Biology 261 – Chapter 2

- A microscope is required for the visualization of microorganisms

- Light microscopes: used to look at intact cells under relatively low magnification
o Resolution = 0.2 microns
 .2 microns = 2 cells
 .1 microns = 1 cell
- Resolution: ability to distinguish two objects as separate and distinct
o Determined by the wavelength of light used
o Limit is about 2 nanometers
o Increase resolution by using blue filters because shorter wave lengths or immersion oils
- Immersion oils: decrease the scattering of light
- Always start with scanning because as you move up working distance gets smaller and field
of view
- Compound light microscope: use in labs, have light bulbs
o Compound = more than one lens
o Uses visible light to illuminate cells
o Compound because it has an ocular and objectives
- Types of light microscopy
o Bright-field
o Phase-contrast
 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
o Dark-contrast
 Light reaches the specimen from the sides
 Light reaching the lens has been scattered by specimen
 Image appears light on a dark background
 Excellent for observing mobility
o Fluorescence
 Used to visualize specimens that fluoresce
 Emit light of one color when illuminated with another color of light
 Cells fluoresce naturally (auto fluorescence) or after they have been stained with
a fluorescent dye like DAPI
 Widely used in microbial ecology for enumerating bacteria in natural samples
 Used often with antibiotics
 Ultraviolent light
- Improving contrast results in a better final imagine
- Staining is an easy way to improve contrast
o Dyes are organic compounds that have different affinities for specific cellular materials
 Examples: methylene blue, safranine, and crystal violet
- Staining
o Spread culture in thin film over slide
o Dry in air
o Pass slide through flame to heat fix
o Flood slide with stain, rinse, and dry
o Place drop of oil on slide, and examine with microscope
o Simple stains: one die
 Stains because stain has positive charge and cell has negative charge
 Crystal violet – purple
 Safranine – red
 Methalene blue – light blue
 Morphology – size and shape
o Differential stains: more than one kind of die
 More information and most frequently used
  Gram stain: renders different kinds of cells different colors
o Bacteria can be divided into two different groups (gram positive,
which appear purple, and gram negative, which appear red)
 Acid fast stain
 Endospore stain
o Use stain to create contrast to distinguish from background
 Differential stains (pos/neg)
 Gram staining (stains the cell): multiple stains
o Crystal violet: 1% stain (purple)
o Gram iodine: mordant (combines with violet)
o 70% ethanol: decolorizer
 Gram neg: loses CVI (colorless because of thin cell wall)
 Gram pos: purple (thick cell wal)
o Safranine: red pink (basic)
 Basic staining (pos) chromophore (part of stain that is color) stains cell
 Acidic staining (neg) chromophore – repelled by cell, stains background
- Three dimensional imagining
o Differential interference contrast (DIC) microscopy
 Uses a polarizer to create two distinct beams of polarized light
 Gives structures such as endospores, vacuoles, and granulose, a 3D appearance
 Structure not visible using bright-field microscopy are sometimes visible using
DIC
o 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 imagine based on the data received from the stylus
o Confocal scanning laser microscopy (CSLM)
 Uses a computerized microscope coupled with a laser source to generate a three-
dimensional image
 Computer can focus the laser on single layers of the specimen
 Different layers can then be compiled for a 3D image
- Electron microscope
o Uses electrons instead of photons to image cells and structures
o Shorter wave lengths in these gives us better resolution than light microscopes
o Two types of microscopes
 Transmission electron microscopes (TEM): internal structure, cut specimen
 Electromagnets function as lenses
 System operates in a vacuum
 High magnification and resolution
 Used to see internal structures of cells
 Specimen must be very thin and be stained
 Specimens are fixed and cut with diamond knives into thin specimens.
