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Today: Microscopes (Ch. 3), begin Prokaryotic cells (Ch. 4)
Ch. 3 - Microscopes
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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: 10-6m
nanometer: 10-9m
Light Microscopy
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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
Electron Microscopy
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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
Staining
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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
 Basic dyes
 Acid dyes
o Hydrophobic - Sudan black, stains lipids
Stains
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Simple Stain: uses one staining agent
Differential Stain: divides bacteria into separate groups based on different staining
properties
Gram stain
 most important staining procedure
 Dr. Christian Gram, 1884
o Procedure:
 Crystal Violet - 1 minute
 Gram's Iodine - 1 minute
 Decolorize with 95% ethanol (5-10 sec), Rinse
 Safranin - 1 minute
Stains (continued)
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Gram stain (continued)
o Gram positive - traps crystal violet-iodine complex, due to thick layer of
peptidoglycan in cell wall
 PURPLE
o 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
Stains (continued)
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Acid-fast stain:
o used to stain Mycobacterium, which have high mycolic acid content
(waxy)
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 reveals capsule layer around cells
o mix bacteria with nigrosin, spread on clean slide, dry
o useful to observe Klebsiella pneumonia
Stains (continued)
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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
not easily decolorized
o heat smear over steam, rinse with water
o 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)
Ch. 4 - Procaryotic and Eucaryotic Cells
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General Differences
o Procaryotes
 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
Procaryotic and Eucaryotic Cells (continued)
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General Differences (cont.)
o Eucaryotes
 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)
Prokaryotic Cells: Shape and Arrangement
Cell (Plasma) membrane
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Structure:
o phospholipid bilayer (hydrophilic head and hydrophobic core)
o contains embedded proteins
o fluid mosaic model
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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
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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 isotonic: equal solute concentration
o hypotonic: lower concentration of solutes
o hypertonic: higher concentration of solutes
Transport
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Passive:
o simple diffusion
o Facilitated diffusion: protein mediated, no energy involved
Active:
o Active transport: energy used to bring in molecules
o Group translocation: active transport where molecule is modified during
transport (example: phosphorylation)