Background and Purpose:  This is probably the most widely utilized staining procedure used in bacteriology.  This technique is essential in diagnostic procedures that require bacterial identification.  Due to differences in the structural and chemical composition of cell walls, bacteria are separated into two groups: gram-positive or gram-negative.  Gram-positive cell walls are comprised of a thick layer of peptidoglycan.  In contrast, gram-negative cell walls have a much thinner layer of peptidoglycan that is accompanied by and outer membrane composed of lipids.  These cell wall types can be distinguished from one another based on their ability to retain a primary stain during decolorization with alcohol.  Gram-positive cells do not decolorize very readily and retain a purple color.  In comparison, gram-negative cells decolorize more readily and will appear pink or red after they are counterstained.  Because this technique can determine differences between microorganisms, Gram staining is classified as a differential stain.  

General Procedure:  A bacterial smear is initially stained with crystal violet.  After being rinsed with water, the smear is then treated with Gram's iodine and then decolorized with alcohol.  After a second rinsing, safranin is used to counterstain the smear.

Materials:  crystal violet, Gram's iodine, ethyl alcohol, safranin, staining rack, slide holder, disposable latex gloves, wash bottle, bibulous paper

Background and Purpose:  This special staining technique allows you to observe the spores of spore-forming bacteria.  These structures are protected by a spore coat that makes them resistant to various chemical and physical agents.   Unfortunately, this same impervious protein coat makes spores very difficult to stain through conventional methods.  This technique uses rather vigorous heat treatment to force a primary stain into the spores.  Therefore, if present, spores can be more readily observed and differentiated from vegetative cells.  The shape and position of the spore within a cell and cell swellings can also be confirmed.  These characteristics are useful  when identifying spore-forming species.  Of medical importance are  Bacillus anthracis (the cause of anthrax), Clostridium tetani (the cause of tetanus), Clostridium botulinum (the cause of botulism), and Clostridium perfringens (the cause of gas gangrene).

General Procedure:  Using heat, a bacterial smear is first stained with malachite green.  After rinsing with water, the smear is counterstained with safranin. 

Materials:  malachite green, safranin, hot plate (or Bunsen burner and tripod), small beaker (150 ml), staining rack, slide holder, forceps, disposable latex gloves, wash bottle, bibulous paper