Cytology: the study of cells and Microscopy: how we study them

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Chapter 6

• Cytology the study of cells and Microscopy how
  we study them

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Properties of Light

• Microscopy depends on two parameters
• Magnification
• Resolution
• Magnification without resolution is useless
• Shorter wavelength greater resolution
• Resolving power of a lens is a numerical measure
  of the resolution
• A microscope with 0.4 nm resolving power views
  two object as separate entities if they are at
  least 0.4 nm apart
• 1000X is the highest magnification allowing good
  resolution with light microscope

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Good resolution
Poor resolution
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Longer wavelengths
Shorter wavelengths
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Light and Objects

• Reflection
• Transmission
• Absorption
• Refraction
• Diffraction

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Compound Light Microscope

• Uses visible light (bright field)
• Magnification up to 1000X Resolution 0.2 µm
• Ocular lens (eyepiece)
• Objective lenses
• Low power 10X
• High power 40X
• Oil immersion 100X
• Total mag. ocular mag. X Objective mag
• 10(ocular)40(high power objective)450(total
  mag)

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• Adding oil between the 100X lens and the specimen
  is essential for resolution

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Darkfield Microscopy

• Used to examine living organisms, to visualize
  more detail, or to avoid staining
• Condenser blocks direct light so only reflected
  and refracted light enters objective
• Bright object in dark background

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Phase Contrast Microscopy

• Uses special condenser to accentuate small
  differences in the refractive index of various
  structures within an organism
• Amplifies variations in density within a specimen
  
• Changes in light are seen as different degree of
  brightness

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Brightfield, Darkfield, andPhase Contrast
Microscopy
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Fluorescence Microscopy

• Fluorescence is the emission of light of a longer
  wavelength as a result of excitation with
  ultraviolet light
• Some organisms fluoresce naturally, some must be
  treated with a fluorochrome (dye)
• Labeled objects look bright against dark
  background.

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Fluorescence Microscopy
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Electron Microscopy

• A beam of electrons is used instead of light
• SEM and TEM
• Used to examine objects smaller than 0.2 µm
• The higher resolving power is due to the short
  wavelength of electron beams
• Electromagnetic lenses are used to focus the
  electron beam

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Scanning Electron Microscope

• Magnification 1000 10,000X
• Resolution 20 nm
• Can be used to study the surface of intact cells
  and viruses
• Primary electron beam knocks secondary electrons
  out of the specimen
• Secondary electrons are collected and used to
  make a 3D picture

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Transmission Electron Microscope

• Magnification 10,000 100,000X
• Resolution 2.5 nm
• Can be used to examine thin sections of a cell or
  macromolecules
• Three electromagnetic lenses
• Final picture generated on a fluorescent screen

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Cytology study of cells

• All living things are composed of cells
• There are 2 types of cells
• Prokaryote
• Bacteria
• Archaea
• Eukaryote
• Plants
• Animals
• Protists
• Fungi

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• All cells have 3 basic components
• Plasma membrane
• Cell boundary
• Nucleic Acids
• genetic information
• Ribosomes
• Site of protein synthesis

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Distinguishing Characteristics of Prokaryotes

• No nucleus or other membrane bound organelles
• DNA is 1 single circular chromosome
• DNA is not associated with histones
• Cell walls contain peptidoglycan (except Archaea)
• Divide by binary fission
• 70s ribosomes

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Distinguishing Characteristics of Eukaryotes

• DNA is found in nucleus
• Have a number of membrane bound organelles
• DNA is found in multiple chromosomes and is
  associated with histones
• Cell walls, if present, have no peptidoglycan
• Divide by mitosis
• 80s ribosomes

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Prokaryotic cell structure
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Structures external to prokaryotic cell wall

• Glycocalyx
• Flagella
• Axial filaments
• Fimbriae and pili

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• Glycocalyx is a polymer made of sugar, protein,
  or both
• Glycocalyx exists in two forms
• Capsule
• Slime layer
• Capsule can be a virulence factor
• Protects against phagocytosis
• Enhances adhesion
• Protects against dehydration

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Flagella and Axial Filaments

• Flagella rotate clockwise or anticlockwise
• Rotation of axial filaments produces a movement
  in the outer sheath that moves the whole organism
• Axial Filaments are present only in spirochetes

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Flagellar Structure
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Axial Filaments
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Pili

• Both consist of a protein called pilin
• Fimriae
• A few to several hundreds/cell
• Attachment
• Sex pili
• Used in conjugation
• 1 or 2/cell

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The Cell Wall

• Complex, semi-rigid structure responsible for
  cell shape
• Protects the cell from adverse changes in
  environment and osmotic pressure
• Bacterial cell walls are composed of
  peptidoglycan (murein)
• Peptidoglycan consists of a repeating
  disaccharide attached by protein side chains

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Structures internal to the cell wall

• Plasma membrane
• Phospholipid bilayer with peripheral and integral
  proteins
• fluid mosaic model
• Functions
• selective permeability contain enzymes capable
  of breaking down nutrients and producing ATP
• Chromatophores and Thylakoids infoldings in
  membrane containing pigments for photosynthesis

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• Cytoplasm
• Thick, aqueous (about 80 water) solution
• Contains nuclear area, ribosomes, and inclusions
• Nuclear area or nucleoid
• Contains the bacterial chromosome
• single, continuous, circular thread of
  double-stranded DNA
• There is no nuclear envelope and no histones

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• Plasmids
• small, circular, double-stranded DNA molecules
• not part of primary chromosome
• replicate independently
• can be gained or lost without harming the cell
  can be transferred between bacteria
• Carry non-essential information
• antibiotic resistance, tolerance to toxic metals,
  production of toxins, and the synthesis of
  enzymes

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• Ribosomes - site of protein synthesis
• Prokaryotes have 70S ribosomes
• Eukaryotic cells have 80S ribosomes
• Several antibiotics inhibit protein synthesis on
  70S ribosomes
• Selective toxicity

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• Inclusions- Reserve deposits for the cell
• Metachromatic granules contain inorganic
  phosphates used in ATP production
• Polysaccharide granules- sugar stores (glycogen
  and starch)
• lipid inclusions lipid storage
• sulfur granules sulfur for energy by oxidation
  
• carboxyzomes contain enzyme carboxylase for
  bacteria that fix carbon dioxide
• gas vacuoles used to maintain buoyancy in
  aquatic organisms
• magnetosomes - contain iron oxide, act like a
  magnet, used for movement