HS2413B_HISTOLOGICAL LAB REPORT GROUP 5

FACULTY OF HEALTH SCIENCE
MLT 428 | HISTOLOGICAL TECHNIQUE

SESSION OCT 2021 - JAN 2022
LABORATORY REPORT

HISTOLOGICAL LABORATORY ROUTINE

PREPARED BY :
ILYA HUSNA BINTI MUHAMMAD YAZID (2020819494)
NUR AFIFAH SYAFIAH BINTI SABRI (2020473342)
NUR AIMI BATRISYIA BINTI MOHD AMIN (2020834216)
NUR ALIA BATRISYIA BINTI MD FAZENI (2020828376)
NURUL AMIRA BINTI MOHD DARUS (2020627954)

GROUP : HS2412B



LECTURERS : MADAM HARTINI BINTI YUSOF
DR. NUR AYUNIE BINTI ZULKEPLI

DATE OF SUBMISSION : 06 JANUARY 2022

INTRODCTION TO Microscopic Accessioning
HISTOLOGY LAB Examination - Specimen handling &

logging

Histology refer to Mounting & Labelling Histology Gross Section
study of tissues Staining Workflow Tissue Fixation

and related Tissue Sectioning Tissue Tissue Processing
disease. Thus, work Embedding

in Histopathology
Laboratory is a

process that
involves in
examining the
process tissue and
detecting the
related diseases.

Objective Tissue grossing Problem :The cassettes

to trim the fixed tissue specimens Principles cannot close properly due
into small blocks before it is to the thickness of the
Grossing important in describing the tissue.
processed using a tissue processor. specimen's size, shape, color and any
apparent abnormalities. Solution: Make sure to not
Equipment Description of margins and their
orientation also important in gross cut the tissue too thick.
Ducted Fume Hood (Iryas) examination Slices the tissue 2-3
mm.
Materials Apparetus Procedures
Conclu
sion
Appendix/tonsil Scalpel holders Personal protective equipment (PPE) worn properly to
specimens fixed Scalpel blades avoid the risk of any hazard Tissue with correct
Forceps Cassettes were labelled with (type of specimen, student orientation was obtained
with Tissue cassettes name, student ID number and date)
commercially Containers Specimen grossing was performed one at a time. tissue Result
prepared 10% Rulers specimen was sliced with correct orientation at ranges
Dissecting boards 2-3 mm thin and placed in the cassette. tissue
formalin Medisheet The macroscopic details of specimens such as size, block
Gloves shape, colour, apparent abnormalities, margin and
Aprons orientation were noted.
Masks The tissue cassette was placed into 10% Neutral
Goggles Buffered Formalin.
Formalin waste was discarded into the formalin waste
container.

Objective: to remove any extractable Procedure

water in tissue and fixed it in TISSUE proceSSING
complete physical and partial chemical
state throughout several main step in

processing which are dehydration,
clearing and infiltration of wax

Conclusion : Apparatus : Materials :Fixed wop5afr0mse%aplrbaeaosdwarafodeglalecduedtod.entihsert1tto.sio0lalw0ll(1ec0a20ods.h00mo0lLl) 701wad%4dia0sdsta0iinpllmclgrAeoledhlp6cooa0ofwlrh0eaAfomdotlberlsrbo2oyltfuLote 810dw6i%0aasd0tsidamlpiAllnelcrlegodcohpof4awohlro0Aaelb0tfdeosmrroblluy2totLe
Tissue undergo several processing step to tissue blocks
prevent tissue autolysis 1. Forceps
(10% formalin)
2. Gloves
Principle : functions to eliminate 3. Aprons Results :
all extractable water from the 4. Masks processed
tissue blocks

tissue, and replace it with a support

medium that provides sufficient
tAipisrlnlasloaucucttectehhotideeanrpdberaariloneenucogadtevongesmetsseroeoxnnarttitttgsehfwhdoetars pA1mr10le0ci0pxo0a0th0urommerledllaxn(bay2dyblLsexa)onyldeuwldetiatennoseg aob2fb9ytLs5odoa%ilws1duta9dtiaes0illnl0cepgaomdrlhce1loowp0lhaoa0orfftmleoedrrl
rigidity to allow sectioning of the PROGRAM FOR ROUTINE OVERNIGHT alcohol

