Microscopy Chapter 4 Microscopy, Staining and Classification General Principles of Microscopy –Wavelength of radiation – uses light or electrons –Magnification – increase in size of object –Resolution - clarity –Contrast – difference between 2 objects or object and background; stain to increase contrast © 2012 Pearson Education Inc.

Figure 4.1 The electromagnetic spectrum Visible light Micro- wave Infra- red UV light X rays Radio waves and Television One wavelength 400 nm 700 nm Gamma rays Increasing wavelength Crest 10 0 m 10 3 m 10 –4 m 10 –8 m Increasing resolving power Trough 10 –12 m

Figure 4.2 Light refraction and image magnification by a convex glass lens-overview Convex lens Inverted, reversed, and enlarged image Focal point Specimen Glass Light Air

Chicken egg Human red blood cell Large protozoan (Euglena) Chloroplasts Flea Typical bacteria and archaea Diameter of DNA VirusesProteins Ribosomes Amino acids Atoms Scanning tunneling microscope (STM) 0.01 nm–10 nm Scanning electron microscope (SEM) 0.4 nm–1 mm Transmission electron microscope (TEM) nm–100 µm Atomic force microscope (AFM) 1 nm–10 nm Compound light microscope (LM) 200 nm–10 mm Unaided human eye 200 µm– Mitochondrion Figure 4.3 The limits of resolution of the human eye and of various types of microscopes

Microscopy Light Microscopy –Bright-field microscopes – uses light –1. Simple –Contain a single magnifying lens –Similar to magnifying glass –Leeuwenhoek used simple microscope to observe microorganisms © 2012 Pearson Education Inc.

Microscopy –2. Compound –Series of lenses for magnification –Light passes through specimen into objective lens –Have one or two ocular lenses –Total magnification (objective lens X ocular lens – 10X) –Low – red – 4X – total 40X –medium - yellow – 10X – total100X –high – blue – 40X – total 400X –Oil immersion – white -100x – total 1000X - Oil immersion lens increases resolution - Most have condenser lens (direct light through specimen) © 2012 Pearson Education Inc.

Microscopy - 3. Darkfield microscope – background dark – used for unstained organisms - 4. Fluorescent microscope – specimen stained with fluorescent dyes - 5. Phase-contrast microscope – allows observation of dense structures in living organisms – no staining needed – sharp contrast

Figure 4.4 A bright-field, compound light microscope-overview Line of vision Ocular lens Path of light Prism Body Specimen Objective lenses Condenser lenses Illuminator Ocular lens Body Objective lenses Condenser Illuminator Remagnifies the image formed by the objective lens Base Fine focusing knob Coarse focusing knob Diaphragm Stage Arm Transmits the image from the objective lens to the ocular lens using prisms Primary lenses that magnify the specimen Controls the amount of light entering the condenser Focuses light through specimen Holds the microscope slide in position Light source Moves the stage up and down to focus the image

Figure 4.5 The effect of immersion oil on resolution-overview Microscope objective Refracted light rays lost to lens Glass cover slip Light source Specimen Slide Without immersion oil Glass cover slip Light source Slide Microscope objective More light enters lens Lenses With immersion oil Immersion oil

Microscopy Electron Microscopy –Light microscopes cannot resolve structures closer than 200 nm –Greater resolving power and magnification –Magnifies objects 10,000X to 100,000X –Detailed view of bacteria, viruses, ultrastructure, and large atoms –Two types –Transmission electron microscopes –Scanning electron microscopes © 2012 Pearson Education Inc.

Figure 4.11 A transmission electron microscope (TEM) -overview Light microscope (upside down) Column of transmission electron microscope Condenser lens (magnet) Lamp Condenser lens Objective lens Eyepiece Final image seen by eye Final image on fluorescent screen Projector lens (magnet) Objective lens (magnet) Specimen Electron gun

Magnetic lenses Electron gun Primary electrons Secondary electrons Specimen holder Vacuum system Specimen Photo- multiplier Detector Scanning circuit Monitor Beam deflector coil Figure 4.12 Scanning electron microscope (SEM)

Figure 4.13 SEM images-overview

Microscopy Probe Microscopy –Magnifies more than 100,000,000X –Two types –Scanning tunneling microscopes –Atomic force microscopes © 2012 Pearson Education Inc.

