QUICK REVIEW Flashcards
(632 cards)
1
Q
-microscopic study of normal tissues
A
Histology
2
Q
Gross /Microscopic Pathology:
A
Gross: macroscopic examination
Microscopic
3
Q
Anatomic Pathology:
A
Surgical
Autopsy
Exfoliative
4
Q
: gross examination of tissue histology obtained during surgery to determine “cause” of disease
A
Surgical
5
Q
: gross examination of tissue histology removed from a identification/interpretation/char dead body to determine
“cause” of death
A
Autopsy
6
Q
: microscopic study of desquamated epithelial cell surfaces.
A
Exfoliative
7
Q
- Diagnosis and monitoring of diseases through examinations of blood, body fluids, secretions, tissue biopsy, chemical, microbiological and immunological abnormalities
A
Clinical Pathology
8
Q
-concerned with acterization of changes occurring in cells, tissues and fluids.
A
Clinical Pathology
9
Q
-able to support physician’s information for appropriate treatments
A
Clinical Pathology
10
Q
- Examination of cells or tissues which are sourced from a living organism
A
Biopsy
11
Q
- Requires surgical materials/specimen
A
Biopsy
12
Q
Biopsy:
A
Excisional
Incisional
13
Q
-entire tumor removal
A
Excisional
14
Q
-maybe partial, complete or by pieces removal
A
Excisional
15
Q
-portion of tumor only
A
Incisional
16
Q
-opening and cutting the tissue to obtain specimen/ through drainage of fluid secretion or exudates
A
Incisional
17
Q
METHODS OF SURGICAL BIOPSY:
A
Fine Needle
Aspiration
Core Needle
Biopsy
Punch Biopsy
Shave Biopsy
Curretings
18
Q
-simples and least invasive
A
Fine Needle Aspiration
19
Q
-remove cells from the area of abnormality
A
Fine Needle Aspiration
20
Q
-remove cells from the area of abnormality
A
Fine Needle Aspiration
21
Q
- Less pain and may be done conveniently
A
Fine Needle Aspiration
22
Q
- Not always adequate
A
Fine Needle Aspiration
23
Q
- Disturbs cell architecture
A
Fine Needle Aspiration
24
Q
-removes cells and small amount of surrounding tissues
A
Core Needle Biopsy
25
-uses vim-silverman needle (cutting needle)
Core Needle Biopsy
26
Provides additional information in lesion exam without surgery
Core Needle Biopsy
27
Trauma to surrounding tissues
Core Needle Biopsy
28
- Preferred for full-thickness skin specimen
Punch Biopsy
29
- Involves a circular blade, rotated down to epidermis dermis subcutaneous fat
Punch Biopsy
30
Punch Biopsy
- Yields ________ cylindrical tissue sample
3-4 mm
31
- No surgery
Punch Biopsy
32
- May result to reproduction of malignant cells when some portion of mass is removed.
Punch Biopsy
33
- Small fragments of tissues are scraped from the surface (skin).
Shave Biopsy
34
- Small fragments of tissues are scraped from the surface (skin).
Shave Biopsy
35
- For diagnosis of non-cancerous skin tumors
Shave Biopsy
36
Cosmetically safe
Shave Biopsy
37
Diagnosis/treatment of benign lesions
Shave Biopsy
38
- May result to excision if malignant lesion due to possible seeding.
Shave Biopsy
39
- Tissues are scooped/spooned from tissues that has grown from body cavities.
Curretings
40
- Most superficial mode of biopsy
Curretings
41
-scarring is minimal
Curretings
42
-lack of surgical margins
Curretings
43
- Incidence of local recurrence of abnormal cells.
Curretings
44
COMMON ORGANS SUBJECTED TO BIOPSY:
Breast
Lymph Nodes
Liver
Spleen
Exudates from cavities (pleural, peritoneal and pericardial)
Cervix Endometrial mucosa (uterus)
45
AUTOPSY PURPOSES:
-Determine the [?]
-Determine the [?]
-Preservation of [?] in cases of future examination or scientific study
- [?]
-Disclosure of [?]
-[?]
cause of death
final diagnosis
tissue from the dead body
Criminal prosecution
public health hazards
Civil justice
46
PHASES OF AUTOPSY
1. External examination
2. Determination of _______________ Length/height and weight
3. Symmetry observation
4. General nutritional status and preservation of tissues
5. Inspection of body parts for identification of recent injuries ;scrutiny of unique marks of deeper significance 6. Posterior lividity Rigor mortisBody temperature 7. Microscopic examination
47
PRE-REQUISITES OF AUTOPSY PROCEDURES
_______________________
_____________________________ is stated and specified according to purpose and completeness.
[?] needed for the procedure
Written consent/permission
Type of autopsy procedure
Apparatuses or instruments needed for the procedure
48
PRE-REQUISITES OF AUTOPSY PROCEDURES
_______________________
_____________________________ is stated and specified according to purpose and completeness.
[?] needed for the procedure
Written consent/permission
Type of autopsy procedure
Apparatuses or instruments needed for the procedure
49
Rule 1
The signer must be:
Mentally competent
At least ______ [21]years old or married, or related to the deceased
50
a. If the deceased was married: ____________
Surviving spouse must sign
51
b. If no surviving spouse:_______________
Children who are at least 21 yrs old may sign
52
c. If no surviving spouse and child is either unmarried/under 21:______________
Guardian of the child may sign; if absent, the judge with jurisdiction may sign
53
d. If no surviving spouse nor child:__________________
• Father of the deceased
• Mother of the deceased
• Guardian of the deceased
• Next of kin
• Person with custody of the body and responsible for the burial
54
Rule 3
Prior to autopsy, there was an objection from a close relative:
Autopsy will NOT proceed. Inform the pathologist imediately
55
Rule 4 In cases of absence due to mental incompetency, age or relationship: (?) should be informed and consulted
Hospital administrator/person-in-charge
56
Rule 5
_______________________________________________must be present when permit is signed.
Two (2) witnesses (hospital employees)
57
TYPES OF AUTOPSY
According to Purpose
Routine Hospital Autopsy
Medico Legal Autopsy
According to completeness of the procedure
Partial
Complete
According to Incision
Straight-cut
Y-shaped
58
-conducted in private hospitals
Routine Hospital Autopsy
59
-conducted in private hospitals
Routine Hospital Autopsy
60
- Conducted at the NBI or government institutions for purposes of prosecution
Medico Legal Autopsy
61
-Examination is only on the permitted part/region of the body
Partial
62
- Examination is done in the whole body.
Complete
63
- Begins at the midline to suprasternal notch down to the pubis
Straight-cut
64
- Begins at the midline to suprasternal notch down to the pubis
Straight-cut
65
- Begins at both of the shoulder regions down to the xiphoid and incised downward to the pubis
Y-shaped
66
AUTOPSY TECHNIQUES
LETULLE'S
GHON'S
ROKITANSKY'S
VIRCHOW'S
67
"En masse"
LETULLE'S
VIRCHOW'S
68
"En bloc"
GHON'S
69
In Situ
ROKITANSKY'S
70
E.g. Oral, cervical, thoracic, abdominal and pelvic organs are removed as one large organ block
LETULLE'S
71
E.g: Cervical and thoracic organs (1 block), abdominal organs (1 block) and urogenital system (1 block) removed as separate blocks.
GHON'S
72
E.g: infected bodies with HIV, Hepatitis-B. Easy to perform in children.
