Microscopy: Prescott's Microbiology, Chapter 2

Mikroskopie/ Microscopy

• Microscopy relates to studying microorganisms, the subject matter of Prescott's Microbiology (11th Edition), specifically chapter 2, pages 20 to 36.

Size Ranges

• Atoms are the smallest entities, followed by amino acids, diameter of DNA, proteins, ribosomes, and viruses
• Size ranges then extend to Chlamydia, mitochondrion, bacteria, Rickettsia, and red blood cells
• Larger entities in this range include chloroplasts, large protozoa, human eggs, ticks, human hearts, and dogs

Visualizing Specimens

• Microscopic observation is essential because most cells are too small to be seen unaided
• Sizes range from nanometers (nm) for viruses, up to 0.5 μm for bacteria, and exceeding 100 μm for plant cells
• There are two primary types of microscopes for viewing cells and their internal structures
• Light microscopes use light to illuminate the specimen.
• Electron microscopes use electrons to illuminate the specimen.

Lenses and Light

• Understanding how a light microscope operates requires considering how lenses bend and focus light to form images.
• Refraction bends a wave's direction as it transitions into a medium where its speed changes.
• Light moves in a straight line through a given medium
• When light crosses from one medium into another, refraction occurs.
• Refractive index measures the extent to which a substance reduces the velocity of light
• In a microscope, lenses work as a collection of prisms operating as a unit
• Light rays from a distant light source are focused at the focal point F
• The distance between the center of the lens and the focal point is called the focal length (f).
• Eyes cannot focus on an object closer than 25 cm
• A convex lens enlarges objects
• Lens strength is determined by focal length
• Short focal lengths magnify more than long focal lengths.

Types of Light Microscopes

• Several types of light microscopes exist
• Bright field microscope
• Dark field microscope
• Phase contrast microscope
• Fluorescence microscope

Microscope Components

• Eyepiece or ocular lens contains the ocular lens, providing 10x to 15x magnification
• Nosepiece holds objective lenses, rotatable for magnification changes
• Objective lenses consist of 4x, 10x, 40x, and 100x magnification
• Total magnification is calculated by multiplying the eyepiece and objective lens powers (e.g., 10x eyepiece with 40x objective lens = 400x magnification)
• Stage clips are there to hold the slide in place
• Stage is the flat platform that supports the slide for analysis
• Diaphragm controls the intensity and size of the light cone on the specimen; transparent specimens require less light
• Light source projects light upwards through the diaphragm, slide, and lenses
• Condenser Lens focuses light onto the sample; useful with high-power objective lenses
• Arm supports the microscope when carried
• Coarse adjustment knob coarsely adjusts the focus by moving the stage up or down
• Fine adjustment knob finely tunes the focus
• Base supports the microscope

Bright Field Microscope

• Bright field microscopes routinely are used in microbiology labs to view stained and unstained specimens
• Specimens appear dark against a bright background
• Several objective lenses are involved
• Parfocal objective lenses must remain in focus as they are moved or changed
• total magnification: Objectives multiplied by the ocular eg., 40x * 10x = 400x

Microscope Resolution

• Resolving power of a lens is its ability to distinguish between closely adjacent small objects
• Resolution depends on the numerical aperture of the lens (n sin θ)
• n is the refractive index of the medium in which the lens works (eg., air = 1)
• e (theta) is 1/2 the angle of the cone of light entering the object
• Higher θ values indicate better resolution and the ability to see smaller objects

Microscope Resolution

• Resolution is mathematically described using Abbe's formula
• d = 0.5λ / n sinθ
• d equals the smallest distance between two objects that reveals that they are separate
• Depends on the wavelength (λ) used to illuminate the specimen and the numerical aperture of the lens (n sin θ)
• Wavelength of light is between 450 to 550 nm, while θ equals the numerical aperture
• Minimum distance: d=0.5λ/n sinθ. At best, a bright-field microscope can distinguish objects 0.2 μm apart; viruses cannot be examined this way with the light microscope

Working Distance

• Defines the distance between the objective lens and cover slip (or a slide)
• Higher resolution = shorter working distance
• Properties of objective lenses include:
  • Magnification: 10x, 40-45x, 90-100x
  • Numerical aperture: 0.25, 0.55-0.65, 1.25-1.4
  • Focal length: 16mm, 4mm, 1.8-2.0 mm
  • Working distance: 4-8 mm, 0.5-0.7 mm, and 0.1mm

Oil Immersion Objectives

• Oil replaces air which allows more light rays to enter the lense
• Oil allows light beams to bend towards objective lens
• Numerical aperture and resolution increases

Dark-Field Microscopy

• An image forms by light reflected or refracted by specimen

• A dark-field stop, placed under the condenser produces a hallow cone of light focusing only on the subject.

