Microscope Notes

Observing Organisms Through a Microscope

Introduction

• This unit covers observing organisms through a microscope.

• Key topics include:

  • Wavelength and the metric system

  • Compound light microscope

    • Magnification

    • Field of view

    • Depth of focus

    • Microscope resolution

    • Increasing contrast

  • Wavelength

  • Numerical aperture

  • Oil immersion

  • Stains (for both dead and live samples)

    • Simple vs. differential stains

  • Phase contrast (for live samples)

  • Dark field (for live samples)

  • Fluorescence

Aims

• Identify the parts of a microscope.

• Understand how a brightfield microscope functions.

• Learn how to use the microscope safely.

Microscope Sizes and Types

• Microbes vary in size, necessitating different types of microscopes.

• The sizes of organisms commonly studied range as indicated:

  • DNA double helix: 0.1 \text{ nm}

  • Atomic force microscope (AFM): 1 \text{ nm} - 10 \text{ nm}

  • Transmission electron microscope (TEM): 10 \text{ pm} - 100 \mu\text{m}

  • Bacteriophages (viruses): 50 \text{ nm}

  • E. coli bacteria: 1 \mu\text{m}

  • Scanning electron microscope (SEM): 10 \text{ nm} - 1 \text{ mm}

  • Red blood cells: 4 \mu\text{m}

  • Light microscope (LM): 200 \text{ nm} - 10 \text{ mm}

  • Tick: 200 \mu\text{m}

  • Unaided eye: Up to 1 \text{ m}

• Key Concept: Microscopes magnify small objects. The size of the specimen determines which microscope can be used effectively due to different resolution ranges.

How Microscopes Work

• Microscopes use lenses to change the path of light (diffraction).

Lenses and Refraction

• Lenses change the path of light rays through refraction.

• Convex lenses are commonly used.

Compound Light Microscope

• Uses a series of lenses and visible light for illumination.

• Lenses magnify the object's size.

• Also known as bright-field microscopy.

• Total magnification depends on the magnification of the ocular and objective lenses.

• Maximum magnification is about 2000X due to the resolution limit of visible light.

Light Path in a Compound Microscope

• Light from a source passes through the condenser.

• Condenser lenses direct light rays through the specimen.

• Light rays pass into the objective lens.

• The image is magnified again by the ocular lens.

Parts and Functions

• Iris diaphragm:

  • Regulates the amount of light that reaches the specimen.

  • Open: Increases the number of photons, increasing resolution.

  • Closed: Decreases the number of photons, increasing contrast.

• Condenser: Focuses light onto the specimen.

• Lamp/light source: Provides illumination.

Lenses and Magnification

• Ocular lens: 10x magnification.

• Objective lenses:

  • Scanning: 4x

  • Low Power: 10x

  • High Power: 40x

  • Oil Immersion: 100x

• Total Magnification: (ocular magnification) x (objective magnification).

  • Example: Scanning: (10\text{x}) \text{ x } (4\text{x}) = 40\text{x}

• Revolving nosepiece: Holds objective lenses and allows for easy switching.

Additional Parts

• Arm: Supports objective/oculars and is used to carry the microscope.

• Stage: Place for the slide/specimen.

• Slide Holder/Stage Clip: Holds the slide and specimen.

• Lamp or Illuminator: Illuminates the specimen.

• Base: Supports the microscope and is used to carry it.

• Power Switch: Turns the light source on and off.

• Light intensity knob/Voltage regulator: Adjusts the brightness of the light source.

• X-Y Axis/Stage Adjustment Knob: Moves the stage right, left, backwards, or forwards.

• Fine Adjustment Knob: Used for detailed focusing.

• Coarse Adjustment Knob: Used for initial focusing (scanning lens only).

Microscopy Theory and Terms

• Magnification

• Depth of field

• Focal point

Size and Magnification

• As magnification increases (4X -> 10X -> 40X), the size of the object in view increases while the field of view decreases.

