MICRB 106/107
Elementary Microbiology & Lab
Read both Contracts!
Finding Me
Goals & Objectives
Evaluation Criteria
Policies, Requirements, etc…
Textbooks
The Schedules
MICRB 106: Evaluation
• Participation (9%)
– Self-evaluation
– Professor’s evaluation
• Quizzes (4 x 2% ea. = 8%)
– Unannounced.
– Only on Mondays.
• Exams I (15%)
• Exams II, III, IV (20% ea. = 60)
• Bad Bug Talk (8%)
– With a partner.
– 15 minute maximum.
MICRB 107 (lab): Evaluation
• Lab Assignment Worksheets (50%)
– Five 10% submissions.
– Due dates announced.
• Quizzes (6 x 2% ea. = 12%)
– Unannounced.
– Only on Wednesdays.
• Practical Exams (2 x 15% ea. = 30%)
• Lab Behavior/Etiquette (8%)
• “Microbiology is a branch of Biology that
uses procedures involving sterile
technique and the use of culture media
which are necessary to isolate and grow
microorganisms” – Roger Stanier, 1978
• “Microorganisms (microbes) are
organisms too small to be seen clearly by
the naked eye” - Prescott et al., 2001
Which are the microorganisms?
• Life forms, or other self replicating entity, that requires
microscopy technology to be clearly visualized.
• Many are unicellular, sometimes cells are organized in
filaments or clumps, and others are complex with only a portion
of their life cycle being microscopic.
• Most can carry out life processes independently from other
cells, others are highly parasitic.
• They often require specialized techniques for their study:
microscopy, culturing, biochemical and/or molecular.
• They include all prokaryotic and many eukaryotic life forms.
Large difference in scale.
Average eukaryotic cell is
about 50x bigger than the
average prokaryote.
We’ll get back to these
differences in more
detail in a latter lecture.
What are some of the
major groups of life that
we call microbes?
3 Domains of Life
(2 Prokaryote & 1 Eukaryote)
“Tree of Life”
Most all of life’s diversity is microbial; represented in all major phyla.
Don’t forget, some animals too!
Flatworms & Roundworms
Trichinella spiralis larva in skeletal muscle (W.M., X260).
The spiral juvenile and its nurse cell are visible in this preparation.
Viruses: An infectious
particle with an acellular
organization of protein and
nucleic acids (RNA or
DNA), and lacking
independent metabolism.
It requires the metabolism
of a host cell in order to
replicate. Viruses are
about 50 to 200 nm in size.
Prion: An infectious aberrant brain protein that causes
abnormal aggregation of similar normal brain proteins; no
nucleic acids.
Note on Classification:
Eukaryote (e.g. Humans)
• Domain: Eukarya
• Kingdom: Animalia
• Phylum: Chordata
• Class: Mammalia
• Order: Primata
• Family: Hominidae
• Group: Homo
• Species: sapiens
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Prokaryote (e.g. E. coli)
Domain: Bacteria
(no kingdom)
Phylum: Proteobacteria
Class: γ-proteobacteria
Order: Enterobacteriales
Family: Enterobacteriaceae
Genus: Escherichia
Species: coli
Binomial nomenclature: Genus species (italic or underlined)
Just like varieties of apples, or races of people, there are strains of
a prokaryote species (e.g. the harmless Escherichia coli K12 versus
the deadly pathogenic E. coli O157:H7). Why so?
Why Study Microbes?
1) Microbes and Man in Sickness and Health
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Parasitism; Pathogens (disease causing)
Infectious disease is leading cause of death in developing countries (45%).
Commensalisms; Natural Microbiota (do no harm)
Mutualisms; Natural Microbiota (do us good; probiotics)
2) Major Developments in Biology:
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Recombinant DNA technology; Cloning
Industrial Applications (antibiotics)
Polymerase Chain Reaction (PCR) for Diagnostics
Genomics (Computer Technology for Studying Life)
3) The Role of Microbes in Ecosystems
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Sources for drug discovery (antibiotics & antiviral drugs)
Cycling of Elements (Biogeochemistry of C, N, S …)
Agriculture (crop diseases; nutrient enhancement)
Pollution Bioremediation (oil, xenobiotics)
4) Microbes in Human Civilization:
• Food and Beverage preservation (pre-history)
• Turns in History - The “Four Horseman”: War, Famine,
Pestilence, Death.
– Moses leads Israelites out of Egypt; facilitated by plague (1500 BC)
– Athenians (Greeks) lost Peloponnesian War in 404 BC due to plague
(Yersinia pestis; bacterial disease)
– Fall of Rome (565 AD) due to overcrowding exacerbated by Attila the
Hun’s (barbarians’) army cutting off water supplies to Rome –
epidemic malaria (protist disease) and other infectious diseases.
– Spanish conquering the Aztec civilization (1500’s) by introducing
small pox and measles (viral disease).
– Salem Witch Hunt (1692): Puritan rye gets infected by Ergot
(Claviceps purpurea; fungal plant pathogen); bread makes them
loony; hallucinations perceived as evil spells.
– Great Famine (Ireland ca 1850’s): potato blight (Phytophthora
infestans; oomycete fungi) starved ten’s of thousands to death; over
a million immigrated to America.
Over 3.5 billion years of “microbes”;
only 400 years of Microbiology!
• First life at 3.8 Ga by evolved chemical complexity; alien entry; or divine intervention.