ROKITANSKY'S
73
E.g. heart, lungs, uterus
VIRCHOW'S
74
Commonly done in medical colleges to teach student
LETULLE'S
75
Block by block dissection
GHON'S
76
-in combination with en bloc.
ROKITANSKY'S
77
- Removal of organ one after another.
VIRCHOW'S
78
TAKE EVERYTHING TOGETHER
LETULLE'S
79
ONE BLOCK AT A TIME
GHON'S
80
ALL AT ONCE THEN ONE BY ONE
ROKITANSKY'S
81
ONE BY ONE
VIRCHOW'S
82
FRESH TISSUE EXAMINATION:
Teasing/Dissociation
Squash Preparation (Crushing)
Smear Preparation
83
-dissection of specimen with a needle
Teasing/Dissociation
84
Teasing/Dissociation -Uses isotonic salt solution (________________________________)
Normal saline/Ringer’s Solution
85
-can be unstained or stained (supravital dye/Methylene blue)
Teasing/Dissociation
86
Teasing/Dissociation -permits cells to be examined in a __________________________
Living state
87
Squash Preparation (Crushing) -done in _____________ small tissues
<1mm
88
-sample is forcibly compressed with another slide or cover glass
Squash Preparation (Crushing)
89
-supravital stain is placed at the junction and absorbed through capillary attraction
Squash Preparation (Crushing)
90
-may be observed as fresh preparation or with supravital staining
Smear Preparation
91
-can be made permanent by fixing the sample while wet.
Smear Preparation
92
-preferred for thick secretions such as serous fluids, concentrated sputum and enzymatic lavage → cancer diagnosis
Smear Preparation
93
-accomplished using applicator stick or platinum loop
Streaking
94
-direct or zigzag line
Streaking
95
-attempts to obtain relatively uniform distribution of secretion
Streaking
96
-utilizes selected portion of the material
Spreading
97
-utilizes selected portion of the material
Spreading
98
-able to maintain cellular interrelationships of the material
Spreading
99
-recommended for fresh sputum, bronchial aspirate and thick mucous
Spreading
100
-a drop of the sample is placed on one slide and disperses upon the placement of another slide.
Pull-apart
101
-two slides are pulled at opposite direction, uninterrupted.
Pull-apart
102
-May be fresh or vital stained.
Pull-apart
103
-the surface of the freshly cut piece of tissue is bought in contact and pressed on to the surface.
Touch preparation (Impression smear)
104
-allows cells to be directly transferred onto the slide without destroying intercellular relationship
Touch preparation (Impression smear)
105
FROZEN SECTION
Slice required: thin slices _________________________
10-15 um
106
Microtome: kept at cold temperature ____________________
-10 to -20 deg celsius
107
Microtome components:
a.Freezing Microtome
b.Cold Microtome
108
Effect of slow freezing:
distortion of tissue (ice crystals)
109
METHODS OF FREEZING:
Liquid nitrogen
Isopentane
Carbon dioxide
Aerosol spray
110
-most common meyhod of freezing
Liquid nitrogen
111
-used with non fatty unfixed tissues at -10 to -25 degrees Celsius
Liquid nitrogen
112
-may result to crack due to rapid expansion; produces ice crystals artifact
Liquid nitrogen
113
-causes vapor phase which acts as an insulator= uneven cooling
Liquid nitrogen
114
-damage to the block and blade occurs if done at -70 degrees Celsius
Liquid nitrogen
115
-liquid at room temperature
Isopentane
116
-small crystals starts to form at -170 degrees Celsius
Isopentane
117
-excellent for freezing muscle tissue
Isopentane
118
-conventional freezing microtome gas
Carbon dioxide
119
-adequate in freezing small pieces of tissue (except muscle)
Aerosol spray
120
-e.g. Fluorinated hydrocarbons (Cryokwik) - rapid freezing
Aerosol spray
121
METHODS OF FROZEN SECTION:
COLD KNIFE PROCEDURE
CRYOSTAT PROCEDURE
122
-utilizes Carbon Dioxide technique
COLD KNIFE PROCEDURE
123
COLD KNIFE PROCEDURE -sample must be _________ thick
3-5mm
124
-applied with drops of gum syrup and several intermittent bursts of CO2 with 1-2 seconds duration, and 4 seconds interval
COLD KNIFE PROCEDURE
125
COLD KNIFE PROCEDURE **Dew Line: point at which sections may be cut at [?] thickness.
10 um
126
COLD KNIFE PROCEDURE ** Controlled environment:
Knife: ___
Tissue: ___
Environment : ___
-40 to -60 deg cel
-5 to -10 deg cel
0 to 10 deg cel
127
CRYOSTAT PROCEDURE
**Consist of: Refrigerated Chamber: __________________
-20 deg cel
128
CRYOSTAT PROCEDURE
Optimum working temperature of cryostat : __________________
-18 to-20 deg cel
129
-tissue must be sufficiently cold to prevent compression and displacement of cell.
CRYOSTAT PROCEDURE
130
-provides the simplest, quickest and least labor intensive
CRYOSTAT PROCEDURE
131
-routine for intraoperative and rapid diagnosis
CRYOSTAT PROCEDURE
132
-only provide individual sections, and does not form ribbons.
CRYOSTAT PROCEDURE
133
MOUNTING OF TISSUE BLOCK (Cryostat)
Recommendation labs:
Optimal Cutting Temperature (O.C.T) compound
Lab-Tek Products
Division of Miles Laboratories
134
MOUNTING OF TISSUE BLOCK (Cryostat)
Temperature settings:
____ for brain, lymph notes, liver, spleen, uterine, curreting, soft cellular tumors.
____for non-fatty breast tissue, ovary, prostate, tongue, and GI tract
____ for fatty breast and omental tissue
-5 to -15 deg cel (soft tissues)
-15 to -25 deg cel (muscle w/ fat)
-35 deg cel (pure fat)
135
Mounting media:
Synthetic water-soluble glycols and resins.
136
FREEZING PREVIOUSLY FIXED TISSUES
Cryostat section of [?] -> attaches easily to the slide without adhesive, with good preservation of enzymes.
fresh, unfixed tissue
137
is recommended for any cold sectioning of fixed material (e.g. Fats and lipids)
Cryostat
138
DO NOT adhere to slide during staining.
Formalin-fixed tissues
139
DO NOT adhere to slide during staining.
Formalin-fixed tissues
140
Formalin-fixed tissues:
-must be coated with [?] or;
-immerse the tissue block in boiling [?] for [?] prior to freezing and sectioning
albumin/ chrome-glycerin jelly
10% buffered formalin ; 1-2 minutes
141
Special fixatives : [?] Histochemistry and lipid demonstration
10% formol calcium at 4°C
142
Alcohol fixed tissues: washed in water for [?] before sectioning.
12-24 hours
143
SPECIAL PROCESSING TECHNIQUE:
FREEZE DRYING
FREEZE SUBSTITUTION
144
FREEZE DRYING
-done through “quenching” of tissue at _______ then subsequently followed by “desiccation” , then “sublimation” through vacuum chamber at _____________.
-160 deg cel
-40 deg cel
145
-produces minimum shrinkage and allows tissue to be processed in a fresh state.
FREEZE DRYING
146
-Used in: Enzyme studies (Hydrolytic) Demonstrating mucous, glycogen, and proteins.
FREEZE DRYING
147
-involves “dehydration” at low temperature.
