METODE STERILISASI

SUTRIYO
Methods of Sterilization
Steam Sterilization

Dry heat sterilization

Filtration

Gas sterilization

Irradiation

NOTE: End products must pass sterility tests.

 STERILIZATION
• Absolute Definition.
– Completely destroys all microbial life.
• Relative Definition.
– In any sterilization process not all microorganisms
die at once.
Their numbers decrease gradually over the time they
are exposed to the sterilizing agent
Sterilization is a Complex Process
 Three Main Groups
1.Contaminating Microorganisms
- Number & Type
2.Object to be Sterilized
– Cleanliness
– Compatibility / method of sterilization
– Contact with all surfaces
– Packaging
3.Sterilizer
– Knowledge & skill of operator
 Contaminating
Microorganisms

Microbial Characteristics and

Microbial Control
 Sterilizer Loading Guidelines
1. Ensure packaging is correct
2. Ensure package placement in the sterilizer
will allow for free circulation of the steriliant
around all packages and into each package.
3. Packages must not be laid on top of one
another.
4. Position items on edge to facilitate air
removal and permit steriliant circulation.
Steam Sterilization
 Moist-heat /steam sterilisation
a. Type : heat sterilization
b. Pasteurization,Tyndallization, Autoclaving
c. Steam is a good vehicle for heat
d. Moist heat kills microorganisms more quickly
than dry heat
e. Pressure, Time & Temperature are the three
main elements required for effective steam
sterilization
f. If temperature is decreased, time must be
increased
• Device : Autoclave
– Autoclave used to sterilize using pressurized
steam
• Heated water  steam  increased pressure

• Preferred method of sterilization

• sterilisation at lower temperatures than dry-
heat sterilisation,
• mode of action is denaturation/coagulation
of microbial proteins
 PHASES of STERILIZATION
• Conditioning Phase
• Exposure Phase
• Exhaust Phase
 CONDITIONNING PHASE
• pulsing in of steam, each time the
temperature rises
• air is being removed at the same time
• this take various times depending on steam
and sterilizer
• Sterilization begins when the chamber is
completely filled with steam
• Exposure / STERILIZATION PHASE
• EXHAUST
– steam is rapidly exhaust from the chamber
– causes severe variations in pressure in chamber
– items in sterilizer must be able to withstand this
After Sterilization

A items must be completely

cool

Hands should be clean and dry

Dust covers may be used after

item has completely dried
Autoclave Cycle
 Moist-heat Sterilization
• is principally used to sterilize materials that are both
– Thermo stable and
– moisture can perfuse
• Application:
 Solutions sealed in containers ampuls, vials,
 Bulk Solutions,
 Glassware,
 Surgical dressing,
 Closures,
 Instruments
 Minimum Standards for
Sterilization-Exposure Period
Degrees F Degrees C Pressure Time (min.)
280 138 0.8
270 132 2
257 125 8
250 121 15 lb or 103.4 kPa 12 -15
245 118 18 - 20
240 115-116 10 lb or 68.91 kPa 30
 Advantages:
• Rapid
• Destroys the most resistant bacterial spores
• No toxic residues
• Most economical sterilizing agent
• Effective
• Large volumes
 Disadvantages:
1. Cannot use for oily preparation (oil base ointment)
2. Cannot use for moisture or high temperature sensitive
preparations or medical devices
 Autoclave Components
• CHAMBER (BODY)
- surrounded by steel case
- extremely strong
- sizes vary from table top, model to floor to ceiling models
DOOR
• very strong
• manual wind up or electric
• steam must not escape around edges
• open outward or up and down
• safety feature… will not open once the cycle is started
JACKET
• steel jacket separated from chamber by 3 or 4 inches
• steam continuously circulates through jacket
• keeps temperature constant
• prevents condensation, speeds drying,keeps steam readily available
BAFFLE
- deflector that covers steam inlet to prevent steam from wetting packs close to
inlet
• DRAIN
– drains away air at the beginning of the cycle
– drains away excess moisture during exhaust phase
– traps debris in screen
• STEAM TRAP
– he steam trap closes when 212°F is reached to
keep the pressure constant in the chamber
– it remains open until pure steam touches it
• VACUUM PUMP
– pumps air from the chamber from the bottom
– speeds the air removal process
 Maintenance
• Sterilizers must be properly maintained by either the
manufacturer or in-house maintenance technicians trained by
the manufacturer.
• Cleaning routines setup for
– sterilizer exterior and carts
– checking door gaskets
– filter baskets in drain
• Weekly clean drain line
• Weekly cleaning of interior

