How do you prepare cotton blue solution from the cotton blue powder?

Answer 1

In order to reduce the possible volatile and long-term exposure effects of L-P CB, a reagent lacking phenol was developed to analyze inertial impaction samples. To date, approximately 20 000 individual spore-trap samples (Air-O-Cell cassette and Allergenco MK-5 Multi Trace Slide, Zefon International, St. Petersburg, Florida) have been analyzed using the new reagent, hereafter called Lacto-Cotton Blue (LCB). The recipe for Lacto-Cotton Blue mounting medium is: glycerol 250 mL (Sigma-Aldrich, St. Louis, Missouri), 85% lactic acid 100 mL (Sigma-Aldrich, St. Louis, Missouri), 3 mL of cotton blue stock, and de-ionized water 50 mL. A stock solution of Cotton Blue stain is made to add to the mounting medium: 85% lactic acid 99 mL, Aniline (Cotton) Blue crystals 1.0 g (Fisher Scientific, Los Angeles, California).

To make the Cotton Blue stock solution, first add cotton blue crystals to lactic acid while stirring vigorously on a stir-plate. Stir until cotton-blue crystals are dissolved. Next, vacuum filter the solution through a #50 Whatmann 90 mm filter disc (Fisher Scientific, Los Angeles, California) in a Büchner porcelain funnel. Cover the orifice of the porcelain funnel to insure airborne particulate matter cannot enter the reagent mixture. After filtration has occurred, check the clarity of the stock dye solution. Place one drop of the dye on a clean microslide with a 22 × 22 mm cover slip and examine at 400×. Particles of dye should be approximately 2 μm or less in diameter. If large clumps of dye are observed, repeat vacuum filtration of the dye solution until the appropriate clarity is obtained.

The next step is to make the LCB mounting medium. First, mix the water, lactic acid, and glycerine (in that order) for one hour on a stir-plate. Once the solution is homogenous, add three mL of Cotton Blue stock solution to the water, lactic acid, and glycerine solution. Stir the entire mixture for an additional hour. Cover the flask with laboratory film (Parafilm, Fisher Scientific, Los Angeles, California) while the mixture is stirring to insure against airborne contamination.