HS2413B_HISTOLOGICAL LAB REPORT GROUP 1

FACULTY OF HEALTH SCIENCES



BACHELOR OF MEDICAL LABORATORY
TECHNOLOGY (HONS)

UNIVERSITI TEKNOLOGI MARA, PUNCAK ALAM



MLT 428
HISTOLOGICAL TECHNIQUES

(LABORATORY REPORT)



CHRONICLES OF HISTOLOGICAL TECHNIQUES



LECTURER: DR. NUR AYUNIE BINTI ZULKEPLI
PREPARED BY:

Name Student ID

Bassey ak James 2020489872
Izzah Syazlina binti Mohd Ashri 2020620566
Nur Allieya binti Mohd Kamal 2020492754

Nurul Aida Shaffiqah binti Mohd Adli 2020473436

Nurin Iffah binti Ely Arman 2020461446
Nurul Fitriyyah binti Mohd Alwi 2020492658

WORKFLOW IN HISTOPATHOLOGY LAB

Fresh Specimen Tissue Grossing Tissue Processing
& Fixation

Tissue Staining Tissue Sectioning Tissue Embedding
(H&E)

Slide Mounting
& Labelling

1.0) Introduction to Histology Equipment

EQUIPMENT:
1.Tissue processor
2.Tissue embedding centre
3.Rotary microtome

1.Tissue processor

Tissue transfer container

Reagent Internal fume hood
container Rotating head

Wax container
Keypad control

2. Tissue embedding centre

Cold Tissue warmer
plate
Wax dispenser
Hot plate
Forceps
3. Rotary microtome warmer

Cold plate

Thickness indicator LCD Arm
Thickness controller Block holder

Knife

Base

1.1) TISSUE GROSSING

MATERIALS :
Human tissue specimens fixed with commercially prepared 10% formalin

(Provided by MLT)

APPARATUS :

1.Scalpel holders 7. Dissecting boards
2.Scalpel blades 8. Medisheet
3. Forceps 9. Gloves
4.Tissue cassettes 10. Aprons
5. Containers 11. Masks
6. Rulers 12. Goggles

EQUIPMENT :
Ducted Fume Hood: Iryas

PRINCIPLE

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deactivated in the fixation process which is done after the grossing steps. The tissues then need to be cut
not more than 4 mm in thickness so that the cassette can be properly closed and can be fixed better.
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RESULT
Figure above shows the specimen was being cut into smaller

pieces so that it can fit properly in the cassette



PROCEDURE

Personal Protective All the equipments were The cassettes were
Equipment (PPE) were prepared and the fume labelled with the
students' name, ID
worn. hood was turned on
before grossing. number, date and cells
used.

All formalin waste was The cassetes then were Specimen grossing was
dicarded into the soaked in a container done one at a time.
formalin waste filled with 10% Neutral Grossed sample then
Buffered Formalin (NBF). was placed into the
container to prevent cassete.
potential hazards.

PROBLEM TROUBLESHOOT
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The specthimiceknowr uansecvuetntoo thin,
Csapreeciismteankewnitbhyadsishsaercptibnlgadthee
Specimen was cut roughly One cassette, one specimen
resulting in specimen trauma

Overloadcsapsesceitmteens in one

CONCLUSION

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useful information about the specimen.

1.2) TISSUE PROCESSING

REAGENTS : EQUIPMENT :
Automated Tissue Processor:
1.10% Neutral Buffered Formalin (NBF) Shandon Citadel 1000
2.Alcohols (Absolute, 95%, 80%, 70%, and
50%) MATERIALS :
Fixed tissue blocks soaked in 10%
3.Mixture of Absolute Alcohol and Xylene
(50:50) Neutral Buffered Formalin

4. Xylene APPARATUS :
5.Paraffin wax 1.Gloves 4.Goggles
6.Distilled water 2.Masks 5.Aprons

PRINCIPLE 3. Forceps

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microscopic examination. The principle used is to remove all extractable water from the tissue and replace it
with another medium which is a paraffin wax. There are three steps used during processing which are
dehydrating, clearing and impregnating.

