Throughout this week in the Histology lab, I was operating the automated hematoxylin and eosin stainer, while also submitting stained slides to the pathologists to be reviewed.

Hematoxylin and eosin is a stain used in routine Histology slides. The hematoxylin stains nuclei in the tissue blue, whereas eosin stains the cytoplasm and other structures varying shades of pink. Hematoxylin is a natural dye originating from the logwood tree, Haematoxylum campechianum. However, it is not hematoxylin that is truly staining the nuclei, but rather its oxidized state of hematein. Oxidation of hematoxylin to hematein can occur using two different methods. One is a natural oxidation process that uses air or light. This process takes a significant amount of time to oxidize the dye, however, it produces a very stable stain. The other method uses chemical oxidation that requires either sodium iodate or potassium iodate. While this process occurs much quicker than the natural method, these stains may deteriorate and require filtering before use. However, this oxidized state of hematoxylin is still not sufficient to stain tissues. Hematoxylin must also be combined with a ‘mordant’, which gives the dye affinity to the tissue. Typical mordants that are added to hematoxylin are aluminum, iron, lead, tungsten, and molybdenum.

It is easy to view the stains empirically to determine their staining reactions. However, theoretically, hematoxylin is a positively charged dye. It binds to negatively charged components of tissue, such as nucleic acids, to stain them blue. Eosin, on the other hand, is a negatively charged dye, and binds to positively charged components of tissue, such as red blood cells, muscle, and collagen, staining them red or pink.

In staining tissues with hematoxylin and eosin, the procedure differs with the type of hematoxylin that is used. For example, hematoxylin that is mordanted to aluminum (also known as alum hemaxoylin) can be stained either progressively or regressively. A progressive stain will stain the tissue with hematoxylin for a specific amount of time. However, a regressive stain will overstain the tissue, then differentiate it with acid alcohol. After slides are differentiated, they are ‘blued’ using a bluing agent, such as lithium carbonate or ammonia water. This ‘bluing’ reaction reforms the mordant and dye link in the tissue and changes the red color of the nucleus to a blue.

All in all, this routine histological stain produces beautiful slides in order for the pathologist to properly diagnose disease!