• Only light reflected and refracted by the specimen forms an image

• Produces images of living organisms

• Field is usually dark

Phase Contrast Microscopy

• Onpigmented cells can be difficult to see with a bright field microscope

• Cells are stained to make them visible

• Phase contrast microscopy makes cells visible by using different refractive indexes that allow the slowing of light

• Two types of light are combined to form an image

• • Deviates light (slowed as passed)
• • Light from surrounding environment

• Two special components are used

• • condenser annulus and phase plate.

Other Forms of Microscopy

• Differential Interference Contrast uses prisms to generate beams of light at right angles
• The beams combine to form images

Fluorescence Microscopy

• Image is made visible through fluorescent light being emitted from the specimen
• Fluorochromes are used to stain specimens which absorbs energy and releases it as light
• UV Light, violet or blue is usually used

Types of Microscopy

• Epifluorescence microsopy employs a lens that acts as a condenser
• Specimen is illuminated from the top not bottom
• A mercury lamp produces an intense beam to pass through an excitation filter

Preparing Specimens

• Increases visibility of the specimen
• Accentuates features
• Preserves specimens

Fixation

• Stained cells should resemble the living cells
• Fixation is process to preserve structures of specimen
• It inactivates enzymes which disrupt cell morphology and toughens cell structures so they do not change during observation
• Kills microorganisms and firmly attaches them to the microscope slide
• Typically a thin film of cells (smear) is gently heated as a slide is passed through flame -Heat Fixation
• used to protect fine cellular substructures and morphology of larger, delicate microorganisms -Chemical fixation
• Fixative mixtures contain ethanol, acetic acid, formaldehyde

Stains

• Dyes have two features in common:

  • Chromophore groups – conjugated double bonds give the dye its colour
  • Can bind to cells with ionic, covalent of hydrophobic bonding

• Dyes are divided into two general classes:

• Basic dyes

  • e.g. methylene blue, crystal violet, safranin, malachite
  • bind to negatively charged molecules (nucleic acids, proteins) -Acidic dyes
  • e.g. eosin, rose bengal, acid fuchsin
  • bind to positively charged cell structures

• Microorganisms can have their shape/arrangement effectively stained with singular dyes

• Staining is simple and easy to use

• The fixed smear is covered for a short period, then the excess stain is washed with water/blotted dry

Gram Staining

• Gram Staining is a Differential stain developed by Hans Christian Gram in 1884.
• Distinguishes organisms into two groups: Gram positive and Gram negative.

Gram Reaction

• Eubacteria divided into 2 compositions: cell walls

  • 50% peptidoglycan and 1-4% lipids

  • <10% peptidoglycan and 10-20% lipids

Acid-fast Staining

• Commonly used to identify Mycobacterium tuberculosis and M. leprae
• • Have cell walls containing lipids constructed from mycolic acids prevent dyes from readily binding to cells Can then be stained by harsh procedures: Uses high concentrations of phenol to drive basic fuchsin into cells

Endospore Staining

• Requires heat to dye into target - endospore
• Structure is exceptionally resistant to stains and is typically developed by bacterial genera, bacillus and clostridium
• Shaeffer-Fulton procedure - endospore stained with malachite green in presence of heat - washed with water and counterstained with safranin
• A blueish-green is endospore while cells are pink-and-red

Capsule Staining

• It also called negative staining
• Capsules create networks that surround capsule structure
• cells mixed with dye can appear lighter among the black

Flagella Staining

• Provides value in the cell
• Bacterial cell, slender
• coat with mordant, then basix fuchsin

Electron Microscopy

• Higher resolution

• TEM- electron pass through specimens to study their internal structures

• SEM- surface area

• The transmission electron microscope (TEM)

• • The Electron transmits and is focused on a speciment

TEM - Sample Preparation

• Specimens should be thin, otherwise there will be knife marks in specialized processes
• After the specimen is removed it is studied through TEM - a detailed process will be shown

Freeze Etching Technique

• Cells are frozen but may break down and exposed to coated layers platinum and carbon

Scanning Electron Microscopy

• SEM produces images for atoms release on a surface of an object
• Air dried material are examined
• Provides a 3D image to specimen

Confocal Microscopy

• Uses a laser beam on a specimen
• A components removes the lost light
• A single point will be iluminatted with a 3D image