1. A Brief Journey to the Microbial World
Chapter 2
A Brief Journey to the Microbial World

2. I. Seeing the Very Small 2.1 Some Principles of Light Microscopy
2.2 Improving Contrast in Light Microscopy
2.3 Imaging Cells in Three Dimensions
2.4 Electron Microscopy
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3. 2.1 Some Principles of Light Microscopy
Compound light microscope uses visible light to illuminate cells
Many different types of light microscopy:
Bright-field
Phase-contrast
Dark-field
Fluorescence
Animation: Light Microscopy
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4. 2.1 Some Principles of Light Microscopy
Bright-field scope (Figure 2.1a)
Specimens are visualized because of differences in contrast (density) between specimen and surroundings (Figure 2.2)
Two sets of lenses form the image (Figure 2.1b)
Objective lens and ocular lens
Total magnification = objective magnification  ocular magnification
Maximum magnification is ~2,000
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5. Specimen on glass slide
Figure 2.1a
Ocular lenses
Specimen on glass slide
Objective lens
Stage
Condenser
Figure 2.1 Microscopy.
Focusing knobs
Light source
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6. Figure 2.2
Figure 2.2 Bright-field photomicrographs of pigmented microorganisms.
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7. Magnification Light path
Figure 2.1b
Magnification Light path
100, 400, 1000
Visualized image
Eye
Ocular lens
10
Intermediate image (inverted from that of the specimen)
Figure 2.1 Microscopy.
10, 40, or 100 (oil)
Objective lens
Specimen
None
Condenser lens
Light source
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8. 2.1 Some Principles of Light Microscopy
Resolution: the ability to distinguish two adjacent objects as separate and distinct
Resolution is determined by the wavelength of light used and numerical aperture of lens
Limit of resolution for light microscope is about 0.2  m
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9. 2.2 Improving Contrast in Light Microscopy
Improving contrast results in a better final image
Staining improves contrast
Dyes are organic compounds that bind to specific cellular materials
Examples of common stains are methylene blue, safranin, and crystal violet
Animation: Microscopy & Staining Overview
Animation: Staining
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10. II. Heat fixing and staining
Figure 2.3
I. Preparing a smear
Spread culture in thin film over slide
Dry in air
II. Heat fixing and staining
Pass slide through flame to heat fix
Flood slide with stain; rinse and dry
III. Microscopy
Figure 2.3 Staining cells for microscopic observation.
Slide
Oil
Place drop of oil on slide; examine with 100 objective lens
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11. 2.2 Improving Contrast in Light Microscopy
Differential stains: the Gram stain
Differential stains separate bacteria into groups
The Gram stain is widely used in microbiology (Figure 2.4a)
Bacteria can be divided into two major groups: gram-positive and gram-negative
Gram-positive bacteria appear purple and gram-negative bacteria appear red after staining (Figure 2.4b)
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12. Step 1 Step 2 Step 3 Step 4 Figure 2.4a
Flood the heat-fixed smear with crystal violet for 1 min
Result: All cells purple
Step 2
Add iodine solution for 1 min
Result: All cells remain purple
Step 3
Decolorize with alcohol briefly — about 20 sec
Result: Gram-positive cells are purple;
gram-negative cells are colorless
Figure 2.4 The Gram stain.
Step 4 G-
Counterstain with safranin for 1–2 min
Result: Gram-positive (G+) cells are purple; gram-negative (G-) cells are pink to red G+
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13. Figure 2.4b Figure 2.4 The Gram stain. © 2012 Pearson Education, Inc.13

14. 2.2 Improving Contrast in Light Microscopy
Phase-Contrast Microscopy
Invented in 1936 by Frits Zernike
Phase ring amplifies differences in the refractive index of cell and surroundings
Improves the contrast of a sample without the use of a stain
Allows for the visualization of live samples
Resulting image is dark cells on a light background (Figure 2.5)
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15. 2.2 Improving Contrast in Light Microscopy
Dark-Field Microscopy
Light reaches the specimen from the sides
Light reaching the lens has been scattered by specimen
Image appears light on a dark background (Figure 2.5)
Excellent for observing motility
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16. Figure 2.5
Figure 2.5 Cells visualized by different types of light microscopy.
