Staining and staining problems

Staining and staining problems

There is 2 main types of stains that is used most commonly. They are the Romanowsky and Papanicolaou(and it's derivatives). Romanowsky-type stain are more rewarding and practical, and readily available in practice situations. Example of such stains are Wright's stain, Giemsa stain, Diff-Quik. Romanowsky-type stain are inexpensive, readily available to practicing veterinarian, easy to prepare, maintain and use. They stain organism and cytoplasm of cells very well.

Smears are first air dried, preserving(fix) the cells, and cause them to adhere to the slide so they do not fall off during the staining procedure. Diff-Quik does not undergo the metachromatic reaction. As a result, granules of some mast cells do not stain, When mast cell granules do not stain, the mast cells may be misclassified as macrophages. This can lead to confusion in examination of some mast-cell tumors. Variation between different Romanowsky-type stains should not cause a problem once the evalutator has become familiar with the stain used routinely.

The procedure recommended for each stain should be followed generally. However, adaptations is to be applied according to the thickness of smear. Thick smear, higher protein concentration, longer stain time. Vice-versa for thin smear.

Poor stain quality often confuse both the novice and the experienced cytologist. Most staining problem can be avoided if the following precaution are taken:

• Use new slides, fresh and well filtered(if needed) stain, and fresh buffer(if needed)


• Stain cytologic preparations immediately after drying


• Take care not to touch the surface of slide or smear at any time.
  
  

The table below shows some common problems and solution.

Problem	Solution
Excessive Blue Staining
Prolonged stain contact	Decrese staining time
Inadequate wash	Wash longer
Specimen too thick	Make thinner smears
Stain, diluent, buffer or wash water too alkaline	Check with pH paper and correct pH
Exposure to formalin vapors	Store and ship cytologic preps separate from formalin containers
Wet fixation in ethanol or formalin	Air dry smears before fixation
Delayed fixation	Fix smears sooner
Surface of the slide was alkaline	Use new slides
Excessive Pink Staining
Insufficient staining time	Increase staining time
Prolonged washing	Decrease duration of wash
Stain or diluent too acidic	Check with pH paper and correct pH; fresh methanol may be needed
Excessive time in red stain solution	Decrease stain time in red solution
Inadequate time in blue stain solution	Increase time in blue stain solution
Mounting coverslip before preparation is dry	Allow preparation to dry completely before mounting coverslip
Weak Staining
Insufficient contact with one or more of the stain solutions	Increse staining time
Fatigued(old) stains	Change stains
Another slide coverd specimen during staining	Keep slides separate
Uneven Staining
Variation of pH in different areas of slide surface	Use new slides and avoid touching their surface before and after preparation
Water allowed to stand on some areas of the slide after staining and washing	Tilt slides close to vertical to drain water from the surface or dry with a fan
Inadequate mixing of stain and buffer	Mix stain and buffer thoroughly
Precipitate on Preparation
Inadequate stain filtration	Filter or change the stains
Inadequate washing of slide after staining	Rinse slides well after staining
Dirty slides used	Use clean new slides
Stain solution dries during staining	Use sufficient stain and do not leave it on slide too long
Miscellaneous
Overstained preparation	Destain with 95% methanol and restain
Refractile artifact on RBC with Diff-Quik stain	Change the fixative

*Reference: Cowell & Tyler (1992), Cytology and Haematology of the Horse.

Cheers
Douglas