotoxic toxin)
2. Clinical Infections: a. Pregnant women (stillbirth & spontaneous abortion); b. Newborns (bacteremia &
meningitis) and; c. immunosupressed host (endocarditis)
a. Microscopy: G+ rods or coccobacilli in pairs or in chains
b. Colony: Narrow zone of β-hemolysis (SBA)
c. Grows best at 30 -35 C, but growth occurs at 0.5 -45 C, isolated from tissues by cold enrichment. d. End-
over-end tumbling motility in broth (22-25°C)
e. Umbrella-shaped or inverted christmas tree pattern into a semi-solid tube
f. CAMP Reaction: Positive rectangle lo k type enhance hemolysis observe
g. Hippurate hydrolysis & bile esculin hydrolysis positive
2c. Aerobic Gram Positive Bacilli (Catalase Negative)
A. Erysipelothrix rhusiopathiae (Red skin, red disease)
1. Clinical Infections: Erysipeloid, septicemia, diffuse cutaneous infection
a. Microscopy: Thin, filamentous G(+) rods
b. Colony: α-hemolytic w/ prolonged incubation
3. Identification: Test tube brush-like pattern at 22°C, produces H2S in TSI
B. Arcanobacterium haemolyticum
1. Clinical Infections: Pharyngitis, cervical lymphadenopathy and soft tissue and sepsis
a. Microscopy: Curved, G+ rods w/ pointed ends that becomes coccal after 48 hrs
b. Colony: “ all olo ies, β-hemolytic, pits agar, leaves black dot under a colony
c. Identification: Lipase and Lecithinase positive (EYA)
Exhibit CAMP inhibition reaction(phospholipase D inhibits β-lysin)
C. Gardnerella vaginalis
1. Clinical Infections: Bacterial vaginosis (Excessive vaginal discharge, pH > 4.5 and foul smell) associated in UTI
a. Microscopy: Pleo orphi Clue cells in vaginal fluid.
b. Colony: Ɣ-hemolytic (BAP) and β-hemolytic (HBT)
c. Other Test: Whiff Test (Vaginal secretion + 10% KOH  fishy aminelike odor), urease positive
Characteristic Catalase Motility BEA Hipurate H2S (TSI) Hemolysis Urease
C. diptheriae + - - - - β -
L. monocytogenes + + + + - β -
E. rhusiopathiae - - - - + ɣ,α -
A. haemolyticum - - - - - β -
G. vaginalis - - - - - ɣ +
2c. Aerobic Gram Positive Bacilli (Branching Actinomycetes)

A. Nocardia spp.
1. Virulence Factor: Superoxide dismutase, nocobactin (iron chelating compound)
2. Clinical Infections: Pulmonary (N. asteroides) and cutaneous infection (N. brasiliensis); Actinomycotic mycetomas
3. Lab Diagnosis: a. Microscopy: Beaded, branching bacilli (G/S), partially acid fast (0.5-1% H2SO4)
b. Colony: β-hemolytic (SBA), Chalky, dry, crumbly “a ouraud’s & Mycosel), 22 C & 37 C (3-6 d)
2d. Aerobic Gram Positive Bacilli (Spore-Formers)
General Characteristics: Catalase (+) and forms endospores aerobically

A. Bacillus anthracis
1. Virulence Factor: a. Glutamic acid capsule; b. Exotoxins (Edema Factor or Protease)
2. Clinical Infections:
a. Cutaneous anthrax - most common, least severe, manifests as an erythematous papule to eschar formation
b. Inhalational anthrax - a.k.a wool sorters disease (progress to mild form to respiratory distress)
c. Gastrointestinal anthrax - most severe affecting the abdominal area.
3. Laboratory Diagnosis:
a. Microscopy: Large, G+ rod w/ square ends (in pairs or chains), bamboo rod appearance b. Colony:
• Nonhemolytic, large, gray, flat, irregular ( edusa head )
• Beaten egg white consistency (SBA)
• Large, mucoid colonies in bicarbonate agar
• String of pearls i MHA containing 10 U penicillin
B. Bacillus cereus
1. Virulence Factor and Clinical Infections:
Characteristics a) Diarrheal toxin b) Emetic toxin
Incubation period 8-16 hrs 1-5 hrs
Symptoms Diarrhea Vomiting
Duration of illness 24 hrs 9 hrs
Food Implicated Meat producers Fried & Boiled rice
Stability to heat Negative Positive
2. Laboratory Diagnosis:
a. Microscopy: Large, G+ bacilli
b. Colony: Large, feathery, spreading, wide zo e of β he olysis
c. Identification: Lecithinase Test, Motility test, Penicillin Susceptibility, Presensence of Parasporal crystals
Organism Lecithinase Motility Penicillin Sensitivity Parasporal Crystals
B. anthracis (+) (-) (+) (-)
B. cereus (+) (+) (-) (-)
B. thuringiensis (+) (+) (-) (+)
B. mycoides (+) (-) (-) (-)
3a. Gram (-) Diplococci

Obligate aerobe, Capnophilic, Oxidase (+), Catalase (+), Glucose Fermenter (except for M. catarrhalis)
A. Neisseria gonorrhoeae
1. Virulence Factor: Transferrin receptors, outer membrane proteins, pili, LOS (Endotoxins), capsule, IgA Protease
2. Clinical Infections: Urethritis & cervicitis, PID, sterility, ectopic pregnancy, Fitz-Hugh-Curtis syndrome,
conjunctivitis, disseminated gonoco ccal infection (DGI), endocarditis & arthritis
B. Neisseria meningitides
1. Virulence Factor: Pili, capsule (A, pandemics; B, community acquired; Y, pneumonia; W-135, invasive disease),
Outer membrane proteins, LOS, IgA1 protease
2. Clinical Infections: Epidemic meningitis, meningococcemia (purpura & petechial rash, disseminated intravascular
coagulation (DIC), Waterhouse-Friderichsen syndrome)
C. Moraxella catarrhalis
1. Virulence Factor: Atta h e t to respiratory EC’s
2. Clinical Infections: Localized inf. (otitis media & sinusitis), lower RT inf., Systemic inf. (endocarditis, meningitis)
III. Laboratory Diagonosis
A. Specimen Collection & Transport
1. N. gonorrhoeae: Urethra, cervix, rectum & pharynx (direct plating to selective media using transport swabs)
2. N. meningitidis & M. cattarhalis: CSF, blood, nasopharyngeal swabs & aspirates
B. Direct Microscopy: N. gonorrhoeae: Urogenital specimen (kidney or coffee bean shaped G- diplococci)
C. Culture D. Incubation: 35°C (↑ CO2, 72 hrs.)
E. Identification
1. Microscopy: G(-) diplococci
2. Colony Morphology
a. N. gonorrhoeae and N. meningitides:
Medium Inhibitory Agents BACTERIOLOGY
Suppressed HANDOUTS
Organisms
Vancomycin G(+)
Thayer-Martin Colistin G(-) S small, tan, translucent and
raised
Nystatin Yeast
b. M. catarrhalis: Colony can be swept
Modified TM Trimethoprin Swarming of Proteus intact (hockey puck), resembles
Martin Lewis Anisomycin Yeast wagon wheel (48 hrs)
New York City Amphothericin B Yeast
F. Identification
Blood MTM, ML, Superoxol
Organisms Glucose Maltose Lactose DNase / TH
Agar NYC (30% H2O2 )
N. gonorrhoeae (-) (+) (+) (+) (-) (-) (-)
N. meningitidis (+) (+) (-) (+) (+) (-) (-)
M. catarrhalis (+) (-) (-) (-) (-) (-) (+)
4a. Fastidious Gram Negative Bacilli

