MICROBIOLOGY REVIEW HANDOUTS

Objectives

BACTERIOLOGY

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 hosts 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 100C
Labile
Stable
Ab neutralization
Detoxified
Not detoxified
Biologic Activity
Individual to toxin
Same for all toxins

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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
Plastic polymers or cellulose esters (0.22 m)
HEPA filters (0.3 m)
c. Radiation
X rays, gamma rays and UV
2. Chemical Methods
a. Antiseptics
Type
Agents
Alcohol (50%-70%) ethanol, isopropanol
Halogens
iodophors
Heavy Metals
AgNO3 & HgCl2
Phenolics
chlorhexidine, triclosan

Applications
parenteral and antibiotic solutions & vaccines.
biological safety cabinets

disposables: syringes, catheters or gloves

b. Disinfectant (Sterilizers)
Type
Agents
Aldehydes
formaldeyde (1-8%), glutaraldehyde (2%)
Halogen
chlorine and chlorine compounds (Bleach)
Detergents
quaternary ammonium compounds
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).

MICROBIOLOGY REVIEW HANDOUTS

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 organisms 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; patients room number or location;
requesting physician; culture site; date and time of collection
b. Requisition Form: Patients name, age and gender; patients room number or location; physicians 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. (4C)
Room Temp. (22C)
Body Temp. (37C)
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 & Amies (Dacron, Rayon or Calcium Alginate swabs) or JEMBEC System

MICROBIOLOGY REVIEW HANDOUTS

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 <25WBCs 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
a. Slide warmer at
Wet Preps
Fluids or semisolids
60C for 10 mins.
Drop
Clear, Pus, swab rinse, tissue homogenate
b. Flooding with 95%
Pellet
Blood culture, Dilute
methanol for 1 min.
Rolled
Swab material (Pus)
Pull-apart
Thick, granular, mucoid
C. Stains
1. Simple stain: colors the forms and shapes. E.g. Methylene Blue
2. Differential stain: colors specific components. E.g. Gram Stain, Acid Fast Stain
3. Antibody or Probe-mediated stain: directed specifically at identification of an organism
General Morphology
Wright-Giemsa
Sample with cellular background
Selected Morphology
Methylene Blue
Metachromatic granules
Acid Fast Stains
Mycolic Acid
Gram Stain
Cell Wall
India Ink
Capsules
Schaefer Fulton
Endospores
Leifson
Flagella
D. Microscopes
Microscope
Bright Field
Dark Field
Phase-contrast
Fluorescence

Magnification
10-1000
10-400
10-400
10-400

Genus (Species)-Specific Stains

Intracellular organisms________
Antibody or DNA
Lacks cell wall_______________
probe stains
Not resolved by light microscope

Application
Stained cells
For cells not readily stained
Living or unstained cells
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

MICROBIOLOGY REVIEW HANDOUTS

E. Terminology for Direct Examinations
Gram Positive Cocci
Chains
Streptococcus
Cluster
Staphylococcus, Viridans Strep.,
Diplococci
Streptococcus pneumoniae
Encapsulated S. pneumoniae, S. pyogenes
Gram Negative Cocci
Neisseria spp.,
Diplococci
Moraxella catarrhalis
Gram Positive Bacilli
Small
Listeria, Corynebacterium
Large
Clostridium, Bacillus
Diptheroid
Corynebacterium
Beaded
Mycobacterium, Corynebacterium
Branched
Nocardia, Actinomyces
Bifid/V forms
Bifidobacterium

Gram Negative Coccobacilli

Singly
Fastidious G (-) Bacilli
Chains, Masses Gram (-) anaerobes
Gram Negative Bacilli
Small
Haemophilus, Legionella, Bordetella,
Brucella, Francisella, Pasteurella
Medium
Enterics, Pseudomonads
Curved/Spiral
Vibrio, Campylobacter, Helicobacter
Spiral

Borrelia, Leptospira, Treponema

F. Examination of Prepared Material

Cells & structures
Associations
Necrosis, heavy protein fluid
Epithelial cells
Cell surface in collection site
Mononuclear cells Chronic inflammation
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%)

MICROBIOLOGY REVIEW HANDOUTS

2. Temperature, pH & Moisture
a. Temperature: 35-37C (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.
II.
A.

B.

C.