• Earth then was extreme: no oxygen, intense radiation, very hot, & electrical discharge.
• Energy for earliest life from reduced (electron rich) organic and inorganic compounds.
• Later came the ability to capture light energy for synthesis of organic matter from CO2.
• Oxygenic photosynthesis slowly oxygenated the atmosphere; greater energy yield
from metabolizing organic matter by aerobic respiration helped accelerate evolution.
Over 400 years of Microbe Hunting!
What fundamental discoveries started it all?
• First Light Microscopes (1600s)
• Origins of New Life and the Debunking
“Spontaneous Generation” (1660’s to 1860s)
Louis Pasteur
• Linking Microbes to Human Disease (late 1800s)
Robert Koch
• Discovering Vaccines and Immunity (late 1800s)
Edward Jenner, Elie Metchnikoff , Emil von Behring
• Agricultural and Environmental Microbiology
(late 1800s) Martinus Beijerinck, Sergei Winogradsky
First Light Microscopes
Zacharias Jannsen (1595),
Robert Hooke (1665):
first “cells” from cork;
Micrographia, 1665
First Microscopes
Antonie van Leeuwenhoek (1676): excellent simple microscopes;
discovered and observed microbes, called them “animalcules”.
Size?
See Table 3.1
Microscopy:
• Light Microscopy
- Specimen illuminated with visible
or ultraviolet light.
- Used for seeing specimens from
0.5 µm to 0.5 cm. (10,000-fold
difference)
• Electron Microscopy
- Specimen “illuminated” with
electrons.
- Used for specimens from 0.5 nm
to 50 µm. (100,000 – fold difference)
Light Microscopes:
Anatomy & Optics:
• Light source is from underneath.
• Condenser concentrates the light;
forming a “cone shaped” beam pointing
into the specimen.
• Note that the image gets flipped after the
sample.
• The prism redirects the path of light to the
oculars, during the path from prism to
oculars the image flips again – back to
“normal”.
Magnification:
- Objective lenses
- Ocular lenses
- Total magnification is their product.
Blurring and dimming of the image at higher objective
magnification (e.g. 100 x):
• Light changes its pathway
angle, or bends, when is
passes through materials of
different consistencies (optical
density). This is called
refraction.
• It happens when light passes
through glass into air. Thereby,
some light illuminating a sample
may not pass into the objective
(hence dimming), or the path
may be shifted out of focus
(hence blurring).
• The problem appear worse
with a 100x objective.
• Immersion oil between
specimen and objective is the
solution; it’s similar to glass.
Brightfield
Illumination:
* Uses full spectrum, or
white light (“all the
colors of the rainbow”),
directed to pass
through the specimen.
* Often very small nonpigmented specimens
makes them appear
nearly featureless,
transparent, even
invisible.
* Colored stains are
used to enhance the
image of very small
specimens
Fluorescence
Microscopy:
Another way to see cells and
structures difficult to see with brightfield
illumination, and more sensitive than
colored dyes.
• The microscope illuminates the
sample with ultraviolet light (UV), and
the sample must be dyed with a
compound that will fluoresce a color
when illuminated with UV light; call
fluorochromes.
• Immuno-fluorescence:
-Antibodies are immune system
proteins that can bind to a target
specimen very specifically. They
are very useful in fluorescent
microscopy when they have a
fluorescent dye bound to them.
Darkfield Microscopy:
Brightfield
• The image is like a
photographic negative of
the brightfield image.
•Stains are typically not
used with darkfield.
• Special optics enhance
the contract so more
details are seem.
• Instead of a solid
“cone of light”
condensed onto the
specimen, brightfield
optics create a hollow
cone that illuminate the
specimen’s sides.
Salamander egg;
500 µm
Human cheek cells;
50 µm (stained)
Darkfield
Darkfield Optics
The annular stop blocks light from most of the condenser lens, except for
the edges; hence a “hollow cone” of light points onto the sample. Without
the extra light coming through the sample, the background is dark.
Other Kinds of Light Microscopy
• Phase Contrast:
partly like darkfield, but the optics create a
light wavelength shift as it passes through denser parts of the
specimen; resulting in even greater contrast enhancement so to see
even more detail.
• Differential Interference Contrast (DIC):
Uses two
light sources condensing on the sample to create more contrast than
phase contrast. Furthermore, the light passes through a prism,
resulting in a separation of color and more color seen in the image.
• Confocal Microscopy:
Uses lasers and fluorochrome dyes.
Laser illumination permits focusing on specific layers within a
specimen. When coupled with computer software, 3D images can
be generated.
Image Resolution: Light’s Limitation
• Resolution refers to the
ability to distinguish between
two points in space.
• Higher resolution means
you distinguish between two
objects that are closed
together.
• Maximum resolution is the
closest two point in space
can be apart from each and
still distinguish them.
• Illuminating a sample with
a smaller wavelength of light
can improve maximum
resolution. UV light is a little
better that white light, and
electrons are the best!
White Light
has a
maximum
resolution of
about 0.2 µm.
Two cells or
viral particle
smaller than
this will appear
as a single
blurred object
when next to
each other.
Electron Microscopy
The Maximum Resolution is 0.5 nm, which is 400-times better than light.
Transmission EM creates 2D images, and specimens must be very thin for
electrons to pass through. Scanning EM makes 3D images of any surface.
TEM (2D)
SEM(3D)
Both require special staining of a non-living specimen.
Be able to compare the differences between light and electron microscopy.