General Characteristics
• Pleomorphic, small G(-) bacilli, MacConkey (-) I. Haemophilus spp.
• Facultative anaerobes, ferment carbohydrates (exp. H. ducreyi), oxidase & catalase (+)
• Requires growth factors: Hemin/hematin (X Factor) and nicotinamide adenine dinucleotide (NAD or V Factor)
II. Clinically significant species
1. H. influenzae (Pfeiffer’s a illus)
a. Virulence factor: Capsule [ a-f (b, most common), lacks adherent capability, associated with systemic &
invasive infections], IgA protease and LPS
b. Clinical Manifestations of Infections:
Encapsulated Strains Nonencapsulated Strains
Systemic & RT Infections Otitis Media
Septicemia, Septic arthritis Conjunctivitis
Tracheitis, Meningitis, Pericarditis Bacteremia
Pneumonia, Epiglotitis Sinusitis, Pneumonia
2. H. aegyptius (Koch-Weeks bacillus) or H. influenzae bio. aegyptius: o ju ti itis pi k eye & Brazilian Purpuric Fever
3. H. ducreyi: Chancroid (soft chancre)
1. Specimen Processing & Isolation
a. Specimen: Premoised/transport swab, blood & body fluids
b. Culture Media: Chocolate Agar (w/ Bacitracin or 1 % IsoVitaleX for H. ducreyi or H. aegypticus)
c. Incubation: i. Most Haemophilus spp.: 5-10% CO2 at 35-37°C (2-3 days), ii. Haemophilus ducreyi: 5-10%
CO2 at °C / high hu idity days
2. Microscopy
a. Coccobacilli & filamentous
b. H. ducreyi - o o a illi that appear as school of fish railroad tracks or finger prints
3. Culture
a. H. influenzae: Translucent, smooth and convex; mousy / bleach like odor; encapsulated (larger & mucoid)
b. H. ducreyi - Small, flat, smooth, transparent to opaque; colonies can pushed intact; clumpy in saline
4. Identification
a. Neufeld Quellung Rxn: antisera is reacted with the antigens in the capsule making the capsule more prominent
b. Staphylococcus Streak: Haemophilus is streaked w/ S. aureus, S. pneumoniae, Neisseria and certain yeasts
• Positive: Satellitism dewdrop colonies surrounding S. aureus
c. X and V requirement; d. Hemolytic patterns
e. Porphyrin Test: Test for the ability to produce ALA (ALA  porphobilinogen/porphyrin  Hemin)
• Porphobilinogen (add Ko a ’s rgt a d Porphyri s emit with 360nm UV, red-orange)
Species X factor V factor Hemolysis ALA
H. influenzae (+) (+) (-) (-)
H. aegyptius (+) (+) (-) (-)
H. haemolyticus (+) (+) (+) (-)
H. parahaemolyticus (-) (+) (+) (+)
H. parainfluenzae (-) (+) (-) (+)
H. ducreyi (+) (-) (-) (-)
A. aphrophilus (-) (-) (-) (+)
4b. Other Fastidious Gram Negative Bacilli
I. Clinically significant species

A. HACEK Group: Dysgonic fermenter, normal biota of the oral cavity, oral infections, subacute bacterial endocarditis 1.
Aggregatibacter aphrophilus: foa lo i g eeds ↑ o . of CO2
2. A. actinomycetemcomitans: 4-6 point star in the center of the colony (48 h).
3. Cardiobacterium hominis: G(-) rod in rosettes or long filaments
4. Eikenella corrodens: Infections from human bites or fights, chlorine bleachlike odor, Assacharolytic 5. Kingella
spp.: Coccobacilli w/ square ends in pairs
B. Capnocytophaga
1. Infections: Periodontitis, Local to fulminant infection (from animal bites)
2. Lab Diagnosis: Microscopy (thin, fusiform, spindle-shaped); Colony (haze, gliding motility)
C. Pasteurella multocida
1. Infections: Pasteurellosis (systemic, pneumonic & cutaneous infections from animal bites)
2. Lab Diagnosis. Microscopy (Bipolar staining ) ; Colony (musty odor, mushrooms) D. Brucella
spp.
1. Infections: Brucellosis or undulant fever (transmitted trough aerosol, percutaneous and oral routes)
2. Others: Facultative intracellular, BSL-3
3. Lab Diagnosis
Species Natural Host Serum Aggln. H2S (Pb Acetate) Urease Thionine Fuchsin
B. melitensis Goat/sheep + - +<2 hrs. - -
B. abortus Cattle + + +<2 hrs. + -
B. suis Swine + + +<0.5 hrs. - +
B. canis Dogs - +<0.5 hrs. - -
E. Francisella tularensis
1. Infections: Tularemia (Animal bite or scratch or arthropod)
2. Others: Facultative intracellular, BSL-3
3. Lab Diagnosis: Culture: Blood-cystine-glucose agar (require cysteine, cystine, thiosulfate)

F. Legionella pneumophila
1. Virulence Factor: Multiply within macrophages and free-living protozoa, multiply at 20-43°C (40-60°C), adhere &
persist in piped water systems (biofilms)
2. Epidemiology: Aquatic sources (lakes, rivers, hot springs), mad made distribution systems (hot water systems,
cooling towers, etc.), humidifiers and respiratory therapy equipments
3. Infections: Legio aire’s disease and Pontiac Fever
4. Specimen: Respiratory, body fluids, blood
5. Microscopy: Direct fluorescent Ab (weakly staining in G/S)
6. Culture: Requires Iron & L-cysteine (BCYE), Ground-glass
H. Bordetella pertussis
1. Manifestation: Pertussis: a. catarrhal; b. paroxysmal; c. convalescent
2. MOT: droplets or direct contact w/ secretions
3. Lab Diagnosis: Bordet-Gengou, Regan-Lowe, Charcoal-horse lood mercury droplets or pearls
Characteristics B. pertussis B. parapertussis B. bronchiseptica
Charcoal-horse blood + (3-5 d) + (2-3 d) + (1-2 d)
Blood agar - + +
Mac/ Catalase/ Motility - - +
Oxidase + - +
Urease - + (24 hrs) + (4 hrs)
5. Gram Negative Bacilli (MacConkey Positive)

Fermentative: Enterobacteriaceae, Vibrio, Aeromonas, Plesiomonas
Oxidative: Acinetobacter baumannii, Burkholderia, Pseudomonas
Asaccharolytic: Stenotrophomonas Acinetobacter iwoffii
5a. Gram Negative Bacilli (MacConkey Positive, Oxidase Negative, Fermentative)
I. Enterobactericeae
Medium Gram (-) bacilli, Oxidase negative (except P. shigelloides), MacConkey positive, glucose fermenters, reduces
nitrate to nitrite (except. P. agglomerans), motile (except Klebsiella & Shigella)
II. Opportunistic Members
A. Escherichiae coli: Pneumonia, septicemia, appendicitis, peritonitis, meningitis, wound infections, etc.
Disease Syndromes i. Virulence Factor ii. Diseases / Symptoms iii. Other Information
1. Uropathogenic E. coli Pili & cytolysins Most common cause of UTI

Fimbriae & enterotoxins Epidemic & tra eler’s Mo tezu a’s re e ge
2. Enterotoxigenic E. coli (ETEC)
(heat stable/labile) diarrhea or turista
Shigella like infection Non-motile & non-lactose
3. Enteroinvasive E. coli (EIEC) Shiga-Toxin
(dysenteric stools) fermenter
4. Enteropathogenic E. coli Pili Watery w/ mucus but no
Infantile diarrhea
(EPEC) blood
5. Enterohemorrhagic Hemorrhagic colitis Associated w/ 0157:H7
Cytotoxin (Verotoxin)
Verotoxic E. coli (EHEC) Hemolytic uremic syndrome Sorbitol (-) & MUG (-)
6. Enteroadherent E. coli
a. Diffusely adherent E. coli (DAEC) UTI & diarrhea
b.Enteroaggregative E. coli (EAEC) Diarrhea (watery)
B-C. Klebsiella (Friedlander) & Enterobacter

1. Pneumonia, UTI, septicemia, wound infections, etc.
2. E apsulated, u oid olo ies that stri g .
3. Klebsiella (plasmid- ediated E“BL’s
Species (IMViC Rxns) Indole MR VP Citrate
E. coli + + − −
K. pneumoniae subs. pneumoniae − − + +
subs. oxytoca + − + +
Enterobacter spp. − − + +
Species (Decarboxylase Rxns) LDC ODC ADH

K. pneumoniae + − −
E. aerogenes + + −
E. cloacae − + +
D-E. Serratia & Citrobacter

1. Bacteremia, septicemia, UTI, pneumonia, wound infections , etc.
2. ONPG (+); S. marcescens: Red pigment (prodigiosin) and lipase, gelatinase & DNase +
S. marcescens C. freundii
TSI A/A or K/A A/A or K/A
H2S − +
F. Proteus, Morganella & Providencia 1.

