Step 64 – Understand the Stain
Know what you are trying to demonstrate with the stain you are performing.
Just “following the method” and not really knowing what should be seen in the finished section will lead to poor results.
Step 65 – Use a Positive Control
Always use a control slide known to contain the structure/ substance you are trying to demonstrate.
“If the structure/substance we are staining for is not visible in a slide we assume it is not present.”
Step 66 – Use Accurate Timing
Use accurate timing.
Timing is always approximate. Inaccurate timing produces inconsistent results.
Step 67 – Consider Reagent Stability
Be aware of the shelf life of the reagents you are using. Some reagents or dye solutions deteriorate slowly while others are very unstable and must be made up fresh and used immediately. Others have to be left for some time to oxidize (ripen) before they can be used at all.
We assume all reagents can be used for an indefinite period.
Step 68 – Store Reagents Correctly
Store reagents correctly. Some require refrigeration because they are inclined to support the growth of fungi or molds. Others are light sensitive and require storage in the dark.
“All our reagents are stored on the shelf above the staining bench. Sometimes we see stray organisms in our sections.”
Step 69 – Adhere to the Method
Follow the protocol exactly.
Staff members achieve different results when supposedly using the same protocol.
Step 70 – Record Any Changes
Document any departure from the method you are using.
Sometimes when results are poor it is difficult or impossible to work out why because protocol changes have not been recorded.
Step 71 – Standardize Washing Steps
Take particular care with washing steps. Standardize them as far as possible as they are frequently the cause of variable results.
Lab staff members use different washing techniques – some use vigorous agitation, others are much more gentle.
Step 72 – Set Up Microscope Carefully
Use microscopic control at crucial stages such as differentiation steps. Be aware of the effect of the microscope setup on the appearance of un-coverslipped (wet) sections; it can produce the appearance of false background staining.
For all methods the level of staining is assessed by looking at the slide with the naked eye.