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histotechnology > Staining techniques and troubleshooting > Flashcards

Staining techniques and troubleshooting Flashcards

1
Q

General Staining

A

DEWAXATION - xylene is hydrophobic
▪Xylene min
▪ Xylene 2 min

REHYDRATION- descending grades of alcohol
▪ 100% alcohol2 min
▪ 95% alcohol 1 min
▪ 70% alcohol 1 min
▪ RTW or dH2O 2 min

STAINING HAPPENS HERE
DEHYDRATION
▪ 95% alcohol 5-15dips
▪ 100% alcohol 5-15dips
▪ 100% alcohol 5-15dip
CLEARING
▪Xylene 1 min
▪ Xylene 1 min

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2
Q

Reagent Set what do they contain
how to handle

A
  • contain volatile and toxic vapors keep in FLAMMABLE cabinet
    -ensure ventilation is turned on
    -have proper PPE
    -Label slides prior to staining and put a permenant label on after staining
    -put slides in a carrier when transferring
    -when using the slide carrier make sure its dry.If you are going to take the slides into the water
    bucket, it must be xylene-free. If you are taking
    the slides to xylene, it must be water-free. Water
    and xylene are not miscible. It will contaminate
    the reagents. If the slide carrier is wet, dip it into
    a fresh bucket of 100% alcohol. It is miscible with
    both water and xylene.
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3
Q

What is the purpose of staining? and what are the THREE MAJOR STEPS IN STAINING PROCESS

A

 To aid in the visualizing tissue components.
 To demonstrate tissue components differently

  1. DEWAXATION AND REHYDRATION: “Taking sections to water”
  2. STAINING: Applying reagents and stains/dye.
  3. DCM: Dehydrate, Clear and Mount.
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4
Q

What is DEWAXATION AND REHYDRATION

A

-dewax in xylene as it removes infiltration medium (paraffin) so the tissue is prepped for staining
-the paraffin wax remaining after being taken out of the oven will be removed by xylene
-descending grades of alcohols from 100-95-70 clear out the xylene and introduce the tissue to water with RTW in a pre filled bucket
-use a low flow rate so tissue doesnt break off and all alcohol is rinsed off the slide

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5
Q

What happens in the staining phase

A

-apply reagents in aqueous environment
-after each step in staining reagent the slides have to be rinsed out in RTW bucket
-After blueing with Scott’s TWS and RTW, take your
slide to the microscope and check to make sure
the nuclei are blue and not red.

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6
Q

What are you check for when you CHECK DIFFERENTIATION MICROSCOPICALLY

A

-in the bluing step the red color of HX is converted into a blue color
-alkaline pH of bluing solution causes a mordant dye-lake to reform in the tissue and become more permanent.
-dont let slides dry out - take it out of the water and put it on the mic
-if slide if left out for too long itll look grainy. dip it back into water and rehydrate to check microscopically

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7
Q

What is the DEHYDRATION phase

A

slides are preserved by removing water/aqueous stains by process of DCM

 Ascending grades of alcohol.
 Gradual removal of water from tissue.
 Very important to avoid contamination of alcohols and xylenes

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8
Q

What is the CLEARING phase

A

 Removal of alcohol from tissue slides.
 Has high refractive index .
 Tissues are left clear and crisp without a cloudy haze over tissue (unless contaminated.
 Xylene must not be contaminated with water
(immiscible with each other).W

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9
Q

What is the purpose of mounting

A

-protect stained tissue from damage
-better for microscopic examination stops refraction of light under the mic
-preserves for storage

-need glass coverslips
-permount mounting media - make sure to check viscosity. If exposed to air itll harden ; add drops of xylene to dilute if too thin add permount reagent

-wear ppe and work in ventilated area
-make sure there is no haze
-opaque spots show incomplete dehy and clearing
-wipe frosted side first to check where the tissue is
-dont allow tissue section to dry out, return slides to xylene if dried out
-lower coverslip at 45 degree angle, permount is drawn up by capillary action and dab to remove excess mountant on paper towel

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10
Q

staining results what you should see - microscopic examination

A

Nuclei Blue
Muscle and collage - pink
RBCs Orange-pink

do tissue id

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11
Q

REAGENTS IN THE DEWAXATION SET
what are their properties

A
  • XYLENE - HYDROPHOBIC
  • ALCOHOL - HYDROPHILIC
  • WATER
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12
Q
  • Which bucket do the slides go into first?
  • What would happen if the slides were placed in
    the wrong reagent…what is your next course of
    action?
A

xylene

take slide and dip into 100% alcohol work back 70, 90, 100 and back into xylene to dewax

not anyother grade because itll have water in it

-dry slide carrier or only one that has been 100% alcohol

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13
Q

What would you do if slides were exposed to
water first?

A

dip in 100% alcohol

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14
Q

What would you do if slides were exposed to
rehydrating alcohol first? 100%, 95%, 70%, and not
xylene

A

dip in 100% alcohol

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15
Q

What would you do if slides were not dewaxed or
rehydrated and stain was applied to the tissue?

A

rinse in water, remove the H&E stain, then in ascending grades of alcohol, xylene to dewax and follow steps as normal

tissue is not affected because there is still wax on the tissue

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16
Q

WATER AND SLIDES TURN MILKY WHEN
SLIDES ARE PLACED IN WATER FOLLOWING ALCOHOL DURING DEPARAFFINIZATION
What would cause this and the Corrective action

A

-Presence of xylene on the slides.
-Inadequate time in rehydrating alcohols to
remove xylene.