FREEZE SUBSTITUTION
148
-fixed in Rossman’s or 1% Acetone then dehydrated in absolute alcohol.
FREEZE SUBSTITUTION
149
FREEZE SUBSTITUTION
-rapid freezing (3:1 propane-isopentane) at __________ is followed by substitution of water –free acetone and cooled to ____________
-175 deg cel
-70 deg cel
150
For best preservation: Substituting fluid should contain fixing agent and solvent for ice (e.g. 1% osmium tetroxide in acetone, mercuric chloride in ethanol or picric acid in ethanol)
FREEZE SUBSTITUTION
151
For best preservation: Substituting fluid should contain fixing agent and solvent for ice (e.g. 1% osmium tetroxide in acetone, mercuric chloride in ethanol or picric acid in ethanol)
FREEZE SUBSTITUTION
152
MANUAL TISSUE PROCESSING:
I. FIXATION
II. DECALCIFICATION
III. DEHYDRATION
IV. CLEARING
V. IMPREGNATION/INFILTRATION
VI. EMBEDDING (Casting/Blocking)
➢ Fixation
➢ Dehydration
➢ Clearing
➢ Infiltration
➢ Embedding
➢ Sectioning (+ Floating, Fishing-out, Drying)
➢ Staining
➢ Mounting
➢ Labelling
153
First and most critical step of tissue processing
FIXATION
154
FIXATION Purpose:
1. To [?] the tissue by stopping all cellular activity and let it be examined in a “life-like” state
2. To [?] of cellular elements by arresting autolysis and putrefaction.
3. To [?] protoplasmic substances.
preserve
prevent breakdown
coagulate or precipitate
155
FIXATION Purpose:
1. To [?] the tissue by stopping all cellular activity and let it be examined in a “life-like” state
2. To [?] of cellular elements by arresting autolysis and putrefaction.
3. To [?] protoplasmic substances.
preserve
prevent breakdown
coagulate or precipitate
156
FIXATION Rationale: To (?) the tissue from trauma of further handling.
harden and protect
157
Methods of Fixation:
Heat Fixation
Microwave Fixation
Cryopreservation
Chemical Fixation
158
-rarely used, application is restricted to smears e.g. boiling, poaching
Heat Fixation
159
-a form of heat fixation,
Microwave Fixation
160
-utilized best with commercial glycol-based fixatives not evaporate at 55 degrees Celsius.
Microwave Fixation
161
-usually in the form of freeze drvino
Cryopreservation
162
-useful in histochemistry (preserve cancer markers)
Cryopreservation
163
Classification of Fixation:
Additive Fixation
Non-Additive Fixation
164
- Fixative becomes part of the tissue due to cross linking or formation of molecular complexes = stable proteins
Additive Fixation
165
-not incorporated to the tissue but alters tissue composition and stabilizes the tissue by means of water removal (H-molecule) from proteins to allow new cross links to occur.
Non-Additive Fixation
166
-Prevents autolysis and makes tissue unsuitable for bacterial decomposition
Non-Additive Fixation
167
E.g. Formalin, mercury and osmium tetroxide
Additive Fixation
168
E.g. Alcoholic fixatives
Non-Additive Fixation
169
Classification of fixing agents:
1. Coagulant Fixatives
2. Non-Coagulant fixatives
170
-result in a permeable meshwork of protein strands
1. Coagulant Fixatives
171
-result in a permeable meshwork of protein strands
1. Coagulant Fixatives
172
-additive in nature; produce a less permeable gel
2. Non-Coagulant fixatives
173
-MOST COMMON fixing agent
2. Non-Coagulant fixatives
174
Mechanism of Fixation:
A. Denaturation
B. Addition and Cross-Link Formation
175
Most common mechanism of fixation
A. Denaturation
176
Non-coagulant fixing
B. Addition and Cross-Link Formation
177
Alcohol-induced/Acetone-induced dehydration
A. Denaturation
178
Chemicals react with proteins and cells to increase surface area
B. Addition and Cross-Link Formation
179
Removal of free water, then replacement by dehydrants in tissues
A. Denaturation
180
Binds to natural chemical in tissues
B. Addition and Cross-Link Formation
181
Irreversible
A. Denaturation
182
Staining is stable
B. Addition and Cross-Link Formation
183
FIXATIVES: General Consideration
1.Fixation of tissue after removal from the body: ____________________
2.Sufficient volume of fixative: _____________________ (best penetration)
3.Removal of anatomical barriers:[?]
4.Sectioning of large specimen: [?]
5. Replacement of contaminated fixatives: [?]
6. Reduction of tissue distortion by filling in the tubular structure with [?]
7. Tissue blocks: [?] for permeation of fixative
8. Avoid [?]: loss of immunohistochemical antigenicity
9. Maintaining the [?] prior to and after fixation is important
<1h
20:1 or 10:1
fascia, bone, feces, thick tissues
GIT tract, lungs (inflated with fixative)
Bile, blood, feces
corkboard or gauze
Small enough ( 4-6mm)
prolonged fixation
shape
184
FACTORS INVOLVED IN FIXATION:
Volume
Hydrogen ion concentration
Temperature
Thickness of section
Osmolality
Concentration
Time Interval
185
Fixation Volume __________________________
20:1 or 10:1
186
enhances fixative penetration
Agitation
187
Fixation Hydrogen ion concentration __________________________
pH of 6-8
188
Fixation Acidity: formalin heme pigment (?)
black deposits
189
Fixation Ideal buffers:
PO4, HCO3, Cacodylate and Veronal
190
Fixation Temperature
_________________________ _________________________
40 deg cel
0-4 deg cel
191
Formalin heated at __________________________
60 deg cel: used for urgent biopsy (but distorted)
192
deteriorate quickly
Brain cells
193
BM continues to undergo mitosis up to _______________when refrigerated.
30 mins
194
does not react in room temp
Nucleic acid
195
Thickness of section
______________ (EM)
______________ (LM)
[?] (solid material, e.g.liver)
1-2mm2
2cm x 0.4mm
10-15 mm
196
Rate:
______________ (EM)
______________ (LM)
2 cm2x 4mm thick completes at 4-6 hours
2-3 mm/hour
197
must be removed prior to fixation
Fecal matter and stomach contents
198
must be removed prior to fixation
Fecal matter and stomach contents
199
Brain is fixed in _________________for 2-3 weeks due to its structure.
10% buffered formalin
200
: best penetration
: Worst penetration
: in between
Formalin and Alcohol: best penetration
Glutaraldehyde: Worst penetration
Mercurials: in between
201
Slightly hypertonic solution:
Isotonic solution:
400-450 mOsm.
340 mOsm.
202
: Cell shrinkage
: Swelling, poor fixation
Hypertonic solutions
Hypotonic solutions
203
added to osmium tetroxide for electron microscopy
Sucrose
204
Concentration Lowest level possible
Formalin:
Formaldehyde: ______
Glutaraldehyde: _________________(for immuno electron micrsocopy)
10%
35%-40%
3% or 0.25
205
Too High Concentration:
produces artifacts
206
Fixation is done immediately (<1 hr) after removal from body/death to prevent
autolysis or putrefaction.