tissue without damaging or TISSUE PROCESSING

distorting the parenchyma

Reagents; 1.10% Buffered Formalin (2 Hour)
2.50% Alcohol (1 Hour)
10% Neutral Buffered 3.70% Alcohol (1 Hour)
Formalin- commercially 4.80% Alcohol (1 Hour)
prepared 5.95% Alcohol (1 Hour)
6.Absolute Alcohol (2 Hour) Equipment : Automated
Alcohol (absolute, 95%, 7.Absolute Alcohol (2 Hour) Tissues Processor:
80%, 70%, 50%) 8.Mixture of Absolute Alcohol and Xylene Shandon Citadel 1000
(1 Hour)
Absolute alcohol and Xylene 9. Xylene (2 Hour) Troubleshooting
Tissue
mixture (50:50) transfer
Xylene 10. Xylene (2 Hour) Problems; Tissue basket
Paraffin wax 11.Paraffin Wax (3 Hour) feels soft or mushy attachment
Distilled water Paraffin Wax (3 Hour) during embedding
10% Neutral Buffered slots
Formalin Solution:
Alcohol (absolute, 95%, 80%, Note: Change reagents and Rotating
70%, 50%) Total processing time= reprocess tissue head
Absolute alcohol and Xylene 21 Hour Tissue was process Keypad
mixture (50:50) in a short time control
Xylene
Paraffin wax
Distilled water

TISSUE EMBEDDING Result
Embedding How-To

Paraffin 2.Open cassettes

blocks 1.With PPE worn, 3.A small amount

Objec
tive equipment, apparatus and an appropriate of molten paraffin
and materials were mould was chosen were poured into
To embed the processed tissue
blocks with paraffin wax Equipment
s prepared. mould

Princ
iple Paraffin Wax Tissue

To orientate tissue specimen Embedding Centre 5.The mould was transferred 4.With warm clean
into paraffin block precisely - Slee Mainz MPS/C to a cold plate. The tissue forceps, tissue was
/ MPS/P / MPS/W transferred into mould,
Apparatus Materials
was firmly and gently cut surface downward.
Paraffin chamber pressed with forceps. (orientated accordingly)
1. Gloves Processed (solidified paraffin hold
2. Aprons tissue
tissue in place)

3. Masks blocked Forceps warmer 6.Labelled cassette 7.Molten paraffin was
4.Tissue paper poured into the mould to
5. Forceps Reagent
s

6. Scraper Paraffin Cold plate Hot plate was placed on top fully cover the face of the

7. Gauze wax Storage of the mould. cassette and labeled paper

8. Steel block mouTldroubleshooting compartment was placed on top of the

cassette.
Conclusion
Problem b-u-wPbPaobruxleeSrPssioirlnneoloogtunnwboctllpeteyimhaomeroonafubflfaldioinrck n-eTairspPse-ucramSaRerooasbeblsfpe-lufeeodiettmsndtmiiietonbbingoleaondfcetkedr 9.Paraffin tissue block was
-Tissue not A satisfactory separated from its mould
Scoelnuttrieodn block was 8.The mould
ce-nFctiroxeldtfiipsrlsmauteley at obtained after the paraffin was immediately was
on solidified and excess wax placed on the cryo
from the paraffin tissue
block cassette was removed console.

TROUBLESHOOTING RESULT tissue sections
SOLUTION
PROBLEM CONCLUSION
- Ensure to use the right mode between
- Thick and thin ribbon tissue A perfect tissue ribbon was
during sectioning macro and micro unit obtained and the fishing skills

