jadwal praktikum & kuliah pembahasan hasil … · jadwal kuliah dan praktikum ketrampilan dasar...

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



Kelompok 4



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


sblmnya 77.4 181.8 44.3


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

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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

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