Practical: Microscopy Technique
Microscopy is the technique of using a microscope to view objects that cannot be
seen clearly by the naked eye. It allows scientists, especially in medical
laboratories, biology, and research, to study the structure and details of tiny
organisms, cells, and tissues.
Basic Parts of a Light Microscope
Part Function
Eyepiece (Ocular lens) Magnifies the image (usually 10x).
Objective lenses Different magnifications (e.g., 4x, 10x, 40x, 100x).
Stage Holds the slide.
Condenser & Iris diaphragm Focus and adjust light.
Light source/Mirror Provides illumination.
Coarse & Fine focus knobs Bring the image into clear focus.
Arm & Base Support and stability.
Principle of Microscopy
Works on magnification and resolution.
o Magnification = enlarging the image.
o Resolution = ability to distinguish two close points as separate.
Uses lenses to bend (refract) light to produce an enlarged image.
Objective
To learn:
The correct technique for focusing a light microscope at low and high
power.
Preparation and observation of a red blood cell suspension using normal
saline.
Observation of stained Peripheral Blood Films (PBF) for different
hematological conditions.
Materials Required
✅ Light microscope (functional and clean)
✅ Clean glass slides
✅ Normal saline (NS)
✅ Fresh whole blood
✅ Pre-stained PBF slides (Anaemia, Leukaemia, Leukopenia)
✅ Droppers
✅ Immersion oil
✅ Lens tissue / lens paper
Part A: Focusing the Microscope
1. Place the microscope on a flat, stable surface.
2. Plug in and switch on the light source (or adjust the mirror for natural light).
3. Place a slide on the stage and secure with stage clips.
4. Use the low power objective (10x):
o Bring the objective lens close to the slide using the coarse focus knob
while looking from the side.
o Look through the eyepiece and slowly move the coarse knob away
from the slide until the image appears.
o Fine-tune with the fine focus knob.
5. Switch to high power objective (40x):
o Center the area of interest.
o Use only the fine focus knob to sharpen the image.
6. For oil immersion (100x):
o Rotate the nosepiece half-way between 40x and 100x.
o Place a small drop of immersion oil on the slide.
o Click the 100x lens into place so it touches the oil.
o Focus gently with the fine focus only.
Part B: Preparation & Observation of Red Cell Suspension
1. Place a clean slide on the bench.
2. Add 1 drop of normal saline near the center of the slide.
3. Using a clean stick or pipette, add 1 small drop of fresh blood into the
saline.
4. Mix gently using a corner of another slide or a stick.
5. Cover with a cover slip if available (optional).
6. Place on the stage.
7. Observe under 10x, then 40x:
o Look for the shape, size, and arrangement of red blood cells.
Part C: Observation of Stained Peripheral Blood Films
1. Select the stained PBF slide (e.g., anaemic, leukemic, leukopenic).
2. Place on the microscope stage.
3. Focus first under 10x to locate the monolayer area (where cells are evenly
spread).
4. Switch to 40x to examine cell distribution.
5. Apply a drop of immersion oil directly onto the smear.
6. Rotate to 100x oil immersion objective.
7. Use only the fine focus knob to clearly view the morphology:
o Examine red cells, white cells, and platelets.
o Note abnormalities (e.g., reduced RBCs, blast cells, low WBC count).
Results Recording
Slide Type Objective Lens Used Observations
Red Cell Suspension 10x, 40x e.g., normal size, shape, arrangement
Anaemic PBF 100x (Oil) e.g., Size, staining characteristic
Leukemic PBF 100x (Oil) e.g., blast cells, increased WBCs
Leukopenic PBF 100x (Oil) e.g., decreased WBCs
Care and Maintenance of a Microscope
Proper care keeps the microscope in good working condition, ensures clear images,
and extends its lifespan.
1. General Handling
Always carry the microscope with both hands — one hand holding the
arm and the other supporting the base.
Place it on a flat, stable surface, away from edges to prevent falls.
Keep it away from water, chemicals, and direct sunlight.
2. Before Use
Make sure lenses are clean and free from dust or oil.
Check that the light source or mirror is clean and properly aligned.
Adjust the light and condenser properly for best illumination.
3. During Use
Start focusing with the lowest power objective (10x) before moving to
higher powers.
Use coarse focus knob only with low power lenses; use fine focus knob
with high power and oil immersion lenses.
Never force the knobs — they should turn smoothly.
Do not drag the oil immersion lens through oil when switching
objectives — rotate carefully.
4. After Use
If you used immersion oil, clean the 100x objective lens immediately:
o Use special lens paper or soft lint-free tissue — never rough cloth
or tissue.
o Gently wipe off the oil, then clean with a drop of lens cleaning
solution if needed.
Lower the stage or raise the objectives fully.
Switch to the lowest power objective (10x) for storage.
Unplug the microscope if it has an electrical light source.
5. Cleaning
Part How to Clean
Lenses (eyepiece & Use only lens paper or lens cleaning solution. Wipe
objectives) gently in circular motion.
Body and stage Wipe with soft, damp cloth. Avoid harsh chemicals.
Clean carefully with lens paper if dusty. Avoid
Mirror
scratching.
6. Storage
Cover the microscope with a dust cover or store it in its box/cabinet when
not in use.
Store in a dry, clean place — moisture can lead to fungal growth on lenses.
Keep away from corrosive chemicals.
7. Precautions
Never attempt to dismantle lenses or internal parts.
Report mechanical or optical problems to the lab technician or supervisor.
Do not touch lenses with fingers — skin oils can damage optical surfaces.
Handle slides carefully to avoid scratching the stage.
