Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

MLSCI 250 > 350 - Topic 6 (Tissue Processing & Embedding) > Flashcards

350 - Topic 6 (Tissue Processing & Embedding) Flashcards

1
Q

Four distinct stages in tissue processing:

A
  • fixation: fully fixed before further processing
  • dehydration: remove all unbound water and fixative from tissue
  • clearing: removes dehydrating agent and acts as solvent for paraffin wax; also raises refractive index of tissue closer to that of glass = high power magnification
  • infiltration: support medium; paraffin wax = allows cutting of thin sections
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

T or F. Dehydrating solutions are hydrophobic

A

F! hydrophilic! allows them to draw out water form the tissue and into the solution

solutions frequent exchanged with increasingly concentrated solutions which will dilute any remaining water

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

ethyl alc as dehydrating agent

A
  • routinely used
  • infinitely soluble with water
  • fast-acting
  • non-toxic
  • may cause hardening and shrinkage
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

isopropyl alc as dehydrating agent

A
  • miscible in water but IMMISCIBLE in salt solutions = tissue must be washed following fixation
  • isopropyl can be mixed w paraffin so clearing agent not used/required
  • not toxic
  • does not harden/shrink tissue
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

methyl alcohol as dehydrating agent

A
  • similar to ethanol (ethyl alc)
  • more hazardous/toxic
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What happens if a large concentration gradient exists between water and alcohol during dehydration?

A

cells may be distorted or damaged by flow of solutions
tissue should be subjected to 50-70% alc immediately after fixation, followed by increasingly concentrated solutions = 80%, 95%, then absolute alc

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

what do clearing agents have in common?

A

they are soluble in both dehydrants and paraffin

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

once tissue is dehydrated, tissue is filled with alcohol which does not dissolve in wax, so what does clearing agent do?

A

acts as a link between these steps = dissolves any remaining alcohol

leaves tissue receptive to non-polar substances = molten wax

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

T or F. Clearing agents have a high refractive index

A

T! 1.4-1.51

helps make tissue appear transparent (1.51 to 1.62)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

xylene

A

routinely used
- fast-acting
- miscible with most solvents as well as paraffin
- over-exposure will harden tissue
- flammable, moderately toxic

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

toulene

A
  • similar to xylene; unlikely to harden tissues
  • more volatile than xylene (more vapours)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

chloroform

A
  • used for CNS that may become brittle when cleared w xylene; esp. brain and eyes
  • non-flammable
  • highly toxic
  • in presence of oxygen = phosgene = exceptionally hazardous; poisonous gas
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Xylene substitute

A
  • short chain aliphatic hydrocarbons; like butane and petroleum jelly
  • less toxic and work almost as well as xylene
  • intolerant of water so final alcohol must be completely anhydrous
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

universal solvents

A

chemicals capable of dehydrating AND clearing tissue
two most commonly used = tetrahydrofuran (THF) and dioxane
neither recommended due to safety concerns and tendency to over-harden tissue

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

lower melting points =

A

softer wax

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

higher melting points =

A

harder waxes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

wax additives and their purpose:

A
  • plasticizers: used to make wax harder and facilitate ribboning
  • beeswax: lowers the melting point and makes section ‘sticky’
  • rubber: facilitates ribboning by increasing elasticity
  • resins: makes wax harder/increase the melting point
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

factors affecting tissue processing:

A

agitation
heat
vacuum/pressure
viscosity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

how does agitation affect tissue processing?

A
  • ensures solutions surrounding tissue do not become locally saturated
  • helps avoid ‘dead zones’ where solutions are not effectively exchanged during processing
  • most use magnetic stir bars; some blow air bubbles through solutions; other repeated drain and fill the retort
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

how does heat affect tissue processing?

A
  • heat will speed up; use w caution
    = sustained temps above 40C may alter microscopic appearance of delicate tissue
  • most recommend no heat at al during F,D,C steps for highest quality and only enough heat to keep paraffin wax molten
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

how does vacuum/pressure affect tissue processing?

A
  • pressure = force solutions into tissues
  • vacuum = open porous structures
  • vacuum effective aid during infiltration; lowering pressure in retort = clearing agent more easily replaced by hot wax
22
Q

how does viscosity affect tissue processing?

A
  • measure of how thick liquid is
  • water is standard for low-viscosity
  • alcohol and xylene = low-viscosity
  • wax = significantly higher
  • lower viscosity solvents work more quickly than higher = so timing in each station varied accordingly
23
Q

how does an enclosed tissue processor work?

