MLT428 HISTOLOGY TECHNIQUES E-REPORT (GROUP 5)

UNIVERSITI TEKNOLOGI MARA (UiTM)
KAMPUS PUNCAK ALAM





FACULTY OF HEALTH SCIENCES





HISTOLOG(MICLTA4L2T8E)CHNIQUE



CLASS : HS2413B



GROU
P : 5
TITLE : TISSUE ANALYSIS IN HISTOLOGY LAB




No. NAME STUDENT ID

1.
NURUL SYAZA ATHIRAH BINTI MEHAMAD FISOL 2021829676

2. NURUL NADIA BINTI NORDIN 2021454336

3. RAMA AN NUUR BINTI ABDUL JALAL 2021809754

4. SITI NURFARZANA BINTI HALIK 2021886984

5. NUR AMNI AQILAH BINTI ROSLI 2021817296

PREPARED FOR : MADAM HARTINI YUSOF

INTRODUCTION

Histopathology refers to the examination Instrument in Histopathology Lab
of a biopsy or surgical specimen by a
pathologist. Histological techniques Tissue transfer
provide a visual means for the bYoausrkpaertagraph text
examination and analysis of cell/tissue
physiology and morphology at the Integral fume hood

microscopic level. Histology is invaluable Reagent containers
for studying and understanding the Rotating head
microscopic three-dimensional Wax container

organization, structure, and function of Keypad control
cells and tissues and is especially useful
for the diagnosis and understanding of AUTOMATED TISSUE PROCESSOR
Brand : Shandon Citadel 1000
disease at the cellular level. To dehydrate tissue, and replace
the water with a support medium
AuYtoourmpaaratgerdaph text that gives it sufficient rigidity.
Microtome Setting
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Hand wheel
Wax reservoir
Cassette holder
Mold bin
Blade holder
Forceps warmer
Blade angle Wax dispenser
adjustment clamp
Square cold spot
Waste tray LED screen
Cooling plate
MICROTOME
Brand : Slee Mainz (CUT5062) TISSUE EMBEDDING CENTRE
Brand : Slee Mainz MPS/C
To cut thin slices of tissue
known as sections, to be cut To produce paraffin block of tissue by
embedding the tissue using melted
paraffin that provides strong external
support during sectioning

01 FLOW CHART OF
GENERAL PROCEDURE
TISSUE SEND TO
LABORATORY 02 03 04
STRAIGHT AFTER
OPERATION. TISSUE TISSUE TISSUE
GROSSING PROCESSING EMBEDDING

08 07 06 05

IMAGE MOUNTING TISSUE TISSUE
ANALYSIS AND STAINING SECTIONING
LABELLING

TISSUE
OBJECTIVE
GROSSING
To cut the fixed tissue samples


into small pieces before running


them through a tissue


processor.

PRINCIPLE PROCEDURE

When preparing a paraffin block,
Wear appropriate
Turn on the grossing

tissue samples like these are placed
protective equipmen station fume hood

in a small plastic cassette. The
Add 10% neutral
Label the cassette with the

cassettes are initially submerged in a
buffered formalin
name of student, student

identification number,date

fixative. Fixing and processing the
to the tissue. and type of tissue specimen

tissue will allow for a reliable


histopathological diagnosis. eg. uterus using pencil

PRINCIPLE After cutting, the
Take out the tissue

tissue specimen
specimen from the

Fixation : Proteins in the tissues get distorted
10% neutral buffered

and adhere to one another as a result of
is put in the

labeled cassette. formalin
fixation.Fixatives have the ability to cross-link


proteins, converting a semi-liquid state into a

semi-solid state that facilitates the movement
Measure the tissue
specimen using a ruler and

of tissue.
cut in the range 3~4 mm

ADDITIONAL INFO using a scalpel on the

cutting board
MATERIALS:

Scalpel holders, scalpel

blades, forceps, tissue

cassettes, containers, rulers,

dissecting boards, Grossing or

Medisheet, gloves, masks,

goggles, box,tissue specimen

REAGENT:

10% neutral bufferred

formalin

EQUIPMENT :

Duct Fume Hood :

Iryas

RESULTS PROBLEM &
CONCLUSION
SOLUTION
A precise histological

PROBLEM : Cut the
diagnosis can be made

tissue specimen too
by treating the tissue

small gently during grossing


SOLUTION : Make sure to
and using the

measure the tissue specimen
appropriate fixation.
thoroughly using a ruler before

cutting. .