207
: poor quality of tissue
Longer blood supply interruption
208
Drying: Artefacts (moist with)
saline
209
A. SIMPLE FIXATIVES:
1. ALDEHYDES
a. Formaldehyde
b. Glutaraldehye
2. METALLIC
a. Mercuric chloride
b. Chromate fixatives
-Potassium dichromate
-Chromic acid
c. Lead fixative
-Picric acid
-Acetic acid
-Acetone
-Alcohol
-Osmium tetroxide (osmic acid)
d. Heat
210
-made up of two or more fixative; has combined effect that acts on the cell and tissue constituents
B. COMPOUND FIXATIVES
211
B. COMPOUND FIXATIVES:
Haidensusa's Fkuid
Carnoy's Fluid Bouin's
Fluid Formol saline
Buffered formalin
212
B. COMPOUND FIXATIVES:
Haidensusa's Fkuid
Carnoy's Fluid Bouin's
Fluid Formol saline
Buffered formalin
213
TYPES OF FIXATIVES: According to composition
SIMPLE FIXATIVES
COMPOUND FIXATIVES
214
TYPES OF FIXATIVES: According to Action
215
• Lipids:
mercuric chloride or potassium dichromate
216
• Phospholipids:
Baker’s formol-calcium
217
• Cholesterol:
digitonin
218
• Carbohydrates:
alcoholic fixatives
219
• Glycogen:
Rossman’s fluid or cold absolute alcohol
220
• Proteins:
neutral buffered formol saline or formaldehyde vapor
221
• Electron microscopy:
double fixation
222
Used for electron and light microscopy
Glutaraldehyde
223
Made up of two formaldehyde residues (larger) slower penetration
Glutaraldehyde
224
Glutaraldehyde composition:
2% Glutaraldehyde + Osmium Tetroxide= EM
225
Must be cold and buffered (3 months life span)
Glutaraldehyde
226
Glutaraldehyde Recommended size:
227
Glutaraldehyde Concentrations:
2.5% for small fragments
4% for larger tissues (<4mm)
228
• Rapid and irreversible changes (fixes quickly and at 4 deg cel)
Glutaraldehyde
229
• Not good for immunohistochemical
Glutaraldehyde
230
• Gives best overall cytoplasmic and staining nuclear detail
Glutaraldehyde
231
• More stable effect, less shrinkage
Glutaraldehyde
232
• More expensive
Glutaraldehyde
233
• Makes tissue brittle
Glutaraldehyde
234
* MOST COMMON FIXATIVE
35-40% Formaldehyde; 10% Formalin
235
(lung metaplasia with long term expo)
paraformaldehyde
236
Possible explosion if not neutralized/buffered (Calcium or
Magnesium carbonate)
35-40% Formaldehyde; 10% Formalin
237
Change regularly to prevent bleaching
Formalin
238
is added as preservative to formaldehyde
Methanol
239
: restores natural tissue color after fixation
70% alcohol
240
Formation of "formic acid": Counteracted by
alcoholic picric acid or 1% KOH in 80% alcohol/ buffered formalin
241
Optimal choice
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
242
Recommended for preservation of surgical post-mortem and
research specimen
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
243
• Prevent alterations
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
244
• Less shrinkage
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
245
Hardens tissue better
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
246
Tissues are stored indefinitely
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
247
• Prolonged fixation causes bleach tissues
10% Neutral Buffered Formalin/ Phosphate Buffered formalin
248
Fixative of choice for immunohistochemistry, molecular tests
10% Neutral Buffered Saline
249
Readily available, time consuming, stable
10% Neutral Buffered Saline
250
Compatible with stains
10% Neutral Buffered Saline
251
Preserves fat, mucin, glycogen
10% Neutral Buffered Saline
252
Used to preserve cadavers
10% Formol Saline Solution
253
Recommended for CNS tissues and general post-mortem.
10% Formol Saline Solution
254
Slow fixative >24h (35 deg cel), 48h (20-25 deg cel)
10% Formol Saline Solution
255
Preservation and storage of surgical, post mortem and research specimen
Sodium Dihydrogen Phosphate/Disodium Hydrogen Phosphate
256
Best for iron pigments and elastic fibers
Sodium Dihydrogen Phosphate/Disodium Hydrogen Phosphate
257
is recommended for routine post-mortem tissues.
Formol Corrosive (Formol-Sublimate)
~ Formol-mercuric chloride solution
258
Polymerized form of formaldehyde
Paraformaldehyde
259
From a fine white powder and depolymerizes back to formalin when heated.
Paraformaldehyde
260
Suitable for paraffin embedding and sectioning
Paraformaldehyde
261
Allows subsequent immunotherapy-detection of certain antigens
Paraformaldehyde
262
Reversible mode of action by excess water
Paraformaldehyde
263
Suitable for LM in resin embedding and sectioning,
and for electron microscopy.
Karnovsky's Fixative (4% paraformaldehyde - 1% glutaraldehyde in 0.1M P4)
264
Indications of Incomplete Fixation:
[?]has not been maintained
Poor[?]
Lack of [?] b/w cell nucleus and cytoplasm
Tissue structure
staining
contrast
265
Indications of Well fixed : 10% NBF
Nucleus has [?]
[?]is seen
[?]nuclear membrane
bubbling artifacts
Contrast
Crisp
266
: Contains potassium salts and formalin
**Kaisserling’s animal cells
267
Common metallic fixative; widely used as secondary fixative
Mercury Containing Fixatives
268
Recommended concentration: 5-7 deg cel (sat. aqueous solutions)
Mercury Containing Fixatives
269
Mode of action: Additive and Coagulative
Mercury Containing Fixatives
270
Good with TRICHOME STAIN
Mercury Containing Fixatives
271
Recommended for renal tissue, fibrin, CT, muscles
Mercury Containing Fixatives
272
Fixative of choice for tissue photography
Mercury Containing Fixatives
273
• Excellent nuclear detail preservation
Mercury Containing Fixatives
274
• Good penetration during intense immunostaining
Mercury Containing Fixatives
275
Fixation of hematopoietic and reticuloendothelial tissues
Mercury Containing Fixatives
276
• Poor penetration (hardens outer layer only)
Mercury Containing Fixatives
277
•Produces shrinkage of tissue
Mercury Containing Fixatives
278
• Mercury deposits
Mercury Containing Fixatives
279
Recommended for fixing small pieces of liver, spleen, connective tissue fibers and nuclei (fragile organs)
Zenker's Fluid (HgCl2, glacial and acetic acid)
280
Good general fixative for adequate preservation of all kinds of tissue and gives excellent staining result
Zenker's Fluid (HgCl2, glacial and acetic acid)
281
Recommended for Trichrome staining
Zenker's Fluid (HgCl2, glacial and acetic acid)
282
Excellent micro-anatomic fixative for pituitary gland, BM, and blood-containing tissue (liver and spleen)
Zenker's Formol /Hellys Solution (HgCI2 + Formaldehyde)
283
Produce brown/black pigment
Zenker's Formol /Hellys Solution (HgCI2 + Formaldehyde)
284
May be used in urine preservation
Haidenhein's Susa Solution (Magnesium Chloride, glacial acetic acid and formalin)
285
Recommended for skin tumor biopsy
Haidenhein's Susa Solution (Magnesium Chloride, glacial acetic acid and formalin)
286
Excellent cytology fixative
Haidenhein's Susa Solution (Magnesium Chloride, glacial acetic acid and formalin)
287
Commonly used for bone marrow biopsy
B-5 Fixative (HgCI2 + Anhydrous Sodium Acetate)
288
Used for wet smear preparation and connective tissue
Schaudinn's Fixative (Alcohol containing)
289
Precipitates all protein and fixes CHO
Chromic Acid
290
Preserves lipid and mitochondria
Potassium Dichromate (K2Cr207)
