Teknologi Mikrobiologi

Dr. Velma Buntuan, M.Kes

Sasaran umum
Menjelaskan teknologi pemeriksaan mikrobiologi Dapat menggunakan teknologi mikrobiologi

sasaran khusus
Dapat menjelaskan perkembangan teknologi mikrobiologi Dapat menjelaskan teknologi untuk sterilisasi Dapat menjelaskan teknologi pemeriksaan konvensional di bidang mikrobiologi Dapat menjelaskan teknologi moderen dibidang mikrobiologi

Perkembangan teknologi pemeriksaan mikrobiologi

Mikrobiologi

Ilmu mempelajari makhluk hidup yang sangat kecil (mikroorganisme yang tidak dapat dilihat dengan mata biasa tanpa bantuan dari suatu peralatan khusus

Disiplin ilmu

Bakteriologi, imunologi, virologi, mikologi dan parasitologi Mikroorganisme yang berkaitan dengan penyakit infeksi

Purba : Kutukan Dewa terhadap dosa-dosa, sehingga penyembuhanya perlu pengorbanan Infeksi Hipocrates : Faktor intrinsik Faktor eksttrinsik

Geratio spontanea: Makluk hidup berasal dari benda mati

Anton Van Leeuwenhoek: Alat Mikroskop dapat dilihat benda-benda kecil dari berbagai cairan (gugur teori diatas)

 Louis Pasteur (1860) memanfaatkan penemuan leeuwenhoek membantah teori generatio spontanea Robert Koch (1876) dokter Jerman - Teknologi kultur (Media pembenihan ditemukan) menumbuhkan kuman Anthrax yang berasal dari Binatang percobaan - Hasil dari penemuan ini dikenal postulat Koch:

Postulat Koch
Kuman harus dapat selalu ditemukan didalam tubuh binatang yang sakit tetapi tidak pada binatang yang sehat Kuman tersebut harus dapat diasingkan dan dapat dibiakan dalam bentuk biakan murni diluar tubuh binatang yang sakit Biakan murni kuman tersebut harus mampu menimbulkan penyakit yang sama pada binatang percvobaan Kuman tersebut haru s dapat diasingkan lagi dari binatang percobaatb tersebut

 Pada tahun 1900: semua jenis kuman penyebab berbagai penyakit penting akhirnya dapat ditemukan: 1. Bacillus antrachis 2. Corrynebacterium diphteria, 3. Salmonella thyposa 4. Clostridium tetani 5. Treponema Pallidum

 Kamajuan teknologi selanjutnya: 1. ditemukan jasad renik yang lebih halus dari kuman 2. dapat menembus saringan kuman yaitu: VIRUS 3. Virus mosaik tembakau (Iwanowsky 1892) 4. Virus penyebab foot and mouth disease pada ternak (Loffler & Froch, 1898)

Bidang Imunologi
Orang yang sembuh dari penyakit, tidak mudah lagi kena penyakit yang sama Kemajuan bidang kekebalan tubuh (Imunologi mulai dikembangkan) Pelopor Edward Jenner (1749-1823)

Teknologi Sterilisasi
Suatu usaha untuk membebaskan alat atau bahan dari segala macam kehidupan, terutama kehidupan mikroorganisme Mengunakan teknologi tertentu (Cara fisik dan cara kimia

Sterilisasi

 Zaman dulu: Membakar luka dengan logam yang membara dapat mencegah infeksi, meskipun meninggalkan jaringan parut Bahan kimia (Halogen, Yodium, klorin, Alkohol, fenol, peroksida, zat warna, deterjen dll) Gas (Oksigen Etlen (ETO), Uap formaldehida

 Cara Fisik: 1. Panas : - Basah (Otoklaf) - Merebus - Pasteurisasi 2. Kering: - Pembakaran - Udara panas - Radiasi - Penyaringan Zat-zat kemotherapetik (Antimikroba)

Teknologi konvensional
Pemeriksaan menggunakan alat-lat konvensional untuk melakukan pemeriksaan mikrobiologi Identifikasi bakteri: - Pewarnaan - Media pembenihan dan reagen

Zat warna
1. Kinyoun Gabbet (Tan Thiam Hok)

2. Ziehl Neelsen

Larutan Kinyoun (Fukhsin karbol 4 %) Larutan Gabbet ((H2SO4 + alkohol + biru metilen 1 %)

- 1,5 gr basic fuchsin dalam 30 ml ethanol - 15 gr phenol dalam 285 ml Aquadest - Asam alkohol 3 % - Larutan Methileen blue 0,1%

 Pewarnaan yang lain : - Fluorochrom

(mikroskop fuorosensi) - M. TBC
-

warna kuning orange

J Clin Microbiol. 2004 February

Metode

- Mycobaterium

berbentuk batang

- Warna merah dengan latar belakang biru

M.tuberculosis Cultur
M. tuberculosis is grown on a selective medium known as 1. Lowenstein-Jensen medium 2. Ogawa medium

1. Lowenstein-Jensen medium
Lowenstein-Jensen medium popularly known as LJ medium is a growth medium specially used for culture of Mycobacterium notably
Time growth (25 day)

Mycobacterium tuberculosis.

