Paul Ehrlich's acid-fast staining test, developed in 1882, is useful as a differential stain. Certain bacterial cell walls contain lipoidal mycolic acids. Under normal circumstances, these acids prevent dyes from coloring the cell. Therefore, gram staining is essentially useless on such bacteria. However, these mycolic acids are semipermeable to dye dissolved in alcohol and phenol and applied to heat. It is theorized that after cooling, mycolic acids coagulate and form an impassible barrier. Acid alcohol is unable to decolorize the bacteria. On a sterile slide, two circles are drawn with a wax pen. A single drop of water is placed in the first circle. Using aseptic technique, a small amount of bacteria is transfered from the slant into the first circle containing water and mixed around. Then using aseptic technique, a loop full of this diluted bacteria is transfered to a second drop of water located in the second circle. This is done to further dilute the bacteria. The slide is allowed to air dry for about 5 minutes and then is ran once or twice over the bunsen burner to 'heat fix' the sample to the slide. The slide is then set on top of a ring stand. A piece of paper towel the size of a slide is placed on top of the slide. Carbolfuchsin is added until the paper towel is fully saturated and then heat is applied through the use of a bunsen burner. It is important not to boil the carbolfuchsin, just to allow steaming. Therefore, periodically throughout the heating more carbolfuchsin must be added. After 5 or 6 minutes, the heating is removed and the slide is cooled for 1 minute. Then the paper towel is removed with tweezers and water is used to wash the slide of excess carbolfuchsin. Then the slide is rinsed quickly with acid alcohol. The slide is then covered with acid alcohol and allowed to rock gently for 15 seconds to remove the remainder of freely rising red carbolfuchsin. The slide is washed once more with water, and then is covered with methylene blue for 1 minute. Then the slide is washed with water, blotted dry, and looked at under the light microscope using oil immersion. Acid-fast rods appear bright red and are often in clumps. Non-acid fast bacteria stain blue.