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17. 2.2 Improving Contrast in Light Microscopy
Fluorescence Microscopy
Used to visualize specimens that fluoresce
Emit light of one color when illuminated with another color of light (Figure 2.6)
Cells fluoresce naturally (autofluorescence) or after they have been stained with a fluorescent dye like DAPI
Widely used in microbial ecology for enumerating bacteria in natural samples
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18. Figure 2.6 Figure 2.6 Fluorescence microscopy.
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19. 2.3 Imaging Cells in Three Dimensions
Differential Interference Contrast (DIC) Microscopy
Uses a polarizer to create two distinct beams of polarized light
Gives structures such as endospores, vacuoles, and granules a three-dimensional appearance (Figure 2.7a)
Structures not visible using bright-field microscopy are sometimes visible using DIC
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20. Nucleus Figure 2.7a Figure 2.7 Three-dimensional imaging of cells.
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21. 2.3 Imaging Cells in Three Dimensions
Atomic Force Microscopy (AFM)
A tiny stylus is placed close to a specimen
The stylus measures weak repulsive forces between it and the specimen
A computer generates an image based on the data received from the stylus (Figure 2.7b)
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22. Figure 2.7b Figure 2.7 Three-dimensional imaging of cells.
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23. 2.3 Imaging Cells in Three Dimensions
Confocal Scanning Laser Microscopy (CSLM)
Uses a computerized microscope coupled with a laser source to generate a three-dimensional image (Figure 2.8)
Computer can focus the laser on single layers of the specimen
Different layers can then be compiled for a three-dimensional image
Resolution is 0.1  m for CSLM
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24. Figure 2.8 Figure 2.8 Confocal scanning laser microscopy.
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25. 2.4 Electron Microscopy
Electron microscopes use electrons instead of photons to image cells and structures (Figure 2.9)
Two types of electron microscopes:
Transmission electron microscopes (TEM)
Scanning electron microscopes (SEM)
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26. Electron source Evacuated chamber Sample port Viewing screen
Figure 2.9
Electron source
Evacuated chamber
Sample port
Figure 2.9 The electron microscope.
Viewing screen
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27. 2.4 Electron Microscopy Transmission Electron Microscopy (TEM)
Electromagnets function as lenses
System operates in a vacuum
High magnification and resolution (0.2 nm)
Enables visualization of structures at the molecular level (Figure 2.10a and b)
Specimen must be very thin (20–60 nm) and be stained
Animation: Electron Microscopy
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28. Cytoplasmic membrane DNA (nucleoid) Cell wall Septum Figure 2.10a
Figure 2.10 Electron micrographs.
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29. Figure 2.10b Figure 2.10 Electron micrographs.
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30. 2.4 Electron Microscopy Scanning Electron Microscopy (SEM)
Specimen is coated with a thin film of heavy metal (e.g., gold)
An electron beam scans the object
Scattered electrons are collected by a detector and an image is produced (Figure 2.10c)
Even very large specimens can be observed
Magnification range of 15–100,000
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31. Figure 2.10c Figure 2.10 Electron micrographs.