Septicemia, UTI, pneumonia, etc.
2. Rapid urease (+), Deaminase (+)
3. Proteus produces swarming motility, burnt chocolate odor
Reaction P. mirabilis P. vulgaris Prov. stuartii Prov. retgerri M. morganii
Indole – + + +
H2S + – –
ODC + – – +
Motility S/+ +
III. Primary Intestinal Pathogens
A. Salmonella enterica subsp. enterica: Reservoir - GI tracts of poultry, pets and domestic animals
1. Classification: 7 subspecies, clinical isolates are subgroup 1, E.g . Salmonalla Typhi, Paratyphi and Chloraesuis
2. Virulence Factors: Fimbria, enterotoxin, transverse intestinal mucosa
3. Clinical Infections: Acute gastroenteritis or food poisoning, Enteric Fever or Typhoid fever (S. Typhi & S.
Paratyphi). Isolated in blood (week 1-2), urine (3-4), stool (2-3). Bacteremia (other serotypes)
4. Other Characteristics: Metallic colonies w/ black ring in Bismuth sulfite agar, Diagnosed with Widal’s test.
B. Shigella: Reservoir - Humans only
1. Virulence Factors: neurotoxin & enterotoxins (S. dysenteriae), Other species: only enteroxin 2. Disease: Shigellosis
or bacillary dysentery
Test S. dysenteriae S. flexneri S. boydii S. sonnei
Mannitol – + + +
ONPG / ODC – – – +
Serogroup A B C D
Test Salmonella Shigella

Motility, H2S (+) (-)
Infectious Dose 106 100-200
C. Yersinia pestis
1. Disease: Bubonic and Pneumonic Plaque
2. Laboratory Diagnosis: Bipolar staining "safety pin" , Cauliflower (SBA), Stalactite (broth), Grows best at 25-30°C
D. Yersinia enterocolitica
1. Disease: Acute enteritis (infants and children)
2. Laboratory Diagnosis: Bipolar staining", ull’s eye colonies (CIN, 48 hrs), Grows at 25-30°C, Motile at 25°C
Test Y. pestis Y. enterocolitica
TSI Yellow/Orange
Motile (25°C) – +
Sucrose – +
5b. Laboratory Diagnosis

I. Serologic Grouping
A. For identification and antigenic determination of E. coli, Klebsiella, Shigella, Salmonella (Kaufmann-White classification)
B. Types of antigen:
1. O or somatic Ag: Associated with the cell wall, heat stable, LPS of the outer membrane, Endotoxin
2. K or envelope Ag: Consist of the capsular polysaccharide, heat labile (100 C for 10 min), Vi Ag of Salmonella
3. H or flagellar Ag : In motile members. For Salmonella spp.
II. Selective Media for Enterobactericeae
A. MacConkey Agar - Selects G(-) bacteria. Differentiates lactose from non-lactose fermenters
• Bile salts, neutral red, crystal violet: inhibit gram (+) bacteria ; Lactose: Carbohydrate source
• Neutral red: Indicator (Brown at pH 6.8-8.0, Pink-red at pH <6.8)
B. Eosin Methylene Blue - Selects G(-) bacteria. Differentiates lactose from non-lactose fermenters
• Eosin and methylene blue and Lactose
C. Hektoen Enteric Agar (HEA) - Selects for stool pathogens. Differentiates lactose from non-lactose fermenters
• Bile salts (high amounts): inhibit G(+) bacteria, G(-) coliforms ; Lactose, Bromthymol blue
• Sodium thiosulfate: sulfur source ; Ferric ammonium citrate: H2S Indicator
D. Xylose-Lysine-Deoxychlate (XLD) Agar - Selects for stool pathogens. Differentiates lactose from non-lactose fermenters
• Bile salts (high amounts); Xylose, Lactose; Phenol Red; Sodium thiosulfate and Ferric ammonium citrate
E. Salmonella-Shigella Agar) - Selects for stool pathogens. Differentiates lactose from non-lactose fermenters
• Bile salts, Brilliant Green Agar, Lactose, Neutral Red, Sodium thiosulfate and Ferric ammonium citrate
F. Cefsulodin-irgasan-novobiocin (CIN) Agar - Selective media for Yersinia species
• Cefsulodin – Inh. G(+) bacteria & most G(-) bacilli ; Novobiocin inh. G(+) cocci ; Crystal violet – inh. G(-)
bacteria
G. Selinite, G(-) Broth - Enrichment broth for cultivation of GI pathogens. Selects for stool pathogens
5b. Biochemical Identification
I. Carbohydrate Utilization Test A.
Oxidation-Fermentation Tests
• Differentiating glucose fermenters from glucose oxidizers
• Hugh-Leifson O/F Basal Medium (OFBM)
• Contents: 1% Carbohydrates & 0.2% peptone
• Fermenter: Change in color in both tubes Enterobacteriaceae
• Oxidizer: Change in color in open tube Pseudomonad
• Nonoxidizer: No change in color in both tubes
B. Triple Sugar Iron Agar
• Test whether a G(-) rod utilizes glucose, lactose and sucrose
• Contents: 1% lactose, 1% sucrose & 0.1% glucose; Na thiosulfate & Ferrous sulfate (1st Bacteria + Na thiosulfate
 H2S gas, 2nd H2S gas + Ferrous sulfate  ferrous sulfide); Phenol red
Reactions Possible Organism(s)
K/K Pseudomonas
K/ A Serratia, Providencia, Morganella, Shigella
+
K/ A , H2S Citrobacter freundii, Proteus, Salmonella
A/ A Escherichia coli, Enterobacter, Klebsiella
A/A Citrobacter, Serratia
+
A/ A , H2S Citrobacter freundii
C. ONPG: Test capacity to produce β-galactosidase and hydrolyzes ONPG to form о-nitrophenol
• Positive: Yellow (Lactose and dLFs, S. sonnei ); Negative: Colorless (Non-lactose fermenters)
II. Glucose Metabolic Ends Products
A. MR-VP (Clark and Lubs)
• Determines the products of glucose fermentation
• 1st pathway produces mixed acid (MR - red)
• 2nd pathway produces acetoin (VP - pink-red)
a. Methyl Red Test: Glucose (fermentation)  Mixed acid (acetic, lactic, succinic & formic) + MR
• Positive: Bright red color (E. coli) ; Negative: Yellow color (Klebsiella and Enterobacter)
b. Voges-Proskauer Test: Glucose (fermentation)  Acetoin + VP (α-naphthol & 40% KOH)
• Positive: Red (pink-red) at the surface (Klebsiella and Enterobacter)
• Negative: Yellow Color (copper like) at the surface (E. coli)

III. Amino Acid Utilization
A. Decarboxylase Test
• Determines capacity to decarboxylate Amino Acid to form diamines
• Content: Glucose, bromcresol purple & cresol red, 1% amino acid
a. Lysine –Lysine decarboxylase Cadaverine
b. Ornithine –Ornithine decarboxylase Putrescine
c. Arginine –Arginine dihydrolase Citrulline  Ornithine
Positive: Alkaline (purple) , Negative: Acid (yellow)
B. Deaminase Test
• Determines capacity to deaminate phenylalanine to phenylpyruvic acid (+ 10% FeCl3 -> Green)
• Positive: Green or brown slant (Proteus, Providencia, Morganella) ; Negative: Slant color remains
IV. Miscellaneous Test

