1
Q
Steps in Tissue processing
A
Fixation
Dehydration
Clearing
Infiltration
Embedding
Trimming
Sectioning
Staining
Mounting
Labelling
2
Q
first and most important step
A
fixation
3
Q
two purpose of fixation
A
- Preserve the morphological and chemical integrity of cell
- Harden and protect tissue for further handling
4
Q
primary purpose of fixation
A
preserve morphological and chemical integrity
5
Q
tissues must be fixed _____
A
within 1hr to prevent putrefaction and autolysis
6
Q
penetration rate of formalin
A
1mm/hr
7
Q
ratio of fixative to tissue (routine)
A
20:1
8
Q
ratio of fixative to tissue (Oste)
A
5-10 x
9
Q
ratio of fixative to tissue (museum preparations)
A
≥50 x
10
Q
if not fixed ASAP, autopsy materials must be:
A
- placed in mortuary ref at 4C
- undergo arterial embalming
11
Q
if not fixed ASAP, surgical specimens must be:
A
refrigerated, but do not freeze
12
Q
must be fixed before grossing (suspended whole in 10% NBF for 2-3 weeks)
A
brain
13
Q
tissues tend to float (put in cotton or open completely before fixation)
A
hollow organs (stomach, intestines)
14
Q
tissues tend to float (cover with gauze)
A
air-filled lungs
15
Q
fixed (injection with formol alcohol) before grossing, then immerse in fixative
A
eyes
16
Q
water must not be used in tissues with _____ , because it is water-soluble
A
glycogen
17
Q
must be washed with water overnight, then immerse in tissue softeners
A
hard tissues
18
Q
example of hard tissues:
A
cervix
fibroids
hyperkeratotic skin
fingernails
19
Q
factors involved in fixation:
pH
A
6 - 8
20
Q
factors involved in fixation:
temperature
*routine: _____
*autotechnicon: _____
*EM and histochem: _____
*rapid fixation: _____
*tissues with TB: _____
A
room temperature
40C
0-4C
60C
100C
21
Q
factors involved in fixation:
size of tissue
*routine (LM): _____
*edematous lungs: _____
*EM: _____
A
2cm^2 (<5mm)
1 - 2 cm thick
1 - 2 mm^2
22
Q
factors involved in fixation:
osmolality
A
slightly hypertonic
(400-450 mOsm)
23
Q
factors that accelerates/hastens fixation
A
- smaller tissues
- heat (37-56C)
- agitation
- vacuum
24
Q
factors that retards/slows down fixation
A
- larger tissues
- cold temperature
- mucus & blood (flush w/NSS)
- fatty tissues (slice thinly)
25
mechanism of fixative that becomes part of the tissue; form cross-links/complexes; example is aldehyde fixatives
additive fixatives
26
mechanism of fixative that does not become part of the tissue; removes bound water; example is alcohol fixatives
non-additive fixatives
27
composition of fixative where there is only 1 fixative
simple fixative
28
composition of fixative where there are 2 or more fixatives
compound fixative
29
action of fixative that permits the microscopic study of tissue without structural pattern and intercellular relationship alteration
microanatomical
30
action of fixative that preserves specific cell parts/elements at the expense of other cellular components
cytological
31
categories of cytological fixatives:
nuclear
cytoplasmic
histochemical
32
preserves nucleus and chromatin material
nuclear fixative
33
preserves cytoplasm and membrane-bound organelles
cytoplasmic fixative
34
preserves chemical components
histochemical fixative
35
example of nuclear fixative:
Heidenhain's
Newcomer's
Bouin's
Flemming's with acetic acid
Carnoy's
36
example of cytoplasmic fixative:
Helly's
Orth's
Regaud's
Flemmings w/o acetic acid
Formalin w/ post chroming
37
example of histochemical fixative:
Absolute ethanol
Newcomer's
Acetone
10% Formol saline
38
example of microanatomical fixative:
Heidenhain's
Brasil's
Bouin's
10% Formol saline
10% NBF
Zenker's
Formol sublimate
39
acid fixative fixes _____
basic fixative fixes ______
acid stain stains _____
basic stain stains _____
nucleus
cytoplasm
cytoplasm
nucleus
40
List of fixatives:
1. aldehyde
2. metallic
3. alcoholic
4. picric acid
5. glacial acetic acid
6. osmium tetroxide
7. trichloroacetic acid
8. acetone
41
aldehyde fixatives:
1. formalin/formaldehyde
2. 10% NBF
3. 10% Formol saline
4. formol corrosive
5. formol calcium