Rotary microtome: Sleep - Readjust the knife and the block. Cool was improved.
Mainz CUT5062 - Section tissue wrinkled or curved the block in the refrigerator. Re-trim the


block

OBJEC
TIVE Tissue SECTIONING APPARATUS

to produce very thin - Slide racks
tissue sections that are - Clean and frosted glass slides
placed on the microscope - High profile microtome blades
- Scraper
slides for staining. - Clean brush
- Clean gauze
MATERIAL EQUIPMENT - Paper
- Tissue paper
paraffin blocks - Rotary microtome: Slee Mainz - Pasteur pipettes
CUT5062 - Applicator sticks
PRINCIPLE - Tissue floatation bath: XH- - Pencils
1001, Thermo Scientific 3120058 - Biohazards bags
- Cut the tissue section at 3-4 microns - Freezer
- Prevent wrinkles or bubbles and folding - Oven

under the tissue section

The tissue floatation bath was prepared PROCEDURES Using a glass slide, fishing of selected sections
by heating up distilled water to 42 was performed onto the slide. The tissue was
degree Celcius
prevent from moving using applicator stick

The slide was labeled with name, student The residual wrinkles or bubble was The glass slide was gently raised
ID, type of specimen and date. removed using pipette slightly above water surface
The finger protection guard was
The best section from the ribbon sections The slide was placed onto a slide
removed and the blade was fixed into was selected and gently separated than rack.
place.
other by using and applicator stick the slide was deparaffinized on the hot
The ‘M’ button was selected until ‘Macro’ The ribbons was laid on the water plate with the temperature 60 degree
word appeared on the display panel for carefully to prevent wrinkles or bubbles
trimming mode at 10 macro. Celcius
Start trimming until the complete tissue forming on tissue
section appears on the tissue ribbon. An applicator stick of forceps was used to

pull the ribbon sections from the blade
and transferred into the water bath

By using brush, the residual tissue The ‘M’ button was selected until the
ribbons were removed ‘Macro’ word disappeared on the display

panel for sectioning mode at 3 micron.

After the tissue block is cooled in the
freezer, it is placed in the object
clamp in previous orientation.

TISSUE STAINING Objective: Materials

Principles to stain the tissue section using Unstained tissue slides
hematoxylin and eosin (H&E)
cgtwandHtafhShoihueyshietvlebegaceoaemelwltuhirusceutntalreeeiiihiri.tasislglneoatlhslsahgsx'tunsaatyecedosisloonsitfiplsmcnagutoiioutionicunpnhnvisklrosseteeeei,srrsudnsttaathtahsahsittinastbehnoa.tnsaesnucsmdeeic Resu
lt
Reagents Equipment
Apparatus Processed tissue blocks
Hematoxylin 3G Oven: Memmert
Slide racks (Sakura) - Hotplate & stirrer: Pro, HP-7 Lab Conclusion
Forceps Commercially prepared Plus Series
Filter paper Eosin ( Sakura) - A good H&E stained tissue
Tissue paper Commercially prepared was obtained
Funnel Xylene
Measuring cylinder Alcohol (Absolute, Problem :
Tripod stand 95%)
Distilled water Nuclei of the tissue is too pale
Tap water due to light hematoxylin stain on
the tissue
Procedures Clear patches of tissue after
hydration sequence
A. Various concentration alcohol was prepared as
follow: Solution:
95% alcohol (250 ml) - 380 ml of absolute
alcohol was added to 20 ml of distilled water. The effectiveness of hematoxylin
B. Tissue staining: is reduced. Discard the old
The unstained slides were removed from the hematoxylin and replace with the
oven. new one.
The slide was stained according to the procedure Change reagent regularly to avoid
xylene from contaminated with
water

Objectives Principle Materials Apparatus Reagents Equipment

To preserve the To preserve and H&E - stained Slide racks Mounting Ductless fume hood,
stained tissue so it support the stained slides FGmCooamrvuceze/repssl2i4p (x2260xm4m0 ) medium Model: ESCO,
can be studied
ACSepoplpfll-iincaadjthaoerrssivtiecklabels (Coverseal-X) AscentMAX
microscopically section to light sticker Xylene
To label the slides microscopy
for identification

purposes

Conclusion

a clean and satisfactory SLIDE MOUNTING
slide tissue were obtained

Problem & LABELLING Procedures

The presence of bubble
coverslip was chosen according to
during mounting slide The task was performed in a the size of the tissue section
The excess of mounting
media smudge the fume hood
appearance of slide
The coverslip was gently lowered until mounting media was placed on one edge
Solution it touched the mounting media. of the slide so that it will run parallel

Ensure to have a clean The slide was examined macroscopically to the edge.
surface of coverslip from to ensure there are no air bubbles If air bubbles appear, an applicator
any contaminant stick was used by gently press the
Use appropriate drops of
mounting media to the coverslip to remove them.
size of tissue




Results: H&E - stained slides Lastly, the slide was placed on the A written sticker label was
slide rack to be air-dried pasted at the frosted end

of the slide.

