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The page below is a sample from the LabCE course Theoretical and Practical Aspects of Routine H&E Staining. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Poor Contrast Between the Hematoxylin and the Eosin

If the hematoxylin or the eosin is too dark or too pale the contrast between the nuclear staining and the cytoplasmic staining will be poor.

Decreasing the time in the hematoxylin and/or eosin solution will decrease the intensity of the stain. Conversely, increasing the time will increase the intensity.

The time in the differentiating solutions can be adjusted as well. Increasing the time in the acid alcohol or the dehydrating alcohols will decrease the intensity of the hematoxylin and eosin respectively. Whereas decreasing the times in these will increase the intensity of the hematoxylin and eosin.

With these factors in mind, the optimal times in both the dyes and the differentiators need to be determined to achieve the proper balance between the nuclear and cytoplasmic staining.

Keeping the reagents fresh by exchanging them with new reagents at regular intervals is also important to ensure good results.

The top image shows a very poorly stained slide with very little contrast between the hematoxylin and the eosin. A well-stained slide is shown in the bottom image.