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32. II. Cell Structure and Evolutionary History
2.5 Elements of Microbial Structure
2.6 Arrangement of DNA in Microbial Cells
2.7 The Evolutionary Tree of Life
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33. 2.5 Elements of Microbial Structure
All cells have the following in common:
Cytoplasmic membrane
Cytoplasm
Ribosomes
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34. 2.5 Elements of Microbial Structure
Eukaryotic vs. Prokaryotic Cells
Eukaryotes (Figures 2.11b and 2.12c)
DNA enclosed in a membrane-bound nucleus
Cells are generally larger and more complex
Contain organelles
Prokaryotes (Figures 2.11a and 2.12a and b)
No membrane-enclosed organelles, no nucleus
Generally smaller than eukaryotic cells
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35. Eukaryote Cytoplasmic membrane Endoplasmic reticulum Ribosomes Nucleus
Figure 2.11b
Cytoplasmic membrane
Endoplasmic reticulum
Ribosomes
Nucleus
Nucleolus
Nuclear membrane
Golgi complex
Figure 2.11 Internal structure of cells.
Cytoplasm
Mitochondrion
Chloroplast
Eukaryote
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36. Eukaryote Cytoplasmic membrane Nucleus Cell wall Mitochondrion Eukarya
Figure 2.12c
Eukaryote
Cytoplasmic membrane
Nucleus
Figure 2.12 Electron micrographs of sectioned cells from each of the domains of living organisms.
Cell wall
Mitochondrion
Eukarya
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37. Prokaryote Cytoplasm Nucleoid Ribosomes Plasmid Cytoplasmic membrane
Figure 2.11a
Cytoplasm
Nucleoid
Ribosomes
Plasmid
Cytoplasmic membrane
Cell wall
Figure 2.11 Internal structure of cells.
Prokaryote
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38. Prokaryotes (a) Bacteria Figure 2.12a
Figure 2.12 Electron micrographs of sectioned cells from each of the domains of living organisms.
(a) Bacteria
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39. Prokaryotes Archaea Figure 2.12b
Figure 2.12 Electron micrographs of sectioned cells from each of the domains of living organisms.
Archaea
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40. 2.5 Elements of Microbial Structure
Viruses (Figure 2.13)
Not considered cells
No metabolic abilities of their own
Rely completely on biosynthetic machinery of infected cell
Infect all types of cells
Smallest virus is 10 nm in diameter
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41. Figure 2.13
Figure 2.13 Viruses.
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42. 2.6 Arrangement of DNA in Microbial Cells
Genome
A cell’s full complement of genes
Prokaryotic cells generally have a single, circular DNA molecule called a chromosome
DNA aggregates to form the nucleoid region (Figure 2.14)
Prokaryotes also may have small amounts of extra-chromosomal DNA called plasmids that confer special properties (e.g., antibiotic resistance)
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43. Figure 2.14 Figure 2.14 The nucleoid. © 2012 Pearson Education, Inc.43

44. 2.6 Arrangement of DNA in Microbial Cells
Eukaryotic DNA is linear and found within the nucleus
Associated with proteins that help in folding of the DNA
Usually more than one chromosome
Typically two copies of each chromosome
During cell division, nucleus divides by mitosis
During sexual reproduction, the genome is halved by meiosis
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45. 2.6 Arrangement of DNA in Microbial Cells
Escherichia coli Genome
4.64 million base pairs
4,300 genes
1,900 different kinds of protein
2.4 million protein molecules
Human Cell
1,000 more DNA per cell than E. coli
7 more genes than E. coli
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46. 2.7 The Evolutionary Tree of Life
The process of change over time that results in new varieties and species of organisms
Phylogeny
Evolutionary relationships between organisms
Relationships can be deduced by comparing genetic information in the different specimens
Ribosomal RNA (rRNA) is excellent for determining phylogeny
Relationships visualized on a phylogenetic tree (Figure 2.16)
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47. Figure 2.16 DNA DNA sequencing Aligned rRNA gene sequences Cells
Gene encoding ribosomal RNA
Isolate DNA
PCR
Sequence analysis
Generate phylogenetic tree
Figure 2.16 Ribosomal RNA (rRNA) gene sequencing and phylogeny.
