Nile red can be prepared through an acid hydrolysis by boiling a solution of
Nile blue with
sulfuric acid. This process replaces an
iminium group with a carbonyl group. Alternatively, Nile red and its
analogs (naphthooxazine dyes) can be prepared by acid-catalyzed
condensation of corresponding 5-(dialkylamino)-2-nitrosophenols with
2-naphthol. The yields are generally moderate as no co-oxidant is used in this procedure. Since the reaction to generate Nile red does not usually completely exhaust the supply of Nile blue, additional separation steps are required if pure Nile red is needed. File:Nile red 01.jpg|Nile red under visible and ultraviolet (366 nm) light in different solvents. From left to right: 1.
water, 2.
methanol, 3.
ethanol, 4.
acetonitrile, 5.
dimethylformamide, 6.
acetone, 7.
ethyl acetate, 8.
dichloromethane, 9. n-
hexane, 10.
methyl-tert-butylether, 11.
cyclohexane, 12.
toluene. File:Bacillus subtilis stained with Nile Red.jpg|
Bacillus subtilis stained with Nile red as a membrane dye (shown in red). This strain grows partly as cell chains, so a membrane dye may be useful to distinguish internal cell boundaries. ==References==