Dr. Mudatsir, M.

Kes
Program Studi Pendidikan Pendidikan Dokter
Fakultas Kedokteran Unsyiah
Darussalam Banda Aceh
25 Mei 2015
 Antisepsis
 Antiseptik
 Desinfeksi
 Desinfektan
 Sterilisasi
 Cide (sid)
 Statik
 Mencegah terjadinya infeksi
 Mencegah makanan menjadi rusak
 Mencegah kontaminasi mikroorganisme
dalam industri
 Mencegah kontaminasi terhadap bahan-
bahan yg dipakai dalam pengerjaan biakan
murni.
Spora (endospora)
Different Kinds of Bacteria “Death”
Total cell count

1. Bacteriostatic
Viable cell count

2. Bacteriocidal

3. Bacteriolytic

Time
 Pengendalian dengan cara Panas/Fisik
Pengendalian dengan cara Sterilisasi Kimia
Pengendalian dengan cara Sterilisasi Radiasi
Pengendalian dengan cara Sterilisasi Filtrasi

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1. Panas Kering
a. Pembakaran(incenerator)
Teknik Sterilisasi pada Perbenihan Mikroba
- Menggunakan oven
suhu 160-180 C
- Waktu relatif lama; 1-2
jam
- Digunakan untuk alat-
alat yang tahan panas
( petridis, pipet, tabung
reaksi, labu erlenmayer,
dll)
a. Otoklaf
- menggunakan suhu 121 C dan tekanan 15 lbs (1 atm), waktu
15 menit
- Cara kerjaterjadi 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
Autoclave
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
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.
Beberapa hal yang perlu diperhatikan pada
disinfeksi 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
• Faktor-faktor yang mempengaruhi
sterilisasi dengan cara kimia:
1. Jenis bahan yang digunakan
2. Konsentrasi bahan kimia
3. Sifat Kuman
4. pH
5. Suhu
 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
 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
• Hidrogen Peroksida (H2O2)
- Efektif dan nontoksid
- Molekulnya tidak stabil
- Menginaktif enzim mikroba
• Gas Etilen Oksida
Mensterilkan bahan yang terbuat dari plastik
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
Forms of Radiation
 Sinar Gamma
• 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”
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
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.
Filtration

Figure 9.9a
 Kelemahan Filtrasi
- Banyak filtrat tersisa pada saringan
- Virus lolos dan
- Filter hanya sekali pakai
 2. Menyaring udara

• 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.
Laminar Air Flow
What is Biosafety?

• Safety from exposure

to Infectious Agents

Smallpox
Table: Animal facility containment levels

Biosafety is related to several fields
 ECOLOGY: referring to imported
life forms not indigenous to the
region (Reggie the alligator)
 AGRICULTURE: reducing the risk
of alien viral or transgenic genes,
or prions such as BSE/"MadCow“;
reducing the risk of food bacterial
contamination
 MEDICINE: referring to organs
or tissues from biological origin,
or genetic therapy products,
virus; levels of lab containment
protocols BSL-1, 2, 3, 4 in rising
order of danger
 CHEMISTRY: i.e., nitrates in
water, PCB levels affecting
fertility
 EXOBIOLOGY: i.e., NASA's
policy for containing alien
microbes that may exist on
space samples - sometimes
called "biosafety level 5"
  Research Universities:
Promoting safe laboratory
practices, and procedures;
proper use of containment
equipment and facilities;
provides advice on
laboratory design and risk
assesment of experiments
involving infectious agents,
rDNA in-vitro and in-vivo.

Bottom Line: Risk & Containment

 Biohazard Symbol

• Charles Baldwin at
National Cancer
Institute at NIH.
• Symbol to be
“memorable but
meaningless” so it
could be learned.
• Blaze orange – most
visible under harsh
conditions
 Laboratory Safety
 Bloodborne pathogens (BBP)
 Recombinant DNA (rDNA)
 Biological waste disposal
 Infectious substance and
diagnostic specimen shipping
 Respiratory Protection
 Bioterrorism and Select agents
 Mold and indoor air quality
 Occupational safety and health in the use
of research animals
 Biohazards used in animal models
 Viruses
 Bacteria
 Fungi
 Chlamydiae/Rickettsiae
 Prions
 Recombinant DNA
 Transgenic Plants, Animals and Insects
 Human and Primate Cells, Tissues, and
Body Fluids
 Brain Tissue from Demented Patients
 Viral Vectors
 Replication deficient viruses
 Biosafety Concepts

Biosafety In Microbiological
and Biomedical Laboratories
“BMBL” (acronym)

CDC/NIH Publication
Safety “Guidelines”
Regulations of Institution receives
NIH funding
Code of Practice and “Gold” Standard in
Industry anl
Gold Standard

Clinical & Research Lab.

Lab. Animal Facilities

HHS Publication No. (CDC) 93-8395

 The New BMBL

• Early print edition….