M. Tuberculosis bacterial colonies

From Wikipedia, the free encyclopedia

 composition - Malachite green - Glycerol - Asparagine - Potato flour - Coagulation eegs - Mineral salt solution
Potassium dihydrogen phosphate

Magnesium sulfate Sodium citrate

2. Media Ogawa
Komposisi media - Larutan garam : Monopotasium (KH2PO4) Sodium glutamat Aquades - Glycerol - Malachit hijau - Telur yang di kocok Lama pertumbuhan 6-8 minggu

New Diagnostic methods

1. Automated culture method - Bactec TB-460 - Bactec MGIT 960 - VersaTREK - Bact/Alert 3D 2. Nucleic Acid amplification method 3. Genetic Identification methods - PCR restriction-enzyme analysis - DNA Probe - Genetic sequencing

4. Non-Conventional Phenotyping Diagnostic Methods - Phage-Based Assay - The Micro-Colony Method - Microscopic Observation broth-Drug Susceptibility Assay (MODS) - Analysis of Cell Wall Mycolic Acids

Teknologi Moderen di bidang Mikrobiologi

1. Automated culture method

Although known for decades,the ability of a liquid medium to support a faster growth was heavily hampered by its susceptibility to contamination The use of antimicrobial combination potential contaminants (Gram-positive, gram negative bacteria) During the same period, automation was taking its first step in microbiology with blood cultures leading the field (for diagnostic mycobacteriology)

Cara pengambilan darah / sampel

Alat Kultur Automatik

Alat identifikasi bakteri

Konvensional

Mikrovact system

Teknologi identifikasi bakteriBakteri

Sodium Dodecyl SulfatePolyacrilamide gel Electroforesis

The principle (Bactec-TB-460)

modified Middlebrook 7H9

In use Palmitic acid (radiolabeled)

Contamination is controlled (PANTA)

- Polymyxin B - Amphotericin B - Nalidixic acid - Trimethoprim - Azlocillin

The vials containing the medium remain sealed through the whole culture process and the specimen is inoculated by puncturing the rubber septum with a needle
The instrumen: Once paired needles have perforated the rubber septum of the vial. The gaseous phase is aspirated and replaced with air containing 5% CO2 Aspirated gas is analyzed by -counter to quantify the eventual present of radiolabeled CO2
www. Tuberculosis Textbook.com.

 When viabel mycobacteria are present in the culture vial, the radiolabeled palmitic acid is metabolized and radioactive CO2 is liberated into gaseous phase The vials, which are held in anexternal incubator, must be loaded into the instrument for reading The reading is usually performed twice a week during the first 15 day of incubation, and weekly thereafter, until the 42 day

 The principle - The medium a modified Middlebrook 7H9 medium - Medium in which a supplement is added at the moment of use (OADC) enrichment: Oleic acid Albumin Dextrose Catalase - Contamination is controlled (PANTA) - Polymyxin B - Amphotericin B - Nalidixic acid - Trimethoprim - Azlocillin - As the tubes containing the medium are screw-capped, no needle is needed for inoculation

 A silicon film embedded with a ruthenium salt is present at the bottom of the tube as a flourecence indicator

VersaTREK
The versaTREK use technology of previously development blood culture system and is commercialized Trek diagnostic system
VersaTREK bottle (Courtesy Diagnostic System

The principle
The medium a modified Middlebrook 7H9 To which the OADC enrichment must be added Two diffrent antimicrobial mixtures are available The first one,also known as AS include : (OADC)

- Oleic acid - Albumin - Dextrose - Catalase

The secound contains (PVNA) - Polymyxin B - Vancomycin - Nalidixic acid - Amphotericin B

 The instrumentation : - Incubator and reader - Which also shakes the bottle during the incubation - The pressur within each bottle is monitored by a manometer through a proper connector - Cultures precenting a decreased headspace presure Positive

