Microbiology PDF

MICROBIOLOGY REVIEW HANDOUTS
BACTERIOLOGY
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 immune
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)
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 m) 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. Safety Program
1. Standard Precaution: Blood and body fluids from all patients should be considered infectious
2. 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
B. Safety from Infectious Agents
1. Routes of Infection
a. Mucous Membrane contact - rubbing the eyes (conjunctiva) or nose with contaminated hands
b. Airborne - inhalation of aerosols produced during centrifugation of unstoppered tubes (M. tuberculosis, Brucella)
c. Ingestion - failure to wash hands after work, eating, drinking and mouth pipetting (Salmonella and Shigella)
d. Direct Inoculation - puncture by contaminated needles and broken glass (Hepatitis virus)
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 adult humans. Work
done 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 container.
c. Biosafety Level-3 - For handling organisms that can be transmitted by aerosols. Controlled access. Ducted air
ventilation with special lab clothing & 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. Package the specimen that will maintain the organism’s viability
2. Collection Procedures
a. Blood Culture - highest concentration occurs before the fever spikes. Draw 2 sets from right and left arms,
respectively ≥1 hr apart. ≥20 ml per set is collected in adults and 5-10 ml per set in children
b. CSF & Body Fluids - collect ≥1 mL 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 then 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 number; patient’s room number or location;
requesting physician; culture site; date and time of collection
b. Requisition Form: Patient’s name, age and gender; patient’s room number or location; physician’s name; 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 & Amie’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. Sputum with <25WBC’s and >10 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 ≥10 mins.
Pellet Blood culture, Dilute b. Flooding with 95%
Rolled Swab material (Pus) methanol for 1 min.
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-Giemsa Sample with cellular background
Selected Morphology Genus (Species)-Specific Stains
Methylene Blue Metachromatic granules Intracellular organisms________
Antibody or DNA
Acid Fast Stains Mycolic Acid Lacks cell wall_______________
probe stains
Gram Stain Cell Wall Not resolved by light microscope
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 Cocci Gram Negative Coccobacilli
Chains Streptococcus Singly Fastidious G (-) Bacilli
Cluster Staphylococcus, Viridans Strep., Chains, Masses Gram (-) anaerobes
Diplococci Streptococcus pneumoniae
Gram Negative Bacilli
Encapsulated 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
Gram Positive Bacilli Spiral Borrelia, Leptospira, Treponema
Small Listeria, Corynebacterium
F. Examination of Prepared Material
Large Clostridium, Bacillus
Cells & structures Associations
Diptheroid Corynebacterium
Necrosis, heavy protein fluid
Beaded Mycobacterium, Corynebacterium
Epithelial cells Cell surface in collection site
Branched Nocardia, Actinomyces
Mononuclear cells Chronic inflammation
Bifid/V forms Bifidobacterium
Irritation of glandular surfaces
Purulence Acute inflammation
Red blood Cells Trauma, hemorrhage
1g. Principles of 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, Gamma - 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 Medi - 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. Obligate anaerobe____ - cannot grow in the presence of oxygen
c. Facultative anaerobe__ - can grow either with or without oxygen
d. Microaerophile_______ - requires a reduced level of oxygen for growth
e. Aerotolerant anaero__ - can survive in the presence of oxygen but do not use oxygen for metabolism
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
2. Macroscopic Morphology (Colony appearance)
3. Environmental Requirements
4. Susceptibility to Antimicrobial Agents
5. Nutritional Requirements and Metabolic Capabilities
a. Enzyme capabilities (Enzyme based test)
b. Presence of Metabolic Pathways
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
1. Virulence Factor
a. Enterotoxins: A, B & D: food poisoning; B: pseudomembrane colitis
b. Toxic Shock Syndrome Toxin-(Enterotoxin F): Menstruating-associated toxic shock syndrome
c. Exfoliative Toxin: Scalded skin syndrome
d. Cytolytic Toxins: Hemolysins (α, β, δ), Panton-Valentine Leucocidin (ɣ-Hemolysin)
e. Enzymes: i. Staphylocoagulase (Coagulase) - fibrinogen  fibrin ;
ii. ; Staphylokin (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 - folliculitis & furuncles, boils & carbuncles, bullous impetigo
b. Scalded skin syndrome , toxic shock syndrome, food Poisoning
B. Staphylococcus epidermidis
1. Virulence Factor: Exopolysaccharide “slime” 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
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____ - detects bound coagulase “clumping factor”.
Negative: No clumping. Positive: Macroscopic 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: zone diameter >16 mm Resistant: zone diameter ≤16 mm
2b. Gram (+) Streptococci

 Introduction: G(+) cocci in pairs / chains, Catalase (-), Aerotolerant anaerobes, Some are capnophilic
Species A. Lancefield B. 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, γ 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)
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, 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: Nosocomial infection, UTI, bacteremia, endocarditis
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 hemolytic
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 strep. & 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:___________
6. Salt Tolerance Test - Test the ability to grow in 6.5% NaCl; Positive -turbidity/color change (purpleyellow):________
7. Optochin (Taxo P) Susceptibility - Test for susceptibility to Taxo P; Positive - zone of inhibition:_____________
8. Bile Solubility Test - Test for solubility to bile salt (2% Na desoxycholate)
Positive - Colony disintegrates: ______________ , Negative - Colonies remains intact
1. β hemolytic Bacitracin PYR SXT CAMP/Hippurate
A S + R -
B R - R +
Not A/B R - S -
2. α hemolytic 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 hemolytic 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
A. 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
“pseudomembrane”) and 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: SBA (Narrow zone of β-hemolysis); i. Cystine-tellurite blood agar & Tinsdale's agar
(black colonies w/ brown halo); ii. Loeffler Serum / Pai Agar (produces metachromatic granules)
4. Toxigenicity Test – Elek Test
5. Identification
Characteristic C. diptheriae C. ulcerans C. pseudotuberculosis C. jeikeium
Tinsdale’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 and joint)
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
e. Umbrella-shaped or inverted christmas tree pattern into a semi-solid tube
f. CAMP Reaction: Positive rectangle “block” 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: Small colonies, β-hemolytic, pits agar, leaves black dot under
c. Identification: Lipase and Lecithinase positive (EYA), Exhibit CAMP inhibition reaction (phospholipase D)
C. Gardnerella vaginalis
1. Clinical Infections: Bacterial vaginosis (Excessive vaginal discharge, pH > 4.5 and foul smell) associated in UTI
a. Microscopy: Pleomorphic “Clue cells” in vaginal fluid.
b. Colony: ϒ-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 - - - - + γ,α -
A.haemolyticum - - - - - β -
G. vaginalis - - - - - γ +
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%)
b. Colony: β-hemolytic (SBA), Chalky, dry, crumbly (Sabouraud’s & Mycosel), 22C & 37C (3-6 d)
2d. Aerobic Gram Positive Bacilli (Spore-Formers)