N.B. All the above must be documented

Internal Chemical Indicators
Biological Incubation
 Pasteurization
•This process was originally employed by Louis Pasteur.
•Currently this procedure is employed in food and dairy
industry.
• It can be used on heat sensitive liquids and medical
devices.
There are two methods of pasteurization;

•The Holder method (heated at 62.9oC for 30 minutes).

•Flash method (heated at 72oC for 15 seconds) quickly followed by cooling to
13oC so that protein becomes coagulated

– Ultra-high-temperature: 140°C for <1 sec

 TYNDALLIZATION
• Also called Fractional Sterilization.
• John Tyndall gave the idea about tyndallization.
• The substance is heated at temp. of about 80-100oC
for 30 mins and then the material is incubated. If
spores are present, they will germinate and becomes
vegetative cell.
• The procedure is repeated for 3 successive days.
• On heating between 80-100oC for 30 mins on
successive days with incubation will result in
resistant spores to germinate and the vegetative cells
are killed on second and third days.
• The success of process depend on the
germination of spores.
• Time consuming.
• Heat sensitive materials such as
microbiological media, solution of chemicals
and biological materials can be sterilized by
this process
Dry Heat Sterilization
 Dry-heat sterilisation
• Ovens
• method
– Flaming
– Incineration
– Hot-air sterilization
• mode of action is cellular dehydration and then
pyrolysis/oxidation.
• Examples of dry-heat sterilisation cycles include:
– 170°C for 1 hour
– 160°C for 2 hours
– 140°C for 4 hours.
• A lag time is therefore required to ensure that
the article/container has achieved this
temperature.
• Application
– oils and other aqueous vehicles (e.g. glycerin,
propylene glycol)
– heat-stable therapeutic agents/excipients
– glassware (e.g. bottles).
 Advantages & Disadvantages
• Sterilization by heat requires higher
temperatures and longer exposures than
sterilization by steam.
• Heat transfer is slow, small volumes of oil and
thin layers of powder should be used.
FLAMING
STERILISASI FILTRASI
SINTERED GLASS MEMBRANE FILTER
SYRINGE FILTER
 FILTRATION
 Membrane filtration used to remove microbes from fluids
and air
 Liquid filtration
 Used for heat sensitive fluids
 Membrane filters allow liquids to flow through
 Traps microbes on filter
 Depth filters trap microbes using electrical charge
 Filtration of air
• High efficiency particulate air (HEPA) filter removes nearly all
microbes from air
– Filter has 0.3µm pores to trap organisms
 Penyaring Ideal
• Tidak menghilangkan konstituen yang diinginkan.
• Tidak membawa komponen-komponen yang
diinginkan.
• Tidak mengubah larutan atau gas.
 JENIS PENYARING
• Screen Filters
– Penyaring Membran
– Penyaringan Lapis Ganda
– Penyaring tidak Simetris
– Penyaring Catridge
• Depth Filters
– Penyaring Zeta-Plus
– Penyaring Sinter Glass
– Penyaring Selulosa-Asbes
 Keuntungan Sterilisasi Filtrasi
• Efektif digunakan untuk bahan obat yang termolabil
• cepat, terutama pada penyaringan dengan sejumlah
kecil larutan.
• Cocok untuk menghilangkan kuman dari larutan
sejati dengan viskositas redah
• Dapat menyaring mikroba hidup dan mati, bakteri,
bentuk vegetatif/ spora, sehingga dapat mengurangi
resiko pirogen.
 Kerugian Sterilisasi Filtrasi
• Beberapa jenis penyaring dapat mengabsorbsi bahan
aktif tertentu.
• Kemungkinan terjadi kerusakan bentuk penyaring,
sehingga terjadi ketidakpastian sterilisasi.
• Penyaring bakteri kadang masih dapat meloloskan
virus dan spora.
Mechanisms
Structure of a Depth Filter
Structure of a Conventional
Membrane Filter
CARTRIDGE FILTER
 BUBBLE POINT TEST
• Uji ini dilaksanakan dengan menggunakan tekanan
udara atau tekanan gas lain pada bagian aliran ke
atas dari penyaring hidrofilik dimana lubang-lubang
itu diisi dengan air.
• Tekanan dinaikkan bertahap hingga gelembung-
gelembung melewati penyaring dan dideteksi pada
aliran ke arah bawah cairan.
• Tekanan titik gelembung ini berbanding terbalik
dengan garis tengah lubang, dan merupakan ukuran
lubang paling besar.
BUBBLE POINT TEST
• Misalnya 55 pound per inci persegi untuk
suatu penyaring membran hidrofilik dengan
porositas 0,2 μm. Bila terjadi lubang kecil atau
cacat pada penyaring, penggelembungan
terjadi pada tekanan yang jauh lebih rendah
darpada yang diharapkan
• Kadang masih membutuhkan zat bakterisid.
• Beberapa penyaring sukar dicuci.
• Beberapa penyaring ada yang bersifat alkalis
dan dapat melepaskan serabutnya sehingga
menimbulkan pencemaran.
• Filtrat yang diperoleh belum bebas dari virus
karena tidak menggunakan panas.
STERILISASI RADIASI

