Microbiology

Master bacteriology, virology, mycology, and laboratory identification methods for the MedTech board exam.

1. Gram-Positive Bacteria

Staphylococci

Species	Coagulase	Key Features	Diseases
S. aureus	+	Beta-hemolytic, golden colonies, mannitol fermenter	Skin infections, SSTI, endocarditis, osteomyelitis, food poisoning, TSS
S. epidermidis	-	Novobiocin sensitive, biofilm former	Catheter infections, prosthetic device infections
S. saprophyticus	-	Novobiocin resistant	UTI in young women

MRSA (Methicillin-Resistant S. aureus)

• Carries mecA gene encoding PBP2a
• Resistant to all beta-lactams
• Treatment: Vancomycin, linezolid, daptomycin
• Detection: Oxacillin screen agar, cefoxitin disk test

Streptococci

Group/Species	Hemolysis	Key Tests	Diseases
S. pyogenes (GAS)	Beta	Bacitracin S, PYR +	Pharyngitis, impetigo, necrotizing fasciitis, rheumatic fever, PSGN
S. agalactiae (GBS)	Beta	CAMP +, hippurate +	Neonatal meningitis, sepsis, UTI in pregnancy
S. pneumoniae	Alpha	Optochin S, bile soluble, quellung +	Pneumonia (#1 cause CAP), meningitis, otitis media
Viridans streptococci	Alpha	Optochin R, bile insoluble	Subacute bacterial endocarditis, dental caries
Enterococci	Gamma/Alpha	Bile esculin +, 6.5% NaCl +, PYR +	UTI, endocarditis, nosocomial infections

Other Gram-Positive Bacteria

Corynebacterium diphtheriae

• Chinese letter/palisade arrangement
• Metachromatic granules (Loeffler's)
• Tellurite agar: Black colonies
• Elek test: Toxin production
• Causes: Diphtheria (pseudomembrane)

Listeria monocytogenes

• Tumbling motility at 25°C
• Cold enrichment (4°C)
• Beta-hemolytic, catalase positive
• CAMP test with S. aureus
• Causes: Meningitis in neonates/elderly, listeriosis in pregnancy

Bacillus anthracis

• Large, boxcar-shaped rods
• Non-hemolytic, non-motile
• Medusa head colonies
• Capsule: Poly-D-glutamic acid
• Causes: Cutaneous, pulmonary, GI anthrax

Clostridium species

• C. perfringens: Gas gangrene, double zone hemolysis
• C. tetani: Tetanus, drumstick spores
• C. botulinum: Botulism, flaccid paralysis
• C. difficile: Pseudomembranous colitis, toxin A/B

2. Gram-Negative Bacteria

Enterobacteriaceae (Lactose Fermenters)

Organism	IMViC	Key Features	Diseases
E. coli	++--	Most common GNR, pink on MAC, green sheen on EMB	UTI (#1), neonatal meningitis, gastroenteritis (ETEC, EHEC O157:H7)
Klebsiella pneumoniae	--++	Mucoid colonies, large capsule	Pneumonia (currant jelly sputum), UTI, nosocomial
Enterobacter	--++	Motile (vs Klebsiella), ornithine +	Nosocomial infections, UTI, pneumonia

IMViC = Indole, Methyl Red, Voges-Proskauer, Citrate

Non-Lactose Fermenters

Organism	Key Tests	Diseases
Salmonella typhi	H2S +, citrate -, lactose -, motile	Typhoid fever, rose spots, Widal test
Salmonella enterica	H2S +, citrate +, motile	Gastroenteritis, bacteremia
Shigella	H2S -, non-motile, lysine -	Bacillary dysentery, bloody diarrhea
Proteus	Swarming, urease +, H2S +, PD +	UTI (alkaline urine, struvite stones)
Yersinia enterocolitica	Cold enrichment (4°C), urease +	Pseudoappendicitis, mesenteric adenitis

