Exercise 4

Name: DAYAGAN, GWYNETH MARIE M. Yr. & Sec.
: BSMT - 2C
EXERCISE 4: DEHYDRATION and CLEARING

Dehydration is the process wherein water and fixative are removed from the tissue
and replaced with a dehydrating fluid in preparation of impregnation. Reagents for this
purpose are called dehydrating agents. Water is immiscible with the medium where the tissues
will be embedded, therefore dehydration must be performed prior to subsequent processing.
The most common dehydrating agent is the ascending grades of alcohol. The volume of the
dehydrating agent should not be less than 10 times the volume of the tissue. Ethyl alcohol
mixes with and has affinity for water which allows it to penetrate easily between the tissue
cells.
While clearing or dealcoholization is performed so that the alcohol in the tissues

are removed and replaced a fluid that will dissolve the wax with which the tissue is to be
impregnated or the medium in which the tissue is to be mounted. The clearing agent serves to
mix with alcohol and removes it from the tissues. The volume of the clearing agent should not
be less than 10 times the volume of the tissue. Xylene as a clearing agent removes and
replaces water from the tissues, leaving the tissue with a translucent appearance
1. OBJECTIVES
At the end of the laboratory exercise, the students should be able to:
1.1 Accurately prepare 70% and 80% ethyl alcohol from 95% (stock) ethyl alcohol.
1.2 Properly dehydrate tissues using manual methods of processing.
1.3 Demonstrate properly manual method of clearing.
2. MATERIALS
2.1 Fixed Liver, Lung & Kidney tissue 2.8 Funnel

2.2 Dental floss 2.9 Calculator
2.3 Pasture pipette 2.10 Marker pen
2.4 Masking tape / gum label 2.11 Ruler
2.5 10ml Reagent bottles / Vials 2.12 Litmus paper
2.6 Serologic pipette 2.13 Aspirator
2.7 Stirring rod
3. REAGENTS
3.1 Absolute ethyl alcohol ……….….. 100 ml
3.2 95% Ethyl alcohol ……………… 100 ml
1.2 Distilled water ………….. 1000 ml
1.3 Xylene ………………………………….. 1000 ml
1. PROCEDURE
1. DEHYDRATION:
4.1 Prepare 250ml of 70% and 80% ethyl alcohol from 95% ethyl alcohol using the
formula: C1V1 = C2V2. Show your computation to your instructor before you
proceed. Label the container the bottle correctly with your group.
4.2 Immerse the previously fixed and decalcified tissue in 70% for 6 hours, 95% ethyl
alcohol for 12 hours, absolute ethanol for 2 hours and another 2 changes of absolute
ethanol for 2 hours.
2. CLEARING:
2.1. Immerse the previously dehydrated tissue specimen in two changes of xylene for 1
hour each.
5. RESULT/OBSERVATION
5.1 Draw and label the set up for dehydration.
A. LIVER
1
B. KIDNEY
C. LUNGS
2
5.1 Draw and label the set up for Clearing.
A. LIVER
B. KIDNEY C. LUNGS
3
1.2 Computations:
Compute for the volume of alcohol and distilled water in preparing 70%, 80% and 95% alcohol
from the alcohol stock solution (95%).
A. 70% ALCOHOL
GIVEN:
SOLUTION:
ANSWER: To obtain 250mL of 70% alcohol, dilute 184.21mL of 95% alcohol with
65.75mL distilled water.
B. 80% ALCOHOL
GIVEN:
SOLUTION:
ANSWER: To obtain 250mL of 80% alcohol, dilute 210.53mL of 95% alcohol with
39.47mL distilled water.
4
6. STUDY QUESTIONS
6.1 What are the characteristics of an ideal dehydrating agent?

