3rd Internal Assessment MBBS Practical Exam - Microbiology

Q2. Gram Stain from the Provided Culture Plate

a) How to Identify the Culture Plate

• Type of medium (Blood Agar, MacConkey, Chocolate Agar, CLED, etc.)
• Colony characteristics visible on the plate (size, color, hemolysis, etc.)
• Smell (e.g., sweet grape-like smell of Pseudomonas)

b) Colony Characteristics to Note

1. Size - pin-point, small, medium, large (in mm)
2. Shape - circular, irregular, filamentous, rhizoid
3. Elevation - flat, raised, convex, umbonate, umbilicate
4. Edge/margin - entire, undulate, lobate, filamentous, curled
5. Color - creamy white, golden yellow, grey, pink, etc.
6. Consistency/texture - smooth (S-form), rough (R-form), mucoid
7. Surface - glistening, dull, matte
8. Opacity - opaque, translucent, transparent
9. Hemolysis (on blood agar): Alpha (partial/greenish), Beta (complete/clear), Gamma (none)
10. Odor if distinctive

c) Gram Stain Procedure

1. Prepare smear - Transfer a loopful of colony material onto a clean glass slide; spread thinly
2. Air dry the smear
3. Heat fix - Pass slide through Bunsen flame 2-3 times (or methanol fix)
4. Apply Crystal violet (primary stain) - flood slide for 1 minute; rinse with water
5. Apply Gram's iodine (mordant) - 1 minute; rinse with water
6. Decolorize with acetone-alcohol - 15-30 seconds until no more violet color runs; rinse immediately with water
7. Counterstain with Safranin - 1 minute; rinse with water
8. Blot dry gently; examine under oil immersion (100x objective)

• Gram-positive organisms = Purple/violet (retain crystal violet-iodine complex)
• Gram-negative organisms = Red/pink (lose crystal violet; take up safranin)

          [Oil Immersion 100x]
  
  ●●●●  ← Gram +ve cocci (purple) e.g. Staphylococcus
     ≈≈≈≈≈ ← Gram -ve bacilli (pink/red) e.g. E. coli
  
  Background = red (Gram -ve stained material)
  WBCs = red/pink

d) Provisional Diagnosis and Further Tests

• Provisional diagnosis: Staphylococcal infection
• Further confirmatory tests:
  1. Catalase test (differentiates Staphylococcus from Streptococcus - positive = Staph)
  2. Coagulase test (tube and slide) - positive = S. aureus
  3. Mannitol salt agar fermentation
  4. DNase test
  5. Antibiotic sensitivity testing (AST/ABST)

• Provisional diagnosis: Gram-negative enteric infection
• Further tests:
  1. Oxidase test
  2. IMViC reactions (Indole, Methyl Red, Voges-Proskauer, Citrate)
  3. Triple Sugar Iron (TSI) agar
  4. Urease test
  5. Biochemical identification panel / API strips
  6. Antibiotic susceptibility testing

Q3. Z-N (Ziehl-Neelsen) Stain

b) Perform Z-N Stain - Procedure

1. Prepare and fix the smear - Air dry, then heat fix
2. Flood with Carbol fuchsin (concentrated phenol + basic fuchsin dye)
3. Gently heat the slide by passing a flame under until steam rises (do NOT boil) - 3-5 minutes. This drives the stain into the waxy mycobacterial cell wall
4. Rinse with water
5. Decolorize with 20% H₂SO₄ or 3% acid-alcohol - until only a slight pink tinge remains
6. Rinse with water
7. Counterstain with methylene blue - 1-2 minutes
8. Rinse, blot dry, and examine under oil immersion (100x)

• Acid-fast bacilli (AFB) = Bright red/magenta rods against a blue background
• Non-acid-fast organisms and background = Blue

[Oil Immersion 100x Field]

  ═══  ←  AFB (acid-fast bacilli): bright RED, slender rods, 
           may be beaded, slightly curved
           e.g. Mycobacterium tuberculosis
  
  Background, WBCs, non-AFB = BLUE (methylene blue counterstain)

b) Type of Stain

• The Kinyoun method is the cold variant (no heating; uses more concentrated carbol fuchsin)
• The fluorochrome (auramine-rhodamine) method is the fluorescent variant

c) Different Acid-Fast Structures

d) Modified Z-N Stain

• Standard Z-N uses: 20% H₂SO₄ (strong acid)
• Modified Z-N uses: 1% H₂SO₄ (weak acid) or 0.5-3% acetic acid

Q4. Stool Microscopy

a) Identify the Parasite - Morphological Forms

        Outer wall (smooth)
         ___
        /   \
       | *  * |  ← 4 nuclei with central karyosome
       |  ## |  ← Chromatoid body (cigar-shaped)
        \___/
        
   Size: 10-20 µm

Summary Reference Table for Viva