In a recent post we talked about Congo red, an amyloid stain with a pretty fascinating history, but there are two other stains that can be used for amyloid detection which are crystal violet and Thioflavin T.
Crystal violet, also known as methyl violet, was the first stain used for the detection of amyloids, back in 1875. When it was first discovered crystal violet had a variety of uses, including being used as an antiseptic, though now it has largely been replaced in that capacity by more modern medicines.
As you’ll recall from the Congo red post, Congo red works by hydrogen bonding to amyloid’s beta pleated sheet, which means that it can only be used for amyloids that have the beta pleated sheet intact. In the crystal violet stain however, violet dye binds to the amyloid’s surface carboxyl ions. Crystal violet is not the most specific stain for amyloids however, with other tissue structures often showing similar colors, so small amyloid deposits can sometimes be missed with this stain.
An aqueous mounting media must be used with crystal violet as it is soluble in solvents. The results are amyloid staining purple/violet and other tissue structures blue.
In addition to being used as an amyloid screening stain, crystal violet is used in Gram staining for the distinction between Gram-positive and Gram-negative bacteria. Gram-positive bacteria, such as Streptococcus pneumoniae, the bacteria Hans Christian Gram was first trying to identify in the lungs of pneumonia patients, will stain violet with crystal violet and remain so after decolorization with ethanol. Other bacteria, referred to as Gram-negative, such as Salmonella Typhi that is associated with Typhoid fever, were decolorized after the application of ethanol. We now know that this differentiation has to do with the composition of the bacteria’s cell wall, with Gram-positive bacteria having a cell wall containing thick layers of peptidoglycan, while Gram-negative have thin layers of peptidoglycan and high lipid content.
Outside of the histology laboratory, crystal violet dye has also been used for the visualization of latent fingerprints on nonporous surfaces, and as a DNA stain in conducting DNA gel electrophoresis.
Finally, there is one more stain used for amyloid detection which is thioflavin T. Thioflavin T, which sounds like it should be the name of an eccentric 90’s rapper, is actually a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils. Its potential as a fluorescent probe for amyloid was first recognized by Vasser and Culling in 1959, but it wasn’t until 1989 that amyloid fibrils in vitro were fully quantified by fluorescence emission of thioflavin T.
During thioflavin T staining, hematoxylin and acetic acid rinses are used to reduce autofluorescence, however thioflavin T can also react with other molecules like DNA and certain proteins that have a similar molecular groove as that of amyloid fibrils, which is why Congo red remains the more commonly used stain for amyloid diagnosis.
Want to know more? Register for the HT Prep Course taking place live during the NSH Convention, September 13th.
References:
https://www.sciencedirect.com/topics/medicine-and-dentistry/crystal-violet
https://asm.org/getattachment/5c95a063-326b-4b2f-98ce-001de9a5ece3/gram-stain-protocol-2886.pdf
https://makezine.com/laboratory-86-revealing-latent-fing/
https://link.springer.com/chapter/10.1007/978-1-60761-389-3_14
https://www.tandfonline.com/doi/pdf/10.1080/13506129.2017.1304905
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