Power Point Lecture Presentations prepared by Mindy Miller-Kittrell

Microscopy and Staining: Overview PLAY © 2014 Pearson Education, Inc. Microscopy and Staining: Overview

Microscopy • General Principles of Microscopy • Wavelength of radiation • Magnification • Resolution

Figure 4. 1 The electromagnetic spectrum. © 2014 Pearson Education, Inc.

$Figure 4. 2 Light refraction and image magnification by a convex glass lens. Light$

Figure 4. 3 The limits of resolution (and some representative objects within those ranges)

Microscopy • General Principles of Microscopy • Contrast • Differences in intensity between two objects, or an object and its background • Important in determining resolution • Staining increases contrast • Use of light that is in phase increases contrast © 2014 Pearson Education, Inc.

Microscopy • Light Microscopy • Bright-field microscopes • Simple • Contain a single magnifying lens • Similar to magnifying glass • Leeuwenhoek used simple microscope to observe microorganisms © 2014 Pearson Education, Inc.

Microscopy • Light Microscopy • Bright-field microscopes • Compound • Series of lenses for magnification • Light passes through specimen into objective lens • Oil immersion lens increases resolution • Have one or two ocular lenses • Total magnification = magnification of objective lens X magnification of ocular lens • Most have condenser lens (direct light through specimen) © 2014 Pearson Education, Inc.

Figure 4. 4 A bright-field, compound light microscope. Line of vision Ocular lens Remagnifies the image formed by the objective lens Body Transmits the image from the objective lens to the ocular lens using prisms Arm Objective lenses Primary lenses that magnify the specimen Stage Holds the microscope slide in position Condenser Focuses light through specimen Diaphragm Controls the amount of light entering the condenser Illuminator Light source Coarse focusing knob Moves the stage up and down to focus the image Fine focusing knob Base © 2014 Pearson Education, Inc. Ocular lens Path of light Prism Body Objective lenses Specimen Condenser lenses Illuminator

Figure 4. 5 The effect of immersion oil on resolution. Microscope objective Lenses More light enters lens Refracted light rays lost to lens Immersion oil Glass cover slip Slide Specimen Without immersion oil © 2014 Pearson Education, Inc. Light source With immersion oil

Microscopy • Light Microscopy • Dark-field microscopes • Best for observing pale objects • Only light rays scattered by specimen enter objective lens • Specimen appears light against dark background • Increases contrast and enables observation of more details © 2014 Pearson Education, Inc.

$Figure 4. 6 The light path in a dark-field microscope. Objective Light refracted by$

Microscopy • Light Microscopy • Phase microscopes • Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining • Light rays in phase produce brighter image, while light rays out of phase produce darker image • Contrast is created because light waves are out of phase • Two types • Phase-contrast microscope • Differential interference contrast microscope © 2014 Pearson Education, Inc.

Figure 4. 7 Principles of phase microscopy. Rays in phase Rays out of phase Phase plate Bacterium Ray deviated by specimen is 1/4 wavelength out of phase. © 2014 Pearson Education, Inc. Deviated ray is now 1/2 Wavelength out of phase.

Figure 4. 8 Four kinds of light microscopy. Nucleus Bacterium Bright field Dark field

Microscopy • Light Microscopy • Fluorescent microscopes • Direct UV light source at specimen • Specimen radiates energy back as a longer, visible wavelength • UV light increases resolution and contrast • Some cells are naturally fluorescent; others must be stained • Used in immunofluorescence to identify pathogens and to make visible a variety of proteins © 2014 Pearson Education, Inc.

Figure 4. 9 Fluorescence microscopy. © 2014 Pearson Education, Inc.

Figure 4. 10 Immunofluorescence. Antibodies Bacterium Cell-surface antigens Bacterial cell with bound antibodies carrying

Microscopy • Light Microscopy • Confocal microscopes • Use fluorescent dyes • Use UV lasers to illuminate fluorescent chemicals in a single plane • Resolution increased because emitted light passes through pinhole aperture • Computer constructs 3 -D image from digitized images © 2014 Pearson Education, Inc.

Light Microscopy PLAY © 2014 Pearson Education, Inc. Light Microscopy

Microscopy • Electron Microscopy • Light microscopes cannot resolve structures closer than 200 nm • Electron microscopes have greater resolving power and magnification • Magnifies objects 10, 000 X to 100, 000 X • Detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms • Two types • Transmission electron microscopes • Scanning electron microscopes © 2014 Pearson Education, Inc.

Figure 4. 11 A transmission electron microscope (TEM). Light microscope (upside down) Lamp Column of transmission electron microscope Electron gun Condenser lens Specimen Objective lens (magnet) Eyepiece Projector lens (magnet) Final image seen by eye © 2014 Pearson Education, Inc. Final image on fluorescent screen

Figure 4. 12 Scanning electron microscope (SEM). Electron gun Magnetic lenses Primary electrons Beam deflector coil Scanning circuit Secondary electrons Specimen holder Vacuum system © 2014 Pearson Education, Inc. Photomultiplier Detector Monitor

Figure 4. 13 SEM images. © 2014 Pearson Education, Inc.

