Simple Stains

1
Simple Stains

• Bacteria have nearly the same refractive index as
  water, therefore, when they are observed under a
  microscope they are opaque or nearly invisible to
  the naked eye.
• Different types of staining methods are used to
  make the cells and their internal structures more
  visible under the light microscope.
• Simple stains use one dye that stains the cell
  wall.
• The cells are then visible against a light
  background.
• Steps
• Place the slide on the staining rack.
• Flood the slide with a basic stain either
  crystal violet (1 min.), Safranin (2 min.), or
  Methylene blue (2 min.).
• Wash the stain off the slide with deionized
  water.
• Blot the slide with bibulous paper.

2
Differential Staining

• Differential Stains use two or more stains and
  allow the cells to be categorized into various
  groups or types.
• Both techniques allow the observation of cell
  morphology, or shape, but differential staining
  usually provides more information about the
  characteristics of the cell wall (Thickness).
• The most common differential stain used in
  microbiology is the Gram Stain.
• Basic stains, due to their positive () charge
  will bind electrostatically to negatively charged
  molecules such as many polysaccharides, proteins
  and nucleic acids.
• Acid stains ( - ) bind to positively charged
  molecules which are much less common, meaning
  acidic stains are used only for special purposes.
  
• Some commonly encountered basic stains are
  crystal violet, safranin (a red dye) and
  methylene blue.
• Basic stains may be used alone (a simple stain)
  or in combination (differential stain) depending
  on the experiment involved.

3
Gram Staining

• The Gram Stain is a differential stain.
• Four different reagents are used and the results
  are based on differences in the bacterial cell
  wall.
• Gram Positive bacteria have a relatively thick
  cell wall composed of a special carbohydrate
  called Peptidoglycan.
• Gram Negative bacteria have a much thinner cell
  wall composed of the same carbohydrate,
  Peptidoglycan, but with certain chemical
  differences, such as the presence of
  Lipopolysaccharides (LPS).
• Notice that both Gram Positive and Gram Negative
  bacteria have a cell wall composed primarily of
  Peptidoglycan.
• Gram Staining Steps
• Crystal violet acts as the primary stain. Crystal
  violet may also be used as a simple stain because
  it dyes the cell wall of any bacteria.
• Grams iodine acts as a mordant (Helps to fix the
  primary dye to the cell wall).
• Decolorizer is used next to remove the primary
  stain (crystal violet) from Gram Negative
  bacteria (those with LPS imbedded in their cell
  walls). Decolorizer is composed of an organic
  solvent, such as, acetone or ethanol or a
  combination of both.)
• Finally, a counter stain (Safranin), is applied
  to stain those cells (Gram Negative) that have
  lost the primary stain as a result of
  decolorization.

4
Gram Staining Procedure
5
Gram Staining Results

• When reporting the results of the Gram stain you
  indicate the type of stain used, the reaction,
  and the morphology of the cells observed.
• Round (spherical), purple (or dark blue) cells
  are reported as Gram positive cocci (GPC).
• Rod-shaped, purple (or dark blue) cells are
  reported as Gram positive bacilli (GPB).
• The standard abbreviations for the four types of
  Gram stain and morphology are
• Gram Positive Cocci (GPC)
• Gram Positive Bacilli (GPB)
• Gram Negative Cocci (GNC)
• Gram Negative Bacilli (GNB)
• Notice that both Gram Positive and Gram Negative
  bacteria have a cell wall composed primarily of
  Peptidoglycan.
• Spiral-shaped bacterial cells do not Gram stain
  well and are usually observed using dark-field
  microscopy. There are no standard abbreviations
  for Gram stain reactions of the spirilla of
  medical importance.
• Certain other types of bacteria may not Gram
  stain well, such as, Acid-fast Mycobacteria
  (Mycobacterium tuberculosis).

6
Bacterial Cell Shapes

• Bacteria can have several different shapes, but
  the primary shapes we will be observing are
• Spherical or round cells cocci (plural) or
  coccus (singular)
• Rod shaped bacilli (plural) or bacillus
  (singular)
• Spiral shaped spirilla
• Some bacteria have characteristic clustering or
  arrangements, usually due to how the cells divide
  and whether they remain attached together when
  they divide.
• Diplococci divide in one plane and remain
  attached together after cell division.
• Streptococci divide in one plane and form long
  chains of attached cells.
• Staphylococci divide in many planes and remain
  attached together forming a grape-like cluster