HS2413B_HISTOLOGICAL LAB REPORT GROUP 3

FACULTY OF HEALTH SCIENCE



MLT 428
HISTOLOGICAL TECHNIQUE



OCT 2021 - FEB 2022



LABORATORY REPORT



TITLE: ROUTINE PROCEDURE IN HISTOPATHOLOGY LAB

Prepared by: Student's ID:
Student's name: 2020498516

IEESYA ATIFAH BINTI MOHD FAUZI

NOR ALIAH SYAHIRAH BT MOHD HADZIR 2020498732

NUR ALYAA QALISHA BINTI MOHAMMAD SAID 2020897946

NUR MAISARAH BINTI MOHAMAD 2020853802

WAN ANISSA BINTI WAN MOHD ZAKI 2020498894

LECTURERS:
MADAM HARTINI YUSOF
DR. AYUNIE BINTI ZULKEPLI

WORKFLOW1.0 & the laboratory
equipments.

MLT 428 HISTOLOGICAL TECHNIQUE

TISSUE PREPARATION TISSUE PROCESSING TISSUE EMBEDDING

Trimming or dissection of selected Eliminate water from cells and To position and pour paraffin wax
tissue specimens fixed with fixative replace it with a solidifying into the specific mould holding
solution for preparing proper tissue media the processed tissue specimen,

blocks. resulting in a paraffin-embedded
tissue block.
AUTOMATED TISSUE PROCESSOR:
SHANDON CITADEL 1000 TISSUE SECTIONING

To dehydrate tissue, and Trimming: Exposing an
replace the water with a appropriate amount of tissue and
support medium that gives assuring the top and bottom of
it sufficient rigidity. the block are parallel and
horizontal to the blade's edge
when sectioning

Sectioning: To make thin slices for
staining on microscope slides

TISSUE EMBEDDING CENTRE: SLEE MAINZ MPS/C TISSUE STAINING
To produce paraffin block of tissue by embedding the tissue using
To differentiate the nucleus and
melted paraffin or waxes that provides strong external support cytoplasm by contrasting colors
during sectioning of tissue when its cooled.
by Haematoxylin and Eosin
Staining.

MICROTOME: MOUNTING & LABELLING
To permits
extremely To mount the sample with a
thin slices coverslip using a mounting
medium for protecting the
of
material, sample physically.
known as
sections, FROZEN SECTION
to be cut.
To prepare slide for rapid
tissue interpretation under
microscope using the cryostat.

SLEE Mainz CUT5062

1.0.1

INTRODUCTION

MLT 428 HISTOLOGICAL TECHNIQUE

In the 1700s, the word “tissue” was introduced by Marie-François Xavier Bichat to the
medical dictionary and the composition of organs from tissues was then clearly
understood. The study was then continued by J.J Lister to complete the study by
improving the microscope system and got to work on histology analysis. Histological
staining also developed in the 1700s. However, the first use of hematoxylin was in 1863
by Wilhelm von Waldeyer as a nuclear stain and it is widely used now in tissue staining.

Histology, an alias to microscopic anatomy or microanatomy, is a branch of biological
studies of tissues and their respective structure. It helps hospital laboratories to identify
the disease processes that affect tissues depending on their own classes and the disease
itself. The study also allows laboratories to check for abnormalities in cells that indicate
the patient's illness's true aetiology by conducting specific procedures.

Diagnosis can be made by looking at a little piece of tissue from different organs. As in
this lab report, we mainly focus on the tonsil, appendix and uterus parts in the
laboratory. To examine tissue features and microscopic structures of cells, a variety of
approaches had applied. Histological staining is a methodology in preparing sample
tissues for examination under the microscope by staining them with histological stains.
The stain used in this experiment is a basic dye, Hematoxylin and acidic dye, Eosin.
Mostly known as H&E stain. The stained microscope sections will guide the hospital
laboratories by highlighting the anatomical structure of the cells that appear within the
tissues.

