Microbiology LRR Analysis

ZN Stain (Acid Fast Stain) Protocol:

1. Carbol Fuchsin: Primary stain (Red).
2. Heat: Acts as the mordant.
3. H2SO4 (Sulphuric Acid): Decolorizer.
4. Methylene Blue: Counterstain (Blue background).

H2SO4 Concentrations (Exam Favorite):

• 20-25%: Mycobacterium tuberculosis (TB).
• 5%: Mycobacterium leprae (Fite-Faraco stain).
• 1%: Nocardia, Legionella, Isospora, Cyclospora (Modified ZN stain).
• 0.25-0.5%: Spores.

Gram Staining:

• Gram Positive: Purple (retains Crystal Violet).
• Gram Negative: Pink/Red (takes up Safranin).
• Step Sequence: Crystal Violet → Iodine (Mordant) → Alcohol/Acetone (Decolorizer - Most Critical Step) → Safranin.

Light Microscope Parts:

• Condenser: Located below the stage; focuses light on the sample.
• Arrangement (Eye to Light): Eyepiece → Objective Lens → Condenser → Light Source.

Types of Microscopes:

• Dark Field: For thin spiral bacteria (Spirochetes like Treponema pallidum/Syphilis) and Flagella.
• Electron Microscope: Uses electrons + vacuum. Fixative: 2.5% Glutaraldehyde.
• Fluorescent Microscope: Uses UV light + Dichroic mirror.
  • TB: Auramine-Rhodamine.
  • Malaria: Acridine Orange.
  • Fungus: Calcofluor White.

Capsule Staining:

• M'Fadyean Reaction: Uses Methylene Blue. For Anthrax (Polypeptide capsule). Appearance: Purple capsule.
• Quellung Reaction: For Polysaccharide capsules (Pneumococcus, Klebsiella, etc.).

Bacterial Motility (High Yield):

• Tumbling: Listeria (at 25°C).
• Darting / Shooting Star: Vibrio & Campylobacter.
• Corkscrew: Spirochetes (Treponema).
• Twitching: Trichomonas (Parasite) & Eikenella/Kingella.
• Swarming: Proteus (Most common), Vibrio parahaemolyticus, Serratia, Clostridium tetani.
• Falling Leaf: Giardia.

Bacterial Growth Curve:

• Lag Phase: No division, size increases.
• Log Phase: Maximum division.
• Stationary Phase: Spore formation, toxin production, antibiotic production.

Genetics Transfer:

• Transduction: Via Bacteriophage (Virus). Drug resistance in Staph aureus.
• Transformation: Uptake of free DNA.
• Conjugation: Via sex pilus/contact (Most common method generally).

Sterilization Methods (Heat):

• Dry Heat (Hot Air Oven): 160°C for 2 hours. Items: Glassware, oils, powders, grease. ("Oily greasy liquidy dusty").
• Moist Heat (<100°C):
  • Pasteurization: Holder (63°C/30min), Flash (72°C/20sec - kills Coxiella).
  • Inspissation: (80-85°C for 30 mins x 3 days). For LJ Media (TB) and Loeffler's Serum Slope (Diphtheria).
• Moist Heat (100°C):
  • Tyndallization: 100°C for 20 mins x 3 days. Kills spores.
• Moist Heat (>100°C - Autoclave): 121°C for 15 mins at 15 PSI.
  • Items: Surgical instruments, culture media (except LJ/LSS), gowns.
  • Prions: 134°C for 1.5 hours.

Chemical Sterilization:

• 2% Glutaraldehyde (Cidex): For endoscopes/bronchoscopes.
• Plasma Sterilization (STERRAD): Uses H2O2 plasma. For heat-sensitive items (Arthroscopes, cameras).

Biological Controls (Match the Following):

• Hot Air Oven / ETO: Bacillus atrophaeus (subtilis).
• Autoclave / Plasma: Bacillus stearothermophilus ("Thermo" = heat).
• Radiation: Bacillus pumilus.
• Filtration: Brevundimonas diminuta.

