MC

Chapter 3: Observing organisms through a microscope

Microscopy and Magnification

  • This chapter introduces observing organisms through a microscope, with emphasis on Helicobacter pylori as an example organism context.
  • Key idea: different imaging modalities offer different resolutions, depths, and capabilities. Choose the right tool for the organism size and the structures of interest.

Microscopy Instruments and Resolution Ranges

  • Imaging modalities and typical resolution ranges:
    • AFM (Atomic Force Microscope): ranges from 0.1\,\text{nm} up to 10\,\text{nm}
    • TEM (Transmission Electron Microscope): ranges from 10\,\text{pm} to 100\,\mu\text{m}
    • SEM (Scanning Electron Microscope): ranges from 10\,\text{nm} to 1\,\text{mm}
    • LM (Light Microscope): ranges from 200\,\text{nm} to 10\,\text{mm}
    • Unaided eye: can resolve objects approximately \ge 200\,\mu\text{m}
  • Examples mentioned/illustrative sizes (actual objects that illustrate scale):
    • Red blood cells, E. coli bacteria, DNA double helix, and other items shown in the scale chart
  • Actual size scale notes include different orders of magnitude from nm to mm, illustrating what each instrument can resolve
  • Range of organisms covered in this book spans from the smallest visible scales to visibly larger samples, aligning with the instrument capabilities above

Units of Measurement

  • Microorganisms are measured in micrometers (\mu\text{m}) and nanometers (\text{nm})
  • 1 mm = 10^3 μm = 10^6 nm, etc., illustrating unit conversions as needed

Check Your Understanding (3-1)

  • Question: How many nanometers is 10\ \text{mm}?
  • Calculation: 10\ \text{mm} = 10\times 10^3\ \mu\text{m} = 10^4\ \mu\text{m} = 10^7\ \text{nm}
  • Answer: 10^7\ \text{nm}

Microscopy: The Instruments

  • A simple microscope uses a single lens, similar to a magnifying glass but with a higher-magnification lens
  • Emphasizes the basic principle of magnification with a single optical element rather than a compound system

Light Microscopy

  • Light microscopy uses visible light to observe specimens
  • Types include:
    • Compound light microscopy
    • Darkfield microscopy
    • Phase-contrast microscopy
    • Differential interference contrast (DIC) microscopy
    • Fluorescence microscopy
    • Confocal microscopy

Compound Light Microscopy

  • How it works:
    • The image from the objective lens is magnified again by the ocular lens
  • Example of total magnification:
    • Ocular = 10\times, objective = 25\times
    • Total magnification = O\times O_{b} = 10 \times 25 = 250!\times
  • Quick quiz: If the ocular is 10X and the objective is 25X, the total magnification is 250\times (Option D)

The Compound Light Microscope: Optical Path

  • The path of light from the specimen to the observer involves:
    • Specimen -> condenser -> objective lenses -> body tube -> ocular lens -> eye
    • Light originates from the illuminator beneath the stage and travels through the condenser and objective lenses toward the ocular and observer
  • Visual reference: Figure 3-1b (The Compound Light Microscope) shows the line of vision and components such as the ocular lens, objective lenses, condenser, illuminator, and body tube

Resolution and Limitations in Compound Light Microscopy

  • Resolution (resolving power): the ability to distinguish two points as separate
  • A microscope with resolving power of 0.4\,\text{nm} could distinguish points separated by at least 0.4\,\text{nm}
  • Shorter wavelengths of light provide better resolution
  • The practical limit of resolution for a compound light microscope is such that magnification typically tops out around \approx 1500\times
  • The refractive index of the medium affects light bending; oil immersion is used to minimize refraction and improve resolution
  • If oil is not used with an oil-immersion objective, the image becomes fuzzy due to refraction losses
  • Oil immersion objective (with higher numerical aperture) improves resolution by reducing light refraction at the specimen–air interface

Refraction and Oil Immersion

  • Figure 3-3 illustrates refraction in the compound microscope when using an oil immersion objective
  • Oil immersion helps maintain a higher numerical aperture (NA) by matching the refractive indices of the lens and the specimen medium

