The absorption and transmission characteristics of common biological stains are published in the Sigma-Aldrich Handbook of Stains, Dyes, and Indicators and H. J. Conn's Biological Stains, which include actual spectroscopic data for hundreds of dyes. Typical visible light absorption spectra for red (Congo Red), green (Malachite Green), and blue (Methylene Blue) dyes are presented in Figure 1. In this figure, absorption (diffuse density) is plotted as a function of wavelength from 300 to 700 nanometers.

Congo Red has a strong absorption band at 340 nanometers in the near-ultraviolet region and another at 500 nanometers near the blue-green transition region. This dye transmits red wavelengths above 560 nanometers and thus, appears red to the eye. Appearing visually as a green dye, Malachite Green has a strong absorption band centered at 600 nanometers near the yellow-red transition region, with a wide transmission band (passing blue and green light) between 400 and 550 nanometers. Methylene blue has a strong absorption band centered at 660 nanometers, in the red region of the visible spectrum, and transmits wavelengths below 600 nanometers, bestowing a blue color to the dye.

When the diffuse density (absorption) by a stain exceeds 0.1, the wavelength band falling within the region greater than 0.1 is considered absorbed. This can conveniently be represented as a bar graph, such as the ones illustrated in Figure 2. The darker areas represent regions of absorption by the dyes that exceed a diffuse density of 0.1, and the lighter areas represent wavelengths passed (not absorbed) by the dye. This type of graph provides a simple index for comparing a set of dyes to quickly determine the suitable dye for any particular staining application.

A compilation of bar charts for the common biological stains are listed in Table 1. Use this table as a reference in choosing dyes to stain biological specimens for photomicrography using black & white film.

Spectral Absorption Characteristics of Common Biological Stains Stain Spectral Absorption  Acid Fuchsin   530-560    Aniline Blue   550-620    Azure B   580 +  Azure C   580-640  Basic Fuchsin   520-570    Brilliant Cresyl Blue   550 +  Carmine   500-570    Congo Red 400-560    Crystal Violet   550-610    Darrow Red   450-550    Eosin Y   490-530    Erythrosin B   510-540    Ethyl Eosin   530-550    Fast Green   560 +  Giemsa   500 +  Light Green SF   590 +  Luxol Fast Blue   500-640  Methyl Green   560 +  Methylene Blue   590 +  Neutral Red   480-570    Nigrosin   450 +  Nuclear Fast Red   460-550    Orange G   450-510    Orcein   500-620    Phloxine B   520 +  Prussian Blue   560 +  Pyronin B   510-560    Saffron 350-480    Safranin O   470-550    Sudan IV   470-580    Sudan Red   450-590    Tartrazine 400-460    Toluidine Blue   560 +  Trypan Blue   500 +  Wright's   500 +  Nanometers 400 500 600 < Ultraviolet         Infrared >

Table 1