6. Gendre's (alcoholic formalin)
7. glutaraldehyde
8. 4% paraformaldehyde
9. acrolein
10. glyoxal
42
most commonly used fixative
10% formalin
43
problems using formalin:
1. decomposition to formic acid
2. prolonged storage produces white precipitate
3. brown/black crystalline precipitates on blood-containing tissues
4. fumes are irritating to the eyes, nose, and skin
44
routine and best general tissue fixative
10% NBF
45
best fixative for iron-containing pigments and elastic fibers
10% NBF
46
CNS and postmortem fixative
10% formol saline
47
fixative ideal for silver impregnation
10% formol saline
48
_____ concentration of formol saline for enzyme histochemistry
4%
49
composed of formalin + mercuric chloride
formol corrosive
50
ideal fixative for silver reticulin methods
formol corrosive
51
fixative for lipids in frozen sections
formol calcium
52
formalin + 95% ethanol + picric acid + glacial acetic acid
Gendre's (alcoholic formalin)
53
a sputum fixative, but is also good for glycogen and microincineration studies
Gendre's
54
primary fixative for transmission electron microscopy
Glutaraldehyde
55
a formalin polymer in white powder form; also for EM
4% paraformaldehyde
56
mixture of glutaraldehyde and paraformaldehyde
acrolein
57
smallest aldehyde
glyoxal
58
metallic fixatives:
1. mercuric chloride
2. chromate
3. lead
59
fixative of choice for cell detail preservation in tissue photography
mercuric chloride
60
fixative recommended for renal biopsies
mercuric chloride
61
may produce black deposits except Heidenhain's susa
mercuric chloride
62
to remove black deposits, do _____
dezenkerization
63
mercuric chloride _____ tissues
glacial acetic acid _____ tissues
shrinks
swells
64
examples of mercuric chloride fixative:
1. B5
2. Ohlmacher's
3. Schauddin's
4. Carnoy-Lebrun's
5. Heidenhain's SuSa
6. Zenker
7. Zenker-formol (Helly's)
65
alternative for mercuric chloride
zinc sulfate
66
bone marrow fixative w/ anhydrous sodium acetate
B5
67
rapid fixative that gives excellent nuclear differentiation
Ohlmacher's
Carnoy-Lebrun's
68
fixative for stool
Schaudinn's
69
fixative for skin biopsies
Heidenhain's SuSa
Su= sublimat
Sa= saure
70
fixative for trichrome staining and bone marrow (recommended)
zenker
71
fixative for pituitary glands, intercalated disks, and BM w/ potassium dichromate
zenker-formol (Helly's)
72
fixative that has a fine, yellow-brown pigment (removed w/ acid alcohol)
chromate
73
example of chromate fixatives:
1. chromic acid
2. Regaud's (Moller's)
3. Orth's
4. Potassium dichromate
74
fixative for carbohydrates
chromic acid
75
fixative for chromatin, mitotic figures and mitochondria
Regaud's (Moller's)
76
it demonstrates rickettsia, tissue necrosis and early degenerative processes
Orth's
77
fixative mainly for mitochondria; if acidified, it destroys mitochondria but fixes cytoplasm, chromatin and chromosomes
potassium dichromate
78
fixes mucin and mucopolysaccharides
lead
79
acts as fixative and dehydrating agents
alcoholic fixatives
acetone
80
excellent preservative for glycogen but rapidly denatures proteins
alcoholic fixatives
81
preserves nuclear stans but dissolves fats
alcoholic fixatives
82
example of alcoholic fixatives:
1. methanol
2. ethanol
3. isopropyl alcohol
4. carnoy's fluid
5. newcomer's
83
fixative for blood and BM smears
methanol
84
preserves but does not fix glycogen; useful for PCR
ethanol
85
fixative for touch preparations and Wright-Giemsa stain
isopropyl alcohol
86
most rapid fixative
carnoy's fluid
87
for fixing chromosomes and urgent biopsies
carnoy's fluid
88
fixative for mucopolysaccharides and nuclear proteins
newcomer's
89
highly explosive fixative when dry
picric acid
90
an excellent glycogen fixative
picric acid
91
small tissue fragments can be seen
picric acid
92
major disadvantage: excessive yellow staining
picric acid
93
example of picric acid fixatives:
1. bouin's
2. brasil's
3. hollande's
94
fixative for embryos, pituitary and Masson's trichrome stain; not for kidney biopsies
bouin's
95
less messy than bouin's
brasil's
96
