Microscopy

Microscope Types

light - mag. 1,000x

Brightfield: Widely used (used in UTA labs), bright background, dark specimen.

Darkfield: dark background, light specimen, live organisms.

Phase-Contrast: used for viewing how they function, 3D like images, live specimens and organelles.

Differential Interference Contrast: similar to phase contrast, uses different polarization waves.

Fluorescence: Uses fluorochromes, used to determine which cells are living and which are dead or to find locations in a cell.

Confocal: uses z-planes to scan specimens, thick specimens like biofilm.

Two-Photon: Used to view thicker specimens, like embryos and brain slices. Living cells.

Electron - mag. 20-100,000x

Transmission (TEM): Thin, dehydrated slices, view internal slices, do not utilize wavelengths, electron beams used to increase magnification and resolution of images.

Scanning (SEM): Examine surface specimens on a 3-D like image, uses electrons, special sputter coat to emit electrons from the specimens.

Scanning Probe - mag. 100-100,000,000x

Scanning tunneling (STM): Used on items such as gold and other conducting materials, also used on organic materials such as DNA.

Atomic Force (AFM): useful to observe specimens on atomic level, utilizes a probe that passes through a specimen

Staining

Wet stain: good for viewing live specimens

Fixed amount: smearing, good for staining. specimen is killed.

Basic Stain: colors positively charged ions

Acidic Stain: colors negatively charged ions

Positive Stain: Dye is absorbed into cell

Negative Stain: Dye is absorbed into the background

Simple Stains: single stain, emphasizes structures.

Differential Stain: 2+ stains, differentiates organisms based on stain interactions

Types of stains

Basic: Crystal Violet, Methylene blue, Malachite green, Safranin

Acidic: Rose Bengal, Acid Fuchsin, Congo Red

Negative: India Ink

Staining Technique

Gram Stain: Distinguishes different cell wall components, differentiates positive and negative grams. Steps; Primary stain of crystal violet, Mordant (iodine), decolorize with alcohol, counter stain with safranin. Positive gram will stain purple, Negative gram will stain red.

Acid Fast Stains: Detect mycolic acid. Two methods, Ziehl-Neelson method (uses heat)and Kinyoun method (without heat). Steps; Heat fix smear, primary stain with carbolfuschin, decolorize with alcohol, counterstain with methylene blue. Cell is dyed red, non cells/ background are stained blue.

Capsule Stain: Diagnostic tool for detection of protective coating, dye does not penetrate capsule. Steps; No heat smear, primary stain with India ink. Used to visualize capsules (halos)

Endospore Stain: Identifies dormant/hidden endospores. Uses Schaeffer Fulton method. Steps; Heat fix smear, Primary stain with malachite green, decolorize with water, counter stain with safranin. Endospores aid bacteria through extreme environments.

Flagella Stain: identifies flagella appendages. Used for bacteria, archaea, and eukaryotes. Steps: No heat smear, primary stained with specialized acids, decolorized with water, and counter stained with carbol fuschin.

Prepping for Electron Microscopy -CANNOT VIEW LIVE SPECIMENS

TEM: Samples must be cut into very thin sections. Specimens are embedded into plastic resin and dehydrated. Stained with electron dense heavy metals.

SEM: Even more dehydrated, critical point drying with liquid CO2. Specially sputter coated with metal.