291
Recommended for demonstration of chromatin, mitochondria, mitotic figures, golgi bodies, RBC and colloid-containing tissue
Regaud's (Muller's) Fluid (3% K2Cr207)
292
Recommended for study of early degenerative processes and tissue necrosis (Ex. Neurons)
Orth's Fluid (2.5 K2Cr207)
293
Demonstrates myelin, Rickettsiae, and other bacteria
Orth's Fluid (2.5 K2Cr207)
294
Used for preservation of glycogen mucopolysaccharide, and amyloid
Lillie's Fixative
295
Fixed connective tissue mucin
Lillie's Fixative
296
Forms insoluble lead carbonate
Lillie's Fixative
297
Removed by filtering or adding acetic acid
Lillie's Fixative
298
Fixative and dehydrant at the same time
Alcohol Fixatives
299
Removes excess water
Alcohol Fixatives
300
Mode of action: Denaturation or precipitation of proteins
Alcohol Fixatives
301
For fixing dry and wet smears, blood smears and bone marrow
tissues
Absolute Methyl Alcohol
302
RBC lysis at lower concentration while WBC are inadequately
preserved
Ethyl Alcohol (70-100%)
303
Fixes sputum
Gendre's Fixative (Alcoholic formalin)
304
Most rapid fixative and maybe used for urgent biopsy
Carnoy's Fixative (Alcohol+ Chloroform+ Acetic Acid)
305
Used on frozen section and smears
Clarke's Solution (Absolute ethanol and acetic acid)
306
Preserves nucleic acids and extract lipids
Clarke's Solution (Absolute ethanol and acetic acid)
307
Fixes mucopolysaccharide and nuclear protein
Newcomer's Fluid
308
Better reaction in Feulgen Stain than Carnoy's
Newcomer's Fluid
309
Nuclear and histochemical fixative
Newcomer's Fluid
310
Fixes sputum
Gendre's Fixative (Alcoholic formalin)
311
Less messy than "Bouin's"
Brasil's Alcoholic Picrofomol Fixative
312
Excellent for glycogen
Brasil's Alcoholic Picrofomol Fixative
313
Preserves glycogen but causes considerable tissue shrinkage
Picric Acid
314
Allows brilliant staining with Trichome method
Picric Acid
315
Recommended for fixation of embryos and pituitary biopsy
Bouin's Solution
316
Excellent fixative for glycogen demonstration = Hemolyzes RBC
Bouin's Solution
317
Remove yellow color by 70% ethanol followed by 5% sodium thiosulfate and running water
Bouin's Solution
318
Highly explosive when dry
Bouin's Solution
319
Better and less "messy" than Bouin's solution
Brasil's Alcoholic Picroformol Fixative
320
Fixative Excellent fixative for glycogen demonstration
Brasil's Alcoholic Picroformol
321
• Preserves cytoplasmic structures well
Osmium Tetroxide
322
• Used extensively for neurological tissues
Osmium Tetroxide
323
• Fixes fats for electron microscope
Osmium Tetroxide
324
• Expensive, poor penetration
Osmium Tetroxide
325
• Reduced w/ sunlight → black deposit; dark bottle
Osmium Tetroxide
326
• Acid vapour → conjunctivitis, osmic oxide in cornea → blindness
Osmium Tetroxide
327
Inhibits hematoxylin
Osmium Tetroxide
328
Extremely volatile
Osmium Tetroxide
329
Most common chrome-osmium acetic acid fixative
Flemming's Solution
330
Excellent fixative for nuclear structure for EM
Flemming's Solution
331
Rrecommended for cytoplasmic structures
Flemming's Solution without Acetic acid
332
Fixes brain tissues for diagnosis of rabies
Acetone
333
Dissolves fat and evaporates rapidly
Acetone
334
Preserves glycogen poorly
Acetone
335
Thermal coagulation of tissue proteins
Heat Fixation
336
Employed for frozen tissue sections
Heat Fixation
337
Used in bacteriologic smear
Heat Fixation
338
Fixes chromosomes, lymph glands, and urgent biosies
Carnoy's Fluid (Ethyl alcohol, glacial acetic acid and chloroform)
339
Used to fix brain tissue for diagnosing rabies
Carnoy's Fluid (Ethyl alcohol, glacial acetic acid and chloroform)
340
Fixes mucopolysaccharides and nuclear protein
Newcomer's Fluid (Isopropyl alcohol)
341
Nuclear and histochemical fixative
Newcomer's Fluid (Isopropyl alcohol)
342
DECALCIFICATION PURPOSE
removal of (?) from a bone or calcified tissue
adjusts the hard substance of bones to the softness of paraffin embedding medium
calcium ions
343
DECALCIFICATION PRINCIPLE
Calcium chelation
344
DECALCIFICATION GENERAL CONSIDERATIONS:
Done after ________ and before __________
Fixation
Dehydration
345
DECALCIFICATION Tissue preparation:
> Complete fixation
Reagent Volume: 20:1 volume
346
DECALCIFICATION Concentration:
5-10%
347
DECALCIFICATION: Aids in speeding up the process
Agitation
348
DECALCIFICATION Ideal time:
24-48h, >14 days
349
DECALCIFICATION Optimum temperature:
18-30 degrees celsius
350
DECALCIFICATION: Promotes destructive action of acids on matrix.
Heat
351
- tissues will undergo complete digestion
55 degrees C
352
- Impair nuclear staining w/ Van Gieson’s
37 degrees C
353
- reduced effectiveness of trichrome & PAS
37 degrees C
354
TYPES OF DECALCIFYING AGENTS
-based on strong mineral acids
-based on weaker organic acids
-composed of chelating agents
-Ion exchange resin
-Electrophoresis
355
-most widely used ACID DECALCIFYING AGENTS
(Strong Mineral Acids)
356
-readily available
ACID DECALCIFYING AGENTS (Strong Mineral Acids)
357
-routine for large amount of bony tissues
ACID DECALCIFYING AGENTS (Strong Mineral Acids)
358
-Slower action; with greater distortion
ACID DECALCIFYING AGENTS (Strong Mineral Acids)
359
-cannot be measured by chemical test
ACID DECALCIFYING AGENTS (Strong Mineral Acids)
360
• Most common and fastest acid decalcifying agent
NITRIC ACID
361
• Recommended at 5-10% concentration
NITRIC ACID
362
• Very rapid with minimal distortion
NITRIC ACID
363
• Prevents tissue damage when combined with formaldehyde or alcohol
NITRIC ACID
364
Rapid
Aqueous Nitric Acid Rapid Solution (10%)
365
Produces good nuclear staining
Aqueous Nitric Acid Rapid Solution (10%)
366
Recommended for urgent biopsies
Formol Nitric-Acid
367
Produces lesser tissue destruction
Formol Nitric-Acid
368
Routine purposes
Perenyi's Fluid (Nitric acid+Chromic acid)
369
Decalcifies and soften tissues at the same time
Perenyi's Fluid (Nitric acid+Chromic acid)
370
Not for urgent biopsies
Perenyi's Fluid (Nitric acid+Chromic acid)
371
Most rapid decalcifying agent
Phloroglucin Nitric Acid
372
Recommended for urgent cases
Phloroglucin Nitric Acid
373
Appearance of yellow color → neutralized with 5% Sodium Sulfate
Phloroglucin Nitric Acid
374
For silver impregnation of nerve fibers
De Castro's Fluid (w/ Chloral Hydrate)
375
Inferior to nitric acid
HYDRO-CHLORIC ACID
376
Slower and greater distortion
HYDRO-CHLORIC ACID
377
• Produces good nuclear staining
HYDRO-CHLORIC ACID
378
• Recommended for surface decalcification of tissue blocks
HYDRO-CHLORIC ACID
379
Moderately rapid
Von Ebner's Fluid
380
Does not require washing out before dehydration
Von Ebner's Fluid
381
For teeth and small pieces of bone
Von Ebner's Fluid
382
-Act slowly on dense cortical bones
ACID DECALCIFYING AGENTS (Weak Acids)
383
-ACID DECALCIFYING AGENTS Better suited for BM (e.g. acetic acid and formic acid)
(Weak Acids)
384
• Used in 10% concentration
FORMIC ACID
385
• Moderate acting, weak decalcilying agent at the same time a fixative
FORMIC ACID
386
• Recommended for routine decalcification of postmortem research tissues.
