Gram staining technique for bacterial identification and classification

Gram Lab

Gram Staining Lab

The Gram staining of bacteria is one of the most important tests in identifying specific bacterial strains, and is usually the first test performed when medical or research laboratories need to identify an unknown bacteria (Xu, 1997; AACC, 2011). Named for the inventor of the technique, Hans Christian Gram, Gram staining first came into use in Gram's own Danish medical and research practice in 1882, and became very widely known and used after the details of the technique were published in 1884 (Xu, 1997). Though some adjustments can be made to the original technique without losing effectiveness, the basic process of Gram staining has remained unchanged for over a century, and is as effective and essential a test today as it was in the latter half of the nineteenth century (Xu, 1997).

Simply put, the Gram staining test often (though not always) allows the researcher to determine the broad class of bacteria that an unknown species belongs to by using dyes and washes that are absorbed and retained differently by different bacteria (AACC, 2011). Crystal violet dye is the standard dye used in the first step of Gram stain testing; the cell walls of some bacteria will retain the crystal violet dye after the sample is washed out, and the cell walls of other bacteria will not hold onto the dye (AACC, 2011; Xu, 1997). Those bacteria that retain the dye and thus take on a crystal violet hue are called Gram positive, while those bacteria that do not retain the dye (and are usually stained with another dye to provide better contrast in subsequent steps of the test are Gram negative (AACC, 2011; Xu, 1997).

Steps in Gram Testing

The first step of Gram stain testing, after properly preparing a slide with a smear of bacterial culture, is to add crystal violet stain to the slide containing the culture smear in order to allow Gram positive bacteria to absorb the dye (Xu, 1997). The dye only needs to set for ten seconds to up to a minute, though it can be even more immediate for very thinly prepared slides (Xu, 1997). After the staining, washing away the excess dye with a thin and gentle stream of water (taking care not to wash off the sample itself) is necessary, and the sample is then saturated in an iodine solution and rinsed again, this time in order to bring out the dye staining even more (Xu, 1997). Decolorizer is then used to remove any remaining coloration from the slide and the solution, though it is important not to use too much or the bacteria themselves will not appear stained anymore (Xu, 1997). Finally, a counterstain (typically a basic fuchsin solution) is used to stain Gram-negative bacteria a pink color that contrasts with the purple/violet of the Gram-positive bacteria (Xu, 1997; AACC, 2011).

Materials and Methods

The materials and methods used for this lab are exactly as described in the laboratory manual, with one significant exception. Following the staining, the samples were washed with water for two seconds and in iodine for a full minute before the alcohol was used to decolorize the samples. Other than this adjustment utilized to ensure more reliable results, however, the laboratory manual's instructions were utilized for each step of the experiment.

Discussion

Simply put, Gram-positive bacteria have higher concentrations of peptidoglycan and lower levels of lipids in their cell walls than Gram-negative bacteria, and these peptidoglycans retain the crystal violet dye while the lipids hold the fuchsin solution (Xu, 1997). It is for this reason that the Gram staining technique enables a basic differentiation of bacterial types. The cell wall differences also create significant differences in how the bacteria work as organisms and during infections, so Gram testing provides information that is very useful for treating bacterial infections as well as for determining if a bacterial infection exists at all.