Bacteria are often classified into Gram-positive and Gram-negative strains by staining with crystal violet (CV). The described bioorthogonal modification of CV with trans-cyclooctene (TCO) can be used to render Gram-positive bacteria magnetic with tetrazine-functionalized magnetic nanoparticles (MNP-Tz). This method allows class-specific automated magnetic detection and magnetic separation.
[Credit: Angewandte Chemie, Wiley-VCH]
Gram staining was developed about a hundred years ago by Danish bacteriologist Hans Christian Gram. In this technique, bacterial cultures are colored by a stain known as crystal violet, which enters into the murein layer of the bacterial cell walls. Treatment with an iodine-containing solution forms water-insoluble complexes between the crystal violet and iodine. There are two classes of bacteria that differ in the structures of their cell walls. A thick murein layer surrounds one class; the others have only a thin one. Whereas subsequent treatment with ethanol dissolves the stain complex out of the thin murein layer, it remains firmly lodged in the thick murein layers. Bacteria whose stain can be washed away in this manner are classified as Gram-negative; those that remain dark purple are Gram-positive.
Scientists working with Ralph Weissleder at Harvard University in Boston (USA) have now developed Gram staining into a magnetic diagnostic technique. To achieve this, they attached a “molecular hook” to the molecules of crystal violet. With this modified dye, the staining process works just as it does with the original. After staining, however, “eyes” that correspond to the “hooks” are used to attach magnetic nanoparticles to the stain. This makes it easy to quantify the bacteria: nuclear magnetic resonance (NMR) instruments detect the magnetization of the nanoparticles.
It is possible to take an NMR measurement before washing with ethanol to obtain the total number of Gram-positive and Gram-negative bacteria, and again after the washing step to determine the concentration of Gram-positive bacteria.
The advantage of this magnetic detection method is its high sensitivity. It is possible that samples could be directly magnetized and measured without prior purification or culture of the bacteria. By using the simple but sensitive miniaturized micro-NMR instruments developed by this research group, fast and sensitive on-the-spot diagnosis is conceivable. In addition, the magnetization could be used for the separation of bacteria from the sample.
About the Author
Dr. Weissleder is a Professor at Harvard Medical School, Director of the Center for Systems Biology at Massachusetts General Hospital (MGH), and Attending Clinician at MGH. His research interests include the development of novel molecular imaging techniques, tools for detection of early disease detection, development of nanomaterials for sensing and systems analysis. He is a member of the US National Academies Institute of Medicine.