less lysis than bouin's, with decalcifying properties
hollande's
97
fixative for GI tract and endocrine tissues
hollande's
98
most commonly combined with other fixatives; solidifies at 17C
glacial acetic acid
99
fixes and precipitates nucleoproteins, chromosomes, and chromatin
glacial acetic acid
100
pale yellow powder that dissolves in water
osmium tetroxide
101
excellent lipid fixative
osmium tetroxide
102
fixes myelin and peripheral nerves
osmium tetroxide
103
secondary fixative for EM
osmium tetroxide
104
has a black precipitate crystal pigment (remove with cold water)
osmium tetroxide
105
fixative that does not use hematoxylin, because it inhibits hematoxylin
osmium tetroxide
106
most common chrome-osmium acetic acid fixative for nuclear structures
Flemming's with acetic acid
107
OsTe fixative for cytoplasmic structures, especially mitochondria
flemming's w/o acetic acid
108
fixative and weak decalcifying agent
trichloroacetic acid
109
precipitates protein with a swelling effect that counteracts shrinkage of other fixatives
trichloroacetic acid
110
fixative that has a softening effect on dense tissues
trichloroacetic acid
111
fixative used at ice-cold temperatures (-5 to 4C)
acetone
112
fixative for h2o-diffusable enzymes (phosphatases and lipases) and rabies diagnosis
acetone
113
fixation for bacteriologic smears and frozen sections
heat fixation
114
mechanism of heat fixation
thermal coagulation of tissue proteins
115
technique that can penetrate tissues with 10-15 um thickness with an optimum temperature of 45-55C; it increases movement of molecules and accelerates tissue processing stages
microwave technique
116
a rapid microwave fixative that has a mixture of methanol and polyethylene glycol (PEG); it can recover DNA, RNA and proteins for molecular analysis
Universal Molecular Fixative (UMFIX)
117
fixative for enzyme histochemistry:
1. 4% formalin (formol saline)
2. acetone (cryostat sections)
118
fixative for electron microscopy:
1. glutaraldehyde
2. osmium tetroxide
3. paraformaldehyde
4. zamboni's (immunohistochem)
119
fixative for electron immunocytochemistry:
1. karnovsky's paraformaldehyde-glutaraldehyde
2. acrolein
120
an already fixed tissue is placed into another fixative
secondary fixation
121
an already fixed tissue is fixed using 2.5-3% potassium dichromate (mordant) for one day
post-chromatization (post-mordanting)
122
to remove excess fixative, wash out with:
1. _____: formalin, chromates. osmic acid
2. _____: picric acid (bouin's)
3. _____: mercuric chloride fixatives
tap water
50-70% alcohol
alcoholic iodine
123
transport medium for unfixed tissues (renal, skin, oral mucosa); not a fixative; rather, refrigerated
Michel's solution
124
done after fixation and before dehydration (optional)
decalcification
125
it is done to remove calcium or lime salts from bones or calcified tissue
decalcification
126
ratio of decalcifier to tissue
20:1
127
optimum temperature in decalcification:
*routine
*impairs Van Gieson nuclear staining
tissue digestion
room temperature (18-30C)
37C
55C
128
duration of decalcification
1-2 days
129
types of decalcifying agents:
1. acids
2. chelating agents
3. ion exchange resin
4. electrophoresis
130
example of acid decalcifying agents:
1. 5-10% nitric acid
2. HCl
3. 5% formic acid
4. TCA
5. sulfurous acid
6. chromic acid (flemming's)
131
most common decalcifying agent
5-10% nitric acid
132
type of nitric acid that contains formalin
formol nitric acid
133
type of nitric acid that is also a tissue softener
perenyi's fluid
134
considered as the most rapid decalcifying agent
phloroglucin nitric acid
135
disadvantage of nitric acid
imparts a yellow color due to nitrous acid
136
remedy for yellow color of nitric acid
5% sodium thiosulfate or urea crystals
137
for slower decalcification of block; slower and produces more distortion to tissues
HCl
138
contains 36% HCl or 36% NaCl; used for teeth and small bone pieces
Von Ebner's fluid
139
a fixative and moderate acting decalcifier
5% formic acid
140
best general decalcifying agent, can also be used for teeth and small bone pieces
