Department of Biological Sciences

College of Arts and Sciences

TISSUE PROCESSING
(An Outline for Biotechniques)

Submitted by:
Albarico, Iris
Calipay, Angelica
Empron, Jesarie
Isaac, Karren Listherway
Mejorada, Cilyn Jean
Nodalo, Chyril Mae
Nuenos, Charlotte Jane

Submitted to:
Ms. Merry Joy Tutor
 I. Objectives

At the end of the lesson, the students will be able to:

 Define tissue processing,

II. Body

Tissue processing is the technique of getting fixed tissues into paraffin. This describes
the steps required to take animal and human tissues from fixation to the state where it is
completely infiltrated with a suitable wax i.e. paraffin wax and can be embedded and ready for
section cutting on a microtome.
The aim of tissue processing is to process the fixed tissue into a form in which it can be made
into thin microscopic sections. The steps in tissue processing are as follows: Dehydration,
Clearing and Infiltration.
Dehydration is the process of removing water from tissues. It is important because
paraffin is not miscible with water. Typically, this sequence is a graded series of ethanols (e.g.,
50, 70, 95, 100%). Dehydration is usually complete when less then 3-4% of water remains in the
tissues. Time required for this depends on:
1. Permeability of tissues
2. Continuous rotation of fluid to prevent stagnation of fluid around tissues
3. Temperature
4. Vacuum applied dehydrants: Ethyl alcohol, Methyl alcohol, Butyl alcohol and Isopropyl
alcohol. Acetone is also used as a dehydrant.
The most commonly used dehydrant is ethyl alcohol.
In dehydration, the tissues are passed through a series of progressively more concentrated
alcohol baths. Concentration of first alcohol bath depends on the fixative and size and type of the
tissue, e.g. delicate tissue needs lower concentration of alcohol and smaller interval between two
strengths of alcohol.
Usually 70% alcohol is employed as the first solution and100% as the last solution. After about
40 tissues have passed through the first change of alcohol, it is discarded and all the other
changes are brought one step lower. Absolute alcohol at the end is always fresh.
Usually tissues are kept in each solution for 40 to 60 minutes.
A layer of anhydrous CuSO4 is placed at the bottom of a dehydrating bottle or beaker and is
covered with 2-3 filter paper of approximate size to prevent staining of the tissue. Anhydrous
CuSO4 removes water from alcohol as it in turn removes it from tissues. Anhydrous CuSO4 is
white in colour while the hydrated form is blue. Therefore, it acts as an indicator for the presence
of water.
Advantage of CuSO4:
1. Rapid dehydration
2. Prolongs life of alcohol
3. Blue colouration of CuSO4 indicates that both alcohol and CuSO4 should be changed.
Acetone - Acetone is clear colourless inflammable fluid which is miscible with water, ethanol. It
is used for complete dehydration. Four changes of acetone of half an hour or two changes of one
hour are given to achieve complete dehydration of tissues.
Advantages
1. Rapid action
2. Easily removed by most clearing agents.
3. Less expensive
Disadvantages
1. Highly volatile.
2. Causes shrinkage and brittleness of tissues.
3. Dissolves lipid more than ethanol.

Clearing is a process which leaves the tissues clear and transparent. This term relates to
the appearance of the tissues after the dehydrating agent has been removed. Once absolute
ethanol is achieved, the tissue is placed in an intermediate organic solvent that is miscible with
both the alcohol and the wax (e.g., xylene, toluene, benzene, or other less toxic synthetics).
Since opaque tissue specimens became clear when placed in some of these agents, they were
collectively referred to as “clearing agents” or “clearers.” The end point of clearing can be noted
by the transparent appearance of the tissue. Thus, clearing serves two purposes:
1. Removes alcohol to make paraffin impregnation complete
2. Acts as solvent for the mounting media which renders the tissues transparent and improves the
refractive index, making microscopic examination easier.
Clearing Agents:
Xylene - It is colourless and most commonly used. Two changes of one hour each are given to
get the end point. Prolonged treatment hardens the tissues. It is not preferred for brain tissue.
Carbol-xylene - It clears rapidly, it is kept reserved for material difficult to clear.
Other Clearing Agents:
Toluene Cedarwood Cloroform
oil
Dioxane Benzene