General Characteristics
G(+) cocci in clusters, Catalase (+), Oxidase (-), facultative anaerobes, 7.5-10% NaCl (+)
Clinically Significant Species
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 (, , ), Panton-Valentine Leucocidin (-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 -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
Staphylococcus epidermidis
1. Virulence Factor: Exopolysaccharide slime or biofilms
2. Infections: Hospital acquired UTI, prosthetic valve endocarditis
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

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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. -hemolytic
S. epidermidis: Small to medium. Gray-white. -hemolytic
S. saprophyticus: Small to medium. White-yellow or orange. -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 (5g)
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 g Novobiocin.
Susceptible: zone diameter >16 mm Resistant: zone diameter 16 mm

2b. Gram (+) Streptococci

I. General Characteristics
Introduction: G(+) cocci in pairs / chains, Catalase (-), Aerotolerant anaerobes, Some are capnophilic
Species
A. Lancefield
B. Browns
Common Terms (Group)
A

A
S. pyogenes
S. agalactiae
B

B
S. dysagalactiae, S. equi
C

C
S. bovis group
D
,
D Non Enterococcus
E. faecalis, E. facium
D
,,
D Enterococcus
S. pneumoniae

Pneumococcus
S. anginosus, mutans, mitis
A, C, F, G, N
,,
Viridans streptococcus
II. Clinically Significant Species
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
2. Clinical Infections
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

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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 -hemolytic isolates)
2. S. anginosus group (Small-colony forming -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
III. Laboratory Diagnosis
C. Colony Characteristics
Species
Description
Group A
pinpoint, large zone of hemolysis
Group B
larger, narrow zone of hemolysis
Viridans
small; , , hemolytic
Group D
small; , , hemolytic
Pneumococci
glistening, dome-shaped, mucoid, umbilicated; hemolytic
D.
1a.
1b.
2.

Biochemical Identification
Bacitracin (Taxo A) -Test for susceptibility to 0.04 U Bacitracin. Positive Group A
Sulfamethoxazole-Trimethoprim - Test for susceptibility to 1.25 g of SXT disk. Positive Not Group A or B
PYR Hydrolysis - Test for the ability to hydrolyze the substrate L-pyrrolidonyl--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 (purpleyellow):________
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. hemolytic
Bacitracin
PYR
SXT
CAMP/Hippurate
A
S
+
R
B
R
R
+
Not A/B
R
S
2. hemolytic
S. pneumoniae
D Enterococcus
D Non-enterococcus
Viridans Strep.

Optochin
S
R
R
R

BEA Hydrolysis
+
+
S

6.5% NaCl
+
-

3. , , hemolytic
D Enterococcus
D Non-enterococcus
Viridans Strep.

BEA Hydrolysis
+
+
S

6.5% NaCl
+
-

PYR
+
-

PYR
+
-

MICROBIOLOGY REVIEW HANDOUTS

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 -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
C. diptheriae
C. ulcerans
C. jeikeium
Characteristic
C. pseudotuberculosis
Tinsdales 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)
3. Laboratory Diagnosis
a. Microscopy: G+ rods or coccobacilli in pairs or in chains
b. Colony: Narrow zone of -hemolysis (SBA)
c. Grows best at 30-35C, but growth occurs at 0.5 -45C, 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)

I. Clinically Significant Species
A. Erysipelothrix rhusiopathiae (Red skin, red disease)
1. Clinical Infections: Erysipeloid, septicemia, diffuse cutaneous infection
2. Laboratory Diagnosis
a. Microscopy: Thin, filamentous G(+) rods
b. Colony: -hemolytic w/ prolonged incubation
3. Identification: Test tube brush-like pattern at 22C, produces H2S in TSI
B. Arcanobacterium haemolyticum
1. Clinical Infections: Pharyngitis, cervical lymphadenopathy and soft tissue and sepsis
2. Laboratory Diagnosis
a. Microscopy: Curved, G+ rods w/ pointed ends that becomes coccal after 48 hrs
b. Colony: Small colonies, -hemolytic, pits agar, leaves black dot under
c. Identification: Lipase and Lecithinase positive (EYA), Exhibit CAMP inhibition reaction (phospholipase D)

MICROBIOLOGY REVIEW HANDOUTS

C. Gardnerella vaginalis
1. Clinical Infections: Bacterial vaginosis (Excessive vaginal discharge, pH > 4.5 and foul smell) associated in UTI
2. Laboratory Diagnosis
a. Microscopy: Pleomorphic Clue cells in vaginal fluid.
b. Colony: -hemolytic (BAP) and -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
+

L. monocytogenes
+
+
+
+

E. rhusiopathiae
+
,
A.haemolyticum

G. vaginalis

2c. Aerobic Gram Positive Bacilli (Branching Actinomycetes)

I. Clinically Significant Species
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: -hemolytic (SBA), Chalky, dry, crumbly (Sabourauds & Mycosel), 22C & 37C (3-6 d)

