Lab Exercises Week 1:
#3 Microscope
#5 Simple Staining
#1 Ubiquity of Microorganisms
Pre lab due: None
Post lab due: 1/17/15 & 1/24/15
Principles of Light Microscopy
• Light passes through specimen and then series
of magnifying lenses
• Bright-field microscope is most common type
• Three key concepts
– Magnification: apparent increase in size
• Modern compound microscopes have two lens types:
objective and ocular
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• Magnification is product of
objective (4x, 10x, 40x, and
100x) and ocular lens (10x)
• Condenser lens (between
light source and specimen)
focuses light on specimen,
does not magnify
Ocular lens
(eyepiece)
Magnifies the
image, usually
10-fold (10×).
Objective lens
A selection of lens
options provide
different
magnifications. The
total magnification is
the product of the
magnifying power of
the ocular lens and
the objective lens.
Specimen
stage
Condenser
Focuses
the light.
Light source
Iris diaphragm
Controls the
amount of light
that enters the
objective lens.
Rheostat
Controls the
brightness of the
light.
Courtesy of Leica, Inc., Deerfield, FL
Microscopy-Brightfield
1.5. Size in the Microbial World
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Nucleus
Small
molecules
Atoms
Proteins
Viruses
Mitochondria
Prion fibril
Lipids
Ribosomes Smallest
bacteria
Most
bacteria
Most eukaryotic cells
Adult roundworm
Human height
Electron microscope
Light microscope
Unaided human eye
0.1 nm
1 nm
10 nm
100 nm
1 µm
10 µm
The basic unit of length is the meter (m), and all
other units are fractions of a meter.
nanometer (nm) = 10–9 meter = .000000001 meter
micrometer (µm) = 10–6 meter = .000001 meter
millimeter (mm) = 10–3 meter = .001 meter
1 meter = 39.4 inches
100 µm
1 mm
1 cm
0.1 m
These units of measurement correspond to units
in an older but still widely used convention.
1 angstrom (Å) = 10–10 meter
1 micron (µ) = 10–6 meter
1m
10 m
Oil has same refractive index as glass
3.2. Microscopic Techniques: Dyes and Staining
 Samples can be immobilized, stained to visualize
 Basic dyes (positive charge)
• Attracted to negatively charged cellular components
 Acidic dyes (negative charge)
• Negative staining: cells repel, so colors background
• Can be done as wet mount
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Spread thin film of
specimen over slide.
Allow to air dry.
Pass slide
through
flame to
heat-fix
specimen.
Flood the smear with
stain, rinse, and dry.
Examine with microscope.
Goldilocks technique
 Using a sterile loop place three drops of water
onto one slide.
 Re-sterilize your loop and get some bacteria on
your loop (enough that you can see it with your
eye).
 Mix with water to create a smear.
 1st one you will be able to see the bacteria (very
cloudy).
 2nd one you will only barely be able to see the
bacteria (slightly cloudy).
 3rd one you should not be able to see bacteria in
water drop (clear).
3.3. Morphology of Prokaryotic Cells
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 Two types most common
Coccus
Rod (bacillus)
• Coccus: spherical
• Rod: cylindrical
 Variety of other shapes
(a)
• Vibrio, spirillum, spirochete
• Pleomorphic (many shapes)
• Great diversity often found in low
nutrient environments
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1 µm
Vibrio
(c)
(b)
11.4 µm
Spirillum
15 µm
(d)
15 µm
Spirochete
(a)
1 m
(b)
1 m
a: Courtesy of Walther Stoeckenius; b: Courtesy of James T. Staley
(e)
7.5 µm
(a): © SciMAT/Photo Researchers, Inc.; (b, c, d, e): © Dennis Kunkel Microscopy inc.
Groupings
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 Most prokaryotes divide by
binary fission
• Cells often stick together
following division
• Form characteristic groupings
Chains
Diplococcus
Cell divides
in one plane.
Chain of cocci
(a)
Packets
Cell divides
in two or more planes
perpendicular to one
another.
Packet
(b)
Clusters
Cell divides
in several planes at
random.
Cluster
(c)
(a): (top): © George Musil/Visuals Unlimited; (bottom): © David M. Phillips/Visuals Unlimited; (b): © R. Kessel & C.
Shih/Visuals Unlimited; (c): © Oliver Mecks/Photo Researchers, Inc.
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?
Major Groups of Microbial World
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Microbial World
Infectious agents
(non-living)
Organisms
(living)
Domain
Bacteria
Archaea
Viruses
Eucarya
Eukaryotes
Prokaryotes (unicellular)
Algae
(unicellular or
multicellular)
Protozoa
(unicellular)
Protists
Fungi
(unicellular or
multicellular)
Helminths
(multicellular
parasites)
Viroids
Prions
Microbial Ubiquity
 Prokaryotes found growing in severe conditions
• Ocean depths, volcanic vents, polar regions all harbor
thriving prokaryotic species
• Many scientists believe that if life exists on other
planets, it may resemble these microbes
 Individual species have limited set of conditions
 Important to grow microbes in culture
• Medical significance
• Nutritional, industrial uses
4.5. Environmental Factors That Influence Microbial Growth
 Prokaryotes inhabit nearly all environments
• Some live in comfortable habitats favored by humans
• Some live in harsh environments
• Termed extremophiles; most are Archaea
 Major conditions that influence growth
• Nutrient availability
• Temperature
• Atmosphere- Oxygen
• pH
• Water availability