DECALCIFICATION - DEHYDRATION - CLEARING Flashcards
(110 cards)
1
Q
● Not commonly done in the Histopath Laboratory
● Only certain hard human tissues need decalcification
(bone, teeth, nails)
● Same ratio in fixation and decalcification
A
DECALCIFICATION
2
Q
● High concentrations and greater amounts of fluid will increase the speed of the process
● More concentrated acid solutions decalcify bone more rapidly but may cause more harm to the tissue.
A
DECALCIFICATION
3
Q
FD CIETS SML
A
Fixation → Dehydration → Clearing → Impregnation, → Embedding → Trimming → Sectioning → Staining → Mounting → Labeling
4
Q
● Heat can hasten _____________
but can also damage the tissue
A
decalcification
5
Q
Recommended ratio of fluid to tissue volume for decalcification
A
20:1
6
Q
Ideal time required for Decalcification
A
24-48 hours
7
Q
● Dense bone tissues usually
require up to ___ days or longer
in order to complete the
process.
A
14 days
8
Q
At ___C impaired nuclear staining of Van Gieson’s stain for collagen fibers.
A
At 37 C
8
Q
Optimum temperature = Decalcification
A
Room temp (18-30°C)
8
Q
At __ C = tissue will undergo
complete digestion within
24-48 hours.
A
55 C
9
Q
DECALCIFYING AGENTS (4)
A
● Acids
● Chelating Agents
● Ion Exchange Resins
● Electrical Ionization
10
Q
● Most commonly used, but not the best decalcifying agent
A
Nitric Acid
11
Q
○ EDTA (Ethylene Diamine Tetraacetic Acid)
○ Used as an anticoagulant to bind calcium
A
Chelating Agents
12
Q
2 examples of Nitric Acid
A
○ Perenyi’s Fluid
○ Phloroglucin-Nitric Acid
13
Q
- functions as both decalcifying agent and tissue softener
A
Perenyi’s Fluid
14
Q
most rapid decalcifying agent
A
Phloroglucin-Nitric Acid
15
Q
- best general decalcifying agent
A
5% Formic Acid
15
Q
● Both a fixative and decalcifying agent
● Recommended for small pieces of bones and teeth
A
Formic Acid
16
Q
Example of Hydrochloric Acid
A
Von Ebner’s Fluid
17
Q
What is the best general decalcifying agent?
A
5% Formic acid
17
Q
recommended for teeth and small pieces of bones
A
Von Ebner’s Fluid
18
Q
What kind of acid makes up Von Ebner’s?
A
Hydrochloric acid
18
Q
What is the most commonly used decalcifying agent?
A
Nitric Acid
19
Q
3 WAYS TO MEASURE DECALCIFICATION
A
- Physical/Mechanical Test
- X-ray/Radiological Method
- Chemical Method
20
● Inaccurate since it is very subjective
● Commonly used in schools since it is cheap.
Physical/Mechanical Test
21
● Done by touching or bending tissue with the fingers
● Alternate method: pricking the
tissue with a fine needle or a probe
Physical/Mechanical Test
22
● For unduly hard tissues that may damage the microtome knives
● Utilized to soften tough tissue
TISSUE SOFTENERS
22
● Most ideal, Most Sensitive, Most
reliable method.
● The best way to measure the extent of decalcification
X-ray/Radiological Method
23
● Very Expensive; not commonly done
● Uses X-ray to measure the extent of decalcification
X-ray/Radiological Method
24
● Calcium Oxalate test
● Simple, reliable, recommended for routine purposes
Chemical Method
25
● Must not be skipped so that the next step, Impregnation (Paraffin Wax), to completely fill the spaces in between the specimen.
1. Water and Paraffin Wax does not mix
DEHYDRATION
26
(4) Examples of TISSUE SOFTENERS
● 4% aq. Phenol.