FORMIC ACID
387
• Addition of CITRATE may accelerate the decalcification
FORMIC ACID
388
Better nuclear staining than nitric
Formic Acid-Sodium Citrate Solution
389
Recommended for autopsy materials, bone marrow, cartilage and research tissues.
Formic Acid-Sodium Citrate Solution
390
Slow acting
TRICHLOROACETIC ACID
391
Slow acting
TRICHLOROACETIC ACID
392
Suitable for small spicules of bone
TRICHLOROACETIC ACID
393
minute pieces of bone.
SULFUROUS ACID
394
Very weak decalcifying solution suitable only for minute pieces of bone
SULFUROUS ACID
395
Fixative and decalcifying agent.
CHROMIC ACID (Flemming's Fluid) -environmental toxin
396
Used for decalcifying minute bone spicules
CHROMIC ACID (Flemming's Fluid) -environmental toxin
397
Nuclear staining with hematoxylin is inhibited
CHROMIC ACID (Flemming's Fluid) -environmental toxin
398
pH at 4.5
CITRIC ACID-CITRATE BUFFER
399
Too slow for routine process
CITRIC ACID-CITRATE BUFFER
400
No cell or tissue distortion
CITRIC ACID-CITRATE BUFFER
401
-combines with calcium and salts
CHELATING AGENTS
402
complex, slowly reducing its size
EDTA (Versene)
403
• combines with calcium, forming an insoluble nonionized
EDTA (Versene)
404
• Used as anticoagulant and as water softener
EDTA (Versene)
405
• Disodium salt is adjusted to 7 and 8 for preservation of DNA
EDTA (Versene)
406
Conventional stains are unaffected
Neutral EDTA
407
Act slow but with little tissue damage
Neutral EDTA
408
-Cellular detail is well preserved
ION EXCHANGE RESIN (Ammonium-sulfonated polysterene)
409
-hastens decalcification by removing calcium ions from formic acid thereby increasing solubility from the tissue
ION EXCHANGE RESIN (Ammonium-sulfonated polysterene)
410
-decalcification is shortened
ELECTROPHORESIS (Electrical Ionization)
411
-Temperature: 30-45 degrees Celsius
ELECTROPHORESIS (Electrical Ionization)
412
-Utilizes 90% formic acid and conc. HCI
ELECTROPHORESIS (Electrical Ionization)
413
-positively charged calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution.
ELECTROPHORESIS (Electrical Ionization)
414
-faster than routine decalcification due to the use of microwave irradiation, speeding the process from days to hours.
MICROWAVE OVEN
415
POST DECALCIFICATION TREATMENT
Washing-out
Running water: __________________
3-8 hrs
416
POST DECALCIFICATION TREATMENT
Neutralization:
2% Li carbonate or
5 – 10 % Na bicarbonate
417
POST DECALCIFICATION TREATMENT
Frozen Sectioning:
[?] -> water , or
store in [?] with 15% sucrose or in [?] with 15-20% sucrose at 4oC
acid-decalcified tissues
formol saline; PO4- buffered saline
418
POST DECALCIFICATION TREATMENT
EDTA: wash -> immerse in
alcohol
419
TEST FOR COMPLETENESS OF DECALCIFICATION:
420
PHYSICAL:
Touch
Pliability
Resistance to fingernails or needling
421
X-RAY: Opaque – reflects presence of _________________
Ca
422
Most expensive but most ideal and most reliable
X-RAY
423
CHEMICAL TEST (Calcium Oxalate Test)
Results:
Clear fluid: ________________________________
Cloudiness: ________________________________
Used agent -> alkalinized with [?] -> added with [?]
Complete decalcification
(+) Ca oxalate (incomplete)
NH3 water ; aqueous NH4 sodium oxalate
424
Physical or X-ray method
ION EXCHANGE RESIN
425
-determines the degree of decalcification
ION EXCHANGE RESIN
426
Reactivation of used Resin:
N/10 HCl and Distilled H2O
427
TISSUE SOFTENERS
Perenyi’s
4% Aq. Phenol (Lendrum’s)
Molliflex
2% HCl
1% HCl in 70% alcohol
428
DEHYDRATION PURPOSE:
-removal of ____________ and ____________ water from tissue -Done after __________ and before _____________
Free ; Bound (intracellular fluid)
Decalci, Fixation ; Clearing
429
must have high affinity with water
Dehydrating Agent
430
DEHYDRATION -Sequence of reagent use:
• Normal tissues:
70% alcohol > 95% alcohol> 100% alcohol
431
Used for routine dehydration
Ethanol 70%
432
Used for routine dehydration
Ethanol 70%
433
BEST dehydrating agent
Ethanol 70%
434
Fast acting, not poisonous
Ethanol 70%
435
Acts as a tissue softener in the addition of 4% Phenol
Ethanol 70%
436
For blood and tissue films
Methanol Absolute
437
For blood and tissue films
Methanol Absolute
438
Toxic dehydrating agent
Methanol Absolute
439
For plants and animals microtechniques
Butyl Alcohol
440
For plants and animals microtechniques
Butyl Alcohol
441
Less shrinkage and has hardening effect than EtOH
Butyl Alcohol
442
Slow acting
Butyl Alcohol
443
Efficient for lipid solvent
Isopropanol Absolute (99.8%)
444
Fully miscible with water, organic solvent, and melted paraffin
Isopropanol Absolute (99.8%)
445
Used in microwave processing
Isopropanol Absolute (99.8%)
446
Rapid acting
Acetone
447
Recommended for urgent biopsy
Acetone
448
Removes lipids
Acetone
449
Extremely volatile
Acetone
450
Rapid, but toxic
EGME (Cellosolve)
451
Allows longer storage
EGME (Cellosolve)
452
Combustible at 110-120 degrees Fahrenheit
EGME (Cellosolve)
453
Propylene-based glycol
EGME (Cellosolve)
454
Miscible with water, alcohol, paraffin wax and xylol
Dioxane (Diethylene dioxide)
455
Can act as a fixative
Dioxane (Diethylene dioxide)
456
Removal of water using calcium chloride or oxide is done through: Graupner's Method or Weisenberger's Method
Dioxane (Diethylene dioxide)
457
Miscible w/ alcohol, xylene, benzene, ether, chloroform & acetone.
THF
458
Does NOT dissolve aniline dyes
THF
459
Used for remixing, clearing and dehydrating paraffin sections
THF
460
Used for dehydration of smear
Triethyl Phosphate
461
-placed at the bottom of the container and is covered with filter paper.
