Chapter 3

Microscopy, Staining, and Classification

© 2012 Pearson Education Inc.

Lecture prepared by Mindy Miller-Kittrell North Carolina State University

Microscopy and Staining

ANIMATION Microscopy and Staining: Overview
© 2012 Pearson Education Inc.

Table 4.1 Metric Units of Length


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

© 2012 Pearson Education Inc.

Figure 4.1 The electromagnetic spectrum

400 nm

700 nm

Visible light X UV rays light Infra- Microred wave Radio waves and Television

Gamma rays



Increasing wavelength 10–4m



Crest One wavelength
Trough Increasing resolving power

and enlarged image .2 Light refraction and image magnification by a convex glass lens-overview Light Air Glass Focal point Specimen Convex lens Inverted.Figure 4. reversed.

3 The limits of resolution of the human eye and of various types of microscopes Diameter of DNA Ribosomes Typical bacteria and archaea Flea Atoms Proteins Viruses Chloroplasts Large protozoan (Euglena) Chicken egg Amino acids Mitochondrion Human red blood cell Scanning tunneling microscope (STM) 0.4 nm–1 mm Atomic force microscope (AFM) 1 nm–10 nm Compound light microscope (LM) 200 nm–10 mm Unaided human eye 200 µm– .01 nm–10 nm Transmission electron microscope (TEM) 0.078 nm–100 µm Scanning electron microscope (SEM) 0.Figure 4.

. or between an object and background – Important in determining resolution – Staining increases contrast – Use of light that is in phase increases contrast © 2012 Pearson Education Inc.Microscopy • General Principles of Microscopy – Contrast – Differences in intensity between two objects.

.Microscopy • Light Microscopy – Bright-field microscopes – Simple – Contain a single magnifying lens – Similar to magnifying glass – Leeuwenhoek used simple microscope to observe microorganisms © 2012 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 (objective lens X ocular lens) – Most have condenser lens (direct light through specimen) © 2012 Pearson Education Inc. .

4 A bright-field.Figure 4. compound light microscope-overview Ocular lens Remagnifies the image formed by the objective lens Line of vision Body Transmits the image from the objective lens to the ocular lens using prisms Ocular lens Path of light Prism Body Objective lenses Specimen Condenser lenses Illuminator 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 .

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

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 © 2012 Pearson Education Inc. .

6 The light path in a dark-field microscope Objective Light refracted by specimen Light unrefracted by specimen Specimen Condenser Dark-field stop Dark-field stop .Figure 4.

Microscopy • Light Microscopy – Phase microscopes – Examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining – Contrast is created because light waves are out of phase – Two types – Phase-contrast microscope – Differential interference contrast microscope © 2012 Pearson Education Inc. .

7 Principles of phase microscopy-overview Rays in phase Rays out of phase Phase plate Bacterium Ray deviated by specimen is 1/4 wavelength out of phase. . Deviated ray is now 1/2 wavelength out of phase.Figure 4.

Figure 4.8 Four kinds of light microscopy-overview Nucleus Bacterium Bright field Dark field Phase contrast Nomarski .

. others must be stained – Used in immunofluorescence to identify pathogens and to make visible a variety of proteins © 2012 Pearson Education Inc.Microscopy • Light Microscopy – Fluorescent microscopes – Direct UV light source at specimen – Specimen radiates energy back as a visible wavelength – UV light increases resolution and contrast – Some cells are naturally fluorescent.

Figure 4.9 Fluorescent microscopy-overview .

Figure 4.10 Immunofluorescence-overview Antibodies Fluorescent dye Antibodies carrying dye Bacterium Cell-surface antigens Bacterial cell with bound antibodies carrying dye .

.Microscopy • Light Microscopy – Confocal microscopes – Use fluorescent dyes – Use UV lasers to illuminate fluorescent chemicals in a single plane – Resolution increased because light passes through pinhole aperture – Computer constructs 3-D image from digitized images © 2012 Pearson Education Inc.

Microscopy ANIMATION Light Microscopy © 2012 Pearson Education Inc. .

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

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

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

13 SEM images-overview .Figure 4.

Microscopy ANIMATION Electron Microscopy © 2012 Pearson Education Inc. .

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

14 Probe microscopy-overview DNA Enzyme .Figure 4.

Staining • Principles of Staining – Staining increases contrast and resolution by coloring specimens with stains/dyes – Smear of microorganisms (thin film) made prior to staining – Microbiological stains contain chromophore – Acidic dyes stain alkaline structures – Basic dyes stain acidic structures © 2012 Pearson Education Inc. .

