CHAPTER 10 CLEARING Flashcards
1
Q
is a colorless clearing agent that is most commonly used in histology laboratories.
A
Xylene (Xylol)
2
Q
Clearing time is usually 1/2 to 1 hour.
A
Xylene (Xylol)
3
Q
It is used for clearing, both for embedding and mounting procedures.
A
Xylene (Xylol)
4
Q
It is generally suitable for most routine histologic processing schedules of less than 24 hours, and when the tissue block size is less than 5 mm. in thickness.
A
Xylene (Xylol)
5
Q
is reasonably cost effective and works well for short-term clearing of small tissue blocks.
A
Xylene (Xylol)
6
Q
is one of the routinely used chemical in histology and pathology laboratories because of its vital role in the paraffin wax tissue processing method.
A
Xylene (Xylol)
7
Q
It is mostly used as a clearing agent during tissue processing and as a dewaxing agent during staining.
A
Xylene (Xylol)
8
Q
It is also used in cover slipping, in cleaning tissue processors, as solvent to remove synthetic immersion oil from the microscope objective and in recycling of used slides.
A
Xylene (Xylol)
9
Q
However, several toxicities believed to be caused by intermediate products of its metabolism such as methyl benzaldehyde have been reported. These include central nervous system disorders, respiratory depression, abdominal pain, dryness and redness of skin, dermatitis, liver diseases, nephrotoxicity, conjunctivitis, and teratogenic and fetotoxic effects.
A
Xylene (Xylol)
10
Q
It is the most rapid clearing agent, suitable for urgent biopsies which it clears within 15-30 minutes.
A
Xylene (Xylol)
11
Q
It makes tissues transparent.
A
Xylene (Xylol)
12
Q
It is miscible with absolute alcohol and paraffin.
A
Xylene (Xylol)
13
Q
It does not extract out aniline dyes.
A
Xylene (Xylol)
14
Q
For mounting procedures, it does not dissolve celloidin and can, therefore, be used for celloidin sections.
A
Xylene (Xylol)
15
Q
It evaporates quickly in paraffin oven and can, therefore, be readily replaced by wax during impregnation and embedding.
It is cheap.
A
Xylene (Xylol)
16
Q
It is highly inflammable and should be appropriately stored.
A
Xylene (Xylol)
17
Q
If used longer than 3 hours, it makes tissues excessively hard and brittle.
A
Xylene (Xylol)
18
Q
It causes considerable hardening and shrinkage of tissues; hence, is not suitable for nervous tissues and lymph nodes.
A
Xylene (Xylol)
19
Q
becomes milky when an incompletely dehydrated tissue is immersed in it
A
Xylene (Xylol)
20
Q
may irritate eyes, nose and respiratory tract.
A
Xylene (Xylol)
21
Q
It can be absorbed through the skin and cause dermatitis.
A
Xylene (Xylol)
22
Q
At high concentrations, it is toxic and narcotic.
A
Xylene (Xylol)
23
Q
is better at preserving tissue structure and is more tolerant of small amounts of water left behind in the tissues than xylene
A
Toluene
24
Q
more expensive than xylene and more toxic, so toluene is less commonly used.
A
Toluene
25
may be used as a substitute for xylene or benzene for clearing both during embedding and mounting processes.
Toluene
26
Time recommended for clearing is 1 -2 hours.
Toluene
27
It is miscible with both absolute alcohol and paraffin.
Toluene
28
It acts fairly rapidly and is recommended for routine purposes.
Toluene
29
Tissues do not become excessively hard and brittle even if left in toluene for 24 hours.
Toluene
30
Clears overnight.
Toluene
31
It is not carcinogenic.
Toluene
32
It is slower than xylene and benzene.
Toluene
33
It tends to acidify in a partially filled vessel.
Toluene
34
Highly concentrated solutions will emit fumes that are toxic upon prolonged exposure.
Toluene
35
It is more expensive.
Toluene
36
is preferred by some as clearing agent in the embedding process of tissues because it penetrates and clears tissues rapidly.
Benzene
37
It used to be a popular routine clearing agent until recently when its highly carcinogenic properties were recognized.
Benzene
38
Its use for clearing purposes is therefore strongly discouraged.
Benzene
39
It is rapid acting, hence is recommended for urgent biopsies (15-60 minutes) and routine purposes.
Benzene
40
It volatilizes rapidly in paraffin oven and is therefore easily eliminated from the tissue.
Benzene
41
It is miscible with absolute alcohol.
Benzene
42
It does not make tissues hard and brittle. It causes minimum shrinkage.
Benzene
43
It makes tissues transparent. It clears overnight.
Benzene
44
It is highly flammable.