Anhydrous Copper Sulfate crystals
462
-accelerate dehydration by removing water from the dehydrating fluid
Anhydrous Copper Sulfate crystals
463
TEST FOR COMPLETENESS OF DEHYDRATION
Result (+) for water: _________ = indicates that dehydrating fluids must be changed since it has been saturated with water.
Blue color
464
-Immiscibility of reagents used
CLEARING
465
-An intermediate solvent miscible to prior reagents is required
CLEARING
466
Removal of dehydrating agent from the tissues and replacing it by a solvent
CLEARING
467
transparent & translucent tissue
CLEARING
468
Clearing prior to Embedding
Solvent: dealcoholize and act as solvent of [?]
Agents: [?]
paraffin
xylene, toluene, dioxane and chloroform
469
Clearing in Mounting
microscopic preparations transparent (use of solvents with [?])
Agents must be solvents of the Mounting media: [?]
high refractive index
xylene, toluene, terpineol, carbol-xylene
470
GENERAL CONSIDERATIONS CLEARING:
Should be miscible with _____ and _____.
Could be used in amounts at least [?] the volume of tissue
Must be miscible with both the [?]
Most of the clearing agents are [?]
Alcohol ; paraffin
10X
dehydrant & infiltrating agent.
flammable
471
GENERAL CONSIDERATIONS CLEARING:
Should be miscible with _____ and _____.
Could be used in amounts at least [?] the volume of tissue
Must be miscible with both the [?]
Most of the clearing agents are [?]
Alcohol ; paraffin
10X
dehydrant & infiltrating agent.
flammable
472
FACTORS AFFECTING
-agents with low BP are readily replaced by paraffin (exceptChloroform)
-Higher viscosity leads to slower penetration
- does not affect the rate but affects the quality of cleared tissue
Boiling Point
Viscosity
Refractive Index
473
15 - 30 mins/ 30 min - 1 hr
1. Xylene/Xylol
474
Most rapid; ideal for urgent biopsy
1. Xylene/Xylol
475
Excellent clearing agent but tends to make tissues excessively hard & brittle.
1. Xylene/Xylol
476
Can be used for celloidin sections
1. Xylene/Xylol
477
Turns milky when dehydration is not complete
1. Xylene/Xylol
478
Highly inflammable
1. Xylene/Xylol
479
15 - 60 mins
2. Benzene
480
Rapid agent
2. Benzene
481
Highly inflammable
2. Benzene
482
Doesn't make tissues hard & brittle but may cause considerable shrinkage
2. Benzene
483
Carcinogenic
2. Benzene
484
1-2 hours
3. Tolouene/Toluol
485
Similar to xylene but does NOT harden tissues nearly so much
3. Tolouene/Toluol
486
Slower than xylene or benzene
3. Tolouene/Toluol
487
Not carcinogenic but emits toxic fumes
3. Tolouene/Toluol
488
May acidify if vessel is partially filled
3. Tolouene/Toluol
489
For nervous tissues, lymph nodes & embryos
4. Choloroform
490
Best for large specimens and tough tissues
4. Choloroform
491
Slow & Doesn't make tissues transparent
4. Choloroform
492
Slow & Doesn't make tissues transparent
4. Choloroform
493
Toxic to the liver on prolonged inhalation
4. Choloroform
494
Not volatile in paraffin oven
4. Choloroform
495
Tissues tend to float
4. Choloroform
496
2-3 days
5. Cedarwood Oil
497
Recommended for CNS, smooth muscles & skin
5. Cedarwood Oil
498
Recommended for CNS, smooth muscles & skin
5. Cedarwood Oil
499
Slow but minimal shrinkage
5. Cedarwood Oil
500
For both celloidin and paraffin sections
5. Cedarwood Oil
501
Tissue floats - Absolute alcohol
5. Cedarwood Oil
502
Must be followed by immersion in xylene or benzene to remove oil from tissues
5. Cedarwood Oil
503
Turns milky on prolonged storage
5. Cedarwood Oil
504
Clears 70% alcohol
6. Aniline Oil
505
Recommended for embryos, insects and delicate specimens
6. Aniline Oil
506
Slow ; May be adulterated
7. Clove Oil
507
Removes aniline dyes
7. Clove Oil
508
Similar to chloroform
8. Carbon Tetrachloride (CCI4)
509
Both dehydrating and clearing agent
9. Dioxane
510
For large pieces of tissues & embryonic materials
10. Amyl acetate
511
Substitute for cedarwood oil
11. Terpineol (artificial oil of lilac)
512
Slow ; Used in double embedding
12. Methyl Benzoate/Salicylate
513
Slow ; Used in double embedding
12. Methyl Benzoate/Salicylate
514
Clearing tissues directly from water
13. Glycerin, gum syrup and Brun's solution
515
No alcoholization took place ; Improves RI only
13. Glycerin, gum syrup and Brun's solution
516
for materials that are difficult to clear
14. Carbo-Xylene
517
Should be thoroughly rinsed in xylene
14. Carbo-Xylene
518
Should be thoroughly rinsed in xylene
14. Carbo-Xylene
519
Should be thoroughly rinsed in xylene
14. Carbo-Xylene
520
Should be thoroughly rinsed in xylene
14. Carbo-Xylene
521
Found in citrus peels
15. Limonene based
522
Found in citrus peels
15. Limonene based
523
Similar to ester in clearing action and eliminating from wax
15. Limonene based
524
Sold as a xylene replacement
15. Limonene based
525
Composed of long chain aliphatic Hydrocarbon
16. Clearite
526
Used as a substitute for xylene
17. Coconut Oil
527
Does not lose the quality but has tendency to solidify at room temperature
17. Coconut Oil
528
Others:
Oil of bergamot
Phenol in alcohol
Creosote
529
To remove clearing agent from tissues
IMPREGNATION/INFILTRATION
530
make process shorter; easily removed
• Xylene and benzene
531
are difficult to remove; longer infiltration time
• Chloroform and cedarwood oil
532
may affect infiltration
• Type of clearing agent
533
To fill up cavities or tissue spaces
• Tissue spaces or tissue cavities must be filled up to give the tissue a firm consistency and facilitate easy cutting.
IMPREGNATION/INFILTRATION
534
A step in tissue processing that involves removal of clearing agent from tissues so that it will be replaced by a medium that will fill up all cavities and tissue spaces.
IMPREGNATION/INFILTRATION
535
IMPREGNATION/INFILTRATION
Vol. of medium: ______________________
MP: _________________________
Hard tissues require wax with a higher MP
Type of Microtome
[?]
