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=662340142
http://sciencephoto.com/images/download_lo_res.html?id=670066703
http://sciencephoto.com/images/download_lo_res.html?id=771800109
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 Microscope
Simple – One Lens
http://students.ou.edu/J/Renee.E.Jones-1/Episode%202.html
Compound – Multiple Lenses
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=770900075
http://sciencephoto.com/images/download_lo_res.html?id=770900084
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%2
0STM.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
– 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
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
• 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
Coccus (Sphere)
Bacillus (Rod)
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
Spiral
Important Features of Eukaryotic
Cells
External Structures
• Cell wall (plants)
• Plasma membrane
• Flagella
• Cilia
Internal Structures
• Cytoplasm
• Membranous organelles
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–
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–
–
–
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Nucleus
Mitochondria
Chloroplasts (plants)
Endoplasmic reticulum (R/S)
Golgi apparatus
Lysosomes
Peroxisomes
• Nonmembranous organelles
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–
–
–
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/P470060LM_of_epithelial_cells_from_the_human_mouth-SPL.jpg?id=804700060
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.