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 (“medusa head”+_”), beaten egg white consistency (SBA);
large, mucoid colonies in bicarbonate agar; “string of pearls” in 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 zone of β hemolysis
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 gonococcal infection (DGI), endocarditis & arthritis
B. Neisseria meningitis
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, DIC, Waterhouse
Friderichsen syndrome)
C. Moraxella catarhallis
1. Virulence Factor: Attachment 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
Medium Inhibitory Agents Suppressed Organisms
Vancomycin G(+)
Thayer-Martin Colistin G(-)
Nystatin Yeast
Modified TM Trimethoprin Swarming of Proteus
Martin Lewis Anisomycin Yeast
New York City Amphothericin B Yeast
D. Incubation: 35°C (↑ CO2 , 72 hrs.)
E. Presumptive Identification:
1. Microscopy: G(-) diplococci
2. Colony Morphophology
a. N. gonorrhoeae and N. meningitides: Small, tan, translucent and raised
b. M. catarrhalis: Smooth & opaque. Colony can be swept intact (hockey puck), resembles wagon wheel (48 hrs)
F. Identification
Blood MTM, ML, Superoxol
Organisms Glucose Maltose Lactose DNase / TH
Agar NYC (30% H2O2 )
N. gonorrhoeae (-) (+) (+) (+) (-) (-) (-)
N. meningitidis (+) (+) (-) (+) (+) (-) (-)
M. catarrhalis (+) (-) (-) (-) (-) (-) (+)
Review Questions
4. Which of the following infections is commonly attributed to Staphylococcus aureus?
A) Scalded skin syndrome B) Toxic shock syndrome C) Food poisoning D)All of these
5 .Which reaction is correct for S. pneumoniae?
A) Optochin susceptible C) CAMP reaction positive
B) Bile-esculin hydrolysis positive D) Hippurate hydrolysis positive
6. Which statement incorrectly describes the mode of action of the antibiotic listed for modified Thayer-Martin media?
A) Vancomycin inhibits G(+) bacteria C) Anisomycin inhibits fungi and molds
B) Colistin inhibits G(-) bacteria D) Trimethoprim lactate inhibits swarming of Proteus
4a. Gram Negative Bacilli or Coccobacilli (Fastidious, MacConkey Negative)

Introduction
 Fastidious, 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 bacillus)
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 Sinusitis
Pericarditis Bacteremia
Pneumonia, Epiglotitis Pneumonia
2. H. aegyptius (Koch-Weeks bacillus) or H. influenzae bio. aegyptius: conjunctivitis “pink eye” & BPF
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 33°C w/ high humidity (≤7 days)
2. Microscopy
a. Coccobacilli & filamentous
b. H. ducreyi - coccobacilli 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 Rx__n: 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 Kovac’s rgt) and Porphyrins (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 Gram Negative Bacilli or Coccobacilli (Fastidious, MacConkey Negative)

I. Clinically significant species
A. HACEK Group: Dysgonic fermenter, normal biota of the oral cavity, oral infections, subacute bacterial endocarditis_
1. Aggregatibacter aphrophilus: “foam loving” needs ↑ conc. 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 - G/S); 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
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. Lab Diagnosis: Culture: Blood-cystine-glucose agar (require cysteine, cystine, thiosulfate), BSL-3
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: Legionnaire’s disease and Pontiac Fever
4. Specimen: Respiratory, body fluids, blood
5. Microscopy: Direct fluorescent Ab
6. Culture: Requires Iron & L-cysteine (BCYE), Ground-glass
H. Bordetella pertussis
1. Manifestation: Pertussis: catarrhal, paroxysmal, convalescent
2. Mot: droplets or direct contact w/ secretions
3. Lab Diagnosis: Bordet-Gengou, Regan-Lowe, Charcoal-horse blood (“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)
5a. Gram – Bacilli (Fermentative, MacConkey Positive, Oxidase Negative)

I. Enterobactericeae
 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
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 & traveler’s “Montezuma’s revenge”
2. Enterotoxigenic E. coli (ETEC)
(heat stable/labile) diarrhea or “turista”
Shigella like iinfection 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
Verotoxin
Verotoxic E. coli (EHEC) Hemolytic uremi syndrome Sorbitol (-) & MUG (-)
6. Enteroadheren E. coli
Diffusely adherent E. coli (DAEC) UTI & diarrhea
Enteroaggregative E. coli (EAEC) Diarrhea (watery)
B. Klebsiella (Friedlander) & Enterobacter
1. Pneumonia, UTI, septicemia, pneumonia, wound infections, etc.
2. Encapsulated, mucoid colonies that “string”.
3. Klebsiella (plasmid- mediated ESBL’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 − + +
B-C. 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 − +
D-E. Proteus, Morganella & Providencia
1. Septicemia, UTI, pneumonia, etc.
2. Rapid urease (+), Deaminase (+)
3. Proteus produces swarming motility
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
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
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 (+) (-)
6
Infectious Dose 10 100-200
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
2. Laboratory Diagnosis: Bipolar staining", bull’s eye colonies (CI_N, 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 of E. coli, Klebsiella, Shigella, Salmonella
B. Types of antigen:
1. O or somatic A_: Heat stable, LPS of the outer membrane, Endotoxin
2. K or envelope Ag: Heat labile (100C for 10 min), Polysaccharide, Vi Ag of Salmonella
3. H or flagellar A: In motile members. For Salmonella spp.
II. Selective Media for Enterobactericeae
A. MacConkey_ - 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. Hektoen Enteric Agar (HEA - 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 (SSA) - 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. Enrichment 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
a. Fermenter: Change in color in both tubes Enterobacteriaceae
b. Oxidizer: Change in color in open tube Pseudomonas
c. 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, Ferrous sulfate & Na thiosulfate (1st : Bacteria + Na thiosulfate
 H2S gas, 2nd : H2S gas + Ferric ions  ferrous sulfide - black precipitates), Phenol red
Reactions Possible Organism(s)
K/K
K/ A Citrobacter, Serratia, Providencia, Morganella
K/ A , H2S+ Citrobacter freundii, Proteus, Salmonella
A/ A Escherichia coli, Enterobacter, Klebsiella
A/A Enterobacter, 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 + MR  Red
 Positive: Bright red color (E. coli) ; Negative: Yellow color (Klebsiella and Enterobacter)
b. Voges-Proskauer Test: Glucose (Fermentation)  Acetoin + VP (α-naphthol & 40% KOH)  Red
 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 Cadaverin__e
b. Ornithine –Ornithine decarboxylase Putrescin__e
c. Arginine –Arginine dihydrolase Citrulline  Ornithin__e
 Positive: Alkaline (purple) , Negative: Acid (yellow)
B. Deaminase Test
 Determines capacity to deaminate phenylalanine to phenylpyruvic acid
 Positive: Green 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) ; Negative: no color change / yellow
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
K/A Escherichia coli, Enterobacter cloacae, Citrobacterspp., 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
a. Motile: Growth extending from line of inoculation
b. Indole positive : Pink to wine colored ring after adding Kovac’s
c. 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 (Fermentative, MacConkey Positive, Oxidase Positive)