1.1

TISSUE PREPARATION

tissue grossing and fixation

MATERIAL REAGENT APPARATUS

Human tissue specimens 10% of commercially prepared formalin 1. Mask
2. Gloves
fixed with commercially RESULTS 3. Lab coat/protective clothing
4. Medisheet / Grossing paper
prepared 10% formalin Tissue blocks 5. Dissecting board

PRINCIPLE (prefered to use plastic board)
6. Forceps
GROSSING 7. Scalpel handle
An examination and dissection process of tissue that was an initial and 8. Scalpel blades
essential steps in the histology lab to obtain an accurate diagnosis of 9. Tissue cassettes
patients, macroscopically and microscopically. 10. Containers with appropriate lids
11. Ruler
FIXATION: 12. Pencil
Fixative solution which is a 10% of commercially prepared formalin
preserved the tissues permanently in their natural state as possible by EQUIPMENT
stopping enzyme activity, killing microorganisms, hardened the tissues
and fixed all components of tissues. It also enhanced the staining of 1.Ducted Fume Hood: Iryas
tissue steps.



PROCEDURE:
PROBLEM
1. A complete PPE (personal protective equipment) was worn properly to
avoid the risk of any potential hazard. 1. Thickness of tissue that was more
than 4mm, often have an uneven
2.Cassettes were labeled with name/student ID number, date and type of surface of tissues after soaking the
specimen by using a pencil. tissue in the fixative solution.

3.Grossing workstation in the Ducted Fume Hood were prepared with 2. Some of the tissue parts was not in
instruments that can be readily used for tissue grossing. The fume hood the same layer affect the entire
light and suction vent switches were turned on. The workstation could be process to get a perfect sectioning of
clearly seen and well ventilated to prevent inhalation of formaldehyde tissue.
fumes.
3. The tissue block surface was not
4.Only one specimen container that was opened at one time to avoid mix- smooth due to trimming technique.
ups of specimens.
SOLUTION
5.Grossing or trimming of tissues with proper orientation has been done
using scalpel and forceps according to accepted protocols for the 1. A uniform and thin slices of tissue that
laboratory. It is performed one at a time and ensured the trimmed tissues have 3-4 mm thickness prepared by
were approximately 3-4mm thick by using the ruler. using a ruler.

6.Macroscopic detailed conditions of the tissues such as its color, unusual 2. Excess of tissues were removed if it is not
tissue surface or pigmentation and apparent abnormalities were in the same layer of the entire tissue.
described.
3. Cut the tissue in one way motion. Avoid
7. The trimmed tissue was transferred into a labeled cassette by using a to cut the tissue in forward and backward
forceps. motion.

8.Cassettes containing trimmed tissue were immersed in 10% of CONCLUSION
commercially prepared formalin containers for 6 to 24 hours.
Grossing and fixation main purpose to
9..Formalin waste was discarded into formalin waste container get an accurate and diagnostically
dissection of preserved tissue with
proper orientation was obtained .
In tissue preparation, it is also important
to always replace the fixative formalin
solution when the specimen contain high
percentage of blood.

1.2

TISSUE PROCESSING

PRINCIPLE: EQUIPMENT
Automated Tissue Processor
1. FIXATION (Model: Shandon Citadel 1000)

Fixation denatures proteins, making the cell and its constituents REAGENTS
less susceptible to autolysis. 50% Alcohol (2L)
70% Alcohol (2L)
2. DEHYDRATION 80% Alcohol (2L)
The process where water is removed from the tissue including the 95% Alcohol (2L)
unbound fixative Alcohol + Xylene

3. CLEARING RESULT
Displaces the dehydrating solution, allowing the infiltration
medium to reach the tissue component. Processed Tissue

4. INFILTRATION
Paraffin wax is infiltrated or injected into tissue sections to stabilise
the tissue and allow thin slices to be cut.

