You are given a whole blood sample labeled A. With the materials provided, a) Determine ABO and Rhesus group of sample A. b) Determine the PCV of sample A and comment on your result.

Here are the answers to your practical examination questions:

1. Whole Blood Sample A

• 1.a. Determine ABO and Rhesus group of sample A.

  • Procedure (ABO Forward Grouping):
    1. Place one drop each of anti-A, anti-B, and anti-D sera into separate labeled sections on a tile or in tubes.
    2. Add one drop of a 5% red cell suspension of sample A to each serum.
    3. Mix gently and observe for agglutination macroscopically and microscopically (if on tile).
  • Procedure (ABO Reverse Grouping):
    1. Place one drop each of known A cells and B cells into separate labeled sections on a tile or in tubes.
    2. Add two drops of plasma/serum from sample A to each cell suspension.
    3. Mix gently and observe for agglutination.
  • Interpretation: Agglutination with anti-A indicates A antigen; with anti-B indicates B antigen. Agglutination with anti-D indicates Rh positive. Reverse grouping confirms ABO type by detecting anti-A and/or anti-B antibodies in plasma.

• 1.b. Determine the PCV of sample A and comment on your result.

  • Procedure:
    1. Fill a plain capillary tube (for non-anticoagulated blood) or a heparinized capillary tube (for anticoagulated blood) with sample A, leaving about 15 mm unfilled.
    2. Seal one end of the capillary tube with clay or by heating.
    3. Place the sealed tube in a microhematocrit centrifuge with the sealed end facing outwards.
    4. Centrifuge at 10,000-12,000 rpm for 5 minutes.
    5. Read the Packed Cell Volume (PCV) using a microhematocrit reader.
  • Comment on Result: Compare the obtained PCV value to the normal reference range (e.g., 0.40-0.54 L/L for adult males, 0.37-0.47 L/L for adult females). A value below the range suggests anemia, while a value above the range suggests polycythemia.

• 1.c. Make a suitable film of sample A and stain for determination of red cell morphology.

  • Procedure (Thin Film Preparation):
    1. Place a small drop of blood (sample A) near one end of a clean, grease-free glass slide.
    2. Place a spreader slide in front of the blood drop at a 30-45 degree angle.
    3. Draw the spreader back into the drop, allowing the blood to spread along its edge.
    4. Push the spreader smoothly and rapidly forward to create a thin film with a feathered edge.
    5. Allow the film to air dry completely.
  • Procedure (Staining - e.g., Leishman stain):
    1. Place the dried film on a staining rack.
    2. Flood the slide with undiluted Leishman stain for 1-2 minutes (for fixation).
    3. Add an equal volume of buffered distilled water (pH 6.8) to the stain, mix gently by blowing, and allow to stand for 5-10 minutes (for staining).
    4. Wash gently with buffered distilled water until the film appears pinkish-purple.
    5. Allow to air dry vertically.

• 1.d. What 2 precautions did you take when making the film.

  1. Ensure the glass slides are clean and grease-free to prevent artifacts and ensure even spreading of the blood.
  2. Use the correct angle and speed when pushing the spreader slide to produce a thin, uniform film with a good feathered edge, avoiding thick areas or streaks.

2. Histological Sections B and C

• 2.a. Stain slide B to demonstrate general tissue morphology.

  • Procedure (Hematoxylin and Eosin - H&E stain):
    1. Deparaffinize slide B in xylene (2 changes, 5 minutes each).
    2. Rehydrate through graded alcohols (100%, 90%, 70% ethanol, 2 minutes each) to distilled water.
    3. Stain with Hematoxylin for 5-10 minutes.
    4. Wash in running tap water for 5 minutes.
    5. Differentiate in 1% acid alcohol (1-2 dips).
    6. Blue in Scott's tap water or 0.2% ammonia water for 1-2 minutes.
    7. Wash in running tap water for 5 minutes.
    8. Stain with Eosin for 1-5 minutes.
    9. Dehydrate through graded alcohols (70%, 90%, 100% ethanol, 2 minutes each).
    10. Clear in xylene (2 changes, 5 minutes each).
    11. Mount with a coverslip using a suitable mounting medium.

• 2.b. Stain slide C to demonstrate connective tissue.

  • Procedure (Masson's Trichrome stain):
    1. Deparaffinize slide C in xylene (2 changes, 5 minutes each).
    2. Rehydrate through graded alcohols to distilled water.
    3. Mordant in Bouin's solution at $\text{56}^\circ\text{C}$ for 1 hour or at room temperature overnight.
    4. Wash in running tap water until yellow color disappears (approx. 5-10 minutes).
    5. Stain with Weigert's Iron Hematoxylin for 10 minutes.
    6. Wash in running tap water for 5 minutes.
    7. Stain with Biebrich Scarlet-Acid Fuchsin solution for 10-15 minutes.
    8. Rinse in distilled water.
    9. Differentiate in Phosphomolybdic-Phosphotungstic Acid solution for 10-15 minutes.
    10. Stain with Aniline Blue solution for 5-10 minutes.
    11. Rinse in distilled water.
    12. Differentiate in 1% Acetic Acid solution for 2-5 minutes.
    13. Dehydrate rapidly through 95% and absolute alcohol.
    14. Clear in xylene (2 changes).
    15. Mount with a coverslip.
  • Expected Result: Collagen fibers stain blue, nuclei stain black, cytoplasm, muscle, and red blood cells stain red.

3. Anaerobic Culture Sample D

• 3.a. Make a film and stain by Gram's method. Comment on your findings.

