4. Microscopy and stainning.pdf

Microbial cell Structure and Function Microbes vs Microorganisms https://www.bates.edu/gould-research-lab/research/ Microscopy • Light microscope can magnify 1,000x • Common, important tool in microbiology • Electron microscope (1931) can magnify >100,000x Light Microscopy • Uses light • Li...

Microbial cell Structure and Function Microbes vs Microorganisms https://www.bates.edu/gould-research-lab/research/ Microscopy • Light microscope can magnify 1,000x • Common, important tool in microbiology • Electron microscope (1931) can magnify >100,000x Light Microscopy • Uses light • Light passes through specimen and then series of magnifying lenses • Maximum magnification is 1000X • Bright-field microscope is most common type Light Microscopy • Three key concepts • Magnification: apparent increase in size • Resolution: ability to distinguish two objects that are very close together (0.2um, iphone 1 mm) • Contrast determines how easily cells can be seen • Transparent bacteria lack contrast Principles of Light Microscopy • Too much or too little and you lose information • Microscopy has several methods to add contrast 7 Light Microscopes That Increase Contrast • Dark-Field Microscope • Uses dark background • Directs light toward specimen at angle • Only light scattered by specimen enters objective lens Light Microscopes That Increase Contrast • Phase-Contrast Microscope • Special optics amplify difference between refractive index of dense material and surrounding medium • Makes cells and other dense material appear darker Light Microscopes That Increase Contrast • Differential Interference Contrast Microscope • Specimen appear three dimensional • Depends on differences in refractive index • Uses two beams of light Fluorescence microscope • Fluorescence Microscopes • Uses UV light • Fluorescent substances uses UV light and emit fluorescence • Cells or materials either naturally fluorescent or tagged with fluorescent dyes Electron Microscopes • Electron microscopy • Uses • electromagnetic lenses instead of glass lens • electrons instead of light • Magnifies a million times • Used for viruses • Drawback • Lens and specimen in vacuum Electron Microscopes • Electron microscope – 2 kinds • Transmission Electron Microscope (TEM) • Scanning Electron Microscope (SEM) Preparing Specimens Apply sample to slide  Wet mount  Smear Simple Staining Simple Staining • Uses one dye https://www.quora.com/What-is-the-shape-of-cheek-cells-and-how-can-you-find-out-the-shape-of-cheek-cells Differential Staining • Gram stain most common for bacteria • Two groups: Gram-positive, Gram-negative Differential Staining https://labster-image-manager.s3.amazonaws.com/bd7be05a-dd65-4273-a739ff9801800aad/GRM_The_Gram_stain_procedure_overview_THEORY.en.x1024.png Differential Staining • Acid-fast staining used to detect Mycobacterium • Includes causative agents of tuberculosis and leprosy • Cell wall contains high concentrations of mycolic acid • Waxy fatty acid that prevents uptake of dyes • Harsher methods needed Special Stains - Cell Structures • Capsule stain • India ink added to wet mount is common method Special Stains - Cell Structures • Endospore stain • Members of genera including Bacillus and Clostridium form endospore • Endospore stain uses heat to facilitate uptake of the primary dye malachite green by endospore • Counterstain (usually safranin) used to visualize other cells https://upload.wikimedia.org/wikipedia/commons/7/7a/Bacillus_subtilis_Spore.jpg Special Stains -Cell Structures • Flagella stain • Flagella commonly used for prokaryotic motility • Too thin to be seen with light microscope • Flagella stain coats flagella to thicken and make visible • Presence and distribution can help in identification Special Stains • Fluorescent Dyes and Tags • Some dyes bind to structures in all cells