I. History

• Galileo
  
• Hooke
  
• Leeuwenhoek

  II. Definitions

• Refraction: bending of light as it passes from one medium to another
  
• Refractive index: measure of how greatly a medium slows the velocity of light
  
• focal point: point where parallel rays of light focus
  
• focal length: distance between center of lens and focal point
  
• compound microscope: two or more magnifying lenses, ocular and objective lenses
  
• resolution: ability to reveal fine detail or two points distinctly

III. Light Microscopy

     A.  bright field microscopy

• maximum magnification 1000X
  
  
• oil immersion microscopy

    B. Dark Field Microscopy

• used to visualize living microorganisms e.g. Treponema pallidum
  

    C. Phase Contrast Microscopy

• utilizes differences in refractive indexes

    D. Fluorescent Microscopy

• uses ultraviolet light and fluorochromes

IV. Confocal Microscopy

• Focused laser beam
  
• Narrow depth of field

V. Electron Microscopy

     A. Introduction

• uses electrons instead of light
  
  
• electrons have a shorter wavelength than light (0.0055 nm vs. 550 nm)

    B. Transmission electron microscope

 

    C. Scanning Electron Microscope

 

    D. Scanning Tunneling Electron Microscope
 
 

STAINING

I. Preparation of Staining

• Fixation—maintains appearance of cells as closely as possible.
  
• Fixing bacteria to slide

           1) heat

           2) chemical

II. Dyes

• Most dyes have two things in common:

           1) contain chromophores

           2) bind to molecules in object being stained

• ionizable dyes

          1) basic dyes (cations)

          2) acid dyes (anion)

III. Types of Staining Procedures

   A. Simple Staining

• single dye
  
  

   B. Differential Staining

       1. Gram Stain (1884)

• Procedure

          1) Crystal violet

          2) iodine mordant

          3) decolorize: ethanol, acetone

          4) counterstain: safranin or basic fuchsin

• Results

     2. Acid fast stain

• Mycobacterium, Cryptosporidium, etc.
  
• Two methods: Ziehl-Neelson and Kinyoun’s
  
• Procedure

          1) primary stain: carbol fuchsin with or without phenol

          2) decolorize: acid ethanol

          3) counterstain

• Results
  
  

     3. Structural stains

          a. capsule

• negative stain
  
• Wet mount methods: India ink or nigrosin dye
  
• Dry smear methods: Congo red

          b. spore

• Schaeffer-Fulton stain
  
• Procedure

                     1) 5% malachite green

                     2) decolorize

                     3) counterstain: safranin

• results

              c. flagella

• hard to see
  
• mordants: tanninc acid and potassium alum