  • Procedure (Gram Stain):
    1. Prepare a thin smear of sample D on a clean glass slide and air dry.
    2. Heat fix the smear by passing it through a flame a few times.
    3. Flood the smear with Crystal Violet for 1 minute.
    4. Rinse gently with water.
    5. Flood with Gram's Iodine for 1 minute.
    6. Rinse gently with water.
    7. Decolorize with Acetone-Alcohol for 10-20 seconds until the runoff is clear.
    8. Rinse immediately with water.
    9. Flood with Safranin for 1 minute.
    10. Rinse gently with water, air dry, and examine under oil immersion.
  • Comment on Findings: Report the Gram reaction (Gram-positive or Gram-negative), morphology (cocci, bacilli, spirilla), and arrangement (clusters, chains, pairs). For example, "Gram-positive cocci in clusters" or "Gram-negative bacilli."

• 3.b. Pour a blood agar plate and subculture identical colonies from D on it.

  • Procedure (Pouring Blood Agar Plate):
    1. Prepare blood agar base medium according to manufacturer's instructions, sterilize by autoclaving, and cool to $\text{45-50}^\circ\text{C}$.
    2. Aseptically add 5-10% sterile defibrinated sheep or horse blood to the cooled base medium and mix gently.
    3. Pour approximately 15-20 mL of the blood agar mixture into sterile Petri dishes on a level surface.
    4. Allow to solidify completely.
  • Procedure (Subculturing):
    1. Using a sterile inoculating loop, aseptically pick an isolated, identical colony from the original anaerobic culture of sample D.
    2. Streak the picked colony onto the surface of the freshly prepared blood agar plate using the quadrant streaking method to obtain isolated colonies.
    3. Incubate the plate under appropriate conditions (e.g., $\text{35-37}^\circ\text{C}$ for 18-24 hours, potentially anaerobically if the original culture was strictly anaerobic).

• 3.c. Make and examine a wet preparation from D. Comment on your finding.

  • Procedure (Wet Preparation):
    1. Place a drop of sterile normal saline on a clean glass slide.
    2. Using a sterile inoculating loop, pick a small amount of sample D and emulsify it in the saline drop.
    3. Cover with a coverslip, avoiding air bubbles.
    4. Examine immediately under a microscope, first with 10x and then 40x objective, reducing light for better contrast.
  • Comment on Finding: Observe for bacterial motility (e.g., "motile bacilli observed") and general morphology. Also note the presence of any other elements like yeast or host cells.

4. Urine Sample E

• 4.a. Perform a test to determine the presence of reducing sugars.

  • Procedure (Benedict's Test):
    1. Add 5 mL of Benedict's reagent to a test tube.
    2. Add 8 drops (approximately 0.5 mL) of urine sample E to the reagent.
    3. Mix the contents and heat the test tube in a boiling water bath for 3-5 minutes.
    4. Observe the color change.
  • Expected Result: A color change from blue to green, yellow, orange, or brick-red precipitate indicates the presence of reducing sugars. The intensity of the color/precipitate correlates with the concentration.

• 4.b. Perform a test to determine the presence of proteins.

  • Procedure (Heat and Acetic Acid Test):
    1. Fill a test tube two-thirds full with clear urine sample E.
    2. Heat the upper portion of the urine gently over a flame until it boils.
    3. Observe for turbidity (cloudiness), which indicates protein or phosphates.
    4. Add 3-5 drops of 10% acetic acid to the heated portion.
    5. Re-boil the upper portion.
    6. Observe for changes in turbidity.
  • Expected Result: If turbidity persists or increases after adding acetic acid and re-boiling, protein is present. If turbidity disappears, it was due to phosphates.

5. Blood Sample F and Stool Sample G

• 5.a. Prepare and stain a thick film from blood sample F for examination for malaria parasites.

  • Procedure (Thick Film Preparation):
    1. Place a large drop of blood (sample F) in the center of a clean glass slide.
    2. Using the corner of another slide or an applicator stick, spread the blood in a circular motion to form a thick, opaque film about the size of a dime (approximately 1 cm diameter).
    3. Allow the film to air dry completely in a horizontal position (do not heat fix).
  • Procedure (Staining - e.g., Giemsa stain):
    1. Place the dried thick film in undiluted Giemsa stain for 10-15 minutes (this lyses red blood cells and stains parasites).
    2. Rinse gently with buffered distilled water (pH 7.2) until the film appears bluish-purple.
    3. Allow to air dry vertically.
  • Examination: Examine the stained thick film under oil immersion objective for the presence of malaria parasites (trophozoites, schizonts, gametocytes).

• 5.b. Examine and carry out routine tests on stool Sample G. Comment your findings.

  • Procedure (Macroscopic Examination):
    1. Observe the color (e.g., brown, pale, black), consistency (e.g., formed, soft, watery, hard), and presence of any abnormal components like blood, mucus, adult worms, or proglottids.
  • Procedure (Microscopic Examination - Wet Mount):
    1. Prepare a saline wet mount: Mix a small portion of stool with 0.85% saline on a slide, cover with a coverslip.
    2. Prepare an iodine wet mount: Mix a small portion of stool with Lugol's iodine on a slide, cover with a coverslip.
    3. Examine both mounts under low (10x) and high power (40x) objectives for ova, cysts, trophozoites of parasites, red blood cells, white blood cells, yeast, and undigested food fibers.
  • Procedure (Chemical Examination - Fecal Occult Blood Test):
    1. Apply a thin smear of stool sample G to the test area of a guaiac card.
    2. Add a drop of developer solution to the test area.
    3. Observe for a color change (e.g., blue color indicates presence of blood).
  • Comment on Findings: Report all observations, for example: "Macroscopic: Soft, brown stool, no visible blood or mucus. Microscopic: Few Giardia lamblia cysts observed, no ova or trophozoites. Chemical: Fecal occult blood test negative."