Non-Fermenting Gram-Negative Rods

Pseudomonas aeruginosa

• Obligate aerobe, oxidase positive
• Grape-like/fruity odor
• Pyocyanin (blue-green) + pyoverdin (yellow)
• Grows at 42°C
• Causes: Burns, CF, nosocomial, hot tub folliculitis

Acinetobacter baumannii

• Oxidase negative (unlike Pseudomonas)
• Coccobacillary
• Highly resistant (MDR)
• Nosocomial: Pneumonia, wound infections
• Survives on dry surfaces

Stenotrophomonas maltophilia

• Oxidase negative
• Intrinsic resistance to carbapenems
• Treatment: TMP-SMX
• Nosocomial, immunocompromised

Burkholderia cepacia

• Oxidase positive
• Important CF pathogen
• Highly resistant
• Can grow in antiseptics

Fastidious Gram-Negative Bacteria

Haemophilus influenzae

• Requires X (hemin) and V (NAD) factors
• Chocolate agar (not blood agar)
• Satellitism around S. aureus
• Type b: Meningitis, epiglottitis (vaccine preventable)
• Non-typeable: Otitis media, sinusitis

Neisseria species

• Gram-negative diplococci, oxidase +
• N. gonorrhoeae: Maltose -, gonorrhea
• N. meningitidis: Maltose +, meningitis
• Chocolate agar or Thayer-Martin
• Intracellular in PMNs

Bordetella pertussis

• Bordet-Gengou or Regan-Lowe agar
• Mercury drop colonies
• Causes whooping cough
• DFA for rapid detection

Legionella pneumophila

• BCYE agar (buffered charcoal yeast extract)
• Requires L-cysteine and iron
• Urinary antigen test
• Legionnaires' disease, Pontiac fever

3. Other Clinically Important Bacteria

Spirochetes

Treponema pallidum

• Cannot be cultured in vitro
• Darkfield microscopy
• Screening: VDRL, RPR (non-treponemal)
• Confirmatory: FTA-ABS, TP-PA
• Causes: Syphilis (primary, secondary, tertiary)

Borrelia burgdorferi

• Ixodes tick vector
• BSK medium
• Stage 1: Erythema migrans
• Stage 2: Bell's palsy, carditis
• Stage 3: Arthritis

Leptospira interrogans

• Hook-shaped ends
• EMJH or Fletcher's medium
• Rat urine exposure (rice paddies)
• Weil's disease: Jaundice, renal failure

Mycobacteria

Species	Growth Rate	Key Tests	Disease
M. tuberculosis	Slow (2-8 weeks)	Niacin +, nitrate +, 37°C	Pulmonary TB, extrapulmonary TB
M. leprae	Cannot culture	Fite stain, armadillo/mouse footpad	Leprosy (Hansen's disease)
M. avium complex	Slow	Non-photochromogenic	Disseminated disease in AIDS
M. marinum	Slow	Photochromogenic, 30°C	Swimming pool granuloma

AFB Staining

• Ziehl-Neelsen: Hot stain, carbol fuchsin
• Kinyoun: Cold stain
• Auramine-rhodamine: Fluorescent, more sensitive
• AFB appear red/pink against blue background

Obligate Intracellular Bacteria

Chlamydia

• C. trachomatis: STI, trachoma, LGV
• C. pneumoniae: Atypical pneumonia
• C. psittaci: Psittacosis (birds)
• Elementary body: Infectious, extracellular
• Reticulate body: Replicating, intracellular

Rickettsia

• R. rickettsii: Rocky Mountain spotted fever (tick)
• R. prowazekii: Epidemic typhus (louse)
• R. typhi: Endemic/murine typhus (flea)
• Weil-Felix reaction (cross-reacting with Proteus)
• Treatment: Doxycycline

Curved/Spiral Bacteria

Helicobacter pylori

• Curved, urease strongly positive
• Microaerophilic, 5-7 days growth
• CLO test, urea breath test, stool antigen
• Causes: PUD, gastric cancer, MALT lymphoma

