MODULE 4 - Sectioning Flashcards
1
Q
Involves cutting of tissues into thin slices
A
Sectioning
2
Q
Size of paraffin sections
A
4-6 um
3
Q
Size of celloidin sections
A
10-15 um
4
Q
Size of frozen sections
A
4 um
5
Q
Disadvantage of using cambridge/rocking microtome
A
Difficult in re-orienting tissues and restriction in tissue size of blocks
6
Q
Cambridge/rocking microtome is made by
A
Trekfall
7
Q
Most simple microtome
A
Cambridge/Rocking
8
Q
Cambridge/Rocking microtome is made for
A
serial sections of large paraffin blocks
9
Q
Thickness produced when using cambridge/rocking microtome?
A
10-12 micra
10
Q
Routinely and most common microtome
A
Rotary
11
Q
Rotary microtome is made by
A
Minot
12
Q
Thickness produced when using rotary microtome
A
4-6 micra
13
Q
Rotary microtome is used for
A
paraffin embedded tissues
14
Q
Sliding microtome is made by
A
Adams
15
Q
The most dangerous microtome and for what reason
A
Sliding microtome because of exposed knife
16
Q
Example of wax used in sliding microtome
A
Ester wax
17
Q
Sliding microtome is used for
A
celloidin embedded tissues and extremely hard tissues
18
Q
Thickness produced when using sliding microtome
A
7-9 micra
19
Q
2 types of sliding microtome
A
- Base sledge
2. Standard sliding
20
Q
Most dangerous type of sliding microtome
A
Standard microtome
21
Q
Freezing/cold knife uses
A
intermittent burst of CO2 to immediately harden block holder and tissues without prior fixations.
22
Q
Freezing/cold knife has a
A
second cooling device to lower temp of knife
23
Q
Freezing/cold knife is used for
A
frozen sections and heat-sensitive structures
24
Q
Freezing/cold knife is used for demonstration of
A
fats and neurological structures
25
Freezing/cold microtome was invented by
Queckette
26
Dew line is
point of sections in which it can be cut at 10um
27
thickness and tissue block produced using freezing/cold knife microtome
thickness: 10-15 um
block: 3-5 mm
28
Type of microtome enclosed in a chamber and is primarily composed of rotary microtome
Cryostat/cold microtome
29
Cryostat/cold microtome is used for
fresh frozen tissues for fluorescent antibody staining and histochemical enzyme studies
30
Temperature used in cryostat/cold microtome
-5 to -30 degC
| average -20 degC
31
Microtome used when tissues are usually embedded in plastic
ultrathin microtome
32
ultrathin microtome is used for
EM
33
thickness of tissues when using ultrathin microtome
0.5 um
34
process of removing excess paraffin wax from tissues once properly
fixed on the slide
DEPARAFFINIZATION
35
METHODS of DEPARAFFINIZATION
1. Passing of slide over alcohol lamp
2. Immersing slide in xylene
3. Putting it on oven (55-60 degC)
36
Microtome knives that is 25mm long
Plane concave
37
Microtome knives that is 120mm long
Biconcave
38
Microtome knives that is 100mm long
Plane wedge
39
Plane concave flat side is used for?
| concave side is for?