PROCEDURE: TISSUE PROCESSING CYCLE
1.All the processing reagents were
prepared 10% Buffered Formalin 2:00
2.All the prepared processing reagents 50% Alcohol 1:00
were placed in the corresponding 70% Alcohol 1:00
container 80% Alcohol 1:00
3.All the cassettes containing tissue 95% Alcohol 1:00
specimens were transferred into the tissue Absolute Alcohol 2:00
processor organiser basket using forceps Absolute Alcohol 2:00
4.The tissue processor organizer baskets Mixture of Absolute Alcohol and Xylene 1:00
were placed into the basket hanger. Xylene 2:00
5.The lid of the tissue processor basket was Xylene 2:00
placed on top of the organiser basket Paraffin Wax 3:00
6.The basket was firmly inserted into the Paraffin Wax 3:00
operating head slot
7.Using the hand-held controller, each step PROBLEM CAUSES
for the processing time is properly
programmed Tissue feels soft during Paraffin may be
8.To begin the tissue processing cycle, press embedding. saturated with xylene
“Auto Start” button and close the Tissue was processes
operating door. Tissue bounce out from for too short
9.Tissue Processing Cycle began. block during tissue The processing
sectioning. reagents may have
Tissue processing is a very important step that must saturated with water
be closely monitored as it takes longer to process the
tissue, any mistakes will cause the tissues to change, Water remaining in the
necessitating reprocessing. tissue.
The tissue should not be underprocessed or
CONCLUSION overprocessed, as this will affect the tissue details. Replace reagents and reprocess
It's critical that the tissues are treated properly and
rendered ready for the next phases. SOLUTION tissue according to the correct

protocol.

1.3

TISSUE EMBEDDING

PRINCIPLE: MATERIAL:
The embedding processing concept is to properly and 1. Processed tissue
precisely position a histology specimen into a block of REAGENTS:
paraffin wax. This is to ensure and allow the tissue sample 1.Paraffin wax (SAKURA Tissue-Tek
to be supported and held in place so that precision cutting
of thin sections for histological diagnostic purposes can be Paraform & Surgipath Paraplast Plus)
made for further process APPARATUS:

PROCEDURE: 1. Forceps
2.Steel block mould
1.Processed tissue cassettes were removed from the 3. Scraper
tissue processing machine. 4. Gloves
5.Tissue paper
2.The tissue cassettes were transferred into pre warmer 6. Masks
unit of the tissue embedding centre to be pre- EQUIPMENT:
warmed. 1.Paraffin Wax Tissue Embedding Centre:
SLEE Mainz MPS/C / MPS/P / MPS/W
3.Cassette was opened to view the tissue sample and an
appropriate mould is chosen based on the tissue RESULT
sample size.
Figure 1 : Good paraffin-
4.A small amount of molten paraffin was poured into embedded tissue block of
the mould. appendix

5.With warm forceps, processed tissue was transferred Figure 2: Slight crack along
into the mould and arranged the tissue according to the edges of paraffin-
the sectioning side. This side of the mould is facing embedded tissue block
down
PROBLEM SOLUTION
6.Mould transferred into a cold plate. The tissue was
firmly and gently pressed flat with a pair of warm and - Presence of bubble Wax is dispensed slowly
cleaned forceps.
when dispensing wax into and bubbles is eliminated
7.Paraffin was poured into the mould fully until covered
the face of the cassette. the mould. before cooling the wax.

CONCLUSION 8. The labelled paper was placed on top of the c1a. ssette. - Position of the tissue Tissue specimen is gently
9.Immediately cooled down by placing the mould on the specimen was not centered anchored at the center of
and slightly slanted. the mould when cooling
cryo console. the wax. The flat side is
10.Solidified paraffin wax separated from the mould to - Tissue is incorrectly position downward.
orientated .
form a paraffin tissue block.
11.Excess wax from the paraffin tissue block is removed - Presence of crack at the Gently tapped the mould
side of the cooled block on the palm or by using
by using a scalpel. due to intense slamming scalpel to clean excess
on the table for removal. wax and the side of the
When paraffin wax was in liquid, it covers the tissue and block is picked carefully.
solidifies quickly when cooled. The medium was
penetrated into the tissue, providing a matrix and
avoiding tissue morphology alteration during microtome.
The orientation of the specimen during embedding is
significant for demonstrating appropriate morphology
and more uniform embedding.

