Sterilization is a process that should eliminate virtually all colony forming units (CFUs) of
any kind of microbe present in the food product. It is necessary for the complete destruction
or removal of all microorganisms (including spore-forming and non-spore forming bacteria,
viruses, fungi and protozoa) that could contaminate pharmaceuticals or other materials and
thereby constitute a health hazard. Since the achievement of the absolute state of sterility
cannot be demonstrated, the sterility of a pharmaceutical preparation can be defined only in
terms of probability. The efficacy of any sterilization process will depend on the nature of the
product, the extent and type of any contamination and the conditions under which the final
product has been prepared. The requirements for Good Manufacturing Practice should be
observed throughout all stages of manufacture and sterilization. A sterile product should be
free of any CFU, and microbial spoilage may be caused only by reinfection or
recontamination from the surrounding environment. Autoclaving in hermetically sealed
containers at 121.10 C and 2.013 x 105 Pa pressure for longer than 15 min is the only
technique that is considered to guarantee sterile conditions. This results in the virtually
complete destruction of all CFUs in the initial product, but also leads to a significant
reduction of its nutritional and sensory properties. For aseptic packaging, high-temperature
short-time (HTST) processing is used, especially UHT, to preserve the nutritional and
sensory properties besides achieving a commercially sterile product. In such a case, the
reduction of CFUs follows a logarithmic function as shown in equation:
log S(t) = log (N t /N0 )
The equation shows the time-dependent survival rate S(t) as a function of cell count after a
defined treatment time t ( Nt ) in comparison with the cell count exactly at initial time t = 0
(N0 ). This shows that S cannot exactly reach zero level, an acceptable low number of CFU of
relevant microorganisms (bacteria, yeasts and molds) being able to contaminate, multiply or
survive in the product and being harmful to the consumer or product quality) is achievable,
which ensures the microbial stability of the closed product over a defined period of storage
time. Hence, the number of active cells is reduced to an amount of 1CFU per 1000 or lower.
This means that the risk per package to include one CFU equals 1:1000. If, sometimes
required, the risk for a microbiological failure of package should be less than 10**5 to
achieve higher food safety. But the advantages first become clear by looking on the z values
of microbes have been approximately 100C until the proposal of typical values for quality 1
factors C0 to compare the heating effect on appropriate compounds. It is similar to the F0
value, with the change that the reference temperature is 1000C:
C0 = 10 (T-100)/z T
[2]
Equation [2] reflects that the high temperatures show less effect on the degradation of quality
factors due to their increased z values. UHT processing at temperatures of 130-1500C for very
short heating times 2-6s has very low thermal effect on food but it is quite effective for
The only problem with UHT processing is that several heat resistant enzymes like proteases,
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lipases and peroxidases are still active after high heat treatment, which tends to deteriorate the
food quality. This may lead to quality defects like off- flavors and age gelation during storage.
The methods of sterilization can be divided into three main groups:
1)Physical methods: Heat distribution within the sterilizer chamber or load is usually
measured using thermocouples. Pressure measurement is by means of pressure gauges or
through pressure transducers connected to electronic recording instruments. Physical
measurement of relative humidity in sterilizers is difficult. Microprocessor-controlled
sterilizers utilize multi-channel recording systems, which are able to handle large amounts of
data from physical instruments and give a more detailed control and monitoring of sterilization
cycle.
Dry heat sterilization- In dry-heat process, the primary lethal process is considered to
be oxidation of cell constituents. Dry- heat sterilization requires high temperature and
long exposure time. The method is, therefore, more convenient for heat-stable, nonaqueous materials that cannot be sterilized by steam because of its deleterious effects
or failure to penetrate. Such materials include glassware, powders, oils and some oilbased injectable.
Preparations to be sterilized by dry heat are filled in units that are either sealed or
temporarily closed for sterilization. The entire content of each container is maintained
in the oven (labelled image) for the time and at the temperature given in the table below.
Other conditions may be necessary for different preparations to ensure elimination of
all undesirable microorganisms.
2
TEMPERATURE (0C)
MINIMUM STERILIZATION TIME
(min)
160
180
170
60
180
30
Specific conditions of temperature and time for certain preparations are stated in
individual monographs.
The oven should normally be equipped with a forced air system to ensure even
distribution of heat throughout all the materials processed. This should be controlled by
monitoring the temperature. Containers that have been temporarily closed during the
sterilization procedure are sealed after sterilization using aseptic techniques to prevent
microbial recontamination.
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Labelled image: Hot air oven
Moist heat sterilization- Exposure of microorganisms to saturated steam under
pressure in an autoclave achieves their destruction by the irreversible denaturation of
enzymes and structural proteins. The temperature at which denaturation occurs varies
inversely with the amount of water present. Sterilization in saturated steam thus requires
precise control of time, temperature and pressure. As displacement of the air by steam
is unlikely to be readily achieved, the air should be evacuated from the autoclave before
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admission of steam. This method should be used whenever possible for aqueous
preparations and for surgical dressings and medical devices.
The recommendations for sterilization in an autoclave (labelled image) are 15 minutes
at 121-1240C (200kPa). The temperature should be used to control and monitor the
process; the pressure is mainly used to obtain the required steam temperature.
Alternative conditions, with different combinations of time and temperature are given
below. (1atm = 101 325 Pa)
TEMPERATURE(0C)
APPROXIMATELY
MINIMUM
CORRESPONDING
STERILIZATION TIME
PRESSURE (kPa)
(min)
126-129
250(~2.5atm)
10
134-138
300(~3.0atm)
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Minimum sterilization time should be measured from the moment when all the
materials to be sterilized have reached the required temperature throughout. Monitoring
the physical conditions within the autoclave during sterilization is essential. To probes
placed in the load at the potentially coolest parts of the loaded chamber (as established
in the course of the validation programme). The conditions should be within +/-20C and
+/-10kPa (+/-0.1 atm) of the required values. Each cycle should be recorded on a timetemperature chart or by other suitable means.
-Aqueous solutions: in glass containers usually reach thermal equilibrium within 10
minutes for volumes up to 100ml and 20 minutes for volumes up to 1000ml.
-Porous loads: such as surgical dressings and related products, should be processed in
an apparatus that ensures steam penetration. Most dressings are adequately sterilized
by maintaining them at a temperature of 134-1380C for 5 minutes.
In certain cases, glass, porcelain or metal articles are sterilized at 1210-1240C for 20
minutes.
-Fats and oils: may be sterilized at 1210C for 2 hours but, whenever possible, should be
sterilized by dry heat.
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Labelled images: Autoclave
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Radiation sterilization
The influence of sunlight in preventing the spread of
infection has been known for centuries but it was realized only towards the end of the
19th century that the radiations primarily responsible lie in the ultraviolet range of
wavelengths.
Radiation is commonly classified as electromagnetic and particulate. Electromagnetic
radiation comprises of photons of energy including ultraviolet, gamma, X- and cosmic
radiation. Beta particles or electrons are a type of particulate radiation. Radiation
sterilization is also known as cold sterilization as it is a non-thermal method.
Destruction of microorganisms results from interference in the metabolism of the cell.
- Use of ultra violet rays: Ultraviolet (UV) radiation in the region of 2537Å has been
shown to possess the greatest activity in destroying microorganisms. It is commonly
employed in the reduction of air-borne contamination in the maintenance of aseptic
areas and rooms within the processing environment in pharmaceutical industry. The
penetrating power being negligible, its effectiveness is limited to exposed surfaces
only. The most common source of artificial UV radiation is the UV lamps. The
bactericidal effectiveness is primarily a function of the intensity of radiation and
duration of exposure and varies with the susceptibility of the organism. A dose of
2.5 M rads is generally accepted as adequate.
UV light is absorbed by the nucleic acids of the cell where it does the largest
damage. The absorption and subsequent reactions are predominantly in the
pyrimidines of the nucleic acids causing the formation of pyrimidine dimer in which
two adjacent pyrimidines become bonded in DNA molecule.