Depth of Field

• Depth of Field (or Focus): The thickness of the specimen that is in focus at one time.

Field of View

• Field of View: The area visible through the microscope.

• Examples:

  • High magnification: 28 mm, 35 mm, 50 mm

  • Low magnification: 70 mm

Microscope Resolution

• Resolution is the ability to distinguish between two points.

Resolution Explained

• Microscope Resolution: A microscope's ability to distinguish two points a specific distance apart.

• Higher resolution: Objects closer together can be distinguished as independent entities.

Awareness of Textures

• Resolution allows us to see textures we wouldn't otherwise be aware of.

Resolving Power

• A microscope with a resolving power of 0.4 \text{ nm} can distinguish two points if they are at least 0.4 \text{ nm} apart.

• The shorter the wavelength used, the greater (better) the resolution.

• White light can resolve down to 0.2 \mu\text{m}.

• This limits the magnification of a light microscope to about 2000X.

Numerical Aperture and Resolving Power

• Resolving power depends on the numerical aperture of the lens.

• Numerical aperture indicates the lens’s ability to gather light.

• The higher the numerical aperture, the greater the resolving power of the lens.

• Examples: 4X (NA 0.1), 100X (NA 1.25).

Numerical Aperture

• NA is the light-gathering capacity of the objective.

• Limit of resolution = \frac{0.61\lambda}{NA}

  • Minimum distance between two objects to be seen as independent entities.

  • \lambda = Wavelength of illumination.

• NA \text{ (Numerical Aperture)} = n \sin(\alpha)

  • n = Refractive index of air or liquid.

  • \alpha = Aperture angle between specimen and lens.

• The NA of each objective lens is inscribed in the metal tube, ranging from 0.25-1.4.

• The higher the NA, the better the light-gathering properties and resolution of the lens.

Oil Immersion Lens

• At high magnifications, light rays refract from a straight path by bending away from the specimen.

• Immersion oil is added between the lens and slide to preserve the direction of light rays.

• Immersion oil has the same refractive index as glass, becoming part of the microscope's optics.

• Exercise caution when using oil.

Proper Handling of Microscope

• Always use the microscope that corresponds to your seat.

• Carry using both hands (one on the arm and one under the base).

• Set the microscope down gently and never drag it.

• Ensure the cord is not dangling off the desk when plugged in.

• Clean lenses before and after use.

Microscope Concepts: Getting Started

• Notify your professor if the microscope is not in good shape.

• Quickly clean lenses with lens paper.

• Illumination: Maximum power, iris closed.

• Always start on 4X power and take the stage all the way up.

• Place the object in the light path.

• Use coarse focus (stage down) until the object is in focus (around 45° angle rotation). Center the object in the field of view.

• Move to 10X power. Fine focus and center.

• Move to 40X power. Fine focus and center.

• Move 40X objective away, add oil drop to slide, move to 100X objective (careful not to touch the stage).

• Fine focus and center. Open iris (more light) as needed for a brighter background.

Microscope Concepts: Putting It Away

• Remove any slide.

• Clean lenses, especially the 40X and 100X:

  1. Use lens paper and rotate until no oil comes off.

  2. Apply lens cleaner to paper and rotate on the lens.

• Clean the stage (no oil left).

• Lower the stage all the way.

• Point the 4X lens at the stage.

• Turn the dimmer all the way down.

• Wrap the cord and put it back in the drawer.

Staining

• Staining increases contrast between the sample and background.

• Unstained and stained specimens in brightfield illumination are shown for comparison.

Staining and Visualization

• Allows visualization by light microscopy.

• Stains are salts composed of a positive and a negative ion, one of which is colored (chromophore).

• Basic dyes: Color is in the positive ion.

• Acidic dyes: Color is in the negative ion.

• Negatively charged bacteria attract positively charged (basic) dyes.

• Examples: Crystal violet, methylene blue, malachite green, safranin.

