Microbiology & Immunology

II
Radiation sterilization and Filtration
 Radiation sterilization
Several types of radiation find a sterilizing application in the manufacture of
pharmaceutical and medical products, principal among which are accelerated
electrons (particulate radiation), gamma rays and UV light (both electromagnetic
radiations). The major target for these radiations is believed to be microbial DNA,
with damage occurring as a consequence of ionization and free radical production
(gamma-rays and electrons) or excitation (UV light). This latter process is less
damaging and less lethal than ionization, and so UV irradiation is not as efficient a
sterilization method as electron or gamma-irradiation. Vegetative bacteria generally
prove to be the most sensitive to irradiation (with notable exceptions, e.g.
Deinococcus (Micrococcus) radiodurans), followed by moulds and yeasts, with
bacterial spores and viruses as the most resistant (except in the case of UV light
where mould spores prove to be most resistant). The extent of DNA damage
required to produce cell death can vary and this, together with the ability to carry
out effective repair, probably determines the resistance of the organism to
radiation. With ionizing radiations (gamma-ray and accelerated electrons),
microbial resistance decreases with the presence of moisture or dissolved oxygen
(as a result of increased free radical production) and also with elevated
temperatures.

4th semester 2
Radiation sterilization with high energy gamma rays or accelerated electrons
has proved to be a useful method for the industrial sterilization of heat-
sensitive products. However, undesirable changes can occur in irradiated
preparations, especially those in aqueous solution where radiolysis of water
contributes to the damaging processes. In addition, certain glass or plastic
(e.g. polypropylene, PTFE) materials used for packaging or for medical
devices can also suffer damage. Thus, radiation sterilization is generally
applied to articles in the dried state; these include surgical instruments,
sutures, prostheses, unit-dose ointments, plastic syringes and dry
pharmaceutical products. With these radiations, destruction of a microbial
population follows the classic survivor curves and a D-value, given as a
radiation dose, can be established for standard bacterial spores (e.g. Bacillus
pumilus) permitting a suitable sterilizing dose to be calculated. In the UK it is
usual to apply a dose of 25 kGy (2.5 Mrad) for pharmaceutical and medical
products, although lower doses are employed in the USA and Canada. UV
light, with its much lower energy, causes less damage to microbial DNA. This,
coupled with its poor penetrability of normal packaging materials, renders
UV light unsuitable for sterilization of pharmaceutical dosage forms. It does
find applications, however, in the sterilization of air, for the surface
sterilization of aseptic work areas, and for the treatment of manufacturing-
grade water.
4th semester 3
 Sterilizer design and operation
Gamma-ray sterilizers
Gamma-rays for sterilization are usually derived from a cobalt-60 (60Co) source
(caesium-137 may also be used), with a half-life of 5.25 years, which emits
radiation at two energy levels of 1.33 and 1.17MeV. The isotope is held as pellets
packed in metal rods. For safety reasons, this source is housed within a reinforced
concrete building with walls some 2 m thick, and it is only raised from a sunken
water-filled tank when required for use. Control devices operate to ensure that the
source is raised only when the chamber is locked and that it is immediately lowered
if a malfunction occurs. Articles being sterilized are passed through the irradiation
chamber on a conveyor belt.
Radiation monitors are continually employed to detect any radiation leakage during
operation or source storage, and to confirm a return to satisfactory background
levels within the sterilization chamber following operation. The dose delivered is
dependent upon source strength and exposure period, with dwell times typically
up to 20 hours. The difference in radiation susceptibilities of microbial cells and
humans may be gauged from the fact that a lethal human dose would be delivered
by an exposure of seconds or minutes.
4th semester 4
 Electron accelerators
Two types of electron accelerator machine exist, the electrostatic accelerator
and the microwave linear accelerator, producing electrons with maximum
energies of 5MeV and 10MeV, respectively. Although higher energies would
achieve better penetration into the product, there is a risk of induced
radiation and so they are not used. In the first, a high energy electron beam
is generated by accelerating electrons from a hot filament down an
evacuated tube under high potential difference, while in the second,
additional energy is imparted to this beam in a pulsed manner by a
synchronized travelling microwave. Articles for treatment are generally
limited to small packs and are arranged on a horizontal conveyor belt, usually
for irradiation from one side but sometimes from both. The sterilizing dose is
delivered more rapidly in an electron accelerator than in a 60Co plant, with
exposure times for sterilization usually amounting to only a few seconds or
minutes. Varying extents of shielding, depending upon the size of the
accelerator, are necessary to protect operators from X-rays generated by the
bremsstrahlung effect.

