BackLAB 1: Microscopy in Microbiology: Use, Care, and Observation of Microorganisms
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Observing Microorganisms Through a Microscope
Introduction to the Compound Microscope
The compound light microscope is an essential tool in microbiology, allowing for the visualization of microorganisms that are otherwise invisible to the naked eye. It uses a system of lenses and illumination to magnify specimens, making it possible to study the structure and morphology of bacteria, fungi, algae, and protozoa.
Objective: To learn the parts of the microscope, practice focusing at increasing magnifications, and identify microbial morphologies.
Applications: Used for observing cell size, shape, and arrangement in various microorganisms.
Parts of the Compound Microscope and Their Functions
Understanding the function of each microscope part is crucial for proper use and maintenance.
Base: Provides stability and houses the illuminator.
Ocular/Eyepiece: Usually 10× magnification; remagnifies the image formed by the objective lens.
Arm: Used for carrying the microscope safely.
Body Tube: Transmits the image from the objective to the ocular lens.
Stage: Platform for holding slides.
Coarse Adjustment: Large knob for initial focusing at low power.
Fine Adjustment: Small knob for precise focusing at high power and oil immersion.
Condenser: Focuses light onto the specimen.
Iris Diaphragm: Adjusts the amount and angle of light for contrast and brightness.
Objective Lenses: Primary magnification lenses (typically 4×, 10×, 40–45×, 100×).
Numerical Aperture (N.A.): Engraved on the objective; determines resolving power.
Magnification and Resolving Power
Magnification is the product of the ocular and objective lens powers. Resolving power (RP) is the ability to distinguish two points as separate and is a critical measure of microscope performance.
Total Magnification:
Resolving Power Formula:
Light Microscopes: Resolution ~200 nm.
Electron Microscopes: Resolution <0.2 nm.
Improving RP: Use shorter wavelength light or increase N.A.
Use of Immersion Oil
Immersion oil is used with the 100× objective to increase resolution by reducing light refraction.
Refractive Index: Glass and oil have the same refractive index (N = 1.52), minimizing light loss.
Application: Place a drop of oil on the slide before rotating the 100× objective into position.
Care: Only use oil with the 100× objective and clean immediately after use with lens paper.
Rules for Microscope Use and Care
Proper handling and maintenance are essential for accurate results and instrument longevity.
Carry with two hands (base and arm).
Do not tilt the microscope.
Keep both eyes open to reduce eyestrain.
Focus away from the slide to avoid damage.
Use slow, careful movements, especially at high power.
Start with low power and progress to higher magnifications.
Keep stage and lenses oil-free except for the oil immersion lens.
Use only lens paper for cleaning lenses.
Wipe oil off objectives before storage.
Cover and return the microscope after use.
Report any problems immediately.
Microscope Operation Procedure
Follow these steps for optimal observation and safety:
Place the microscope securely on the bench.
Load the prepared slide into the stage clips.
Adjust binocular eyepieces for a single field of view.
Focus the condenser and adjust the iris diaphragm for optimal illumination.
Focus at low power (10×), center the specimen, and sketch general cell size and shape.
Switch to high-dry (40–45×), use fine adjustment, and increase light as needed.
For oil immersion (100×), add oil, fine-adjust, and record observations.
After observation, clean the oil objective and slide, and return to low power before removing the slide.
Field of Vision and Scale
The field of vision decreases as magnification increases, affecting how much of the specimen is visible at once.
4× (Scanning): ~4.5 mm field
10× (Low): ~1.8 mm field
40–45× (High-dry): ~0.4 mm field
100× (Oil): ~0.18 mm field
Example Calculation: If a Bacillus cell is 2 μm long, then cells fit end-to-end across a 2 mm field.
Observing Microbial Morphologies
The Three Basic Bacterial Morphologies
Bacteria display three primary shapes, each with characteristic arrangements:
Bacillus (Bacilli): Rod-shaped; can be single, paired (diplobacilli), or in chains (streptobacilli). Examples: Escherichia coli, Bacillus subtilis.
Coccus (Cocci): Spherical; can be single, paired, in clusters (staphylo-), or chains (strepto-). Examples: Staphylococcus (clusters), Streptococcus (chains).
Spiral (Spirilla/Spirchetes): Curved or helical rods; rigid (spirilla) or flexible (spirochetes).
Relative Sizes of Microorganisms
Microorganisms vary greatly in size, which affects their visibility under different magnifications:
Bacteria: Smallest cells, typically 1–5 μm in length.
Yeast cells: About 10 μm in diameter.
Algae and Fungi: Larger, often multicellular; visible at lower magnifications.
Protozoa: Single-celled eukaryotes, larger than bacteria.
Prepared Slides: What to Observe
Each type of microorganism requires different objectives for optimal observation:
Algae: High-dry (40–45×) is usually sufficient; look for cell walls, chloroplasts, and filamentous structures.
Fungi: High-dry or oil immersion; observe hyphae (branching filaments) and spores.
Protozoa: Low to high-dry; look for cilia, flagella, or pseudopods.
Bacteria: Oil immersion (100×) only; sketch all three morphologies if present.
Recording Observations and Drawing Conclusions
Accurate record-keeping is essential for scientific study:
Note microscope number and type (monocular/binocular).
Record eyepiece adjustment settings.
Sketch representative cells at each magnification and label total magnification.
For bacteria, sketch all observed shapes and arrangements.
Answer in-procedure questions and summarize findings, including the largest and smallest organisms observed and the effect of magnification on the field of vision.