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

Staining • Simple Stains • Differential Stains – – – – Gram stain Acid-fast stain Endospore stain Histological stain • Special Stains – Negative (capsule) stain – Flagellar stain © 2012 Pearson Education Inc. .

Figure 4.16 Simple stains-overview .

Gram-positive cells remain purple. Result: Iodine acts as a mordant. Slide is flooded with safranin for 1 min. then rinsed with water and blotted dry. all cells remain purple.Figure 4. Result: Smear is decolorized. Gram-negative cells are pink. Result: Gram-positive cells remain purple. then rinsed with water. then rinsed with water. Slide is flooded with iodine for 1 min. but Gram-negative cells are now colorless. Result: All cells are stained purple.17 The Gram staining procedure-overview Slide is flooded with crystal violet for 1 min. Slide is flooded with solution of ethanol and acetone for 10–30 sec. then rinsed with water. .

Figure 4.18 The Ziehl-Neelsen acid-fast stain .

19 Schaeffer-Fulton endospore stain of Bacillus anthracis .Figure 4.

Staining • Differential Stains – Histological stain – Two popular stains for histological specimens – Gomori methenamine silver (GMS) – Hematoxylin and eosin (HE) © 2012 Pearson Education Inc. .

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

21 Flagellar stain of Proteus vulgaris Flagella .Figure 4.

Staining • Staining for Electron Microscopy – Transmission electron microscopy uses chemicals containing heavy metals – Absorb electrons – Stains may bind molecules in specimens or the background © 2012 Pearson Education Inc. .

.Staining ANIMATION Staining © 2012 Pearson Education Inc.

. and identification – Organize large amounts of information about organisms – Make predictions based on knowledge of similar organisms © 2012 Pearson Education Inc. nomenclature.Classification and Identification of Microorganisms – Taxonomy consists of classification.

.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 – Linnaeus proposed only two kingdoms © 2012 Pearson Education Inc.

scapularis (deer tick) l. pacificus (black-eyed tick) l.Figure 4.22 Levels in a Linnaean taxonomic scheme-overview Domain Bacteria Archaea Eukarya Kingdom Animalia Plantae Fungi Phylum Chordata (vertebrates) Arthropoda (joint-legged animals) Platyhelminthes Nematoda (tapeworm) (unsegmented roundworms) Class Insecta Crustacea Arachnida Order Scorpionida Acariformes (mites) Parasitiformes (mites and ticks) Araneida Family Ixodidae (hard ticks) Argasidae (soft ticks) Genus Dermacentor Ixodes Rhipicephalus Species l. ricinus (castor bean tick) .

Protista.Classification and Identification of Microorganisms • Linnaeus and Taxonomic Categories – Linnaeus proposed only two kingdoms – Later taxonomic approach based on five kingdoms – Animalia. Plantae. Fungi. 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 – Emphasis on comparison of organisms’ genetic material – Led to proposal to add domain © 2012 Pearson Education Inc.

Bacteria. .Classification and Identification of Microorganisms • Domains – Carl Woese compared nucleotide sequences of rRNA subunits – Proposal of three domains as determined by ribosomal nucleotide sequences – Eukarya. 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 Serological tests Phage typing Analysis of nucleic acids © 2012 Pearson Education Inc.

23 Two biochemical tests for identifying bacteria-overview Gas bubble Inverted tubes to trap gas Acid with gas Acid with no gas Inert Hydrogen sulfide produced No hydrogen sulfide .Figure 4.

Figure 4. the automated MicroScan system Wells .24 One tool for the rapid identification of bacteria.

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

26 Phage typing Bacterial lawn Plaques .Figure 4.

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.

27 Use of a dichotomous taxonomic key-overview Gram-positive cells? No Rod-shaped cells? No Cocci and pleomorphic bacteria No Obligate anaerobes Yes Gram-positive bacteria Yes Can tolerate oxygen? Yes Ferments lactose? No Non-lactosefermenters Yes Can use citric acid (citrate) as sole carbon source? No Produces gas from glucose? No Shigella Yes Escherichia Yes Produces hydrogen sulfide gas? No Produces acetoin? No Citrobacter Yes Salmonella Yes Enterobacter .Figure 4.

Classification and Identification of Microorganisms ANIMATION Dichotomous Key: Overview ANIMATION Dichotomous Key: Sample with Flowchart ANIMATION Dichotomous Key: Practice © 2012 Pearson Education Inc. .

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