A. Chemicals/reagents preparation PROCEDURE

Various concentration of 1. 1000ml of Absolute Alcohol 2. 1400ml of Absolute 3. 1600ml of Absolute
alcohol was prepared as was added to 1000ml of Alcohol was added to 600ml Alcohol was added to 400ml

follows: distilled water to make 50% of distilled water to make of distilled water to make
Alcohol (2.0L) 70% Alcohol (2.0L) 80% Alcohol (2.0L)

B. Tissue processing 5. 1000ml of Absolute 4. 1900ml of Absolute
Total processing time = 21 hours Alcohol was added to Alcohol was added to 100ml
Full program time = 21 hours 11 minutes, 1000ml Xylene tp make
allowing one (1) minute for every change Alcohol and xylene mixture of distilled water to make
of position 95% Alcohol (2.0L)
(2.0L)
All of the reagents was

placed in the automated 10% Buffered Formalin 50% Alcohol was set 70% Alcohol was set
tissue processor and the was set for 2 hours for 1 hour for 1 hour

time was set as follow:

Two Xylene were Two absolute alcohol Mixture of Absolute 95% Alcohol was 80% Alcohol
set for 2 hours were set for 2 hours alcohol and Xylene set for 1 hour was set for 1 hour
was set for 1 hours

Two Paraffin wax RESULT
were set for 3 hours
Processed tissue: Tissue specimen shrinked
and became slightly smaller

PROBLEM TROUBLESHOOT

Excessive dehydration that leads to micro Shorten the dehydration time and process the smaller
chatter around the edge of the tissue biopsy tissue separately from the larger tissue.


Ensure adequate impregnation of the tissue section in
paraffin wax
Tissue is shrink and dry




CONCLUSION

Tissue processing is a crucial step in order to produce good tissue slides that can be used to diagnose the
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1.3) TISSUE EMBEDDING

MATERIAL: APPARATUS:
Processed tissue blocks
1. Forceps 5. Tissue paper
Reagent: 2. Scraper 6. Gloves
Molten paraffin wax 3.Steel block mould 7. Aprons
4. Gauze 8. Masks
EQUIPMENT:
Paraffin Wax Tissue Embedding
Centre : Slee Mainz MPS/C /

MPS/P / MPS/W

PRINCIPLE

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wax. Paraffin wax is used as the embedding substance as it is cheaper and easy to use. Before putting the
tissue in the mould, it is essential to make sure the tissue orientation is correct and the cutting surface is
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RESULT

Figure shows tissue block : tissue specimen is embedded in molten paraffin
wax, once solidifies.

PROCEDURE The temperature of the Processed tissue was Small amount of molten
embedding machine was kept in the cassette bath paraffin wax were poured
Personal Protective into the mould. The tissue
Equipment (PPE) were set within the range of and an appropriate
worn and noted prior the 54-60°C (melting point) mould size was chosen. then was placed in the
embedding process. center of the molten wax.
for paraffin.

The labelled cassette Warm forceps can be The mould then was The tissue must be
was placed on the molten used to secure the tissue. transferred onto a cold placed by referring to the
The mould then was filled plate to secure the tissue
paraffin. The label then in place, as the paraffin correct specimen
was placed on top of the up with the molten orientation by using
paraffin. solidifies.
cassette. warm forceps.

The cassette then was Once solidifies, the block The cryo console and the
cooled on the cryo then was taken out from lights were switched off
and the work area were
console to fully solidifies. the mould and excess
wax were removed by cleaned off from any
excess wax.
using scalpel.

PROBLEM TROUBLESHOOT

Bubble entrapped
in the block and Embed the tissue in the
molten paraffin wax
around the tissue properly


Melt the paraffin wax

and start over or

Cracked paraf
fin wax block again

re-embed again

Air bubbles entrapp
ed on the surface

of the paraffin block.



Avoid applying too much force while cooling on the
cold plate.

CONCLUSION

The process of embedding tissue in a molten medium as a paraffin block proved to be a necessary step in
thiissstuoelofogricaarlchteicvhanl piquurpeso.seTsh. is step is important as it gives structure, prevents distortion and preserves the

1.4) TISSUE SECTIONING

APPARATUS : EQUIPMENT :
Rotary microtome: Slee Mainz
1.Slide racks 7.Paper CUT5062
2.Clean frosted end 8.Tissue paper Tissue floatation bath: XH-1001,
glass slides 9.Pasteur pipettes Thermo Scientific 3120058
3.High profile 10.Applicator sticks Freezer
microtome blades 11.Pencils Oven
4. Scraper 12.Biohazard bags
5.Clean brush MATERIALS :
6.Clean gauze Paraffin blocks

PRINCIPLE
Tissue sectioning can be considered as the most anticipated process throughout the whole process
of tissue preparation. After the tissue block hardened during the embedding process in the paraffin
wax, the tissue block was then being trimmed and sectioned to get a thin slice of tissue by using a
rotary microtome. The thin slice of tissue is essential in order to view the structure of tissue clearly
and observe what kind of changes occur on the tissue. The fishing technique to pick up the tissue
from the warm water bath is important as it helps in removing wrinkles from the sectioned tissue.