5% formic acid
141
decalcifier of post-mortem research tissues and suitable for immunohistochemical staining
5% formic acid
142
has a better nuclear staining than nitric acid; for BM and cartilage
formic acid-sodium citrate solution
143
a fixative and weak decalcifying agent which permits good nuclear staining but slow; only for small bone spicules
TCA
144
weak decalcifying agent; only for minute bone pieces
sulfurous acid
145
also for minute bone pieces; carcinogenic
chromic acid (Flemming's)
146
it is combined with calcium and other salts to form complexes pH is adjusted to 7-7.4
chelating agents
147
excellent for IHC and EM, but inactivates alkaline phosphatase (remedy: magnesium chloride)
chelating agents
148
ammonium form of polystyrene
ion exchange resins
149
it uses formic acid-containing decalcifying solutions to increase solubility
ion exchange resins
150
complete decalcification can be measured using physical or x-ray method
ion exchange resins
151
it is not recommended for fluids that contain mineral acids (nitric acid, HCl)
ion exchange resins
152
attraction of positively charged calcium ions
electrophoresis
153
makes use of 88% formic acid; requires shorter time to remove calcium and is suitable for small bone fragments
electrophoresis
154
tests to measure extent of decalcification:
1. physical/mechanical
2. x-ray/radiologic
3. chemical/calcium oxalate
4. bubble test
155
bending of tissue or probing using a needle; prone to produce artifacts and destroys cellular details
physical/mechanical
156
most reliable, ideal, and sensitive, but it is very expensive; not for mercuric chloride fixatives due to radiopacity
x-ray/radiologic
157
by calcium hydroxide/calcium oxalate precipitation;
(+) precipitation = incomplete decalcification
chemical/calcium oxalate
158
uses calcium carbonate;
(+) bubbles = incomplete decalcification
bubble test
159
tissue softeners:
for hard tissues to facilitate cutting and processing
1. lendrum's (4% phenol)
2. perenyi's fluid
3. molliflex (tissue may appear swollen and soapy)
4. 2% HCl
5. 1% HCl in 70% alcohol
160
post decalcification:
to remove/neutralize acid from tissues
1. lithium carbonate
2. 5-10% sodium carbonate
3. rinsing in running tap water
4. formol saline with 15% sucrose
5. phosphate buffered saline with 15-20% sucrose
161
to remove fixative and water; done through ascending grades of alcohol
dehydration
162
ratio of dehydrating agent to tissue
10:1
163
initial concentration of alcohol for routine tissues
70%
164
initial concentration of alcohol for delicate tissues
30%
165
types of dehydrating agents:
1. alcohols
2. acetone
3. dioxane
4. cellosolve
5. tri-ethyl phosphate
6. tetrahydrofuran
166
other dehydrating agents for EM
1. ethanol
2. propylene oxide
3. acetonitrile
167
routinely used for dehydration
alcohols
168
<70% alcohol may _____ tissues
macerate
169
dehydration hastens at _____
37C
170
it ensures complete dehydration; it removes water from dehydrating agent;
(+) blue = presence of water
anhydrous copper sulfate
171
most common (routine) and best dehydrating agent; fast-acting, penetrates tissues easily, and not toxic
ethanol
172
for blood and tissue films, and blood smears; can lead to blindness and death
methanol
173
for plant and animal microtechniques; slow-acting, but less shrinkage
butanol
174
a universal solvent (dehydrating and clearing agent)
tertiary butanol
dioxane
tetrahydrofuran
175
alcohol that dissolves paraffin
pentanol
176
excellent ethanol substitute; best clearing agent for microwave techniques
isopropanol
177
mixture of ethanol + methanol
denatured alcohol
178
highly miscible but flammable
acetone
179
fast-acting; for most urgent biopsies; it can cause considerable shrinkage and brittleness; lipids are removed
acetone
180
diethylene oxide
dioxane
181
miscible with paraffin, alcohol, xylene; produces less shrinkage and tissues can be stored for longer period; expensive, tissues tend to ribbon poorly and highly toxic
dioxane
182
uses pure dioxane and paraffin
