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Ketrampilan Dasar LaboratoriumKetrampilan Dasar Laboratorium
Program Studi Ilmu Biomedik 2011
Kuliah 7: Nopember 16
Jadwal praktikum & kuliah
Pembahasan Hasil Metabolisme (tabel 4)
Pembahasan Hasil Elektroforesis
Praktikum 8: Kultur sel (ragi dan bakteri)
Jadwal Kuliah dan Praktikum Ketrampilan Dasar Laboratorium
Nop 16/17 pk 10:00-12:00 KULIAH 7 Teknik Kultur Sel LT pk
08:00-11:00/ pk 12:00-15:00
Tugas: laporan
praktikum
Nop 24 pk 08:00-10:00 KULIAH 8
Histoteknik; Dr Alya
Nop 28-Des 1 Praktek Histoteknik 1
(Pemrosesan Jaringan: Dehidrasi s/d Impregnating
Des 5-8 Praktek Histoteknik 2
(Blocking s/d Perwarnaan dan Mikroskop
Tugas: laporan
praktikum
Kelompok
1 Anwar, Ernawati, Dedy, Imam, Vera
2 Roy, Martina, Lily, Dorra
3 Taya, Leo, Yeni, Musthari, Siti
4 Donny, Dita, Ningrum, Sukaisi
HasilHasil MetabolismeMetabolisme ((TabelTabel 44)) KELOMPOK GLUKOSA TRIGLISERIDA UREA
B ST SA B ST SA B ST SA
KELOMPOK 1
Menu : nasi lengkap+air
putih; 1 jam sblmnya 0 0.275 0.167 0 0.222 0.091 0 0.009 0.017
Kelompok 2
Makan roti coklat + teh
manis; 1 jam sblmnya 0 0.359 0.271 0 0.23 0.256 0 0.398 0.098
Kelompok 3
Menu : nasi lengkap+air
putih; 1 jam sblmnya 0 0.239 0.185 0 0.198 0.18 0 0.037 0.041
Kelompok 4
Menu : nasi lengkap+air
putih; 1 jam sblmnya 0 0.340 0.409 0 0.228 0.065 0 0.006 0.009
Kelompok 5
Menu: segelas susu; 1
jam sblmnya 0 1.472 1.339 0 0.217 0.290 0 0.091 -0.108
BerdasarkanBerdasarkan kurvakurva standarstandar kitakita
mengetahuimengetahui A A berbandingberbanding luruslurus
dengandengan konsentrasikonsentrasi unsurunsur
Untuk menghitung konsentrasi sampel:
Ksampel = Asampel/Astandar*Kstandar
HasilHasil MetabolismeMetabolisme ((TabelTabel 4)4) MENU GLUKOSA
(mg/dl)
TRIGLISERIDA
(mg/dl)
UREA
(mg/dl)
1 nasi lengkap+air putih; 1 jam
sblmnya 60.7 82.0 75.6
2 roti coklat + teh manis; 1 jam
sblmnya 75.5 222.6 9.8
3 nasi lengkap+air putih; 1 jam
sblmnya 77.4 181.8 44.3
4 nasi lengkap+air putih; 1 jam
sblmnya 120.3 57.0 60.0
5 segelas susu; 1 jam sblmnya 91.0 267.3 0.0
Nilai N
orm
al
HasilHasil MetabolismeMetabolisme ((TabelTabel 44))
0
100
200
300
A B C D E
Ko
nse
ntr
asi
(m
g/d
l)
Mahasiswa
Konsentrasi Glukosa, Trigliserida dan Urea
pada sampel Darah Mhs
Glukosa
Trigliserida
Urea
PembahasanPembahasan HasilHasil ElektroforesisElektroforesis:: AgaroseAgarose
10 9 8 7 6 5 4 3 2 1
Hasil positif DNA: sumur 2,3,5,6,9,10
** Kesalahan pada kelompok pagi – pewarna yang dipakai
menyerap cahaya UV dengan akibat hasil DNA kurang jelas
Kenapakah hanya satu pita DNA yang muncul pada
hasil elektroforesis agarose kita?
PembahasanPembahasan HasilHasil ElektroforesisElektroforesis:: SDSSDS--PAGEPAGE
St M S Gp Gs Ep Es P St M S Gp Gs Ep Es P
Standard:
Myosin
-galaktosidase
Fosforilase
Albumin
Ovalbumin
Karbonik anhidrase
Tripsin inhibitor
Lisozim
Aprotinin
200.000
116.250
97.400
66.200
45.000
31.000
21.500
14.400
6.500
Log
bera
t m
ole
kul (D
alto
ns)
jarak dari awal (mm)
KulturKultur JaringanJaringan//SelSel
the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions
In vitro cultivation of organs, tissues & cells at defined temperature using an incubator and supplemented with a medium containing cell nutrients and growth factors is collectively known as tissue culture
SejarahSejarah KulturKultur SelSel//JaringanJaringan
• 1885- Roux: maintained embryonic chick cells in a cell culture with saline
• 1907- Harrison: lymph cells successfully cultured
• 1911- Lewis: used liquid media made from sea water, serum, embryo extract, salts and peptones
• 1913-Carrel: introduced strict aseptic techniques so that cells could be cultured for long periods.
