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Total magnification: calculated by multiplying the objective lens magnification by the ocular lens magnification
Resolution: ability to distinguish between two points a specified distance apart micrometer: 10
-6 m nanometer: 10
-9 m
 Bright-field microscope: dark image against a brighter background
 Dark-field microscope: only refracted light enters objective, making specimen bright, background dark (useful for observing living cells, particularly spirochetes)
 Phase-contrast: converts slight changes in refraction into easily detected
 variations of light intensity (useful in observing living cells)
Fluorescence: specimen exposed to ultraviolet light, compounds in the sample fluoresce, giving off visible light
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
Uses electrons as illumination source (shorter wavelength allows greater resolution)
Two types: o Transmission (TEM): electrons pass through thin section of specimen o Scanning (SEM): image produced by electrons which are emitted from the surface of an object
 Fixation: process by which internal and external structures of cells are preserved o Heat-fixation - fix an air-dried thin film (smear) by passing through flame
 Dyes: have chromophore groups (give color) and bind to cells by ionic, covalent, or hydrophobic bonding o Ionic stains
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
Basic dyes
Acid dyes o Hydrophobic - Sudan black, stains lipids

 Simple Stain: uses one staining agent
 Differential Stain: divides bacteria into separate groups based on different staining
 properties
Gram stain o

 most important staining procedure
Dr. Christian Gram, 1884
Procedure:
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Crystal Violet - 1 minute
Gram's Iodine - 1 minute
Decolorize with 95% ethanol (5-10 sec), Rinse
Safranin - 1 minute
 Gram stain (continued) o Gram positive - traps crystal violet-iodine complex, due to thick layer of o peptidoglycan in cell wall
 PURPLE
Gram negative - large amount of lipids in cell wall are dissolved by the
95% ethanol, allowing crystal violet-iodine complex to escape, must add counterstain to colorless cells
 RED
 Acid-fast stain: o used to stain
(waxy)
Mycobacterium , which have high mycolic acid content
 o Must use steam heat to force carbolfuchsin stain into cells o Acid-alcohol decolorizer removes stain from non-acid fast cells o counterstain with methylene blue
Negative/Capsular stain: o o o reveals capsule layer around cells mix bacteria with nigrosin, spread on clean slide, dry useful to observe Klebsiella pneumonia

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Spore stain: used to stain endospores of Clostridium and Bacillus o endospores do not readily take up dye, but once it penetrates the stain is o o not easily decolorized heat smear over steam, rinse with water counterstain with safranin o Endospores - Green; Vegetative cells - Red
Flagellar stain: flagella of bacteria are coated with tannic acid or potassium alum and stained with basic fuchsin (Gray Method)
 General Differences o Procaryotes
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 generally smaller (0.2 - 2 microns diameter and from 2-8 microns in length) no nucleus or other membrane bound organelles one circular chromosome (most) no histone proteins associated with DNA cell wall generally contains peptidoglycan (complex
 polysaccharide) divide by binary fission
 General Differences (cont.) o Eucaryotes
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 large cells
"true nucleus" and other membrane bound organelles multiple linear chromosomes histone proteins always associated with DNA cell wall does not contains peptidoglycan) divide by mitosis (complex process)
 Structure: o phospholipid bilayer (hydrophilic head and hydrophobic core) o o contains embedded proteins fluid mosaic model

 o some infoldings related to photosynthesis (known as thylakoids or chromatophores)
Permeability: o semi-permeable layer

 small molecules and water pass freely charged molecules and larger ones cannot pass
Moving materials across the membrane:


Osmosis: diffusion of water across a semi-permeable membrane
Osmotic pressure: internal pressure which equalizes water movement in and out of a cell
 Solutions relative to cell solute concentration: o o o isotonic: equal solute concentration hypotonic: lower concentration of solutes hypertonic: higher concentration of solutes
 Passive: o simple diffusion o Facilitated diffusion: protein mediated, no energy involved
 Active: o o
Active transport: energy used to bring in molecules
Group translocation: active transport where molecule is modified during transport (example: phosphorylation)