Graupner's
183
uses dioxane and anhydrous calcium oxide/quicklime
Weiseberger's
184
ethylene glycol monoethyl ether
cellosolve
185
rapid, and tissues can be stored for longer periods w/o distortion; combustible at 110-120F, toxic, and decompose on sunlight
cellosolve
186
for dehydrating sections and smears; removes water very easily, and produces minimum shrinkage and distortion
tri-ethyl phosphate
187
dissolves many substances, including fats; can cause conjunctival irritation and odorous
THF
188
transition fluid; for resin infiltration
propylene oxide
189
propylene oxide substitute
acetonitrile
190
to remove the dehydrating agent; "dealcoholization"
clearing
191
miscible with dehydrating agent and embedding medium; makes the tissues translucent/transparent; flammable liquids with low boiling point
clearing agent
192
types of common clearing agents:
1. xylene (xylol)
2. toluene (toluol)
3. benzene
4. chloroform
5. cedarwood oil
193
other clearing agents:
1. aniline oil
2. clove oil
3. carbon tetrachloride
4. oils of bergamot, origanum, wintergreen
5. carbon disulfide & phenol
6. terpineol
7. limonene
8. high test aviation lead free gasoline
194
clearing agents for double-embedding:
1. methyl benzoate
2. methyl salicylate
3. amyl acetate
195
routine and most rapid clearing agent
xylene
196
Xylene
*clearing time: _____
*refractive index: _____
*for tissue sections with _____
*for _____ sections, but not for _____ and _____ tissues
*turns _____ when tissues are incompletely dehydrated
15-20 mins to 1 hour
1.48 to 1.50
<5mm thick
celloidin; nervous and lymphatic
milky
197
xylene and benzene substitute
toluene
198
tissues do not become excessively hard or brittle even if left for 1 day; not carcinogenic, but toxic upon prolonged exposure
toluene
199
can cause aplastic anemia
benzene
200
for urgent biopsies (15-60 mins); causes minimum shrinkage to tissues
benzene
201
clearing agent for tough tissues
chloroform
202
slower than xylene and hepatotoxic, but causes less brittleness
chloroform
203
for tissue blocks up to 1 cm, and can be used for vacuum embedding
chloroform
204
tissues do not become translucent and tend to float
chloroform
205
clearing agent for CNS, smooth muscles, and skin
cedarwood oil
206
it requires two changes to properly be used, and difficult to remove
cedarwood oil
207
no tissue distortion, but very slow; turns milky upon prolonged storage
cedarwood oil
208
clearing agent for delicate tissues, embryos and insects
aniline oil
209
tissues tend to become aduterated
clove oil
210
everything similar to chloroform, but cheaper
carbon tetrachloride
211
oils of _____ (skin), _____ (skin and smooth muscle, _____
bergamot, origanum, wintergreen
212
clearing agent for smooth muscles
carbon disulfide and phenol
213
clearing agent for eyes
terpineol
214
obtained from citrus fruits and with a faint pleasant odor
limonene
215
it replaces the clearing agent with an infiltrating medium to fill all cavities
infiltration (impregnation)
216
ratio of infiltrating medium to tissue
25:1
217
types of infiltrating media:
1. paraffin
2. celloidin
3. gelatin
4. plastic (resin)
218
simplest, most common and best infiltrating medium
paraffin
219
introduced by Butschlii; not recommended for fatty tissues
paraffin
220
duration of paraffin infiltration
<24 hours
221
melting point of paraffin
56 C
222
low MP: _____
high MP: _____
paraffin is soft
paraffin is hard
223
overheated paraffin temperature: tissues become brittle, with shrinkage and hardening
60 C
224
filtered in wax oven using Green's 904/coarse filter
fresh wax
225
used twice only; water removed by heating wax at 100-105 C
reused wax
226
for extra hardness, paraffin can be mixed with _____
10-20% beeswax or ceresin
227
methods of paraffin wax impregnation:
1. manual
2. automatic
3. vacuum
228
requires 4 changes of wax at 15-minute intervals; temperature: 2-5 C above MP
manual
229
use of automatic tissue processor; with 2 thermostatically controlled wax baths
automatic
230
wax bath thermostat
3 C above MP
231
