3 Microscopy Permits Visualization of Objects Too Small to Be Normally Seen
4 Types of Microscopes Light microscopes Electron microscopes Simple light microscopeCompound light microscopeDissecting light microscopeElectron microscopesTransmission electron microscopeScanning electron microscopeUltra high power microscopeScanning-tunneling microscopeAtomic force microscope
5 Simple vs. Compound Microscope Simple – One LensCompound – Multiple Lenses
11 Important Concepts in Microscopy MagnificationResolving PowerContrastViewing FieldImage orientationDepth of focusSize of the field of viewWorking distance
12 Magnification How much bigger the object under the microscope looks Depends on the lens or lensesTotal magnification = product of lens magnificationsOculars: 10XObjective lenses: 4X, 10X, 40X, 100XTotals: 40X, 100X, 400X, 1000X
13 Resolving PowerAbility to tell the difference between two objects that are close togetherHigher resolution lets us see smaller things clearlyDepends on:Light wavelength – shorter is better (blue filter)Refractive index – keeping constant is better (immersion oil)
15 Contrast Ability to tell the difference between objects and background Can be improved using stainsBauman, 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 reversedDepth of focus – How much thickness of the sample is in focusSmaller as magnification increasesParfocal – stays in focus as magnification increasesField of view – How much area of the slide is seenParcentral – stays centered as magnification increasesWorking distance – How far the objective lens is from the slide
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!
26 Cytology is the Study of Cells Cell = smallest unit of lifeComposed of water and macromoleculesH, C, O, N are most predominant elementsTwo types of cellsProkaryotic cellsEukaryotic cellsOrganisms can be one or many cellsUnicellular – Single-celled organismMulticellular – 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 StructuresInternal StructuresCapsuleCell wallPlasma membraneFlagellaPiliCytoplasmNucleoid (chromosome)Ribosomes
35 Introduction to Microscope Use Light microscopeExercise 7.1 – field of viewExercise 7.2 – depth of focusExercise 7.3 – image orientationDissecting microscopeExercise 7.4 – introduction to dissecting microscopes
37 Elodea Leaf Drop of water on slide Transfer Elodea leaf into drop Place one edge of coverslip against dropGently lower coverslip over dropDrop of safranin on slide next to coverslip – diffuse in4X 10X 40X
38 Onion Epidermis Drop of water on slide Transfer onion epidermis into dropPlace one edge of coverslip against dropGently lower coverslip over dropDrop of iodine on slide next to coverslip – diffuse in4X 10X 40X
39 Cheek Cells Drop of methylene blue on slide Scrape inside of cheek with toothpickSwirl into stain dropPlace one edge of coverslip against dropGently lower coverslip over drop4X 10X 40X
40 Ear Swab Slide Preparation Staining Procedure Roll wet, sterile swab over top of ear and roll onto clean slideRepeat 2 more timesAir-dry (10+ minutes)Hold slide with clothespin1 second per dip, 10 dips per jarOrder of stains:Alcohol (light blue)Eosin (red)Methylene blue (blue)Distilled waterBlot with bibulous paper4X 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.