CORRECTIVE ACTION:
* Change the alcohols in the rehydration section of the dewaxing set.
* Then take the slides through fresh 100% alcohol → 95% alcohol → 70% alcohol → water

17
Q

how will CONTAMINATED REAGENTS IN THE
DEWAXATON SET affect staining

A
  • Can cause poor quality staining of tissue
    components.
  • Can obscure the microscopic image of the tissue.
    *can be caused by inadequate rinsing

in xylene you can see contaminated water droplets

18
Q

LIDES ARE HAZY OR MILKY IN THE LAST XYLENE BEFORE COVERSLIPPING causes and corrective action

A

Causes:
-Incomplete dehydration in alcohols during DCM.
-Contaminated 100% alcohol that carried water
over to the xylene buckets in DCM.

CORRECTIVE ACTION:
-Back up the slides and change the fresh alcohols and xylene solutions.
* Then re-dehydrate in the alcohols and clear the sections in xylene.
* The slides should be clear and transparent.

19
Q

LOW REAGENT LEVEL can lead to

A

-Inadequate dewaxation. If paraffin is not removed from tissue stains will not be able to penetrate

-incomplete rehydration and dehydration in DCM because of the contaminated 100%etoh and xylene

-make sure reagents are filled to the reagent line on bucket and you may need to repeat dewaxation and rehydration steps

20
Q

how to troubleshoot staining problems

A

-check staining of slides when they are in the last xylene bucket before coverslipping.
-skip coverslip removal step if troubleshooting is needed
-if the slides can be troubleshooted “take them back to water”

if you need to take back to water - work backwards from DCM
xylene → xylene → 100% ETOH → 100% ETOH → 95% ETOH → RTW → troubleshoot stain

21
Q

ARTIFACTS ON MOUNTED SECTIONS - not due to staining

A
  • WATER DROPLETS
  • WHITE SPOTS IN TISSUE SECTION – PATCHY STAINING
  • DRY MOUNTING: CORN-FLAKING OR TRAPPED AIR
  • RETRACTION
  • TISSUE NOT CRISP
  • BUBBLES FROM COVERSLIPPING
  • MOUNTANT ON THE COVERSLIP
  • SCRATCHES ON THE TISSUE/TISSUE WIPED OFF.
22
Q

What causes water droplets and how to correct

A

Cause
* Incomplete dehydration.
* Contaminated 100% alcohol in DCM.
* Contaminated xylene
*can look like color leaching

CORRECTIVE ACTION:
* remove coverslip and Soak slide in xylene to remove coverslip.
*xylene will dissolve permount
* Change contaminated reagents – take slides to fresh 100% ETOH and clear in fresh xylene 2x – dip slides until they are no longer streaky.
* Ensure there are no water droplets in xylene.

23
Q

WHITE SPOTS IN TISSUE SECTION
causes and corrective action

A

Causes
* Incomplete deparaffinization and dewaxation.
* Patchy staining due to presence of wax in the tissue prior to application of stains – irregular staining.
* Contaminated dewaxing xylene.
* Section not dried properly in the oven.

CORRECTIVE ACTION:
* Ensure slides stay longer in the dewaxing xylene.
* Avoid using dewaxing xylene contaminated with water.
* Remove stain, remove wax and re-stain.
* make sure slide is completely dry in the oven

24
Q

CORN FLAKING OR TRAPPED AIR - dry mounting artifact
causes and corrective action

A

causes
Too slow in applying the coverslip.
-trapped air in nuclei = cornflake artifact
-high refractile lines in tissue because its dried out BEFORE coverslipping
- ialso Xylene has evaporated from tissue prior to
coverslipping.

CORRECTIVE ACTION:
* Soak slide in xylene until the coverslip comes off.
* Re-coverslip making sure xylene is not dried out

25
Q

RETRACTION
causes and corrective action

A

causes
-Mounting medium was too thin (too much xylene was added to thin it out) can also cause bubbles under the coverslip

corrective action
-Soak slide in xylene until the coverslip comes off.
-Re-coverslip with a mounting medium that has the correct consistency.

26
Q

TISSUE SECTION NOT AS CRISP AS USUAL
causes and ca

A

CAUSES:
* Mounting medium too thick.

CORRECTIVE ACTION:
* Re-coverslip with correct coverslipping technique and correct mountant medium consistency

27
Q

BUBBLES FROM COVERSLIPPING
causes and ca

A

causes
* Incorrect angle when bringing slide close to
coverslip.
* Coverslipping too fast

CORRECTIVE ACTION:
* Soak slide in xylene until the coverslip comes off.
* Re-coverslip and lower slide gently over the coverslip on a 45° angle.

-the tissue can be seen as dark and grainy

28
Q

EXCESSIVE MOUNTING MEDIA causes and corrective action

A

-foggy

causes
* Too much mounting medium applied. one drop is good
-make sure its not too thick with clean and dry gloves
* Incorrect handling of coverslip - only the edges
* Mountant medium on gloves.

corrective action
* Soak slide in xylene until the coverslip comes off.
* Re-coverslip making sure you dont smudge the new coverslip.

29
Q

SCRATCHES ON TISSUE/WIPE OFF
causes and corrective action

A

causes
-improper handling of tissue during staining
-tissue was too close and touched other materials causing it to be wiped off during coverslip or staining. if it got close to staining racks

CA
-handle tissues with care
-put tissue in center of slide when mounting onto slide during microtomy
-cant troubleshoot -tissue needs to be recut and restained

histotechnology (13 decks)

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