done inside an embedding oven (2-4 C above MP)
vacuum
232
impregnation under negative atmospheric pressure; fastest method (time reduced from 25-75% of normal time) and hastens the removal of air bubbles
vacuum
233
paraffin wax substitutes:
1. paraplast
2. embeddol
3. ester wax
4. bioloid
5. tissue mat
6. water-soluble wax
234
paraffin + synthetic plastic polymer (DMSO)
MP: 56-57 C
paraplast
235
similar to paraplast, but MP: 56-58 C
embeddol
236
prior clearing is not required; insoluble to water, but soluble to 95% ethanol
ester wax
237
harder than paraffin (use sliding/sledge type microtome)
MP: 46-48 C
ester wax
238
semisynthetic embedding for eyes
bioloid
239
embedding medium made of rubber
tissue mat
240
MP is 38-42 C or 45-56 C
polyethylene glycols
241
most common example of water-soluble wax
carbowax
242
prior dehydration and clearing is required; used for enzyme histochemistry
water-soluble wax
243
hygroscopic (dissolves when fishing out)
water-soluble wax
244
purified form of nitrocellulose
celloidin
245
embedding medium used for specimen with large, hollow cavities; dissolve in equal parts of ether and alcohol; slow but does not require heat during processing
celloidin
246
methods of celloidin impregnation:
1. wet method
2. dry method
3. nitrocellulose
247
celloidin technique that uses equal parts of ether and 70% alcohol; for bones, brain, teeth
wet method
248
celloidin technique that uses Gilson's mixture (chloroform & cedarwood oil); for eyes
dry method
249
preferred since it produces harder blocks & thinner sections; soluble in equal parts of ether and alcohol
low viscosity nitrocellulose
250
to prevent crackling of LVN, add _____
plasticizer (castor oil)
251
embedding medium that is rarely used; for frozen sections, histochemical and enzyme studies
gelatin
252
embedding medium that is water soluble (prior dehydration and clearing is not required); tissues should be <2-3 mm thick
gelatin
253
to prevent molds, add _____
1% phenol
254
aka. resin; for EM (80nm thick), high resolution light microscopy (renal biopsies and hematopoietic tissues) and extremely hard tissues
plastic
255
plastic classifications according to chemical composition:
1. epoxy
2. polyester
3. acrylic
256
resin with widest application; for LM and choice for TEM; has the ability to produce sections with thickness of 30-40 nm
epoxy
257
epoxy resins:
1. bisphenol A
2. glycerol
3. cyclohexane dioxide (spurr)
258
most stable epoxy resin
bisphenol A
259
fastest epoxy resin, but highly toxic
cyclohexane dioxide
260
resin that is seldom used; for EM
polyester
261
resin preferred for high resolution LM
acrylic
262
acrylic for uncalcified bones
acrylates (methyl methacrylates)
263
acrylic for TEM
methacrylates (polyglycol/glycol methacrylates)
264
it is done after wax impregnation
embedding (casting/blocking)
265
orientation: surface of section should be _____ to the bottom of mold
parallel
266
temperature in embedding
5-10 (6-10) C above MP of wax
267
molds used:
1. leukhart's mold
2. compound unit
3. plastic rings and base molds
4. disposable molds
5. watch glass
6. TIMS
268
2 L-shaped strops of heavy brass or metal; size of mold is adjustable
leukhart's mold
269
made of interlocking grids or compartments
compound unit
270
stainless steel base mold fitted with a plastic embedding ring
plastic rings and base molds
271
disposable molds:
1. peel away - even block w/o trimming
2. ice tray - from ordinary refrigerators
3. paper boat - cheap and easy to make
272
molds used for fragmentary biopsies
watch glass
273
1st infiltration w/ celloidin, 2nd infiltration w/ paraffin
double embedding
274
done hand in hand w/ embedding if individual mode is used due to solidification that produces blocks
blocking
275
process of removing excess wax to allow easy sectioning
trimming
276
formation after trimming
truncated pyramid
277
at least _____ of wax should surround the block
2mm
278
process where cold, embedded tissue is cut into uniformly thin pieces via microtome
sectioning
279
microtome is invented by
Wilhelm His Sr.