TISSUE
OBJECTIVE
PROCESSING
To get ready the chemicals

needed for tissue

processing

PRINCIPLE PROCEDURE

In order to cut tiny pieces of tissue
A. Reagent Preparation
with a microtome, tissue processing

involves extracting water from the
Prepare the medium using
After the

followed the table : preparation of the

tissue and replacing it with a
medium, place the

medium that solidifies after the
medium into the

basket.
removal of the water.
B. Tissue Processing
MATERIALS:
All the prepared
.Take out the tissue

Basket reagent is pour into
cassette from 10%
the basket and put
neutral buffered

REAGENTS: inside the automated
formalin.
tissue processor.
10% Neutral Buffered Formalin,

Alcohol (Absolute, 95%, 80%, 70%,

50%), Absolute Alcohol and Xylene

Mixture (50:50), Xylene, Paraffin

Wax, Distilled Water

EQUIPMENT : .Arranged the

tissue cassette in

the basket..

Automated Tissue
Set the time for
Place the basket that

Processor:
tissue processing
contains cassettes

according to the
into the automated

Shandon Citadel
table above : tissue processor,

1000 close and press

PROBLEM &
'START' button .
RESULTS SOLUTION
CONCLUSION
PROBLEM : Too much heat and time in

liquid paraffin may harden tissue and
Tissue processing requires extreme

create microtome problems, which
caution. Any faults change the tissue,

leads to the drying and shrinking of
needing to be repeated. Tissues must

tissues. be adequately treated for subsequent

processes. Tissue preparation with


proper chemicals and embedding in a

suitable medium for the tissue type

SOLUTION : Make sure the tissue
yields diagnostically valuable sections.
is not exposed too long to heat

and in liquid paraffin.

TISSUE EMBEDDING

OBJECTIVE

tissue is embedded in a medium to create a block that can be used for
tissue sectioning and archival preservation of representative tissue.

PRINCIPLE PROCEDURE 1. Label the cassette by writing the name
of the tissue and student’s name.

tissues or the specimens are 2. Fill half of the mold with paraffin wax,
enclosed in a mass of the then put the mold at the cold spot area.
embedding medium using a mold.
Since the tissue blocks are very 3. Using warm forceps select the tissue, then place the tissue
thin in thickness they need a in the mold according to the side to be sectioned. This side
supporting medium in which the should be facing down against the mold. A small amount of
tissue blocks are embedded. This pressure may be used in order to have more even embedding.
supporting medium is called
embedding medium. 4. Chill the mold on the cold plate, orienting the tissue
and firming it into the wax with warmed forceps. This
MATERIAL ensures that the correct orientation is maintained and

cassette the tissue surface to be sectioned is kept flat.
automated tissue processor
paraffin wax 5. Insert the identifying label and
histology stainless steel embedding molds place the labeled embedding
forceps cassette base onto the mold.
ethyl alcohol
6. Add more paraffin into
RESULTS the mold to fill the
cassette and mold.

7. Cool the block on
the cold plate.

8. Remove the block
from the mold.

EQUIPMENT

Paraffin wax tissue
Embedding Centre: Slee
Mainz MPS/C / MPS/P /

MPS/W

PROBLEM SOLUTION CONCLUSION

Tissues is not embedded at the Press the tissue uniformly. Keep the rcTaerchtlaaiteaaecnfbkmitdbelaepeernattssoidlesbwasvbtuneiautedrhibnabmobellulodoetcskt
same level in embedding step. paraffin molten enough to get all the
pieces embedded at the same level.

Air bubbles traps around the tissues. make sure dispenses the wax slowly during
the process

TISSUE PRINCIPLE:
SECTIONING
sectioning refers to the service of cleanly and
consistently cutting paraffin embedded or frozen
tissue into a thin slice. These thin slices are referred

to as sections and are then mounted to a slide.

PROCEDURE 2. 3. OBJECTIVE :

1. Install the trim the paraffin The paraffin blocks to create an ideal thickness
block using were removed from
microtome. Please the microtome and tissue section
turn on, insert the "macro" setting ( frozen for 5 minutes
blade, adjust the 10 microns ) MATERIALS
angle, and set the in the freezer.
5. a microtome
mode. 6. paraffin (tissue) block
The tissue ribbon was
4. placed in a 42-45 degree The slide was labelled a blade
Celsius water bath, and and allowed to dry at water bath
remove and section the room temperature on
paraffin blocks with a the fishing technique slide
microtome using the was carried out using a the slide rack. paint brush
"micro" setting until a
glass slide. forceps
tissue ribbon was and pencils
obtained.

PROBLEM SOLUTION

Ribbons become compress and crumple when Use consistent wheel rotation to get even ribboning and
sectioning change the blade.