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48. 2.7 The Evolutionary Tree of Life
Comparative rRNA sequencing has defined three distinct lineages of cells called domains:
Bacteria (prokaryotic)
Archaea (prokaryotic)
Eukarya (eukaryotic)
Archaea and Bacteria are NOT closely related (Figure 2.17)
Archaea are more closely related to Eukarya than Bacteria
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49. 2.7 The Evolutionary Tree of Life
Eukaryotic microorganisms were the ancestors of multicellular organisms (Figure 2.17)
Mitochondria and chloroplasts also contain their own genomes (circular, like prokaryotes) and ribosomes
These organelles are related to specific lineages of Bacteria
Mitochondria and chloroplasts took up residence in Eukarya eons ago
This arrangement is known as endosymbiosis
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50. Green nonsulfur bacteria Diplomonads (Giardia)
Figure 2.17
BACTERIA
ARCHAEA
EUKARYA
Animals
Slime molds
Entamoebae
Green nonsulfur bacteria
Euryarchaeota
Fungi
Methanosarcina
Mitochondrion
Methano- bacterium
Extreme halophiles
Plants
Gram- positive bacteria
Crenarchaetoa
Proteobacteria
Thermoproteus
Methano- coccus
Ciliates
Chloroplast
Pyrodictium
Thermoplasma
Cyanobacteria
Thermococcus
Flagellates
Flavobacteria
Marine Crenarchaeota
Pyrolobus
Methanopyrus
Trichomonads
Thermotoga
Figure 2.17 The phylogenetic tree of life as defined by comparative rRNA gene sequencing.
Microsporidia
Thermodesulfobacterium
Diplomonads (Giardia)
LUCA
Aquifex
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51. III. Microbial Diversity
2.8 Metabolic Diversity
2.9 Bacteria
2.10 Archaea
2.11 Phylogenetic Analyses of Natural Microbial Communities
2.12 Microbial Eukarya
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52. 2.8 Metabolic Diversity
The diversity in microbial cells is the product of almost 4 billion years of evolution
Microorganisms differ in size, shape, motility, physiology, pathogenicity, etc.
Microorganisms have exploited every conceivable means of obtaining energy from the environment
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53. 2.8 Metabolic Diversity Chemoorganotrophs Chemolithotrophs
Obtain their energy from the oxidation of organic molecules (Figure 2.18)
Aerobes use oxygen to obtain energy
Anaerobes obtain energy in the absence of oxygen
Chemolithotrophs
Obtain their energy from the oxidation of inorganic molecules (Figure 2.18)
Process found only in prokaryotes
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54. 2.8 Metabolic Diversity Phototrophs
Contain pigments that allow them to use light as an energy source (Figure 2.18)
Oxygenic photosynthesis produces oxygen
Anoxygenic photosynthesis does not produce oxygen
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55. Energy Sources Chemicals Light Chemoorganotrophs Chemolithotrophs
Figure 2.18
Energy Sources
Chemicals
Light
Chemotrophy
Phototrophy
Organic chemicals
Inorganic chemicals
Figure 2.18 Metabolic options for conserving energy.
(glucose, acetate, etc.)
(H2, H2S, Fe2+, NH4+, etc.)
Chemoorganotrophs
Chemolithotrophs
Phototrophs
(glucose  O2
CO2  H2O)
(H2  O2
H2O)
(light)
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56. 2.8 Metabolic Diversity All cells require carbon as a major nutrient
Autotrophs
Use carbon dioxide as their carbon source
Sometimes referred to as primary producers
Heterotrophs
Require one or more organic molecules for their carbon source
Feed directly on autotrophs or live off products produced by autotrophs
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57. 2.8 Metabolic Diversity
Organisms that inhabit extreme environments are called extremophiles
Habitats include boiling hot springs, glaciers, extremely salty bodies of water, and high-pH environments
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58. 2.9 Bacteria
The domain Bacteria contains an enormous variety of prokaryotes (Figure 2.19)
All known pathogenic prokaryotes are Bacteria
The Proteobacteria make up the largest phylum of Bacteria
Gram-negative
Examples: E. coli, Pseudomonas, and Salmonella
Gram-positive phylum united by phylogeny and cell wall structure (Figure 2.21)
Cyanobacteria are relatives of gram-positive bacteria (Figure 2.22)
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59. Proteobacteria Spirochetes Green sulfur bacteria Planctomyces
Figure 2.19
Spirochetes
Green sulfur bacteria
Planctomyces
Deinococcus
Green nonsulfur bacteria
Chlamydia
Cyanobacteria
Thermotoga
Gram-positive bacteria
OP2
Aquifex
Figure 2.19 Phylogenetic tree of some representative Bacteria.