A. Citrate, Malonate or Acetate Utilization: Determine the ability to use Na citrate as the sole source of carbon
• Positive: Growth or Blue color ; Negative : Green
B. Indole Production
• Test for the ability to split tryptophan to form indole (indole + p-dimethylaminobenzaldehyde  red )
• Positive: Pink to wine colored ring ; Negative: No color change C. Urease (Christensens: agar or Stuarts: broth)
• Test the ability to hydrolyzes urea (Urea –Urease ammonia)
• Positive: Red or magenta (rapid urease) or orange (weak urease producer, e.g. Klebsiella and Enterobacter) ;
Negative: no color change / yellow (e.g. E. coli)
D. Lysine Iron Agar
• Determines the ability to decarboxylate or deaminate lysine and form H2S
• Contents: Lysine, Glucose, H2S Ind., bromcresol purple
a. If glucose is fermented, the yellow butt forms.
b. If lysine is decarboxylated, a purple butt forms
c. If lysine is deaminated, a reddish slant forms.
Reactions Possible Organism(s)
R/A Proteus spp., Morganella spp., Providencia spp.
K/A Escherichia coli, Enterobacter cloacae, Citrobacter spp., Shigella spp., Yersinia spp.,
K/K Klebsiella spp., Enterobacter aerogenes, Serratia sp., Salmonella spp., Plesiomonas spp.
+
K/K, H2S Salmonella spp., Edwardsiella spp.
E. Sulfide-Indole-Motility Agar
• Determines the ability to form H2S, indole and observe for motility
• Motile: Growth extending from line of inoculation
• Indole positive : Pi k to i e olored ri g after addi g Ko a ’s

• H2S positive : Blackening of the medium
F. MUG Test
• Determine the ability of an organism to produce β-D-glucuronidase.
• Final product: 4-methylumbelliferyl moiety which is fluorescent under 366-nm UV light.
• Positive: Blue fluorescence Escherichia coli ; Negative: Lack of flourescence Pseudomonas aeroginusa
6a. Gram Negative Bacilli (MacConkey Positive, Oxidase Positive, Fermentative)
I. Vibrio spp.
• Indications of Infection: consumption of raw seafood. Gastroenteritis (cholera-like). Contact with fresh, estuarine, or
marine water.
• Microscopy: Comma-shaped G(-) rods, polar/peritrichous flagella, exhibit darting or shooting star motility
• Physiology: Facultative anaerobe, reduces nitrate to nitrite , oxidase (+), most species are susceptible to O/129, most
species is positive to string test and halophilic (Except V. cholerae & V. mimicus)
A. Vibrio cholerae
1. Virulence Factor and Disease: Cholera enterotoxin (Choleragen) - profuse watery diarrhea (rice water stools).
Leads to dehydration and hypovolemic shock
• O Ag (01 & 0139): Markers for strains capable of epidemic and pandemic spread. Produce cholera toxin
Biogroups Eltor Classical
Hemmaglutination (chicken RBC)
β-hemolysis in SBA
Positive Negative
Voges-Proskauer Test
Resistance to Polymxin B (50 U)
B. Vibrio parahaemolyticus
1. Disease: Gastroe teritis summer diarrhea ; 2. Other Characteristics: Kanagawa toxin positive: β-hemolytic
C. Vibrio vulnificus
1. Disease: Septicemia & wound infection ; 2. Other Characteristics: Lactose positive
D. Vibrio parahaemolyticus
1. Disease: Wound infection ; 2. Other Characteristics: Strict halophile
E. Laboratory Diagnosis
1. Specimen Collection & Transport: Body fluids, tissues, swabs, stool (alkaline peptone H2O, pH 8.5)
2. Culture Media: SBA (greenish hue, α or β hemolytic), Mac (NLF) & TCBS II. Aeromonas hydrophila ater lo i g
1. Virulence: Cytotoxic enterotoxins
2. Disease: Intestinal (five diarrheal syndrome), Extraintestinal (wound infections, septicemia & meningitis, etc.)
3. Lab Diagnosis: β-hemolytic (SBA),Ferments Lactose, pink-centered(CIN)
III. Plesiomonas shigelloides
1. Disease: Gastroenteritis, bacteremia, meningitis, etc.
2. Lab Diagnosis: Pink in Inositol brilliant green bile salt agar; LDC, ODC, ADH Positi e Trio
IV. Laboratory Diagnosis
A. Thiosulfate Citrate Bile Salt Sucrose Agar
Selective and differential media for Vibrio : Contents: Sucrose, bromthymol blue, Na Citrate and Oxgall
a. Growth w/ fermentation (yellow): V. cholerae, V. alginolyticus
b. Growth w/o fermentation (green): V. mimicus, V. parahaemolyticus, V. vulnificus
c. No growth: Aeromonas spp., Plesiomonas sp.
B. String Test: Reagent - 0.5% Na desoxycholate [Positive - Viscous stringing (Vibrio spp.)
Negative - No viscous stringing (Aeromonas, Plesiomonas)]
C. Vibriostatic test: Reagent: 0/129 disks [Susceptible - V. cholerae, P. shigelloides
Resistant - Aeromonas spp., Other Vibrio spp.]
Vibrio cholerae Other Vibrio spp. Aeromonas spp. Plesiomonas
TCBS + + - -
String Test + + - -
Broth w/ 6.5% NaCl + + - -
Broth w/o 6.5%NaCl + - + +
Vibriostatic Test (150 μg 0/129) + - - +
Inositol Fermentation - - - +
Comments LDC, ODC, ADH
6b. Gram Negative Bacilli (Assacharolytic, Oxidase Positive)
Introduction: Oxidase Positive, Small, curved, G(-) bacilli, most species are asaccharolytic, microaerophilic
II. Epidemiology and Disease
A. Campylobacter jejuni
1. Epidemiology: Exposure to animals and ingestion contaminated water and poultry
2. Disease: Most common cause of bacterial gastroenteritis worldwide, Guillain-Barre syndrome
B. Campylobacter fetus subsp. fetus: Bacteremia in Immunocompromised & elderly patient
1. Epidemiology: Immunocompromised & elderly patient
2. Disease: Bacteremia
C. Helicobacter pylori
1. Epidemiology: Gastric ulcer patients
2. Virulence Factors: Urease, adhesin and cytotoxins
3. Disease: Gastric, peptic and duodenal ulcers; type B gastritis and GI carcinoma III. Laboratory
Diagnosis
A. Specimen
1. C. jejuni: Feces (Stuart, Cary-Blair & Campy Thio)
2. C. fetus subsp. fetus: Blood (Blood culture media)
3. H. pylori : Tissue biopsy “tuart’s , Cystei e-Brucella broth)
B. Culture Media and Incubation
1. Campylobacter spp: Butzler, “kirro ’s, ˚C or ˚C Mi roaerophili
2. H. pylori: “kirro ’s, Chocolate or Brucella agar with 5% horse blood, 35- ˚C Mi roaerophili
C. Microscopic Morphology
1. Campylobacter spp: S-shaped wings of seagulls a d produces darting motility
2. H. pylori: Multiple flagella at one pole
D. Colony Morphology: Moist ru y looki g C. jejuni), Convex & translucent (C. fetus), translucent & circular(H.
pylori)
E. Identification
Test C. jejuni C. fetus H. pylori
Hippurate hydrolysis + — —
Growth at 42°C + — —
Growth at 25°C — + —
Urease — — +
Nalidixic acid S R R
Cephalotin R S S
1. Urease Test: Tissue is place in Christensen medium for 37°C for 2 hours
2. Urea breath test : 13/14C labeled urea (oral dose) –Urease 13/14CO2 (Detected by scintillation counter )
7a. Gram Negative Bacilli (MacConkey Positive, Non Fermentative)

• Fail to acidify O-F Media overlaid w/ mineral oil, grow in MacConkey as colorless colonies , fail to acidify TSI or KIA, most
isolates in oxidase (+), resistance to a variety of antimicrobial agents (aminoglycosides & βeta-lactams)
• Contaminants in disinfectants, detergents, collection tube, venous catheters, ventilators, humidifiers, nebulizers, etc. II.
Clinically Significant Species
A. Pseudomonas aeruginosa
1. Clinical Significance: Most commonly isolated nonfermenter (75% of nonfermenters in nosocomial bacteremias,
5-15% of nosocomial inf.) wound inf., pulmonary disease (cystic fibrosis patients), UTI, endocarditis, meningitis
2. Virulence Fator:
i. Enzymes (protease, hemolysins, lecithinase, elastase & DNase)
ii. Exotoxin A ( inhibits protein synthesis),
iii. Alginate (polysaccharide polymer in mucoid strains)
3. Identifying Characteristic: Pigmented (Fluorescein/Pyoverdin, Pyocyanin, Pyorubin, Pyomelanin) ; Fruity grapelike
odor or corn tortilla-like odor (2-aminoacetophenone) ; Grow at 42°C; Cetrimide Agar (+); Acetamide (+)
B. Acinetobacter spp.
1. Clinical significance: 2nd most common nonfermenter
2. Identifying characteristics: Plump, paired G(-) coccobacilli; A. baumanii (saccharolytic) - purplish (MAC) or
cornflower blue (EMB) ; A. iwoffi (assaccharolytic)
C. Stenotrophomonas maltophilia
1. Clinical significance: 3rd most common nonfermenter
2. Identifying characteristic: lavender green (BAP), Ammonia-like smell, oxidizes glucose W(+), oxidizes maltose
S(+) a. Burkholderia cepacia
1. Clinical significance: Pneumonia in patients with cystic fibrosis or chronic granulomatous dse.
2. Identifying characteristics: Dirtlike odor in BAP, ONPG +; dark pink in Mac in 4-7 d b.
Burkholderia pseudomallei
1. Clinical significance: Melioidosis
2. Identifying characteristics: Bipolar staining (G/S), wri kled & deep pi k i Ashdo edia, Earthy
odor c. Burkholderia glandioli: Associated w/ patients w/ CF & CGD,
d. Burkholderia mallei: Glanders, Nonmotile

Organisms Oxidase Pigment Glucose Maltose Growth at 42°
S. maltophilia (-) Y (+) (S+) (+/-)
A. baumannii (-) (-) (+) (+/-) (+)
A. iwoffi (-) (-) (-) (-) (-)
Organisms Oxidase Pyoverdin Pyocyanin Gelatin ONPG

Acetamide hydrolysis
Cetrimide
42° Growth
P. aeruginosa (+) (+) (+) (-)
P. fluorescens (+) (+) (-) (+) (-)
P. putida (+) (+) (-) (-) (-)
B. cepacia (+) (-) (+)
B. pseudomallei (+) (-) (-)
8a. Anaerobic Bacteriology

I. Introduction
A. Aerotolerant anaerobe: Survive O2 exposure but performs metabolic processes only into an anaerobic
environment.
B. Obligate, strict anaerobe: Strict anaerobic requirement (0% O2), killed almost instantly in the presence of oxygen
C. Exogenous
• Contamination of wound or puncture by objects. E.g. C. tetani (tetanus) and C. perfringens (gas gangrene)
• Ingestion of preformed toxins in vegetable or meat. E.g. C. botulinum (botulism), C. perfringens (food poisoning)
• Colonization of GI tract of toxin-producing organism. E.g. C. botulinum (infant botulism)
D. Endogenous
• Gain access to normally sterile site
• Skin: Propionibacterium acnes
• RT: Prevotella, Porphyromonas, Fusobacterium, Anaerobic Cocci
• GIT: Bacteroides fragilis, Clostridium difficile
• GUT: Bacteroides, Prevotella, Fusobacterium
II. Specimen Selection, Collection, Transport and Processing
A. Specimen Quality (Selection)
1. Suitable specimens: Tissue biopsy (necrotic tissues), needle and syringe aspiration (exudates, abscess)
2. Unsuitable Specimens: Swabs, voided urine, feces, coughed sputum, bronchial washings.
3. Fecal specimens for Clostridial illness: Food poisoning (C. perfringens), botulism (C. botulinum),
pseudomembranous enterocolitis (C. difficile), neutropenic enterocolitis (C. septicum)
B. Specimen Transport
1. Rubber-stoppered collection vial: For liquid specimens (i.e. pus & body fluids)
2. Oxygen free transport tubes and anaerobic pouch: For swab specimens and tissue specimens, respectively
3. Contents: i. Reducing agent (thioglycolic acid, Na thioglycolate); ii. redox indicator (resazurin, methylene blue)
C. Processing Clinical Specimens
1. Macroscopic Examination: Foul odor (Anaerobic G- bacilli), brick-red fluorescence and necrotic tissue black
exudates (Porphyromonas, Prevotella), sulfur granules (anaerobic G+ bacilli)
2. Microscopic Examination of the Specimen: To determine a polymicrobic infection, guide for media selection,
provide a presumptive identification and reveal leukocytes and squamous epithelial cells
3. Inoculation to Plated or Tubed Media
a. Anaerobic blood agar: Enriched media
b. PEA & CNA blood Agar: Selective for G(+) anaerobes
c. Anaerobic broth: i . Thioglycollate broth ; ii. Cooked meat broth and iii. peptone-yeast extract glucose (PYG) -
analysis of metabolic end products by GLC
d. Kanamycin-vancomycin-laked blood (KVLB):Selective for Bacteroides & Prevotella
e. Bacteroides bile esculin agar (BBE): Selective & differential for B. fragilis
f. Cycloserine cefoxitin fructose agar (CCFA): Selective & differential for C. difficile
g. Egg-yolk Agar (EYA): For determination of lecithinase and lipase production
• C. perfringens: Lecithinase + (white opaque zone) ; F. necrophorum, C. botulinum: Lipase + (iridescent sheen)
4. Anaerobic Incubation
a. Anaerobe Chamber: Storage & inoculation under anaerobic condition
b. Anaerobic Jars and Bags: Anaerobiosis produced by gas generator envelope
c. Holding Jars: During processing, for inoculated plates pending incubation & examination of cultures
• Contents: gas generator (85-90% N2 , 5% H2, 5-10% CO2), catalyst (palladium pellets, iron powder),
desiccant (silica gel, blue → pink), redox indicator (methylene blue and resazurin)
8b. Clinically Significant Species

I. Gram Positive Spore Forming Bacilli
• Anaerobes and their diseases (1) Virulence Factors, (2) Associated Disease
• Procedures for Identifying anaerobes : (3) Cellular Morphology, (4) Colony Morphology, ( 5) Other Characteristics
A. Clostridium perfringens
1. α-toxin (type C food poisoning), enterotoxin
2. Gas gangrene, myonecrosis and food poisoning
3. Gram-variable large square rods with blunt ends (boxcar)
4. Double zone of hemolysis
alpha toxin (partially hemolyzed outer zone), theta toxin (completely hemolyzed inner zone)
5. a. Lecithinase positive (opaque zone around the colonies in Egg Yolk Agar); b. Nagler reaction (Type A Toxin
demonstration); c. CAMP (Bow-tie) & Reverse CAMP (Arrow-head) positive
B. Clostridium tetani
1. Tetanospasmin (neurotoxin) - causes a spastic type of paralysis with continuous muscular spasms.
2. Tetanus - Trismus (lockjaw), risus sarcodinicus (distorted grin) & backward arching of the back muscles
3. Swollen terminal spore, drumstick appearance
4. Smoothly swarming but slow growing, narrow zone of β-hemolysis
C. Clostridium botulinum
1. Botulism toxin (neurotoxins) - flaccid type of paralysis
2. Food borne botulism - ingestion of preformed toxin
Infant botulism- ingestion of spores
Wound botulism - contamination of wound with spores
3. Swollen subterminal spores (Tennis racket)
4. Usually β-hemolytic (SBA)
5. Lipase positive (iridescent, multicolored sheen, resembling gasoline on water or mother-of-pearl in EYA),
Confirmation by demonstration of neurotoxin in serum, stool, vomitus or gastric contents
D. Clostridium difficile
1. Toxin A (enterotoxin) & B (cytotoxin)
2. Antibiotic-associated diarrhea and pseudomembrane colitis
3. Thin rods, rare spores
4. Horse stable, barnyard odor, chartreuse fluorescence; large, yellow colonies that fluoresces golden-yellow (CCFA)
5. Confirmed by demonstration of toxin B (cytotoxin) and organism in feces