I. Vibrio spp.
 Indications of Infection: consumption of raw seafood causing 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, reduce nitrate to nitrite , Oxidase (+), most species are susceptible to O/129 and
positive to string test and halophilic (Except V. cholera__e & V. mimicus)
A. Vibrio cholerae
1. Virulence Factor and Disease: Cholera toxin (Choleragen)- profuse watery diarrhea (rice water stools) leading to
dehydration and hypovolemic shock
• O Ag (01 & 013__9): Markers for strains capable of epidemic and pandemic spread
Biogroups Eltor Classical
Hemmaglutination (chicken RBC) Positive Negative
β-hemolysis in SBA
Voges-Proskauer Test
Susceptibility to Polymxin B (50 U)
B. Vibrio parahaemolyticus
1. Disease: Gastroenteritis “summer diarrhea”
2. Other Characteristics: Kanagawa toxin positive: β-hemolytic
C. Vibrio vulnificus
1. Disease: Septicemia & wound infection
2. Other Characteristics: Lactose positive Vibrio
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
 Virulence: Cytotoxic enterotoxin
 Disease: Intestinal (five diarrheal syndrome), Extraintestinal (wound nfections, septicemia & meningitis, etc.)
 Lab Diagnosis: β-hemolytic (SBA),Ferments Lactose, pink-centered(CIN)
III. Plesiomonas shigelloides
 Disease: Gastroenteritis, bacteremia, meningitis, etc.
 Lab Diagnosis: Pink in Inositol brilliant green bile salt agar; LDC, ODC, ADH “Positive 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, Negative: No viscous stringing)
C. Vibriostatic test: Reagent: 0/129 disks (Susceptible: zone of inhibition)
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 - - - +
Addt. Comments LDC, ODC, ADH
6b. Gram Negative Bacilli (Assacharolytic, Oxidase Positive)
I. General Characteristics of Campylobacter
 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
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 (Stuart’s , Cysteine-Brucella broth)
B. Culture Media and Incubation
1. Campylobacter spp: Butzler, Skirrow’s, 42˚C or 37˚C (Microaerophilic)
2. H. pylori: Skirrow’s, Chocolate or Brucella agar with 5% horse blood, 35-37˚C (Microaerophilic)
C. Microscopic Morphology
1. Campylobacter spp: S-shaped “wings of seagulls” and produces darting motility
2. H. pylori: Multiple flagella at one pole
D. Colony Morphology: Moist “runny” looking (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 (Oxidative, MacConkey Positive)

 Fail to acidify O-F Media overlaid w/ mineral oil, grow in Mac (colorless) , fail to acidify TSI, most isolates in oxidase (+),
resistance to a variety of antimicrobial agents (aminoglycosides & beta lactams)
 Contaminants in disinfectants, detergents, collection tube, venous catheters, ventilators, humidifiers, nebulizers, etc.
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)
iv. Inherently resistance to a number antimicrobial agents
3. Identifying Characteristic: Pigmented (Fluorescein/Pyoverdin, Pyocyanin, Pyorubin, Pyomelanin) ; Fruity grapelike
or corn tortilla-like odor (2-aminoacetophenone) ; Grow at 42°C and 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(+)
D.a. Burkholderia cepacia
1. Clinical significance: Pneumonia in patients with cystic fibrosis or chronic granulomatous disease.
2. Identifying characteristics: Dirtlike odor in BAP, ONPG +; dark pink in Mac in 4-7 d
D.b. Burkholderia pseudomallei
1. Clinical significance: “Melioidosis”
2. Identifying characteristics: Bipolar staining (G/S), wrinkled & deep pink in Ashdown media, “Earthy odor”
D.c. Burkholderia glandioli: Associated w/ patients w/ CF & CGD,
D.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
• E.g. Skin -> P. acnes; RT -> G(-) bacilli & cocci ; GIT -> G(-) bacilli & C. difficile ; Gut -> G(-) Bacilli
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: Reducing agent (thioglycolic acid, Na thioglycolate, cystine), 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 & Prevotela
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 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), desiccant, redox indicator
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 shape appearance)
4. Double zone of hemolysis
 alpha toxin (partially hemolyzed outer zone), theta toxin (completely hemolyzed inner zone)
5. Lecithinase positive (opaque zone around the colonies in Egg Yolk Agar), Nagler reactionn (Type A Toxin
demonstration), CAMP (Bow-tie) & Reverse CAMP (Arrow-head) positive
B. Clostridium tetani
1. Tetanospasmin - Neurotoxin
2. Tetanus – spastic type of paralysis with continuous muscular spasms.
Trismus (lockjaw), risus sarcodinicus (distorted grin) & backward arching of the back muscles
3. Swollen terminal spore, drumstic kappearance
4. Smoothly swarming but slow growing, narrow zone of β-hemolysis
C. Clostridium botulinum
1. Botulism toxin (neurotoxins) - flaccid type of paralysis
2. Foodborne 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-associate 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 (cyototoxin) and organism in feces
E. Clostridium septicum
1. Associated w/ malignancies (colorectal cancer), neutropenic enterocolitis and myonecrosis
2. Thin rods, subterminal spores
3. Resembles “Medusa head”, β-hemolytic, smoothly swarming
Double Zone Chartreuse Spore

Swarming Lecithinase Lipase
of hemolysis Fluorescence Position
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. Actinomycosis (sinus tracts, which erupt to the surface and drain pus that may contain “sulfur granules”),
2. Branching filamentous rods
3. White, opaque and resemble “molar toot”
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, end of cells may be spatulated or bifurcated “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 fluorescen__ce -> Veillonella parvula
9a. Spirochetes
I. Clinically Signifant Species
A. Leptospires
1. Characteristics: Tightly coiled, thin, spirochetes w/hooked ends; culturable to Fletcher’s, Stuart; visible by dark-
field, phase contrast & immunoflourescence microscopy
2. Leptospira interrogans: Acquired trough contact with urine of animals (e.g. rodents) who carry the organism.
Leptospirosis and Weil disease (systemic disease with renal & hepatic failure)
B. Borreliae
1. Characteristics: Less tightly coile_d (3 -10 coils); culturable to Kelly medium; visualized by bright-field microscopy
2. Borrelia recurrentis: Endemic (tickborne) and epidemic relapsing fever (louse-borne)
3. Borrelia burgdorferi: Lyme disease (1. Localized, 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 chancre (primary), condylomas (secondary),
gummas, neurosyphilis (tertiary) and; ii. congenital syphilis.
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)
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 Ticks
• South African, Israeli spotted fevers
• 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. Giemsa, Wright’s stained buffy coat Murine typhus + V –
4. Weil-Felix rxn. (P. vulgaris OX-19, OX-2, P. mirabilis OX 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 urethratis, 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: Mycoplasm colonies growing on SP4 Agar 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 and 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
 Primary / Reactivation: Tubercles or granuloma (granulomatous lesions from multinucleated cells) and
Caseation (cheese like masses from break down of tubercles) in lungs
 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: resembles “water droplets” in Middlebrook
c. ScreeningTest: Tuberculin Skin Testing (Mantoux test, Pirquet test, PPD test)
 Positive: erythema (redness) and induration (firmness) in 48-72 hours
ii. Nontuberculosis mycobacteria (NTM’s)
A. Photochromogens – Unpigmented when grown in the dark and develop pigment after light
a. M. asiaticum
b. M. kansasii: “Yellow bacillus”, swimming pool granuloma, 2nd 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: Grows best 42°C, “Bird’s nest” in cornmeal 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: Chronic diarrhea (e.g. Johne’s & Crohn’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
v. Noncultivatable
a. M. leprae - Hansen’s Disease
 Tuberculoid Leprosy - Skin lesions and peripheral nerve involvement
 Lepromatous leprosy - Extensive skin lesions and symmetric nerve damage
iii. Laboratory Diagnosis

