microscopy and staining- microbio

Units of measurement

• Microorganisms are measures in micromegers um and nanometers (nm)

• 1 um= 10^-6 m = 10^-3 mm

• 1 nm= 10^-9 m = 10^-6 mm

• 1000 nm = 1 um

• 0.001 um = 1 nm

Anton van leeuwenhoek’s microscopic observations

• A simple microscope has only one lens

• Similar to a magnifying glass, but with a much better ( higher magnification) lens 

light microscopy

• Types of light microscopy 

  • Compound light microscopy

  • Darkfield microscopy

  • Phase-contrast microscopy

  • Differential interference contrast (DIC) microscopy

    • These three use white light

• Fluorescence microscopy

• Confocal microscopy

  • Both use UV light 

The compound light microscope

• The use of any kind of microscope that uses visible light to observe specimen

  • Compound light microscopy

  • Darkfield microscopy

  • Phase-contrast microscopy

  • Differential interference contrast (DIC)

  • Fluorescence microscopy

  • Confocal microscopy

• In a compound microscope, the image from the objective lens is magnified again by the ocular lens

• Total maginiifcation= objective lens x ocular lens 

Compound light microscopy:

• Resolution (resolving power) is:

  • The ability of the lenses to distinguish two point

  • Or the ability to distinguish fine detail and structure

• A microscope with a resolving power of 0.4 nm can distinguishing between two points at least 0.4 nm apart

• Shorter wavelengths of light provide greater resolution

• The limit of resolution of a compound light microscope is 0.2 um 

• This limits the magnification of compound light microscopes to approx 1500x 

•  Refractive index is a measure of the light-bending ability of a medium

• Light may refract after passing through a specimen to an extent that it doesnt pass through the objective lens

• Immersion oil is used to keep light from refracting 

Resolution (resolving power) is:

• The ability of the lenses to distinguish two point

• Or the ability to distinguish fine detail and structure

The limit of resolution of a compound light microscope is

0.2 um

What is the refractive index?

a measure of the light-bending ability of a medium

Brightfield illumination

• Dark objects are visible against a bright
background

• Light reflected off the specimen does not
enter the objective lens

• May be difficult to view unstained cells due
to a lack of contrast with a cell’s
surroundings

Darkfield microscopy

• Light objects are visible against a dark
  background

• Opaque disk placed in condenser

• Only light reflected off the specimen
  enters the objective lens

• Useful for viewing live unstained
  microorganisms

  • Can be used to view Treponema
    pallidum, the very slender spirochete
    that causes syphilis

• you dont need staining or contrast like light microscope does

• useful for live, unstained, microbes

darkfield microscopy can be used to view what bacterium

• Treponema pallidum, the very slender spirochete
  that causes syphilis

Phase-contrast microscopy:

• Allows detailed examination of living organisms
  and internal cell structures

• No fixation or staining necessary

• Brings together two sets of light rays, direct
  rays, and diffracted rays to form an image

Differential interference contrast (DIC) microscopy:

• Similar to phase-contrast

• Uses two light beams and prisms
  to split light beams, giving more
  contrast and color to the specimen

• Images may be brightly colored
  and appear three-dimensional

Fluorescence microscopy:

• Uses UV (short wavelength) light

• Fluorescent substances absorb UV light and emit longer
  wavelength (visible) light

• Cells may be stained with fluorescent dyes
  (fluorochromes) if they do not naturally fluoresce

• Depending on the fluorochrome used, cells may stain
  bright yellow or green or orange against a dark field of view

  • Auramine O: fluorochrome that stains
    Mycobacterium tuberculosis

Fluorescent-antibody (FA) technique:

• Also known as immunofluorescence

• Antibodies specific for a type of microbial pathogen are prepared and tagged with a fluorochrome

• These “fluorescent antibodies” are applied to a
microscope slide bearing a specimen that may contain the pathogenic microbe

• If the pathogenic microbe is present, the fluorescent
antibodies will adhere, causing the microbe to fluoresce when viewed with fluorescence microscopy

• Provides a means of rapid and specific detection of pathogens in patient specimens

Confocal microscopy

• Cells are stained with flurochrome dyes

• Short-wavelenght (blue) light is used to excite a plane of specimen

• Exceptionally clear two-dimensional images can be obtained

• Each plane in a specimen is illuminated and a three-dimenstional image can be constructed with a computer

Two-photon microscopy:

• Cells are stained with fluorochrome dyes

• Two photons of long-wavelength (red) light are used to excite the dyes

• Can study living cells up to 1 mm deep 

• Can track the activivty of cells in real time 

Super-resolution light microscopy:

• Uses two laser beam,s

  • One wavelength stimulates fluorescent molecules to glow

  • Second wavelength cancels out all fluorescence except for that in 1 nm

• A computer scans the specimen nm by nm, then puts the images together 

Scanning acoustic microscopy:

• Measures sound waves that are reflected back from a specimen

• Used to study cells attached to surfaces

  • Ex:

    • Cancer cells

    • Arterial plaque

    • Bacterial films

• Resolution of 1 um 

Electron microscopy:

• Uses electrons instead of light

• The shorter wavelength of electrons give greater resolution

• Used for images too small to be seen with light microscopes, such as viruses and internal cellular structures

• Use electromagnetic lenses to focus electron beans

• Images are black and white but are often enchanced by adding color digitally 

Transmission Electron Microscopy

• A beam of electrons passes through ultrathin
sections of a specimen, then through an electromagnetic lens, then focused by a projector lens

• Images are produced on a viewing screen
and saved digitally

• Specimens may be stained with heavy-metal
salts for contrast

• Magnification: 10,000–10,000,000x

• Limit of resolution: 0.2 nm

• gives (internal structure of cell)

scanning electron microscopy

•An electron gun produces a beam of
electrons that scans the surface of an entire
specimen

• Secondary electrons emitted from the
specimen are transmitted to an electron
collector, amplified, used to produce an
image on a viewing screen, and saved
digitally

• Provides a striking three-dimensional view
of specimens

• Magnification: 1000–500,000x

• Limit of resolution: 0.5 nm

scanned probe microscopy

• Use various kinds of probes to examine the surface of
  specimens with electric current

• Does not modify the specimen in any way

• Enables:

  • mapping of atomic and molecular shapes

  • characterization of magnetic and chemical properties

  • Detection of temperature variations within cell

• Includes:
  • Scanning tunneling microscopy
  • Atomic force microscopy

Scanning tunnel microscopy:

• Uses a tungsten probe to scan a
specimen and reveal details of its surface
• Can resolve features as small as an atom
• No special specimen preparation needed
• Can produce detailed views of molecules
such as DNA

atomic force microscopy

• Uses a metal-and-diamond probe placed
onto a specimen; movements are
recorded
• Produces three-dimensional images at
near atomic detail

Preparing smears for staining:

• Staining: coloring microorganisms with a dye that emphasizes certain structures

• Smear: a thin film of material containing microorganisms spread over a slide

• Fixing a smear precede staining:

  • Attaches microorganisms to the slide

  • Kills the microorganisms

  • preserves parts of microves with minimal distortion

What is  coloring microorganisms with a dye that emphasizes certain structures

staining

What is a thin film of material containing microorganisms spread over a slide

smear

What does fixing do?

• Attaches microorganisms to the slide

• Kills the microorganisms

• Preserves parts of microbes with minimal distortion 

staining

•Stains consist of a positive and negative ion, one of which is
colored (chromophore)

• In a basic dye, the chromophore is a cation

• crystal violet, methylene blue, safranin

• In an acidic dye, the chromophore is an anion eosin, acid
fuchsin, nigrosin

• Bacterial cells have a negative charge, so basic dyes adhere
to them

• Staining the background instead of the cell is called
negative staining—uses acidic dyes

• negative staining- staining something that is not your target

Simple stains:

• Simple stain: use of a single basic dye

• Examples:

  • methylene blue

  • carbolfuchsin

  • crystal violet

  • safranin

• highlights the entire microorganism to visualize cell
  shapes and structures

• A mordant may be used to hold the stain or coat the
  specimen to enlarge it

What may be used to hold the stain or coat the specimen to enlarge it

mordant

Gram stain:

• Classifies bacteria into gram-positive or gram-negative

  • Gram-positive bacteria have thick peptidoglycan cell walls

    • Will stain purple

• Gram-negative bacteria have thin peptidoglycan cell walls and an outer membrane of lipopolysaccharides and phospholipids

  • Will stain pink/red

What bacteria have have thick peptidoglycan cell walls

• Will stain purple

gram positive

What bacteria have thin peptidoglycan cell walls and an outer membrane of lipopolysaccharides and phospholipids

• Will stain pink/red

gram negative

Acid-fast stain:

• Binds only to bacteria that have a waxy material in their cell walls, which is not decolorized by acid-alchol

• Used for the identification of 

  • Mycobacterium

  • nocardia

Negative staining for capsules:

• Capsules are a gelatanious covering that do not accept most dyes

• Suspension of india ink or nigrosin contrasts the background witht the capsule

• The cells are then stained with a simple stain

• The capsule appears as a halo around the stained bacterial cell

Endospore staining:

• Endospores are resistant, dormant structures inside some cells that cannot be stained by ordinary methods

• Shaeffer-fulton endospore stain:

  • Primary stain: malachite green, usually with heath to help dye penetrate the endospore

  • Decolorize cells: water

  • Counterstain: safranin

• Spores appear green withing red or pink cells 

flagella staining

• Flagella are structures of locomotion

• Flagella are too slender and cannot be viewed with a light microscope unless stained

• Flagellar stain uses a mordant and carbolfuchsin to thicken appearance of flagella, making them visible under the light microscope

• Enables determination of the number and
arrangement of flagella