E. Clostridium septicum
1. Associated w/ malignancies (colorectal cancer), neutropenic enterocolitis and myonecrosis 2.
Thin rods, subterminal spores
3. Rese les Medusa head , β-hemolytic, smoothly swarming
Double Zone Chartreuse Spore Position
Swarming Lecithinase Lipase
of hemolysis Fluorescence
C. perfringens - + - + - ST
C. botulinum - - - - + ST
C. tetani + - - - - T
C. difficile - - + - - ST
C. septicum + - - - - ST
II. Gram Positive Bacilli: Outline: (1) Disease, (2) Cellular Morphology, (3) Colony Morphology, ( 4) Other Characteristics
A. Actinomyces israelli
1. A ti o y osis si us tra ts, hi h erupt to the surfa e a d drai pus that ay o tai sulfur granules ),
2. Branching filamentous rods
3. White, opaque and rese le molar tooth
B. Propionibacterium acnes
1. Actinomycosis, acne, subcute bacterial endocarditis, contaminants of blood culture bottles
2. Anaerobic diphtheroids
3. Small & white to large & yellowish tan
C. Bifidobacterium
1. Actinomycosis, in mixed infections of abdomens and GUT
2. Diptheroid, e d of ells ay e spatulated or ifur ated dog bones
3. Small, white, convex, shiny with irregular edge
Branched bacilli Catalase/Indole Comments
A. israelii + - Molar tooth colony
P. acnes - + Diptheroid
Bifidobacterium - - Rods w/ forked ends
III. Gram Negative Bacilli
1. Virulence Factor: Polysaccharide capsules, adherence factors
2. Spectrum of Disease: In mixed infection, brain abscess and peritoneal infections
3. Cellular Morphology; 4. Colonial Characteristics; 5. Other Characteristics
A. Bacteroides fragilis
3. Coccobacili or pleomorphic
4. Gray black colonies on BBE Agar, Grow in KVLB agar

5. Tolerates and hydrolyze 20% bile, saccharolytic and resistant to kanamycin, vancomycin & colistin B. Prevotella
spp.
3. Tiny coccobacilli
4. Fluoresces brick red, Black pigment in KVLB agar.
5. Inhibited by 20% bile, saccharolytic, susceptible to colisten C. Porphyromonas spp.
3. Tiny coccobacilli
4. Fluoresces brick red, No growth in KVLB agar.
5. Inhibited by 20% bile, asaccharolytic, susceptible to vancomycin
D. Fusobacterium nucleatum
3. Spindle-shaped w/ pointed ends
4. Crumblike or Ground glass, Greening on air exposure, Fluoresces chartreuse, Lipase positive (EYA)
5. Asaccharolytic, susceptible to kanamycin and colistin
Bile Esculin Growth on Brick Red Chartreuse Iridescent
Species V K Co
Resistance Hydrolysis KVLB Fluorescence fluorescence sheen (EYA)
B. fragilis R R R + + + - - -
Prevotella R R S - - + + - -
Porphyromonas S R R - - - + - -
Fusobacterium R S S - - - - + +
IV. Cocci
A. Spectrum of Disease: In mixed infection, brain abscess and peritoneal infections
B. Cellular Morphology:
1. G(-) diplococci / in chains -> Veillonella parvula
2. G(+) cocci -> Peptococcus & Peptostreptococcus (Finegoldia magna) C. Colony
Morphology: Red fluorescence -> Veillonella parvula
V. Confirmatory Test
• Gas-liquid chromatography: Metabolic ends products from glucose metabolism
• Gas chromatography: Whole-cell long chain fatty acid methyl ester (FAME) analysis
9a. Spirochetes
I. Clinically Signifant Species

A. Leptospires 1.
Characteristics:
• Tightly coiled, thin, spirochetes w/ hooked ends
• Cultura le to Flet her’s, Stuart, EMJH
• Visible by dark-field, phase contrast & immunoflourescence microscopy
2. Leptospira interrogans:
• Acquired through contact with urine of animals (e.g. rodents) who carry the organism.
• Leptospirosis and Weil disease (systemic disease with intravascular disease, renal & hepatic failure) B.
Borreliae
2. Characteristics:
• Less tightly coiled (3 -10 coils); culturable to Kelly medium; visualized by bright-field microscopy
3. Borrelia recurrentis:
• Endemic (tickborne) and epidemic relapsing fever (louse-borne)

4. Borrelia burgdorferi:
• Lyme disease
1. Localized (Erythema chronicum migrans),
2. Early disseminated
3. Late persistent
C. Treponemes
1. Characteristics
• Coiled (4-14); non culturable, motile with graceful flexuous movements; visible by dark-field
2. Treponema pallidium subsp. pallidum:
i. Syphilis – manifests as
a. Primary (chancre)
b. Secondary (condylomas)
c. Tertiary (gummas, neurosyphilis)
ii. Congenital syphilis
iii. Serologic Tests: VDRL & RPR, FTA-ABS, TP-PA and EIA
3. Treponema pallidum subsp. pertenue - Yaws
4. Treponema pallidum subsp. endemicum - Endemic Syphilis or Bejel 5. Treponema carateum - Pinta
9b. Chlamydiaceae
I. Introduction: Obligate Intracellular Bacteria
A. General Characteristics
C. trachomatis C. pneumoniae C. psittaci
EB Morphology Round Pear-shaped Round
Glycogen in inclusions (+) (-) (-)
Sulfa drug sensitivity (+) (-) (-)
Natural Hosts Humans Humans Birds
B. Chlamydia trachomatis
Biovars Clinical Syndromes Route of Transmission
A,B,Ba,C Ocular (Endemic) Trachoma Hand to eye from fomites
L1,L2,L3 Lymphogranuloma venereum
Sexual
Urogenital Disease (PID , Reiter Syndrome)
D-K
Inclusion conjunctivitis Hand to eye
• Cell Culture, serology , molecular (PCR)
• Frei’s test (intradermal skin test of LGV Ag): Positive: erythema (redness) and induration (firmness) C.
Chlamydophilia pneumoniae:
• Pneumonia & pharyngitis
• Guillain-Barre syndrome D. Chlamydophilia psitacci:
• Psittacosis & ornithosis (parrot fever)
• acquired from birds by inhalation of aerosols
9c. Ricketsiaceae and Similar Organism
I. Introduction: Obligate intracellular bacteria, arthropod borne

A. Characteristics: Arthropod-borne, obligate intracellular. Manifest as triad of fever, headache and rash B.
Rickettsia and Orentia
1. Spotted fever Group
Agent Disease Vector or MOT
Rickettsia akari • Rickettsialpox . Mites

• Bouttonneuse fever
• Mediterranean spotted fevers
Rickettsia conorii South African, Israeli spotted fevers
• Ticks
Indian, Kenyan tick typhus
•
Rickettsia rickettsii • Rocky mountain spotted fever.
2. Typhus Group
• Epidemic typhus Lice