The method had been used to destroy the viruses of homologous serum jaundice in
blood serum and to inactivate viruses and bacteria in vaccine production. It is also
used for irradiation of incoming or internal air of the sterile filling areas for
antibiotics, as an aid to asepsis in manufacturing houses and in hospitals and to
prevent cross-infection in hospitals, schools, etc.
- Ionizing radiation: These are produced directly from the charged particles and
indirectly from gamma rays and can penetrate matter to a significantly greater
degree than UV radiation. Ionizing radiations destroy microorganisms by stopping
reproduction as a result of lethal mutations. Harmful effects of gamma rays on
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microorganisms may be the result of direct or indirect action. The most important
types of ionizing radiations and their nature are listed in the table below:
Radiation
Nature
Electromagnetic waves
Wavelength approximately 10Å to 10-4 Å
X- rays
Gamma rays
Particulate Radiations
Cathode rays
Fast electrons
Beta rays
Fast electrons
Fast neutrons
Mass 1, no charge
Fast protons
Mass 1, +ve charge
Fast deuterons
Mass 2, +ve charge
Alpha rays
Mass 4, 2 +ve charge
Nuclear fission fragments
Heavy atomic particles
Table: Important types of Ionizing Radiations and their Name
All these radiations are obtained either from the electrical generators or radioactive
elements except the last one which is obtained from nuclear reactions.
Roentgen (r) is the fundamental unit used in the measurement of radiation doses and
is defined in terms of the number of ionizations produced per unit volume of air.
Another unit is the rad , which represents energy absorption of 100 ergs per gram
of material, however, for sterilization purposes the term M rad, or 1x106 rad , is the
most practical unit.
The factors that affect the lethal activity of ionizing radiations are the oxygen effect,
sulfhydryl and other protective compounds, sensitizing agents, age and pH of the
culture, freezing, moisture, heat sensitization and recovery conditions.
High- speed electrons are generated by Van de Graaf accelerator. These are the only
means for sterilization of a product by continuous process. They have been used for
sterilization of catgut sutures, plastic administration sets, a number of vitamins,
antibiotics and hormones in the dry state. However, this method of sterilization is
relatively expensive; further, such conditions are not present in manufacturing
houses.
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Gamma radiation sterilizations are most valuable because of their power of
penetration and the absence of any thermal effect. Radiation treatment in the
preservation of foods is the most important use in food industry. Combining an
antibiotic treatment with the radiation also gives promising results. Gamma rays are
commonly obtained using radioactive isotopes of cobalt,
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Co. Two gamma rays
are emitted in a succession as a result of disintegration of almost all of the unstable
atoms of this isotope. The ionizing radiations cause both excitations and ionizations
and their absorption is not affected by the structure of the molecules in the irradiated
matter. Gamma rays resemble X- rays of short wavelength and are emitted by both
natural and artificial radioactive elements. They differ from X- rays mainly in that
they are emitted from a given element at one or two fixed wavelengths. X- and
gamma rays, being electromagnetic radiations, have much greater power of
penetration- an important practical attribute.
Radiation sterilization however involves a considerable capital expenditure and the
safety of all of the personnel engaged in such work needs constant attention and
monitoring.
2)Chemical methods: Chemical monitoring is based on the ability of the sterilization process
to produce sufficient change in the physical or chemical characteristics of chemical substances
to be detected visually or by physically instrumentation. Chemical indicators (CIs) generally
undergo melting or color changes.
Sterilization by heating with bactericide- This method may be used for sterilizing
aqueous preparations which are unstable at higher temperatures attained in the moist
heat sterilization processes. The method has a lower margin of safety and should be
used only when moist heat sterilization is not applicable. It is used for the sterilization
of injections which though reasonably thermostable do show some evidence of
deterioration when heated at 115-1160C for 30 minutes in an autoclave.
The method cannot be used for the sterilization of injections meant for intrathecal or
peridural administration. Similarly, this method is not suitable for intravenous
injections when a single dose exceeds 15ml. In this process the medicament is dissolved
or suspended in a suitable solution of the following bactericide so as to achieve the
concentration of bactericides given in the table.
