Microscopy and Cytology
Transcript of Microscopy and Cytology
Microscopy and Cytology
Introduction to Microscopes
Microscopy Permits Visualization of Objects Too Small to Be Normally Seen
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Types of Microscopes• Light microscopes
– Simple light microscope– Compound light microscope– Dissecting light microscope
• Electron microscopes– Transmission electron microscope– Scanning electron microscope
• Ultra high power microscope– Scanning-tunneling microscope– Atomic force microscope
Simple vs. Compound MicroscopeSimple – One Lens Compound – Multiple Lenses
http://www.scienceeducationonline.com.au/microscopes.htmlhttp://students.ou.edu/J/Renee.E.Jones-1/Episode%202.html
Parts of the Compound Light Microscope
http://academic.pgcc.edu/~kroberts/Lecture/Chapter%204/04-04_CompoundLM_L.jpg
Parts of the Dissecting Light Microscope
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Electron Microscopes Magnify Extremely Small Objects
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Ultra High Power Microscopes Can Resolve Individual Molecules
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Principles of Microscopy
Important Concepts in Microscopy
• Magnification• Resolving Power• Contrast• Viewing Field
– Image orientation– Depth of focus– Size of the field of view– Working distance
Magnification
• How much bigger the object under the microscope looks
• Depends on the lens or lenses• Total magnification = product of lens
magnifications– Oculars: 10X– Objective lenses: 4X, 10X, 40X, 100X– Totals: 40X, 100X, 400X, 1000X
Resolving Power
• Ability to tell the difference between two objects that are close together
• Higher resolution lets us see smaller things clearly
• Depends on:– Light wavelength – shorter is better (blue filter)– Refractive index – keeping constant is better
(immersion oil)
Oil Immersion Improves Resolution
http://academic.pgcc.edu/~kroberts/Lecture/Chapter%204/04-05_OilImmersion_L.jpg
Contrast• Ability to tell the difference between objects
and background• Can be improved using stains
Bauman, R.W. (2010). Microbiology with Diseases by Taxonomy (3rd ed.) New York, NY: Benjamin Cummings.
Considerations for the Viewing Field• Orientation – Image is inverted and reversed• Depth of focus – How much thickness of the
sample is in focus– Smaller as magnification increases– Parfocal – stays in focus as magnification increases
• Field of view – How much area of the slide is seen– Smaller as magnification increases– Parcentral – stays centered as magnification increases
• Working distance – How far the objective lens is from the slide– Smaller as magnification increases
Microscope Care
Use the Coarse Focus Knob for the Lowest Power Only
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Always Store the Microscope With the Lowest Power Objective in Place
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At the Beginning of the Day…• Remove the dust cover from the microscope.• Inspect for damage. Report anything you find!• Plug in the microscope.• Clean all lenses with lens paper ONLY.
– DO NOT clean lenses with anything other than lens paper!– Inform instructor if you find oil on a lens.
• Rotate the 4X objective into position above the stage.• Center the stage, and roll it down to the lowest
position.• Turn on the microscope light source.
Use of the Oil Immersion Lens• Find specimen and focus on 4X using coarse and then
fine focus knobs.• Move up to 10X and focus using FINE FOCUS KNOB
only.• Move up to 40X and focus using FINE FOCUS KNOB
only..• Slide 40X objective partly out of the way.• Place ONE drop of immersion oil on slide.• Gently slide 100X (oil immersion) objective into place.• Focus using FINE FOCUS KNOB only!
Use of the Oil Immersion Lens
• When finished observing under oil immersion:– Rotate from 100X objective to 4X objective and
remove slide.– Clean oil from slide using lens cleaner and lens
paper.– Carefully clean oil from the oil immersion lens
using lens cleaner and lens paper at the end of each class.
At the End of the Day…
• Remove slides from the microscope stage.• Turn off the microscope light source.• Clean oculars, ALL lenses, stage, and base with
lens cleaner and wipe with lens paper.• Rotate the nosepiece until the 4X objective is
in place.• Center the stage, and roll it to the lowest
position.• Unplug the microscope.• Cover the microscope with the dust cover.