Flat- celloidin
| Concave- paraffin
40
Biconcave is used for
paraffin embedded and rotary
41
Plane wedge is for
frozen sections and extremely hard tissues
42
Bevel angle is
angle formed between the cutting edges 27-32 deg
43
Clearance angle is also known as
Tilting/inclination angle
44
Clearance angle is
angle formed between surface of blocks and cutting edge of knife @0-15 deg but in fixation it is 5-10 deg to avoid uneven sections (alternate thin and thick)
45
In Clearance angle >10 indicates and <10 indicates
>10 - low thickness
| <10 - high thickness
46
Wedge angle is
angle formed in sides of wedge knife 14-15 deg
47
Thickness produced by disposable blades
2-4 um
48
Glass and diamond knives are used for
EM
49
Purpose of honing
to remove nicks
50
Honing uses what movement
heel to toe movement, edge first 20-30 times
51
Honing uses
hones/oil stones
| lubricant
52
Oil stones used in honing
Belgium yellow
Arkansas
Fine carborundum
53
Lubricants used in honing
```
soapy water
mineral oil
clove oil
xylene
liquid paraffin
```
54
Belgium yellow
gives best result
55
Arkansas
gives more polishing effect than belgium yellow
56
fine carborundum
for badly nicked knives, has coarser bite
57
Alternatives for oil stones
Glassplates and diamantine
58
What composes glass plates
powdered aluminum oxide with water = paste = ABRASIVE
59
Diamantine is
for final polishing
60
Polishing and sharpening of knife
stropping
61
purpose of stropping
to remove burrss and other deformities formed by honing
62
stropping is done with what movement and how many strokes
toe to heel, edge last 40-120 double strokes
63
Strapping uses _____ made of _______
paddle strop, horse leather
64
Strops are treated with
oil
65
In stropping do not use ______ because it can ________
lubricant, mineral oil - blister and damage leather
66
Purpose of floatation water bath
thermostatically controlled bath to remove folds and wrinkles to flatten ribbons
67
Temperature of floatation water bath when in use
6-10 degC lower than wax melting point
| specifically, 45-50 degC
68
Product of section
thin slices , ribbons
69
what is fishing out
removal of ribbon in float out bath
70
3 methods for Drying of slides
1. leave in incubator @37 degC overnight
2. put in oven @50-60 degC for 2 hours
3. in a hot plate @45-55 degC for 30-45 mins
71
This is used to promote attachment to slides and prevent detachment
adhesives
72
Routine tissue adhesive
Mayer's egg albumin
73
Mayer's egg albumin is composed of
Egg white + glycerin + thymol crystals
74
Thymol crystals is used to
prevent growth of molds
75
Poly-L-Lysine is used for
immunohistochemistry
76
APES (3-aminopropylthriethoxysilane) is used for
cytology
77
With strong adhesive property
sodium silicate
78
Adhesive that is a commercial syrup
sodium silicate
79
Enumerate methods of freezing and their details
```
Liquid nitrogen- most rapid, for histochem
Isopentane- liquid at RT
Aerosol sprays (cryokwik)
CO2- freezing microtome
Freon 2.2- high thermal conductivity
```
80
Microtome parts and their details
1. Block holder/chuck- where tissues are held in position
2. Knife carrier/knife- disposable knives, for actual cutting
3. Rotating/flying wheel- done mechanically to start the cutting process
4. Pawl, ratched feed wheel, adjustment screw- lines up tissues in proper positions with knife
81
A microtome that is theoretically not recommended for serial sections since tissues are cut in slightly curved planes.
Cambridge/rocking microtome
82
Typically, sections are cut between ___________ µm using paraffin wax for diagnostic histology
3 and 5 um
83
It was originally designed for cutting sections of very large blocks (whole brain).
Sliding microtome
84
Microtomes used for serial sections (book)
Rotary
| Sliding- basic sledge
85
It is used to cut undehydrated thin to semi-thin sections of fresh, frozen
tissues
Freezing/cold knife
86
capable of freezing fresh tissues within 2-3 minutes, and cutting sections of 4 µ with ease.
The Cryostat or Cold Microtome
87
It is most commonly used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis.
The Cryostat or Cold Microtome
88
It is often housed in the frozen section room close to the operating room to allow direct consultation between surgeon and pathologist.
The Cryostat or Cold Microtome
89
microtome equipped with a glass or gem grade diamond knife is used to cut very thin sections (typically 60 to 100 nanometer) of tissue embedded in epoxy resin.
Ultrathin microtome
90
Thin sectioning for the TEM is often done with a
gem quality diamond knife
91
After sectioning, all the accumulated paraffin and small pieces of tissues
must be brushed away with a soft brush and not allowed to stay in the
microtome, since this may later interfere with the cutting of tissue blocks.
TRUE OR FALSE
True
92
After carefully drying the machine and knife holder, the parts should be
wiped with
xylol
93
Prolonged and continuous application of the painted parts with xylene will
remove the paint
94
Movable portions should not be oiled to prevent rusting
| TRUE OR FALSE
False
95
The microtome must always be covered when not in use
| TRUE OR FALSE
True
96
The microtome should be placed on an unstable bench, away from air drafts, doorways and passing staff.
