LECTURE PRESENTATIONS

For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION

Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark

Chapter 2
A Brief Journey to
the Microbial World
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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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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
https://images-na.ssl-images-
amazon.com/images/I/41arbIoqZlL._AC_SL230_.jpg

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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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Figure 2.1a A light microscope

Ocular
lenses Specimen on
glass slide

Objective lens

Stage

Condenser

Focusing knobs

Light source

https://www.youtube.com/watch?v=SUo2fHZaZCU
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Figure 2.1b Path of light through a compound light microscope. Besides 10x, ocular lenses are available in
15–30x magnifications

no magnification

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Figure 2.2 Bright-field photomicrographs of pigmented microorganisms

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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

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Figure 2.3 Staining cells for microscopic observation

I. Preparing a smear

Spread culture in thin Dry in air

film over slide

II. Heat fixing and staining

Pass slide through Flood slide with stain;

flame to heat fix rinse and dry

III. Microscopy

Slide Oil

Place drop of oil on slide;

examine with 100
objective lens

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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)»

© 2012 Pearson Education, Inc. Marmara University – Enve3003 Env. Eng. Microbiology
Gram Staining:
• Aim is to identify the type of infecitous
bacteria to decide the treatment process or
medication.

• https://www.youtube.com/watch?v=Jvo6IGKT
vxA&t=189s
• See class notes
Figure 2.4a The Gram stain - Steps in the procedure
Step 1 Flood the heat-fixed
smear with crystal
Result: violet for 1 min
All cells purple

Step 2 Add iodine solution

for 1 min
Result:
All cells
remain purple

Step 3 Decolorize with

alcohol briefly
Result: — about 20 sec
Gram-positive
cells are purple;
gram-negative
cells are colorless

Step 4 G- Counterstain with

Result: safranin for 1–2 min
Gram-positive
(G+) cells are purple; G+
gram-negative (G-) cells
are pink to red

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Figure 2.4b Microscopic observation of gram-positive (purple) and gram-negative (pink) bacteria

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2.2 Improving Contrast in Light Microscopy
• Phase-Contrast Microscopy
– 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 b)

© 2012 Pearson Education, Inc. Marmara University – Enve3003 Env. Eng. Microbiology
2.2 Improving Contrast in Light Microscopy
• Dark-Field Microscopy
– Image appears light on a dark background
(Figure 2.5 c)
– Excellent for observing motility

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Figure 2.5 Cells visualized by different types of light microscopy. The same field of cells of the baker’s yeast
Saccharomyces cerevisiae visualized by (a) bright-field microscopy, (b) phase-contrast microscopy, and (c)
dark-field microscopy. Cells average 8–10 µm wide.

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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 either 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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Figure 2.6 Fluorescence microscopy. Same cells are observed by bright-field microscopy in part a and by
fluorescence microscopy in part b. Cells fluoresce red because they contain chlorophyll a and other pigments.

Fluorescence photomicrograph of cells of Escherichia coli made fluorescent by staining with the
fluorescent dye DAPI.
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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)

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Figure 2.8 Confocal scanning laser microscopy

(a) Confocal image

of a microbial biofilm
community cultivated in the
laboratory.
The green, rod-shaped cells
are Pseudomonas aeruginosa
experimentally introduced
into the biofilm.
Other cells of different colors
are present at different
depths in the biofilm.
(b) Confocal image of a
filamentous cyanobacterium
growing in a soda lake. Cells
are about 5 µm wide.

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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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Figure 2.9 The electron microscope

Electron
source

Evacuated
chamber
Sample
port

Viewing
screen

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2.4 Electron Microscopy
• Transmission Electron Microscopy (TEM)
– Enables visualization of structures at the
molecular level (Figure 2.10a and b)
– Specimen must be very thin (20–60 nm) and
be stained

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Figure 2.10a Electron micrographs. (a) Micrograph of a thin section of a dividing bacterial cell, taken by
transmission electron microscopy (TEM). Note the DNA forming the nucleoid. The cell is about 0.8 µm wide.

Cytoplasmic DNA
Septum Cell wall (nucleoid)
membrane

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2.4 Electron Microscopy
• Scanning Electron Microscopy (SEM)
– Magnification range of 15–100,000

Figure 2.10c Scanning electron micrograph of bacterial cells. A single cell is about 0.75 µm wide.