SejarahSejarah KulturKultur SelSel//JaringanJaringan
• 1940s: antibiotics penicillin and streptomycin used in culture medium to decrease the problem of contamination
• 1948- Earle: isolated mouse L fibroblasts which formed clones from single cells
• 1952- Gey: established a continuous cell line from a human cervical carcinoma known as HeLa (Helen Lane) cells
• 1975- Kohler & Milstein: produced the first hybridoma capable of secreting a monoclonal antibody
PengunaanPengunaan KulturKultur SelSel Model systems for studying basic cell biology interactions between disease causing agents and
cells effects of drugs on cells process and triggering of aging
nutritional studies Toxicity testing study the effects of new drugs
Cancer research Study the function of various chemicals viruses
and radiation to convert normal cultured cells to cancerous cells
PengunaanPengunaan KulturKultur SelSel Virology cultivation of viruses for vaccine production study the infectious cycle of the virus
Genetic Engineering production of commercial proteins (large
scale production possible) “Gene Therapy” Cells with a functional gene can replace
cells with non-functional gene Production of “replacement” organs/tissue
KekuranganKekurangan kulturkultur selsel
Cell characteristics can change over time
“in vitro” not exactly the same as ‘in vivo”
Pada praktikum minggu ini kita akan coba mengkultur sel bakteri serta memonitor aktifitas kultur ragi
• mengukur produksi CO2 • melihatnya dengan mikroskop • mengukur konsentrasi secara indirect dengan teknik McFarland Scale
RagiRagi –– Yeast CultureYeast Culture Sangat berguna sebagai sistem kultur untuk
meneliti banyak hal yang berkaitan dengan
DNA dan protein di sel eukariotik
Banyak resources di web, misalnya:
data dan link: http://genome-www.stanford.edu/Saccharomyces
“buku teks”: http://www.phys.ksu.edu/gene/chapters.html
konprensi: http://www.yeast-meet.org/2012/
YEAST GENETICS AND MOLECULAR YEAST GENETICS AND MOLECULAR BIOLOGYBIOLOGY **diadaptasi dari: **diadaptasi dari: http://www.cmb.gu.se/Department+of+Cellhttp://www.cmb.gu.se/Department+of+Cell--++and+and+
MolecularbiologyMolecularbiology /?/?languageId=languageId=100001100001&contentId=&contentId=--11&disableRedirect= &disableRedirect=
true&returnUrltrue&returnUrl=http %=http %33A%A%2 2 F%F%22Fwww.cmb.gu.se%Fwww.cmb.gu.se%22FF
The yeast Saccharomyces cerevisiae
A yeast cells is about 4-7mm large
The ”eyes” at the bottom are bud scars
The yeast The yeast Saccharomyces Saccharomyces cerevisiaecerevisiae::
Yeast lives on fruits, flowers and other sugar containing substrates
Yeast copes with a wide range of environmental conditions: Temperatures from freezing to about 55°C
Yeasts proliferate from 12°C to 40°C
Growth is possible from pH 2.8-8.0
Almost complete drying is tolerated (dry yeast)
Yeast can still grow at sugar concentrations of 3M (high osmotic pressure) and up to 20% alcohol
The yeast The yeast Saccharomyces Saccharomyces cerevisiaecerevisiae:uses:uses
Saccharomyces cerevisiae is the main organism in wine production - reason is high fermentation capacity, low pH and high ethanol tolerance
Saccharomyces cerevisiae is the main organism in beer production – ferments sugar to alcohol even in the presence of oxygen and at low temperatures (8°C)
Saccharomyces cerevisiae is the yeast used in baking because it produces carbon dioxide from sugar very rapidly
The yeast The yeast Saccharomyces Saccharomyces cerevisiaecerevisiae:uses:uses
Saccharomyces cerevisiae is used to produce commercially important proteins - can be genetically engineered; it is regarded as safe and fermentation technology is highly advanced
Saccharomyces cerevisiae is used for drug screening and functional analysis because it is a eukaryote but can be handled as easily as bacteria
Saccharomyces cerevisiae is the most important eukaryotic cellular model system because it can be studied by powerful genetics and molecular and cellular biology techniques - many important features of the eukaryotic cell have first been discovered in yeast
Saccharomyces cerevisiaeSaccharomyces cerevisiae is a eukaryoteis a eukaryote
belongs to fungi, ascomycetes
divides by budding which results in two cells of unequal size, a mother (old cell) and a daughter (new cell)
yeast life is not indefinite; yeast cells age and mothers die after about 30-40 divisions
Saccharomyces cerevisiaeSaccharomyces cerevisiae is a eukaryoteis a eukaryote
has a eukaryotic structure with different organelles:
◦ Cell wall consisting of glucans, mannans and proteins
◦ Periplasmic space with hydrolytic enzymes
◦ Plasma membrane consisting of a phospholipid bilayer and many different proteins