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

I. Clinically Significant Species
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 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
(+)
(-)
(-)
(-)

MICROBIOLOGY REVIEW HANDOUTS

3a. Gram (-) Diplococci
I. General Characteristics
Obligate aerobe, Capnophilic, Oxidase (+), Catalase (+), Glucose Fermenter (except for M. catarrhalis)
II. Clinically Significant Species
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 ECs
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(+)
Colistin
G(-)
Thayer-Martin
Nystatin
Yeast
Modified TM
Trimethoprin
Swarming of Proteus
Martin Lewis
Anisomycin
Yeast
New York City
Amphothericin B
Yeast
D. Incubation: 35C ( 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

MICROBIOLOGY REVIEW HANDOUTS

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 (Pfeiffers 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)
III. Laboratory Diagnosis
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 35-37C (2-3 days),
ii. Haemophilus ducreyi: 5-10% CO2 at 33C 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 Kovacs rgt) and Porphyrins (emit with 360nm UV, red-orange)
Species
V factor
Hemolysis
ALA
X factor
H. influenzae
(+)
(+)
(-)
(-)
(+)
(+)
(-)
(-)
H. aegyptius
H. haemolyticus
(+)
(+)
(+)
(-)
H. parahaemolyticus
(-)
(+)
(+)
(+)
H. parainfluenzae
(-)
(+)
(-)
(+)
(+)
(-)
(-)
(-)
H. ducreyi
A. aphrophilus
(-)
(-)
(-)
(+)

MICROBIOLOGY REVIEW HANDOUTS

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 20-43C (40-60C), 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: Legionnaires 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)

MICROBIOLOGY REVIEW HANDOUTS

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

1. Uropathogenic E. coli

Pili & cytolysins

Most common cause of UTI

2. Enterotoxigenic E. coli (ETEC)

Fimbriae & enterotoxins

(heat stable/labile)

3. Enteroinvasive E. coli (EIEC)

Shiga-Toxin

Epidemic & travelers

diarrhea
Shigella like iinfection
( dysenteric stools)

4. Enteropathogenic E. coli
(EPEC)
5. Enterohemorrhagic__
Verotoxic E. coli (EHEC)
6. Enteroadheren E. coli
Diffusely adherent E. coli (DAEC)
Enteroaggregative E. coli (EAEC)

Pili
Verotoxin

Infantile diarrhea
Hemorrhagic colitis
Hemolytic uremi syndrome

iii. Other Information

Montezumas revenge
or turista
Non-motile & non-lactose
fermenter
Watery w/ mucus but no
blood
Associated w/ 0157:H7
Sorbitol (-) & MUG (-)

UTI & diarrhea

Diarrhea (watery)

B. Klebsiella (Friedlander) & Enterobacter

1. Pneumonia, UTI, septicemia, pneumonia, wound infections, etc.
2. Encapsulated, mucoid colonies that string.
3. Klebsiella (plasmid- mediated ESBLs)
Species (IMViC Rxns)
Indole
MR
+
+
E. coli

K. pneumoniae subs. pneumoniae

+

subs. oxytoca
Enterobacter spp.

VP

+
+
+

Citrate

+
+
+

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/+
+

MICROBIOLOGY REVIEW HANDOUTS

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 Widals test.
B. Shigella
1. Virulence Factors: neurotoxin & enterotoxins (S. dysenteriae), Other species: only enteroxin
2. Disease: Shigellosis or bacillary dysentery
Test
S. dysenteriae
S. sonnei
S. flexneri
S. boydii
Mannitol

+
+
+
ONPG / ODC

+
Serogroup
A
B
C
D
Test
Salmonella
Shigella
Motility /H2S
(+)
(-)
6
10
100-200
Infectious Dose
C. Yersinia pestis
1. Disease: Bubonic and Pneumonic Plaque
2. Laboratory Diagnosis: Bipolar staining "safety pin" , Cauliflower (SBA), Stalactite (broth), Grows best at 25-30C
D. Yersinia enterocolitica
1. Disease: Acute enteritis
2. Laboratory Diagnosis: Bipolar staining", bulls eye colonies (CI_N, 48 hrs), Grows at 25-30C, Motile at 25_C
Test
TSI
Motile (25C)
Sucrose

Y. pestis

Y. enterocolitica
Yellow/Orange

+
+

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 (100C 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

MICROBIOLOGY REVIEW HANDOUTS

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 -galactosidase and hydrolyzes ONPG to form -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 (-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

MICROBIOLOGY REVIEW HANDOUTS

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 Kovacs
c. H2S positive : Blackening of the medium
F. MUG Test
Determine the ability of an organism to produce -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
-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: -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, or hemolytic), Mac (NLF) & TCBS

MICROBIOLOGY REVIEW HANDOUTS

II.