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
E. Media & Isolation Methods
1. Solid Media: 35°C, 5-10% CO2 ,↑ humidity
i. Egg Based (18-24 d) - Lowenstein-Jensen and Petragnani (↑ malachite green)
ii. Agar based (10-12 d) - Middlebrook 7H10 & 7H11 and Mitchison’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
M. bovis
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. Rifampi: 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 mobilization of donor bacterium’s chromosome, plasmid, or
transposon via sex pili
11b. Antimicrobial Susceptibility Testing

I. Introduction
 Determine whether the organism is capable of expressing resistance and the extent of its acquired resistance.
II. Disk diffusion Testing Methods
Variable Standard
Inoculum Disk diffusion: 1.5 x 108 CFU/mL
Broth microdilution: 5 x 105 CFU/mL
Agar dilution: 1x104 CFU/mL
Formulation Mueller-Hinton
2+ 2+
Ca , Mg content 25 mg/L Ca2+, 12.5 mg/L Mg2+
Thymidine content Minimal or absent
pH 7.2-7.4
Agar depth 3-5mm (4 mm)
Incubation
Atmosphere Humidified ambient air
Temperature 35°C
Length Disk diffusion: 16-18 hr
Others
Colonies Sampled from isolates 4-5
Distance ≥20 mm apart
Maximum stacks Maximum of 4
MYCOLOGY
Objectives
1. Describe the general characteristics and structures
2. Describe the four divisions
3. Enumerate and describe disease and its associated genus/species
4. Enumerate and describe lab methods for diagnosis
Ia General Characteristics
A. Yeasts: Unicellular, forms a bacterial-like colony. Reproduce by budding
B. Molds: Multicellular and has woolly appearance in culture. Made up of mycelium (intertwining structures of hyphae).
1. Parts of Hyphae (tubelike structure, fundamental units of fungi)
a. Aerial (reproductive).
• Extends above the surface
• Produce and supports reproductive structures (conidia or spores)
• Projects above the surface of the medium
b. Vegetative (thallus.
• Extends downward into the medium
• Absorbs water and nutrients
2. Types of Hyphae
a. Septate: With frequent crosswalls
b. Sparsely septate (aseptate): Few cross walls
3. Structures associated to Hyphae
a. Conidiophore / Sporangiophore.
• Hyphal branch that produces and acts as stalks for conidia / sporangium
b. Conidia / Sporangium.
• Asexual structures that form in the sides of hyphae or conidiophores / sporangiophore
c. Phialide / Annellide.
• Secondary segments born from conidiophore (flask or vase-shaped)
• Produces conidia with (annellide) or without (phialides) increasing in length
d. Vesicle / Columella
• Enlarged or dome shaped structure at the tip of conidiophore / sporangiophore
4. Other Hyphael Form
a. Spirals: Corkscrew like hyphae (Trichophyton mentagrophytes)
b. Nodular bodies: Knot of twisted hyphae (Microsporum canis & T. mentagrophytes)
c. Racquet: Enlarged, club shaped (Epidermophyton floccosum)
d. Pectinate body: “Broken comb” (M. audouinii)
e. Favic Chandelier: Antler hyphae (T. schoenleinii and T. violaceum)
5. Hyaline versus Dematiaceous hyphae
a. Hyaline (Moniliaceous): Nonpigmented or lightly pigmented
b. Dematiaceous: Darkly pigmented due to melanin in the cell wall
C. Dimorphism and Polymorphism

a. Dimorphism (Dimorphic Fungi: Ability to exist in two forms)
• Yeast or Spherule phase: When grown at 37°C with increased CO2
• Yeast (Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis,
Penicillium marneffei, Sporothrix schenckii)
• Spherule: Large, round structure that contains spores (Coccidioides immitis)
• Mold phase: When grown at 25°C with ambient air
b. Polymorphism (Polymorphic Fungi).
• Have both yeast and mold forms in the same culture
D. Reproduction
• Blastic conidiogenesis: : parent cell enlarges, a septum forms and an enlarge portion splits off to form a daughter cell
• Thallic conidiogenesis: a septum forms first, and the new growth beyond the septum become the daughter cell
1. Asexual: Results in the formation of conidia. Forms conidia from hyphae of one organism
a. Macroconidia - large and multicelled (Microsporum, Trichophyton, Epidermophyton)
b. Microconidia - small and unicellular
c. Blastoconidia (blastospores)__ - Developed as the daughter cell buds from mother cell, hyphae or pseudohyphae
i. Blastomyces, Histoplasma, Paracoccidioides, Penicillium, Sporothrix
ii. Candida albicans, Geotrichum candidum, Trichosporon beigelii
iii. Cryptococcus neoformans
d. Chlamydoconidia (chlamydospores) - Thick, walled, resting spores produced by “rounding up” and enlargement of
terminal hyphal cells. Terminal (tip), sessile (sides) and intercalary (within)
i. M. audouinii
ii. P. brasiliensis
iii. C. albicans
e. Arthroconidia (arthrospores) - Fragmentation of the hyphae into barrel- or rectangular- shape spores
i. Coccidioides
ii. Geotrichum
iii. Trichosporon
f. Sporangiospores - Produced at tip of sporangiophore
i. Zygomycetes: Absidia, Mucor, Rhizopus
2. Sexual: Results in the formation of spores. Formed by merging of cell and fusion of nuclei from two mating strains
a. Ascospores (Sac Fungi) - contained in a saclike ascus. In molds with septate hyphae
b. Zygospores (Conjugation Fungi) - large spore enclosed in a thick wall from fusion two identical cells. In molds with
aseptate hyphae
c. Basidiospores (Club Fungi) - Spores produced on a basidium. In molds with septate hyphae and clamp connections
d. Oospore - Fusion of two separate non identical cells
3. Phases of Reproduction
a. Teleomorph - If the fungi reproduce sexually
b. Anamorph - If the fungi reproduce asexually
c. Synanamorphs - If more than 1 anamorph is present for the same teleomorph
Ib Taxonomy
A. Zygomycota
• Aseptate (Sparsely septate)
• Presence of Sporangiophore and Sporangium (contains Sporangiospores)
• Mucor, Rhizopus and Absidia
B. Ascomycota
• Septate , Presence of Ascospores
• Yeast (Saccharomyces)
• Mold (Piedra, Microsporum, Trichophyton, Pseudoallescheria, Coccidioides, Blastomyces, Histoplasma)
C. Basidiomycota
• Septate w/ clamp connections
• Presence of Basidiospores
• Filobasidiella neoformans
D. Deuteromycota
• Fungi Imperfecti
• No mode of sexual reproduction
• Largest number of species
Ic. Clinically Significant Species