• Cell Wall: Contains Chitin, Beta-glucan, Mannoproteins.
• Beta-1,3-D-Glucan Test: Positive for most fungi. Negative in "BMC": Blastomyces, Mucor, Cryptococcus.
• Galactomannan: Specific for Invasive Aspergillus.
• Culture Media: SDA (Sabouraud Dextrose Agar). pH 5.6 (Acidic). Antibiotics added to inhibit bacteria.
• Stains:
  • LPCB: Lactophenol Cotton Blue.
  • KOH Mount: 10-20% KOH dissolves keratin (for skin/hair/nail).
  • GMS (Silver stain): Fungi appear black.

• Subcutaneous Infections (MRCS): Mycetoma, Rhinosporidiosis, Chromoblastomycosis, Sporotrichosis.

Pityriasis Versicolor:

• Agent: Malassezia furfur.
• Appearance: Hypopigmented patches.
• Microscopy: Spaghetti and Meatball appearance.
• Culture: SDA with Olive Oil (Lipophilic). Appearance: Fried Egg colonies.

Dermatophytes (Tinea/Ringworm):

• Trichophyton: Macroconidia (Pencil), Microconidia (Abundant). Hair Perforation Test Positive (T. mentagrophytes).
• Microsporum: Macroconidia (Spindle/Fusiform). Ectothrix.
• Epidermophyton: Macroconidia (Club shaped).

Mnemonic: "Body Heat Probably Changes Shape" (Yeast at 37°C/Heat, Mold at 25°C/Cold).

• Blastomycosis: Broad-based budding yeast (Figure of 8).
• Histoplasmosis: Darling's Disease. Bird/Bat droppings (Caves). Intracellular yeast. Tuberculate Macroconidia in mold form.
• Paracoccidioidomycosis: Mariner’s Wheel / Mickey Mouse appearance.
• Coccidioidomycosis: Barrel-shaped arthrospores.
• Sporotrichosis: Rose Gardener’s Disease. Lymphocutaneous spread. Yeast: Cigar bodies (Asteroid bodies). Flower: Rosette arrangement.
• Penicillium marneffei: Bamboo rats. Red pigment.

• Chromoblastomycosis: Trauma (wood/thorn). Copper Penny bodies (Sclerotic/Medlar bodies).

Candida:

• C. albicans: Germ Tube Test (+), Chlamydospores on Cornmeal Agar.
• Microscopy: Pseudohyphae & Budding yeast.

Cryptococcus neoformans:

• Clinical: Meningitis in HIV.
• Source: Pigeon droppings.
• Stain: India Ink (Negative stain - visualizes Capsule).
• Culture: Bird Seed Agar (Brown colonies).
• Rx: Liposomal Amphotericin B + Flucytosine (Induction) -> Fluconazole (Maintenance).

Mucormycosis:

• Risk: DKA (Diabetic Ketoacidosis).
• Morphology: Aseptate (No septa), Broad hyphae, 90° (Right angle) branching.
• Contrast Aspergillus: Septate, Acute angle (45°) branching.

Pneumocystis jirovecii (PCP):

• HIV (CD4 <200).
• Appearance: Crushed Ping-Pong balls (GMS stain).
• Rx: Cotrimoxazole (DOC).

• Entamoeba histolytica: Flask-shaped ulcer. Rx: Metronidazole + Paromomycin (Luminal agent).
• Balantidium coli: Ciliated (motile), Kidney-bean nucleus. Source: Pigs.
• Free Living Amoeba:
  • Naegleria fowleri: "Brain eating amoeba". History of swimming in ponds. Enters via nose (cribriform plate) -> PAM (Primary Amoebic Meningoencephalitis).
  • Acanthamoeba: Contact lens users. Keratitis. Culture: Non-nutrient agar with E. coli overlay.
• Giardia lamblia:
  • "Old man with spectacles" appearance (2 nuclei, 4 pairs flagella).
  • Falling Leaf Motility.
  • Fatty diarrhea (Malabsorption).
  • Dx: String Test (Entero test).
• Trichomonas vaginalis:
  • Sexually transmitted. Green frothy discharge, Strawberry Cervix.
  • Twitching motility.
  • Only Trophozoite (No cyst stage).
• Hemoflagellates:
  • Trypanosoma cruzi: Chagas Disease. Kissing Bug (Reduviid). Romana's Sign (Unilateral eye swelling). Cardiomegaly/Megaesophagus.
  • Leishmania donovani: Kala Azar. Sandfly. Hepatosplenomegaly. LD Bodies (Amastigote form in macrophages). Rx: Liposomal Amphotericin B.
  • Coccidian Parasites (HIV Diarrhea): Acid Fast Oocysts.
    • Cryptosporidium: 4-6 µm (Round). Rx: Nitazoxanide.
    • Cyclospora: 8-10 µm (Round).
    • Cystoisospora: Oval shape. Rx: Cotrimoxazole.