PROCEDURE The microtome blade is placed The specimen is clamped by The "M" button is selected until
by loosening the blade fixation inserting the paraffin block at the "Macro" word appears on
Microtome angle and the blade is inserted from the display panel for trimming
is adjusted. the specimen holder of the mode and the appropriate
one side. After the blade has microtome. The fixing lever is trimming thickness is set
been securely clamped, the adjusted for the orientation of
knife guard is raised into its according to the specimen size.
position before trimming to the specimen.
avoid injury. The tissue block is trimmed until
a suitable area for tissue
Sectioning process The "M" button is selected until The tissue block is placed in the
is done by cutting the word "Macro" disappeared refrigerator for 5-10 minutes to sectioning exposed. Trimming
usually done at thickness of 10-
the block at a on the display panel for cool the block for easier
thickness about 4- sectioning mode. sectioning process. µ30 m.

µ5 m until ribbon

sections appeared.

The ribbon sections are picked up by forceps and The best sections from The glass slide is The glass slide
transferred on the surface of the warm water the ribbon sections are immersed vertically in containing the
the water bath to pick tissue section is
bath. Make sure the ribbons are not folded and gently separated by
no wrinkles on the ribbon after placing them on using an applicator up the sections. placed at an
upright position on
the water bath. stick.
a slide rack.

Switch "Off" the The residual tissue and wax are removed The tissue section allowed to dry
microtome. from the microtome using a brush and overnight at 37°C / dry for 15-30
disposed into biohazard bin.
minutes at 58-60°C.

PROBLEM TROUBLESHOOT

Obvious knife lines on the Replace the knife with a new knife or remove hard
sectioned tissue particles that might be present in the tissue or wax.

Tissue was compressed causing Cool the tissue blocks for 5 minute in the refrigerator,
disorientation when observed as undercooling it will provide soft blocks.
under the microscope.
CONCLUSION
RESULT
Tissue sectioning is a crucial part of the whole process.
A thin slice of tissue was Without good sectioning techniques, the tissue will not
obtained from tissue appear as expected on a slide, thus making it hard to
Tobhseerr
evfeorpea, rtaslloftethchentiiqssuuees clearly under the microscope.
sectioning for microscopic for sectioning need to be
examination. mastered and all troubleshooting must be taken into

account in order to produce a nice viewing tissue section.

1.5) TISSUE STAINING

MATERIALS : REAGENT:
Unstained tissue slides 1.Hematoxylin 3G (Sakura) - Commercially

APPARATUS : prepared
1.Slide racks 2.Eosin (Sakura) - Commercially prepared
2. Forceps 3. Xylene
3.Filter paper 4.Alcohol (Absolute, 95%)
4.Tissue paper funnel 5.Distilled water
5.Measuring cylinder 6.Tap water
6.Tripod stand
EQUIPMENT :
1.Oven: Memmert
2.Hot plate:

PRINCIPLE

Normal tissue is colourless. Thus, the staining process is used to differentiate tissue components and make it visible to
observe under a microscope. Staining usually works by using two dyes which include acidic and basic dyes. The most
common staining dyes used in histology laboratories are H&E stains (Hematoxylin and Eosin) as these stains provide
a very detailed view of the tissue. Hematoxylin is considered as a basic dye and it is used to stain acidic structures such
as nucleus, ribosomes and other structures with a deep blue-purple colour. Meanwhile, Eosin acts as an acidic dye and
stains basic structures such as cytoplasm. Eosin binds to acidic components and stains them orange-pink colour. Next,
in the staining procedure, xylene is used as a clearing agent to remove all the wax away and it is used to make tissues
become transparent to easily view the tissues morphology under the microscope. Increasing concentration of alcohol
is used to remove water from the tissue section.

