J is for Janus Green B

The ability to stain living cells without killing them is termed vital staining. Whether the cells are removed (supravital) or remain in the body (intravital), the terms are interchangeable and often simply referred to as vital staining. Vital stains have been used in diagnostic and surgical techniques and in this article, we take a look at Janus Green B whose chemical structure is shown (C₃₀H₃₁N_6.Cl) in Figure 1. Like many other certified dyes, Janus Green B is known by other synonyms such as Diazin Green S and Union Green B.

What is Janus Green B?

Janus Green B is a dark green to black powder belonging to the phenazine group of dyes. Introduced by Leonor Michaelis in 1900, the interesting feature of this dye is that it must be oxidised to become coloured. In histology, this basic dye is used to stain mitochondria in living tissues. Regarded as the powerhouse of cells, mitochondria are membrane-bound organelles that produce adenosine triphosphate (ATP), the primary carrier of energy. It was Michaelis in 1900 who first selectively stained these organelles with Janus Green B and proposed that mitochondria were cellular oxidising agents. The specificity of this stain for mitochondria is due to the activity of cytochrome oxidase which maintains the dye in its oxidised, blue-green state (Figure 2. Hamster ovarian cell with mitochondria staining blue-green- Courtesy of CL Case, https://accounts.smccd.edu/case/biol230/ex3.html). In the rest of the cell, the dye is reduced to a pink or colourless compound. Studies of lymphatic vessels in rabbits have also shown that the dye highlights the threadlike structures known as Bonghan ducts since they contain a high density of mitochondria. When stained with Janus Green B and viewed under dark ground microscopy, mitochondria appear as bright orange-yellow granules (Figure 3. Mitochondria are seen as bright orange-yellow granules when viewed under high power with dark ground microscopy- Todd & Barnetson, 1988).

Additionally, Janus Green B can be used to stain other living materials such as fungi, yeast cells, chromosomes and nucleic acids. When the dye is used in the presence of oxygen within the tissues, the indicator dye oxidises to a blue-green colour. The use of Janus Green B has proved so useful as a flagella stain that it has been applied to both protozoa and spermatozoa (Figure 4. Spermatazoa stained with Janus Green B- Aksoy et al, 2012).

Other uses

In the laboratory, other uses of Janus Green B have included assays for the diagnosis of amyloid-related diseases and the rapid staining of peripheral nerves of insects.