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

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 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.