Microscopes and culture media

• Microorganisms are too small to be seen with the unaided eye and must be observed with a microscope
• The word "microscope" comes from the Latin word "micro" (small) and the Greek word "skopos" (to look at)
• Modern microbiologists use microscopes that produce magnifications ranging from ten to thousands of times greater than van Leeuwenhoek's single lens microscope
• Helicobacter pylori is a spiral-shaped bacterium first seen in cadaver stomachs in 1886
• Some microbes are more readily visible than others due to their size or observable features
• Microorganisms must undergo staining procedures before their cell walls, capsules, and other structures lose their colorless natural state
• Microorganisms and their components are measured in micrometers (μm) and nanometers (nm)
• A micrometer (μm) is equal to 0.000001 meters (10^-6 m) and a nanometer (nm) is equal to 0.000000001 meters (10^-9 m)
• Light microscopy uses visible light to observe specimens
• A compound light microscope has a series of lenses and uses visible light for illumination
• Total magnification of a specimen is calculated by multiplying the objective lens magnification by the ocular lens magnification
• Resolution in microscopy refers to the ability of lenses to distinguish fine detail and structure
• The resolving power of a microscope is influenced by the wavelength of light used
• To achieve high magnification with good resolution, immersion oil is used with the oil immersion objective lens
• Darkfield microscopy is used to examine live microorganisms that are invisible in ordinary light microscopes or cannot be stained
• Phase-contrast microscopy allows detailed examination of internal structures in living microorganisms without the need for fixing or staining the specimen
• Differential interference contrast (DIC) microscopy uses differences in refractive indexes and two beams of light to produce brightly colored, nearly three-dimensional images
• Fluorescence microscopy takes advantage of substances that absorb short wavelengths of light and emit light at longer wavelengths
• Confocal microscopy is used to reconstruct three-dimensional images by illuminating one plane of a specimen at a time
• Two-photon microscopy (TPM) uses long-wavelength light and requires two photons to excite the fluorochrome to emit light
• Scanning acoustic microscopy (SAM) interprets the action of a sound wave sent through a specimen to study living cells attached to surfaces
• Electron microscopy is used to examine objects smaller than about 0.2 μm, such as viruses or internal cell structures
• Electron Microscopy:

  • Used for objects smaller than about 0.2 μm, such as viruses or internal structures of cells
  • Uses a beam of electrons instead of light
  • Electron microscopes have better resolution than other microscopes due to the shorter wavelengths of electrons
  • Images produced are black and white, but can be artificially colored
  • Two types of electron microscopes: transmission electron microscope (TEM) and scanning electron microscope (SEM)
• Transmission Electron Microscopy (TEM):

  • A finely focused beam of electrons passes through an ultrathin section of the specimen
  • Electromagnetic condenser lens focuses the beam on a small area of the specimen
• Scanning Electron Microscopy (SEM):

  • Provides three-dimensional views of specimens
  • Uses a primary electron beam to knock electrons out of the specimen's surface, producing secondary electrons that are used to create an image
• Staining Procedures for Light Microscopy:

  • Staining is used to color microorganisms with dyes that emphasize certain structures
  • Microorganisms must be fixed to the slide before staining
  • Types of stains: basic dyes (crystal violet, methylene blue, malachite green, safranin) and acidic dyes (eosin, acid fuchsin, nigrosin)
• Simple Stains:

  • Aqueous or alcohol solution of a single basic dye
  • Highlights entire microorganism to show cellular shapes and basic structures
• Differential Stains:

  • React differently with different bacteria to distinguish them
  • Examples: Gram stain and acid-fast stain
• Gram Stain:

  • Classifies bacteria into gram-positive and gram-negative groups
  • Steps: primary stain (crystal violet), mordant (iodine), decolorizing agent (alcohol), counterstain (safranin)
  • Gram-positive bacteria retain purple color, gram-negative bacteria appear pink
• Acid-Fast Stain:

  • Binds strongly to bacteria with waxy material in cell walls
  • Used to identify acid-fast bacteria like Mycobacterium tuberculosis and Mycobacterium leprae
  • Steps: carbolfuchsin, heating, acid-alcohol, counterstain (methylene blue)
• Special Stains:

  • Used to color and isolate specific parts of microorganisms
  • Examples: capsule staining and endospore staining
• Negative Staining for Capsules:

  • Capsules are gelatinous coverings around microorganisms
  • Capsule staining is used to determine the organism's virulence
  • Capsules appear as halos surrounding stained bacterial cells
• Endospore (Spore) Staining:

  • Endospores are resistant, dormant structures formed within bacterial cells
  • Endospores are stained using the Schaeffer-Fulton endospore stain
  • Steps: malachite green (primary stain), heat, water wash, counterstain (safranin)
• Endospores appear green within red or pink cells in a properly prepared smear
• Endospores are highly refractive and can be detected under a light microscope when unstained
• Without a special stain, endospores cannot be differentiated from inclusions of stored material
• Bacterial flagella are structures of locomotion too small to be seen with a light microscope without staining
• Flagella staining procedure uses a mordant and the stain carbolfuchsin to build up the diameters of flagella until they become visible under the light microscope
• Microbiologists use the number and arrangement of flagella as diagnostic aids
• Culture media provide essential nutrients and minerals to support the growth of microorganisms in the laboratory