III.

IV.

Aeromonas
Virulence: Cytotoxic enterotoxin
Disease: Intestinal (five diarrheal syndrome), Extraintestinal (wound nfections, septicemia & meningitis, etc.)
Lab Diagnosis: -hemolytic (SBA),Ferments Lactose, pink-centered(CIN)
Plesiomonas shigelloides
Disease: Gastroenteritis, bacteremia, meningitis, etc.
Lab Diagnosis: Pink in Inositol brilliant green bile salt agar; LDC, ODC, ADH Positive Trio
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)
TCBS
String Test
Broth w/ 6.5% NaCl
Broth w/o 6.5%NaCl
Vibriostatic Test (150 g 0/129)
Inositol Fermentation
Addt. Comments

Vibrio cholerae
+
+
+
+
+
-

Other Vibrio spp.

+
+
+
-

Aeromonas spp.
+
-

Plesiomonas
+
+
+
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
III. Laboratory Diagnosis
A. Specimen
1. C. jejuni: Feces (Stuart, Cary-Blair & Campy Thio)
2. C. fetus subsp. fetus: Blood (Blood culture media)
3. H. pylori : Tissue biopsy (Stuarts , Cysteine-Brucella broth)
B. Culture Media and Incubation
1. Campylobacter spp: Butzler, Skirrows, 42C or 37C (Microaerophilic)
2. H. pylori: Skirrows, Chocolate or Brucella agar with 5% horse blood, 35-37C (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)

MICROBIOLOGY REVIEW HANDOUTS

E. Identification
Test
C. jejuni
C. fetus
H. pylori
+

hydrolysis
Hippurate
+

Growth at 42C

Growth at 25C
Urease

+
S
R
R
Nalidixic acid
Cephalotin
R
S
S
1. Urease Test: Tissue is place in Christensen medium for 37C 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)

I. General Characteristics
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.
II. Clinically Significant Species
A. Pseudomonas aeruginosa
1. Clinical Significance: Most commonly isolated nonfermenter (75% of nonfermenters in nosocomial bacteremias,
5-15% of nosocomial inf.) wound inf., pulmonary disease (cystic fibrosis patients.), UTI, endocarditis, meningitis
2. Virulence Fator:
i. Enzymes (protease, hemolysins, lecithinase, elastase & DNase); ii. Exotoxin A ( inhibits protein synthesis),
iii. Alginate (polysaccharide polymer in mucoid strains)
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 42C 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
III. Laboratory Diagnosis
Organisms
S. maltophilia
A. baumannii
A. iwoffi

Oxidase
(-)
(-)
(-)

Pigment
Y
(-)
(-)

Glucose
(+)
(+)
(-)

Maltose
(S+)
(+/-)
(-)

Growth at 42
(+/-)
(+)
(-)

MICROBIOLOGY REVIEW HANDOUTS

Organisms

P. aeruginosa
P. fluorescens
P. putida
B. cepacia
B. pseudomallei

Oxidase

Pyoverdin

(+)
(+)
(+)
(+)
(+)

(+)
(+)
(+)
(-)
(-)

Pyocyanin
Acetamide
Cetrimide
42 Growth
(+)
(-)
(-)

Gelatin
hydrolysis

(+)
(-)

ONPG

(-)
(-)
(-)
(+)
(-)

8a. Anaerobic Bacteriology

I.

II.

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
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

MICROBIOLOGY REVIEW HANDOUTS

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. -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 -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 -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, -hemolytic, smoothly swarming
Swarming
C. perfringens
C. botulinum
C. tetani
C. difficile
C. septicum

II.

+
+

Double Zone
of hemolysis
+
-

Chartreuse
Fluorescence
+
-

Lecithinase

Lipase

+
-

+
-

Spore
Position
ST
ST
T
ST
ST

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

MICROBIOLOGY REVIEW HANDOUTS

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
Species

Co

B. fragilis
Prevotella
Porphyromonas
Fusobacterium

R
R
S
R

R
R
R
S

R
S
R
S

Bile
Esculin Growth on Brick Red
Chartreuse
Iridescent
Resistance Hydrolysis
KVLB Fluorescence fluorescence sheen (EYA)
+
+
+
+
+
+

+
+

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

MICROBIOLOGY REVIEW HANDOUTS

9a. Spirochetes
I. Clinically Signifant Species
A. Leptospires
1. Characteristics: Tightly coiled, thin, spirochetes w/hooked ends; culturable to Fletchers, Stuart; visible by darkfield, 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
(+)
(-)
(-)
in
inclusions
Glycogen
Sulfa drug sensitivity
(+)
(-)
(-)
Natural Hosts
Humans
Humans
Birds
B. Chlamydia trachomatis
1. Clinical Infections
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
2. Laboratory Diagnosis
Cell Culture, serology , molecular (PCR), Freis 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

MICROBIOLOGY REVIEW HANDOUTS

Rickettsia rickettsii
2. Typhus Group
Rickettsia prowazekii

Rocky mountain spotted fever.

