Microbiology Chapter 2: Observing Microbial Cells PDF

CHAPTER 2 Observing the Microbial Cell Copyright © 2024 by W. W. Norton & Company, Inc. Chapter Objectives (Ch 2.1 – 2.4) 1. Describe what is needed to detect and observe microbes. 2. Describe microbial shapes. 3. Describe types of microscopy. Introduction § Since Leeuwenhoek, powerful microscopes h...

CHAPTER 2 Observing the Microbial Cell Copyright © 2024 by W. W. Norton & Company, Inc. Chapter Objectives (Ch 2.1 – 2.4) 1. Describe what is needed to detect and observe microbes. 2. Describe microbial shapes. 3. Describe types of microscopy. Introduction § Since Leeuwenhoek, powerful microscopes have been devised to search for microbes in familiar and unexpected habitats. 3 2.1 Observing Microbes § Resolution is the smallest distance by which two objects can be separated and still be distinguished. 4 2.1 Observing Microbes § Resolution is the smallest distance by which two objects can be separated and still be distinguished. § Detection is the ability to determine the presence of an object. § Magnification means an increase in the apparent size of an image to resolve smaller separations between objects. 5 Microbes Differ In Size, Over A Range Of A Few Orders Of Magnitude Think of the area difference! 6 Microscopy at Different Size Scales § Different microscopes are required to resolve various cells and subcellular structures. § Light microscopy is the simplest. 7 Microbial Size § Eukaryotic microbes Protozoa, algae, fungi 2 µm–20 cm Structures can be seen under a light microscope. 8 Microbial Size § Eukaryotic microbes § Protozoa, algae, fungi 2 µm–20 cm Structures can be seen under a light microscope. Prokaryotes Bacteria, archaea 0.4–10 µm Subcellular structures are too small to resolve by light microscopy. 9 Prokaryotic Cell Structures are Generally Simple § Certain shapes of bacteria are common to many taxonomic groups: Bacilli: rods Cocci: spheres Spiral forms: – Spirochetes – Spirilla Sometimes the name gives it away! 10 2.2 Optics and Properties of Light § Light is part of the spectrum of electromagnetic radiation. Wavelength of visible light = ~400–750 nm 11 Conditions for Resolution § For electromagnetic radiation to resolve an object, certain conditions must exist: 1. Contrast between object and its medium 12 Conditions for Resolution § For electromagnetic radiation to resolve an object, certain conditions must exist: 1. Contrast between object and its medium 2. Wavelength smaller than the object 13 Conditions for Resolution § For electromagnetic radiation to resolve an object, certain conditions must exist: 1. Contrast between object and its medium 2. Wavelength smaller than the object 3. Appropriate magnification 14 Light Interacts with an Object § Absorption means that the photon’s energy is acquired by the absorbing object. 15 Light Interacts with an Object § Reflection means that the wavefront bounces off the surface of an object. 16 Light Interacts with an Object § Refraction is the bending of light as it enters a substance that slows its speed. 17 Light Interacts with an Object § Scattering occurs when the wavefront interacts with an object smaller than the wavelength of light. 18 Magnification by a Lens § Magnification requires the bending of light rays, as in refraction. § Glass with curvature (lens) bends light rays to intersect at a focal point. focal point 19 2.3 Bright-Field Microscopy § Generates a darker image of an object over a light background § To increase resolution: Use shorter-wavelength light Reduce contrast Use immersion oil. Use wider lens closer to specimen 20 The Compound Microscope § A compound microscope is a system of multiple lenses designed to compensate for aberration. Ocular lens (4X) Objective lens (10X) § Total magnification = magnification of the ocular multiplied by that of the objective (4X * 10X = 40X) 21 Bacteria Observed at Different Levels of Focus 22 Fixation and Staining § The detection and resolution of cells under a microscope are enhanced by: Fixation – Cells are made to adhere to a slide in a fixed position. Staining – Cells are given a distinct color. 23 Different Kinds of Stains § A simple stain adds dark color specifically to cells, but not to the external medium or surrounding tissue. § A differential stain stains types of cell differently to distinguish them. The most famous differential stain is the Gram stain. 24 The Gram Stain § The Gram stain differentiates between two types of bacteria – Gram-positive bacteria retain the crystal violet stain because of their thicker cell wall. – Gram-negative bacteria do not and are counterstained 25 2.4 Fluorescence Microscopy, Super-Resolution Imaging, and Chemical Imaging § Specimen absorbs light of a defined wavelength and then emits light of lower energy 26 2.4 Fluorescence Microscopy, Super-Resolution Imaging, and Chemical Imaging § Specimen absorbs light of a defined wavelength and then emits light of lower energy § Can be used to label specific cells even in environmental samples § Fluorescence can be observed in live organisms. 27 Molecules with Fluorescence Properties (Fluorophores) for Labeling