Infiltration is the process that follows after clearing. After clearing, tissues are
transferred to molten paraffin wax for filtration and impregnation. In order for the paraffin wax
to mix with the tissue specimen, the wax is heated to its melting temperature. The specimen is
then placed in several changes of molten wax, often under vacuum, in order to replace the
clearing agent and completely infiltrate the tissue with the wax embedding medium. Since the
paraffin melting temperature is significantly elevated over room and body temperatures, various
artifacts (e.g., shrinkage) are introduced by this method. When infiltration is complete, the tissue
is embedded in fresh wax, which is then allowed to harden at reduced temperatures within a
special embedding mould (e.g., paraffin boats).
Factors to be considered in Infiltration:
1. Size and type of tissues. Longer time is required for thicker tissues. Vacuum reduces the time
required for complete impregnation.
2. Clearing agent employed.
3. Use of vacuum embedding tissue processing may be performed manually or with the help of
automated tissue processor. Routinely 12 containers containing different solutions are used for
processing in the following order:
10% formalin – container no 1, 2
50% alcohol – container no 3
90% alcohol – container no 4 & 5
Absolute Alcohol – container no 6
Acetone – container no 7 & 8
Xylene – container no 9 & 10
Paraffin Wax – container no 11 & 12
Tissue processing can be performed either manually or through automated tissue processor. The
device can handle larger number of tissues, process more quickly and produces better quality
outcome.
Two types of devices are available:
1. Tissue transfer or dip dunk
2. Fluid transfer or enclosed
Advantages of automated tissue processor - Saves time, decreases human error, effective fluid
circulation, Temperature can be adjusted and vacuum/pressure can also be incorporated.
Tissue Transfer Type:

The machine consist of a time clock, a circular superstructure that contains basket carrier, a
receptacle basket and receptacles (stainless steel or plastic capsules), and a circular deck which
holds the reagent beakers and plastic baths. Small blocks of tissue are enclosed in the perforated
capsules. These capsules are placed in the basket which in turn is attached to one of the yokes in
the superstructure, while it is in the raised position. When the superstructure descends the basket
is immersed in the first solution and other reagent beakers are covered preventing evaporation of
reagents. To move the basket from one reagent to the next the entire superstructure ascends and
descends at scheduled intervals controlled by the time clock. During immersion the basket
rotates so the infiltration of fluid into the tissues is optimum. The entire process takes about 16
hours. The machine is started in the evening so that the process is complete in the morning,and
embedding is done.

Enclosed Type:

In this type of tissue processor the tissues remain in one container but reagents get changed at
scheduled interval.
Advantages:
1. Can be used when the number of tissue blocks is limited.
2. Non-availability of automated tissue processor
Disadvantages:
1. Difficult to use when large number of tissue blocks are to be processed.
2. Proper agitation of reagent not achieved.
3. More evaporation of reagents.
4. Process is tedious and requires constant attention
Precautions:
1. Labels should be written with graphite pencil, India ink or typed.
2. The fluid used in complete dehydration and clearing tend to become contaminated with fluid
carried over from previous vat by the tissue. Every alternate day daily the last solution is the
series are replaced by fresh solution of 100% alcohol, acetone and xylene and the previously
used once one moved forward while the first one is discarded. Other reagents are changed twice
a week or earlier with an average work load. It is far better to change the reagents a day earlier
than to have a precious surgical specimen improperly infiltrated.

Manual: In this process the tissue is changed from one container of reagent to another by hand.
III. Summary
Tissue processing is the technique of getting fixed tissues into paraffin. This describes the
steps required to take animal and human tissues from fixation to the state where it is completely
infiltrated with a suitable wax i.e. paraffin wax and can be embedded and ready for section
cutting on a microtome.
The aim of tissue processing is to process the fixed tissue into a form in which it can be made
into thin microscopic sections. The steps in tissue processing are as follows: Dehydration,
Clearing and Infiltration.
Dehydration is the process of removing water from tissues. The tissues are passed through a
series of progressively more concentrated alcohol baths. Dehydration is usually complete when
less than 3-4% of water remains in the tissues. Clearing is a process which leaves the tissues
clear and transparent. This term relates to the appearance of the tissues after the dehydrating
agent has been removed. Once absolute ethanol is achieved, the tissue is placed in an
intermediate organic solvent that is miscible with both the alcohol and the wax (e.g., xylene,
toluene, benzene, or other less toxic synthetics). Infiltration is the process that follows after
clearing. After clearing, tissues are transferred to molten paraffin wax for filtration and
impregnation. In order for the paraffin wax to mix with the tissue specimen, the wax is heated to
its melting temperature in order to replace the clearing agent and completely infiltrate the tissue
with the wax embedding medium is then allowed to harden at reduced temperatures within a
special embedding mould (e.g., paraffin boats).
Tissue processing is very important and must be done carefully following the protocols. Poorly
processed tissue will result into faulty sections that will be observed under the microscope.
References
 www.nios.ac.in/media/documents/dmlt/HC/Lesson/07.pdf-retrieved on March 2017
 https://www.ncbi.nlm.nih.gov/pubmed/25015141-retrieved on March 2017