Brightfield Illumination

  • Brightfield microscopy produces an image with dark objects against a bright background
  • Light reflected off the specimen does not enter the objective lens
  • Unstained cells can be difficult to view due to low contrast

Darkfield Microscopy

  • Visualizes light objects against a dark background
  • An opaque disk in the condenser blocks central light, so only diffracted or scattered light enters the objective
  • Particularly useful for viewing live, unstained microorganisms (e.g., Treponema pallidum, the slender spirochete that causes syphilis)

Phase-Contrast Microscopy

  • Allows detailed examination of living organisms and internal cell structures without fixation or staining
  • Combines direct and diffracted light rays to enhance contrast

Differential Interference Contrast (DIC) Microscopy

  • Similar to phase-contrast but uses two light beams and prisms to split light
  • Produces higher-contrast images with vivid color and a three-dimensional appearance

Fluorescence Microscopy

  • Uses ultraviolet (UV) light to excite fluorescent substances, which then emit longer-wavelength visible light
  • Cells may be stained with fluorescent dyes (fluorochromes) or may naturally fluoresce
  • Typical appearance: bright colors (yellow/green/orange) against a dark background
  • Example fluorochrome: Auramine O stains Mycobacterium tuberculosis

Fluorescence Microscopy: Fluorescent Antibody Technique (FA or Immunofluorescence)

  • Antibodies specific to a microbe are tagged with a fluorochrome
  • When applied to a slide, if the pathogen is present, antibodies bind and the microbe fluoresces under fluorescence microscopy
  • Advantage: rapid, specific detection of pathogens in patient specimens

Confocal Microscopy

  • Cells stained with fluorochrome dyes
  • Short-wavelength blue light excites a single plane of the specimen
  • Produces exceptionally clear two-dimensional images and allows construction of three-dimensional images by compiling successive planes with computer assistance

Two-Photon Microscopy

  • Uses fluorochrome dyes excited by two photons of long-wavelength (red) light
  • Can study living cells up to about 1 millimeter deep
  • Enables real-time tracking of cellular activity in thick samples

Super-Resolution Light Microscopy

  • Breaks the diffraction limit by using two laser beams: one excites fluorescence, the other suppresses it except in a narrow nm band
  • A computer scans nm-by-nm and reconstructs a higher-resolution image

Scanning Acoustic Microscopy

  • Measures sound waves reflected from a specimen
  • Used to study cells attached to surfaces, such as cancer cells, arterial plaque, and bacterial biofilms
  • Provides mechanical and acoustic information in addition to optical data

Electron Microscopy

  • Replaces light with electrons; electrons have much shorter wavelengths, enabling far higher resolution
  • Used for imaging very small objects (e.g., viruses, internal cellular structures) and ultrastructures
  • Images are typically black and white and are often color-enhanced digitally after capture
  • Two main types:
    • Transmission Electron Microscopy (TEM): electrons pass through a thin specimen section to form an image
    • Scanning Electron Microscopy (SEM): electrons scan the surface to produce a three-dimensional surface image
  • Figure 3-12a illustrates both TEM and SEM with labeled components (electron gun, lenses, specimen, detectors, viewing screen)

Scanned Probe Microscopy

  • Uses physical probes to examine surfaces at the atomic/molecular level
  • Does not modify the specimen
  • Capabilities include:
    • Mapping atomic and molecular shapes
    • Characterizing magnetic and chemical properties
    • Detecting temperature variations within cells
  • Includes:
    • Scanning Tunneling Microscopy (STM)
    • Atomic Force Microscopy (AFM)

Preparing Smears for Staining (1–3 of 3)