Characteristics of an Ideal Dehydrating Solution:
1. It should dehydrate rapidly without producing considerable shrinkage or distortion of
tissues.
2. It should not evaporate very fast.
3. It should be able to dehydrate even fatty tissues.
4. It should not harden tissues excessively.
5. It should not remove stains.
6. It should not be toxic to the body.
7. It should not be a fire hazard.
6.2 Give examples of dehydrating agents that also act as clearing agent?
Dehydrating Agents that also act as clearing agents are Dioxane (Diethylene Dioxide)
and THF or Tetrahydrofuran.
6.3 Aside from alcohol, what are the other dehydrating agent which can be used in tissue
processing? Cite their advantages and disadvantages.
ACETONE
Advantages:
• It is cheap, rapid acting dehydrating agent utilized for most urgent biopsies.
• It dehydrates in ½ to 2 hours.
• It is freely miscible in water and organic solvents.
Disadvantages:
• It is not recommended in routine dehydration purpose due to considerable tissue
shrinkage produced.
• It penetrates the tissue poorly.
• It causes brittleness in the tissue when exposed to acetone in a prolonged period of time.
DIETHYLENE DIOXIDE (Dioxane)
Advantages:
• It is an excellent dehydrating and clearing agent.
• It is readily miscible with water, melted paraffin, alcohol, and xylol.
• It produces less tissue shrinkage compared to alcohol dehydration.
Main Disadvantage:
It is extremely dangerous because its vapor produces a cumulative and highly toxic action in
man.
5
ETHYL GLYCOL MONOETHYL ETHER (Cellosive)
Advantages:
• It dehydrates rapidly.
Disadvantages:
• At 110° - 120°F, it is combustible.
• It is toxic by inhalation, skin contact, and ingestion.
TRIETHYL PHOSPHATE
Advantages:
• It removes water very readily.
• It is used to dehydrate sections and smears following certain stains and produces
minimum shrinkage.
Disadvantages:
• It produces very little distortion and hardening of tissues.
TETRAHYDROFURAN (THF)
Advantages:
• Have similar properties to dioxane: it is used as a dehydrating and clearing agent since it
is miscible with water, alcohol, and paraffin.
• It can dissolve many substances including fats.
• Most staining procedures give improved results with THF.
Disadvantages:
• It is toxic when ingested or inhaled.
6.4 What are the essential oils that can act as clearing agent? Give their functions.
CEDARWOOD OIL
• Clears both paraffin and celloidin sections during the embedding process only.
• Recommended for CNS tissues & cytological studies (smooth muscles and skin)
• Improves cutting of the sections
• Tissues may be left in oil indefinitely without considerable damage and distortion.
TERPINEOL (Artificial Oil of Lilac)
• Working substitute/ replacement for oil of cedarwood in the paraffin embedding
technique
• It is used in clearing celloidin section in the mounting procedure.
ANILINE OIL
• It is NOT routinely used as a clearing agent.
• It is used ONLY for clearing embryos, insects, and very delicate specimens due to its
ability to clear 70% alcohol without excessive tissue shrinkage and hardening.
CLOVE OIL
• Causes minimum shrinkage of tissues
6
• Quality is not guaranteed due to its tendency to become adulterated because wax
impregnation after clearing with clove oil is slow and difficult
• Tissues become brittle, aniline dyes are removed, and celloidin is dissolved
OIL OF BERGAMOT
• Slow clearing agent but not harmful to the tissue
OIL OF ORIGANUM (Spanish Hop Oil or Thyme Oil)
• Only for embedding process
OIL OF WINTERGREEN (Methyl Salicylate or Methyl Benzoate)
• Nitrocellulose solvent
• It is applicable only when double embedding is required.
6.5 What will happen to a clearing agent if water is not completely removed from tissues?
Incomplete removal of water or incomplete dehydration would impair the penetration of

the clearing agent into the tissue. If they are not completely removed, the clearing agent will
become cloudy, cannot act properly and cannot penetrate the tissue which makes it non-receptive
to infiltration or impregnation since both paraffin and water are immiscible. As a result, the
specimen would be soft or mushy tissue blocks.
7
Student’s Name: DAYAGAN, GWYNETH MARIE M. Group& Sec: 2C Date: 7/6/2021
Exercise No. 4: DEHYDRATION and CLEARING
CRITERIA BEGINNING DEVELOPING ACCOMPLISHED EXEMPLARY SCORE

(5) (10) (15) (20)
Ability to Answer Able to answer Able to answer Able to answer 75% Able to answer
Questions 25% of the 50% of the of the item required. 100% of the
Able to answer all the items required. items required. item required.
required items in the
activity.
Content/Knowledge Presents 25% Presents 50% of Presents 75% of the Presents 100%
Presented of the the knowledge knowledge required. of the
Presents knowledge required. knowledge
scientific/practical required. required.
facts based on
knowledge of the
subject.
Finding Relevant 25% of the 50% of the 75% of the answers 100% of the
Information answers were answers were were supported by answers were
Presents facts and supported by supported by facts & relevant supported by
relevant information to facts & facts & relevant information. facts & relevant
support the answers. relevant information. information.
information.
Sentence Fluency 25% of the 50% of the 75% of the 100% of the
& Organization of sentences were sentences were sentences were sentences were
Thoughts clear & clear & clear & clear &
Sentences are very understandable. understandable. understandable. understandable.
clear & Lacks Few ideas Most of the ideas All ideas
understandable. spontaneity of showed showed spontaneity. showed
ideas. spontaneity. spontaneity.
Ideas were presented
in order and
spontaneous.
Punctuality Did not able to Submitted on Submitted on the Submitted on or
Submitted the output on submit on the the closing date closing and a day before the due
the specified date & due date & but exceed 1 after the due date. and closing
time. closing date. day on the due dates.
date.
TOTAL SCORE
Preceptor’s Signature: __________________
Clearing agent will be cloudy

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Name: DAYAGAN, GWYNETH MARIE M. Yr. & Sec.

EXERCISE 4: DEHYDRATION and CLEARING

While clearing or dealcoholization is performed so that the alcohol in the tissues

1.2 Properly dehydrate tissues using manual methods of processing.

1.3 Demonstrate properly manual method of clearing.

2.1 Fixed Liver, Lung & Kidney tissue 2.8 Funnel

5.1 Draw and label the set up for dehydration.

6.1 What are the characteristics of an ideal dehydrating agent?

Incomplete removal of water or incomplete dehydration would impair the penetration of

CRITERIA BEGINNING DEVELOPING ACCOMPLISHED EXEMPLARY SCORE

Preceptor’s Signature: __________________

Clearing agent will be cloudy

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