New Spin on Gram Stain Bacteria

Different characteristics between Gram-negative and Gram-positive bacteria. Image obtained from:

Different characteristics between Gram-negative and Gram-positive bacteria. Image obtained from: Chemical and Engineering News

An old but reliable method: The Gram Stain. Invented by Hans Christian Gram in 1884, Gram stain has changed the course of science. Often times, it is the first step in attempting to identify the unknown bacteria. As soon as you get a sample in the microbiology lab, Gram stain – whatever you do gram stain!

In general, Gram stain-ing will yield two results under the microscope: Gram Negative GNB (Pink in colour) and Gram Positive GPB (Purple in colour).These two categories can help laboratory professionals or researchers to now begin a series of test depending on their colour  – saves a lot of time and resources!

How does it work? More importantly, this is how I learned the theory behind Gram Stains.

The procedure of Gram Stain on a heat-fixed smear of bacterial culture:
(1) Crystal Violet (Purple) for 1 minute
(2) Iodide (mordant) for 1 minute
(3) Acetone / Ethanol (decolourization)
(4) Safranin for 1 minute

The theory behind obtaining a Gram positive identification or Gram negative identification has been based upon its physical components in the outer membrane layer of the bacterium. As seen from the picture above, GPB has a thicker peptidoglycan layer, whereas GNB has a layer of lippolysaccharides.

Once crystal violet ion (CV+) penetrate through the cell wall and membranes of both GPB and GNB, the CV+ ions will interact with negatively charged components of the cell and thus staining purple. Then when mordant is applied, the CV+ ions interact with the mordant ions (iodide I-) and forms large complexes that traps the CV+ ions in between the outer and inner layer of the cell. The next step, decolourization is obtained by the use of alcohol or acetone that loses the outer lipopolysaccharide membrane thus exposing the CV+ and mordant complexes [GNB will lose the purple at this step, GPB will remain purple]. Safranin is used as a counter-stain to yield a colour from the removed CV+ ions [GNB will now be stained pink, GPB will remain purple].

This theory has been learned for many years, until now. A new study conducted by Hai-Lung Dai from the Department of Chemistry at Temple Univerisity suggests another mechanism. With the use of second-harmonic light-scattering and bright-field transmission microscopy, they observed E.coli uptake of crystal violent (CV). They demonstrated that CV is barely able to diffuse beyond the GN thin peptiodoglycan layer, and CV does not cross the cytoplasmic layer. Therefore, this proposes that the distinguish between GPB and GNB is based on how well the dye attaches to the bacteria’s peptidoglycan layer.

Read the research paper here (ACS Chem. Biol. 2015, DOI: 10.1021/acschembio.5b00042)

Wilhelm, M. J., Sheffield, J. B., Sharifian Gh, M., Wu, Y., Spahr, C., Gonella, G., … & Dai, H. L. (2015). Gram’s stain does not cross the bacterial cytoplasmic membrane. ACS chemical biology.

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