(Lab) Bacterial Smear

• Good smears are critical for discerning:

  • The morphology of cells (shape)The arrangement of cells (clustering)
  • Internal structures
  • The specimens we can create bacterial smears from come from growth in culture in our petri dishes or culture media tubes
• SIMPLE STAINING

  • The use of a single stain to color a bacterial cell
  • Commonly used dyes for performing simple staining are methylene blue, basic fuchsin, and crystal violet
  • These are referred to as basic dyes because they have color-bearing ionic groups (chromophores) that are positively charged (cationic)
• DIFFERENTIAL STAIN

  • Two kinds of cells, gram positive [colored purple] and gram negative [colored red or pink], are differentiated
  • Will only affect specific bacteria
• Several factors can affect the outcome of the "Differential Staining":

  • It is important to use cultures that are 16-18 hours old
  • It is critical to prepare thin smears
  • Decolorization is the most critical step in the
  • Gram stain procedure [Decolorization is what separates the organism that will be stained]
• Reagents used in gram staining:

  • Crystal violet – primary stain
  • Gram’s Iodine – Mordant [help enhance the primary stain]
  • Acetone Alcohol – decolorizer [decolorize gram positive organisms
  • Safranin – Secondary stain [stain the other gram organisms]