● Molliflex
● 2% HCl
● 1% HCl in 70% alcohol
27
● We want the impregnating medium to be able to penetrate
the spaces between the organ and tissue
● ___________ fluids are generally used in increasing strengths (all the aqueous tissue fluids are removed but with little disruption to the tissue due to diffusion currents)
DEHYDRATION
27
● Aims to remove fixative and water from the tissue and replacing them with dehydrating fruit in preparation for impregnation
DEHYDRATION
28
● Used in increasing strengths or
ascending grades
○ In order for the tissue to not be
distorted
○ 70% Alcohol → 80% Alcohol →
90% Alcohol → 100% Alcohol (2x)
Alcohol
29
● MOST COMMONLY USED DEHYDRATING AGENT
● Also used for fixation
Alcohol
30
For routine dehydration of
tissues. BEST DEHYDRATING
AGENT
Ethyl Alcohol
31
● Used for blood and tissue films
Methyl Alcohol
32
● Used for plant and animal microtechniques
Butyl Alcohol
33
● Less shrinkage and hardening than ethanol
Isopropyl Alcohol
33
● Denatured Alcohol
● Ethanol + Small amount of Methanol
Industrial Methylated Spirit
34
● Both Fixative and Dehydrating Agent
● Also used for diffusible enzymes, brain (for rabies diagnosis)
Acetone
35
● Sometimes, instead of using 100% again at the end of dehydration, ___________ can be used.
Acetone
36
● Both dehydrating and clearing agent at the same time
● Diethylene Dioxide and
Tetrahydrofuran
37
(6) COMMONLY USED DEHYDRATING AGENTS
1. Alcohol
2. Acetone
3. Dioxane
4. Tetrahydrofuran (THF)
5. Cellosolve (Ethylene Glycol Monoethyl Ether)
6. Triethyl Phosphate
38
● Acts as a tissue softener when added with 95% ethanol
● If the tissue is already soft, no need to add this
4% Phenol
39
ADDITIVES TO DEHYDRATING AGENTS (2)
1. 4% Phenol
2. Anhydrous Copper Sulfate
40
● BOTH dehydrating and indicator of H2O (water) content (100% ETOH)
Anhydrous Copper Sulfate
41
Anhydrous Copper Sulfate
● If copper sulfate is dry = ______ in color
● Has water/Hydrated = ________ in color (incomplete dehydration)
White
Blue
42
A typical dehydration sequence for specimens NOT more than 4 mm thick would be:
1. 70% ethanol - 15 min
2. 90% ethanol - 15 min
3. 100% ethanol - 15 min
4. 100% ethanol - 15 min
5. 100% ethanol - 30 min
6. 100% ethanol - 45 min
43
NOTE AND RECALL:
Higher concentration of alcohol = water concentration will decrease (slowly removing the water from interstitial spaces so tissues will not wrinkle)
After you remove the water, alcohol is now inside the interstitial spaces after the dehydration process
44
What is the Best General Decalcifying Agent?
5% Formic Acid
45
What is considered to be the Best General Tissue Fixative?
10% Buffered Neutral Formalin
46
What is the Best Way to Measure the Extent of Decalcification?
X-Ray or Radiological Method
47
What is the Best Dehydrating Agent?
Alcohol
48
Most commonly used Impregnating Medium?
Paraffin Wax
49
■ Not soluble to water
■ Not soluble to alcohol
■ Needs to be the one to fill the spaces in the specimens so that when it is cut in the microtome, there will be no empty holes.
Paraffin Wax
50
Aka: DEALCOHOLIZATION
CLEARING
51
● Making the tissues transparent
● Process of replacing the dehydrating fluid with a fluid that
is miscible with BOTH the dehydrating fluid and the
impregnating/embedding medium
CLEARING
52
CLEARING AGENTS SUITABLE FOR ROUTINE USE:
● Most commonly used
● Most rapid
Xylene/Xylol
53
● Some of the other uses:
○ Removes alcohol from tissue
section, fat, and paraffin
○ For cleaning tissue processors
Xylene/Xylol
54
If xylene turns MILKY =
___________ DEHYDRATION
INCOMPLETE DEHYDRATION
55
● Toxic to the liver after prolonged inhalation and it does not make the tissues transparent
Chloroform
56
● Used for TOUGH tissue
specimens
Chloroform
57
especially recommended for CNS tissues and cytological studies,
particularly of smooth muscles and skin
Cedarwood oil and clove oil
58
Can be utilized to replace Xylene
because it is not as toxic and is
cheaper
Citrus Fruits Oils
59
59
Recommended for clearing
embryos, insects, and very
delicate specimens
Aniline Oil
60
Why do we need to remove the alcohol?
