MODULE NO.

: 3
MODULE TITLE: “Tissue Processing made EASY”
TOPIC: Tissue Processing (Trimming to Labeling)
WRITER: Ruby G. Meim, RMT, MSMT
SUBMITTED BY: CHRISTINE JOY DOLERA 3BSMT1

PRETEST
1. The tilt of the angle of the knife is set at:
C.
2. The tilt of the angle is set at 10 O, what is the rake angle?
B.
3. Adhesive of choice for tissue ribbons, EXCEPT:
A.
4. Dirt or grit on the microtome knife is best cleansed with:
C.
5. Thymol is added to Meyer's egg albumin to prevent growth of:
A.

STUDY:
Dehydrating Use Composition Advantage/s Disadvantage/s
Agents
Methyl alcohol Blood and tissue A Methyl group  Can be used as a Toxic to the
films (CH3) linked substitute for ethanol body
Smear preparation with a hydroxy Used as a fixative
group (OH)
Tetrahydrofuran Used for demixing, Oxalane  Most staining Toxic
dehydrating and procedures give
paraffin sections improved results
 Miscible with lower
alcohols, ethers,
chloroforms, acetone
and benzene
Dissolve many
substances
Acetone Used in small pieces Composed of  Cheap, rapid acting  Highly
of tissue the elements dehydrating agent flammable
carbon, More miscible when  Penetrates
hydrogen, and epoxy resins than tissue poorly
oxygen alcohol  Causes
brittleness
 Most lipids
are removed
from tissues
 Extremenly
 volatile and
inflammable
 Produces
considerable
tissue
shrinkage

Dioxane It is used as both Ethylene oxide  Produces less tissue  Expensive

dehydrating and shrinkage  Extremely
clearing agent Tissues can be left for dangerous
long period of time  Ribbon poorly
without affecting Vapor produce
consistency and is highly toxic
staining property of the
specimen
Cellosolve Ethylene glycol  Cellosolve dehydrates Toxic
monoethyl ether rapidly
Stored in it for months
without producing
hardening or distortion.
Triethyl Used to dehydrate Triester of  Removes water very None
sections and ethanol and readily and produces
smears following phosphoric acid very little distortion
certain stains and hardening
Produce minimum
shrinkage

Clearing agent Composition Advantage/s Disadvantage/s

Xylene Isomer dimethylbenzene  most rapid clearing – for
urgent biopsies (15-
30mins)
 makes tissues transparent
 miscible with absolute
alcohol and paraffin
 does not extract out aniline
dyes
 for mounting procedures-
does not dissolve celloidin
 during embedding and
impregnation -evaporates
quickly in paraffin oven –
can be readily replaced by
 highly inflammable
 makes tissue
excessively hard if
used over 3 hours
 not suitable for
nervous tissues and
lymph nodes –
causes considerable
hardening and
shrinkage
 wax
 cheap
Toluene Methylbenzene  miscible with both absolute
alcohol and
 paraffin
 acts fairly rapidly
recommended for routine
purposes
 does not make tissues
excessively hard and brittle
if left for 24 hours
Benzene Composed of six carbon  rapid acting – for urgent
atoms joined in a planar biopsies
ring with one hydrogen  (15-60mins) and routine
atom attached to each purposes
 volatilizes rapidly in
paraffin oven – easily
eliminated from the tissue
 does not make tissues hard
and
 brittle
 causes minimum shrinkage
 makes tissues transparent
Chloroform Trichloromethane  for routine work (6-24
hours)
 miscible with absolute
alcohol
 causes minimum
shrinkrage - for tough
tissues
 for large tissue specimens
 not inflammable
Cedarwood oil Largely composed of  very penetrating
 slower than xylene and
benzene
 acidifies in a partially
filled vessel
 highly concentrated
solutions will emit fumes
that
 are toxic upon
prolonged exposure
 more expensive
 High inflammable
 Causes considerable
tissue shrinkage if left
for a long time
 Excessive exposure may
be extremely toxic to
man and may damage
bone marrow causing
aplastic anemia
 toxic to the liver after
prolonged inhalation
 wax impregnation after
chloroform clearing
relatively slow
 does not make tissues
transparent
 not very volatile in
paraffin oven
 vapor may attack rubber
seal used in vacuum
 impregnating bath
 complete clearing is
difficult to evaluate
 tissues tend to float in
chloroform
 evaporates quickly from
a water bath
 extremely slow – not for
 cedrene  miscible with 96% alcohol
which it removes readily
 clears celloidin in 5-6 days
 causes minimal shrinkage
and hardening
 tissues may be left
indefinitely without causing
considerable damage and
distortion
 makes tissues transparent
 improves cutting of
sections
Aniline oil 6 carbon atoms, 1 • recommended for clearing
nitrogen atom and 7 embryos, insects, and very
hydrogen atoms delicate specimens
 ability to clear 70% alcohol
w/o excessive shrinkage
and hardening
Clove oil Mixture of eugenol, • causes minimum shrinkage
eugenyl acetate and
caryophyllene
Carbon Tetrachloromethane  May be used in clearing
tetrachloride tissues for embedding
 Properties similar to that of
chloroform but relatively
cheaper
routine purposes
 hard to be eliminated
from tissues in paraffin
bath
 quality not always
uniform and good
 becomes milky upon
prolonged storage,
should be filtered before
use
 Cedarwood oil
previously used to clear
acetic-
 alcohol fixed tissues may
produce crystals
 very expensive
 not normally utilized as
a routine clearing agent
 quality not guaranteed
– tendency to become
adulterated
 wax impregnation slow
and difficult
 tissues become brittle,
aniline dyes
 removed, celoidin is
dissolved
 expensive
 unsuitable for routine
clearing purposes
 produces considerable
tissue
 hardening
 highly toxic- dangerous
to inhale on prolonged
exposure
DRAW THE DIFFERENT TYPES OF EMBEDDING MOLDS:

ANALYSIS:
ACTIVITY 1: Given the following pictures, discuss briefly the causes/ of possible
problem/s in sectioning and the remedy/remedies/trouble shooting.
A.

Presence of vertical scratches

Cause: defect in the blade edge, calcium, bone or hard
material in the specimen.
Prevention:
- Block holder must be adjusted at the beginning
of sectioning so that the block face and the blasé
are perfectly parallel.
B.

Holes in the section

Cause: Block is faced too aggressively. Specimen is either
dehydrated or improperly processed.
Prevention:
- Chill the block with ice before cutting and discard
ribbons until the hole disappear.
- Face the block less aggressively, with smaller
micrometer advances of the block for each
section removed.

C.

Chatter or microscopic vibration

Cause: Over dehydration or lack of moisture in the
tissue. Dull blade or too much blade tilt. Cutting too
rapidly.
Prevention:
- Proper processing
- Restore moisture by facing the block down on an
ice tray
- Decrease the blade tilt.
- Decrease cutting speed: one wheel per second is
a reasonable speed.

ACTIVITY 2: Answer the following questions:

A. What are some implications of improperly labels tissue slides?

Labeling is a very crucial part of the tissue processing because it serves as the key towards a
successful treatment of patients thus it is very costly therefore, we cannot afford making any
errods. Improperly labeled specimen would harm the patient due to failure to provide proper
and immediate care to the patient based on the inaccurate test and results.

B. Describe a well-mounted tissue sample?

A well-mounted tissue sample has a well-defined three-dimensional structure of the sample.
Its mounting media have a refractive index as close as possible providing good clarity and
contrast. Its stain is not faded even after the mounting process and it must be stable to
preserve and support a stained section for light microscopy. The slide must be free of optical
distortions to avoid viewing artifacts.
 C. What are the possible causes of understaining? Overstaining?
Understaining may be due to insufficient exposure of the slide to the stain, exhausted (over
oxidized or depleted), and over differentiation. Some stains are also affected by the pH,
sections may be too thin, or left long in the dehydration. On the other hand, overstaining may
be caused too long exposure of the slide to the stain, sections are too thick, and the
differentiation step is too short/poor.

POST TEST:
1. Eukitt is diluted using:
C.
2. In preparation of hematoxylin stain, the following must be added:
D.
3. The bevel angle is maintained by:
C.
4. In EA-50 for Pap's, the number 50 represents:
A.
5. The substance that hastens the binding affinity of the dye to the tissue is called:
C.