Epidemic typhus
Sporadic typhus
Brill-Zinsser disease
Murine typhus, Endemic typhus

Rickettsia typhi
3. Scrub Typhus Group
Orientia tsutsugamushi

C.

Ehrlichia / Anaplasma
Ehrlichia chaffeenis
Ehrlichia ewingii
A. phagocytophilum

D. Coxiella
Coxiella burnetii

Lice
Flying squirrels
Recrudescent-Reactivation
Ticks

Scrub typhus

Chiggers

Monocytic ehrlichiosis
Granulocytic ehrlichiosis
Granulocytic anaplasmosis
Q (query) fever

E. Laboratory Diagnosis
1. Immunohistology and PCR
2. Embryonated eggs and tissue culture
3. Giemsa, Wrights stained buffy coat
4. Weil-Felix rxn. (P. vulgaris OX-19, OX-2, P. mirabilis OX

Ticks

Inhalation of dried birthing fluid

Disease
Brill-Zinsser
Epidemic typhus
Murine typhus
Rickettsialpox
RMSF
Scrub Typhus

OX-19
V
+
+

OX-2
V
V
V

OX-K

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
+

MICROBIOLOGY REVIEW HANDOUTS

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 (NTMs)
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 30-32C
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 (22C) & scotochromogen (37C)
c. M. scrofulaceum: Cervical lymphadenitis in children
d. M. xenopi: Grows best 42C, Birds 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. Johnes & Crohns 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 42C, 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 - Hansens Disease
Tuberculoid Leprosy - Skin lesions and peripheral nerve involvement
Lepromatous leprosy - Extensive skin lesions and symmetric nerve damage

MICROBIOLOGY REVIEW HANDOUTS

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: 35C, 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 Mitchisons (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 (68C) Catalase Test
Test for the ability of catalase enzyme to remain active after heating
Stable or inactivated at 68C 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
M. tuberculosis
M. bovis

Niacin

Growth on T2H

Nitrate reduction 68 & SQ Catalase

11a. Principle of Antimicrobial Action & Resistance

I.

II.

Introduction
Antibiotics - Substance obtained from microbes to kill an infecting pathogen.
Bacteriostatic - Inhibit microbial growth
Bactericidal - Kills the microbe leading to lysis and death
Antimicrobial Action
1. Inhibitors of Cell Wall Synthesis
a. Beta-lactams: Binds to the enzyme (penicillin-binding proteins). E.g. penicillins, cephalosporins

MICROBIOLOGY REVIEW HANDOUTS

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 bacteriums 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)

MICROBIOLOGY REVIEW HANDOUTS

Incubation
Atmosphere
Temperature
Length

Humidified ambient air

35C
Disk diffusion: 16-18 hr

Others
Colonies Sampled from isolates
Distance
Maximum stacks

4-5
20 mm apart
Maximum of 4

Objectives
1.
2.
3.
4.

MYCOLOGY

Describe the general characteristics and structures

Describe the four divisions
Enumerate and describe disease and its associated genus/species
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

MICROBIOLOGY REVIEW HANDOUTS

C. Dimorphism and Polymorphism
a. Dimorphism (Dimorphic Fungi: Ability to exist in two forms)

Yeast or Spherule phase: When grown at 37C 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 25C 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

MICROBIOLOGY REVIEW HANDOUTS

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 (Athletes / 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

MICROBIOLOGY REVIEW HANDOUTS

Species
M. audouinii
M. canis
M. gypseum

Growth in Rice Grains

Poor (Brown discoloration)
Good
Good

Fluorescence in Woods Lamp

Positive
Positive
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
Positive (2 d)
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

MICROBIOLOGY REVIEW HANDOUTS

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-handlers 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 (22-30C) or yeast (35-37C)
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, Spelunkers, 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
1. Blastomyces
dermatitidis

2. Coccidioides
immitis
3. Histoplasma
capsulatum
4. Paracoccidioides
brasiliensis

22C (Mold Phase)