RESULT

Figure above shows the result after staining, which is stained tissue slides.

PROCEDURE

400ml of 95% alcohol The slides were After 4 minutes, the The unstained slides
were prepared before heated on the hot slides were removed then were stained
plate at 60°C for according to these
proceeding with about 4 minutes. from the hot plate. order.
staining.

Xylene Xylene Xylene Absolute Alcohol Absolute Alcohol
(3 minutes) (3 minutes) (3 minutes) ( 1 minute) (1 minute)

Distilled water Running tap Hematoxylin 3G Distilled water Running tap 95% Alcohol
(30 seconds) water [Sakura] (1 minute) water (1 minute)

(5 minutes) (5 minutes) (1 minute)

Eosin Distilled water 95% Alcohol 95% Alcohol Absolute Alcohol
(2 minutes) (10 seconds) (10 seconds) (10 seconds) (1 minute)

Xylene Xylene Absolute Alcohol Absolute Alcohol
(2 minutes) (1 minutes) (1 minute) (1 minute)

PROBLEM TROUBLESHOOT





Wax cannot be removed by using the Use hot plate at 60 for 3 minutes and dip it in the
oven as the oven is not functioning well. xylene solution to remove the wax residue.
Stained slides show

ed uneven staining
due to inadequate volume of reagents in


the container. eRnesfuilrlinthgeerqeuaaglesnttasinaitnpgroonpethrevoslluidmees.




CONCLUSION
Tissue staining is important because it can help to visualize the cellular and structure of the tissue. The
process of tissue staining should be done correctly as it can affect the microscopic examination. A wrong
step of staining could lead to way too much or too little of tissue stained which will be difficult to view
ournddeerrtothme amkiecrsousrceotphee.pTrhoceeressfoisred,oint eispirmoppeorrltya.nt to take precautions during the staining of the tissue in

1.6) SLIDE MOUNTING & LABELLING

APPARATUS : MATERIALS :
1.Slide racks H&E-stained slides
2.Applicator stick
3.Cover slip REAGENT:
Xylene mounting medium
(22X40mm / 24X60mm)
4.Glass slide with stained

tissue
5.Self adhesive label sticker

PRINCIPLE
Tissue mounting is the last step in the histology technique. The purpose of this step is to hold the specimen
in place when observing it under a microscope, protect the specimen physically, and preserve the specimen
for a long period. This is because the mounting medium will bind the specimen, slide, and coverslip
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a higher-grade optical clarity. The xylene mounting medium is used when a permanent mount is needed
and it is a mounting medium that is frequently used in Hematoxylin and Eosin stain tissue. In this step air
bubbles need to be avoid as it can interfere the microscopic examination of the specimen.

PROCEDURE Appropriate personal The appropriate size of On the slide, adequate
protective equipment (PPE) coverslip must be chosen amount of mounting
Fume hood must be used must be worn while carrying correctly according to the medium is poured.
while mounting the slide
out the procedure size of the tissue

A sticker labeled with the student's name, If air bubbles are present, The coverslip must be
student's ID number, date, type of specimen, and applicator stick can be used lowered and released slowly
staining method used need to be placed on the
to remove them to prevent the formation of
frosted end of the slide air bubbles




RESULT

Figure above shows the mounting Figure above shows the result from a good
process getting done mounting with no air bubbles present

PROBLEM TROUBLESHOOT


Presence of air bubbles when the
coverslip has been put on top of the slide Use applicator stick and tap lightly on top of the coverslip
to remove the air bubbles




Poured too much of the mounting
medium on the slides Use laboratory tissue to clean the excess of the of the
mounting medium on the side of the slides when it is still


wet or use razor blade when it is dry

CONCLUSION

The process of tissue mounting is the final determinant of a successful tissue preparation before the tissues are
oprbescearvuetidonusnmdeursttbheetalikgehnt smo iaclrlothsceotpises.ueTshtruus,ctiutreiss cimanpboertvainetwteod chlaevaerlya. proper mounting technique and

