Teknologi MikrobiologiDr. Velma Buntuan, M.Kes

Sasaran umumMenjelaskan teknologi pemeriksaan mikrobiologiDapat menggunakan teknologi mikrobiologi

sasaran khususDapat menjelaskan perkembangan teknologi mikrobiologiDapat menjelaskan teknologi untuk sterilisasiDapat menjelaskan teknologi pemeriksaan konvensional di bidang mikrobiologiDapat menjelaskan teknologi moderen dibidang mikrobiologi

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

Disiplin ilmuBakteriologi, imunologi, virologi, mikologi dan parasitologiMikroorganisme yang berkaitan dengan penyakit infeksi

Infeksi Purba : Kutukan Dewa terhadap dosa-dosa, sehingga penyembuhanya perlu pengorbananHipocrates : Faktor intrinsik Faktor eksttrinsikGeratio spontanea: Makluk hidup berasal dari benda matiAnton Van Leeuwenhoek: Alat Mikroskop dapat dilihat benda-benda kecil dari berbagai cairan (gugur teori diatas)

Louis Pasteur (1860) memanfaatkan penemuan leeuwenhoek membantah teori generatio spontaneaRobert 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 KochKuman harus dapat selalu ditemukan didalam tubuh binatang yang sakit tetapi tidak pada binatang yang sehatKuman tersebut harus dapat diasingkan dan dapat dibiakan dalam bentuk biakan murni diluar tubuh binatang yang sakitBiakan murni kuman tersebut harus mampu menimbulkan penyakit yang sama pada binatang percvobaanKuman 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 antrachis2. Corrynebacterium diphteria,3. Salmonella thyposa 4. Clostridium tetani5. 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 ImunologiOrang yang sembuh dari penyakit, tidak mudah lagi kena penyakit yang samaKemajuan bidang kekebalan tubuh (Imunologi mulai dikembangkan)Pelopor Edward Jenner (1749-1823)

Teknologi SterilisasiSterilisasiSuatu usaha untuk membebaskan alat atau bahan dari segala macam kehidupan, terutama kehidupan mikroorganismeMengunakan teknologi tertentu (Cara fisik dan cara kimia

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

Cara Fisik:1. Panas : - Basah (Otoklaf) - Merebus - Pasteurisasi2. Kering: - Pembakaran - Udara panas - Radiasi - Penyaringan Zat-zat kemotherapetik (Antimikroba)

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

Zat warna1. Kinyoun Gabbet (Tan Thiam Hok)2. Ziehl Neelsen- 1,5 gr basic fuchsin dalam 30 ml ethanol- 15 gr phenol dalam 285 ml Aquadest Asam alkohol 3 % Larutan Methileen blue 0,1% Larutan Kinyoun (Fukhsin karbol 4 %)Larutan Gabbet ((H2SO4 + alkohol + biru metilen 1 %)

Pewarnaan yang lain :- Fluorochrom

(mikroskop fuorosensi) - M. TBC

warna kuning orangeJ Clin Microbiol. 2004 February +-

Metode

Mycobaterium berbentuk batang Warna merah dengan latar belakang biru

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

1. Lowenstein-Jensen medium M. Tuberculosis bacterial coloniesFrom Wikipedia, the free encyclopedia Lowenstein-Jensen medium popularly known as LJ medium is a growth medium specially used for culture of Mycobacterium notably

Mycobacterium tuberculosis.Time growth (25 day)

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 kocokLama pertumbuhan 6-8 minggu

New Diagnostic methodsAutomated culture method - Bactec TB-460 - Bactec MGIT 960 - VersaTREK - Bact/Alert 3DNucleic Acid amplification methodGenetic 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 methodAlthough known for decades,the ability of a liquid medium to support a faster growth was heavily hampered by its susceptibility to contaminationThe 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

Mikrovact systemKonvensional

Teknologi identifikasi bakteriBakteri

Sodium Dodecyl Sulfate-Polyacrilamide gel Electroforesis

The principle (Bactec-TB-460)modified Middlebrook 7H9In 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% CO2Aspirated 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 phaseThe vials, which are held in anexternal incubator, must be loaded into the instrument for readingThe 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

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

The principleThe medium a modified Middlebrook 7H9To which the OADC enrichment must be addedTwo diffrent antimicrobial mixtures are availableThe first one,also known as AS include : (OADC) - Oleic acid- Albumin- Dextrose- CatalaseThe 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 bottleThe oxygen consumption due to their metabolismReduces the internal pressure

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

Bact/Alert 3DThe tecnology of a previously developed blood culture systemMedium : - 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 yellowPositive-Negativecc

2. Nucleic Acid amplification methodWhen the Polymerase Chain reaction methodology took insto its first steps inti diagnostic microbiology1. in house method for dianosis for TBC2. Commercial method : - Ampflified MTD - Amplicor MTB tet - BD ProbeTec ET3. Genetic Identification method - PCR restriction-Enzym analisys (PRA) - DNA Probe - INNO LiPA Mycobacterium4. Genetic squensing

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

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

Kegunaan PCRDalam riset kedokteran maupun kedokteran klinis, kegunaan PCR secara garis besarnya terbagi 2 :Mendeteksi organisme penyebab infeksiMendeteksi variasi dan mutasi gen

Kelebihan PCRSangat sensitifDilakukan secara cepatMenggunakan komponen dalam jumlah yang relatif sedikit

4. Non-Conventional Phenotyping Diagnostic MethodsIn 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 proporsedRappid 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 sputuWhich broth cuture are examined microscopically to detected growth characteristic

Microcopic Observation Drug susceptibility (MODS)Pionereed Robert Gilman in PeruLiquid cultur method for detection of M. TBCMicroscopic detection of bacteri coeding that is caracteristic for M. TBCCan be adapted for drug susceptibility testingRelatively simple, relatively inexpensiveNo radioactivity

Kultur

Kepustakaan1.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 Reserved3. Caviedes L, Moorr ,2007, Introducing MODS: A low-coast, Low tech tool for high performance detection of tuberculosis and multi drug resistant tuberculosis, Indian journalMycrobiology. www.ijmn.org4. 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 janeiro6. Mycobacterium tuberculosis - Wikipedia, the free encyclopedia.htm, 20087. Friedland, 2007, MODS assay for the diagnosis of TB, The new EnglandJournal of TBLowenstein-Jensen medium

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