Beta Particle

Gamma Rays
(photons)
 STERILSASI RADIASI
• Ionizing radiation is used extensively for terminal sterilization
of heat-sensitive medical devices and for heat-sensitive
pharmaceutical packaging components prior to aseptic
processing
 STERILSASI RADIASI
suatu proses sterilisasi dengan memaparkan
produk pada sinar gamma atau elektron
berenergi tinggi, selama waktu tertentu
sehingga dicapai faktor keamanan /Sterility
Assurance Level (Sal) 10-6.
 SUMBER RADIASI
1. Co- 60 yaitu radioisotop yang menghasilkan
sinar gamma dengan waktu paruh 5.6 tahun,
energi 1.17 dan 1.37 Mev
2. Elektron berenergi tinggi ( umumnya antara
5 – 10 Mev) yang dihasilkan oleh Mesin
Berkas Elektron ( MBE).
3. Sinar X yang dihasilkan oleh mesin sinar X,
yaitu suatu gelombang elektromagnetik,
dengan sifat yang hampir sama dengan sinar
gamma
 JENIS RADIASI
a.Radiasi sinar Alpha ( partikel)
b.Radiasi sinar Beta / electron (partikel)
c.Radiasi sinar Gamma dan sinar- X ( Gelombang
Elektromagnetik )

d.Neutrons
SUMBER RADIASI UNTUK STERILISASI:
a.Elektron dari Electron Beam Machine
b.Gamma rays dari Co-60 atau Cs-137
c.X-Rays from X-rays machine
Spektum Energi Gelombang Elektromagnetik