A. Superficial Mycoses
• General Characteristics: Affects the outermost layer (stratum corneum) of the skin or hair
1. Name of agent
a. Name of Disease and Clinical Manifestations
b. Laboratory Diagnosis (direct microscopy skin scrapings or hair shaft)
1. Malassezia furfur
a. Tinea versicolor (pityriasis versicolor): brownish (fawn), scaly patches if light skinned or pale, nonpigmented,
untanned patches if dark-skinned
b. Tight clusters of spherical, budding yeast cells with hyphal segments described as “spaghetti and meatballs “
2. Hortaea werneckii
a. Tinea nigra: Black to brown scaly macules in the palms of the hand and soles of the feet
b. Laboratory Diagnosis: Dark one- to two-celled blastoconidia or annelloconidia is seen
3. Piedraia hortae
a. Black Piedra: Hard, brown-black crusts or nodules on the outer side of the hair shaft
b. Dark, thick walled hyphae with swellings that is ascoscarp in appearance
4. Trichosporon beigelii complex
a. White piedra: Light-brown, soft nodules on the scalp, pubic, beard, etc. T. ovoides (scalp) T. inkin (pubic)
b. Hyaline hyphae, blastoconidia and arthroconidiaa when grown on cornmeal-Tween 80 agar
B. Cutaneous Mycoses
1. General Characteristics: Agents of dermatophytoses; hyaline, keratinophilic (hair, nails, skin) & monomorphic molds
2. Infections
i. Scalp : Tinea favosa Trichophyton schoenleinii
Tinea capitis: Gray-patch ringworm Microsporum spp.
Black-dot ringworm Trichophyton spp.
ii. Beard: Tinea barbae Trychophyton spp.
iii. Body: Tinea corporis Trychophyton spp., Microsporum spp.
iv. Groin: Tinea cruris Epidermophyton sp.
v. Feet: Tinea pedis (Athlete’s / Moccasins Foot) Trychophyton spp., Epidermophyton sp.
vi. Nail: Tinea unguium (Onychomycosis) Trychophyton spp. Epidermophyton sp.
Ringworm Site Affected Agent

Tinea capitis Head (hair) Microsporum Trichophyton
Tinea corporis Body (skin) Microsporum, Trichophyton, Epidermophyton
Tinea unguium Nails Trichophyton, Epidermophyton
Agents and Diagnosis
1. Epidermophyton floccosum :
a. Numerous club-shaped , smooth-walled macroconidia with 2 to 5 cells occurring singly or in clusters of 3 to 4
2. Microsporum canis:
• Thick-walled, spindle shaped, multiseptate macroconidia with spiny surfaces and curved tip
3. Microsporum gypseum :
• Numerous, thick walled, cigar-shaped multiseptate macroconidia with spiny surfaces and rounded tips
4. Microsporum audouinii :
• Bizarre-shaped macroconidia: thick walled, club or spindle shape with rough surface. Produces chlamydospore
Species Growth in Rice Grains Fluorescence in Woods Lamp

M. audouinii Poor (Brown discoloration) Positive
M. canis Good Positive
M. gypseum Good Negative
5. Trichophyton mentagrophytes.
• Microspores: numerous, globe-shape, and arranged in grapelike clusters; coiled spiral hyphae is observed
6. Trichophyton rubrum.
• Tear-shaped microconidia borne laterally from hyphae; thin-walled, smooth, pencil-shaped macroconidia
7. Trichophyton tonsurens.
• Abundant tear-, club-, or ballon-shaped microconidia; rare smooth-walled, cylindrical macroconidia
8. Trichophyton schoenleinii.
• Rare conidia (sterile hyphae); characteristic moose antler “favic chandelier” hyphae may be observed
9. Trichophyton violaceum.
• Sterile tortuous hyphae, “favic chandelier” hyphae may be observed
Species Thiamine Requirement Urease Hair Baiting
Trichophyton mentagrophytes Negative Positive (2 d) Positive
Trichophyton rubrum Negative Negative (7 d) Negative
Trichophyton tonsurans. Positive Negative (4 d) Negative
C. Subcutaneous Mycoses
General Characteristics: Result from traumatic puncture of thorns or vegetation contaminated with fungi
2. Name of agent
c. Name of Disease and Clinical Manifestations
d. Laboratory Diagnosis (direct microscopy skin scrapings or hair shaft)
1. Chromoblastomycosis
• Verrucous dermatitidis and chromomycosis manifesting as hard, warty, tumor like cutaneous lesion
• Caused by dematiaceous fungi
• Diagnosed by the presence of sclerotic bodies (copper pennies)
a. Phialophora verrucosa.
• Septate hyphae with short conidiophores that give rise to flask- or cup-shaped phialides;
with collarettes; oval to cylindrical conidia occurs in balls or clusters at ends of phialides.
b. Cladophialophora carrionii.
• Septate hyphae with erect, long-branching conidiophores that give rise to chains of conidia
c. Fonsecaea pedrosoi .
• Mixed sporolation; dark, septate hyphae with primary conidia developing at condiophore tip.
Secondary and tertiary conidia are also formed and result in a loosely organized conidial head.
2. Mycetoma
• A granulomatous infection of subcutaneous tissues with abscesses, draining sinuses and granulomatous pus
a. Pseudoallescheria boydii (Scedosporium apiospernum: Anamorphic form)
• A saclike cleistothecia containing asci and ascospores, which are oval and pointed
b. Exophiala jeanselmei
• Septate hyphae with conidiophores forming cylindrical annelids; conidia gather at tips of annelids
3. Phaeohyphomycosis.
• A superficial, cutaneous, subcutaneous or systemic infection caused by any dematiaceous fungi
a. Alternaria
• Septate, with conidiophores that may branch with chains of conidia, which are muriform and tapered
b. Bipolaris
• Septate, with hyphae that are branching and conidiophores with multicelled, oblong to cylindrical conidia
c. Culvularia
• Septate, with conidiophores twisted at point of attachment to a curved, swollen, multicelled conidia
4. Sporotrichosis
• Infection associated to gardening, exposure to rose thorns (rose-handler’s disease) and sphagnum moss
a. Sporothrix schenckii
• Narrow, septate hyphae with pyriform conidia arranged in rosette floweret or sleeve arrangement
• Small, budding, yeast cell that may radiate an eosinophilic star-like projection (asteroid body) on biopsy
D. Systemic Mycoses
General Characteristics: Dimorphic: mould (22-30°C) or yeast (35-37°C)
1. Ecology and Disease
Species Ecology Disease / Manifestations
B. dermatitidis River valleys and basins, soil Gilchrist, Chicago
H. capsulatum Bird, bat guano, alkaline soil Cave, Spelunker’s, Darling
C. immitis Soil, Semiarid regions Desert bumps, Valley fever, Desert rheumatism
P. brasiliensis Soil S. American Blastomycosis, Lutz-Splendore-Almeida
2. Clinically Significant Species
Species 22°C (Mold Phase) 37°C (Tissue Phase)

Septate hyphae with round or
Thick-walled, large yeast cells with
1. Blastomyces pyriform conidia borne on
single bud on a broad base; broad
dermatitidis conidiophores or directly on hyphae
isthmus at constriction
resembling “lollipops”
Septate, branched hyphae that
Large, round thick walled spherules with
2. Coccidioides produce barrel shaped, arthroconidia
endospores observed in tissue and direct
immitis that alternate w/ empty disjuncter
examination
cells
Septate hyphae with large round, Small, budding; round to oval yeast cells;
3. Histoplasma
thick walled echinulate, tuberculate intracellular to macrophages possible
capsulatum
macroconidia on hyphal stalk with Giemsa or Wrights stain
Small, septate, branched hyphae with Large, round oval, yeast cells with
4. Paracoccidioides intercalary and terminal multiple buds attached to mother cell by
brasiliensis chlamydoconidia; few pyriform narrow constrictions. “Mariner’s wheel”
macroconidia “Mickey Mouse cap”
E. Oppurtunistic Mycoses
3. General Characteristics: Saprophytes and Opportunistic
1. Zygomycetes (Aseptate)
Absidia Sporangiophore arise between nodes from which rhizoids are formed
Mucor Non septate hyphae with no rhizoids formed
Rhizopus Sporangiophores clusters in a stolon. Rhizoids is at the base of sporangiophores
2. Septate and Hyaline (Septate)

