Phase Contrast Microscopy

Microscopy

• Animation: Light Microscopy allows examination of living organisms and internal cell structures by bringing together two sets of light rays, direct rays, and diffracted rays to form an image.
• Phase-Contrast Microscopy accentuates diffraction of the light that passes through a specimen, increasing contrast by combining direct and reflected light rays at the eye.
• Darkfield Microscopy makes light objects visible against a dark background by placing an opaque disk in the condenser, allowing only light reflected off the specimen to enter the objective lens.

Light Microscopy

• Bright-field microscope produces a dark image against a brighter background, as light reflected off the specimen does not enter the objective lens.
• Dark-field microscope produces a bright image against a dark background, as only refracted light enters the objective lens, making it useful for observing living cells, particularly spirochetes.

Fluorescence Microscopy

• Uses UV light to excite fluorescent substances that absorb UV light and emit visible light.
• Cells may be stained with fluorescent dyes (fluorochromes).

Electron Microscopy

• Uses electrons instead of light, with a shorter wavelength that provides greater resolution.
• Used for images too small to be seen with light microscopes, such as viruses.
• Two types: Transmission (TEM) and Scanning (SEM).

Electron Microscopy (continued)

• Transmission (TEM) Microscopy:
• Electrons pass through a thin section of the specimen.
• Light passes through the specimen, then an electromagnetic lens, to a screen or film.
• 2-dimensional structure, with specimens stained with heavy metal salts.
• Magnifies objects 10,000 to 10,000,000x; resolution of 10 pm.
• Scanning (SEM) Microscopy:
• Image produced by electrons emitted from the surface of an object.
• Electron gun produces a beam of electrons that scans the surface of a whole specimen.
• Secondary electrons emitted from the specimen produce the image, with 3-dimensional structure.
• Magnifies objects 1,000 to 500,000x; resolution of 10 nm.

Staining

• Staining: coloring microorganisms with a dye that emphasizes certain structures.
• Smear: a thin film of a material containing microorganisms spread over a slide.
• Fixation: the process by which internal and external structures of cells are preserved.
• Heat-fixation: fix an air-dried thin film (smear) by passing through a flame.

Staining (continued)

• Dyes have chromophore groups that give color and bind to cells by ionic, covalent, or hydrophobic bonding.
• Ionic stains:
• Basic dyes: chromophore is a cation.
• Acid dyes: chromophore is an anion.
• Hydrophobic stains: Sudan black stains lipids.
• Stains consist of a positive and negative ion, one of which is colored (chromophore).
• Negative staining: staining the background instead of the cell.

Simple Stains

• Simple stain: uses a single basic dye to highlight the entire microorganism and visualize cell shapes and structures.
• A mordant may be used to hold the stain or coat the specimen to enlarge it.

Differential Stains

• Differential stain: divides bacteria into separate groups based on staining properties.
• Gram stain:
• Most important staining procedure.
• Dr. Christian Gram, 1884.
• Gram positive: traps crystal violet-iodine complex, due to thick layer of peptidoglycan in cell wall.
• Gram negative: lipids in cell wall are dissolved by ethanol, allowing crystal violet-iodine complex to escape; must add counterstain to colorless cells.

Differential Stains (continued)

• Acid-fast stain:
• Used to stain Mycobacterium, which have high mycolic acid content (waxy).
• Must use steam heat to force carbolfuchsin stain into cells.
• Acid-alcohol decolorizer removes stain from non-acid-fast cells.
• Counterstain with methylene blue.
• Negative/Capsular stain:
• Reveals capsule layer around cells.
• Mix bacteria with nigrosin, spread on clean slide, dry.
• Useful to observe Klebsiella pneumonia.

Special Stains

• Spore stain:
• Used to stain endospores of Clostridium and Bacillus.
• Heat smear over steam, rinse with water.
• Counterstain with safranin.
• Endospores - Green; Vegetative cells - Red.
• Flagellar stain:
• Flagella of bacteria are coated with tannic acid or potassium alum and stained with basic fuchsin (Gray Method).

This animation explains the principles of phase-contrast microscopy, allowing the examination of living organisms and internal cell structures. It brings together direct and diffracted light rays to form an image, accentuating diffraction of light that passes through a specimen.