1. Microscopy and Cytology

2. Introduction to Microscopes

3. Microscopy Permits Visualization of Objects Too Small to Be Normally Seen

4. 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

5. Simple vs. Compound Microscope
Simple – One Lens
Compound – Multiple Lenses

6. Parts of the Compound Light Microscope

7. Parts of the Dissecting Light Microscope

8. Electron Microscopes Magnify Extremely Small Objects

9. Ultra High Power Microscopes Can Resolve Individual Molecules

10. Principles of Microscopy

11. Important Concepts in Microscopy
Magnification
Resolving Power
Contrast
Viewing Field
Image orientation
Depth of focus
Size of the field of view
Working distance

12. 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

13. 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)

14. Oil Immersion Improves Resolution

15. 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.

16. 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

17. Microscope Care

18. Use the Coarse Focus Knob for the Lowest Power Only

19. Always Store the Microscope With the Lowest Power Objective in Place

20. 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.

21. 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!

22. 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.

23. 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.

24. NEVER CLEAN THE MICROSCOPE WITH ANYTHING OTHER THAN LENS PAPER!

25. Introduction to Cytology

26. 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

27. 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.

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

29. Overview of a Prokaryotic Cell

30. Bacterial Cell Morphologies
Coccus (Sphere)
Bacillus (Rod)
Spiral

31. 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)

32. Overview of an Animal Cell

33. Overview of a Plant Cell

35. 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

36. 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

37. 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

38. 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

39. 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

40. 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

41. Order of Experiments Prepare all slides (Exercise 7.6)
Introduction to microscopy (Exercises )
View wet mount slides and prepared bacterial slides under microscope (Exercise )
Exercise 7.5 can be performed whenever you have spare time.