1.4

TISSUE SECTIONING

MATERIAL: PRINCIPLE:
1.Paraffin tissue blocks
The tissue sectioning process is a technique
APPARATUS: in cutting a paraffin-embedded or frozen
1.High profile microtome slides tissue into a thin slice in a clean and
2.Clean frosted end glass slides consistent manner. The tissue sections are
3.Clean brush thin slices that are later placed on a glass
4.Applicator sticks slide. The paraffin sectioning and frozen
5. Forceps sectioning are two basic types of sectioning.
6.Slide racks
7.Pasteur pipettes PROCEDURE: Date: 16 October
8.Clean gauze
9.Tissue paper 1.A tissue floatation bath was prepared by filling the tissue floatation bath
with distilled water.
10. Pencils
EQUIPMENT: 2.The tissue floatation bath temperature is set at 42°C . By using the
calibrated thermometer, the temperature of the tissue floatation bath was
1.Rotary microtome: SLEE Mainz CUT5062 checked.
2.Tissue floatation bath: XH-1001, Thermo
3.The microtome was switched on. The fixing lever for the orientation of the
Scientific 3120058 specimen was adjusted and closed after getting its desired angle.
3. Freezer
4. Oven 4.The slide is labelled with name, student identification number, type of
specimen and date.
RREESSUULLTT
5.The paraffin-embedded tissue block is placed vertically or horizontally
Figure 3: Thin section of Figure 4: Ribbon-like thin onto the standard object clamp.
appendix on glass slide tissue sectioning
6.The finger protection guard was removed and the blade fixation loosened
PROBLEM SOLUTION by turning the right lever counterclockwise.

- Visible lines can be The microtome blade is 7.The blade was inserted from one side and tightened by turning the right
seen on the ribbon when moved to the untouched lever clockwise.
sectioning due to a blunt area or changed into a
blade from trimming. new blade and returned 8.The paraffin-embedded tissue block was trimmed until a complete tissue
to sectioning. section appears on the tissue ribbon.

- The sectioning is seen to Refreeze the paraffin 9.The residual tissue ribbon was removed by using a brush.
be creased and pressed block for another 5 10.Specimen block was removed after putting the finger protection guard back
together due to warm minutes or the paraffin
block or rapid cutting. block is slowly and evenly into its place.
cut. 11.The trimmed paraffin block was cooled by putting it into the freezer for 5

- The sectioning failed to The angle of the blade is minutes.
form a ribbon due to adjusted to desired angle 12.The cooled block is taken out from the freezer to be sectioned.
incorrect or inappropriate 13.Tissue block was ensured fixed in position and new clean or unused area of
angle of the blade.
microtome blade is used for sectioning purposes.
14.Blade fixation was tightened by turning the right lever clockwise.
15.The “M” button was selected until the “Macro” word disappears on the

display panel for sectioning mode.
16.The finger protection guard was removed and the specimen block was

moved towards the blade by pressing the motorized coarse advance or
motorized fine advance button.
17.Sectioning of the paraffin tissue block started to produce a ribbon section.
18.By using applicator sticks or forceps, the ribbon section was pulled from the
blade.
19.The ribbon sections were transferred into the water bath.
20.The ribbons were laid on the water bath carefully to avoid the formation of
wrinkles or bubbles on the tissue.
21.The best section from the ribbon sections was selected and separated
gently by using the applicator stick.
22.Residual wrinkled or bubble forming tissue sections were removed from the
floatation by using a Pasteur pipette.
23.Floating out (fishing) was performed by using a glass slide on the selected
sections. The applicator stick is used to prevent the tissue section from
moving.
24.The glass slide was gently raised above the water surface and the glass
slide containing the tissue section is placed onto the slide rack.
25.A glass slide was placed on the slide rack at room temperature to remove
water from the slides.
26.Dried slides were placed into the oven at 60°C for dewaxing purposes
before continuing with staining.