Tissues become expanded and torn because Do not cut the paraffin block too fast when performing
prolong in the water bath in high temperature tissue sectioning. Don't put the tissue in the water bath
too long, should be put only for 15 seconds. Make sure

the temperature of the water in the water bath not
too high and too low

RESULT tissue
section
on the Conclusion
glass
A thin and very well cut
slide tissue section which is
perfectly tied towards
the glass slide will
make staining process
become easier.

TISSUE STAINING

OBJECTIVE

To stain the tissue section using
haematoxylin and eosin (H&E) stain

PRINCIPLE MATERIALS: unstained tissue slides

Histological staining is a series of technique REAGENTS: Hematoxylin 3G (Sakura), Eosin (Sakura),
processes undertaken in the preparation of Xylene , Alcohol (absolute, 95%), Distilled
sample tissues by staining using histological water , Tap water
stains to aid in the microscope study. Staining
is used to highlight important features of the EQUIPMENTS: Oven (Memmert), Hotplate &
tissue as well as to enhance tissue contrast. Stirrer (Pro, HP-7 Lab Plus Series
Hematoxylin is a basic dye that is commonly
used in this process and stains the nucleus APPARATUS: Slide racks, forceps, filter paper,
giving it a bluish or purple color while Eosin tissue paper, funnel, measuring
(another stain dye used in histology) stains cylinder
the cell's nucleus giving it a pinkish stain.

PROCEDURE RESULTS

1. DEWAXING/ 2. HYDRATION 3. STAINING Thyroid tissue stained using H&E:

DEPARAFINNIZATION 3

The unstained slides were Reagent: Time: Reagent: Time: 2 1. follicles
removed from the oven 100% alcohol 1 mins Haematoxylin 3G 5 mins 2.epithelial cells
and then dipped into : (Sakura) 5 mins 1 3.endothelial cells
100% alcohol Running tap water 30 secs
Reagent: Time: 1 mins Distilled water 2 mins
Xylene 3 mins Eosin 10 secs
Xylene 3 mins 95% alcohol 1 mins Distilled water
Xylene 3 mins
Running tap water 1 mins 40X
Stained microscope glass sildes:
1 mins

4. DEHYDRATION Distilled water

5. CLEARING

Reagent: Time: Reagent: Time:
95% alcohol 10 secs Xylene 1 mins
95% alcohol 10 secs Xylene
100% alcohol 1 mins 2 mins
100% alcohol 1 mins
100% alcohol 1 mins

PROBLEM SOLUTION CONCLUSION

Tissues was stained too dark, hard Make sure timing for each staining Staining of the tissue
to observe under microscope. reagent is correct and do not leave using H&E stains provides
the tissue too long in reagent.
colour contrast to allow
Tissue being washed away from the Change the reagents - certain observation of the cell and
slide, no tissue left on the slide. manufacturers reagent can only
tolerate only certain number of tissue structure under
slides. Change the reagents if it microscope.
already being used too many times.

SLIDE
MOUNTING & LABELLING

OBJECTIVE 1.To mount a tissue slides with a coverslip using a mounting medium.
2.To label the stained slide clearly with specimen and stain details.

MATERIALS: PRINCIPLE

H&E stained slides Sections are mounted under coverslip to maintain
high refractive index necessary for critical
REAGENTS: microscopy and to protect the section during
storage. Staining and mounting reagents that are
Mounting medium (CoverSeal-X), Xylene stable over prolonged time, have strong light
fastness and are resistant to oxidative changes
MATERIALS: should be used. Labelling provides information and
can eliminate the errors that happen when slides
slide racks, coverslips, gauze, forceps, applicator are mis-identified or cannot be properly read.
sticks, coplin jar, self adhesive sticker, pencil

EQUIPMENTS:

ductless fume hood

PROCEDURE The appropriate An adequate The slide was
mounting media (1
coverslip was or 2 drops) was gently lowered at
placed on one edge
chosen according of the coverslip. 45 degree until it

to the size of the touched the

tissue section. mounting

medium.

The stained and The sticker was The slides was labelled with The slide was
labelled slide was placed at the the following details: examined under
observed under frosted end of the micrscope to
the micrscope. the slide. Type of specimen enusre there are
Staining method no air bubbles
Student name trapped.
Student id number
Date

RESULTS PROBLEM SOLUTION

Mounted and labelled H&E slide Air bubble trapped inside the coverslip Press the coverslip with an applicator stick to
during slide mounting. allow the air bubble to escape. But, if there were
CONCLUSIONS: many air bubbles present, the slide was placed in
xylene to remove the coverslip and the
The mounting cover the samples and fills procedure was repeated.
the space between the slides and the
coverslip. When the mounting medium Debris trapped under the coverslip. Clean the coverslip before using with
hardens it will provide rigidity and Kimwipes.
durability of the slides.