1.7) FROZEN SECTION RESULT

APPARATUS : REAGENTs :
Tissue Freezing Medium /
1.Clean glass slides Frozen Section Compound
2. Coverslip 10% Neutral Buffered Formalin
Hematoxylin 3G (Sakura)
3.Microtome blade Eosin (Sakura)
4. Forcep Xylene
5.Applicator stick Alcohol (Absolute, 95%)
6. Brush Distilled Water
7.Coplin Jar ir2n1aT..sptaCOirdpurvmyehwoniessa:nttottaMelotur:EgeseyQmSdPUmlpReItrPoeoeIMcrNfetMErseCsNae
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MATERIALs/specimen :
Fresh tissue

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microscopic analysis. When the tissue sample undergoes a rapid freezing, it converts water into ice and acts
as an embedding media to allow the tissue to be sectioned. The cryostat should be maintained at
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pathologists such as it shortens the time for preliminary diagnosis and less equipment required to perform the
procedure.

PROCEDURE Cryostat was prepared on the scheduled date A Coplin jar containing 10%
of the frozen section. The temperature of the formalin was placed in the oven
Personal protective
equipment (PPE) was wore cryostat was set to -20°C. The forceps, low at 60°C one hour before the
profile microtome blade, brush and applicator expected specimen arrival. It was
while performing the
procedure sticks were placed in the appropriate tissue removed before being used.
sections in the cryostat.

The tissue was The specimen disc was Fresh tissue was embedded on a The specimen was grossed
placed in the orientable specimen disc with Tissue Freezing and the frosted glass slide(s)
µtrimmed at 10 m Medium/Frozen Section Compound
specimen head and was froze in designated area in was labelled with a type of
thick until the specimen and date.

specimen exposed. cryostat.

Then, sectioning was The section was placed onto the Rapid H&E staining was The specimen
labelled slide(s). Unstained slide was performed. After that, the disc was
µperformed at 5 m immediately placed in the pre-heated
slide was mounted and removed from the
thick 10% formalin for about 1 minute microscopic examination orientable

was performed. specimen head

The cryostat was cleaned The residual tissue was removed The specimen disc
after each used and the from the specimen disc and was was left at room
placed in a specimen container temperature to
remaining tissue was allow the
processed for routine containing 10% formalin. It was embedding
processing on the next day then be left aside to fix. medium to melt

PROBLEM TROUBLESHOOT

Tissue curling or folding Flatten the curled tissue with a brush or change the
blade to a sharper blade

Tissue sticked on the brush or forcep Handle the tissue with care by brushing it gently

CONCLUSION

TFrozen section is very useful for quick diagnosis, surgical consultation and also allows for immunofluorescence
and immunohistochemistry studies. There are several techniques that should be done and be aware of in order to
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REFERENCES

Anderson, J. (n.d.). An Introduction to Routine and Special Staining . Retrieved
from Leica Biosystem : https://www.leicabiosystems.com/knowledge-
pathway/an-introduction-to-routine-and-special-staining/

Datson, J. D. (2018, November 29). Uses of Xylene . Retrieved from Sciencing:
https://sciencing.com/alternative-solvents-benzene-8074379.html

Manan Shah. (2019). Frozen section overview and application.Slideshare.net.
https://www.slideshare.net/MananShah133/frozen-section-overview-and-
application

Peters, S. R. (Ed.). (2010). A practical guide to frozen section technique.
Springer Science & Business Media.
https://link.springer.com/content/pdf/10.1007/978-1-4419-1234-3.pdf

Ravikumar, S., Surekha, R., & Thavarajah, R. (2014). Mounting media: An
overview. Journal of Dr. NTR University of Health Sciences, 3(5), 1.
https://doi.org/10.4103/2277-8632.128479

REFERENCES

Rolls, G. (2016, June 16). Steps to Better Grossing. Leica Biosystems; Leica
Biosystems. https://www.leicabiosystems.com/knowledge-pathway/steps-to-
better-grossing/

Sectioning of Paraffin-Embedded Tissue Video Protocol. (n.d.). Retrieved from
Abcam : https://www.abcam.com/protocols/sectioning-of-paraffin-embedded-
tissue-video-protocol

Sim, J. (2018, December 19). Biorepository. Biorepository.
https://www.geneticistinc.com/blog/what-is-a-frozen-section

Troubleshooting Sectioning Problems . (n.d.). Retrieved from LabCE:
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Spencer, L. T., & Bancroft, J. D. (2017, December 13). Tissue processing.
Basicmedical Key. Retrieved December 19, 2021, from
https://basicmedicalkey.com/tissue-processing/

THANK YOU