Microbial Preparation

• Microbial Preparation for examination of Microorganism


  • Wet Mount
  • Hanging Drop Preparation
  • Fixed- stained smear
  • Staining Method
• Staining Method


  • Direct Staining
  • Indirect/ Negative Staining
• Direct Staining


  • Simple Staining
  • Differential Staining
  • Selective Staining
• Differential Staining


  • Gram Staining
  • Acid fast staining
• Simple Staining


  Only one type of dye is used for general study of microorganism
• Differential staining


  Used to contrast 2 or more organism of the same or different species
• Gram Staining


  Differentiate Gram (+) from Gram (-)
• Acid Fast Staining


  Differentiate the Genus Mycobacteria which are acid fast from the non-acid fast bacteria
• Selective Staining


  Specific cell structures are selectively colored by special dyes such that these are distinguished from the vegetative cell
• Selective Staining Examples


  • Fultron-Schaeffers method (spores)
  • Loeffler's method (flagella)
• Indirect/ Negative Staining


  • Background is colored while organism remain unstained
  • Sharp contrast between the capsule and vegetative cell
• Indirect/ Negative Staining Example


  • Indian Ink
• Dye


  An organic compound responsible for staining or coloring certain materials
• Dye Groups


  • Chromophore
  • Auxochrome
• Chromophore Group


  Responsible for giving a specific color to a compound
• Auxochrome Group


  Responsible for transferring the color of the dye to a substance or material to which the dye will act
• Reagents Used in Differential and Selective Staining


  • Initial Stain
  • Mordant
  • Decolorizer
  • Secondary Stain
• Initial Stain


  First stain that is applied on the specimen where in the cell will appear colored
• Mordant


  Substance which will form a bridge between the cell and the initial stain, so that the cell will retain its stain
• Types of Mordant


  • Physical (such as heat or cold)
  • Chemical (such as Iodine, ferrous Sulfate)
• Decolorizer


  • Substance that may be used to remove the initial stain
  • Important for contrast staining of cell parts
  • Decolorized stain will be replaced by secondary stain
• Secondary Stain


  • Counter stain
  • Stain that is applied to the decolorized cell parts
  • Stain the special structure of cell
• Gram Staining


  Developed by Hans Christian Gram in 1884
• Gram Staining Reagents


  • Crystal Violet
  • Iodine Solution
  • Alcohol/ acetone
  • Safranin
• Crystal Violet


  • Gram (+) cell remain violet
  • Gram (-) cell remain violet
• Iodine


  • Gram (+) CV-1 complex formed within cells, cell remain violet
  • Gram (-) CV-1 complex formed within cells, cell remain violet
• Alcohol/ Acetone


  • Gram (+) cell walls dehydrated, shrinkage of pores and decrease permeability, CV-1 complex can't pass out of cell, cell remain violet
  • Gram (-) lipid extracted from cell walls increase porosity, CV-1 complex is removed, cell become colorless
• Safranin


  • Gram (+) cell are not affected, remains violet
  • Gram (-) cell take up the stain, becomes red