Septate hyphae with round or
pyriform conidia borne on
conidiophores or directly on hyphae
resembling lollipops
Septate, branched hyphae that
produce barrel shaped, arthroconidia
that alternate w/ empty disjuncter
cells
Septate hyphae with large round,
thick walled echinulate, tuberculate
macroconidia on hyphal stalk
Small, septate, branched hyphae with
intercalary and terminal
chlamydoconidia; few pyriform
macroconidia

37C (Tissue Phase)

Thick-walled, large yeast cells with
single bud on a broad base; broad
isthmus at constriction
Large, round thick walled spherules with
endospores observed in tissue and direct
examination
Small, budding; round to oval yeast cells;
intracellular to macrophages possible
with Giemsa or Wrights stain
Large, round oval, yeast cells with
multiple buds attached to mother cell by
narrow constrictions. Mariners wheel
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

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F. Yeast and Yeastlike Fungi
4. General Characteristics: Unicellular, budding & round to oval organisms
1. Ecology and Disease
Species
Cryptococcus
neoformans
Candida
albicans
Geotrichum
candidum

Ecology
Pigeon, bat droppings
Decaying vegetation
GI tract
Mucus membranes
Soil
Decaying foods

2. Laboratory Diagnosis
Species
a. Cryptococcus
neoformans
b. Candida albicans
c. Geotrichum candidum
Species
Cryptococcus neoformans
Candida albicans
Geotrichum candidum

Disease / Manifestations
Systemic
Meningitis
Thrush, vulvovaginitis
Diaper rash, onychomycosis, Paronychomycosis
Oral, lung, skin, etc.

Laboratory Diagnosis
Direct Examinations: Round to oval yeast w/ capsule & narrow-base budding
Colony (Niger Seed Agar) : Phenol oxidase (+), Brown-black colonies
Direct Examination: Blastoconidia (budding yeast / pseudohyphae)
Cornmeal (RT, 24-48 hrs): Chlamydoconidia
Serum (35-37C, 1-3 hrs): Germ tubes, hyphalike extension of yeast cell
Direct examination: Rectangular yeast cell (in pairs / chains)
Cornmeal: Fragmented hyphae, Rectangular arthrospores with rounded ends
Capsules
+

Germ Tubes Blastoconidia

+
+
+
+

Arthroconidia

Chlamydoconidia
+

3a. Laboratory Diagnosis

1. Safety Issues
2. Specimen Collection
Agent

Specimen

Specimen
Respiratory
Subcutaneous Tissue
Blood / BM / CSF
Hair, Skin and Nails
Throat, Urine, Vaginal, Cervical

Collection Notes
Primary Isolation Media
Pulled or cut (forceps and scissors)
A. Hair
Woods Lamp
KOH & Culture (SDA
Cleansed with 70% alcohol
B. Skin & Nails
Scrap outer edge / discolored
KOH & Culture (SDA)
Lysis centrifugation
C. Blood & BM
Culture (SPS/BHI)
Wrights and Giemsa
Concentration
D. CSF
India Ink, Culture (BHI)

Specimen

E. Abscess & Lesions

F. Sputum

G. Urine
H. Throat, Vaginal
Cervical

Collection Notes
Primary, Isolation Media
Biopsy or Needle Aspiration
Sulfur granules
Homogenized, Culture (SDA, BHI)
3 consecutive early morning
Giemsa/India Ink/
Culture (SDA, BHI)
3 consecutive early morning
Clean-catch midstream
Culture (SDA)
2 swabs
KOH/Culture (SDA)

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3. Direct Examination
Method
A. KOH (10-20%)
B. KOH w/
Calcofluor white
C. India Ink /
Nigrosin
D. Lactophenol
Cotton Blue
(Aman Medium)

Application
Dissolves keratinin skin,
hair & nails
Bind chiti_n
Flouresce blue white
For Encapsulated ye_ast
(C. neoformans) in CSF

Method
E.i. PAS
E.ii. Fontana-Masson:

Application (Tissue Stains)

Polysaccharides
(purplish-red, pink)
Melanin
(brown)

E.iii. Grocott
methenamine-silver

Fungal morphology Lactic

acid, phenol, aniline blue

E.iv. Gram/Giemsa/
Wrights Stain

Black
Purple or Purple blue

4. Isolation Methods
1. Growth Requirements
i. Nutrients and Moisture: Nitrogen, carbon, vitamins & minerals
ii. Temperature: 25C / 30C (mold) or 37C (yeast)
iii. Time: 2-4 weeks (mold), 2-3 d (yeasts)
B1. Primary Isolation
Media
i. Brain-Heart Infusion
Agar (BHI)
ii. Saborauds Dextrose
Agar (SDA)
iii. BHI-CC
iv. Mycosel or
Mycobiotic
(SDA-CC )
v. Dermatophyte Test
Medium or Pfizer