Benzene
45
If a section is left in benzene for a long time, considerable tissue shrinkage may be observed. Hence, tissues should be transferred to paraffin wax as soon as possible.
Benzene
46
Excessive exposure to benzene may be extremely toxic to man and may become carcinogenic or it may damage the bone marrow resulting in aplastic anemia. If ever benzene is to be used for clearing, the laboratory should be well-ventilated.
Benzene
47
slower in action than xylene, but causes less brittleness
Chloroform
48
Thicker tissue blocks, even those up to I cm. in thickness, can be processed.
Chloroform
49
However, tissues placed in it do not become translucent.
Chloroform
50
It is recommended for routine work (6-24 hours).
Chloroform
51
It is miscible with absolute alcohol.
Chloroform
52
It is recommended for tough tissues (e.g. skin, fibroid and decalcified tissues) for nervous tissues, lymph nodes and embryos because it causes minimum shrinkage and hardening of tissues.
Chloroform
53
It is suitable for large tissue specimens.
Chloroform
54
It is not inflammable.
Chloroform
55
It is relatively toxic to the liver after prolonged inhalation; this may be prevented by adequate room ventilation.
Chloroform
56
Wax impregnation after clearing is relatively slow.
Chloroform
57
It does not make tissues transparent.
Chloroform
58
It is not very volatile in paraffin oven; hence, it is difficult to remove from paraffin sections.
Chloroform
59
It may even produce considerable deterioration of the wax.
Chloroform
60
Its vapor may attack the rubber seal used in vacuum impregnating bath.
Chloroform
61
Complete clearing is difficult to evaluate.
Chloroform
62
Tissues tend to float in chloroform; this may be avoided by wrapping the tissues with absorbent cotton gauze to facilitate sinking of the section in solution.
Chloroform
63
It evaporates quickly from a water bath.
Chloroform
64
is used to clear both paraffin and celloidin sections during
the embedding process.
Cedarwood Oil
65
It is especially recommended for central nervous system tissues and cytological studies, particularly of smooth muscles and skin. It requires two changes in clearing solution.
Cedarwood Oil
66
Clearing is usually complete in 2-3 days.
Cedarwood Oil
67
It is very penetrating.
Cedarwood Oil
68
It is miscible with 96% alcohol which it removes readily.
Cedarwood Oil
69
It clears celloidin in 5-6 days.
Cedarwood Oil
70
It causes minimal shrinkage of tissues.
Cedarwood Oil
71
Tissues may be left in oil indefinitely without considerable damage and distortion.
Cedarwood Oil
72
It does not dissolve out aniline dyes.
Cedarwood Oil
73
It makes tissues transparent.
Cedarwood Oil
74
It does not harden tissues.
Cedarwood Oil
75
It does not interfere too seriously with paraffin penetration if it is not completely removed.
Cedarwood Oil
76
Clearing with cedarwood oil often improves cutting of the sections.
Cedarwood Oil
77
It is an extremely slow clearing agent, hence, it is not recommended for routine purposes.
Cedarwood Oil
78
It is slightly slower in penetrating than benzene.
Cedarwood Oil
79
It is hard to eliminate from the tissues in paraffin bath, making the wax impregnation process very slow.
Cedarwood Oil
80
This may be improved or hastened by transferring the specimen from oil to benzene for 1/2 hour before finally placing the tissue in wax.
Cedarwood Oil
81
Quality is not always uniform and good. Tissues cleared in it initially float before gradually staying to the bottom as clearing proceeds. Hence, the tissue may dry out before it is completely cleared. This can be prevented by superimposing absolute alcohol on the surface of the clearing agent. Once saturated, the specimen should then be transferred to a fresh solution.
Cedarwood Oil
82
It becomes milky upon prolonged storage and should be filtered before use.
Cedarwood Oil
83
It that has been previously used to clear acetic-alcohol fixed tissues may produce crystals with a melting point of approximately 35°C and therefore interfere with adequate clearing of tissue. The solution must be heated to 200°C in order to dissolve the crystals and restore the solution to its normal state.
Cedarwood Oil
84
It is very expensive.
Cedarwood Oil
85
This is not normally utilized as a routine clearing agent but it is
recommended for clearing embryos, insects and very delicate specimens, due to its ability to clear 70% alcohol without excessive tissue shrinkage and hardening.
Aniline oil
86
This reagent causes minimum shrinkage of tissues.
Clove oil
87
However, its quality is not guaranteed due to its tendency to become adulterated.
Clove oil
88
Wax impregnation after clearing with it is slow and difficult.
Clove oil
89
Tissues become brittle, aniline dyes are removed, and celloidin is dissolved.
