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Microscopy and Cytology. Introduction to Microscopes.

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Presentation on theme: "Microscopy and Cytology. Introduction to Microscopes."— Presentation transcript:

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 – 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 LensCompound – 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 0STM.jpg

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 (3 rd 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 – 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

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 Capsule Cell wall Plasma membrane Flagella Pili Internal Structures 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 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)

32 Overview of an Animal Cell

33 Overview of a Plant Cell

34

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 LM_of_epithelial_cells_from_the_human_mouth-SPL.jpg?id=

40 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

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.


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