B2. Differential Test

Media

Application
For
general isolation of
pathogenic &
saprophytic fungi
For isolation of
pathogenic fungi
Cycloheximide: :
inhibits saprophytes
Chloramphenicol:
inhibits bacteria
With CC plus
Phenol Red :
Dermatophytes (red)

i. Birdseed (Niger seed)

agar
ii. Cornmeal agar w/
Tween 80
iii. Cottonseed agar

iv. Potato dextrose agar

v. Rice Medium
vi . Urea Agar
vii. Trichophyton agars
viii. Czapeks

C. Macroscopic Examination
i.
Growth Rate
ii.
Topography Reverse Side (flat, heaped, folded,
wrinkled, umbonate)
iii.
Texture - Length of aerial hyphae (cross sections)
(Wolly/cottony; velvety/silky; powdery/granular;
glabrous/smooth)

Application
C. neoformanss
Detects phenol oxidase
(Black-brown colonies)
Candida spp
Stimulates conidia &
chlamydospore production
B. dermatitidiss
Induces conversion of mold to
yeast
Induces pigment production
of T. rubrum
Stimulates conidia production
Differentiation of
Microsporum spp
Differentiate of T. rubrum &
T. mentagrophytes
7 agars w/ casein or NH4 nitrate
base but different enrichment
Species identification of
Aspergilli

5. Examination of Growth
A. Tease Mount
B. Cellophane tape mount
C. Slide Culture

MICROBIOLOGY REVIEW HANDOUTS

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
2.
Sample in the infected site
3.
Use transport medium (swab, tissue)
B. Specimen Collection
Body system
Disease/manifestation
Croup, Bronchiolitis
Respiratory tract
Pneumonia
Gastrointestinal
Cutaneous
Ocular
CNS
Genital
Congenital

Gastroenteritis
Lesions &
Exanthems (rashes)
Conjunctivitis
Keratitis
Encephalitis
Aseptic meningtis
Urethritis, etc.
Penile lesions
Lesions &
Exanthems (rashes)

4.
5.
6.

Swab (dacron, rayon, cotton)

Aspirates (w/o transport media)
Store at 4C (short), -70C (long term)

Specimen
Nasal aspirate, NP/throat
swabs, BAL

Conjunctival scrapings,
Corneal swabs
CSF,
NP swabs

Common agents
Influenza, PIV, RSV, HSV
Picornaviridae, Rotavirus, Adenovirus
Rotavirus, Norwalk,
Adenovirus 40 & 41, Enterovirus
HHV1-3, Adenovirus, Rubella,
Enterovirus, Rotavirus
HSV, Adenovirus,
Coxsakievirus, Enterovirus
HSV, Arbovirus
Enterovirus, Mumps

Vesicle aspirate/ swab

HSV

Throat, Urine
Serum

CMV, HSV
Rubella

Stool, Rectal swab

Vesicle aspirate, lesion swab

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C. Methods of Diagnostic Virology
1. Direct Detection
a. Cytopathic Effects (CPE) Cowdry Type A HSV & VZV
Alteration, disruption
Intranuclear inclusions
Owl eyes
CMV
or damage to the cells
Cytoplasmic inclusions Negri bodies
Rabies
resulting from virus
Nuclear
enlargement
Koilocytosis
HPV
infection
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
HDFs

Ic. Outline (Clinically Significant Species)

dsDNA
Enveloped

Icosahedral

Complex

1.
2.
Herpesvirdae Poxviridae

ssDNA
Naked

Naked

Icosahedral

Icosahedral

3.
Papilloma
viridae

4.
Adenoviridae

5.
Parvoviridae

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ssRNA
Enveloped
Helical

Icosahedral

1. Orthomyxoviridae
2. Paramyxoviridae
3. Arenaviridae
4. Bunyaviridae
5. Coronaviridae
6. Filoviridae
7. Rhabdoviridae

dsRNA
Naked
Icosahedral

Naked
Icosahedral

11.
13.
8.
Flaviviridae Picornaviridae Reoviridae
12.
9.
Retroviridae Caliciviridae
10.
Togaviridae

Id. Clinically Significant Species

A. Double-stranded DNA viruses
1. Herpesviridae: dsDNA, Enveloped, Icosahedral, Latency and lifelong persistence
Species

MOT

Herpes Simplex
Virus 1 & 2

Contact to
ulcerations

Varicella-Zoster
Virus

Droplet inhalation,
Direct contact with
lesions

Epstein-Barr
Virus

Oral contact w/
saliva

Cytomegalovirus
Salivary gland
virus

Close contact w/
secretions &
excretions, blood &
organ transplants
Close contact w/
saliva

