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Teknik- Teknik Analisis Sel
5/25/2017
KI-1213-2
3
5 Struktur Sel di bawah Mikroskop

Mikroskopi:
◦ Magnification, Contrast, Resolution
◦ Light Microscopy (200 nm)
 Brightfield
 Fluorescent
 Advanced
◦ Electron Microscopy (1 nm)
 Transmission
 Scanning
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5.1 Microskop cahaya
0,61
N sin 
D  resolusi; N  indeks refraksi;   angularape rture
D
  panjang gelombang sinar datang
5.1.1 Brightfield microscopy
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Masalah: Sel umumnya tak berwarna & transparan
◦ Agar strutkturnya terlihat  perlu diwarnai
◦ Dapat melekat dan mudah dibuat irisan yang sangat tipis
Masalah baru yang timbul akibat tindakan di atas:
◦ Mengubah struktur sel/molekul
◦ Hanya memberikan gambaran “sel mati”
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5.1.2 Fluorescent microscopy
Memungkinkan lokalisasi molekul sel spesifik
 Pewarna Fluorescent “berpendar” di dalam gelap
 Zat warna tsb dapat langsung atau tidak langsung berasosiasi
dengan molekul sel:

 Eg: Rhodamin (merah)
 Fluorescein (hijau)
Beberapa pewarna fluoresen dapat digunakan secara
berturutan
 Dengan mikroskop ini dapat ditampilkan sel mati atau hidup

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Bagan alat Mikroskop fluoresen
Aktin dari kultur sel
fibroblast
Figure 5-5
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Figure 5-6
Mikroskopi cahaya untuk objek 3-D
 Confocal Scanning or Deconvolution Microscopy
 Generates 3D images of living cells
 Removes out-of-focus images  optical sectioning
 Can look inside thick specimens (eggs, embryos, tissues)
Figure 5-9
A mitotic ferilized egg of sea urchin (Psammechinus): a)
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fluorescent microscopy, b) confocal microscopic
image
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5.1.3 Advanced light microscopy

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Memungkinkan pengamatan sel hidup yang transparan
Pergeseran fasa cahaya (Light phase shifts) yang
disebabkan oleh specimen digunakan untuk menciptakan
contrast
◦ Phase contrast (refracted and unrefracted light)
◦ Differential interference contrast (two light beams)
Figure 5-14
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H. Freeman and
Time-lapse micrographs of cultured fibroblast
cell
movement
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along a glass surface
5.2 Mikroskop Elektron
5.2.1 Transmission electron microscopy (TEM)
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Operates in vacuum
Specimen usually fixed, embedded, sectioned, and stained
with an electron-dense material
Special techniques:
 Metal shadowing: visualize surface structures, cell components
 Cryoelectron: visualize unfixed, unstained samples
 Freeze fracture, freeze etch: visualize membrane interior
 Freeze etch: visualize cell interior
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Bagan alat Transmission electron
microscope
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Figure 5-15
Figure 5-16
5.2.2 Scanning electron microscopy
Can visualize surfaces of tissues, cells, isolated cell parts
 Specimen is fixed and coated with thin layer of heavy metal
 Images secondary electrons, resolution = 10 nm

Figure 5-20
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5.3 Pemurnian sel dari campurannya
dengan flow cytometry
Membutuhkan
penanda yang
berfluorescent
untuk sel target
Figure 5-21
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
Understanding the roles of each each cell component
depends on methods to break open (lyse) cells and
separate cell components for analysis

Cell lysis is accomplished by various techniques:
blender, sonication, tissue homogenizer, hypotonic
solution
Separation of cell components generally involves
centrifugation