The paraffin wax that will be utilized later on is not soluble to alcohol
60
Aka: INFILTRATION
IMPREGNATION
61
● Process of replacing the clearing agent with the infiltrating medium
● The medium used to infiltrate the tissue is usually the same medium used for embedding
IMPREGNATION
62
FOUR TYPES OF TISSUE IMPREGNATION AND EMBEDDING MEDIA
1 Paraffin
2 Celloidin (Collodion)
3 Gelatin
4 Plastic
63
● The man who introduced paraffin wax embedding:
Butschlii
64
● Simplest, most common and the BEST infiltrating/embedding medium
● NOT recommended for fatty tissues
PARAFFIN
65
Temperature of paraffin oven =
55-60 C
66
Paraffin oven must be maintained at a temperature
2-5 C above the MP (Melting Point) of the paraffin wax
67
● MP: 56-57 C
● Mixture of highly purified paraffin and synthetic plastic polymers
● More elastic and resilient than
paraffin
● SPECIMEN: For large dense tissue blocks such as bones and brain
Paraplast
68
● MP: 56-58 C
● Less brittle and less compressible than paraplast
Embeddol
69
● Recommended for embedding
eyes
Bioloid
70
A product of paraffin, containing
rubber, with the same property as paraplast
Tissue Mat
71
● MP: 46-48 C
● Harder than paraffin
● NOT soluble in water
● Soluble in 95% ETOH (ethyl alcohol) and other clearing agents
● Can be used for impregnation
without prior clearing of the tissue
○ as it is soluble in ETOH
Ester Wax
72
● MP: 38-42 C or 45-56 C
● Mostly polyethylene glycols
○ No need to perform dehydration and clearing
Water-soluble waxes
72
73
Suitable for many enzyme
histochemical studies
Carbowax
73
● Purified form of nitrocellulose
CELLOIDIN (COLLODION)
74
● Suitable for specimens:
○ With large hollow cavities
○ Hard and dense tissues (bones and teeth)
○ Large tissue sections of the whole embryo
CELLOIDIN (COLLODION)
75
2 METHODS FOR CELLOIDIN IMPREGNATION
1. Wet celloidin
2. Dry celloidin
76
Recommended for bones, teeth, large brain sections, and whole organs/organs with large cavities
Wet celloidin
77
Preferred for processing whole eye sections
Dry celloidin
78
Name of the other impregnating medium that may also be for the eye section?
Bioloid
79
Fixative For Embryos
Bouin’s solution
80
Impregnation Medium
For Embryos
Celloidin
81
Impregnating Medium
For Eyes
Bioloid or Celloidin
82
● Share the same characteristic as the carbowax
● Rarely used except when dehydration is to be avoided
● Used when tissues are for histochemical and enzyme
studies
GELATIN
83
● Embedding medium for delicate specimens and frozen
tissue sections
○ When we are using frozen tissues usually it is fresh meaning there is still a water inside
■ Even though there is still water inside the gelatin can still pass through
GELATIN
84
● Water-soluble impregnating medium
○ Not a wax
○ No need for dehydration
Gelatin
85
Why are we allowed not to do dehydration if we intend to use gelatin as our impregnating
medium?
Because gelatin is a water soluble
impregnating medium
Gelatin → Water-Soluble Impregnating Medium
86
Why do we remove water?
○ To prepare the tissue into the paraffin wax impregnation
○ Paraffin wax is not soluble to water
87
What is the impregnating medium that we can use for impregnating frozen sections?
Gelatin
87
Used for Electron Microscopic Studies
PLASTIC / RESIN
88
PLASTIC / RESIN are CLASSIFIED INTO 3 categories
1 Epoxy
2 Polyester
3 Acrylic
89
Brand name: Bisphenol A
Araldite
90
Brand name: Glycerol
Epon
90
Brand name: Cyclohexene dioxide
Spurr
91
Most commonly used routine tissue impregnation medium
Paraffin wax
92
Impregnation medium for large dense tissues like bones, brain and whole organs (embryo
Celloidin
92
Impregnation medium for
frozen sections
Gelatin
93
Impregnation medium for
electron microscopic studies
Plastic / Resin
94
Since Epon is a glycerol, and a
glycerol is a form of epoxy, Epon
therefore is used for _______
microscopic studies
Electron
94
What is the use/purpose of EPON?
Utilize for electron microscopic studies
■ One of the impregnating medium to utilize for that purpose
■ A kind of glycerol epoxy plastic
impregnating medium
■ A brand name of glycerol epoxy
plastic impregnating medium