Rickettsia prowazekii • Sporadic typhus Flying squirrels
• Brill-Zinsser disease Recrudescent-Reactivation
Rickettsia typhi • Murine typhus, Endemic typhus Ticks
3. Scrub Typhus Group
Orientia tsutsugamushi • Scrub typhus Chiggers
C. Ehrlichia / Anaplasma
Ehrlichia chaffeenis • Monocytic ehrlichiosis
Ehrlichia ewingii • Granulocytic ehrlichiosis Ticks
A. phagocytophilum • Granulocytic anaplasmosis
D. Coxiella
Coxiella burnetii Q (query) fever Inhalation of dried birthing fluid
E. Laboratory Diagnosis Disease OX-19 OX-2 OX-K
1. Immunohistology and PCR Brill-Zinsser V V –
2. Embryonated eggs and tissue culture
Epidemic typhus + V –
3. Gie sa, Wright’s stai ed uffy oat
4. Weil-Felix rxn. (P. vulgaris OX-19, OX-2, P. mirabilis OX Murine typhus + V –
Rickettsialpox – – –
RMSF + + –
Scrub Typhus – – V
9d. Mycoplasma and Ureaplasma
I. Characteristics
• Do not possess a cell wall
• Small cell, genome size and colonies
• Pleuropneumonia-like organisms (PPLOs)
A. Mycoplasma pneumoniae
1. Flora: Oropharnyx and upper RT tract
2. MOT: Contact with urine of animals (e.g. rodents) who carry the organism
3. Clinical infections: Asymptomatic infection, primary atypical or walking pneumonia
4. Others: Eaton Agent
B. Mycoplasma hominis and Ureaplasma spp.
1. Flora: GUT
2. MOT: sexual and vertical
3. Clinical infections: Urogenital tract infections (Non gonococcal urethritis, PID); systemic infections in neonates
and in immunosupressed patients
C. Laboratory Diagnosis
1. Specimen Collection and Transport
a. Body fluids, wound and blood
b. Swabs (dacron, polyester and calcium alginate)

c. Inoculated at bedside
2. Direct Examination
a. PCR, serology
b. Culture, Isolation and Identification: Colonies growing on SP4 Agar. Mycoplasma appear as fried egg
while Ureaplasma appear as dark brownish clumps .
c. Differentiated on their ability to ferment glucose, utilize arginine and hydrolyze urea
Species Glucose Arginine Urease
M. hominis – + –
M. pneumoniae + – –
U. urealyticum – – +
10a. Mycobacteria
General Characteristics
Slender, slightly curved rods, high lipid content (mycolic acid), acid fast (stained by basic fuchsin dye but resist
decolorization by 3% HCl), slow growth (2-6 weeks)
1. Mycobacterium tuberculosis complex
A. Mycobacterium tuberculosis
a. Epidemiology: >1 billion persons are infected, 8-10 million new cases / year, 15-20% develops disease b.
Transmission: Inhalation of aerosols or close contact
c. Spectrum of disease: Tuberculosis
i. Primary / Reactivation: Tubercles or granuloma (granulomatous lesions from multinucleated cells) and
Caseation (cheese like masses from break down of tubercles) in the lungs ii.
Extrapulmonary (Miliary): Kidney, joints, CNS, GUT, body cavities, larynx, etc.
d. Culture: Raised, dry, rough, buff (2-3 weeks in LJ, 5-10 days in Middlebrook)
B. Mycobacterium bovis and BCG
a. Transmission: M. bovis (Ingestion of milk from infected cattle)
M. bovis / Bacille Calmette-Guerin (Immunization of immunocompromised individuals)
b. Culture: rese les water droplets i Middle rook
c. ScreeningTest: Tuberculin Skin Testing (Mantoux test, Pirquet test, PPD test)
Positive: erythema (redness) and induration (firmness) in 48-72 hours
2. No tu er ulosis y o a teria NTM’s
• Runyon Classification of NTM: Based on the ability to produce carotenoids

A. Photochromogens – Unpigmented when grown in the dark and develop pigment after light a. M. asiaticum
b. M. kansasii: Yellow bacillus , s i i g pool gra ulo a, nd most common NTM in lungs
c. M. marinum: of the sea, grows best 30-32°C
d. M. simiae
B. Scotochromogens – Pigmented when grown in the dark but may intensify with exposure to light a. M.
gordonae: Tap water bacillus
b. M. szulgai: Photochromogen (22°C) & scotochromogen (37°C)
c. M. scrofulaceum: Cervical lymphadenitis in children
d. M. xenopi: Gro s est °C, Bird’s est i or eal agar
C. Nonphotochromogens – Nonpigmented when grown in the dark and exposed to light
a. M. avium complex Battey bacillus : Most commonly isolated NTM;
Most common cause of systemic bacterial infection in AIDS patients
b. M. avium subs. paratuberculosis: Chro i diarrhea e.g. Joh e’s & Croh ’s dse.
c. M. celatum: Frequent isolate in respiratory specimen
d. M. genavense: Associated with infections of AIDS patients
e. M. haemophilium: Requires hemoglobin and hemin
f. M. malmoense: Coccobacilli without cross-bands
g. M. terrae complex: Radish bacillus
h. M. ulcerans: Grows best 42°C, 3rd most common Mycobacteria
D. Rapid-growers ≤7 days growth
a. M. fortuitum and M. chelonae – abscessus group: Non photochromogen, Arylsulfatase (+) in 3 d, MacConkey (+)
b. M. smegmatis – Schotochromogen
E. Noncultivatable
a. M. leprae - Ha se ’s Disease
i. Tuberculoid Leprosy - Skin lesions and peripheral nerve involvement
ii. Lepromatous leprosy - Extensive skin lesions and symmetric nerve damage
A. Laboratory Safety Considerations
1. Specimen is collected in sterile, leak proof container.
2. BSL 2 - for preparing AFB smears and culture, BSL 3 - For propagation
3. Aerosol - generating procedures is performed in Class II or III BSC
B. Specimen Collection
1. Sputum and gastric lavage - early morning specimens in 3 consecutive days
2. Urine - early morning urine in 3 consecutive days, midstream clean catch
3. Stool - for isolation of M. avium in AIDS patients
4. Blood - for isolation of M. avium complex, can be recovered by BACTEC or by Isolator lysis centrifugation
5. Tissue and body Fluids - homogenized and concentrated), respectively
C. Specimen Preparation
1. n-Acetyl-L-cysteine (NALC) or dithioreitol: liquefy specimen (splits disulfide bonds)
2. 2-4% NaOH: both digestant (mucolytic) and decontaminating (antibacterial) agent
3. Trisodium phosphate & Benzalkonium chloride: acts as digestant and as decontaminating agent, respectively.
4. Oxalic Acid: for Pseudomonas contaminated samples
D. Staining of Acid Fast Bacilli: Acid-Fast Stain (AFB Stain)

1. Ziehl-Neelsen (hot stain) & Kinyuon (cold)
2. Auramine-Rhodamine fluorochrome stain. Examined at (250-400x). More sensitive
AFB (1000x) Flourochrome (450x) Report
0 0 No AFB seen
1-2/300 fields 1-2/70 fields Repeat test
1-9/100 fields 2-18/50 fields 1+
1-9/10 fields 4-36/10 fields 2+
1-9/field 4-36/field 3+
>9/field >36 field 4+
E. Media & Isolation Methods
1. Solid Media: 35°C, 5-10% CO2 ,↑ hu idity
i. Egg Based (18-24 d) - Lowenstein-Jensen and Petragnani ↑ ala hite gree
ii. Agar based (10-12 d) - Middlebrook 7H10 & 7H11 and Mit hiso ’s (Selective Middlebrook) 2.
Liquid Media: Middlebrook broth (10 d)
F. Laboratory Identification
1. Niacin (Accumulation) Test
• Test for the ability to produce and accumulate Niacin (niacin  niacin ribonucleotide)
• Positive (Formation of yellow liquid w/ addition of cyanogen bromide); Negative (Liquid remains milky
white)
2. Nitrate Reduction
• Test for the ability of to reduce nitrate (nitrates –nitroreductase  nitrite)
• Positive (Development of pink to red color); Negative (No color development)
3. Semiquantitative Catalase Test : >45 mm of bubbles or <45 mm of bubbles
4. Heat stable (68ºC) Catalase Test
• Test for the ability of catalase enzyme to remain active after heating Stable or inactivated at 68°C
for 20 mins.
5. Growth Inhibition by T2H (TCH)
• Test for the ability for tolerance to Thiophene-2-Carboxylic Acid Hydrazide Positive (No Growth);
Negative (Growth)
Species Niacin Growth on T2H Nitrate reduction 68° & SQ Catalase

