Microscopes: Light and Electron

• There are two main types of microscopes: light microscopes and electron microscopes

Light Microscopes

• Visible light observes specimens
• A compound light microscope uses two lenses
• The objective lens is located closest to the specimen and magnifies its image by 10x-100x
• The ocular lens is within the eyepiece and magnifies the specimen 10x

Calculating Magnification

• Total magnification in a compound microscope equals objective lens magnification multiplied by ocular lens magnification
• For example: An ocular lens of 10x and an objective lens of 100x yield a total magnification of 1000x

Resolution

• Resolution is the ability to distinguish fine detail and structure, and two points at a certain distance
• A microscope with 6nm resolving power can distinguish two points if they are at least 6nm apart
• Light must pass between two objects to be seen as distinct
• Shorter wavelengths of light offer better resolution

Electron Microscopes

• Electron microscopes use beams of electrons instead of light
• Electrons also travel in waves much shorter than light waves, thus achieving a greater resolution
• Electron microscopes achieve high magnification, as high as 500,000x
• Electron microscopes allow viewing of internal cell structures and viruses

Light vs Electron Microscope

• The smallest object visible to the human eye is 0.10mm
• A compound microscope can view objects as small as 0.20µm
• An electron microscope can view objects as small as 0.20nm
• An electron microscope provides better resolution than a light microscope at the same magnification

Types of Electron Microscopes

• Transmission Electron Microscopy (TEM) examines internal cell structure
• Electron beams cannot penetrate thick cells so it must be cut and thin sectioning done
• Thin sections must be stained before they can be viewed under the TEM
• Uranium is an example of a stain that can be used
• Stains improve contrast between different cell structures

Scanning Electron Microscopy

• Scanning Electron Microscopy (SEM) is only used to view the surface of objects
• Specimens must be coated with a thin film of heavy metal such as gold
• Allows for magnifications (15x-100,000x)

Scanning Tunneling Microscopy

• Scanning Tunneling Microscopy (STM) is the most powerful electron microscope
• STM is used to visualize individual atoms
• A thin metal probe scans specimens, revealing surface irregularities

Clinical Use of Light Microscopes

• Stains make microorganisms visible
• Microorganisms are normally colourless
• Stains are composed of positive and negative ions with one type colored, called the chromophore

Staining Procedure

• A thin film of material called a smear which contains the microorganism of interest is 'smeared' on a slide
• The sample is then fixed by passing it through a flame
• Stain is applied to the sample
• The stain is removed from the sample by rinsing
• The stained sample is now viewed under a microscope

How Stains Work

• Bacteria's outer surface has a net negative charge
• Negative charge attracts positive charge and repels negative charge, stains use this principle

Positive Stains

• Positively charged stains adhere to the negatively charged bacterium
• Bacteria will appear as the color of the stain, the background appears clear, such as Crystal Violet

Negative Stains

• Negative stains are repelled by the negatively charged bacterium
• This repelling is called negative staining
• The bacteria will appear clear and the background will appear colored, such as Nigrosin

Staining Techniques

• Simple stains use a single colored basic dye
• Basic dyes have a positively charged color ion that binds to the organism
• Sometimes a mordant is used to increase the stain intensity

Differential Stains

• Differential Stains are used to differentiate between types of bacteria, and stains react differently with bacteria types
• Very important for diagnostics, differential stains exploit differences in cell wall structure and composition
• Examples: gram stain and acid-fast stain

Gram Stain

• The Gram stain determines if a bacterium of interest is gram positive or gram negative

Acid Fast Stain

• This stain binds strongly to bacteria containing a waxy cell wall such as Mycolic acid
• Used to identify bacteria within the genus Mycobacterium such as Mycobacterium tuberculosis and Mycobacterium leprae
• The waxy cell wall of Mycobacterium retains carbol fuschin dye
• A counterstain with methylene blue leaves tissues and non-acid fast bacteria blue

Capsule Stain

• Capsule stain reveals a thick polysaccharide layer outside the bacterial cell
• Capsule presence indicates bacterium with increased virulence
• The background is colored with a negative stain like nigrosin making it black
• The bacterial cell is stained with a positive stain like safranin making it colored pink
• The capsule does not take up dye and remains colorless appearing as a halo around the cell

Endospore Stain

• Highlights intracellular structures that make bacteria resistant to adverse conditions
• Ordinary stains cannot penetrate the bacterial cell wall
• A primary stain with malachite green colors the endospore green
• A counterstain with safranin colors the rest of the cell pink
• An example of an endospore forming bacteria is Bacillus anthracis

Flagella Stain

• Flagella stains highlight extracellular bacterial structure used for motility
• Flagella are extremely small to observe under a normal light microscope without stain
• A mordant and stain combination increases flagella thickness, making them visible under a light microscope