A
  • tissue remains stationary in retort
  • reagents pumped in and out according to a programmed processing schedule
  • reduces tech exposure to reagents and allows use of vacuum/pressure to speed processing/infiltration
24
Q

microwave processors

A
  • speeds up processing and improve TAT
  • heat tissues from the inside = speeds up diffusion of solutions
  • processes in minutes
    -formalin and xyelene free = safer
  • EXPENSIVE
25
Q

xylene-free processing

A

xylene substitutes
- safer
- aliphatic hydrocarbons dont clear alcohols as quickly so duration of clearing steps increased (up to 50%)
- less tolerant of water contamination than xylene; anhydrous ethanol must be at least 97% pure to be effectively cleared

some labs = mixture of isopropanol and ethanol; no clearing agnet at all

  • fixation
  • ethanol dehydration (50%)
  • continued dehydration/clearing (80% ethanol/20% isopropanol)
  • isopropanol clearing (100% isopropanol)
  • high temp paraffin infiltration = first wax bath should be held at 85C to effectively eliminate isopropanol
26
Q

this is used to monitor alcohol quality

A

hydrometer

27
Q

clean or purge cycle

A

before another processing cycle can begin, paraffin must be removed from the retort and lines of instrument

flush lines w xylene (to dissolve wax) and then alcohol to dissolve xylene

alcohol remaining in lines is no issue; can mix w fixative at beginning of next run

xylene and alcohol used for cleaning are not used during processing and have non-rotating or dedicated positions

28
Q

most common cause of inadequate processing

A

incomplete dehydration
- microtomy = soft, mushy area at centre of tissue (wax failed to infiltrate) = hole on the finished slide ; staining = interfere with nuclear staining

overprocessed = esp small biopsies; brittle, and easily fragments when sectioning; microchatter is a frequent microscopic finding

29
Q

embedding purpose

A
  • support medium for microtomy; thin sections
  • provides elasticity to resist compression caused by microtomy
30
Q

hard wax

A
  • higher MPs
  • great support for harder tissues
  • better suited for cutting thin (2-3 um) sections
31
Q

soft waxes

A
  • lower MP
  • easier to ribbon = ideal for serial sectioning
32
Q

appropriate mold size

A

2-3 mm bigger than tissue on all sides to ensure good support

33
Q

the most critical step in the embedding process

A

orientation

34
Q

tissue with lumens

A

cut and embedded to show a cross-section of lumen

vas deferens
fallopian tube
appendix
arteries
ducts

35
Q

T or F. cut side down is what will be cut first

A

T

36
Q

tissues with layers

A

should be embedded on edge = all layers demonstrated on each slide

skin
intestine
gallbladder

37
Q

embedding tissue with soft layers

A

embed so that tissue is oriented in a way that softest layer will contact blade first = reducing compression

38
Q

embedding elongated tissue

A

best to offset tissue slightly (up to 30 degree angle from horizontal)
- important for tougher/harder tissues like bone and uterus to prevent compression-type artifacts

39
Q

T or F. Paraffin blocks are stored indefintely

A

T! wet specimens (in formalin) discarded after 8 weeks

40
Q

T or F. Paraffin blocks are stored indefinitely

A

T! wet specimens (in formalin) discarded after 8 weeks
blockss retained for at least 30 yrs

41
Q

T or F. Paraffin blocks are stored indefinitely

A

T! wet specimens (in formalin) discarded after 8 weeks
blocks retained for at least 30 yrs

42
Q

these are used to section undecalcified bone, teeth, or to cut thin sections (<2 um) for light microscopy

A

acrylic resins such as methyl methacrylate (MMA) or glycol methacrylate (GMA)

methacrylates more convenient as the formulation can be varied to make a harder or softer block

43
Q

T or F. acrylic resins not useful for electron microscopy

A

T! they are unstable in the electron beam

44
Q

epoxy resins

A

used for electron microscopy
also ideal for cutting semi-thin (0.5-1.0 um) ad ultra thin (~800 nm) sections

45
Q

3 rules to achieve high quality processing results

A
  • must b completely fixed prior to dehydration
  • tissues must be thin <5mm to allow adequate penetration
  • processing schedules must be tailored to sizes and types of tissue processed
46
Q

high volume labs = reagent schedule

A

processor always full
reagents changed on a set schedule

47
Q

lower volume labs = reagent schedule

A

reagents should be changed according to how much tissue has been processed

48
Q

potential causes of incomplete dehydration

A
  • condensation (heat used during processing)
  • absolute alcohol is not absolute
  • wrong processing schedule
  • processor fault (mechanical failure)
49
Q

potential causes of over-processing

A
  • excessive dehydration
  • processing biopsy samples using overnight/routine schedule
  • extended contact w paraffin wax
50
Q

crunchy tissue

A

over-processing

51
Q

variation in staining, periphery of specimen lighter than centre

A

zonal fixation

formaldehyde binds to end terminal AAs as does eosin

formalin fixation reduces eosin binding sites

BUT if incomplete fixation = alcohol finishes fixation so more eosin binding sites = darker eosin in centre

52
Q

xylene on tissue processor is xanthochromic and turbid

A

water contamination

turbid is the big thing! clear and yellow = ok!

replace absolute alcohol as well

MLSCI 250 (35 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions