Download - Biosavety and Sterilization
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Dr. Mudatsir, M. Kes
Program Studi Pendidikan DokterFakultas Kedokteran
Darussalam Banda Aceh26 Mei 2014
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WHO describes this is as: ◦ containment principles◦ technologies ◦ practices
implemented to prevent unintentional exposure to pathogens and toxins, or their accidental release
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To protect:◦ the patient ◦ yourself◦ the environment
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Biosafety levels ◦ Level 1& 2: basic laboratories
◦ Level 3: containment laboratories
◦ Level 4 : high containment laboratories
Each level associated with appropriate ◦ Equipment, practices, work procedures
Diagnostic and health-care laboratories must be biosafety level 2 or above
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Biological Hazards are divided into 4 Biosafety Levels◦ BSL 1◦ BSL 2◦ BSL 3◦ BSL4
Biosafety levels define the lab requirements, protective clothing, and work practices
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BSL 1 (BMBL)◦ Not known to consequently cause disease in
healthy human adults◦ Pose minimal hazards under ordinary conditions
of handling
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Standard Practices◦ Use Mechanical Pipetting
devices◦ No Eating, Drinking, Smoking
in Lab◦ Minimize splashes and
aerosols◦ Decontaminate work surfaces◦ Safe handling of sharps◦ Wash Hands before leaving lab
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Protective Clothing◦ Lab Coat◦ Gloves◦ Eye Protection
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BSL 2 (BMBL)◦ suitable for work involving well-characterized
agents not known to cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment
Examples: Measles Virus, Salmonella, Hepatitis B Virus
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Other Materials Handled at BSL 2(Requires compliance with OSHA BBP Standard)
◦ Human Blood◦ Human Blood Components◦ Human Tissues◦ Human Cell Lines
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Standard Practices◦ All requirements for BSL 1 plus:
Access to laboratory is limited or restricted when work is being conducted
Personnel have specific training in handling pathogenic agents
Biohazard Sign posted on the door Extreme precautions are taken with contaminated
sharp items
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Standard Practices cont. Written Biosafety Procedures Report Spills Minimize aerosol generation Personnel receive appropriate immunizations or test
(e.g. Hep B vaccine or TB skin Testing)
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Written Biosafety Procedures◦ Prepared specifically for lab by PI◦ Incorporated into Lab Safety Plan◦ Personnel are advised of special hazards◦ Personnel are required to read and follow
procedures
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Personal Protective Equipment◦ Lab Coats◦ Gloves
Double Gloving when appropriate◦ Eye Protection - Safety Glasses/Shield
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BSL 3 (BMBL)◦ applicable to clinical, diagnostic, teaching,
research, or production facilities in which work is done with indigenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route.
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Standard Practices◦ All requirements for BSL 2 plus:
Limited lab access 2 doors in a series to access lab Able to wash entire lab Special exhaust ventilation (Not re circulated,
negative pressure)
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Standard Practices –cont.◦ High level of training◦ Personnel receive vaccinations if available◦ Work in Biosafety cabinets
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Examples◦ Mycobacterium Tuberculosis◦ Hanta virus◦ SARS
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BSL 4 (BMBL)◦ required for work with dangerous and exotic
agents which pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease.
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Requirements◦ All requirements for BSL3 plus:
Class III Biosafety cabinet or positive pressure suits Shower/Change rooms Clothing Autoclaved before laundering Air Locks
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Examples◦ Ebola Virus◦ Monkey B Virus◦ Marburg Virus
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Risk Group
Individual risk Community risk
1 no, low no, low
2 moderate low
3 high low
4 high high
Risk group classification Risk group classification
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Unlikely to cause animal or human disease Non pathogenic agent
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Pathogenic for humansUnlikely a serious hazardTreatment and preventive measures availableLimited risk of spread of infection
CDC, Yersinia pestis laboratory
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Pathogenic, cause serious diseaseEffective treatment and preventive measures usually availableLittle person-to-person spread
Laboratory in Lyon France
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Lethal, pathogenic agentReadily transmittable
◦ direct, indirectEffective treatment and preventive measures not usually available
National Institute for Infectious Diseases, Rome, Italy
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BSL Laboratory type Laboratory practices
Safety equipment
1 Basic teaching, research
Good microbiological techniques
None
Open bench work
2 Primary health
services; diagnostic
services, research
Good microbiological techniques,
protective clothing,
biohazard sign
Open bench PLUS
biological safety cabinet for potential aerosols
3 Special diagnostic
services, research
As BSL 2 PLUS
special clothing,
controlled access,
directional airflow
Biological safety cabinet and/or other primary devices for all activities
4 Dangerous
pathogen units
As BSL 3 PLUS
airlock entry, shower exit, special waste
Class III biological safety cabinet, positive pressure suits, double ended autoclave (through the wall), filtered air
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Antisepsis Antiseptik Desinfeksi Desinfektan Sterilisasi Cide (sid) Statik
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Mencegah terjadinya infeksi Mencegah makanan menjadi rusak Mencegah kontaminasi mikroorganisme
dalam industri Mencegah kontaminasi terhadap bahan-
bahan yg dipakai dalam pengerjaan biakan murni.
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Different Kinds of Bacteria “Death”
1. Bacteriostatic
2. Bacteriocidal
3. Bacteriolytic
Log
Cel
l #
Time
Total cell count
Viable cell count
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Pengendalian dengan cara Panas/FisikPengendalian dengan cara Sterilisasi KimiaPengendalian dengan cara Sterilisasi RadiasiPengendalian dengan cara Sterilisasi Filtrasi
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I. Cara Panas1. Panas Kering a. Pembakaran(incenerator)
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Teknik Sterilisasi pada Perbenihan Mikroba
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- Menggunakan oven suhu 160-180 C
- Waktu relatif lama; 1-2 jam
- Digunakan untuk alat-alat yang tahanpanas ( petridis, pipet, tabung reaksi,
labu erlenmayer, dll)
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a. Otoklaf - menggunakan suhu 121 C dan tekanan 15 lbs
(1 atm), waktu 15 menit - Cara kerjaterjadi koagulasi - Untuk mengetahui autoklaf berfungsi dengan
baik digunakan Bacillus stearothermophilus - Bila media yang telah distrerilkan diinkubasi selama 7 hari berturut-turut selama 7 hari: - Media keruh otoklaf rusak - Media jernih otoklaf baik
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Autoclaving• Pressure applied to boiling water prevents
steam from escaping• Boiling temperature increases as pressure
increases• Autoclave conditions – 121ºC, 15 psi, 15
minutes
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Autoclave
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b. Boiling (Memasak Air) - Teknik disinfeksi termurah - Waktu 15 menit setelah air mendidih - Beberapa bakteri tidak terbunuh dengan teknik ini: Clostridium perfingens dan Cl. botulinum
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c. Pasteurisasi• High Temperature Short Time (HTST) yaitu pemanasan
dengan suhu tinggi sekitar 75 C dalam waktu 15 detik, menggunakan alat yang disebut Heat Plate Exchanger.
• Low Temperature Long Time (LTLT), yaitu pemanasan dengan suhu rendah sekitar 60C dalam waktu 30 menit.
• Ultra High Temperature (UHT), yaitu pemanasan dengan suhu tinggi 130 C selama hanya 0,5 detik saja, dan pemanasan dilakukan dengan tekanan tinggi. Dalam proses ini semua MIKROBA mati , sehingga susunya biasanya disebut susu steril.
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Beberapa hal yang perlu diperhatikan padadisinfeksi kimia Rongga (space) Sebaiknya bersifat membunuh (germisid) Waktu (lamanya) disinfeksi harus tepat Pengenceran harus sesuai dengan anjuran Solusi yang biasa dipakai untuk
membunuh spora kuman biasanya bersifat sangat mudah menguap
Sebaiknya menyediakan hand lation merawat tangan setelah berkontak dengan disinfekstan
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Faktor-faktor yang mempengaruhi sterilisasi dengan cara kimia:1. Jenis bahan yang digunakan2. Konsentrasi bahan kimia3. Sifat Kuman4. pH5. Suhu
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Bekerja dengan cepat untuk menginaktivasi mikroorganisme pada suhu kamar
Aktivitasnya tidak dipengaruhi oleh bahan organik, pH, temperatur dan kelembaban
Tidak toksik pada hewan dan manusia Tidak bersifat korosif Tidak berwarna dan meninggalkan noda Tidak berbau/baunya disenangi Bersifat biodegradable/mudah diurai Larutan stabil Mudah digunakan dan ekonomis Aktivitas berspektrum luas
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Alkohol - Paling efektif utk sterilisasi dan desinfeksi - Mendenaturasi protein dengan jalan dehidrasi membran sel rusak & enzim tdk aktif Halogen - Mengoksidasi protein kuman Yodium - Konsentrasi yg tepat tdk mengganggu kulit - Efektif terhadap berbagai protozoa
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Klorin - Memiliki warna khas dan bau tajam - Desinfeksi ruangan, permukaan serta alat non bedah Fenol (as. Karbol) - Mempresipitasikan protein secara aktif, merusak membran sel menurunkan tegangan permukaan - Digunakan sebagai standar pembanding untuk menentukan aktivitas suatu desinfektan
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Hidrogen Peroksida (H2O2) - Efektif dan nontoksid - Molekulnya tidak stabil - Menginaktif enzim mikroba Gas Etilen Oksida Mensterilkan bahan yang terbuat dari
plastik
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Sinar Ungu Ultra (Ultraviolet) - Memiliki daya antimikrobial sangat kuat - Daya kerja absorbsi as. nukleat - Panjang gelombang: 220-290 nm paling efektif 253,7 nm - Kelemahan penetrasi lemah
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Daya kerjanya ion bersifat hiperaktif Sering digunakan pada sterilisasi bahan
makanan, terutama bila panas menyebabkan perubahan rasa, rupa atau penampilan
Sterilisasi dengan sinar gamma disebut juga “sterilisasi dingin”
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1. Menyaring cairan - Digunakan untuk bahan yang peka terhadap panas: serum, urea, enzim,
toxin - Menggunakan berbagai filter -Saringan Sietz asbes -Berkefeld tanah diatomae -Chamberland porselen -Fritted glass filter serbuk gelas -Cellulose Asetat pada industri
minuman
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Figure 1. Filtration of a mixture of bacteria and viruses. If a mixture of viruses and bacteria are filtered through a bacterial-proof filter (red), the viruses will pass through into the filtrate in the flask. Filtered beer is produced by a similar process.
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Filtration
Figure 9.9a
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- Banyak filtrat tersisa pada saringan - Virus lolos dan - Filter hanya sekali pakai
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Menggunakan penyaring HIPA (High-Efficiency Particulate Air)
Filter terdiri dari lipatan selulose asetat Memungkinkan udara tersaring bebas dari
debu dan bakteri Sistem pengaliran udara menggunakan
laminar flow bench udara yang masuk tersaring lebih dahulu.
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1. Menghambat sintesis dinding sel mikroba (Basitrasin, penisilin,)
2. Merusak keutuhan membran sel mikroba (amfoterisin B, polimiksin)
3. Menghambat sintesis protein sel (Kloramfenikol, eritromisin)
4. Menghambat sintesis atau merusak asam nukleat sel mikroba (quinolon, rifampisin)
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Metode difusi Metode Dilusi (Pengenceran)
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Metode Uji Sensitivitas Antibiotik secara in vitro
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Untuk menguji berapa dosis terendah antibiotik dapat membunuh mikroba
Menghitung KHM (MIC) Tidak praktis dan
jarang dilakukan Keuntungan
diketahui kuantitatif antibitik yg mampu menghambat atau mematikan mikroba
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Uji Difusi• Metode Kirby-Bouer• Menggunakan disk
antibiotik• Larutan pembanding
Mc Farland 0,5• Interpretasi
menggunakan tabel CLSI/NCCLS
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pH Lingkungan Komponen-komponen perbenihan Stabilitas antibiotik Besarnya inokulum Masa pengeraman Akrivitas metabolik mikroba
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