5.2 Pemurnian bagian-bagian sel/organel
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5.3.2 Faraksinasi sel dengan sentrifugasi
differensial
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Figure 5-23
5.3.3 Pemisahan Organel dengan
sentrifugasi kesetimbangan gradien densitas
Figure 5-24
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5.4 Struktur sel hewan
Figure 5-42
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5.4 Struktur sel tanaman
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Figure 5-43
Lysosomes
 Peroxisomes
 Mitochondria
 Chloroplasts
 the Endoplasmic Reticulum (ER)
 the Golgi complex
 the Nucleus
 the Cytosol
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5.4 Organel-organel sel eukaryot
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Responsible for degrading
 certain cell components
 material internalized from
the extracellular
environment
Key Features
◦ single membrane
◦ pH of lumen  5
◦ acid hydrolases carry out
degradation reactions
5.4 Lysosomes
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Figure 5-44a
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Responsible for degrading
 fatty acids
 toxic compounds
Key Features
◦ single membrane
◦ contain oxidases and catalase
5.4 Peroxisomes
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Site of ATP production via
aerobic metabolism
Key Features
◦ outer membrane
◦ intermembrane space
◦ inner membrane
◦ matrix
5.4 Mitochondria
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Figure 5-45
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Site of photosynthesis in plants
and green algae
Key Features
◦ outer membrane
◦ intermembrane space
◦ inner membrane
◦ stroma
◦ thylakoid membrane
◦ thylakoid lumen
5.4 Chloroplasts
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Figure
Company5-46
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Responsible for
 most lipid synthesis
 most membrane protein
synthesis
++ ion storage
 Ca
 detoxification
Key Features
◦ network of interconnected
closed membrane tubules
and vesicles
◦ composed of smooth and
rough regions
5.4 The endoplasmic reticulum (ER)
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Figure 5-47
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Modifies and sorts most ER
products
Key Features
◦ series of flattened
compartments & vesicles
◦ composed of 3 regions:
cis (entry), medial, trans (exit)
◦ each region contains different
set of modifying enzymes
5.4 The Golgi complex
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Figure 5-49
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Separates
◦ DNA from cytosol
◦ transcription from translation
Key Features
◦ outer membrane
◦ inner membrane
◦ nuclear pores
◦ nucleolus
5.4 The nucleus
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Figure 5-50
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The portion of the cell enclosed by
the plasma membrane but not part of
any organelle
Key Features
◦ the cytoskeleton
◦ polyribosomes
◦ metabolic enzymes
5.4 The cytosol
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Figure 5-52
Sel dalam Bioteknologi modern
Kultur sel, bakteri dan virus
5/25/2017
KI-1213-2
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6.1 Keunggulan bekerja dengan kutur sel
dibandingkan dengan organisme utuh
 Lebih homogen dibaningkan dengan sel-sel dalam
jaringan
 Dapat disesuaikan dengan kondisi percobaan
 Dapat mengisolasi sel tunggal dan ditumbukan menjadi
koloni degan materi genetik yang seragam
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Pertumbuhan mikroba dalam kulturnya
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Contoh: E. coli dan yeast S. cerevisiae
Memiliki pertumbuhan yang cepat dan hanya memerlukan
nutrisi yang sederhana
Dapat ditumbuhkan dalam agar semipadat
Strain mutan dapat diisolasi dengan cara replica plating
Yeast colonies
Figure 6-1
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6.1 Replica plating
Figure 6-2
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2 Pertumbuhan sel hewan dalam kulturnya
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Memerlukan media yang kaya, termasuk asam amino
esensial, vitamin, garam, glukosa dan serum
Kebanyakan hanya tumbuh dalam permukaan padat
yang khusus
A single mouse cell
A colony of human cells
Many colonies in a petri dish
Figure 6-3
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6.2 Sel Primer dan galur sel (Primary cells
and cell lines)
Kultur Sel Primer (Primary cell cultures) diperoleh dari
jaringan hewan
 Tipe sel tertentu mudah dikulturkan, yang lain tidak
 Jika sel dipisahkan dari hewan utuhnya dan berhasil
membelah diri, umumnya hanya sampai periode terbatas
(sekitar 50 doublings), dan akhirnya mati
 Sel “transformant tertentu” dapat menjadi immortal dan dapat
digunakan untuk membentuk galur sel (cell line)
 Transformant ini bisa berasal dari tumors atau timbul secara
spontan
 Laju transformasi spontan bervariasi antar species
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6.2 Pembentukan kultur sel
Figure 6-5
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2 Beberapa kultur sel dapat berdeferensiasi
membentuk struktur seperti jaringan
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Figure 6-7b,c
6.2 Fusi Sel
 Dua sel yang berbeda dapat diinduksi untuk bergabung
menghasilkan sel hibrida (hybrid cell, heterokaryon)
 Hibrida Interspesifik dapat digunakan untuk genetika sel
somatik
 Sel hibrida tertentu (hybridomas) digunakan untuk produksi
antibodi monoklonal
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Figure 6-8
Figure 6-10
6.2 Produksi antibodi monoklonal terhadap
protein X
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3 Viruses: struktur, fungsi, dan kegunaan
Virus adalah parasit kecil yang tidak dapat bereproduksi
sendiri
 virus menginfeksi sel tertentu dan menggunakan mesin
reproduksi sel inang untuk memperbanyak virus
 Virus terdiri atas asam nukleat (RNA atau DNA) dikelilingi
oleh lapisan/kulit protein
 Viruse dapat menginfeksi sel prokaryot atau eukaryot dan
rentang sel yang dapat bertindak sebagai sel inang dari
viruses terbatas (sempit)
 Study tentang virus memberikan pemahaman mengenai
aspek dasar biologi sel dan pembentukan cancer

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6.3 Bentuk virus
Figure 6-11
The protein coat (capsid) of a virus is constructed of multiple copies of a single
or a few different proteins
Some virus capsids are also surrounded by a envelope consisting of a lipid bilayer
and a few glycoproteins
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6.3 The lytic replication cycle of E. coli
bacteriophage T4
Early proteins replicate viral
DNA and induce expression
of late proteins
Late proteins include capsid and
assembly proteins and enzymes to
degrade the host cell DNA
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Figure 6-16
6.3 The lytic replication cycle of an enveloped virus
Figure 6-17
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6.3 Virus Bakteri yang sering digunakan pada
penelitian biokimia dan genetika
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T phages of E. coli
Temperate phages (bacteriophage )
Small DNA phages
RNA phages
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6.3 Bacteriophage  undergoes either lytic
replication or lysogeny following infection of E. coli
Figure 6-19
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6.3 Commonly used Class I, II, and III
viruses
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6.3 Commonly used Class IV, V, and VI
viruses
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6.3 The retroviral life cycle
Figure 6-22
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