By Andy D’Ewart, Microbial Identifications Study Director
With dozens of tests required to get your product to market, it might be tempting to leave out Gram stain testing. At Nelson Labs, we recommend adding a Gram stain to your genetic identification testing to save you time and money in the long run while providing a more comprehensive identification. A Gram stain with a colony morphology description will reinforce the identification and can eliminate some of the most common failures in genetic testing.
The Gram stain is a test that incorporates dyes into the cell walls of organisms in order to separate them into two main groups: gram positive and gram negative. A microscope is used to observe the stain and shape of the cells (cocci, rods, etc.).
According to the United States Pharmacopeia (USP) <1113>, the Gram reaction is a “critical step for many phenotypic identification schemes” and can eliminate common failures. The most common failure in genetic testing is trying to identify a mixed culture, which contains multiple organisms. Different organisms have different DNA sequences and the genetic analyzers cannot distinguish between the sequences. Mixed cultures cannot always be discovered just by looking at them. A Gram stain allows easy detection of multiple cell types, meaning further work must be done to achieve a pure culture.
To provide a genetic identification, we need to know if we are working with bacterial cells or yeast cells. Because bacterial colonies and yeast colonies can look identical without the use of a microscope, a Gram stain can be used to determine which type of cell is present. This is recommended by USP <1113> which states that without a Gram stain, “subsequent testing may be conducted using the wrong microbial identification kit, resulting in an incorrect result.”
For genetic identifications, a small portion of the ribosomal DNA (rDNA) is sequenced. For bacterial cells, the test reagents target the 16S rDNA, a gene universal among bacteria. Yeast cells are fungi and they do not share this portion of their DNA, so different test reagents must be used. These reagents target a gene shared among all fungi. If the wrong test reagents are used, a sequence will not be generated, resulting in a failed identification attempt.
In addition to eliminating failures, the Gram stain can be used to confirm the genetic identification. USP <1113> recommends that “any microbial identification should be reviewed for reasonableness in terms of the microorganism’s morphology.” The Gram stain is the best way to determine the microorganism’s morphology and to verify the identification.
A Gram stain provides important information to back up your identification. Genetic failures are avoidable and can lead to extra costs and delayed turnaround times. Simple and inexpensive, the Gram stain test can save you time and money.