  • Staining is the process of coloring microorganisms with a dye to emphasize certain structures
  • A smear is a thin film of material containing microorganisms spread on a slide
  • Fixing a smear precedes staining to:
    • Attach microorganisms to the slide
    • Kill the microorganisms
    • Preserve parts of microbes with minimal distortion
  • Fixing methods:
    • Heat fixing: pass the slide through a Bunsen burner flame several times
    • Chemical fixing: cover smear with methanol for 1 minute
  • Stains basics:
    • Stains consist of a positive and negative ion; one is colored (chromophore)
    • Basic (cationic) dyes: crystal violet, methylene blue, safranin
    • Acidic (anionic) dyes: eosin, acid fuchsin, nigrosin
    • Bacterial cells typically have a negative charge, so basic dyes adhere to them
  • Negative staining is a technique where the background is stained instead of the cell (uses acidic dyes)

Simple Stains

  • Simple stain uses a single basic dye
  • Common examples: methylene blue, carbolfuchsin, crystal violet, safranin
  • Purpose: highlights the entire microorganism to visualize cell shapes and structures
  • Mordant: may be used to hold the stain or coat the specimen to enlarge it

Gram Stain (Differential Stain)

  • Purpose: Classifies bacteria into Gram-positive or Gram-negative
  • Key differences:
    • Gram-positive bacteria have thick peptidoglycan cell walls and stain purple
    • Gram-negative bacteria have thin peptidoglycan walls and an outer membrane containing lipopolysaccharides and phospholipids; they stain pink/red
  • Historical method developed by Hans Christian Gram
  • Procedure (4 steps):
    1) Application of crystal violet (primary stain, purple)
    2) Application of iodine (mordant)
    3) Alcohol wash (decolorization)
    4) Application of safranin (counterstain)
  • Visual outcome:
    • Gram-positive: purple
    • Gram-negative: pink/red
  • Gram stains are most consistent when used on young, actively growing bacteria
  • Gram staining is a foundational diagnostic technique in medical microbiology and often the first step in identifying unknown bacteria

Gram Stain: Quick Reference Table

  • Primary Stain: Crystal Violet → Purple (both Gram-positive and Gram-negative initially)
  • Mordant: Iodine → Purple (forms a crystal violet–iodine complex that adheres to thick cell walls)
  • Decolorizing Agent: Alcohol or Acetone–Alcohol → Purple for Gram-positive; Colorless for Gram-negative after decolorization
  • Counterstain: Safranin → Purple (Gram-positive); Pink/Red (Gram-negative)

Gram Stain: Practical Notes

  • Consistency improves when using young, actively growing bacteria
  • Consider clinical relevance: Gram stain informs treatment decisions due to differences in cell wall structure between Gram-positive and Gram-negative bacteria

Special Stains

  • Capsule stain: visualizes capsules surrounding some bacteria
  • Endospore stain: distinguishes endospores from vegetative cells
  • Flagella stain: thickens slender flagella to make them visible under light microscopy

Acid-Fast Stain

  • Binds only to bacteria with a waxy cell wall that resists decolorization by acid-alcohol
  • Used to identify organisms such as Mycobacterium and Nocardia
  • Staining results (typical):
    • Primary stain: Carbolfuchsin → Red
    • Decolorizing agent: Acid-Alcohol → Red (retains stain in acid-fast organisms)
    • Counterstain: Methylene Blue → Red in acid-fast organisms; Blue in non–acid-fast organisms

Negative Staining for Capsules

  • Capsules are gelatinous wraps around some bacteria that do not readily take up most dyes
  • Process: prepare a suspension with India ink or nigrosin to darken the background; then stain the cells with a simple stain
  • Capsule appears as a halo around the stained bacterial cell

Endospore Staining (Schaeffer–Fulton)

  • Endospores are resistant, dormant structures inside some cells
  • Procedure: Primary stain with malachite green (often with heat to aid penetration) → decolorize with water → counterstain with safranin
  • Result: Spores appear green within red or pink cells

Flagella Staining

  • Flagella are thin and difficult to see with ordinary light microscopy
  • Staining uses a mordant and carbolfuchsin to thicken the flagella, making them visible
  • Enables determination of the number and arrangement of flagella

Next Class

  • Chapter 4, Part 1: pre-class reading requested
  • Keep up with homework and questions
  • Bring any problems, concerns, or questions to the next class