25x
56 deg cel
Fixed-knife microtomes
Heavier knives
536
Simplest, most common & best embedding medium ; Shrinks about 10% on cooling
PARAFFIN WAX
537
PARAFFIN WAX
Melting point for normal routine work: ______________________
56-58 deg cel
538
PARAFFIN WAX
Melting point for normal routine work: ______________________
56-58 deg cel
539
PARAFFIN WAX
Paraffin oven: ____________________ higher
2-5 deg cel
540
Permits many staining procedure
PARAFFIN WAX
541
May cause brittle tissues (overheating) or shrinkage (prolonged infiltration), or soft and shrunken (inadequate)
PARAFFIN WAX
542
Not recommended for fats
PARAFFIN WAX
543
PARAFFIN WAX Time depends on
nature & size of tissue
Type clearing agent
544
Paraffin wax must be pure; used only [?]
twice
545
Water - removed by heating paraffin to _________________________
100 (bp of water) -105 deg cel
546
Requires 4 changes with agitation
MANUAL PROCESSING
547
Requires 2-3 changes w/ constant agitation
AUTOMATIC PROCESSING
548
Requires 2-3 changes w/ constant agitation
AUTOMATIC PROCESSING
549
Wax bath: at least 3 degrees Celsius higher than MP
AUTOMATIC PROCESSING
550
Wax bath: at least 3 degrees Celsius higher than MP
AUTOMATIC PROCESSING
551
Done at negative atmospheric pressure (400500 mmHg)
VACUUM EMBEDDING
552
Utilizes Heat and Vacuum
VACUUM EMBEDDING
553
Wax bath: at least 2-4 degrees Celsius above MP
VACUUM EMBEDDING
554
Effects of heat are prevented
VACUUM EMBEDDING
555
Recommended for urgent biopsies, delicate tissues , lung, brain, connective tissue, decalcified bone, eyes, spleen, CNS
VACUUM EMBEDDING
556
Doesn't require cooling
1. Paraplast
557
More elastic & resilient, Doesn't crumble
1. Paraplast
558
Better ribboning with ease
1. Paraplast
559
MP= 56 - 58 ОС
Embeddol
560
Less brittle than paraplast
Embeddol
561
For eyes
Bioloid
562
Contains rubber (from paraffin)
Tissue Mat
563
MP: 46 - 48 OC
2. Ester Wax
564
Soluble in 95% EA & other clearing agents
2. Ester Wax
565
Harder than paraffin
2. Ester Wax
566
Polyethylene glycols
3. Water Soluble Waxes
567
3. Water Soluble Waxes MP of
38-42 deg or 45-56 deg cel
568
Does not require dehydration & clearing
Carbowax
569
Does not remove neutral fats & lipids
Carbowax
570
CARBOWAX Processing: 70 % - 90% - 100% (2x) at
56 degrees C
571
CARBOWAX Blocking at
50 degrees C
572
CARBOWAX Floating solutions:
PEARSE : Diethyl glycol, Dist. H2O
BLANK & MC CARTHY: gelatin & K2CrO4
573
-highly soluble in water; hygroscopic
CARBOWAX
574
-cannot be floated in water
CARBOWAX
575
-avoid overheating-crumbly blocks
CARBOWAX
576
pure nitrocellulose, suitable for large hollow cavities,calcified tissues and whole embryo
Celloidin
577
Concentrations: 2%, 4%, 8% dissolved in equal parts of ether and alcohol
Celloidin
578
Has rubbery consistency
Celloidin
579
Has rubbery consistency
Celloidin
580
-for bones, teeth and large brain sections or whole organs
WET CELLOIDIN
581
-kept in a jar/dessicator
WET CELLOIDIN
582
-uses 70% alcohol for storage
WET CELLOIDIN
583
-for whole eye sections
DRY CELLOIDIN
584
-Chloroform and Cedarwood oil is added for better transparency
DRY CELLOIDIN
585
-Uses Gilson’s mixture (chloroform + Cedarwood) for storage
DRY CELLOIDIN
586
-Another form of celloidin soluble in equal concentrations of ether and alcohol
NITROCELLULOSE METHOD (Low Viscosity Nitrocellulose)
587
Highly explosive
NITROCELLULOSE METHOD (Low Viscosity Nitrocellulose)
588
-No heat, slow
NITROCELLULOSE METHOD (Low Viscosity Nitrocellulose)
589
-Used in high concentration, produces harder tissue blocks and allows cutting of thinner sections
NITROCELLULOSE METHOD (Low Viscosity Nitrocellulose)
590
-Tissues tend to crack -> To prevent this, add Plasticizers: Oleum ricini & castor oil
NITROCELLULOSE METHOD (Low Viscosity Nitrocellulose)
591
Done when dehydration is to be avoided
GELATIN IMPREGNATION
592
Recommended for frozen tissue, histochemical and enzyme studies
GELATIN IMPREGNATION
593
For delicate specimen, friable tissues and frozen sections
GELATIN IMPREGNATION
594
GELATIN IMPREGNATION Volume: [?] the volume of tissue
25x
595
GELATIN IMPREGNATION Added reagents:
10% Formalin: to harden tissue
1% phenol: to prevent growth of molds
596
GELATIN IMPREGNATION Process: After fixation -> washing out -> [?] w/ 1% phenol (24 hours) -> 20% gelatin with 1% phenol (12 hours) ->twice
10% gelatin
20% gelatin
597
Placing infiltrated tissue in a mold containing the embedding medium which is allowed to solidify
EMBEDDING (Casting/Blocking)
598
EMBEDDING (Casting/Blocking) Requirements:
Embedding mold, medium and infiltrated tissue
599
Commonly used embedding medium is
paraffin wax
600
EMBEDDING (Casting/Blocking)
Temperature of melted paraffin used for embedding [?] above its melting point
5-10°C
601
EMBEDDING (Casting/Blocking)
To allow solidification of the resulting block: -place inside the ref at [?]
-5 degrees Celsius
602
: cutting of excess paraffin wax to form four-sided prism
- trimming
603
: 1st embedding is done with agar or nitrocellulose 2nd embedding is done with paraffin wax
Double embedding
604
infiltrate, support and enclose tissue specimen.
EMBEDDING MEDIA
605
SAME in the infiltrating and embedding General
EMBEDDING MEDIA
606
EMBEDDING MEDIA Characteristics:
-Must be capable of being converted readily from liquid to solid form:
1. Crystallization –
2. Evaporation of the solvent –
3. Polymerization –
(paraffin & carbowax)
(celloidin)
(plastics)
607
Consist of 2 L-shaped strips of heavy brass or metal
Leuckhart's
608
Need to assemble prior to use
Leuckhart's
609
Produce blocks of different sizes
Leuckhart's
610
Embed more than one specimen
Compound embedding unit
611
Consist of a special stainless steel base mold fitted with a plastic embedding ring, which later serves as the block holder during cutting
Plastic embedding rings and base
612
Has a warm plate to manage the impregnated specimen and a cold plate at -5°C for rapid solidification of the block
Tissue Tek
613
Ease of use
Tissue Tek
614
Less paraffin wax needed
Tissue Tek
615
Faster embedding
Tissue Tek
616
Firmly attached tissue and holder
Tissue Tek
617
Permanent identification
Tissue Tek
618
Produce perfect blocks even without trimming
Peel away molds
619
Economical and easy to prepare
Paper boat
620
Need to smear inner mold with glycerin prior to use
*Glycerin: facilitate easy removal
Plastic ice trays
621
• Embedding for EM
PLASTIC (RESIN) EMBEDDING
622
• High resolution light microscopy
PLASTIC (RESIN) EMBEDDING
623
• For extremely hard tissues
PLASTIC (RESIN) EMBEDDING
624
(i.e., Bisphenol, Glycerol and Cyclohexene dioxide)
Epoxy
625
– for EM, seldom used
Polyester
626
– for LM (i.e., MMA, GMA)
Acrylic
627
not universally successful
Limonene based
628
not less than 10 x the tissue volume
Dehydrating Agent
629
increasing strengths
Dehydrating Agent
630
offensive odor
THF
631
may cause conjunctivitis
THF
632
Unsuitable for brain & lymph nodes
Xylene