Mycobacteria are present in the bottle

The oxygen consumption due to their metabolism

Reduces the internal pressure

 VersaTREK is a typical walk-away instrumentation Which constinously monitors the bottles, alert when they become positive and signals the end of the incubation period

Bact/Alert 3D
The tecnology of a previously developed blood culture system Medium : - modified Middlebrook 7H9 - in use suplemen OADAC - - Contamination is controlled (PANTA) - Polymyxin B - Amphotericin B - Nalidixic acid - Trimethoprim - Vancomysin - Azlocillin

 If viable mycobacteria a present in the bottle, the CO2 produced by their metabolism causes a change of the color of the sensor, from green to yellow

Negative

Positive -

cc

2. Nucleic Acid amplification method

When the Polymerase Chain reaction methodology took insto its first steps inti diagnostic microbiology 1. in house method for dianosis for TBC 2. Commercial method : - Ampflified MTD - Amplicor MTB tet - BD ProbeTec ET 3. Genetic Identification method - PCR restriction-Enzym analisys (PRA) - DNA Probe - INNO LiPA Mycobacterium 4. Genetic squensing

Apakah PCR itu ?

Polymerase Chain Reaction (PCR) adalah suatu metode secara enzimatis melipatgandakan secara eksponensial suatu sekuen nukleotida tertentu dengan cara in vitro Ditemukan pertama kali oleh Kary Mullis, 1983

Prinsip PCR
PCR berdasarkan pada 3 tahap yang diperlukan dalam reaksi pembentukan DNA : 1. Denaturasi yaitu : untai ganda DNA dipisahkan menjadi rantai tunggal, 2. Annealing (penempelan) primer pada rantai tunggal DNA 3. Extension : pemanjangan rantai DNA (pembentukan rantai DNA yang baru)

Kegunaan PCR
Dalam riset kedokteran maupun kedokteran klinis, kegunaan PCR secara garis besarnya terbagi 2 : 1. Mendeteksi organisme penyebab infeksi 2. Mendeteksi variasi dan mutasi gen

Kelebihan PCR
1. Sangat sensitif 2. Dilakukan secara cepat 3. Menggunakan komponen dalam jumlah yang relatif sedikit

4. Non-Conventional Phenotyping Diagnostic Methods

In addition to on the so-called conventional method for TB diagnosis basides the automated and molecular diagnosic methods descrebed above, Some new technology gies have been proporsed Rappid dtection of growth by microscopic observation of microcolonies in solid or liqued media

Microcopic Observation Drug susceptibility (MODS)

New diagnostic stool are urgently needed Rapid, sensitive detection of tuberculosis and multidrug resistance tuberculosis in sputu Which broth cuture are examined microscopically to detected growth characteristic

Microcopic Observation Drug susceptibility (MODS)

Pionereed Robert Gilman in Peru Liquid cultur method for detection of M. TBC Microscopic detection of bacteri coeding that is caracteristic for M. TBC Can be adapted for drug susceptibility testing Relatively simple, relatively inexpensive No radioactivity

Kultur

Kepustakaan
1.Nendrosuwito, 2000, Standard Operating Procedures (SOP) In NMicrobiology. 2. Acid-Fast (Mycobacteria) Broth-Based Culture and Smear and Susceptibility 2007 by Laboratory Corporation of America Holdings and Lexi-Comp Inc. All Rights Reserved 3. Caviedes L, Moorr ,2007, Introducing MODS: A low-coast, Low tech tool for high performance detection of tuberculosis and multi drug resistant medium tuberculosis, Indian Lowenstein-Jensen journalMycrobiology. www.ijmn.org 4. Wikipedia,2008, Lowenstein-Jensen medium, From Wikipedia, the free encyclopedia 5. Dorman SD, Kritski AL,2006, The MODS Assay for Detection of TB and TB Drug Resistance A Multy Center Study, John Hopskins University, Federal University of Rio the janeiro 6. Mycobacterium tuberculosis - Wikipedia, the free encyclopedia.htm, 2008 7. Friedland, 2007, MODS assay for the diagnosis of TB, The new EnglandJournal of TB

8. Wahongan P, 2008, Polymerase chain reaction (PCR), Parasitologi UNSRAT 9. Palamino, Leao,Ritacco, 2007, Tuberculosis from basic science to patient care, www. Tuberculosis Textbook.com. 10. Cidlia Pina-Vaz, at all,2004, Novel Method Using a Laser Scanning Cytometer for Detection of Mycobacteria in Clinical Samples, American Society of Microbiology