Clove oil
90
All of these, in addition to the expensiveness of the solution, make it unsuitable for routine clearing purposes.
Clove oil
91
may be used in clearing tissues for embedding.
Carbon tetrachloride
92
Its properties are very similar to that of chloroform although it is relatively cheaper.
Carbon tetrachloride
93
Its disadvantage is the same as that of chloroform.
Carbon tetrachloride
94
It produces considerable tissue hardening, and is dangerous to inhale on prolonged exposure due to its highly toxic effects.
Carbon tetrachloride
95
is superior to ordinary dehydrating and clearing agents due
to its ability to perform two processes at the same time, thereby shortening the total processing time and allowing more time for fixation.
Tetrahydrofuran
96
It is non-toxic but has offensive odor and should be used in a well-ventilated room.
Tetrahydrofuran
97
is miscible both with water and paraffin.
Dioxane
98
It is used primarily when time is important because the tissues may be embedded with paraffin within 4 hours after fixation.
Dioxane
99
The tissues are transferred to dioxane straight from Bouin's fluid or a formalin fixative.
Dioxane
100
is changed 3 times within 4 hours and the tissues are transferred directly to paraffin (3 changes are made in a total of 90 minutes)
Dioxane
101
causes greater shrinkage than xylene does.
Dioxane
102
In addition, it is dangerous.
Dioxane
103
Fumes are toxic to human especially to the liver.
Dioxane
104
Other Xylene Substitutes:
105
are isoprene polymers found in essential oils originally derived from plants, though some are now synthesized.
Terpenes
106
They are the earliest transition solvents to be used in histology and include turpentine and oils of bergamot, cedarwood, clove, lemon, oreganum and sandalwood.
Terpenes
107
In general the natural oils are not highly pure compounds but contain several substances.
Terpenes
108
clear tissues and celloidin sections from 80%-95% alcohol, render tissues transparent and have a slow gentle non-hardening action.
Terpenes
109
Most are generally regarded as safe though some have particularly strong odors which can be overpowering, requiring good laboratory ventilation.
Terpenes
110
are moderately effective solvents, but they too are considered toxic.
Terpenes
111
Solvents in this class also dry slowly, leave an oily residue on slides and are relatively expensive.
Terpenes
112
One of the recommended xylene substitutes from the terpene family
Limonene
113
a volatile oil found in citrus peels which goes by several trade names.
Limonene
114
It is a natural oil found in the skins of citrus fruits, such as lemons or oranges, and in cooking is usually referred to as lemon or orange zest.
Limonene
115
is obtained industrially by the steam distillation of orange peel which is a byproduct of the orange juice industry.
Limonene
116
It is a clear, colorless fluid with a distinctly citrus aroma, not unpleasant to most people, although some do not like it.
Limonene
117
is often sold as a xylene replacement and some technologists substitute it for xylene in other uses, but this is not universally successful.
Limonene
118
When used as the clearant immediately prior to cover slipping, there are some reports that the mounting medium, usually dissolved in either toluene or xylene, does not mix well with it.
Limonene
119
In such cases, replacing the it with xylene or toluene, or quickly dipping the section in either one just prior to cover slipping should be effective. This does, of course, defeat the purpose of the replacement to a certain degree.
Limonene
120
offer the clearing action with the lowest hazard rating of all xylene alternatives.
Orange oil based clearing agents
121
It is excellent for preserving fine tissue structure, and can often be used in place of xylene with no alteration of protocol.
Orange oil based clearing agents
122
In using a product containing orange oils, it is important to use a product which has been rigorously purified then stabilized.
Orange oil based clearing agents
123
[?] that are neither pure nor stable can break down to produce compounds which will interfere with staining procedures.
Orange oil based clearing agents
124
can be effective solvents, but they are considered toxic chemicals, posing serious health risks.
Chlorinated hydrocarbons
125
Government regulations have restricted most of the effective solvents in this class.
Chlorinated hydrocarbons
126
is an efficient substitute for xylene, as it is non-hazardous, less expensive and causes less shrinkage of the tissue.
Coconut oil
127
It can be used as a de- alcoholization agent in the histopathological laboratory, without losing the quality of the histological details.
Coconut oil
128
The only drawback associated with coconut oil, is its tendency to get solidified at a lower temperature.
Coconut oil
129
However, this can be overcome by performing the clearing procedure in an incubator, maintaining the required temperature.
Coconut oil
130
Substitution of the conventional xylene with [?] as a clearing agent during tissue processing and as a dewaxing agent during staining gives good tissues, sections and histological slides.
bleached palm oil
131
is nontoxic, nonhazardous, nonflammable, bio-degradable, economic, easy to handle, and readily available.
bleached palm oil