Hyphae that terminate in a conidiophore and expands into a vesicle.
Aspergillus
Vesicle is covered with phialides
F. Yeast and Yeastlike Fungi

4. General Characteristics: Unicellular, budding & round to oval organisms
1. Ecology and Disease
Species Ecology Disease / Manifestations

Cryptococcus Pigeon, bat droppings Systemic
neoformans Decaying vegetation Meningitis
Candida GI tract Thrush, vulvovaginitis
albicans Mucus membranes Diaper rash, onychomycosis, Paronychomycosis
Geotrichum Soil
Oral, lung, skin, etc.
candidum Decaying foods
Species Laboratory Diagnosis
a. Cryptococcus Direct Examinations: Round to oval yeast w/ capsule & narrow-base budding
neoformans Colony (Niger Seed Agar) : Phenol oxidase (+), Brown-black colonies
Direct Examination: Blastoconidia (budding yeast / pseudohyphae)
b. Candida albicans Cornmeal (RT, 24-48 hrs): Chlamydoconidia
Serum (35-37ᵒC, 1-3 hrs): Germ tubes, hyphalike extension of yeast cell
Direct examination: Rectangular yeast cell (in pairs / chains)
c. Geotrichum candidum
Cornmeal: Fragmented hyphae, Rectangular arthrospores with rounded ends
Species Capsules Germ Tubes Blastoconidia Arthroconidia Chlamydoconidia

Cryptococcus neoformans + +
Candida albicans + + +
Geotrichum candidum + +
3a. Laboratory Diagnosis

1. Safety Issues
2. Specimen Collection
Agent Specimen
Respiratory
Subcutaneous Tissue
Blood / BM / CSF
Hair, Skin and Nails
Throat, Urine, Vaginal, Cervical
Specimen Collection Notes Specimen Collection Notes
Primary Isolation Media Primary, Isolation Media
Pulled or cut (forceps and scissors) Biopsy or Needle Aspiration
A. Hair Wood’s Lamp E. Abscess & Lesions Sulfur granules
KOH & Culture (SDA Homogenized, Culture (SDA, BHI)
Cleansed with 70% alcohol 3 consecutive early morning
B. Skin & Nails Scrap outer edge / discolored F. Sputum Giemsa/India Ink/
KOH & Culture (SDA) Culture (SDA, BHI)
Lysis centrifugation 3 consecutive early morning
C. Blood & BM Culture (SPS/BHI) G. Urine Clean-catch midstream
Wrights and Giemsa Culture (SDA)
Concentration H. Throat, Vaginal 2 swabs
D. CSF
India Ink, Culture (BHI) Cervical KOH/Culture (SDA)
3. Direct Examination
Method Application Method Application (Tissue Stains)
A. KOH (10-20%) Dissolves keratinin skin, Polysaccharides
E.i. PAS
hair & nails (purplish-red, pink)
B. KOH w/ Bind chiti_n Melanin
E.ii. Fontana-Masson:
Calcofluor white Flouresce blue white (brown)
C. India Ink / For Encapsulated ye_ast E.iii. Grocott
Black
Nigrosin (C. neoformans) in CSF methenamine-silver
D. Lactophenol
Fungal morphology Lactic E.iv. Gram/Giemsa/
Cotton Blue Purple or Purple blue
acid, phenol, aniline blue Wrights Stain
(Aman Medium)
4. Isolation Methods
1. Growth Requirements
i. Nutrients and Moisture: Nitrogen, carbon, vitamins & minerals
ii. Temperature: 25ᵒC / 30ᵒC (mold) or 37ᵒC (yeast)
iii. Time: 2-4 weeks (mold), 2-3 d (yeasts)
B1. Primary Isolation B2. Differential Test

Application Application
Media Media
C. neoformanss
i. Brain-Heart Infusion i. Birdseed (Niger seed)
For Detects phenol oxidase
Agar (BHI) agar
general isolation of (Black-brown colonies)
pathogenic & Candida spp
ii. Saborauds Dextrose ii. Cornmeal agar w/
saprophytic fungi Stimulates conidia &
Agar (SDA) Tween 80
chlamydospore production
For isolation of B. dermatitidiss
iii. BHI-CC pathogenic fungi iii. Cottonseed agar Induces conversion of mold to
Cycloheximide: : yeast
iv. Mycosel or inhibits saprophytes Induces pigment production
Mycobiotic Chloramphenicol: iv. Potato dextrose agar of T. rubrum
(SDA-CC ) inhibits bacteria Stimulates conidia production
With CC plus
v. Dermatophyte Test Differentiation of
Phenol Red : v. Rice Medium
Medium or Pfizer Microsporum spp
Dermatophytes (red)
Differentiate of T. rubrum &
vi . Urea Agar
T. mentagrophytes
7 agars w/ casein or NH4 nitrate
vii. Trichophyton agars
base but different enrichment
Species identification of
viii. Czapek’s
Aspergilli
C. Macroscopic Examination 5. Examination of Growth

i. Growth Rate A. Tease Mount
ii. Topography Reverse Side (flat, heaped, folded, B. Cellophane tape mount
wrinkled, umbonate) C. Slide Culture
iii. Texture - Length of aerial hyphae (cross sections)
(Wolly/cottony; velvety/silky; powdery/granular;
glabrous/smooth)
VIROLOGY
Objectives
1. Describe the general characteristics and structures
2. Enumerate and describe disease and its associated genus/species
3. Enumerate and describe lab methods for diagnosis
Ia. Introduction
A. General Characteristics: Obligate intracellular parasites, cannot multiply by binary fission, cannot generate ATP, lack
ribosomal RNA, size of 20-250 nm
B. Structure:
1. Components
1. Virion (nucleocapsid) - virus particle
2. Capsid - protein coat, composed of capsomer (protein subunit)
3. Nucleic acid: DNA or RNA
4. Envelope: Phospholipid bilayer with adhesion molecules (spikes)
5. Enveloped nucleocapsid: with envelope
6. Naked nucleocapsid: without envelope
2. Capsid Morphology: 1. Helical (rodlike); 2. Icosahedral (cubic); 3. Complex
3. Nomenclature
1. Family end in –viridae (E.g. Herpesviridae)
2. Genus end in –virus (Simplexvirus)
3. Species - Common names (Human Herpes Virus 1, Human Herpes Virus 2)
4. Taxonomy: a. Type of Genome; b. Strandedness of genome; c. Capsid Morphology; d. Presence & absence of envelope
5. Viral replication
1. Absorption: attachment of the virus to the host cell receptor
2. Penetration: injection (naked), fusion and endocytosis (enveloped)
3. Uncoating: virus loses its capsid exposing the viral nucleic acid
4. Eclipse Stage: replication and expression of the genetic material
5. Assembly: genetic material combines with the protein coat
6. Release: lysis (naked) and budding (enveloped)
Ib. Laboratory Diagnosis