NEVER CLEAN THE NEVER CLEAN THE MICROSCOPE WITH MICROSCOPE WITH ANYTHING OTHER ANYTHING OTHER THAN LENS PAPER!THAN LENS PAPER!
Introduction to Cytology
Cytology is the Study of Cells
• Cell = smallest unit of life– Composed of water and macromolecules– H, C, O, N are most predominant elements
• Two types of cells– Prokaryotic cells– Eukaryotic cells
• Organisms can be one or many cells– Unicellular – Single-celled organism– Multicellular – Organism composed of many cells
Robert Hooke and the Cell Theory
• The cell is the smallest unit of life.
• All living organisms are composed of cells.
• All cells arise from other cells.
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Important Features of Prokaryotic Cells
External Structures• Capsule• Cell wall• Plasma membrane• Flagella• Pili
Internal Structures• Cytoplasm• Nucleoid (chromosome)• Ribosomes
Overview of a Prokaryotic Cell
http://micro.magnet.fsu.edu/cells/bacteriacell.html
Bacterial Cell Morphologies
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Coccus (Sphere) Bacillus (Rod) Spiral
Important Features of Eukaryotic Cells
External Structures• Cell wall (plants)• Plasma membrane• Flagella• Cilia
Internal Structures• Cytoplasm• Membranous organelles
– Nucleus– Mitochondria– Chloroplasts (plants)– Endoplasmic reticulum (R/S)– Golgi apparatus– Lysosomes– Peroxisomes
• Nonmembranous organelles– Nucleoli– Ribosomes– Cytoskeleton– Centrioles (animal cells)
Overview of an Animal Cell
http://millville.sps.edu/allaccess/divisions/science/jdonnelly/Cell%20Page.htm
Overview of a Plant Cell
http://millville.sps.edu/allaccess/divisions/science/jdonnelly/Cell%20Page.htm
Introduction to Microscope Use
• Light microscope– Exercise 7.1 – field of view– Exercise 7.2 – depth of focus– Exercise 7.3 – image orientation
• Dissecting microscope– Exercise 7.4 – introduction to dissecting
microscopes
Cytology
• PREPARE ALL SLIDES FIRST!• Exercise 7.5 – Models• Exercise 7.6 – Wet mounts
– Cyanobacteria – prepared slide– Elodea leaf + safranin– Onion epidermis + iodine– Cheek cells + methylene blue– Ear swab + Romanowsky stain
• Exercise 7.7 – Prepared bacterial slides
Elodea Leaf• Drop of water on slide• Transfer Elodea leaf
into drop• Place one edge of
coverslip against drop• Gently lower coverslip
over drop• Drop of safranin on
slide next to coverslip – diffuse in
• 4X 10X 40Xhttp://lima.osu.edu/biology/images/chlorop.jpg
Onion Epidermis• Drop of water on slide• Transfer onion epidermis
into drop• Place one edge of coverslip
against drop• Gently lower coverslip over
drop• Drop of iodine on slide next
to coverslip – diffuse in• 4X 10X 40X
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Cheek Cells
• Drop of methylene blue on slide
• Scrape inside of cheek with toothpick
• Swirl into stain drop• Place one edge of
coverslip against drop• Gently lower coverslip
over drop• 4X 10X 40X
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Ear Swab
Slide Preparation• Roll wet, sterile swab over
top of ear and roll onto clean slide
• Repeat 2 more times
• Air-dry (10+ minutes)
Staining Procedure• Hold slide with clothespin• 1 second per dip, 10 dips
per jar• Order of stains:
– Alcohol (light blue)– Eosin (red)– Methylene blue (blue)– Distilled water
• Blot with bibulous paper• 4X 10X 40X 100X
w/oil
Order of Experiments
• Prepare all slides (Exercise 7.6)• Introduction to microscopy (Exercises 7.1-7.4)• View wet mount slides and prepared bacterial
slides under microscope (Exercise 7.6-7.7)
• Exercise 7.5 can be performed whenever you have spare time.