Electron Microscopy PLAY © 2014 Pearson Education, Inc. Electron Microscopy

Microscopy • Probe Microscopy • Magnifies more than 100, 000 times • Two types

Figure 4. 14 Probe microscopy. © 2014 Pearson Education, Inc. DNA Enzyme

Staining • Most microorganisms are difficult to view by brightfield microscopy • Coloring specimen with stain increases contrast and resolution • Specimens must be prepared for staining © 2014 Pearson Education, Inc.

Figure 4. 15 Preparing a specimen for staining. © 2014 Pearson Education, Inc.

Staining • Principles of Staining • Dyes used as stains are usually salts • Chromophore is the colored portion of the dye • Acidic dyes stain alkaline structures • Basic dyes stain acidic structures • More common since most cells are negatively charged © 2014 Pearson Education, Inc.

Staining • Simple stains • Differential stains • Gram stain • Acid-fast stain •

Figure 4. 16 Simple stains. © 2014 Pearson Education, Inc.

Figure 4. 17 The Gram staining procedure. © 2014 Pearson Education, Inc.

Figure 4. 18 Ziehl-Neelsen acid-fast stain. © 2014 Pearson Education, Inc.

Figure 4. 19 Schaeffer-Fulton endospore stain of Bacillus anthracis. © 2014 Pearson Education, Inc.

Staining • Differential Stains • Histological stains • Two common stains used for histological specimens • Gomori methenamine silver (GMS) stain • Hematoxylin and eosin (HE) stain © 2014 Pearson Education, Inc.

Figure 4. 20 Negative (capsule) stain of Klebsiella pneumoniae. Bacterium Capsule Background stain ©

Figure 4. 21 Flagellar stain of Proteus vulgaris. Flagella © 2014 Pearson Education, Inc.

Staining • Staining for Electron Microscopy • Chemicals containing heavy metals used for transmission electron microscopy • Stains may bind molecules in specimens or the background © 2014 Pearson Education, Inc.

Staining PLAY © 2014 Pearson Education, Inc. Staining

Classification and Identification of Microorganisms • Taxonomy consists of classification, nomenclature, and identification • Organize large amounts of information about organisms • Make predictions based on knowledge of similar organisms © 2014 Pearson Education, Inc.

Classification and Identification of Microorganisms • Linnaeus and Taxonomic Categories • Linnaeus • His system classified organisms based on characteristics in common • Grouped organisms that can successfully interbreed into categories called species • Used binomial nomenclature © 2014 Pearson Education, Inc.

Figure 4. 22 Levels in a Linnaean taxonomic scheme. © 2014 Pearson Education, Inc.

Classification and Identification of Microorganisms • Linnaeus and Taxonomic Categories • Linnaeus proposed only two kingdoms • Later taxonomic approach based on five kingdoms • Animalia, Plantae, Fungi, Protista, and Prokaryotae • Linnaeus's goal was classifying organisms to catalog them • Modern goal is understanding relationships among organisms • Goal of modern taxonomy is to reflect phylogenetic hierarchy • Greater emphasis on comparisons of organisms' genetic material led to proposal to add domain © 2014 Pearson Education, Inc.

Classification and Identification of Microorganisms • Domains • Carl Woese compared nucleotide sequences of r. RNA subunits • Proposal of three domains as determined by ribosomal nucleotide sequences • Eukarya, Bacteria, and Archaea • Cells in the three domains also differ with respect to many other characteristics © 2014 Pearson Education, Inc.

Classification and Identification of Microorganisms • Taxonomic and Identifying Characteristics • Physical characteristics • Biochemical tests • Serological tests • Phage typing • Analysis of nucleic acids © 2014 Pearson Education, Inc.

Classification and Identification of Microorganisms • Taxonomic and Identifying Characteristics • Physical characteristics • Can often be used to identify microorganisms • Protozoa, fungi, algae, and parasitic worms can often be identified based only on their morphology • Some bacterial colonies have distinct appearance used for identification © 2014 Pearson Education, Inc.

Figure 4. 23 Two biochemical tests for identifying bacteria. Gas bubble Inverted tubes to

Figure 4. 24 One tool for the rapid identification of bacteria, the automated Micro.

Figure 4. 25 An agglutination test, one type of serological test. © 2014 Pearson

Figure 4. 26 Phage typing. Bacterial lawn Plaques © 2014 Pearson Education, Inc.

Classification and Identification of Microorganisms • Taxonomic and Identifying Characteristics • Analysis of nucleic acids • Nucleic acid sequence can be used to classify and identify microbes • Prokaryotic taxonomy now includes the G + C content of an organism's DNA © 2014 Pearson Education, Inc.

Classification and Identification of Microorganisms • Taxonomic Keys • Dichotomous keys • Series of paired statements where only one of two "either/or" choices applies to any particular organism • Key directs user to another pair of statements, or provides name of organism © 2014 Pearson Education, Inc.