micrb 106/107 elementary microbiology & lab read both contracts! finding me goals &...
Post on 22-Dec-2015
219 views
TRANSCRIPT
MICRB 106/107Elementary 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.
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?
Large difference in scale. Average eukaryotic cell is about 50x bigger than the average prokaryote.
3 Domains of Life(2 Prokaryote & 1 Eukaryote)
Most all of life’s diversity is microbial; represented in all major phyla.
“Tree of Life”
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
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• 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:• 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• 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”.
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 non-pigmented 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 Darkfield
• 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 OpticsThe 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 MicroscopyThe 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.