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BIOL 260-General Microbiology
Instructor: Seana Davidson
Welcome to BIOL 260: Microbiology!
•  First day:
–  Review of Syllabus
–  Sign-in
–  Introduce the course, review course
expectations
–  Begin with first lab
•  Exercise 3: Microscope Lab
What is microbiology?
•  The scientific discipline which studies
microbes or microorganisms
–  Biology of microbes
–  The interaction of microbes with other
microbes, the environment, and humans
What are examples of microbes?
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Algae
Fungi
Protozoa
Bacteria
Viruses
1.
Algae
2.
Fungi
3.
Protozoa
4.
Bacteria
5.
Viruses
•  Which microbes are eukaryotes?
•  Which are prokaryotes?
•  Which can perform
photosynthesis?
•  Which are classified based on
locomotion?
•  Which have cell walls?
•  Which have some type of
nucleic acid?
Types of Microbes
fungi
protozoa
algae
viruses
Types of Microbes: Algae
Microscopic
Diatoms and Volvox
Macroscopic
Volvox, microscopic aglae that
form colonies
By Tomachi from http://www.funk.co.nz/blog/science/volvox
Types of Microbes: Protozoa
Types of Microbes: Fungi
mold
yeast
mushrooms
Can form macroscopic
structures
Types of Microbes: Bacteria
Viruses, Viroids, Prions
Microorganisms are associated with
•  Disease
–  Cause of many epidemics in history
–  Bubonic plague (1346-1350)-bacteria-still
around-Yersinia pestis transmitted by fleas
–  Small pox--virus
–  HIV--virus
–  Malaria--protozoa
Bacteria are also associated with
healthy conditions
•  Normal microbiota (normal flora)
–  The bacteria that are present on our bodies
Bacteria are everywhere and provide many services
3.5 billion years of diversification and evolution
•  the environment
•  Soils, Rhizobium—associated with plants
•  Food products—much of our food is
fermented with bacteria in some way
•  Recombinant DNA products
•  Enzymes for many uses
–  Fading of blue jeans, degradation of cellulose, synthesis
of nylon
History of Microbiology
•  It all started with the microscope!
–  Zacharis Janssen (1600 or so) Dutch spectacle
maker. Possibly first telescope and microscope
–  Antoni van Leewenhoek (1632-1723)-crafted
lense and saw ‘animalcules’
–  Robert Hooke (1665)—microscopic
mushrooms (penicillum mold)
Zacharis Janssen’s microscope
•  Modeled after the
telescope
•  Consisted of two
lenses
•  Magnified images
3-10X
Leewenhoek’s microscope
Where do organisms come from?
•  Debunking Spontaneous generation –  Francesco Redi (1668)—flies come from eggs of other flies, not
generation from meat ---200 years more to prove the same for microbes.
–  John Needham (1745)-boiled broth, microbes still grew
–  Lazzaro Spallanzani (1765)-boiled longer, melted necks
of flasks, remained sterile until neck broken
–  Louis Pasteur (1861) –showed microbes were in the air,
cotton ball experiment, and swan necked flask experiment.
What were these?
•  Biogenesis
–  Rudolf Virchow (1858)—father of modern medicine-discredit
humorism, bringing more science to medicine. Diseases are caused by
organisms.
Pasteur’s flasks
Tyndall had trouble repeating this. Why? What was in the lab?
What did he discover? John Tyndall questions Pasteur’s
experiments
•  Could not reproduce Pasteur’s results
•  Found that there were heat resistant forms of
microbes
•  Same year (1876) Ferdinand Cohn discovers heat
resistant forms of bacteria called endospores
•  1877 Robert Koch demonstrates that anthrax
caused by Bacillus anthracis
His method of proof became known at Koch’s
Postulates. New cells need to be placed in categories
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Aristotle-plant or animal kingdom
Kingdom Protista (1866)
Electron microscope (1940’s)
Kingdom Procaryotae (1968)
Carl Woese proposed 3 Domains (1978)
Three Domain System
Prokaryotes (Bacteria)
•  Bacteria Domain (Eubacteria)
–  Gram negative (thinner cell wall)
–  Gram positive (cell wall)
–  No cell walls
•  Archaea Domain (Archaebacteria)No cell
walls
–  Methanogens
–  Halophiles
–  Thermophiles
Binomial system of nomenclature
•  Genus and species
–  Escherichia coli, E. coli
•  Both names are in italics or underlined
•  Note that there are higher levels of
divisions. Order, Family, then Genus and
Species.
Relationship of size and
resolution
Types of microscopes
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Brightfield
Darkfield
Phase Contrast
Fluorescent
Electron
Microscopy-Brightfield
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 µ m –  Shorter wavelengths give higher resolution
Electron Microscopy
•  Transmission Electron Microscopy (TEM)
–  Electromagnets function as lenses
–  System operates in a vacuum
–  High magnification and resolution (0.2 nm)
(traveling electrons have very short wavelength)
–  Enables visualization of structures at the
molecular level (Figure 2.10a and b)
–  Specimen must be very thin (20–60 nm) and
be stained
Microscopy
Electron microscopes - maximum magnification ∼100,000X
Scanning EM: picture of surfaces
Transmission EM: picture of a
slice through the cell: interior
Oil has same refractive index as
glass High magnification lenses use oil to improve image
(oil immersion lens)
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Ways to improve contrast and
visualization
DIFFERENT METHODS
OF LIGHTING
–  Phase contrast / differential interference
contrast(DIC)
–  Darkfield
•  STAINING
–  Simple: cells all stained the same –  Differential: stains different types of cells so that
they can be distinguished (Gram stain)
Microscopy
Stained specimen
Wet mount
Examples of:
Bright field
Phase contrast
Dark field
© 2012 Pearson Education, Inc.
Microscopic Techniques: Dyes and
Staining
• Simple stains
• Basic dyes with positive charge
stick to cells
• Acid dyes provide background
stain
• Differential stains
Gram stain - separates
bacteria into two categories
based on type of cell wall
Acid Fast Stain
Gram-positive
Gram-negative
Figure 2.4a
Step 1
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
Step 4
G-
Result:
Gram-positive
(G+) cells are purple;
gram-negative (G-) cells
are pink to red
© 2012 Pearson Education, Inc.
Counterstain with
safranin for 1–2 min
G+
Gram-positive Cell Wall Thick layer of peptidoglycan
Teichoic acids
Gram-negative Cell Wall
Thin layer of peptidoglycan
Outer membrane – and cytoplasmic membrane
lipopolysaccharide
(LPS)
Differential Stain: Acid Fast
Stains particular group of bacteria based on their
cell membrane composition
(Mycobacterium)
Mycolic acid (waxy)
makes it difficult to get
dye in, so stain with
carbol fuschin, heat,
then wash with acid
alcohol. Dye is
retained only in the
mycolic acidcontaining cells. (pink)
Special stain: Capsule Stain
India ink excluded by the gel-like exopolysaccharide capsule
Special stain: Endospore Stain
Malachite green stains the spores,
Safranin is used as counter stain for cells (pink).
Gram stain does not stain spores.
Special stain: Flagella Stain
Stain with basic stain that will stick to the cells and the flagella,
making them visible. Morphology of
Prokaryotic Cells:
Cell Shapes
Coccus
Rod
Vibrio
Spirillum
Spirochaete
Morphology of
Prokaryotic Cells:
Cell Groupings