Cells and Methods to Observe Them

Microscopy and Cell Structure

Chapter 3

Part I: Observing Cells

Figure 4.1B

  • Measurement Scale:

    • 10 m

    • 1 m

    • 100 mm (10 cm)

    • 10 mm (1 cm)

    • 1 mm

  • Examples of Cell Sizes:

    • Human height

    • Length of some nerve and muscle cells

    • Chicken egg

    • Frog egg

    • Human egg

    • Paramecium (100 μm)

    • Most plant and animal cells (10 μm)

    • Nucleus (1 μm)

    • Most bacteria (100 nm)

    • Mitochondrion (10 nm)

    • Smallest bacteria (1 nm)

    • Viruses (0.1 nm)

    • Ribosome (nanometer scale)

    • Proteins and lipids (atoms scale)

    • Unaided eye (visible scale)

    • Light microscope (10 μm to 100 nm range)

    • Electron microscope (sub-nanometer scale)

Microscope Techniques

Overview of Microscopes

  • Microscopes

    • Most important tool for studying microorganisms.

    • Use visible light to observe objects.

    • Ability to magnify images approximately 1,000x.

    • Electron microscope, introduced in 1931, can magnify images in excess of 100,000x.

    • Scanning probe microscope, introduced in 1981, can view individual atoms.

Principles of Light Microscopy

  • Light passes through a specimen then through a series of magnifying lenses.

Types of Light Microscopes

  1. Bright-field microscope - Most common and easiest to use.

  2. Dark-field microscope.

Important Factors in Light Microscopy

  • Magnification

  • Resolution

  • Contrast

Principles of Light Microscopy: Compound Light Microscope

  • Magnification:

    • Achieved through two magnifying lenses:

    • Ocular lens

    • Objective lens (4x, 10x, 40x, and 100x)

    • Magnification formula: Magnification = Ocular x Objective (e.g., 10x x 100x = 1,000x)

  • Condenser lens:

    • Focuses illumination on specimen but does not affect magnification.

Resolution in Light Microscopy

  • Resolution: Minimum distance between two objects that still appear as separate objects.

  • Resolving power:

    • Measures ability to distinguish two objects that are close together.

    • Determines the usefulness of a microscope.

  • Factors affecting resolution:

    • Lens quality and type.

    • Wavelength of light used.

    • Amount of light released from the lens.

    • Magnification.

    • Sample preparation method.

  • Maximum resolving power of most bright-field microscopes is 0.2 μm - sufficient for most bacteria, but too low to see viruses.

Enhancing Resolution

  • Higher magnification lenses (100x) enhance resolution through the use of immersion oil, which reduces light refraction.

Contrast in Light Microscopy

  • Contrast: Difference in color intensity between an object and the background.

  • Reflects the number of visible shades in a specimen.

  • To increase contrast:

    • Use special microscopes.

    • Utilize specimen staining (note: staining typically kills microbes, thus cannot observe living cells).

Examples of Light Microscopes

  1. Dark-field Microscope:

    • Produces a reverse image, similar to a photographic negative.

    • A modified condenser directs light at an angle; only light scattered by the specimen enters the objective lens.

  2. Phase-Contrast Microscope:

    • Amplifies differences between refractive indexes of cells and surrounding medium, enhancing the appearance of unstained cells.

  3. Differential Interference Contrast:

    • Utilizes two light beams transmitted through the specimen, creating a three-dimensional effect.

  4. Fluorescence Microscope:

    • Observes organisms that are either naturally fluorescent or tagged with fluorescent dyes; absorbs ultraviolet light and emits visible light against a dark background.

  5. Confocal Scanning Laser Microscope:

    • Constructs three-dimensional images of thicker structures, providing detailed sectional views of internal structures of intact organisms.

    • Uses a laser to scan through sections of the organism, with the computer generating a 3D image from the sections.

Electron Microscope (EM)

  • Utilizes electromagnetic lenses, electrons, and a fluorescent screen to produce images.

Key Features of EM

  • Resolution increased by about 1000-fold compared to bright-field microscopes (to approximately 0.3 nm).

  • Magnification increases to 100,000x.

  • Two types of electron microscopes:

    1. Transmission Electron Microscope (TEM):

    • Used to observe fine details.

    • Directs beam of electrons at specimen; electrons either pass through or scatter at surface.

    • Specimen is prepared using thin sectioning or freeze fracturing.

    1. Scanning Electron Microscope (SEM):

    • Used to observe surface details.

    • The specimen is coated with metal (usually gold); electrons are released and reflected into the viewing chamber.