A. Specimen Collection
1. Early stage of infection 4. Swab (dacron, rayon, cotton)
2. Sample in the infected site 5. Aspirates (w/o transport media)
3. Use transport medium (swab, tissue) 6. Store at 4°C (short), -70°C (long term)
B. Specimen Collection
Body system Disease/manifestation Specimen Common agents
Croup, Bronchiolitis Nasal aspirate, NP/throat Influenza, PIV, RSV, HSV
Respiratory tract
Pneumonia swabs, BAL Picornaviridae, Rotavirus, Adenovirus
Rotavirus, Norwalk,
Gastrointestinal Gastroenteritis Stool, Rectal swab
Adenovirus 40 & 41, Enterovirus
Lesions & HHV1-3, Adenovirus, Rubella,
Cutaneous Vesicle aspirate, lesion swab
Exanthems (rashes) Enterovirus, Rotavirus
Conjunctivitis Conjunctival scrapings, HSV, Adenovirus,
Ocular
Keratitis Corneal swabs Coxsakievirus, Enterovirus
Encephalitis CSF, HSV, Arbovirus
CNS
Aseptic meningtis NP swabs Enterovirus, Mumps
Urethritis, etc.
Genital Vesicle aspirate/ swab HSV
Penile lesions
Lesions & Throat, Urine CMV, HSV
Congenital
Exanthems (rashes) Serum Rubella
C. Methods of Diagnostic Virology

1. Direct Detection
a. Cytopathic Effects (CPE) -
Alteration, disruption Cowdry Type A HSV & VZV
Intranuclear inclusions
or damage to the cells Owl eyes CMV
resulting from virus Cytoplasmic inclusions Negri bodies Rabies
infection Nuclear enlargement Koilocytosis HPV
b. Immunoflourescence - Specimen is stained with fluorescein-labeled antibody then observed under fluorescence
microscope (E.g. Direct Fluorescent Antibody, DFA)
2. Virus isolation
• Viral replication are detected by Cytopathic Effects (CPE) , rounding, clumping, vacuolation, granulation, giant
multinucleate cell formation, syncytial formation, cell destruction or lysis or Immunoflourescence
a. Cell Culture
1) Primary Cell Cultures - Have the same karyotype 3) Continuous, Heteroploid or immortal cell lines -
and chromosome number as the original tissue <75% of cells have the same karyotype as normal cells
Human embryonic kidney HEK Human cervical carcinoma HeLa
Rabbit embryonic kidney RK Human laryngeal carcinoma Hep-2
Primary monkey kidney PMK Nasopharyngeal carcinoma KB
Rhesus monkey kidney RMK Human lung carcinoma A-549
Cytomolgus monkey kidney CMK AGMK Vero
African green monkey kidney AGMK
2) Diploid Cell line - A subcultivated primary cell culture, ≥75% of cells have the same karyotype as the normal cells
Human embryonic lung WI-38, MRC-5 Human diploid fibroblast HDF’s
Ic. Outline (Clinically Significant Species)
dsDNA ssDNA
Enveloped Naked Naked
Icosahedral Complex Icosahedral Icosahedral
1. 2. 3. 4. 5.
Herpesvirdae Poxviridae Papilloma Adenoviridae Parvoviridae
viridae
Page 36
ssRNA dsRNA
Enveloped Naked Naked
Helical Icosahedral Icosahedral
Icosahedral
1. Orthomyxoviridae 8. 11. 13.

2. Paramyxoviridae Flaviviridae Picornaviridae Reoviridae
3. Arenaviridae 9. 12.
4. Bunyaviridae Retroviridae Caliciviridae
5. Coronaviridae 10.
6. Filoviridae Togaviridae
7. Rhabdoviridae
Id. Clinically Significant Species
A. Double-stranded DNA viruses
1. Herpesviridae: dsDNA, Enveloped, Icosahedral, Latency and lifelong persistence
Species MOT Disease Specimens for Culture

Oral Herpes (gingivostomatitis & fever
Aspirates (vesicles & lesions),
Herpes Simplex Contact to blister), Genital Herpes, Neonatal Her-
Conjunctival scrapings
Virus 1 & 2 ulcerations pes, Herpetic whitlow, Herpes Encepha-
CSF
litis, Ocular Herpes (Herpes Keratitis )
Droplet inhalation,
Varicella-Zoster Varicella (Chickenpox)
Direct contact with Lesions (vesicles)
Virus Zoster (Shingles)
lesions
Infectious Mononucleosis
Epstein-Barr Oral contact w/ Atypical Lymphocytes (B cells)
Burkitt’s lymphoma, Hodgkin disease
Virus saliva Serum (Paul-Bunnell)
Nasopharyngeal carcinomaa
Close contact w/ Disseminated
Cytomegalovirus
secretions & Infant (Congenital CMV) Urine, blood,
“Salivary gland
excretions, blood & Transplant Patient (40 day fever) body fluids & tissues
virus”
organ transplants HIV Patient
Human Herpes- Close contact w/ Roseola Infantum, Exanthem subitum
T cells
virus 6 & 7 saliva Sixth disease
Human
Sexual contact Kaposi Sarcoma Noncultarable
Herpesvirus 8
2. Poxviridae: dsDNA, Enveloped, Complex
Species MOT Disease Others
Direct Extinct in nature
Variolla Smallpox
inhalation Vaccinia (vaccine strain)
Page 37
3. Papillomaviridae: dsDNA, Naked, Icosahedral

Laryngeal Wart (Papillomatosis)
Human
Close contact Venereal Wart (Condylomata acuminate) Koilocytes (Pap Smears)
Papilloma Virus
Cervical Carcinoma
4. Adenoviridae: dsDNA, Naked, Icosahedral, Posses a long fiber
Epidemic of gastroenteritis (serotype 40 &
Fecal-oral
41) in young children in warmer months
Secretions, CPE (swollen in
Adevinovirus Respiratory infection (Pneumonia)
aerosols, Close grapelike clusters)
Acute respiratory disease (serotype 14)
contact
Epidemics of keratoconjunctivitis
B. Single-stranded DNA viruses
5. Parvoviridae: ssDNA, Enveloped, Icosahedral
Erythema infectiosum, Fifth disease
Contact with
Parvovirus B19 Transient aplastic crises (anemia) Smallest DNA virus
secretions
Hydrops fetalis
C. Single-stranded RNA viruses
1. Orthomyxoviridae: ssRNA, Enveloped, Helical
Species MOT Virulence Factor Disease Lab Diagnosis
Hemagglutinin (16): Culturable to Embryonated
Influenza Aerosols Zoonotic (Birds & mammals)
mediates Attachment chicken eggs
virus Respiratory Respiratory inf.
Neuraminidase (9): PMK (Hemadsorption &
droplets (pneumonia/common colds)
mediates Entry Hemagglutination)
2. Paramyxoviridae: ssRNA, Enveloped, Helical
Species MOT Disease Diagnosis
Respiratory PIV-1 & 2: Croup in children (1°)
Parainfluenza
secretions PIV-3: Bronchiolitis & pneumonia infants (2°) PMK (Hemadsorption)
virus
Aerosols PIV-4: Mild upper RT infections
Saliva & swabs of Stensen’s duct
Mumps
Mumps virus Saliva droplets Hemadsorption &
Sterility
Hemmaglutination inhibition (+)
Measles (Rubeola) Spindle / multinucleated cells
Measles virus Aerosol
Pneumonia & encephalitis (SSPE) (PMK)
Large particle Lower RT infections (Croup, bronchitis,
RSV and hMPV Syncytia in HEp2 (RSV)
droplets interstitial pneumonia) in infants (RSV -1°)
3. Arenaviridae: ssRNA, Enveloped, Helical
LCM, Febrile illness, Hemmorhagic fever,
Rodent borne Arena (sand)
Lassa Virus, etc. Encephalitis
4. Bunyaviridae: ssRNA, Enveloped, Helical
LaCrosse, etc. Arboviral Febrile illness, Hemmorhagic fever,
Hantavirus Rodent borne Encephalitis
5. Coronaviridae: ssRNA, Enveloped, Helical, Club shaped projections
Direct contact, Cold-like infection in adults, EM and molecular methods
Corona virus
droplet or Pediatric diarrhea Treatment:
(SARS-CoV)
airborne SARS (respiratory distress syndrome) UV/Formalin inactivated vaccine
6. Filoviridae: ssRNA, Enveloped, Helical, Club Shaped projections
Page 38