Proteobacteria
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60. Figure 2.21
Figure 2.21 Gram-positive bacteria.
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61. Figure 2.22 Figure 2.22 Filamentous cyanobacteria.
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62. 2.9 Bacteria Many Other Phyla of Bacteria
Green sulfur bacteria and green nonsulfur bacteria are photosynthetic (Figure 2.25)
Deinococcus is extremely resistant to radioactivity (Figure 2.26)
Chlamydia are obligate intracelluar parasites
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63. Figure 2.25 Figure 2.25 Phototrophic green bacteria.
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64. Figure 2.26
Figure 2.26 The highly radiation-resistant bacterium Deinococcus radiodurans.
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65. 2.10 Archaea Two Phyla of the Domain Archaea
Euryarchaeota (Figure 2.28)
Methanogens: degrade organic matter anaerobically, produce methane (natural gas)
Extreme halophiles: require high salt concentrations for metabolism and reproduction
Thermoacidophiles: grow in moderately high temperatures and low-pH environments
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66. 2.10 Archaea Crenarchaeota (Figure 2.28)
Vast majority of cultured Crenarchaeota are hyperthermophiles
Some live in marine, freshwater, and soil systems
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67. Halophilic methanogens
Figure 2.28
Marine group
Crenarchaeota
Halobacterium
Marine group
Euryarchaeota
Natronobacterium
Sulfolobus
Methanobacterium
Halophilic methanogens
Methanocaldococcus
Thermoproteus
Pyrococcus
Methanosarcina
Figure 2.28 Phylogenetic tree of some representative Archaea.
Pyrolobus
Thermoplasma
Methanopyrus
Desulfurococcus
Hyperthermophiles
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68. 2.11 Phylogenetic Analyses of Natural Microbial Communities
Microbiologists believe that we have cultured only a small fraction of the Archaea and Bacteria
Studies done using methods of molecular microbial ecology, devised by Norman Pace
Microbial diversity is much greater than laboratory culturing can reveal
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69. 2.12 Microbial Eukarya
Eukaryotic microorganisms include algae, fungi, protozoa, and slime molds (Figure 2.32)
Protists include algae and protozoa
The algae are phototrophic (Figure 2.33a)
Protozoa NOT phototrophic (Figure 2.33c)
Fungi are decomposers (Figure 2.33b)
Algae and fungi have cell walls, whereas protozoa and slime molds do not
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70. Early-branching, lack mitochondria
Figure 2.32
Flagellates
Slime molds
Trichomonads
Diplomonads
Ciliates
Animals
Green algae
Plants
Red algae
Figure 2.32 Phylogenetic tree of some representative Eukarya.
Fungi
Diatoms
Brown algae
Early-branching, lack mitochondria
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71. Figure 2.33a Figure 2.33 Microbial Eukarya.
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72. Figure 2.33b Figure 2.33 Microbial Eukarya.
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73. Figure 2.33c Figure 2.33 Microbial Eukarya.
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74. 2.12 Microbial Eukarya
Lichens are a mutualistic relationship between two groups of protists (Figure 2.34)
Fungi and cyanobacteria
Fungi and algae
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75. Figure 2.34
Figure 2.34 Lichens.
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