MIC 21 Lab

• Stains

  Generally salts in which one of the ions is colored
• Types of microbiological stains

  • Basic stains
  • Acidic stains
• Basic dye

  A stain that is cationic (positively charged) and will react with material that is negatively charged
• Acid dye

  Dyes with negatively charged chromophores that are repelled by the bacterial surface forming a deposit around the organism
• Acid dyes


  • nigrosin
  • congo red
• Types of staining

  • Simple staining
  • Indirect staining
  • Differential staining
  • Structural staining

  
• Dye

  A general-purpose coloring agent
• Stain

  An organic compound containing a benzene ring plus a chromophore and an auxochrome group
• Chromophore

  A chemical group that imparts color to benzene
• Auxochrome group


  A chemical compound that conveys the property of ionization of chromogen (ability to form salts) and bind to fibers or tissues
• Requirements for staining

  • Stain
  • Mordant
  • Accentuater
  • Decolorizer
• Mordant


  A chemical that forms an insoluble complex with the stain and fixes it or causes the stain to penetrate more deeply into the cell
• Examples of mordants

  • Gram's iodine
  • Phenol
• Accentuater


  A chemical which when added to a stain makes the reaction more selective and intense
• Example of accentuater


  • Potassium hydroxide
• Decolorizer


  A chemical used to remove the excess stain in indirect regressive staining
• Example of decolorizer


  • Ethanol
• Simple staining


  A staining method that uses only a single dye that does not differentiate between different types of organisms

  
• Types of simple staining

  • Direct / Positive staining
  • Indirect / Negative staining

  
• Direct staining (Positive staining)


  A simple staining technique that stains the bacterial cells in a single color

  
• Indirect staining (Negative staining)


  A staining process where the background is stained instead of the cells

  
• Gram stain


  A differential staining procedure that defines two bacterial groups: those which retain the primary dyes ("Gram-Positive") and those which are easily decolorized ("Gram-Negative")

  
• Gram-Positive bacteria


  • Have thick, dense, relatively non-porous walls
• Gram-Negative bacteria


  • Have thin walls surrounded by lipid-rich membranes
• Acid fast staining

  Bacterial cells resist decolorization with acidified organic solvents and are therefore called ACID FAST
• Acid fast staining property


  Depends upon the lipid-rich cell walls of the genus Mycobacteria which are relatively impermeable to various basic dyes unless the dyes are combined with phenol

  
• Capsule staining


  Diagnostically useful since capsules are a virulent factor (e.g. in pneumococci)
• Capsule staining methods

  Negative staining (Nigrosin or India ink) or special staining (e.g. Hiss' method, Anthony's method)

  
• Endospore staining


  Spores are normally impervious to stains and have high light refractivity indicative of high protein content
• Compound light microscope


  Instrument that considers three parameters: magnification, resolution, and contrast

  
• Compound light microscope


  • Magnification extends our ability to observe the details 1000 times, so that we can see objects as small as 0.1 micrometer in diameter
  • Resolution or resolving power is the ability to distinguish two adjacent objects as distinct and separate, which also depends on the wavelength of light used and on the numerical aperture (NA)
  • Improving contrast typically improves the final image, as this will accentuates differences in parts of the specimen

  
• Types of microscopes

  • Light microscope (uses light waves and lenses)
  • Electron microscope (employs electron beams and magnetic fields)
• Magnification

  Extends our ability to observe the details
• Resolution


  Ability to distinguish two adjacent objects as distinct and separate
• Contrast

  Accentuates differences in parts of the specimen
• Parts of a compound light microscope

  • Eyepiece/Ocular Lens
  • Body Tube
  • Objective Lenses
  • Stage
  • Substage
  • Light Source
  • Focusing knobs
  • Base
  • Arm

  
• Eyepiece/Ocular Lens


  Lens where you look through, has different magnifications
• Body Tube


  Contains a prism that bends the light rays so that they will pass through the oculars
• Objective Lenses

  Group of 3 or 4 objective lenses attached to a revolving nosepiece at the base of the body tube, with different magnifying powers: Scanning power (4X), low power (10X), high power (40X), and oil immersion (100X)
• Focal length


  Distance from the center of the lens to the point where parallel rays entering the lens are brought to a focus