Techniques

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Techniques Tools
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• I. Types of Microscopes
• 1. Light Microscopes
• Simple
• B. Complex
• Visual Light.
• 3 objectives, low, high, oil immersion.
• Magnification
• Resolving Power

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Light Microscopes
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Review

• Why is a light microscope called a light
  microscope?
• Name the two lenses in a compound microscope.

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• 2. U.V.
• light for illumination
• Magnification - .
• Resolving .
• 3. Fluorescent
• light with specimens thats been stained
  with
• Magnification - .
• Resolving - .
• 4. Dark-field
• of visible light, light is
  refracted by specimen as light enters the
  objective.
• Mag Resolving -

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• 5. Phase Contrast Microscope
• Observe matter, unstained.
• Detects changes (phases) in visible light as it
  through a specimen.
• Mag -
• Resolving -

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• 6. Transmission Electron Microscope (TEM)
• Beam of
• Thin specimen thats been stained with electrons
  opaques heavy metal.
• Travels specimen.
• Viewed on screen.
• Mag -
• Resolving -

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• 7. Scanning Electron Microscope (SEM)
• Beam of .
• surface produces a
  image of specimen.
• Mag -
• Resolving

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Scanning Tunneling Electron Micrograph
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Review

• What is the difference between magnification and
  resolution?
• Which microscope gives the greatest
  magnification?
• What is the magnification limit most light
  microscopes?

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• II. Techniques for Microscopic Staining
• 1. Living Microscopic Organisms.
• A. Some Bacteria are mobile, others are not
  but appear to move.
• 1. movement due to
  air or water molecules hitting the
  microorganism.
• B. Non Staining Techniques - hard to see.
• Ex.

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• 2. Stained Microorganisms
• Colored by a chemical dye to make visible.
• General Techniques for all staining.
• 1. Smear - slide out the drop with the bacteria
  in it.
• 2. Stained - reveal size, shape, arrangement
  presence of some internal structures.
• Types of Staining
• 1. Simple
• A. Smear.
• B. Single stain is used (Sudan Black).

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• 2. Differential Staining
• A. Gram
• 1.
• 2.
• 3.
• 4. .
• 5. .
• -
  decolorizes after alcohol wash, need a counter
  stain (red, safranin).
• - retains
  blue stain.
• B. Acid Fast - Staining
• Used for the genus
  .
• Retain carbolfuchsin (red)when washed with
  acidic alcohol.
• Counter with
  .

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Gram Staining
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Review

• What is a differential stain?
• Why is a Gram stain so useful?
• What structure of the cell determines its Gram
  staining properties?

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• III. Bacterial Morphology
• Shapes are recognized, but others
  exist.
• 1. - round
• A. pairs -
• B. chains -
• C. cubes -
• Depending when they divide and then adhere to
  each other.
• 2. - rod or cylinder shape.
• 3. Spiral
  Forms - twisted rods or cylinders
• A. - actual
  spirals (corkscrew) rigid.
• B. -
  flexible.

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Acid Fast Stain

• Carbol Fuchsin - 5minutes
• Wash with tap water.
• Acid 2 minutes.
• Wash with tap water.
• Methylene Blue 2 minutes.
• Wash with tap water.
• Blot dry.
• Acid Fast bacteria do no decolorize with acid
  alcohol. They appear red against a blue
  background.

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• 4. - Extensions on
  their surface which gives them a star appearance.
• With age bacteria morphology may change -
  - swell or
  show rudimentary branching.

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• IV. Reproduction
• (
  reproduction) - one cell divides into two
  identical daughter cells.
• Each cells lives on
  their own after replication even though they may
  not be

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Prokaryotes
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Surface Layers - Capsule
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Review

• How do bacteria mainly reproduce? What is the
  advantage of that type of reproduction? What is
  the disadvantage of that type of reproduction?

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• 2. Cell Wall
• Function is to hold the cell together.
• Composed of sub-units found nowhere else in
  nature.
• Produces symptoms of diseases.
• Site of action of some of the most effective
  antibiotics.
• - Common
  structural component made up of sugar amino
  acids that makes the cell wall rigid.
• Components, two different sugars.
• A.
• B.
• Differences in
  determines the gram-staining properties.
• effective against
  cell walls of bacteria.

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• V. Bacterial Structure
• A. -
  Collectively known as the
• 1. - material
  secreted by bacteria that adheres to the exterior
  of the bacterial cell.
• Functions Possible
• Waste products
• Antigenic
• A. - organized,
  thickened material around each cell or pairs of
  cells.
• Ex. - Streptococcus pneumoniae - can not survive
  in a host unless it can synthesize a capsule,
  because the host will quickly destroys it.
• B. - Unorganized
  loosely attached polysaccharide.
• C. - oral bacteria
  that secrete glycocalyx.

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Review

• What is the function of the cell wall?
• What are the affects of penicillin on bacterial
  cell walls?
• What are the cell walls made of? Where is the
  only place on earth that this component is found
  at?

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• A. Gram Positive
• ( )
• 1.Numerous layers of peptidoglycan (up to
  ).
• 2.
• associated in the peptidoglycan layer.

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• B. Gram Negative ( )
• 1. Outer layer consists of a
  that consists of several other
  molecules.
• 2. Lipopolysaccharides (LPS), ,
  Porin proteins.

