Nile blue

Nile blue (or Nile blue A) is a stain used in biology and histology. It may be used with live or fixed cells, and imparts a blue colour to cell nuclei. It may also be used in conjunction with fluorescence microscopy to stain for the presence of polyhydroxybutyrate granules in prokaryotic or eukaryotic cells. Boiling a solution of Nile blue with sulfuric acid produces Nile red (Nile blue oxazone). Nile blue is a fluorescent dye. The fluorescence shows especially in nonpolar solvents with a high quantum yield. The absorption and emission maxima of Nile blue are strongly dependent on pH and the solvents used: The duration of Nile blue fluorescence in ethanol was measured as 1.42 ns. This is shorter than the corresponding value of Nile red with 3.65 ns. The fluorescence duration is independent on dilution in the range 10−3 to 10−8 mol/L. Nile blue is used for histological staining of biological preparations. It highlights the distinction between neutral lipids (triglycerides, cholesteryl esters, steroids) which are stained pink and acids (fatty acids, chromolipids, phospholipids) which are stained blue. The Nile blue staining, according to Kleeberg, uses the following chemicals: Nile Blue A 1% acetic acid Glycerol or glycerol gelatin The sample or frozen sections is/are fixated in formaldehyde, then immersed for 20 minutes in the Nile blue solution or 30 sec in nile blue A (1% w/v in distilled water) and rinsed with water. For better differentiation, it is dipped in 1% acetic acid for 10–20 minutes or 30 sec until the colors are pure. This might take only 1–2 minutes. Then the sample is thoroughly rinsed in water (for one to two hours). Afterwards, the stained specimen is taken on a microscope slide and excess water is removed. The sample can be embedded in glycerol or glycerol gelatin. Unsaturated glycerides are pink, nuclei and elastins are dark, fatty acids and various fatty substances and fat mixtures are purple blue. The PHB granules in the cells of Pseudomonas solanacearum can be visualized by Nile blue A staining.