IMAGE ANALYSING

01 PRINCIPLE: 02 MATERIALS:

The process by which a visualized scene is analyzed Microscope
comprises specific steps that lead the user to either
an enhanced image or data that can be used for
further interpretation.

03 PROCEDURES:

1. Connect the light microscope to a power source.
2. Turn the revolving nosepiece so the 10x objective lens is in position.
3. Mount your specimen onto the stage.
4. Use the metal clips to keep your slide in place.
5. Look into the eyepiece and slowly rotate the coarse adjustment knob and fine
adjustment knob to bring your specimen to focus.
6. After you're done viewing with the 10x objective lens, switch to the 40x
objective lens and examine the slide again.

04 RESULTS (X40):

uttisesr
uues
a b
a - myometrium

c b - uterine gland
c - stroma

atppises
nudeix a - lumen
tthisysr
uoeid
a b b - mlamucinoasaplrgo
lparnida
c - with lymphatic nodules
ab
c a - thyroid follicle

d

b - colloid
c
c - epithelium
d - parafollilular cell

05 PROBLEM SOLUTION 06 CONCLUSION

Tissue become folded when Put the slide into a 1:15 alcohol solution for Significant amounts of visual data
observing under microscope 30 seconds . Then, put the slide into a water
bath for not more than 15 seconds. Gently may now be automatically analysed
Tissues become expanded and press the folded tissue area with a stick
torn because prolong in the while the tissue in the water bath. Do not thanks to the current trend of
water bath in high temperature press the folded tissue too hard since it can
make the tissue torn. image analysis. It enables the use of

Do not cut the paraffin block too fast when computer tools and high-resolution
performing tissue sectioning. Don't put the
tissue in the water bath too long, should be images to examine pathologic tissue
put only for 15 seconds. Make sure the
temperature of the water in the water bath samples. This creates the
not too high and too low
opportunity to create image

analysis techniques that support

pathologists' picture descriptions. In

this regard, numerous techniques

for the detection, classification,

and segmentation of the underlying

tissue primitives in histopathology

pictures have been developed.

FROZEN OBJECTIVE:

SECTIONING to study the frozen
section using a cryostat

PRINCIPLE PROCEDURE

Frozen sectioning is the procedure of The cryostat cuts individual The sections should be cut The section will glide
cutting thin sections of frozen tissue sections, unlike the ribbons with a slow and even smoothly and flat beneath
and is conducted in a cryostat. While of section, with the paraffin
frozen sections are physically less motion at about 10 to 15 um the anti roll plate.
stable than paraffin, they are preparation. thickness.
especially superior in the preservation The cutting of a frozen
of antigenicity and lipid retention. If it has become too Do not freeze the section requires
soft the sections will tissue too hard or
MATERIALS the sections may experience and touch.
also shatter or
cryostat fracture. shatter. The glass slide is lowered gently,
fresh tissue the section will automatically
brush, forcep, coverslip, clean glass Frozen section The anti roll plate is
slide, applicator stick, microtome should be handled flipped back after transfer from the knife to a warm
blade. the section is cut. (room temperature) clear glass
tissue freezing medium/frozen carefully.
section compound slide, where it will instantaneously
10% neutral buffered formalin the slide then mounted. rapid H&E staining melt and adhere.
hematoxylin 3G finally microscopic was performed.
eosin. examination was the section was placed onto
performed. a labelled slide, then directly

placed in pre heated 10%
formalin for 1 minutes.

RESULT

tissue specimen after sectioning tissue specimen is lost
during staining

PROBLEM SOLUTION CONCLUSION

Ice crystal artifacts can be the freezing time of the tissue should be When a disease requires
observed on the tissue increased as it can reduce the size of ice immediate diagnosis, a
frozen section is completed.
crystal artifacts. The tissue will be frozen at
the suitable temperature
the tissue sectioned is missing fix the tissue slide in cold
during the staining ethanol and sectioned for
microscopic examination

using a cryostat.

REFERENCES

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Gandhi, R. (2018, December 18). Grossing of tissue specimens in oral
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Mallick, I. (2022, October 22). Histopathology: Definition, Techniques,
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Shamala Ravikumar, R Surekha, Rooban Thavarajah. (2014, March 10).
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