Figure 4.14 Probe microscopy-overview Enzyme DNA

© 2012 Pearson Education Inc. Staining Principles of Staining –Staining increases contrast and resolution by coloring specimens with stains/dyes –Smear of microorganisms (thin film) made prior to staining –Acidic dyes stain alkaline (base) structures –Nigrosin, India Ink –Basic dyes stain acidic structures –Crystal violet, methylene blue, safranin, malachite green

Spread culture in thin film over slide Pass slide through flame to fix it Air dry Figure 4.15 Preparing a specimen for staining

Simple Stains – single basic dye Differential Stains – use more than 1 dye –Gram stain – for positive or negative –Acid-fast stain – for waxy cell walls –Endospore stain – uses heat to force stain –Histological stain – for tissue (cancer or fungi) Special Stains –Negative (capsule) stain – stains background –Flagellar stain © 2012 Pearson Education Inc. Staining

Figure 4.16 Simple stains-overview

Figure 4.17 The Gram staining procedure-overview Slide is flooded with crystal violet for 1 min, then rinsed with water. Result: All cells are stained purple. Slide is flooded with solution of ethanol and acetone for 10–30 sec, then rinsed with water. Result: Smear is decolorized; Gram-positive cells remain purple, but Gram-negative cells are now colorless. Slide is flooded with safranin for 1 min, then rinsed with water and blotted dry. Result: Gram-positive cells remain purple, Gram-negative cells are pink. Slide is flooded with iodine for 1 min, then rinsed with water. Result: Iodine acts as a mordant; all cells remain purple.

Figure 4.18 The Ziehl-Neelsen acid-fast stain

Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis

Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae Background stain Bacterium Capsule

Figure 4.21 Flagellar stain of Proteus vulgaris Flagella

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 © 2012 Pearson Education Inc.

Classification and Identification of Microorganisms Linnaeus and Taxonomic Categories –Linnaeus –Classified organisms based on characteristics in common –Organisms that can successfully interbreed called species –Used binomial nomenclature in his system – genus and species © 2012 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 © 2012 Pearson Education Inc.

Classification and Identification of Microorganisms Linnaeus and Taxonomic Categories –Linnaeus’s goal was to classify organisms to catalogue them –Modern goal is to understand relationships among groups of organisms –Reflect phylogenetic hierarchy – grouping organisms reflecting their evolution from common ancestors –Emphasis on comparison of organisms’ genetic material –Led to proposal to add domain © 2012 Pearson Education Inc.

Classification and Identification of Microorganisms Domains –Proposal of three domains as determined by ribosomal nucleotide sequences –Eukarya, Bacteria, and Archaea –Cells in the three domains differ by other characteristics © 2012 Pearson Education Inc.

Classification and Identification of Microorganisms Taxonomic and Identifying Characteristics –Physical characteristics –Biochemical tests – microbes ability to utilize or produce certain chemicals –Serological tests – use antiserum (serum containing antibodies); clumping of antigen with antibodies (agglutination) indicates presence of targeted cells –Phage typing – reveals if 1 bacterial strain is/is not susceptible to a particular phage – plaques (clear area) form where bacteria killed by phage –Analysis of nucleic acids © 2012 Pearson Education Inc.

Gas bubble Inverted tubes to trap gas Acid with gas Acid with no gasInert Hydrogen sulfide produced No hydrogen sulfide Figure 4.23 Two biochemical tests for identifying bacteria-overview

Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system Wells

Negative result Positive result Figure 4.25 An agglutination test, one type of serological test-overview

Figure 4.26 Phage typing Bacterial lawn Plaques

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 © 2012 Pearson Education Inc.

Figure 4.27 Use of a dichotomous taxonomic key-overview Gram-positive cells? Rod-shaped cells? Gram-positive bacteria Obligate anaerobes Ferments lactose? Cocci and pleomorphic bacteria Can tolerate oxygen? Can use citric acid (citrate) as sole carbon source? Non-lactose- fermenters Produces gas from glucose? Produces hydrogen sulfide gas? Produces acetoin? Salmonella Enterobacter Citrobacter Escherichia Shigella Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No