Microscopy and Cytology

Introduction to Microscopes

Microscopy Permits Visualization of Objects Too Small to Be Normally Seen http://sciencephoto.com/images/download_lo_res.html?id=670066703 http://sciencephoto.com/images/download_lo_res.html?id=662340142 http://sciencephoto.com/images/download_lo_res.html?id=771800109

Types of Microscopes Light microscopes Electron 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 Microscope Simple – One Lens Compound – Multiple Lenses http://students.ou.edu/J/Renee.E.Jones-1/Episode%202.html http://www.scienceeducationonline.com.au/microscopes.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 http://i.ehow.com/images/a04/jq/h2/use-stereo-microscope-200X200.jpg

Electron Microscopes Magnify Extremely Small Objects http://sciencephoto.com/images/download_lo_res.html?id=770900084 http://sciencephoto.com/images/download_lo_res.html?id=770900075 http://serc.carleton.edu/images/research_education/geochemsheets/techniques/UWSEM.jpg

Ultra High Power Microscopes Can Resolve Individual Molecules http://www.ounqpi.org/Websites/nqpi/Images/facilities/_full_0_low%20temp%20STM.jpg http://cdn.physorg.com/newman/gfx/news/2005/Yan_pressrelease_fig2d.jpg

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 Parcentral – stays centered as magnification increases Working distance – How far the objective lens is from the slide

Microscope Care

Use the Coarse Focus Knob for the Lowest Power Only http://www.olympusamerica.com/seg_section/product.asp?product=1032

Always Store the Microscope With the Lowest Power Objective in Place http://www.scienceeducationonline.com.au/microscopes.html

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 MICROSCOPE WITH ANYTHING OTHER 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. http://sciencephoto.com/images/download_lo_res.html?id=725050013

Important Features of Prokaryotic Cells External Structures Internal Structures Capsule Cell wall Plasma membrane Flagella Pili Cytoplasm Nucleoid (chromosome) Ribosomes

Overview of a Prokaryotic Cell http://micro.magnet.fsu.edu/cells/bacteriacell.html

Bacterial Cell Morphologies Coccus (Sphere) Bacillus (Rod) Spiral http://www.microbelibrary.org/images/atlas-gram/streptococcus%20oralis%20fig5.jpg http://www.microbelibrary.org/images/uploads/0/jpg/1571_bacillus%20subtilis%20fig4.jpg http://www.scientificpsychic.com/health/spirochete.jpg

Important Features of Eukaryotic Cells External Structures Internal Structures Cell wall (plants) Plasma membrane Flagella Cilia 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  40X http://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 http://sciencephoto.com/images/download_lo_res.html?id=670052466

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 http://sciencephoto.com/images/showFullWatermarked.html/P470060-LM_of_epithelial_cells_from_the_human_mouth-SPL.jpg?id=804700060

Ear Swab Slide Preparation Staining Procedure Roll wet, sterile swab over top of ear and roll onto clean slide Repeat 2 more times Air-dry (10+ minutes) 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.