280
it is used for unfixed, unfrozen tissue and uses a vibrating blade
vibratome
281
essential parts of microtome
1. block holder (chuck)
2. knife carrier with knife
3. pawl, ratched feed wheel, and adjustment screws
282
types of microtome:
1. rocking/cambridge
2. rotary
3. sliding
4. freezing
5. ultrathin
283
simplest microtome used for large paraffin tissues, but not serial sections
rocking/cambridge
284
invented by Trefall
rocking/cambridge
285
routinely used microtome for paraffin and serial sections
rotary
286
invented by Minot
rotary
287
microtome used for celloidin and hard, rough tissue blocks
sliding
288
invented by Adams, further developed by Alexander Cummings
sliding
289
preferred type of sliding microtome; knife is held rigidly in clamps
base sledge
290
dangerous type of sliding microtome; knife is moving
standard
291
microtome for frozen section; with a propellant (CO2)
freezing
292
invented by Queckett
freezing
293
also known as cold microtome
cryostat
294
temperature of cryostat
-5 to 30 C (ave 20 C)
295
microtome used for electron microscopy; tissues are 0.5 um thick embedded in resin
ultrathin
296
knife used in ultrathin microtome
diamond knife
297
types of microtome knives:
1. plane concave
2. biconcave
3. plane wedge
4. disposable knives
5. glass (diamond knives)
298
knife used for rocking and rotary microtome
plane concave
299
plane concave knife size
25 mm
300
less concave side is for
celloidin
301
more concave side is for
paraffin
302
knife used for rotary microtome, paraffin sections; both sides are concave
biconcave
303
biconcave knife size
120 mm
304
knife used for sliding microtome, and for very hard tissues and frozen sections
plane wedge
305
plane wedge knife size
100 mm
306
knife coated with polytetrafluoroethylene for easy ribboning
disposable knives
307
has an angle of 27-32; cutting facet; on the tapered edge of knife
bevel angle
308
has an angle of 15; with maximum tissue penetration and less distortion
cutting angle
309
has an angle of 0-15; between the block surface and cutting edge of knife
clearance angle
310
angle between upper surface of facet and surface of block
rake angle
311
angle between sides of knife
wedge angle
312
done for sharpening and removing nicks; from heel to toe by 10-20 strokes
honing
313
types of hones:
1. belgian yellow (best result)
2. arkansas
3. fine carborundum (badly nicked)
4. plate glass (stone substitute)
314
done for polishing and removing burrs; from toe to heel by 40-120 double strokes
stropping
315
it is made of horse leather
(3-4 x 18 inch); should be oiled in the back (do not use mineral oil)
paddle strops
316
types of sections produced:
*paraffin
*celloidin
*renal biopsy
*ultrathin
*frozen
4-6 um
10-15 um
2 um (plastic embedding medium)
80 nm (silver/straw colored)
4 um (rotary microtome)
317
done to flatten tissues after sectioning
water bath
318
done to obtain sections from water bath to the slide
fishing out
319
duration of tissues in water bath
30 secs to 1-2 mins
320
drying the slide can be done by:
* drain at an angle of 60-85 for 2-5 mins
*hot plate at 45-55C for 30-45 mins
* overnight incubator: best for nervous tissues
*wax oven at 56-60C for 20-30 mins
*urgent method: above a bunsen burner until the wax melt
321
adhesives
1. Mayer's Egg Albumin
2. 1% gelatin
3. cellulose
4. poly L-lysine
5. sodium silicate
6. resins
322
most commonly used adhesive; not recommended for exfoliative cytology
Mayer's egg albumin
323
components of mayer's egg albumin
1. egg white
2. glycerol: increases viscosity and prevent drying
3. thymol: prevent mold growth
324
with firmer attachment than albumin; gently heated before use to melt; disadvantage: stains with many dyes
1% gelatin
325
general purpose adhesive for immunohistochemistry
poly L-lysine
326
a commercial syrup with 1:10 dilution; not affected by mild alkaline solutions
sodium silicate
327
adhesive disadvantages to staining:
1. blackening in silver impregnation and reticulin
2. reddening in methyl green-pyronin
sodium silicate
328
with great adhesion
resins
329
made of epoxy resins diluted with 1:10 acetone
araldite
330
MTLE
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