• Teknik untuk memproses

jaringan, apakah Biopsi,

Spesimen yang lebih
besar dikeluarkan saat
operasi, Atau Jaringan
dari otopsi dijelaskan di
bawah ini. Orang yang
melakukan pemrosesan
jaringan dan membuat
slide mikroskopis kaca
adalah HISTOTEKNOLOGI.
 II. Fixation: preservation of tissue
structure

A. Hindari autolisis
B.Fiksatif umum:
1. formaldehyde, buffered
2. 2. glutaraldehyde
3. 3. 70% alkohol
4. 4. panas: air mendidih, microwave
II. Fixation

C. Penerapan fiksatif
1. perendaman
2. perfusi Sebuah. perfusi intrakardiak
3. pertimbangan ukuran sampel
4. waktu pemaparan
II. Fixation

D. Prosedur
1. Pembedahan
2. Pemangkasan dan
orientasi
3. Fiksasi perendaman
Sebuah. kaset tisu
III. Dehydration
A. Definisi: pembuangan air B. Rasional: untuk
parafin embedding / sectioning C. Langkah-
langkah
B. Cuci fiksatif
C. seri alkohol bertingkat Sebuah. 70%, 95%,
100%, 100%
D. mengganti air dengan difusi
E. tidak terlalu panjang, tidak terlalu pendek
III. Dehydration
D. Prosedur
1. pemroses jaringan
otomatis Sebuah.
semalam
2. Mandi: air, alkohol
70,95,100,100%
3. Agen kliring: 2 bak
xylene
IV. Clearing
A. Paraffin solvent
B. Xylene, “clearing
agent”
C. Makes tissue
appear “clear”
V. Infiltration
A. Replace xylene with
paraffin
B. Immerse in melted
paraffin
1. ~55o C MP
C. Remove all bubbles,
xylene
D. Procedure
1. Two baths of melted
paraffin
 VI. Embedding
A. Orient tissue
1. cross section
2. longitudinal section
B. Dissection orientation
C. Avoid bubbles

Fig. 1-30
VI. Embedding
D.Procedure
1. Place tissue cassette
in melted paraffin
2. Fill mold with
paraffin
3. Place tissue in mold
4. Allow to cool
 VII. Sectioning – Trimming
the Block
Untrimmed tissue block

Trimmed block with

excess paraffin
removed and block
face in a trapezoid
shape
 VII. Sectioning
A. Rotary microtome
1. 5-10 mm
2. resolution vs. staining
B. Cryostat
C. Freezing microtome
D. Vibratome

1-1
VII. Sectioning
E. Procedure
1. Place tissue block in microtome with wide
edge of trapezoid lowest, and parallel to knife
2. Advance blade toward block
3. Begin sectioning
 VII. Sectioning
NOTE: Many of the figures in the text are of
plastic embedded sections cut at 1 mm
thickness, and thus showing better resolution
than 5-10 mm paraffin sections seen in lab.
VIII. Mounting sections
A. 40o C water bath
1. Flattens paraffin section
2. Permits mounting on slide
B. Gelatin & albumin
C. Glass slides
D. Oven / air dry
IX. Staining
A. Basic dye: hematoxylin
1. basophilic structures: DNA, RNA
2. differentiation: sodium bicarbonate
B. Acid dye: eosin
1. acidophilic (eosinophilic) structures
a. mitochondria, collagen
C. Water soluble dyes (paraffin sections)
D. Clearing agent (remove paraffin)
E. Rehydrate
F. Stain (trial & error timing)
 IX. Staining
NOTE: most figures in the text are not stained
with H & E, unlike the slides in our collection
(and most collections).
IX. Staining
G. Procedure
1. Slide rack
2. Solutions
a. rehydration
b. stain
c. dehydration
X. Coverslipping
A. Coverslip & mounting medium (not miscible
with water)
B. Dehydrate
C. Clearing agent
D. Permount
 Tissue Processing
• The term tissue processing refers to treatment of
the tissue necessary to impregnate it into a solid
medium, so that the tissue is rendered sufficiently
firm yet elastic for the tissue sections of desirable
thickness to be cut on microtome.
Tissue processing can be performed manually
(hand processing), but where multiple
specimens have to be dealt with it is more
convenient and much more efficient to use an
automated tissue processing machine ( a
“tissue processor”).
 Cont.
These devices have been available since the 1940’s1
and have slowly evolved to be:
1. Safer in use,
2. handle larger specimen numbers,
3. process more quickly,
4. produce better quality outcomes.
 Types of processors:
1) The tissue-transfer (or “dip and dunk”)
machines where specimens are transferred
from container to container to be processed.
2. The fluid-transfer (or “enclosed”) types
where specimens are held in a single
process chamber or retort and fluids are
pumped in and out as required.
 Histopathological Technique
• Histopathological technique deals with the preparation
of tissue for microscopic examination.

• The aim of good histological technique to preserve

microscopic anatomy of tissue and make them hard, so
that very thin section (4 to 5 micron) can be made.

• After staining, the section should represent the

anatomy of the tissue as close to as possible to their
structure in life.
These processes are
Fixati
on
Dehy
Staini
dratio
ng
n

Cuttin Cleari
g ng
Embe
dding
 1. Fixation
• This is the process by which the constituents of cells
and tissue are fixed in a physical and partly also in a
chemical state so that they will withstand
subsequent treatment with various reagents with
minimum loss of architecture.

• This is achieved by exposing the tissue to chemical

compounds, call fixatives.