- Water trapped under the Making sure the slide is CONCLUSION The tissue section will be needed to be cut at 3 to 4 microns
thin section after fishing at 45° angle and with no wrinkles, folds, or air bubbles under the sections.
out from water bath due to fished out at the same Tissue thickness may pick up the extra stains, making
improper fishing angle slowly for better interpretation more challenging. Additional reagents can
technique. positioning. be trapped in wrinkles, folds, and air pockets, causing extra
artifacts on the slides

1.5

TISSUE STAINING

Haematoxylin & Eosin Staining

REAGENTS: Xylene, alcohol (Absolute, 95%), tap water, distilled Water, MATERIAL: Unstained Slide
Haematoxylin 3G (Sakura), Eosin
HAEMATOXYLIN
APPARATUS: Reagent containers, slide racks, forceps, tissue paper, oven, hotplate
BLUE-PURPLE: Stains the nucleus of
PRINCIPLE the cells (chromatin within the
nucleus and nucleus membrane,
DEPARAFFINIZATION: Xylene (x3) - To completely remove the wax. ribosomes, cytoplasmic regions
HYDRATION: 1. Decreasing alcohol concentration + water - To drain rich in RNA).
previous xylene and hydrate tissue. 2. Distilled water - Hydrate tissue.
NUCLEAR STAINING: 1. Haematoxylin - Stains nucleic structure (DNA, RNA). EOSIN

2. Running tap water - Remove excess stain. PINK: Stains basic elements
BLUING: Distilled water - Uses alkalinity to change reddish-purple (erythrocytes, cytoplasm, muscle
and collagen). Stains in different
haematoxylin to a blue/purple-blue colour. intensity of pink, according to
COUNTERSTAIN: 1. Eosin - Acidic counterstain to stain basic elements different types of connective tissue.
(cytoplasm, muscle, collagen). 2. Distilled water - Remove excess eosin.
DEHYDRATION: Increasing alcohol % - To remove all traces of water.
CLEARING: Xylene - Rinse tissue and render it completely transparent.

PROCEDURE DEPARRAFINIZATION Why hydrate tissue?

1.Reagents were prepared The sectioned tissues were baked Hydrated tissue makes it
in reagent containers. in the drying oven at 59-60°C for easier for aqueous reagents
15-30 minutes to deparaffinize to readily penetrate the cells
2.Deparaffinization in its paraffin parts and leave only Hotplate: Pro, HP-
oven/hotplate. the tissues adhered to the slides. 7 Lab Plus Series and tissue elements.

3.Tissue staining.

STAINING CYCLE

DEPARAFFINIZATION. PROBLEMS SOLUTIONS

Xylene 3 min Contaminated solutions The reagents were
and dyes - reagents filled replaced every 2-4 cycles
Xylene 3 min with excess wax and dyes. to ensure sufficient purity
of reagents.
Xylene 3 min

HYDRATION.


Absolute alcohol 1 min

Absolute alcohol 1 min

95% alcohol 1 min Overstain of haematoxylin. Solutions and

Wash in running tap water 1 min The blue-purple dye is too dyes were

Distilled water 1 min intense under replaced and the timings

NUCLEAR STAINING.
the microscope. of staining were timed

Haematoxylin 3G (Sakura) 5 min correctly.

Wash in running tap water 5 min

BLUING.
Tissue detached from slide The microtomb setting

Distilled water 30 sec during staining due to thick was made sure on 3 µm

COUNTERSTAIN.
tissue from sectioning. (for sectioning) to avoid

Eosin 2 min too thick tissues.

Distilled water 10 sec

DEHYDRATION.
CONCLUSION AFTER
95% alcohol 10 sec

95% alcohol 10 sec The H&E procedure stains

Absolute alcohol 1 min the nucleus and cytoplasm BEFORE

Absolute alcohol 1 min contrasting colors to

Absolute alcohol 1 min

CLEARING.
readily differentiate
1 min
Xylene cellular components.

Xylene 2 min

1.6

SLIDE MOUNTING AND

LABELLING

PRINCIPLE: PROBLEM:
Present of air bubble after placing
A mounting medium is used the coverslip on the mounting
on the stained tissue slide to medium
adhere the coverslip to the
slide. SOLUTION:
A mounted stained tissue
slide with coverslip can The coverslip was gently pressed with an
preserve and support applicator stick to remove the bubbles. If too
sections for light microscopy. many bubbles were present, the slide was
placed in the xylene to remove the coverslip
and repeat the mounting procedure.

