Types of Dyes

• Dyes used as biological stains are typically organic salts comprising a colored ion and an uncolored ion. In a basic dye, the colored organic ion is positively charged, so it's drawn toward negatively charged substances. The cytoplasm of bacterial cells tends to soak up basic dyes. Acidic dyes, by contrast, contain a negatively charged organic ion that the bacterium's surface repels; these dyes tend to stain around the organism, creating a colored background against which the bacterium is transparent. Other dyes, like India ink, are pH-neutral.
  
  

Gram Stain

• The Gram stain divides bacteria into two broad classes: those that test positive on the Gram stain, and those that don't. This distinction reflects a structural difference. Gram-positive bacteria have a thick cell wall that is rich in peptidoglycans, while Gram-negative bacteria have a thin layer of peptidoglycans sandwiched between an inner and outer membrane. The first step in Gram staining is to apply crystal violet, followed by Gram iodine; the bacteria soak up the crystal violet dye, which aggregates with the iodide ions. Next, a brief treatment with acetone removes the crystal violet from Gram-negative cells; it does not affect the stain in Gram-positive cells, because their peptidoglycan cell wall is much thicker. Finally, an application of safranin counterstain turns Gram-negative bacteria pink. The Gram-positive bacteria, by contrast, retain the crystal violet, and hence appear purple under the microscope.
  
  

Ziehl-Neelsen Stain

• Due to the composition of their cell walls, some dyed bacteria are "acid-fast," meaning that they will not lose their color if exposed to a dilute acid. Mycobacterium tuberculosis, the bacterium that causes tuberculosis, is a prominent example. Staining a specimen with carbol fuchsin dye while heating it, decolorizing the sample with an acid, and then counterstaining it with another dye will reveal whether acid-fast bacteria are present. This procedure is called the Ziehl-Neelsen stain.
  
  

Methylene Blue Stain

• In this process, bacteria are placed on a slide, heat-killed and stained with a basic dye called methylene blue. This stain makes it easier to inspect the morphology, or shape, of the bacteria. Some bacteria are rod-shaped, while others are spherical or even spiral; methylene blue staining helps microbiologists recognize these structural differences.
  
  

India Ink

• This technique involves suspending microbes in saline solution, and then staining them with India ink. Under the microscope, the India ink is very dark; if a microbe has a gelatinous capsule, it will stand out plainly against this background. This is known as a negative stain, because rather than coloring the microbe, it colors the microbe's surroundings on the slide. This technique is particularly useful when working with certain fungi, especially Cryptococcus neoformans.