◦ Nucleus with nucleolus
◦ Vacuole as storage and hydrolytic organelle
◦ Secretory pathway with endoplasmic reticulum, Golgi apparatus and secretory vesicles
◦ Peroxisomes for oxidative degradation
◦ Mitochondria for respiration
Yeast genetics: the genetic materialYeast genetics: the genetic material
The S. cerevisiae nuclear genome has 16
chromosomes
In addition, there is a mitochondrial genome
The yeast chromosomes contain centromeres and
telomeres, which are simpler than those of higher
eukaryotes
The haploid yeast genome consists of about 12,500
kb and was completely sequenced as early 1996
(first complete genome sequence of a eukaryote)
The yeast genome is predicted to contain about
6,200 genes,
Yeast Yeast genomic analysis:genomic analysis:
• A joint goal of the yeast research community: determination of the function of each and every gene
• Micro array analysis to determine the binding sites in the genome for all transcription factors
• Yeast deletion analysis: more than 6,000 deletion mutants is available for research
• All yeast genes have been tagged to green fluorescent protein (GFP) to allow protein detection and microscopic localisation
• Different global protein interaction projects are ongoing
Yeast: Model OrganismYeast: Model Organism
The yeasts Saccharomyces cerevisiae and
Schizosaccharomyces pombe are regarded as model
organisms in molecular biology
principal cellular systems function in a similar way in
yeasts and human, i.e. across eukaryotes; many
principal molecular mechanisms are conserved
BUT yeasts are not just simple human cells
yeasts are unicellular and hence lack an important
level of complexity, i.e. that of a multicellular
organism
Yeast: Model OrganismYeast: Model Organism Cell cycle control is an example where genetic
analysis in yeasts has provided fundamental insight
The actin cytoskeleton
during the cell cycle
•S. cerevisiae has at least six signal transduction
pathways and genetic analysis in yeast has been key to
understanding how these pathways function • Control of gene expression - the principles of the control of transcription are well conserved across eukaryotes
Yeast: Model OrganismYeast: Model Organism • Vesicular transport - control of protein and
lipid trafficking
• Proteasome - control of the degradation of proteins that have been ubiquitinated
Prions
Ageing
Cell type determination
Functional genetics -heterologous expression in yeast can be used to functionally clone genes form other organisms
dll
PraktikumPraktikum 88
KulturKultur BakteriBakteri dandan RagiRagi Tgl 17 Nopember :
Kelompok praktikum seperti biasa
Kultur Bakteri Teknik steril Penggunaan mikroskop dengan benar Perwarna sel
Ragi Mengukur produksi CO2 dengan perubahan warna pewarna
bromophenol blue Mengukur “Absorbance Spectrum” bromophenol blue (alat
spektrofotometer) Memperkirakan konsentrasi sel melalui kekeruhannya (alat
spektrofotometer) Penggunaan mikroskop dengan benar serta pewarna sel
BakteriBakteri….….
RagiRagi
Mengukur produksi CO2 dengan perubahan warna pewarna bromophenol blue
◦ Setiap meja kerja akan menentukan jumlah ragi, kadar sukrosa serta temperatur (di atas es, temperatur ruangan, waterbath) untuk peparat ragi mereka.
◦ Produksi CO2 diamati melalui perubahan warna indikator pH bromophenol blue
◦ Perubahan tersebut dicatat pada interval tertentu (5-10 menit)
RagiRagi
Mengukur “Absorbance Spectrum” bromophenol blue (alat spektrofotometer)
o Satu meja kerja akan menyiapkan 4 sampel bromophenol blue pada 3 nilai pH diantara 4,6 dan 3,0
o Setiap meja kerja akan menggunakan alat spektrofotomter untuk buat “scan” atau spektra serapan di antara λ = 400nm s/d 700nm
RagiRagi
Memperkirakan konsentrasi sel (CFU – “colony forming units” melalui kekeruhannya (alat spektrofotometer)
◦ Setiap meja akan menyiapkan standar-standar untuk McFarland Scale (dari larutan 1% BaCl2 (yang di sediakan) dan larutan 1% H2SO4 (yang perlu dibuat))
◦ Dengan alat spektrofotometer setiap meja akan mengukur serapan standar-standar dan menyiapkan kurva standar.
◦ Konsentrasi (jumlah sel) dapat diperkirakan dari kurva standar tersebut.
RagiRagi
Penggunaan mikroskop dengan benar serta pewarna sel
o Selain kegiatan dengan alat spektrofotometer, peparat sel ragi diencerkan dan dilihat dengan mikroskop cahaya.
ReferencesReferences::
Sutton, S 2006. Measurement of Cell Concentration in
Suspension by Optical Density, http://www.linkedin.
com/in/scottvwsutton
University of Gottenburg Department of Cell and
Molecular Biology, http://www.cmb.gu.se/
Department+of+Cell-+and+Molecularbiology
/?languageId=100001&contentId=-1&disable
Redirect= true&returnUrl=http %3A%2 F%2F
www.cmb.gu.se%2F