Simple Stain

• Aqueous or alcohol solution of a single, basic dye.

• Stain is applied to a fixed smear and then washed off.

Purpose of Simple Stain

• Determine the shape of the cells (cocci, bacilli, spirilla).

• Look at the arrangement of the cells (singles, pairs, tetrads, etc.).

• Estimate the size of cells (using an ocular micrometer if available).

• Estimate size based on field of view diameter.

Bacterial Morphology

• Cocci (spherical)

• Bacilli (rods)

• Spiral

Smear Preparation and Fixation

1. Oil is the worst enemy. Wash slides with soap and hot water; clean with alcohol and dry with paper. Avoid touching the slide with bare hands.

2. Label one side with stain, microbe, name, and date (abbreviate).

3. Smear and methanol fixation (or heat fixation as an alternative).

   • Methanol Fixation: Apply methanol to the smears for 3-10 seconds; discard excess and air dry.

   • Heat Fixation: Pass the slide three times over the flame. Avoid overheating.

Simple Stain Procedure

• Aqueous or alcohol solution of a single, basic dye.

• Stain is applied to a fixed smear and then washed off.

Simple Stain Preparation

1. Begin with a heat-fixed emulsion.

2. Cover the smear with stain (30 seconds to 2 minutes, depending on the dye). Use a staining tray to catch excess stain. Dispose of excess stain according to lab practices.

3. Grasp the slide with a slide holder. Rinse the slide with water until runoff is clear.

4. Gently blot dry in a tablet of bibulous paper or paper towels. Do not rub. Observe under oil immersion.

Morphology of Prokaryotes

• Several shapes: coccus/cocci, bacillus (bacilli)/rods, atypical.

• Wide size range: 0.2 - 2.0 \mu\text{m} diameter, 2 - 8 \mu\text{m} length.

• Other shapes: Spiral, star-shaped, rectangular.

Arrangements of Cocci

• Diplococci = pairs (e.g., Streptococcus pneumoniae and Neisseria gonorrhoeae).

• Streptococci = chainlike patterns (e.g., Streptococcus pyogenes).

• Tetrads = groups of 4 (Micrococcus sp.).

• Sarcinae = groups of 8.

• Staphylococci = grapelike clusters or broad sheets (Staphylococcus aureus).

Arrangements of Bacilli (Rods)

• Most are single rods.

• Diplobacilli = pairs.

• Streptobacilli = chains (e.g., Bacillus megaterium, Bacillus subtilis).

• Coccobacilli = small, oval-shaped bacilli (e.g., Bordetella pertussis).

Types of Spiral Bacteria

• Vibrios = curved rods (comma-shaped).

• Spirilla = helical shape like a corkscrew.

• Spirochetes = helical and flexible.

Unusual Shapes of Prokaryotes

• Genus Stella: star-shaped cells.

• Genus Haloarcula: rectangular, flat cells.

Differential Stains

• React differently with different kinds of bacteria and can be used to distinguish among them.

  • Gram stain: most frequently used differential stain (based on cell wall composition).

  • Acid-fast stain: based on presence/absence of mycolic acids.

  • Endospore stain: based on presence/absence of endospores.

• Additionally, determine size, morphology, and arrangement of bacteria of interest.

Simple Stain Procedure

• Each student performs a stain using either methylene blue, malachite green, or safranin.

  • One prokaryotic cell (lacking a nucleus):

    • Choose one of the following: Staphylococcus epidermidis, Serratia marcescens, Enterobacter aerogenes, or Bacillus laterosporus.

    • Spread on the slide, dry, heat-fix, and stain with any of the three dyes for 3 minutes.

  • One eukaryotic cell (displaying a proper nucleus):

    • Cheek cells: Add one drop of either methylene blue, malachite green, or safranin to a drop of water. Scrape your inner cheek with a toothpick (no blood), and distribute the cells in the staining solution. Spread and let it dry.

    • Observe on oil immersion.

• Record the results in the lab manual and answer the questions.