M. tuberculosis + R (-) + (-)
M. bovis (-) S (+) (-) (-)
11a. Principle of Antimicrobial Action & Resistance
I. Introduction
• Antibiotics - Substance obtained from microbes to kill an infecting pathogen. • Bacteriostatic -
Inhibit microbial growth
• Bactericidal - Kills the microbe leading to lysis and death
II. Antimicrobial Action
1. Inhibitors of Cell Wall Synthesis
a. Beta-lactams: Binds to the enzyme (penicillin-binding proteins). E.g. penicillins, cephalosporins

b. Glycopeptides: Binds to the precursors, interfering PBD activity. E.g. vancomycin
2. Inhibitors of Cell Membrane Function
a. Lipopeptides: G(+) bacteria. E.g. daptomycin
b. Polymyxins: G(-) bacteria. E.g. polymyxin B & colistin
3. Inhibitors of Protein Synthesis
A. Binds to 30s ribosomal subunits
a. Aminoglycosides: E.g. gentamicin, tobramycin, amikacin
b. Tetracyclines: Broad spectrum including intracellular bacteria. E.g. doxycycline
c. Glycylglycine: For tetracycline resistant bacteria. E.g. tigecycline
B. Binds to 50s ribosomal subunits
a. Macrolide – Lincosamid – Steptogramin:
E.g. Macrolide (erythromycin), Lincosamide (clindamycin), Steptogramin (quinupristin) b.
Oxazolidinones: E.g. linezolid
c. Chloramphenicol: Toxic (bone morrow aplasia)
4. Inhibitors of DNA and RNA Synthesis
a. Fluoroquinolones: Binds and interferes with DNA gyrase. E.g. ciprofloxacin
b. Metronidazole: Breaks DNA strands. Requires anaerobiosis
c. Rifampin: Binds the RNA polymerase. Inhibits RNA synthesis
5. Inhibitors of Folic Acid Synthesis
a. Sulfonamides: Inhibits dihydropteroate synthase. i.e. sulfamethoxazole
b. Trimethoprim: Inhibits dihydrofolate reductase
6. Nitrofurantoin: May inhibit bacterial protein and enzyme synthesis (Only used treat UTI)
III. Mechanisms of Antibiotic Resistance
A. Intrinsic (Natural) Resistance: Results from the normal genetic structural or physiologic state
1. Anaerobes vs. aminoglycosides : Lack of oxidative metabolism to drive uptake
2. Gram (-) bacteria vs. vancomycin: Lack of uptake from inability of vancomycin to penetrate outer membrane
3. Aerobes vs. metronidazole: Inability to aerobically reduce the drug to its active form
B. Acquired Resistance: Results from changes in genetic make-up. Genetic mutation and acquisition of genes via gene
transfer
1. Transformation: Uptake and incorporation of naked DNA into a bacterial cell.
Competent - able to take up free DNA (H. influenzae, S. pneumoniae and N. gonorrhoeae )
2. Transduction: Transfer of bacterial genes by a bacteriophage
3. Conjugation: Due to cell-cell-cell contact leading to mo ilizatio of do or a teriu ’s hro oso e, plas id, or transposon
via sex pili
11b. Antimicrobial Susceptibility Testing
I. Introduction
•
Variable Standard Note:
Inoculum Disk diffusion: 1.5 x 108 CFU/mL
Broth microdilution: 5 x 105 CFU/mL
Agar dilution: 1x104 CFU/mL
Formulation Mueller-Hinton
25 mg/L Ca2+, 12.5 mg/L Mg2+ ↑ Concentration: ↓ a ti ity of
Ca2+, Mg2+ content
Aminoglycosides and Tetracyclines
Minimal or absent ↑ Concentration: ↑ a ti ity of
Thymidine content
Sulfonamide and SXT
7.2-7.4 ↑ pH : ↑ a ti ity of A i ogly osides,
pH
Erythromycin ; ↓ a ti ity of Tetra y li es
Agar depth 3-5mm (4 mm)
Determine the extent of its acquired resistance
II. Disk diffusion Testing Methods (Kirby-Bauer)
Incubation
Atmosphere Humidified ambient air

Temperature 35°C
Length Disk diffusion: 16-18 hr
Others
Age of the Colony 24 hours

Colonies Sampled
4-5
from isolates
Distance 20 mm apart
Maximum stacks 5

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BACTERIOLOGY HANDOUTS

1a. Bacterial Structure

2b. Host-Microorganisms Interactions

Type Agents Type Agents

1d. Clinical Laboratory Safety

1e. Specimen Collection & Processing

C. Specimen Receipt and Processing

1f. Microscopic Examination of Infected Materials

Smear Specimen Fixation

Selected Morphology Genus (Species)-Specific Stains

1. All of the following sites contain normal flora, EXCEPT:

Gram Positive Bacilli

Gram Positive Cocci Gram Negative Coccobacilli

1g. Traditional Cultivation

D. Use of Colonial Morphology

1h. Phenotyping Scheme

2. Gram (+) Cocci

2a. Gram (+) Staphylococci

2b. Gram (+) Streptococci

• Cell Wall (Polysaccharide) Structure Pattern of hemolysis

II. Clinically Significant Species

Group B larger, narrow zone of β hemolysis

2. α he olyti Optochin BEA Hydrolysis 6.5% NaCl PYR

3. Β, α, ɣ he olyti BEA Hydrolysis 6.5% NaCl PYR

2a. Aerobic Gram Positive Bacilli (Catalase Positive)

I. Clinically Significant Species

2. Clinical Infections: a. Respiratory (pharyngitis characterized by the development of an exudate

2c. Aerobic Gram Positive Bacilli (Branching Actinomycetes)

2d. Aerobic Gram Positive Bacilli (Spore-Formers)

General Characteristics: Catalase (+) and forms endospores aerobically

3a. Gram (-) Diplococci

4a. Fastidious Gram Negative Bacilli

I. Clinically significant species

3. Lab Diagnosis: Culture: Blood-cystine-glucose agar (require cysteine, cystine, thiosulfate)

5. Gram Negative Bacilli (MacConkey Positive)

1. Uropathogenic E. coli Pili & cytolysins Most common cause of UTI

B-C. Klebsiella (Friedlander) & Enterobacter

Species (Decarboxylase Rxns) LDC ODC ADH

D-E. Serratia & Citrobacter

F. Proteus, Morganella & Providencia 1.

III. Primary Intestinal Pathogens

Test Salmonella Shigella

5b. Laboratory Diagnosis

• Negative: Yellow Color (copper like) at the surface (E. coli)

IV. Miscellaneous Test

• Indole positive : Pi k to i e olored ri g after addi g Ko a ’s

6a. Gram Negative Bacilli (MacConkey Positive, Oxidase Positive, Fermentative)

6b. Gram Negative Bacilli (Assacharolytic, Oxidase Positive)

7a. Gram Negative Bacilli (MacConkey Positive, Non Fermentative)

III. Laboratory Diagnosis

Organisms Oxidase Pyoverdin Pyocyanin Gelatin ONPG

8a. Anaerobic Bacteriology

8b. Clinically Significant Species

5. Confirmed by demonstration of toxin B (cytotoxin) and organism in feces

4. Gray black colonies on BBE Agar, Grow in KVLB agar

I. Clinically Signifant Species

• Endemic (tickborne) and epidemic relapsing fever (louse-borne)

9c. Ricketsiaceae and Similar Organism

I. Introduction: Obligate intracellular bacteria, arthropod borne

Agent Disease Vector or MOT

Rickettsia akari • Rickettsialpox . Mites

• Epidemic typhus Lice

9d. Mycoplasma and Ureaplasma

b. Swabs (dacron, polyester and calcium alginate)