• Emphasis on “Risk
& Containment”
 Biosafety Concepts
The BMBL
The BMBL continues
to be published by
the
CDC and the NIH

5th edition is now at

the printers

http://www.cdc.gov/od/ohs/biosfty/b
mbl5/bmbl5toc.htm
Principles of Biosafety
 Practice and Procedures
 Standard Practices
 Special Practices & Considerations
 Safety Equipment
 Facility Design and Construction
 Increasing levels of protection
Biosafety Levels 1-4 (BSL)
 Increasing levels of employee and environmental
protection
 Guidelines for working safely in research & medical
laboratory facilities

Animal Biosafety Levels 1- 4 (ABSL)

 Laboratory animal facilities
 Animal models that support research
 Guidelines for working safely in animal research facilities
(1) Standard Microbiological Practices
 Most important concept / Strict adherence
 Aware of potential hazard
 Trained & proficient in techniques
 Supervisors responsible for:
 Appropriate Laboratory facilities
 Personnel & Training
 Special practices & precautions
 Occupational Health Programs
 Biosafety Issues
The BMBL

(2) Safety Equipment

• Primary Containment Barrier
• Minimize exposure to hazard
– Prevent contact / Contain aerosols
• Engineering controls/ equipment
• Personal Protective Equipment (PPE)
– Gloves, gowns, Respirator, Face shield, Booties
• Biological Safety Cabinets
• Covered or ventilated animal cage systems
(3) Facility Design and
Construction
 Secondary Barrier/ Engineering
controls
 Contributes to worker protection
 Protects outside the laboratory
 Environment & Neighborhood
 Ex. Building & Lab design, Ventilation,
Autoclaves, Cage wash facilities, etc.
“Warehouse Type Lab”
Biosafety Level-1 (BSL-1 or ABSL-1)
 Well characterized agents
 Agents not known to cause disease (in healthy human
adults; now healthy immunocompetent adults)
 Prophylactic treatment available
 Open bench procedures
 Animals in open cage system or open
environment (outdoors)
 Good laboratory practices
Desain Lab.Biosafety Levels 1
 Risk Group 1 Agents
• E.coli K-12
• Transgenic Plants
• Plasmids
• Fungi
• Mold
• Yeast
 Bench-top work allowed
 Daily Decontamination
 Manual pipetting
 Required Handwashing
 Red bag waste
 Bio cabinet not required
(unless creating aerosols)
 2˚ containment
Practices & Procedures
 Agents associated w/ human disease
 Treatment for disease available
 Agent poses moderate hazard to personnel
and environment
 Direct contact or exposure
 Percutaneous exposure
 Scratch, Puncture, Needle stick
 Mucus membrane exposure
 Eyes, Mouth, open cut
Desain Lab.Biosafety Levels 2
 Limited access to lab
when work in progress
 Daily decontamination
 Mechanical pipetting
 Labcoat, safety glasses
and gloves required
 Red bag & sharps
containers required
 Biohaz. Sign posted at
entrance to lab
 Label all equipment
(incubators, freezers, etc.)
 TC room – negative air flow
 Documented training
 Baseline serology or pre-
vaccination may be required
 Human or Primate
Cells
 Herpes Simplex Virus
 Replication
Incompetent
Attenuated Human
Immunodeficiency
Virus
 Patient specimens
 Biosafety Level 3
Working in High Containment

Biosafety Level-3 (BSL-3 or

ABSL-3)
• Indigenous or exotic agents
• Aerosol transmission
• Serious health effects
• Treatment may or may not exist
Desain Lab.Biosafety Levels 3
BSL 3 Lab. Litbangkes
 Public access NOT permitted
 Daily decontamination after spill and
upon completion of experiment
 Autoclave required and waste is
disposed at the end of day
 Required foot activated handwashing
sink and controls
 No sharps unless absolutely necessary
 Aerosol minimization procedures required
 Wrap around disposable clothing is required.
Specialized equipment may be required
depending upon procedures
 Biohaz. Signs and labels posted
 Air flow from low hazard to high hazard
“Pressure Mapping”
 Bench top work not permitted
 Documented training and personnel competency
certification (for BSL-3 procedures)
 Baseline serology
 Spills – report immediately and treat accordingly
 Vaccinations/post exposure protocols and SOP’s,
Biosafety Manual, Biosafety Officer
UCSD’s BSL-3
 Human
Immunodeficiency
Virus
 Mycobacterium
tuberculosis
 Coxiella burnetii
Biosafety Level-4
 Builds on BSL-3/ ABSL-3 practices
 Maximum containment facilities
 Pressurized Containment Suite
 BSL-3 + Class III Biosafety Cabinet
 Chemical decontamination showers
 Liquid effluent collection / decontamination
 No BSL-4 labs exist at UCSD
Desain Lab.Biosafety Levels 4

 Prinsip desain laborotoriumnya sama BSL 3

 Tingkat pengamanannya lebih tinggi dari BSL
3
 Tidak boleh bekerja sendiri
 Personil lab telah dilatih prosedur darurat jika
personal terjadi kecelakaan atau sakit
 Mempunyai sistem komunikasi antar
personal di laboratorium dan di luar lab.
 Biosafety Level 4
• Lassa Fever Virus
• Ebola Hemmorrhagic
Fever Virus
• Marburg Virus
• Herpes B Virus
 Biosafety Concepts
Working in High Containment

Biosafety Level-4 (BSL-4 or ABSL-4)

• Dangerous/exotic agents
• Life threatening disease
• Aerosol transmission
• Agents of unknown risk
of transmission or health affects
• No known treatment
Animal Biosafety Level-4
Working in High Containment
 Hygienic Practices
 No Smoking, Eating, Applying cosmetics, lip
balm, contacts
 Wash hands after procedures
 Decontaminate lab bench before and after work
 Proper attire
 Minimum – lab coat, safety glasses, gloves
 Plan your work