PROCEDURE:

1.Appropriate coverslip 2. Adequate mounting
was chose according to medium was placed on one
the size of the tissue edge of the slide.
section.

MATERIAL: 4. The slide was 3. The coverslip was
examined macroscopically gently lowered until it
H&E stained slide to ensure there are no touched the mounting
REAGENT: air bubbles. medium. Mounting medium
Mounting medium (CoverSeal-X) flowed upward due to
5. Label slide with the capillary attraction.
APPARATUS: following details:
6. The sticker
1.Slide rack Type of specimen labeled was
2. Coverslip Staining method placed at the
3.Applicator stick Student name frosted end of
4. Label sticker Student id the slide.
number
Date

RESULT: CONCLUSION:

H&E stained In slide mounting, it is important to
slide choose a good mounting medium in
order to have the best viewing and
preserving the slide during the
examination under the microscope.
Unlabeled or mislabeled of specimen
may lead to great clinical risk.

1.7

FROZEN SECTIONING
Frozen section technique is a rapid and useful histopathological

technique in preparing tissue for microscopic analysis. 5
PRINCIPLE:

The frozen section is the rapid

tissue section using cryostat in PROCEDURE: Embedding
cooling the tissue for immediate
report of tissue sample. 1. Chemicals/Reagents Preparation
95% alcohol (400 ml) was prepared.

1.380ml of Absolute Alcohol added to 20ml of distilled water.

SPECIMEN: 2. Tissue Sectioning
1. The cryostat was prepared by setting the temperature to
Fresh tissue

APPARATUS: Chuck -20°C. The forceps, microtome blade, brush and applicator
stick were placed in the appropriate tissue section in the
1.Clean glass slide cryostat.
2. Coverslip 2. A coplin jar containing 10% formalin was placed in the
3.Microtome blade oven at 60°C one hour before the specimen arrival. Before
4. Forcep using, it was removed from the oven.
5. Brush 3. The specimen was grossed.
6.Applicator stick 4. A glass slide was labelled with a type of specimen and
7.Coplin jar date.
5. The fresh tissue was embedded on a specimen disc with
EQUIPMENT:

1. Cryostat Tissue Freezing Medium and it was froze in the cryostat.

2. Oven 6. The specimen disc was placed in the orientable specimen

CHEMICALS/REAGENTS: head.

1.Tissue Freezing Medium 7. The tissue was trimmed at 10µm thick until specimen is
2.10% Neutral Buffered Formalin
3.Hematoxylin 3G exposed.
4. Eosin
5. Xylene 8. Sectioning was performed at 5µm thick.
6.Alcohol (Absolute, 95%)
7.Distilled water 9. The section was placed onto the labelled slide.
8.Tap water
Immediately, it was placed in the pre-heated 10% formalin for
RESULT:
about 1 minute.
H & E stained
slide 10. Rapid H&E staining was performed. 8

REAGENT TIME
1. Absolute alcohol 10 dips
2. Running tap water Few dips
3. Hematoxylin 3G 1 minute
4. Running tap water Few dips
5. Distilled water 15 seconds 9
6. Eosin 1 minute
PROBLEM: SOLUTION: 7. 95% Alcohol 10 dips

Introduced of ice The chuck covered 8. 95% Alcohol 10 dips
9. Absolute alcohol 10 dips
crystal artifact into with masking tape is

the tissue. used at room 10. Absolute alcohol 10 dips
11. Xylene 10 dips
Shattering artifact in temperature on the 12. Xylene 10 dips
13. Xylene 10 dips
frozen section due to block face to warm

the overcooled block the tissue

CONCLUSION: 11. The slide was mounted and ready for microscopic
Frozen section is the best technique examination.
for rapid tissue interpretation. 12. The specimen disc was removed from the oriented specimen
FFPE is better than frozen section: head and lei it melted at room temperature
- tissue is preserved 13. The residual tissue was removed from the specimen disc and
- the storage of block in longer and can placed in the container containing 10% formalin
be keep at room temperature. 14. The cryostat was cleaned after used.

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