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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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2.5 Elements of Microbial Structure
• All cells have the following in common:
– Cytoplasmic membrane
– Cytoplasm
– Ribosomes

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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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Figure 2.11b Internal structure of eukaryotic cells

Cytoplasmic
membrane
Endoplasmic
reticulum

Ribosomes
Nucleus

Nucleolus
Nuclear
membrane
Golgi
complex
Cytoplasm
Mitochondrion
Chloroplast
Eukaryote

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Figure 2.12c Electron micrograph of sectioned eukaryotic cells

Eukaryote
Cytoplasmic
membrane
Nucleus

Cell
wall

Eukarya Mitochondrion

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Figure 2.11a Internal structure of prokaryotic cells

Cytoplasm Nucleoid Ribosomes

Plasmid

Cytoplasmic
Cell wall
membrane
Prokaryote

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Figure 2.12a Electron micrograph of sectioned prokaryotic cells

Prokaryotes Prokaryotes

(a) Bacteria Archaea

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2.5 Elements of Microbial Structure
• Viruses
– 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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2.7 The Evolutionary Tree of Life
• Evolution
– The process of change over time that results in
new varieties and species of organisms

• Phylogeny : Evolutionary relationships between

organisms
– Ribosomal RNA (rRNA) is excellent for
determining phylogeny
– Relationships visualized on a phylogenetic tree

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Figure 2.17 The phylogenetic tree of life as defined by comparative rRNA gene sequencing

BACTERIA ARCHAEA EUKARYA

Animals
Slime
Entamoebae
molds
Green nonsulfur Euryarchaeota
bacteria Fungi
Methanosarcina
Mitochondrion Methano- Plants
Extreme
Gram- Crenarchaetoa bacterium halophiles
Proteobacteria positive Thermoproteus
Methano- Ciliates
bacteria
Pyrodictium coccus Thermoplasma
Chloroplast
Cyanobacteria Thermococcus
Flavobacteria Flagellates
Marine Pyrolobus
Crenarchaeota Methanopyrus
Trichomonads

Thermotoga

Microsporidia
Thermodesulfobacterium
LUCA Diplomonads
Aquifex (Giardia)
(Last universal
common ancestor)

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2.7 The Evolutionary Tree of Life
• Comparative rRNA sequencing has defined three
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
• Eukaryotic microorganisms were the ancestors of
multicellular organisms (Figure 2.17)

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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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 Classifications of microorganisms based upon how
they obtain energy:
Energy Sources

Chemicals Light

Chemotrophy Phototrophy

Organic Inorganic
chemicals chemicals
(glucose, acetate, etc.) (H2, H2S, Fe2+, NH4+, etc.)

Chemoorganotrophs Chemolithotrophs Phototrophs

(glucose + O2 CO2 + H2O) (H2 + O2 H2O) (light)

Figure 2.18 Metabolic options for conserving energy

2.8 Metabolic Diversity
• Chemoorganotrophs
– 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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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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2.8 Metabolic Diversity
Organisms are also classified according to their
source of cell carbon for biosynthesis:

– 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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2.8 Metabolic Diversity
Extremophiles: Organisms that inhabit
extreme environments
Habitats include
– Boiling hot springs
– Glaciers
– Extremely salty bodies of water
– High-pH environments

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2.9 Bacteria
• The domain Bacteria contains an enormous
variety of prokaryotes (Figure 2.19)
• All known pathogenic prokaryotes are Bacteria

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Figure 2.19 Phylogenetic tree of some representative Bacteria

Spirochetes
Green sulfur Planctomyces
Deinococcus bacteria
Green nonsulfur Chlamydia
bacteria
Cyanobacteria
Thermotoga
Gram-positive
OP2 bacteria

Aquifex

Proteobacteria

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2.10 Archaea

Examples:

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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2.12 Microbial Eukarya
Figure 2.33a Microbial Eukarya – (a) Algae (b) Fungi (c) Protozoa
Figure 2.34 Lichens. (a) An orange-pigmented lichen growing on a rock, and (b) a yellow-pigmented lichen
growing on a dead tree stump,

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2.12 Microbial Eukarya
• Eukaryotic microorganisms include:
* fungi
* algae
* protozoa
* slime molds

- Fungi are decomposers.

- Protists include algae and protozoa:

•The algae are phototrophic (Figure 2.33a)
•Protozoa NOT phototrophic (Figure 2.33c)
- Algae and fungi have cell walls, whereas protozoa
and slime molds do not
2.12 Microbial Eukarya
• Lichens are a mutualistic relationship
between two groups of protists
– Fungi and cyanobacteria
– Fungi and algae

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