Pico

Reduced wave lengths/ increased energy

Penetration of  ,  and  rays
 Electromagnetic Spectrum

High Frequency Low Frequency

Short Wavelengths Long Wavelengths
 UV-RAYS
a. Greatest antimicrobial activity occurs at 250-
260nm.
b. Bacterial spores are quite resistant and require a
dose up to 10 times greater than the vegetative
bacteria.
c. Rays are harmful to skin & eyes.
d. It doesn't penetrate glass, paper or plastic.
e. Used in hospitals to kill air-borne organisms esp. in
operating rooms when they are not in use.
 X-Rays (Roentgen Rays)
• Have higher energy and penetrating power
than UV radiation.
• Damages DNA.
• For sterilization of heat sensitive solids such as
sutures, surgical gloves and plastic items such
as syringes.
 GAMMA IRRADIATION
 Disrupts DNA and RNA in living organisms.
 Have shorter wavelength and higher energy.
 Great penetration.
 Co-60 is used which produces gamma rays.
 Materials that can be sterilized include;
• Media for growth of microorganisms.
• All disposable plastic materials.
• Syringes etc.
 RADIASI PENGION
• Radiasi pengion (radiasi sinar gamma, partikel
elektron, sinar X) jika berinteraksi dengan
bahan akan menimbulkan ionisasi dan
eksitasi, dengan menghasilkan spesi radikal
bebas yang reaktif, ion-ion dan spesi
tereksitasi.
• Spesi tersebut akan bereaksi dengan cepat
dengan senyawa kimia/ bahan yang dilaluinya
• Pada mikroba, reaksi tersebut menimbulkan
akibat yang nyata pada DNA
• Reaksi akan dipercepat oleh lingkungan yang
mengandung air (H2O), oksigen (O2), suhu
tinggi serta kondisi pertumbuhan mikroba
(spora dan vegetatif).
• Reaksi tersebut sampai batas tertentu dapat
menyebabkan mikroba kehilangan
kemampuan membelah diri.
 INTERAKSI RADIASI
• Interaksi radiasi dengan bahan terjadi melalui
dua cara yaitu cara langsung dan tidak
langsung.
– cara tidak langsung : reaksi dipercepat oleh spesi
radikal bebas dan ion-ion yang dihasilkan oleh
ionisasi air (*H, *OH dan e-ag) serta ionisasi O2
(*O2-), serta dapat diperlambat/ dilindungi oleh
berbagai jenis spesi misalnya spesi dari protein
dan alcohol.
• Suhu rendah akan mengurangi gerakan spesi,
yang akan menurunkan kecepatan reaksi.
Kerusakan yang ditimbulkan oleh cara tidak
langsung tersebut, jauh lebih besar
dibandingkan dengan kerusakan akibat radiasi
langsung.
• Kerusakan berlaku untuk mikroba dan alat
kesehatan/ bahan yang diiradisi
 The Parenteral Drug Association
(PDA)
• Overkill: dimana dosis paling tidak dua kali
dosis radiasi yang dibutuhkan untuk mencapai
enam logaritma inaktivasi dari Bacillus
pumilus baik yang berada diluar atau didalam
produk (25 kGy)
• bioburden
 HARGA D10
• efek radiasi pengion untuk mengeliminasi
mikroba bergantung kepada jumlah,dan daya
tahan mikroba tersebut.
• Daya tahan mikroba dinyatakan dengan harga
D10 yaitu dosis absorbsi yang dapat
mengeliminasi satu logaritma (90%)
kandungan mikroba.
 D10 = D / log N0– log N
D = Dosis Radiasi dalam kGy
N0 = Jumlah kandungan mikroba (bioburden)
sebelum iradiasi
N = Jumlah bioburden setelah radiasi*)
*) Jumlah bioburden ditentukan menurut ISO

11737-1
• Radiasi mengeliminasi mikroba secara
eksponensial, mengikuti system logaritma,
dan tidak pernah mencapai harga nol,
• jika harga D10 mikroba sebesar 0,3 kGy maka
untuk mengeliminasi 106 sel mikroba tersebut
dibutuhkan dosis radiasi 6 x 0,3 = 1,8 kGy.
• Daya tahan sangat dipengaruhi oleh suhu,
lingkungan dan bahan yang akan diiradiasi.
 contoh
• Suatu alat yang terkontaminasi spora sebesar
103 sel per alat, dosis sterilasi dihitung sebagai
berikut:

• Dosis sterilisasi = {log ( bioburden ) + log SAL}

x D10
= (log 103 + log 106 ) x D10 = (3+6) x 1.8 kGy
= 6.2 kGy
 Ada lima jenis bentuk/ stadium mikroba yang harus
diperhatikan dengan urutan ketahanan terhadap
radiasi
• prion (disebut juga slow virus, sejenis protein, bukan
organisma hidup karena tidak mempunyai DNA, ),
sangat tahan terhadap radiasi dan panas.
Mikroorganisme D10
virus 4 – 13 kGy
spora bakteria 2 kGy
bentuk vegetatif bakteria 1 kGy
kapang dan khamir dibawah 0.5 kGy
 Mikroorganisme (Virus/Bakteri) D10 (kGy)
Virus / Bakteria
HIV 1-2* 4 - 7.09, rata-rata 5

BPV (bovine parvovirus)** 7.27

PV-1 (polio virus)** 7.13
HAV (hepatitis A virus )** 5.31
PRV (pseudorabies virus )* 5.29
BDBV (bovine virus diarrhoea virus)* < 3.0
Spora Bacillus pumilus*** 1.8 – 2 kGy
Ragi dan jamur*** < 0.5 kGy
*Envelope virus; ** non-envelope virus;
***Harga D10 untuk bakteri, ragi dan jamur umumnya < 2 kGy
Mechanism
of DNA
disruption
by X-Ray &
Gamma
Rays
 How ionizing radiation works

• Electrons disrupt the DNA chain either destroying or

preventing reproduction of the organism
Gamma () Ray Processing Facility
Electron-beam

Dosimeter
 STERILISASI GAS
1. JENIS GAS
2. MEKANISME AKSI
3. APLIKASI
 STERILISASI GAS
Gas Etilen Oksida

Beta-Propiolakton

Hidrogen Peroksida

Asam Perasetat (Peracetic Acid)

Chlorine Compounds

Formaldehid
 MEKANISME AKSI
alkilasi gugus sulfhidril, amino,
karboksil, atau hidroksil pada
metabolit esensial yang
mempengaruhi proses reproduktif
dengan suatu radikal hidroksi etil

Metabolit yang telah diubah tidak tersedia bagi

mikroorganisme, sehingga mikroorganisme itu
mati tanpa reproduksi.
 Ethylene oxide
O a. cyclic ether (C2H40)
b. boiling point 10.7oC
H C C H
c. Colorless, odorless.
H H d. In its pure form ethylene
oxide is highly flammable
in air;
e. explosive.
FAKTOR YANG MEMPENGARUHI

Konsentrasi gas dan lama paparan

Temperatur

Kelembaban relatif udara

 KONSENTRASI GAS
Makin tinggi konsentrasi , makin tinggi
kecepatan inaktivasi
KONSENTRASI

Pada konsentrasi rendah < 300 mg/L, tidak

cukup mencapai sterilitas dalam waktu
sterilisasi yang praktis

Pada konsentrasi yang sangat tinggi,

menyebabkan tekanan meningkat.

Konsentrasi optimum : 500-800 mg/L

 TEMPERATURE

Reaksi alkilasi mengikuti reaksi orde

satu
TEMPERATURE

Kecepatan inaktivasi meningkat setiap

kenaikan 10C

Temperatur optimum : 50-60 C

 KELEMBABAN/HUMIDITY
Kelembaban adalah faktor yang paling penting yang
mempengaruhi efek etilen oksida pada populasi mikroba
KELEMBABAN

Etilen oksida tidak cukup efektif melawan

mikroorganisme terdehidrasi dalam lingkungan kering

Etilen oksida adalah sterilisasi kimia dan harus

bersentuhan dengan sasaran molekul (DNA dan RNA)
yang secara fisik terletak inti sel