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• 3. Cell Membrane or Plasma membrane.
• Located inside the cell wall.
• Composition is 60 protein 40 Lipids.
• Functions
• 1. Osmotic regulator.
• 2. Enzymes necessary for the synthesis
  transport of peptidoglycan, teichoic acid
  other membrane components.
• 3. Secretes extracellular hydrolytic enzymes.
• 4. Ensures segregation of nuclear material
  during cell division.
• 5. Transport of electrons protons that are
  released during aerobic oxidation turns it
  into chemical energy that can be used by the
  cell.
• 6. Barrier to the entry of most molecules into
  the cell (nutrients entering wastes leaving).

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• A. Semi-permeable allows some things to pass, but
  not others.
• 1. Diffusion - do not use energy.
• a. Passive or Simple Diffusion -molecules flow
  freely in out of the cell (Small).
• b. Facilitated Diffusion - Use membrane
  transport proteins (Permeases) for molecules to
  move in out of the cell (Large).
• 2. Active transport - uses energy to cross the
  membrane.
• Several proteins are required for this.
• Surface binding proteins.
• Membrane transport proteins.

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• B. -structures that extend
  from the surface.
• 1. - long slender ,
  protein used for locomotion.
• Bacteria can move lengths /minute (6ft man
  running 82 mph).
• A. Composed of three parts.
• 1.
• 2.
• 3.

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• B. Types of flagella arrangements.
• C. - movement
  toward food substances or away from harmful
  substances.
• Toward- straight line -
• Away - tumble away -
• Due to the of the flagella
  movement (
  
• )
• Possible -
  proteins on the cytoplasm

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• D. - responds to various amount
  of light.
• E. - responds to various
  amount of oxygen.
• F. - reacts to the earths
  magnetic field due to a row of magnetic
  particles. Line up north-south direction.

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Chemotaxsis
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• 2. - shorter
  thinner than flagella.
• Functions
• 1. Movement of
  during sexual
  reproduction.
• 2. to
  surfaces.

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• C. Cytoplasm
• 1.Cytoplasm - water, other 20
  Nucleic Acids, Proteins, Carbohydrates, Lipids.
• Primary site of synthetic synthesis of proteins
• 2. - singular
  circular double strand of DNA that is
  supercoiled.
• that the
  Bacteria.
• , but
  histonelike structures.
• No or useless
  DNA.

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• 3. - Small circular
  pieces of double stranded DNA that contain
  genetic information to resistance of particular
  antibiotics.
• 4. - Site of protein
  synthesis
• Composed of 60 RNA 40 Protein.
• 5. - Not integral
  parts of the cell structure
• A. -
  reserve source of phosphate energy (Volutin).
• B. Granulose -
• C. Others - Sulfur, lipids, glycose.
• 6.
  - special membranes systems found in certain
  photosynthetic bacteria cyanobacteria that
  contain pigment concerned with photosynthesis.

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• D. Special Structures
• 1. - Minute
  highly durable body formed with in the cell
  capable of development into a new vegetative
  organism.
• to
  adverse conditions
• Outer layers
• Cortex layers
• Lack many enzymes but may have some in small
  amounts.
• amounts of ,high
  calcium, protein Polysaccharide antigens.

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Endospore Formation
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Capsule Endospore Staining Directions

• CAPSULE
• Crystal Violet 2-5 minutes
• Drain extra crystal violet in sink.
• Wash with copper sulfate for 30 seconds.
• Dry.

• Endospore
• Stain with toluidine blue for 5-15 minutes.
• Rinse with tap water.
• Dry.

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• VI. Preparation of a Pure Culture
• Culture is a medium in which microorganisms can
  grow on.
• Pure Cultural Techniques
• 2 Techniques are commonly used.
• 1.
  -General preparation.
• 2.
  .
• Inoculating a dilution of the mixed cultures in
  to a melted agar.
• Poured onto a sterile petri dish.
• Bacteria grow sedately (isolated) in a solid
  medium. Help ensure pure culture.

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• VII. Types of Culture Media
• pH and temperature must be controlled in all
  media
• 1.
  Common
• Boiling ground meat with water and filtering off
  the solid material-leaves a clear liquid
  infusion.
• Make solid by adding agar to it.
• Artificial or Complex Medium - beef extracts,
  yeast, blood
• Synthetic or Defined Medium - can write the
  chemical formula for.
• 2. - Various
  sugars are added to the nutrient broth.
• 3. Selective Differential Media
• A. - Chemicals
  are added to the medium to inhibit the growth of
  some bacteria while letting others grow.
• B. - Acid
  indicator is added so that many colonies that
  formed acid will turn colors.
• 4. (Liquid) - Help the
  growth of certain bacteria and not others.

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• VII. Oxygen Requirements
• 1. -
  require free oxygen to grow.
• 2. -
  will not grow in the presence of free oxygen,
  may even be killed.
• 3. -prefers the
  presence of low oxygen.
• 4.
  - lives in the
  presence of both.
• a. -
  produce lactic acids.
• b.
  - carbon dioxide.
• 5. -
  will grow in the presence of oxygen but do not
  posse an oxidative metabolism.

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• VIII. Sterilization Methods
• No living organisms are in the media when
  inoculated.
• 1. Autoclave-steam under pressure 15lbs/in2.
• 2. Filter.