Close Contact
Lake Victoria Rarely cause human infections
with secretions Margburg hemmorhagic fever
Marburg & Ebola but high mortality rates
and excretions Ebola hemorrhagic fever
(EBO-Z, -S, -R) Isolation, PCR and IF
(E.g. monkeys)
7. Rhabdoviridae: ssRNA, Enveloped, Helical
Species MOT Disease Laboratory
Bite of infected Flulike symptoms (prodromal) -> Isolation and IF (Negri bodies)
Rabies virus
animal Fatal encephalitis from the brain of the animal
D. Single-stranded RNA viruses

8. Flaviviridae: ssRNA, Enveloped, Icosahedral, Zoonotic (Arboviral)
Species Vector Virulence Factor and Disease Endemicity
Japanese Most common cause of viral encephalitis in
East, South and Southeast Asia
Encephalitis Virus the world
Dengue Fever (break-bone fever),
Dengue Virus A. aegypti and Hemorrhagic Fever & Shock Syndrome Tropical and subtropical
(DEN 1-4) A. albopictus Thrombocytopenia, hemorrhage, countries
plasma leakage and Shock
Yellow Parts of Africa and South
A. aegypti Systemic Toxic Phase (Jaundice)
Fever Virus America
St. Louis
Culex spp. Meningoencephalitis USA
Encephalitis Virus
West
“classic WNV infection”
Nile Virus
9. Retroviridae: ssRNA, Enveloped, Icosahedral, Requires Reverse Transcriptase (RNA DNA)
Virulence Factor and
Species MOT Others
Disease
Sexual contact, Injection
(Drug Use), Pregnancy, Screening:
Human Acquired Immune
Childbirth and breast Anti-HIV Ab
immunodeficiency Deficiency Syndrome
feeding, Occupational, Western Blot Assay
virus (HIV) (AIDS)
Blood transfusion and gp120/gp160, gp 41, p31, p24
organ transplant
10. Togaviridae: ssRNA, Enveloped, Icosahedral
Eastern-, western-,
Nonspecific febrile illness
Venezuelan- Mosquito-borne
Encephalitis
Equine Encephalitis
German measles
Rubella virus Droplet inhalation Congenital rubella May lead to fetal death and
syndrome spontaneous abortion
11. Picornaviridae: ssRNA, Naked, Icosahedral, Smallest virus
Species MOT Disease Others Manifestations
Aerosols,
Poliovirus Polio (Paralysis) Exanthems, aseptic meningitis
fecal-oral & fomites
(in summer months), pharnygitis
Coxsackie-, Entero-, Prevalent overcrowded Diarrhea
and pneumonia
Echo-, Parechovirus areas w/ poor hygiene Hand, foot and mouth dse.
Rhinovirus Aerosols and fomites Common cold Acid labile
Page 39
12. Caliciviridae: ssRNA, Naked, Icosahedral

Norwalk
Food-borne, water-borne, Most common in the US
“small round Gastroenteritis (outbreaks)
Person-to-person Feces, EM (non culturable)
structured virus”
13. Reoviridae: dsRNA, Naked, Icosahedral
Most common cause of
Fecal-oral route Double-shelled resembling
Rotavirus Gastroenteritis in infants
Food-, waterborne a wheel
and children
Page 40

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MICROBIOLOGY REVIEW HANDOUTS

1a. Bacterial Structure

2b. Host-Microorganisms Interactions

1c. Control of microorganism

1d. Clinical Laboratory Safety

1e. Specimen Collection & Processing

C. Specimen Receipt and Processing

E. Terminology for Direct Examinations

1g. Principles of Traditional Cultivation

2. Temperature, pH & Moisture

1h. Phenotyping Scheme

2a. Gram (+) Staphylococci

III. Laboratory Diagnosis

2b. Gram (+) Streptococci

2a. Aerobic Gram Positive Bacilli (Catalase Positive)

2c. Aerobic Gram Positive Bacilli (Catalase Negative)

2c. Aerobic Gram Positive Bacilli (Branching Actinomycetes)

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

3a. Gram (-) Diplococci

4a. Gram Negative Bacilli or Coccobacilli (Fastidious, MacConkey Negative)

4b. Other Gram Negative Bacilli or Coccobacilli (Fastidious, MacConkey Negative)

5a. Gram – Bacilli (Fermentative, MacConkey Positive, Oxidase Negative)

III. Primary Intestinal Pathogens

5b. Laboratory Diagnosis

5b. Biochemical Identification

IV. Miscellaneous Test

C. Urease (Christensens: agar or Stuarts: broth)

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

7a. Gram Negative Bacilli (Oxidative, MacConkey Positive)

III. Laboratory Diagnosis

Organisms Oxidase Pyoverdin Pyocyanin Gelatin ONPG

8a. Anaerobic Bacteriology

8b. Clinically Significant Species

Double Zone Chartreuse Spore

Bile Esculin Growth on Brick Red Chartreuse Iridescent

9c. Ricketsiaceae and Similar Organism

Rickettsia rickettsii • Rocky mountain spotted fever.

E. Laboratory Diagnosis Disease OX-19 OX-2 OX-K

9d. Mycoplasma and Ureaplasma

iii. Laboratory Diagnosis

11a. Principle of Antimicrobial Action & Resistance

b. Glycopeptides: Binds to the precursors, interfering PBD activity. E.g. vancomycin

2. Inhibitors of Cell Membrane Function

11b. Antimicrobial Susceptibility Testing

C. Dimorphism and Polymorphism

Ic. Clinically Significant Species

Ringworm Site Affected Agent

Species Growth in Rice Grains Fluorescence in Woods Lamp

Species 22°C (Mold Phase) 37°C (Tissue Phase)

2. Septate and Hyaline (Septate)

F. Yeast and Yeastlike Fungi

Species Ecology Disease / Manifestations

Species Capsules Germ Tubes Blastoconidia Arthroconidia Chlamydoconidia

3a. Laboratory Diagnosis

B1. Primary Isolation B2. Differential Test

C. Macroscopic Examination 5. Examination of Growth

Ib. Laboratory Diagnosis

C. Methods of Diagnostic Virology

Ic. Outline (Clinically Significant Species)

Enveloped Naked Naked

Icosahedral Complex Icosahedral Icosahedral

1. Orthomyxoviridae 8. 11. 13.

Species MOT Disease Specimens for Culture