Pertemuan 7MIKROSKOPI DAN PENYEDIAAN

SPESIMEN/PREPARAT MIKROSKOPIS

Microscope, 18th Century

Hooke’s Microscope

Hooke’s Microscope

• English scientist Robert Hooke built this microscope in the 17th century and used it to conduct pioneering research. He discovered the cell structure of plants by observing a thin slice of cork under his microscope.

• Photo Researchers, Inc./Cecil Fox/Science Source

• Microsoft ® Encarta ® Reference Library 2005. © 1993-2004 Microsoft Corporation. All rights reserved.

ISTILAH – ISTILAH

• Refraksi/ pembiasan

• Indek refraksi

• Titik Fokus (F)

• Panjang fokus (f)

© Microsoft Corporation. All Rights Reserved.Microsoft ® Encarta ® Reference Library 2005. © 1993-2004 Microsoft Corporation. All rights reserved.

UNIT PENGUKURAN

Unit Singkatan Nilai

1 centimeter cm 10-2 meter

1 milimeter mm 10-3 meter

1 micrometer μm 10-6 meter

1 nanometer nm 10-9 meter

1 Angstrom Ǻ 10-10 meter

1 picometer ρm 10-12 meter

Mikroskop Cahaya

• Fluoresensi ( fluorescence )

• Kontras- fase ( phase-contrast )

• Medan Terang ( bright – field )

• Medan Gelap (dark – field )

Mikroskup Elektron

• SEM ?

• TEM?

• Lihat contoh hasilnya di Biologi Sel

Electron Micrograph

• This electron micrograph shows a T4 bacteriophage, a virus that infects only bacteria. This bacteriophage measures 300 nanometers in length.

• Photo Researchers, Inc./M. Wurtz/Univ. of Basel/Science Source

• Microsoft ® Encarta ® Reference Library 2005. © 1993-2004 Microsoft Corporation. All rights reserved.

Scanning Electron Microscope

• This scanning electron microscope (SEM) at the University of California, Berkeley is located to the left with the computer images of the specimen on the computer screens to the right.

Image of a Fruit Fly

• This false color image of the head of a fruit fly illustrates the level of detail that electron microscopes are capable of resolving. The magnification of this image is about 200 times. Electron microscopes achieve much greater magnifications than light microscopes by using electrons with wavelengths considerably shorter than those associated with visible light. Light microscopes are capable of maximum magnifications of about 2,000 times, whereas magnifications approaching 1,000,000 times are possible with electron microscopes.

Scanning Auger Microprobe (SAM)

Scanning Auger Microprobe (SAM)

• Modern scientists use a variety of high-powered microscopes to perform research and test their findings. A Scanning Auger Microprobe (SAM), shown here, uses a highly focused electron beam to allow the scientist to look below the surface of the sample.Encarta Encyclopedia

• Microsoft ® Encarta ® Reference Library 2005. © 1993-2004 Microsoft Corporation. All rights reserved.

Salt Crystals

Salt Crystals

• This scanning electron micrograph shows pure salt, or sodium chloride, that has been recrystallized from distilled water. The crystal is built up from a cubic lattice of sodium

• Microsoft ® Encarta ® Reference Library 2005. © 1993-2004 Microsoft Corporation. All rights reserved.

Scanning Tunneling Microscope: Diagram

Scanning Tunneling Microscope: Diagram

• A scanning tunneling microscope (STM) measures the small current produced by electrons moving, or tunneling, between a stylus and a conducting sample. An STM can resolve images at the atomic level. As the stylus moves across the sample, the current produced is monitored and kept constant to a value set by a gauge (depicted by the arrow). To keep the current constant, the height of the stylus changes at it scans the sample. The display shows the height of the stylus as changes in brightness or color as the tip is scanned across the display.Encarta Encyclopedia

Scanning Tunneling Microscope

Pengertian Penting

• Resolusi : kemampuan lensa untuk memisahkan / membedakan diantara 2 obyek kecil yang saling berdekatan.

• d = distance = jarak minimum 2 obyek atau benda

0.5 λ

d = -------------

n Sin θ

λ = Panjang gelombang

n Sin θ = N A ( Numerical Aperture ) Jadi jika d semakin kecil RESOLUSINYA

NAIK. Pada mikroskup cahaya, sering digunakan

λ = 450 – 500 nm

Pada mikroskop cahaya:d mendekati 0.2 μm

( 0.5 ) x (530) nm

d = ----------------------- = 212 nm = 0.2 μm

1.25

Dalam praktek :

λ d mikroskop = -----------------------------------

( NA obyektif + NA kondensor)

PENYIAPAN DAN PENGECATAN SPESIMEN

• Mikrobia perlu difiksasi dan dicat, agar lebih mudah diamati

• Fiksasi : Proses, dimana struktur luar dan dalam sel serta mikrobia diawetkan, sehingga posisinya tetap

Dua cara fiksasi :

1. Fisik : dengan PEMANASAN

2. Kimia : dengan campuran senyawa kimia : etanol, asetat, merkuri klorida, formal dehid, dll.

Cat / pewarna

• Secara kimia didefinisikan sebagai : senyawa organik mengandung CINCIN BENZENA ditambah KROMOFOR dan GUGUS AUKSOKROM

CAT ( STAIN )

1. BENZENA

2. KROMOGEN

3. AUKSOKROM

1 dan 2 disebut Kromofor

KROMOFOR • Senyawa berwarna , terdiri dari:

1. Benzen

2. Kromogen.

Namun belum bisa disebut cat atau pewarna.

• Benzena : suatu pelarut organik yang tidak berwarna

• Auksokrom : gugus kimia yang membawa sifat ionisasi pada KROMOGEN (berkemampuan membentuk garam) dan mengikat pada serat atau jaringan.

• Misalnya: -NH2, -OH, -OCH3, -J, -Br, -Cl.• Kromofor : gugus kimia yang memberi warna

pada benzena.Gugusnya : azo : -N=N- nitroso : -N=0 tio : =C=S- nitro : -NO2 DLL.

Kromogen Terionisasi Kromogen : Senyawa berwarna, namun bukan

cat. Terionisasi,+ = kationik, cat basa

Kromogen terbentuk muatan

listrik- = anionik = cat asam

Prinsip :

suatu substrat sel mikrobia akan tercat bila muatannya berlawanan dengan muatan catnya

Contoh : Cat asam (-) : eosin, rose bengal, acid fuchsin. Sel (+) : struktur sel yang bermuatan + ( Sitoplasma) Cat basa (+) : methylen blue, basic fuchsin, crystal violet, safranin, malachit green. Sel (-) : asam nukleat, protein, permu- kaaan sel bakteri

Faktor-faktor yang mempengaruhi pengecatan

(Jutono)

1. Fiksasi

2. Substrat, apa muatannya

3. Peluntur (Decolorizer)

4. Intensifikasi (mordant, menguatkan)

5. Cat Penutup atau Lawannya = COUNTER STAIN

Tehnik Pengecatan

VISUALISASI Ciri Morfologi DIFFERENSIASI

SEPARASI Struktur Sel

1. Sederhana: menggunakan 1 macam cat melihat bentuk morfologi : bulat, batang, spiral. melihat susunan : rantai, kluster, pasangan, tetrad.

2. Differensial: menggunakan 2 cat kontras

MACAM TEHNIK PENGECATAN

Differensial

1. Melihat struktur• flagella stain• capsula stain• spore stain• nuclear stain

2. Memisahkan dalam kelompok :• Gram + • Gram -