• Malaria:
  • Blackwater Fever: P. falciparum (Hemolysis -> Dark urine).
  • Banana shaped gametocyte: P. falciparum.
• Echinococcus (Hydatid Cyst):
  • E. granulosus: Dog tapeworm. Liver cysts.
  • E. multilocularis: Invasive/Infiltrative cysts.
  • Cyst Layers: Ectocyst (Laminated), Endocyst (Germinal), Pericyst (Host derived).
• Eggs (Identification):
  • Operculated (Lid): Diphyllobothrium, Trematodes (Fasciola).
  • Spines (Schistosoma):
    • S. mansoni: Lateral spine.
    • S. haematobium: Terminal spine (Hematuria/Bladder CA).
    • S. japonicum: Rudimentary/No spine.
  • Plano-convex (D-shaped): Enterobius vermicularis (Pinworm).
  • Barrel/Drum-shaped: Trichuris trichiura (Whipworm).
• Skin Penetration: Hookworm, Strongyloides, Schistosoma.
  • Strongyloides: Parthenogenesis (Female reproduces alone). Larva currens.
  • Enterobius: Perianal itching. NIH swab / Scotch tape test.
• Filaria:
  • Wuchereria bancrofti: No nuclei at tail tip. Causes Elephantiasis.
  • Brugia malayi: Two distinct nuclei at tail tip.
  • Loa Loa: Nuclei till the tip (sheathed).

• Staphylococcus:
  • Catalase (+), Coagulase (+ for S. aureus).
  • S. aureus: Golden pigment. Mannitol Salt Agar (Yellow).
  • Food Poisoning: Dairy/Pastry. Incubation < 6 hrs. Mechanism: Vagal stimulation (preformed toxin).
  • Toxins: Enterotoxins (Food poisoning), TSST-1 (Toxic Shock Syndrome - associated with tampons).
• Streptococcus:
  • Catalase (-). Chains.
  • S. pneumoniae (Pneumococcus): Alpha hemolytic (Green). Diplococci. Optochin Sensitive. Bile Soluble. Capsule (+). "Carrom Coin" / Draughtsman colonies.
  • S. pyogenes (Group A): Beta hemolytic. Bacitracin Sensitive. Rheumatic fever/PSGN.
  • S. agalactiae (Group B): CAMP Test (+) (Arrowhead hemolysis). Neonatal meningitis.
  • Enterococcus: Bile Aesculin hydrolysis (+).

• Neisseria:
  • N. meningitidis: Ferments Maltose & Glucose (M for Meningitis & Maltose).
  • N. gonorrhoeae: Ferments Glucose only.
  • Media: Thayer Martin / NYC medium. Transport: Amies medium.

• Corynebacterium diphtheriae:
  • Appearance: Chinese Letter / Cuneiform.
  • Granules: Volutin/Babes-Ernst (Metachromatic).
  • Stains: Albert Stain (Green bacteria, Black granules). Ponder/Neisser stains.
  • Culture: Loeffler's Serum Slope (Rapid/Enriched), Potassium Tellurite (Selective/Black colonies).
  • Toxin Test: Elek's Gel Precipitation test.
  • Susceptibility: Schick Test.
• Bacillus anthracis:
  • Bamboo Stick appearance.
  • Medusa Head colonies.
  • Inverted Fir Tree (Gelatin stab).
  • String of Pearls (on Penicillin agar).
  • Capsule: Polypeptide (P for Polypeptide).
• Clostridium:
  • C. perfringens: Gas Gangrene. Subterminal spores. Double zone hemolysis (Target hemolysis). Nagler Reaction (Opalescence on Egg Yolk Agar). Reverse CAMP test.
  • C. tetani: Terminal spores (Drumstick/Tennis racket). Swarming motility.
  • C. difficile: Antibiotic-associated diarrhea (Clindamycin/Cephalosporins). Pseudomembranous Colitis. Toxin A & B. Rx: Oral Vancomycin.