Frozen Objective Equipment Procedures All the equipment such as forceps,
Section The cryostat low profile microtome blade,
To learn and to 1. Cryostat: was set to
Principle practice the Slee Mainz -20 degree brush, and applicator sticks was
frozen section Celsius. placed in the appropriate tissue
method by using MEZ

the cryostat. 2. Oven: sections in the cryostat.


Chemicals/Reagents Memmert
The water in the The fresh tissue was embedded on a The specimen that was
tissue sample specimen disc with Tissue Freezing grossed was placed on
freezes quickly, Medium/ Frozen Section Compound and specimen holder with
converting it to freezed in the appropriate area in the
ice. the embedding
cryostat. medium.




The firm ice Sectioning was
within the tissue performed at 5 µm
functions as 1.Tissue Freezing Medium/ The tissue was trimmed at
embedding media, Frozen Section Compound 10 µm thick until the thick.
allowing the specimen was exposed.

2.10% Neutral Buffered

tissue to be Formalin- Commercially
sectioned. prepared A rapid H&E stinging was The section was placed onto the labeled
performed and then slide then the unstained slide was
Mafprapeetsnhedirtxoinatsiisllsssu/e 3.Hematoxylin 3G
Apparatus (Sakura)- commercially followed by mounting the immediately placed in the pre-heated
prepared slide. 10% formalin in 1 minute.



4.Eosin (Sakura)-
commercially prepared A microscopic examination
was performed.
5. Xylene

6.Alcohol (Absolute,95%)
7.Distilled water Problems
8.Tap water
1.Clean glass slides The tissue cracked while sectioning it.
The tissue does not stick to the slide.
2. Coverslip Presence of bubbles on the slide.
3. Microtome blade Solution


4. Forceps Conclusion Cracking is probably due to over freezing. Check the cryostat
and make sure the temperature is around -20 degree Celsius.
5. Brush A slide of the frozen section Put the glass slide outside the cryostat. The temperature
6. Applicator stick for rapid microscopic difference helps the tissue to melt and stick to the slide.
7. Coplin jar The slide does not complete dehydration. Dip the slide with the
examination is obtained. coverslip into xylene and press out the bubbles.

REFERENCES

Pathology research core - tissue sectioning. Mayo Clinic. (2019, April 26). Retrieved December 16, 2021, from
https://www.mayo.edu/research/core-resources/pathology-research-core/services/tissue-sectioning
Rolls, G. (2016, June 16). Steps to better grossing. Leica Biosystems. Retrieved December 16, 2021, from
https://www.leicabiosystems.com/knowledge-pathway/steps-to-better-grossing/
Rolls, G. (2019, April 15). An introduction to specimen processing. Leica Biosystems. Retrieved December 16, 2021,
from https://www.leicabiosystems.com/knowledge-pathway/an-introduction-to-specimen-processing/
Rolls, G., & Anderson, J. (2012, May 30). An introduction to routine and special staining. Leica Biosystems.
Retrieved December 16, 2021, from https://www.leicabiosystems.com/knowledge-pathway/an-introduction-to-
routine-and-special-staining/

Rolls, G. (2016, June 30). Introduction to microtomy: Preparing & sectioning paraffin embedded tissue. Leica
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better-microtomy-flotation-section-drying/
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https://www.leicabiosystems.com/knowledge-pathway/he-basics-part-4-troubleshooting-he/
Theoretical and Practical Aspects of Routine H&E Staining. (2013). MediaLab.
https://www.labce.com/routine_h_and_e_staining.aspx

Troubleshooting Processing Problems - LabCE.com, Laboratory Continuing Education. (2008). Media Lab.
https://www.labce.com/spg572653_troubleshooting_processing_problems.aspx
Peters. S. R. (2010). A practical guide to frozen section technique. Springer Science+Business Media, LLC.
10.1007/978-1-4419-1234-3.