Air bertindak sebagai pembawa etilen oksida melalui

hambatan permeabel

Kelembaban optimal 50 – 60 % RH
 GAS ETILEN OKSIDA
tersedia secara komersial dalam bentuk murni
atau sebagai campuran dengan hidrokarbon
GAS ETILEN OKSIDA

terfluorinasi atau karbon dioksida

Etilen oksida murni sangat mudah terbakar di

udara

Dalam sterilisasi itu biasanya digunakan

bersama dengan gas inert seperti karbon
dioksida atau nitrogen dari sumber yang
terpisah
 Kondisi Pemaparan yang Digunakan dengan
Campuran Etilen Oksida pada Suhu 55°C (131 F)

Waktu
Konsentrasi Tekanan
Isi campuran pemaparan
Nama dagang EtO kamar
(%) minimum
(mg/L) (Psig)
(jam)
Carboxide 10 etilen oksida
450 28 6
90 karbon oksida
Oxyfume-20 20 etilen oksida 670 18 4
80 karbon oksida 920 30 3
Cry- oxcide 11 etilen oksida
(Benvicide) 54 triklorofluorometan 450 5 5
35 diklorodifluorometan 850 18 3

Pennoxide 12 etilen oksida

88 diklorodifluorometan 650 7 4
 Ethylene Oxide Sterilization Processes

• For sterilization purposes it is commercially available in the

pure form or as mixtures with fluorinated hydrocarbons or
carbon dioxide.
• Pure ethylene oxide is highly flammable in air.
• In sterilization it is normally used in conjunction with an
inert gas such as carbon dioxide or nitrogen from a
separate source.
• Commercially available gas mixtures are nonflammable
under normal operating conditions of temperature and
pressure.
• Mixtures of 12% ethylene oxide: 88%
dichlorodifluoromethane and 20% ethylene oxide : 80%
carbon dioxide have been commonly used
Ethylene Oxide Sterilization Chamber
Ethylene Oxide Sterilizer
Ethylene Oxide Sterilization Cycles
Ethylene Oxide Sterilization Cycles
Pressure

Exposure
CHAMBER

Gassing Evacuation
0
Vacuum

Humidification
Air Washes

TIME
 GAS ETILEN OKSIDA
Waktu
Konsentrasi Tekanan
Nama Isi campuran pemaparan
EtO kamar
dagang (%) minimum
(mg/L) (Psig)
(jam)
Carboxide 10 etilen oksida 450 28 6
90 karbon oksida
Oxyfume-20 20 etilen oksida 670 18 4
80 karbon oksida 920 30 3
Cry- oxcide 11 etilen oksida 450 5 5
(Benvicide) 54 triklorofluorometan 850 18 3
35 diklorodifluorometan
Pennoxide 12 etilen oksida 650 7 4
88 diklorodifluorometan
 Applications Of Ethylene Oxide Sterilization

a. small-scale and large-scale applications.

b. cold sterilization
c. In routine sterilization.
a. gas concentrations of 500-800 mg/L,
b. temperatures of 50—60oC and
c. relative humidity of at least 50% RH
d. for cold sterilization : Sterilization by gamma radiation is more
reliable than ethylene oxide
e. Ethylene oxide is penetrative (but less penetrative than gamma
radiation).
f. Penetration is important to the selection of packaging materials
 Applications Of Ethylene Oxide Sterilization

• small-scale and large-scale applications.

• cold sterilization
• In routine sterilization. gas concentrations of 500-800 mg/L,
temperatures of 50—60oC and relative humidities of at
least 50% RH are used
• Sterilization by gamma radiation is more reliable than
ethylene oxide for cold sterilization
• Ethylene oxide is penetrative (but less penetrative than
gamma radiation).
• Free movement of the gas to all parts and internal cavities
of each item being sterilized is an essential prerequisite of
the process.
• Penetration is important to the selection of packaging
materials