• Enterobacteriaceae:
  • Lactose Fermenters (Pink on MacConkey): E. coli, Klebsiella.
  • Non-Lactose Fermenters (Pale/Yellow): Salmonella, Shigella, Proteus.
• E. coli Types:
  • EPEC: Pediatric diarrhea.
  • ETEC: Traveler's diarrhea.
  • EHEC: Hemorrhagic. HUS (Hemolytic Uremic Syndrome) - Anemia, Thrombocytopenia, Renal failure. Strain O157:H7.
  • EAEC: Persistent diarrhea (>14 days). Stacked Brick appearance.
• Vibrio cholerae:
  • Comma shaped. Darting motility.
  • Media: TCBS (Yellow colonies - Sucrose fermenter).
  • Rice Water Stools.
  • Toxin: ADP ribosylation (Increases cAMP).
• Proteus: Swarming motility (Differentiate from C. tetani by Gram stain). Urease (+).

• Haemophilus ducreyi: Chancroid. Painful ulcer. School of Fish appearance.
• Klebsiella granulomatis (Donovanosis): Painless beefy red ulcer. Safety pin appearance.
• Rickettsia (Scrub Typhus): Organism: Orientia tsutsugamushi. Vector: Chiggers (Mite larvae). Eschar (Black scab).

• Rotavirus:
  • Most common cause of diarrhea in children.
  • Double Stranded RNA (Exception: Most RNA are ss).
  • Segmented (11 segments). Wheel shape.
  • Toxin: NSP4.

General Rules (Exceptions):

• All DNA viruses are dsDNA EXCEPT Parvovirus B19 (ssDNA).
• All RNA viruses are ssRNA EXCEPT Reovirus/Rotavirus (dsRNA).
• All RNA viruses are single copy EXCEPT Retrovirus (2 copies).
• Segmented Viruses (Mnemonic: BORA/BIRA): Bunyavirus, Orthomyxovirus (Influenza), Reovirus, Arenavirus.

• Influenza: Orthomyxovirus. 8 Segments. HA (Entry) and NA (Exit) spikes.
• Herpes Viruses:
  • HHV-8: Kaposi Sarcoma (Vascular tumor, HIV patients).
  • EBV (HHV-4): Infectious Mononucleosis (Glandular fever). Target: B Cells (CD21). Downey Cells (Atypical lymphocytes - "Ballerina skirt"). Test: Paul Bunnell (Heterophile Ab). Cancers: Burkitt's Lymphoma, Nasopharyngeal CA.
• Parvovirus B19:
  • Slapped Cheek rash (Fifth Disease/Erythema Infectiosum).
  • Aplastic Crisis in Sickle Cell/Thalassemia patients.
• Hepatitis B Markers:
  • HBsAg: First marker (Infection).
  • HBeAg: High Infectivity/Replication.
  • Anti-HBc IgM: Acute infection.
  • Anti-HBc IgG: Chronic infection or Past infection.
  • Anti-HBs: Immunity (Vaccine or Recovery).
• Measles: 3 Cs (Cough, Coryza, Conjunctivitis). Koplik Spots. Rash starts behind ears. Subacute Sclerosing Panencephalitis (SSPE) is a complication.
• Rubella: German Measles. Congenital Rubella Syndrome (Cataract, Deafness, PDA).
• Ebola: Filovirus. Vector: Bats.
• Nipah: Vector: Fruit Bats.
• Japanese Encephalitis: Vector: Culex mosquito. Amplifier host: Pig. Vaccine: JENVAC.