Gram Positive Bacteria Identification

Far and away, the most important step in identifying Gram positive bacteria is the Gram stain, which was named after Danish scientist Hans Christian Gram and has four basic steps: creating a slide smear, applying a stain, applying a counterstain and viewing the stain. Following a Gram stain, researchers probe for additional characteristics.

Creating a Slide Smear

• The Gram stain begins with a slide smear. With an inoculation loop or wooden stick, researchers collect a small sample of the bacteria (usually grown in a Petri dish) and smear it onto a slide. Next, they will allow the sample to dry, sometimes using a low flame to speed up the process.

Applying a Stain

• Once the sample is dry, researchers apply a stain with crystal violet, a dark purplish chemical. After a minute or so, researchers rinse away any excess crystal violet with water or alcohol. Then comes an iodine smear, which acts as a mordant. That is, iodine helps the crystal violet "stick" to the bacteria. Researchers often refer to this step as "fixing the dye."

Applying a Counterstain

• Once the crystal violet is fixed, researchers apply another rinse and then a counterstain with safranin, a pinkish chemical. Bilbuous paper can help dry the slide, which is now ready for close viewing.

Viewing the Stain

• Under a microscope, Gram positive bacteria appear brown or dark purple, the color of crystal violet. This is because Gram positive bacteria contain high amounts of peptidoglycan, a mesh-like substance, in their cell walls. Peptidoglycan gives cell walls strength and shape, and it distinguishes them from Gram negative bacteria, which will appear pink, the color of safranin. (Without the safranin counterstain, they would be transparent and difficult to view at all.)

  Many Gram negative bacteria do contain some peptidoglycan, but not nearly as much as Gram positive bacteria. The location of the peptidoglycan also differs: In Gram positive bacteria, peptidoglycan forms part of the cell walls' outer layer. The outer layer of Gram negative bacteria, on the other hand, resists crystal violet. For that reason, it is the Gram positive bacteria that appear purple.

After the Gram Stain

• After a Gram stain, researchers can identify Gram positive bacteria more precisely through additional testing. They may investigate, for example, an unknown bacterium's morphology, structure, movement and genetics. Prior research has divided Gram positive bacteria into four families---the Bacillaceae, Micrococcaceae, Mycobacteriaceae and Peptococcaceae---and each of these families has telltale characteristics:

  Bacillaceae are rod-shaped bacteria. If they can move by themselves, they have flagella covering their entire surface.

  Micrococcaceae are circle-shaped. They cannot move by themselves, and when they divide, they form groups of fours, or tetrads.

  Mycobacteriaceae are shaped like gently-curved rods. When they divide, they may either form branches or long, crisscrossing shapes.

  Peptococcaceae are also circle-shaped. Because of their structure, they require environments without oxygen, and some cannot grow where there is salt.

  Considerable variety exists within each family.