Ketrampilan 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 Teknik Kultur Sel LT pk Tugas: laporan 08:00-11:00/ pk 12:00-15:00 praktikum
Nop 16/17
pk 10:00-12:00 KULIAH 7
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 2 3 4
Anwar, Ernawati, Dedy, Imam,Vera Roy, Martina, Lily, Dorra Taya, Leo,Yeni, Musthari, Siti Donny, Dita, Ningrum, Sukaisi
Hasil Metabolisme (Tabel 4) KELOMPOK
GLUKOSA B
KELOMPOK 1 Menu : nasi lengkap+air putih; 1 jam sblmnya
ST
SA
TRIGLISERIDA B
ST
SA
B
ST
SA
0
0.009
0.017
0
0.275 0.167
0
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
0
1.472 1.339
0
0.217 0.290
0
0.091
-0.108
Kelompok 2 Makan roti coklat + teh manis; 1 jam sblmnya
Kelompok 5 Menu: segelas susu; 1 jam sblmnya
0.222 0.091
UREA
Berdasarkan kurva standar kita mengetahui A berbanding lurus dengan konsentrasi unsur Untuk menghitung konsentrasi sampel:
Ksampel = Asampel/Astandar*Kstandar
Hasil Metabolisme (Tabel 4) MENU
1
2
3
4
5
GLUKOSA (mg/dl)
TRIGLISERIDA (mg/dl)
UREA (mg/dl)
nasi lengkap+air putih; 1 jam sblmnya
60.7
82.0
75.6
roti coklat + teh manis; 1 jam sblmnya
75.5
222.6
9.8
nasi lengkap+air putih; 1 jam sblmnya
77.4
181.8
44.3
nasi lengkap+air putih; 1 jam sblmnya
120.3
57.0
60.0
91.0
267.3
0.0
segelas susu; 1 jam sblmnya
Hasil Metabolisme (Tabel 4) Konsentrasi Glukosa,Trigliserida dan Urea pada sampel Darah Mhs
300
Trigliserida 200
Urea
Nilai Normal
Konsentrasi (mg/dl)
Glukosa
100
0
A
B
C Mahasiswa
D
E
Pembahasan Hasil Elektroforesis Elektroforesis:: Agarose
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?
Pembahasan Hasil Elektroforesis Elektroforesis:: SDS SDS--PAGE
St M S Gp Gs Ep Es
P St M
S Gp
Gs Ep Es P
Log berat molekul (Daltons)
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
jarak dari awal (mm)
Kultur Jaringan Jaringan//Sel 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
Sejarah Kultur Sel Sel//Jaringan 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. •
Sejarah Kultur Sel Sel//Jaringan 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 •
Pengunaan Kultur Sel 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
Pengunaan Kultur Sel 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
Kekurangan kultur sel 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
Ragi – Yeast 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 BIOLOGY **diadaptasi dari: http://www.cmb.gu.se/Department+of+Cell http://www.cmb.gu.se/Department+of+Cell--+and+ Molecularbiology /? /?languageId= languageId=100001 100001&contentId= &contentId=--1&disableRedirect= true&returnUrl=http true&returnUrl =http %3 %3A% A%22 F% F%22Fwww.cmb.gu.se% Fwww.cmb.gu.se%22F
The yeast Saccharomyces cerevisiae
A yeast cells is about 4-7mm large The ”eyes” at the bottom are bud scars
The yeast Saccharomyces cerevisiae cerevisiae::
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 Saccharomyces cerevisiae cerevisiae: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 Saccharomyces cerevisiae cerevisiae: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 cerevisiae is 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 cerevisiae is 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 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 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 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 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 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
Praktikum 8 Kultur Bakteri dan Ragi
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
Bakteri…. Bakteri ….
Ragi
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)
Ragi
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
Ragi
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.
Ragi
Penggunaan mikroskop dengan benar serta pewarna sel o Selain kegiatan dengan alat spektrofotometer, peparat sel ragi diencerkan dan dilihat dengan mikroskop cahaya.
References:: References 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
