Ch 2 Part 2. The Microscope
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1 Ch 2 Part 2 The Microscope
2 SLOs for Microscopic Analysis of Microorganisms Convert among the different units of the metric system. List and describe three elements of good microscopy. Differentiate between principles of light microscopy and electron microscopy. Give examples of simple, differential, and special stains.
3 Review of Metric Units of Measurement 1 m = 1 mm = µm = nm = cm 1µm = m = mm 1 nm = m = mm 1000 nm = µm ; µm = nm
4 Microbial Size Cells Alive How big is a...? Macroscopic organisms: cm and m Microscopic organisms: to to Protozoa & algae ~ 3 4 Smallest bacteria ~ 200 most between 1 and 8 Viruses ~ 20 to 800
5
6 Learn all parts in Lab
7 Three Properties of the Compound Light Microscope 1) Magnification due to 2 lenses: and lenses of the compound microscope Calculate total magnifications! Fig ) Resolution: Also known as resolving power. Ability of lens to distinguish between 2 points. Maximum resolving power depends on (=... For eye: mm For light microscope: m Fig 2.13 Fig 3.1
8 Refraction Refractive Index measures lightbending ability of a medium Light may bend in air so much that it misses the small high-magnification lens Immersion oil is used to keep light from bending Role of Immersion Oil: Replaces Fig 2.14 Fig 3.1
9 3) Contrast: Stains change refractive index contrast between bacteria and surrounding medium Too much light can reduce contrast! The substage iris diaphragm controls the amount of light coming into the condenser.
10 Different Types of Lightmicroscopes Brightfield Microscopy Simplest of all the optical microscopy illumination techniques Dark objects are visible against a bright background. Darkfield Microscopy Light objects visible against dark background. used to enhance the contrast in unstained samples. Instrument of choice for spirochetes Fig 3.4
11 Spirochetes (Treponema pallidum) viewed with darkfield microscope
12 Fluorescence Microscope Uses UV light. Fluorescent substances absorb UV light and emit visible light. Cells may be stained with fluorescent chemicals (fluorochromes). Immunofluorescence
13 Principle of Immunofluorescence
14 Electron Microscopy: Detailed Images of Cell Parts Uses electrons, electromagnetic lenses, and fluorescent screens Electron wavelength ~ 100,000 x smaller than visible light wavelength Specimens may be stained with heavy metal salts Two types of EMs:?
15 SEM or TEM? Bacterial division Leaf surface
16 Preparing Specimens for the Light Microscope Hanging drop preparation for living specimens (see lab!) Staining Techniques Provide Contrast Smear air-dry heat-fix Basic (cationic) dyes are charged Acidic (anionic) dyes are charged negative staining (good for capsules) Two types of staining techniques: Simple, and differential
17 Negative Stain Observe cell shape and size Used for bacteria with capsules
18 Simple vs. Differential Staining Simple stains: only require a single dye and an uncomplicated procedure - Cause all the cells in the smear to appear more or less the same color, regardless of type - Reveal shape, size, and arrangement Differential stains - Use two differently colored dyes: the primary dye and the counterstain - Distinguish cell types or parts - More complex and require additional chemical reagents to produce the desired reaction
19 Simple Stains Use a single basic dye. A mordant may be used to hold the stain or coat the specimen to enlarge it. Differential Stains React differently with different bacteria Gram stain Acid fast stain Endospore stain
20 Gram Stain (see lab for more detail) Gram-positive Gram-negative Application of crystal violet (purple dye) Application of iodine (mordant) Alcohol wash (decolorization) Application of safranin (counterstain)
21 Acid Fast Stain Cells that retain a basic stain (usually carbol fuchsin) in the presence of acid-alcohol are called acid-fast. Non acid-fast cells lose the primary stain when rinsed with acid-alcohol, and are counterstained with a different color basic stain (usually blue) Specifically used to detect
22 Endospore Stain Heat is required to drive a stain into the endospore. Two medically important bacteria that form endospores: 1) 2)
23 Gram-positive vs. Gram-negative Bacteria (p.54)
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