TRUE OR FALSE
False
97
Any air movement from air conditioners or other causes will not affect section handling
TRUE OR FALSE
False
98
No fluid must enter the inside of the instrument during cleaning.
TRUE OR FALSE
True
99
Have the instrument inspected at least __________ by a qualified service technician.
once a year
100
Microtome that uses plane concave
rotary or rocking microtome
101
Each knife should have its own corresponding back which should be interchanged with another, to keep the bevel angle.
TRUE OR FALSE
False
102
Too soft cutting edges are likely to
dull easily
103
too hard edges are likely to produce
nicks or jagged edges and irregularities on the knife edge
104
A good cutting edge must be able to cut good sections from a paraffin wax block about
2-3 microns thick
105
may be used for partially calcified materials, paraffin and frozen sections.
Safety razor blades
106
Theoretically, the perfect and optimum cutting angle is obtained when the sides of the wedge knife are inclined at an angle of about
15 deg
107
The degree of sharpness is __________ to the fineness of the abrasive used in sharpening
proportional
108
Hones for manual sharpening when cutting edge has been rendered blunt or nicked
belgium yellow
109
The surface of the hone is wiped clean with a soft cloth moistened with ______ in order to remove the scattered small particles of stones and metal.
xylene
110
In the case of the Minot or plane-wedge knife, the knife is turned over so as to sharpen the other surface every
I0-20 strokes
111
Speed in stropping should be avoided
| True or False
True
112
Leather strops are usually dry and require oiling before they are used.
Strops are usually treated with
vegetable oil (castor)
113
The strop should not be used for at least ________ after treatment.
24-48 hrs
114
generally used for trimming and semi-thin sectioning of tissue blocks for electron microscopy.
Glass Knives
115
used to cut any type of resin block for electron microscopy.
diamond knives
116
used to cut any type of resin block for electron microscopy.
Diamond Knives
117
These tools are needed for handling sections during cutting, and for removing folds and creases on the sections during "floating out" in water bath.
Forceps (fine pointed or curved) and squirrel hair brush
118
The quality of sections cut on a microtome suffer badly from these several (avoidable) causes.
Fecal material and hair
119
Coarse facing is done on the microtome at approximately
30 microns at a time
120
Cooling both the tissue and the wax will give them a similar consistency, and make sectioning easier
TRUE or FALSE
true
121
In fine trimming, the knife is usually tilted at
0-15 deg
122
provides better support for the harder elements in a specimen allowing thinner sections to be obtained.
cold wax
123
Generally a fast, uniform cutting stroke produces the best results and the least compression.
TRUE or FALSE
False
124
Do not stop and restart during a cutting stroke as this will produce bands of different thickness across the section.
TRUE OR FALSE
True
125
Sections should not be left on the water bath for a long time (30 seconds will be enough)
to avoid undue expansion and distortion of tissue.
126
When flattening out sections, 20% alcohol will produce
convection current
127
expand the section to its original dimensions and ensure
| that it is completely flat.
floatation
128
not recommended because they can cause overheating and there is a risk of dust falling onto the section during the drying period.
hot plate
129
Excessive heat can cause
boiling of water droplets underneath the section which will cause damage
130
Dry sections for between
5 and 30 minutes
131
Staining of serial sections should never be attempted unless they are completely dried.
TRUE OR FALSE
True
132
charged slides or an adhesive must be used in
```
antigen retrieval (IHC)
enzyme pretreatment (ISH)
prolonged incubation steps
```
133
Extended storage (usually more than 3 days) of unstained formalin-fixed paraffin embedded slides will cause
loss of antigens
134
When cutting sections for DNA or RNA extraction, all instruments and equipment must be pre-cleaned and wiped down with
RNAse
135
type of water used for floating sections for RNA extraction.
molecular grade water
136
Hardening of the celloidin block may be hastened by placing
chloroform
137
To avoid dehydration and shrinkage in section cutting, what should be done?
lubricated with 60-70% ethanol
138
essential for methods that require exposure of sections to acids and alkalis (especially ammoniacal silver solutions) during staining
adhesives
139
One disadvantage of using albumin
retention of stain which causes dirty background