Then stained with heavy dyes making the internal structure visible
 Scanning electron microscopes (SEM): surface, gold
 Specimen is coated with a thin film of heavy metal
 An electron beam scans the object
 Scattered electrons are collected by a detector and an imagine is produced
 Even very large specimens can be observed
 Used to look at the surfaces of cells
- Elements of cells
o Microbial cells have this in common
 Cytoplasmic membrane
 Cytoplasm
 Ribosomes
o Prokaryotic
  Cytoplasm (water), nucleoid (chromosome), ribosome (where mRNA goes through
protein synthesis), plasmid (DNA, carry genes that are usually resistant to
antibiotics, replicates, cell may or may not have this), cytoplasmic membrane
(phospholipid bilayer) cell wall
o Eukaryotic
 Cytoplasmic membrane, ribosome, nucleus, nucleolus, nuclear membrane,
cytoplasm, endoplasmic reticulum, Golgi, mitochondria, chloroplast
(photosynthesis)
o Difference between pro and eu
 Eukaryotic
 DNA enclose in a membrane bound nucleus
 Cells are generally larger and more complex
 Contain organelles
 Many chromos
 Linear DNA and found within nucleus
o More than one chromo
o Two copies of each chromo
 Prokaryotic
 No membrane enclosed organelles
 No nucleus
 Generally smaller
 Single, circular DNA molecule – chromo
o DNA aggregates to form the nucleoid region
o Plasmids: extra chromosomal DNA that confer special properties
o Viruses: nucleic acid; DNA or RNA, protein coated capsule surrounds nucleic acid may
have a few enzymes
 Not considered cells
 No metabolic abilities of their own
 Rely completely on biosynthetic machinery of infected cell
 Infect all types of cells (hosts)
 Smallest virus in 10 nm in diameter
 Very small compared to bacteria
 Not able to reproduce independent of a host cell so they are not free living cells
(require a host to reproduce)
o Genome: a cells full complement of genes
o Mutation: change in DNA chromos shows up very fast in bacteria as compared to
eukaryotic cells
- Evolution: the process of change in a line of descent over time that results in new varieties
and species of organisms (result of changes in an organism DNA which results in changes in
the phenotype of the organism)
o Phylogeny: evolution relationship between organisms
 Relationships can be deduced by comparing genetic information (nucleic acid or
amino acid sequences) in the different specimens
 Ribosomal RNA (rRNA) are excited excellent molecules for determining phylogeny
because it doesn’t change often
 Can visualize relationships on a phylogenetic tree
o Comparative rRNA sequencing has defined three distinct lineages of cells called
domains
 Bacteria (pro)
 Archaea (pro)
 Eukarya (eu)
o Archaea and Bacteria are NOT closely related
o Archaea are more closely related to Eukarya than Bacteria
 Humans are more related to Eukarya
o Eukaryotic microorganisms were the ancestors of multicellular organisms
 o Mitochondria and chloroplasts also contain their own genomes (circular, like pro) 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
o Endosymbiotic theory: Eukaryotic cells came into being because one prokaryotic cell
engulfed another one but didn’t destroy it. Engulfed cell became what we know as
mitochondria and chloroplasts
- Diversity of microorganisms
o Chemoorganotrophs: obtain their energy from the oxidation of organic molecules
 Use organic molecules like glucose (humans)
 Found in both pro and eu organisms
o Chemolithotrophs: obtain their energy from the oxidation of inorganic molecules (sulfur
nitrogen)
 Process found only in pro (bacteria)
 Organisms are all pro
o Phototrophs: contain pigments that allow them to use light as an energy source (to eat)
 Oxygenic photosynthesis produces oxygen
 Anoxygenic photosynthesis does not produce oxygen
o All cells require carbon as a major nutrient
 Autotrophs: plants, cyanobacteria (can feed themselves)
 Use carbon dioxide as their carbon source
 Sometimes referred to as primary producers
 Heterotrophs: humans (must be fed)
 Require one or more organic molecule for their carbon source
 Feed directly on autotrophs or live off products produced by autotrophs
o Organisms that inhabit extreme environments are called extremophiles (archea  hot
springs)
o Habitats include boiling springs, glaciers, extremely salty bodies of water, and high pH
environments
- Bacteria
o The domain bacteria contains an enormous variety of pro
o All known pathogenic pro are bacteria
o The proteobacteria make up the largest division of bacteria
 Gram negative = [Link], pseudomonas and salmonella
o Cyanobacteria are relatives of gram positive bacteria
 Critical to evolution of life; they oxygenated the Earth’s atmosphere
 Important for oxygen and nitrogen fixation
 Undergo photosynthesis  chlorophyll; make glucose and H2O
 Heterocyst: nitrogen fixation – take free N2 from ammonia, nitrate, nitrite
 Found – pond scum, gunk between sidewalk
o Many other phyla of bacteria
 Green sulfur bacteria and green nonsulfur bacteria are photosynthetic (like
cyanobacteria and plants)
 Deinococcus are extremely resistant to radioactivity (nuclear waste site)
 Chlamydia are inside the cell bacteria that cause STD’s in urinary track
- Archaea (found in extreme environments)
o Two phyla of the domain archaea
 Euryarchaeota
 Methanogens: anaerobically degrade organic matter; produce methane
(natural gas) – swamps
 Extreme halophiles: requires high salt concentrations for metabolism and
reproduction – salt lakes
 Thermoacidophiles: grow in moderately high temperatures and low pH
environments
  Crenarchaeota
 Vast majority of cultured Crenarchaeota are hyperthermophiles (hot
springs, ocean)
 Some live in marine, freshwater, and soil systems
- Microbial communities
o Microbiologists believe that we have cultured only a small fraction of the archaea and
bacteria
o Studies done using methods of molecular microbial ecology, devised by Norman Pace
 Microbial diversity is much greater than labs culturing can reveal
- Microbial Eukarya
o Eukaryotic microorganisms include algae, fungi, protozoa, and slime molds
 Protists: algae and protozoa
 Algae are phototrophic, protozoa are not
 Fungi: decomposers
 Algae and fungi have cell walls, whereas protozoa and slime molds do not
o Lichens are mutualistic relationship between two groups of protists
 Fungi and cyanobacteria
 Fungi and algae