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Section 7: Solving Problems
Problem
The slides are too red.
Cells are too orange.
Faint staining on top 1/16 inch of slide.
Light, uneven, or no staining.
An empty Reagent E (alcohol wash) reservoir prevents slides
from rinsing well, leaving residual Reagent A (EA-50) on the slide.
Check Reagent E level, and if necessary, run the Spray Pattern
test to make sure there is proper reagent delivery. See Section 6.6
Check drain tube for improper installation, or for any
blockage in the tubing (such as stain build-up) This can
cause staining reagents to accumulate in the bowl leading
to incomplete washing of slides. See Section 2.1
Overall, or predominantly red staining can also occur if the
cytoplasmic stain setting is too low. Because the fast-green
molecules of Reagent A (EA-50) are larger than the eosin
y molecules, they penetrate the cell membrane and stain
more slowly, allowing the cells to stain red from the eosin y.
To remedy this problem, use a higher cytoplasmic setting.
Check nozzle delivery and spray patterns before staining
and whenever staining problems occur. (See Section 6.6).
Inadequate reagent delivery is a primary cause of poor staining.
When smears or cytocentrifuge preparations are not fixed
properly, the cytoplasm hardens and shrinks, eventually
becoming impermeable to the EA-50 staining. The cells stain
orange because the orange G molecule is considerably smaller
than the eosin y or fast green molecules and can therefore
permeate the dense cell membranes more easily and quickly.
If a specimen has not been fixed, and then air dries.
Soaking the slide in 50% glycerol for 1 hour may
rejuvenate some of the cells. If the glycerol does not
help, a new smear will have to be prepared.
Orange staining can also be due to incomplete carbowax
removal. Use the pre-wash option or presoak the slides
in alcohol before loading them into the stainer. Check
to see if the Reagent A (EA-50) nozzle is plugged.
Check reagent delivery system as outlined in Sections 2, 4, and 6.
Use the double staining procedure described in Section 13.3
If staining is even, but too light, increase
nuclear or cytoplasmic stain intensity.
If the staining is uneven, check all nozzles (see Preventive
Maintenance chart.) A plugged nozzle may deliver
stain, but application will be sporadic and uneven.
Check for incomplete carbowax removal. Commercially available
spray fixatives have varying amounts of polyethylene glycol
(carbowax). Carbowax protects cells from air drying during
fixation, but if not completely removed can inhibit staining. To
remove the polyethylene glycol, soak slides in 95% alcohol
or 50% glycerol for 10 to 30 minutes before staining.
Check to see if slides were placed in the carousel backward or in
the wrong position (if staining less than 7 slides). See Section 4.1.
Solution
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