Campylobacter jejuni

• Curved/S-shaped, seagull wings
• Microaerophilic, 42°C
• Campy BAP or Skirrow's medium
• #1 bacterial cause of diarrhea worldwide
• Guillain-Barré syndrome association

Vibrio cholerae

• Comma-shaped, oxidase positive
• TCBS agar: Yellow colonies
• Rice-water stools
• O1 and O139 cause epidemic cholera

Vibrio parahaemolyticus

• TCBS agar: Blue-green colonies
• Halophilic (requires salt)
• Kanagawa phenomenon positive
• Seafood gastroenteritis

4. Virology

DNA Viruses

Family	Key Viruses	Diseases
Herpesviridae	HSV-1, HSV-2, VZV, CMV, EBV, HHV-6, HHV-8	Cold sores, genital herpes, chickenpox, shingles, mono, Kaposi sarcoma
Adenoviridae	Adenovirus	Pharyngoconjunctival fever, keratoconjunctivitis, gastroenteritis
Papillomaviridae	HPV (16, 18 high-risk; 6, 11 low-risk)	Warts, cervical cancer, condylomata
Hepadnaviridae	Hepatitis B virus	Hepatitis, cirrhosis, HCC
Parvoviridae	Parvovirus B19	Erythema infectiosum (fifth disease), aplastic crisis

RNA Viruses

Family	Key Viruses	Diseases
Picornaviridae	Poliovirus, Coxsackie, Rhinovirus, HAV, Enterovirus	Polio, hand-foot-mouth, common cold, hepatitis A
Flaviviridae	HCV, Dengue, Yellow fever, Zika, West Nile	Hepatitis C, dengue fever, encephalitis
Orthomyxoviridae	Influenza A, B, C	Flu (antigenic drift/shift)
Paramyxoviridae	Measles, Mumps, RSV, Parainfluenza	Measles, mumps, bronchiolitis, croup
Retroviridae	HIV-1, HIV-2, HTLV	AIDS, adult T-cell leukemia
Rhabdoviridae	Rabies virus	Rabies (Negri bodies in neurons)
Coronaviridae	SARS-CoV-2, SARS, MERS	COVID-19, common cold, SARS, MERS

Hepatitis Viruses Comparison

Feature	HAV	HBV	HCV	HDV	HEV
Genome	RNA	DNA	RNA	RNA	RNA
Transmission	Fecal-oral	Blood, sexual	Blood	Blood	Fecal-oral
Chronic	No	Yes	Yes (most)	Yes	No*
Vaccine	Yes	Yes	No	HBV vaccine	Limited

*HEV can cause chronic infection in immunocompromised; dangerous in pregnancy

HIV Diagnosis

• 4th generation: Combination Ag/Ab test (p24 + HIV-1/2 Ab)
• HIV-1/HIV-2 differentiation: If screening positive
• Confirmatory: Western blot or IFA (older algorithm)
• Viral load: HIV RNA PCR for monitoring
• CD4 count: Immune status, AIDS if <200 cells/µL

5. Mycology

Superficial & Cutaneous Mycoses

Dermatophytes (Tinea)

• Trichophyton: Skin, hair, nails
• Microsporum: Skin, hair (Wood's lamp +)
• Epidermophyton: Skin, nails only
• KOH prep: Septate hyphae
• DTM (Dermatophyte Test Medium): Red color

Malassezia furfur

• Tinea/pityriasis versicolor
• Spaghetti and meatballs (KOH)
• Lipophilic yeast
• Hypo/hyperpigmented patches

Systemic/Dimorphic Fungi

Mold at 25°C (room temp) → Yeast at 37°C (body temp)

Fungus	Endemic Area	Microscopy	Disease
Histoplasma capsulatum	Mississippi/Ohio River valleys, bird/bat droppings	Small yeast in macrophages	Pulmonary histoplasmosis
Blastomyces dermatitidis	Great Lakes, SE USA	Broad-based budding yeast	Blastomycosis, skin lesions
Coccidioides immitis	Southwestern USA, desert soil	Spherules with endospores	Valley fever, San Joaquin fever
Paracoccidioides brasiliensis	South America	Captain's wheel (multiple buds)	Paracoccidioidomycosis
Sporothrix schenckii	Worldwide (rose thorns)	Cigar-shaped yeast	Lymphocutaneous sporotrichosis