Dyes and Staining Techniques

Importance of Dyes and Staining

  • Stains are crucial for observing organisms, allowing differentiation between cell types.

  • Stains are composed of organic salts.

  • Basic dyes: carry a positive charge and bond to negatively charged cell structures; typically used for staining cells.

  • Acidic dyes: carry a negative charge and are repelled by negatively charged cell structures; usually stains the background.

  • Staining divides into categories:

    • Simple Stains: Uses one basic stain to color the cells.

    • Differential Stains: Distinguish between different bacterial types (e.g., Gram stain, Acid-fast stain).

    • Special Stains: Target specific components of the cell (e.g., capsule, endospore, flagella, fluorescent tags).

Overview of Stains
Simple Stains

  • Employ a basic dye to impart color; enhances contrast between colorless cells and transparent backgrounds.

Differential Stains

  • Distinguish one group of microorganisms from another.

  • Commonly utilized in clinical settings, notably Gram stain and Acid-fast stain.

Gram Stain Procedure

  1. Primary Stain: Crystal violet.

  2. Mordant: Iodine (enhances the crystal violet retention).

  3. Decolorizer: Alcohol or acetone (differentiates Gram-positive from Gram-negative).

  4. Counter Stain: Safranin (stains Gram-negative cells red or pink).

  • Gram-positive bacteria retain the purple color due to a thick peptidoglycan layer, while Gram-negative bacteria appear red or pink after decolorization due to a thin peptidoglycan layer.

Acid-fast Stain

  • Targets Mycobacterium due to their lipid-rich cell wall that resists conventional staining.

  • Uses carbol fuchsin as the primary stain, acid-alcohol as the decolorizer, and methylene blue as the counterstain.

Special Stains

  • Capsule Stain: Negative stain that allows the capsule to stand out against a stained background.

  • Endospore Stain: Utilizes heat to facilitate staining, visualizing the endospore as green against a pink background.

  • Flagella Stain: Involves a mordant that coats flagella, increasing their visible diameter.

  • Fluorescent Dyes: Absorb ultraviolet light and emit light at longer wavelengths, allowing observation of cells with specific surface proteins.

Morphology of Prokaryotic Cells

Basic Cell Shapes

  • Coccus: Spherical shape.

  • Bacillus: Rod shape (do not confuse with Bacillus genus).

  • Coccobacillus: Short, round rod.

  • Vibrio: Short, curved rod.

  • Spirillum: Long, curved spiral shape.

  • Spirochete: Helical shape.

  • Pleomorphic: Bacteria that can alter shape.

Grouping of Bacteria

  • Arrangement reflects division along certain planes:

    • Pairs (Diplococci): e.g., Neisseria gonorrhoeae.

    • Chains (Streptococci): e.g., species of Streptococcus.

    • Cubical packets: e.g., Sarcina.

    • Clusters: e.g., Staphylococcus.

External Structures of Prokaryotic Cells

  • Pili: Many types, including fimbriae for surface attachment and sex pili for DNA transfer.

  • Flagella: Long protein structures for motility; can be polar (at one or both ends) or peritrichous (distributed everywhere).

Intracellular Structures

  • Chromosome: Typically one circular double-stranded molecule found in a region called the nucleoid.

  • Ribosomes: Composed of ribonucleic acid (RNA) and proteins (70S - 30S and 50S subunits).

  • Plasmids: Circular DNA molecules that can replicate independently, contributing to traits like antibiotic resistance.

Storage Granules and Endospores

  • Storage Granules: Accumulations of polymers synthesized from excess nutrients.

  • Endospores: Dormant, resistant structures formed through sporulation; critical for survival under harsh conditions.

Summary of Prokaryotic Cell Structures

Structure

Characteristics

Filamentous appendages

Flagella for mobility and pili for attachment

Glycocalyx

Capsule and slime layer for protection

Cell wall

Gram-positive or Gram-negative based on structure

Cytoplasmic membrane

Semi-permeable barrier; environment separation

DNA

Chromosomal DNA, plasmid

Ribosomes

70S ribosomes, protein synthesis

Summary of Eukaryotic Cell Structures

Membrane-bound Organelles

  • Distinct features such as nucleus, mitochondria, chloroplasts (in plants), endoplasmic reticulum, Golgi apparatus, lysosomes, and peroxisomes set eukaryotic cells apart from prokaryotic.

  • Cytoskeleton: Composed of microtubules, intermediate filaments, and actin filaments; provides structural support.

  • Eukaryotic plasma membranes contain sterols like cholesterol for strength.

  • Transport across membranes includes endocytosis and exocytosis processes, with varying mechanisms for nutrient uptake and waste expulsion.