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Name of the bactericide
Concentration of bactericide in
per cent w/v
For injections
1. Chlorocresol
2. Phenyl
mercuric
0.2
acetate or
0.002
Phenyl mercuric nitrate
For eye drops
1. Benzalkonium chloride
0.01
2. Phenyl mercuric acetate or
0.002
Phenyl mercuric nitrate
3. Thiomersal
0.01
4. Chlorohexidine acetate
0.01
The preparation is then transferred into the final container which is then sealed so as to
exclude microorganisms and heated at 98-1000C for 30 minutes by heating it in boiling
water. The bactericide used should be non-toxic, compatible with medicaments, stable
and active at various pH, stable during heating and storage. The lethal effect of
bactericide increases with the rise of temperature.
Gaseous sterilization- The active agent of the gas sterilization process can be ethylene
oxide or another highly volatile substance. The highly flammable and potentially
explosive nature of such agents is a disadvantage unless they are mixed with suitable
inert gases to reduce their highly toxic properties and the possibility of toxic residues
remaining in treated materials. The whole process is difficult to control and should only
be considered if no other sterilization procedure can be used. It must only be carried
out under supervision of highly skilled staff.
The sterilizing efficiency of ethylene oxide depends on the concentration of the gas, the
humidity, the time of exposure, the temperature and the nature of the load. In particular,
it is necessary to ensure that the nature of the packaging is such that the gas exchange
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can take place. It is also important to maintain sufficient humidity during sterilization.
Records of gas concentration and of temperature and humidity should be made for each
cycle. Appropriate sterilization conditions must be determined experimentally for each
type of load.
After sterilization, time should be allowed for the elimination of residual sterilizing
agents and other volatile residues, which should be confirmed by specific tests. Because
of the difficulty of controlling the process, the efficiency must be monitored each time
using the proposed bio indicator strains: spores of Bacillus subtilis or of Bacillus
stearothermophilus.
3)Mechanical methods: Sterilization involves the filtration of parenteral preparations through
the following bacteria proof filters:
Ceramic filters
Seitz filter
Sintered glass filters
Sintered metals filters
Membrane filters
Sterilization by filtration is employed mainly for thermolabile solutions. These may be
sterilized by passage through sterile bacteria-retaining filters (cellulose derivatives, etc.),
plastic, porous ceramic or suitable sintered glass filters or combination of these. Asbestoscontaining filters should not be used.
Appropriate measures should be taken to avoid loss of solute by adsorption onto the filter and
to prevent the release of contaminants from the filter. Suitable filters will prevent the passage
of microorganisms, but the filtration must be followed by an aseptic transfer of the sterilized
solution to the final containers which are then immediately sealed with great care to exclude
any recontamination.
Usually membranes not greater than 0.22 µm nominal pore size should be used. The
effectiveness of the filtration method must be validated if larger pore sizes are employed.
To confirm the integrity of filters, both before and after filtration, a bubble point or similar test
should be used, in accordance
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prescribed pressure to force air bubbles through the intact membrane previously wetted with
the product, with water or with a hydrocarbon liquid.
downs
withstanding heat may be sterilized in the assembly before use by autoclaving at 1210C for 1545 minutes depending on the size of the filter assembly. The effectiveness of this sterilization
should be validated. For filtration of a liquid in which microbial growth is possible, the same
filter should not be used for procedures lasting than one working day.
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BIBLIOGRAPHY:
International Pharmacopoeia, (2019), Methods of sterilization (9th ed.) 5.8.,WHO
Jain,N.K,(2001),Pharmaceutical microbiology(9th ed.)95-110.Delhi:Prakashan
Götz,A.,Wani,A.,Langowski,H.,&Wunderlich,J.(2014).FoodTechnologies:Aseptic
Packaging. Encyclopedia Of Food Safety,124-134.doi:10.1016/b978-0-12-378612-8.00274-2
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