Opportunistic Fungi

Candida albicans

• Germ tube test positive (2-3 hrs at 37°C)
• Chlamydospores on cornmeal agar
• Causes: Thrush, vaginitis, esophagitis, candidemia
• CHROMagar: Green colonies

Cryptococcus neoformans

• India ink: Encapsulated yeast
• Urease positive, no germ tube
• Bird droppings (pigeon)
• Causes: Meningitis in AIDS
• Cryptococcal antigen test

Aspergillus

• Septate hyphae, 45° angle branching
• A. fumigatus most common
• Galactomannan antigen
• Causes: ABPA, aspergilloma, invasive aspergillosis

Mucor/Rhizopus (Mucormycosis)

• Broad, non-septate (pauciseptate) hyphae
• 90° angle branching
• Diabetic ketoacidosis association
• Rhinocerebral infection

Pneumocystis jirovecii

• Fungus (formerly classified as protozoan)
• Cannot be cultured
• GMS stain: Crushed ping-pong balls
• PCP (Pneumocystis pneumonia) in AIDS

6. Culture Media & Techniques

Types of Culture Media

Enriched Media

• Blood agar (BAP)
• Chocolate agar
• Thioglycolate broth
• Brain heart infusion

Selective Media

• MacConkey (GNR)
• Thayer-Martin (Neisseria)
• CNA (Gram positive)
• SS, XLD, HE (Salmonella/Shigella)

Differential Media

• MacConkey (lactose +/-)
• EMB (lactose fermenter)
• Mannitol salt (S. aureus)
• TSI/KIA (sugar fermentation)

Common Culture Media by Purpose

Medium	Purpose/Target	Key Features
Blood Agar	General purpose, hemolysis	Alpha (green), Beta (clear), Gamma (none)
Chocolate Agar	Fastidious organisms (Haemophilus, Neisseria)	Lysed blood releases X and V factors
MacConkey Agar	Gram-negative, lactose fermentation	Pink = lactose +, colorless = lactose -
EMB Agar	Differentiate coliforms	E. coli = green metallic sheen
Thayer-Martin	Neisseria	VCN antibiotics (vancomycin, colistin, nystatin)
Lowenstein-Jensen	Mycobacteria	Egg-based, malachite green
Sabouraud Dextrose	Fungi	Low pH (5.6), inhibits bacteria

Biochemical Tests

Common Biochemical Tests

• Catalase: Bubbles with H₂O₂ (Staph +, Strep -)
• Oxidase: Purple with oxidase reagent (Pseudomonas +)
• Coagulase: Clumping (S. aureus +)
• Indole: Red ring with Kovac's (E. coli +)
• Urease: Pink/red (Proteus +, H. pylori +)
• PYR: Red color (GAS +, Enterococcus +)

TSI/KIA Reactions

• A/A = Acid/Acid (lactose and glucose fermenter)
• K/A = Alkaline/Acid (glucose only, e.g., Shigella)
• K/K = Alkaline/Alkaline (non-fermenter, e.g., Pseudomonas)
• H₂S = Black precipitate (Salmonella, Proteus)
• Gas = Cracks/bubbles in agar

Staining Techniques

Stain	Purpose	Result
Gram Stain	Differentiate bacteria	Gram + = purple, Gram - = pink
Ziehl-Neelsen/Kinyoun	Acid-fast bacteria	AFB = red, background = blue
India Ink	Capsule (Cryptococcus)	Clear halo around yeast
GMS (Grocott)	Fungi, Pneumocystis	Black fungi on green background
PAS	Fungi, glycogen	Magenta/pink
Giemsa	Blood parasites, Chlamydia	Blue/purple organisms

7. Antimicrobial Susceptibility Testing

Disk Diffusion (Kirby-Bauer)

