Step 52 – Use Accurate Timing

 Each step in the staining protocol is accurately timed.

 Step times in staining are approximate and “if we are in a hurry” some steps are skipped. This can produce in consistent results.

Step 53 – Regularly Monitor Quality

 Control slides are regularly stained to monitor stain quality.

 Control slides are never used for H&E stains. This can make it very difficult to determine whether a staining problem is due to poor reagents, an inappropriate protocol or poor fixation.

Step 54 – Standardize Staining Conditions

 Agitation, wash and drain times are optimized for all steps during staining.

 Agitation, wash and drain times are inconsistent. Solvents and reagents rapidly become contaminated. Staining becomes inconsistent.

Step 55 – Ensure Complete Dewaxing

 Slide dewaxing is optimized.

 Slide dewaxing is sometimes incomplete and slides contain patches of residual wax. This produces unstained, or unevenly stained areas in sections.

Step 56 – Renew Reagents Regularly

 Solvents and staining reagents are regularly replaced based on the number of slides stained or racks processed.

 Replacement of solvents and staining reagents is haphazard. They are not replaced until stain quality declines.

Step 57 – Hydrate Sections Thoroughly

 Slides are thoroughly hydrated prior to hematoxylin staining.

 Hematoxylin solution rapidly becomes contaminated with alcohol and sometimes xylene. This causes uneven staining.

Step 58 – Monitor Hematoxylin Quality

 The performance of hematoxylin solutions is carefully monitored. During their working life hematoxylin solutions are progressively diluted by carry over from slides and racks and also affected by continuing oxidation.

 Hematoxylin staining is variable from day-to-day and no attempt is made to understand why. For example, the staining bath surface area, the extent of aeration during staining, and the ambient temperature can all affect the oxidation rate.

Step 59 – Ensure Complete Nuclear “Blueing”

 Thorough “blueing” of nuclei with Scott’s alkaline tap water substitute or ammonia water is always performed after hematoxylin staining. This requirement is influenced by the natural pH of the local tap water.

 Sometimes nuclei appear pinkish in completed sections due to incomplete “blueing” in alkaline tap water after hematoxylin staining. Nuclei that are under-stained with hematoxylin (or over-differentiated) and over-stained with eosin also appear pink.

Step 60 – Avoid Uneven Eosin Staining

 “Blueing” is followed by a very thorough wash in tap water to remove residual alkali that can impede eosin staining and cause weak and uneven staining.

 Inefficient washing after “blueing” (leaving residualal kali) causes eosin staining to be weak and uneven.

Step 61 – Monitor Eosin pH

 The pH of the eosin solution is monitored. It is kept close to pH 5.0 to maintain optimal staining. The addition of a couple of drops of acetic acid can be used as a convenient means of lowering pH.

 No attempt is made to monitor the pH of eosin. When staining intensity falls away the solution is replaced (carry over of alkaline tap water can cause the pH of eosin solutions to rise).

Step 62 – Thoroughly Dehydrate Before Clearing and Cover slipping

 Sections are thoroughly dehydrated before being placed in xylene for clearing.

 Sections are sometimes rushed through alcohol to xylene. Clearing in xylene contaminated with water can result in the presence of tiny water droplets in the tissue that are seen microscopically as opaque areas lacking detail.

Step 63 – Avoid Drying and Crystal Formation

 The coverslip is always applied before the section has a chance to dry and a high quality mountant is used. The long-term storage qualities of the mountant must be known because crystals can appear in poor quality mountant–sometimes after a long period (months or years).

 Sections are allowed to partially dry before the coverslip is applied causing some nuclei to appear black. Mountant chosen on the basis of price alone may develop crystals during long-term storage and coverslips may lift.