chapter 2

1. Chapter 2 A Brief Journey to the Microbial World

2. Microscopy Microscopes are essential for microbiological studies. A microscope is required for the visualization of microorganisms Light microscopy is used to observe less detailed features of intact cell under low magnification than advanced microscopy (i.e. electron and laser) Various types of light microscopes exist, including bright-field, dark-field, phase contrast, and fluorescence microscopes.

3. Magnification vs. Resolution Magnification Increase in apparent size Two sets of lenses form the image Objective lens and ocular lens Total magnification is a product of the magnification of the two sets of lenses Objective magnification X ocular magnification Resolution = clarity Ability to see 2 nearby objects as distinct objects All compound light microscopes optimize image resolution by using lenses with high light-gathering characteristics (numerical aperture). The limit of resolution for a light microscope is about 0.2 ?m.

4. Microscopy

5. Improving contrast results in a better final image Staining is an easy way to improve contrast Dyes are organic compounds that have different affinities for specific cellular materials Examples of common stains are methylene blue, safranin, and crystal violet Simple and/or differential cell staining are used to increase contrast in bright-field microscopy.

6. Staining Cells for Microscopic Observation

7. Staining Cells for Microscopic Observation

8. Staining Cells for Microscopic Observation

9. Differential Stains:The Gram stain The Gram stain is widely used in microbiology On the basis of the Gram stain, bacteria can be divided into two major groups: gram-positive and gram-negative The Gram stain renders different kinds of cells different colors Gram-positive bacteria appear purple and gram-negative bacteria appear pink to red after staining

10. The Gram Stain: Steps in the Gram-stain Procedure

11. The Gram Stain: Steps in the Gram-stain Procedure

12. The Gram Stain

13. Phase Contrast Microscopy Invented in 1936 by Frits Zernike May be used to visualize live samples and avoid distortion from cell stains Can see some internal features Resulting image is dark cells on a light background

14. Electron Microscopy Electron microscopes have far greater resolving power than light microscopes, with limits of resolution of about 0.2 nm. Electron microscopes use electrons instead of photons to image cells and structures Two types: Transmission electron microscopy (TEM) For observing internal cell structure down to the molecular level Scanning electron microscopy (SEM) For three-dimensional imaging and examining surfaces

15. Electron Micrographs

16. Electron Micrographs

17. All microbial cells share certain basic structures in common Cytoplasm Cytoplasmic membrane Allows passage of needed molecules (nutrients, water, etc.) and barrier to harmful chemicals Ribosomes Site of protein synthesis Cell wall (usually) Cell shape

18. Prokaryote vs Eukaryote Two structural types of cells are recognized: Prokaryotic Archaea and bacteria Eukaryotic: plants, algae, fungi, protists, and animals (variety) Comparing prokaryotic and eukaryotic cells Prokaryote comes from the Greek words for prenucleus. Eukaryote comes from the Greek words for true nucleus.

19. Prokaryote Eukaryote Simpler internal structure Absence of nucleus One circular chromosome, not in a membrane No histones No membrane enclosed organelles Peptidoglycan cell walls Binary fission for cell division Smaller Contain nucleus Paired chromosomes, in nuclear membrane Histones Membrane enclosed organelles Simple (polysaccharide) cell walls Cell division by mitosis or meiosis Larger

20. Structure of Prokaryotic vs. Eukaryotic cell

21. Viruses Non cellular Obligate intracellular parasites They must live inside another cell to survive Have only one type of nucleic acid DNA or RNA (never both) Single or Double stranded Protein coat (no plasma membrane) Few to no enzymes Takes enzymes and use host cell metabolic machinery No metabolic activity They require a host cell to exhibit the characteristics of life. Virus diversity Different viruses have different hosts Only some viruses cause disease

22. Size Typical prokaryote: ?1 - 5 ?m long Typical Eukaryotic cell: ?10 - 100 ?m Typical virus: ? 50 - 80 nm

23. Phylogeny The study of the evolutionary relationionships of distinct organisms Although species of Bacteria and Archaea share a prokaryotic cell structure, they differ dramatically in their evolutionary history. Molecular based Compare sequences from common molecules from organisms of interest Relationships can be deduced by comparing genetic information (nucleic acid or amino acid sequences) in the different specimens Carl Woese (1970s) rRNA comparison Ribosomal RNA (rRNA) are excellent molecules for determining phylogeny Can visualize relationships on a phylogenetic tree

24. Ribosomal RNA (rRNA) Gene Sequencing and Phylogeny

25. Phylogeny Comparative ribosomal RNA sequencing has defined the three domains of life: Bacteria (prokaryotic) Archaea (prokaryotic) Eukarya (eukaryotic) Common ancestor - over 3.8 billion years ago Bacteria and archaea are prokaryotes but archaea are more closely related to eukaryotes Eukaryotic microorganisms were the ancestors of multicellular organisms Mitochondria and chloroplasts also contain their own genomes (circular, like prokaryotes) and ribosomes These organelles are ancestors of specific lineages of Bacteria Mitochondria and chloroplasts took up residence in Eukarya eons ago This arrangement is known as endosymbiosis

26. The Tree of Life Defined by rRNA Sequencing

27. 2.8 - Physiological Diversity of Microorganisms The phylogenetic diversity we see in microbial cells is the product of almost 4 billion years of evolution Microorganisms also have a tremendous amount of metabolic diversity Microorganisms have exploited every conceivable means of �making a living� consistent with the laws of chemistry and physics

28. Physiological Diversity All cells need carbon and energy sources. Different species use different strategies Chemoorganotrophs obtain their energy from the oxidation of organic compounds. Chemolithotrophs obtain their energy from the oxidation of inorganic compounds. Phototrophs contain pigments that allow them to use light as an energy source. Oxygenic versus anoxygenic photosynthesis

29. Metabolic Options for Conserving Energy

30. Autotrophs vs. Heterotrophs All cells require carbon as a major nutrient Autotrophs Use carbon dioxide as their carbon source Use sunlight for energy Primary producers Most phototrophs and lithotrophs are autotrophs Heterotrophs Use organic carbon as their carbon source Use products of autotrophs or the autotrophs themselves for energy All organotrophs and SOME lithotrophs are heterotrophs

31. Extremophiles Thrive under environmental conditions in which higher organisms cannot survive. Prokaryotes thrive in habitats that are too cold, too hot, too salty, too basic for any eukaryote Many prokaryotes are extremophiles No environment is devoid of prokaryotic life Salt concentrations up to 30% for some halophiles pH 0-12 Acidophiles and alkaliphiles Temps below 0 �C to above 100 �C Psychrophiles and hyperthermophiles High pressure Barophiles

32. Classes and Examples of Extremophiles