• Medium: Mueller-Hinton agar (MHA)
• Inoculum: 0.5 McFarland standard (~1.5 × 10⁸ CFU/mL)
• Incubation: 35°C, 16-18 hours
• Reading: Measure zone diameter in mm
• Interpretation: Compare to CLSI breakpoints (S, I, R)

MIC and MBC

MIC (Minimum Inhibitory Concentration)

• Lowest concentration that inhibits visible growth
• Methods: Broth dilution, agar dilution, E-test
• Gold standard for susceptibility

MBC (Minimum Bactericidal Concentration)

• Lowest concentration that kills 99.9%
• Subculture from MIC tubes with no growth
• Important for endocarditis, meningitis

Special Susceptibility Tests

Test	Purpose	Method
MRSA Screen	Detect methicillin resistance	Cefoxitin disk (30 µg), oxacillin agar
D-test	Inducible clindamycin resistance	Erythromycin + clindamycin disks
ESBL Detection	Extended-spectrum beta-lactamase	Cephalosporin ± clavulanate
Hodge Test	Carbapenemase detection	Cloverleaf pattern
Beta-lactamase	Penicillinase detection	Nitrocefin (chromogenic cephalosporin)

Common Resistance Mechanisms

• MRSA: mecA gene → altered PBP2a
• VRE: vanA/vanB genes → altered cell wall target
• ESBL: Hydrolyzes 3rd-gen cephalosporins (inhibited by clavulanate)
• AmpC: Chromosomal beta-lactamase (not inhibited by clavulanate)
• CRE: Carbapenem-resistant Enterobacteriaceae (KPC, NDM)
• Efflux pumps: Pump out antibiotics (MDR)

8. Infection Control & Safety

Biosafety Levels

Level	Agents	Requirements
BSL-1	Non-pathogenic (E. coli K-12)	Standard microbiological practices
BSL-2	Moderate risk (most clinical specimens)	BSC, limited access, PPE
BSL-3	Serious/lethal (TB, Brucella, Coccidioides)	Negative pressure, respirators, double doors
BSL-4	Dangerous (Ebola, Marburg)	Full suit, shower exit, isolated building

Transmission-Based Precautions

Contact Precautions

• MRSA, VRE, C. difficile
• Scabies, lice
• Gown and gloves
• Private room preferred

Droplet Precautions

• Influenza, pertussis, mumps
• Meningococcal disease
• Surgical mask within 6 feet
• Private room or cohorting

Airborne Precautions

• TB, measles, varicella, COVID-19
• N95 respirator required
• Negative pressure room
• Keep door closed

Sterilization and Disinfection

Sterilization Methods

• Autoclave: 121°C, 15 psi, 15-20 min
• Dry heat: 160-180°C, 1-2 hours
• Ethylene oxide: Heat-sensitive items
• Gamma radiation: Disposables
• Filtration: Heat-labile solutions (0.22 µm)

Disinfection Levels

• High-level: Glutaraldehyde (kills spores)
• Intermediate: Alcohol, chlorine
• Low-level: Quaternary ammonium
• Most resistant: Prions > Spores > Mycobacteria

Quality Control in Microbiology

• ATCC strains: E. coli ATCC 25922, S. aureus ATCC 25923, P. aeruginosa ATCC 27853
• Media QC: Test with appropriate control organisms
• Stain QC: Include positive and negative controls
• AST QC: Weekly (at minimum) with control strains
• Temperature: Daily monitoring of incubators, refrigerators
• Proficiency testing: External quality assessment programs

Key Takeaways

✓S. aureus is coagulase positive; S. epidermidis/saprophyticus are negative
✓S. pneumoniae: Optochin sensitive, bile soluble
✓E. coli IMViC: ++-- ; Klebsiella: --++
✓Pseudomonas: Oxidase +, grape odor, pyocyanin

✓Dimorphic fungi: Mold at 25°C, yeast at 37°C
✓Candida albicans: Germ tube positive
✓MIC is gold standard for susceptibility testing
✓Autoclave: 121°C, 15 psi, 15-20 minutes

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