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Results Embryos treated with nicotine exhibited stunted growth and some exhibited physical deformities of the head (Figure 1). Those treated with 0.2 mL of the nicotine were smaller than those treated with 0.1 mL of nicotine, which were, in turn, smaller than the control. After being stained with Alcian Blue, embryos treated with 0.2 mL of nicotine solution exhibited a larger amount of cartilage in bones of the limbs and smaller gaps between the areas where the cartilage was stained blue than those treated with 0.1 mL of nicotine solution. Embryos treated with 0.1 mL of nicotine solution had a larger area of the limb bones stained blue with smaller gaps between the stained areas that the control. After being stained with Alizarin Red, however, the colors of stained skeletal structures could no longer be observed (Figure 2). Discussion Treatment of chick embryos with nicotine appears to have negative effects their development, the severity of which increases with the amount of nicotine to which the embryos are exposed. The treated embryos exhibited stunted growth and the embryos treated with the highest amount (0.2 mL) were the smallest. The treated embryos were also deformed, but there was no noticeable consistency in the deformities across the different treatment levels. For example, with the exception of their size, some of the embryos treated with 0.1 mL of the nicotine solution looked relatively similar to the control, but one of them was smaller and with a ventral midline that failed to fuse, so that the vicera were left completely exposed. Comparative bone and cartilage development could not be fully examined because the staining procedure did not result as expected within the expected amount of time. For future experiments, it may be necessary to amend or allow more time to complete the staining protocol. Up until the point where the specimen is stained in Alizarin red, all steps proceed relatively quickly. After being stained with Alcian Blue and cleared in 2% KOH, the stained cartilaginous structures can be readily seen. The staining with Alizarin Red, however, makes the embryos a dark purple color, and the destaining process takes much longer. If this experiment is repeated, Alcian Blue staining alone should be considered as a means of examining bone and cartilage development.