Disease
Oral Herpes (gingivostomatitis & fever
blister), Genital Herpes, Neonatal Herpes, Herpetic whitlow, Herpes Encephalitis, Ocular Herpes (Herpes Keratitis )
Varicella (Chickenpox)
Zoster (Shingles)
Infectious Mononucleosis
Burkitts lymphoma, Hodgkin disease
Nasopharyngeal carcinomaa
Disseminated
Infant (Congenital CMV)
Transplant Patient (40 day fever)
HIV Patient
Roseola Infantum, Exanthem subitum
Sixth disease

Human Herpesvirus 6 & 7

Human
Sexual contact
Kaposi Sarcoma
Herpesvirus 8
2. Poxviridae: dsDNA, Enveloped, Complex
Species
MOT
Disease
Direct
Variolla
Smallpox
inhalation

Specimens for Culture

Aspirates (vesicles & lesions),
Conjunctival scrapings
CSF
Lesions (vesicles)
Atypical Lymphocytes (B cells)
Serum (Paul-Bunnell)
Urine, blood,
body fluids & tissues
T cells
Noncultarable
Others
Extinct in nature
Vaccinia (vaccine strain)
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3. Papillomaviridae: dsDNA, Naked, Icosahedral
Species
MOT
Disease
Laryngeal Wart (Papillomatosis)
Human
Close contact
Venereal Wart (Condylomata acuminate)
Papilloma Virus
Cervical Carcinoma
4. Adenoviridae: dsDNA, Naked, Icosahedral, Posses a long fiber
Species
MOT
Disease
Epidemic of gastroenteritis (serotype 40 &
Fecal-oral
41) in young children in warmer months
Secretions,
Adevinovirus
Respiratory infection (Pneumonia)
aerosols, Close
Acute respiratory disease (serotype 14)
contact
Epidemics of keratoconjunctivitis
B. Single-stranded DNA viruses
5. Parvoviridae: ssDNA, Enveloped, Icosahedral
Species
MOT
Disease
Erythema infectiosum, Fifth disease
Contact with
Parvovirus B19
Transient aplastic crises (anemia)
secretions
Hydrops fetalis

Others
Koilocytes (Pap Smears)

Others
CPE (swollen in
grapelike clusters)

Others
Smallest DNA virus

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 Stensens 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
Species
MOT
Disease
Others
LCM,
Febrile illness, Hemmorhagic fever,
Rodent borne
Arena (sand)
Lassa Virus, etc.
Encephalitis
4. Bunyaviridae: ssRNA, Enveloped, Helical
Species
MOT
Disease
Others
LaCrosse, etc.
Arboviral
Febrile illness, Hemmorhagic fever,
Hantavirus
Rodent borne
Encephalitis
5. Coronaviridae: ssRNA, Enveloped, Helical, Club shaped projections
Species
MOT
Disease
Others
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
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Species

MOT
Disease
Close Contact
Lake Victoria
with secretions
Margburg hemmorhagic fever
Marburg & Ebola
and excretions
Ebola hemorrhagic fever
(EBO-Z, -S, -R)
(E.g. monkeys)
7. Rhabdoviridae: ssRNA, Enveloped, Helical
Species
MOT
Disease
Bite of infected
Flulike symptoms (prodromal) ->
Rabies virus
animal
Fatal encephalitis

Others
Rarely cause human infections
but high mortality rates
Isolation, PCR and IF
Laboratory
Isolation and IF (Negri bodies)
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
Species
MOT
Disease
Others
Eastern-, western-,
Nonspecific febrile illness
VenezuelanMosquito-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-,
Diarrhea
Prevalent overcrowded
and pneumonia
Echo-, Parechovirus
Hand, foot and mouth dse.
areas w/ poor hygiene
Rhinovirus
Aerosols and fomites
Common cold
Acid labile

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12. Caliciviridae: ssRNA, Naked, Icosahedral
Species
MOT
Norwalk
Food-borne, water-borne,
small round
Person-to-person
structured virus
13. Reoviridae: dsRNA, Naked, Icosahedral
Species
MOT
Rotavirus

Fecal-oral route
Food-, waterborne

Disease

Others

Gastroenteritis (outbreaks)

Most common in the US

Feces, EM (non culturable)

Disease
Most common cause of
Gastroenteritis in infants
and children

Others
Double-shelled resembling
a wheel

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