4th semester 5
 Ultraviolet irradiation
The optimum wavelength for UV sterilization is around
260nm. A suitable source for UV light in this region is a
mercury lamp giving peak emission levels at 254 nm. These
sources are generally wallor ceiling-mounted for air
disinfection, or fixed to vessels for water treatment. Operators
present in an irradiated room should wear appropriate
protective clothing and eye shields.

4th semester 6
 Filtration sterilization
The process of filtration is unique among sterilization techniques in that it removes, rather
than destroys, microorganisms. Further, it is capable of preventing the passage of both
viable and nonviable particles and can thus be used for both the clarification and
sterilization of liquids and gases. The principal application of sterilizing-grade filters is the
treatment of heat-sensitive injections and ophthalmic solutions, biological products and air
and other gases for supply to aseptic areas.
They may also be required in industrial applications where they become part of venting
systems on fermenters, centrifuges, autoclaves and freeze driers. Certain types of filter
(membrane filters) also have an important role in sterility testing, where they can be
employed to trap and concentrate contaminating organisms from solutions under test.
These filters are then placed in a liquid nutrient medium and incubated to encourage
growth and turbidity.
The major mechanisms of filtration are sieving, adsorption and trapping within the matrix
of the filter material. Of these, only sieving can be regarded as absolute as it ensures the
exclusion of all particles above a defined size. It is generally accepted that synthetic
membrane filters, derived from cellulose esters or other polymeric materials, approximate
most closely to sieve filters; while fibrous pads, sintered glass and sintered ceramic
products can be regarded as depth filters relying principally on mechanisms of adsorption
and entrapment. The potential hazard of microbial multiplication within a depth filter and
subsequent contamination of the filtrate should be recognized.

4th semester 7
Some characteristics of membrane and depth
filters

4th semester 8
 Filtration sterilization of liquids
In order to compare favorably with other methods of sterilization the microorganism removal
efficiency of filters employed in the processing of liquids must be high. For this reason, membrane
filters of 0.2–0.22mm nominal pore diameter are chiefly used, while sintered filters are used only in
restricted circumstances, i.e. for the processing of corrosive liquids, viscous fluids or organic
solvents. It may be tempting to assume that the pore size is the major determinant of filtration
efficiency and two filters of 0.2mm pore diameter from different manufacturers will behave
similarly. This is not so because, in addition to the sieving effect, trapping within the filter matrix,
adsorption and charge effects all contribute significantly towards the removal of particles.
Consequently, the depth of the membrane and its charge are all factors which can make the
performance of one filter far superior to that of another.
The major criterion by which filters should be compared, therefore, is their titre reduction values,
i.e. the ratio of the number of organisms challenging a filter under defined conditions to the
number penetrating it.
In all cases, the filter medium employed must be sterilizable, ideally by steam treatment; in the case
of membrane filters this may be for once-only use, or, in the case of larger industrial filters, a small
fixed number of resterilizations; sintered filters may be re-sterilized many times.
Filtration sterilization is an aseptic process and careful monitoring of filter integrity is necessary as
well as final product sterility testing.
Membrane filters, in the form of discs, can be assembled into pressure-operated filter holders for
syringes. Filtration under pressure is generally considered most suitable, as filling at high flow rates
directly into the final containers is possible without problems of foaming, solvent evaporation or air
leaks. To increase the filtration area, and hence process volumes, several filter discs can be used in
parallel in multiple-plate filtration systems. Membrane filters are often used in combination with a
fiber glass depth pre-filter to improve their dirt-handling
4th semester capacity. 9
 Filtration sterilization of gases
The principal application for filtration sterilization of gases is in the provision of sterile air
to aseptic manufacturing areas, hospital isolation units and some operating theatres.
Filters employed generally consist of pleated sheets of glass microfibres supported by
sheets of aluminium; these are employed in ducts, wall or ceiling panels, overhead
canopies, or laminar airflow cabinets. These high efficiency particulate air (HEPA) filters
can remove up to 99.997% of particles >0.3mm in diameter and thus are acting as depth
filters. In practice their microorganism removal efficiency is rather better as the majority of
bacteria are found associated with dust particles and only the larger fungal spores are
found in the free state. Air is forced through HEPA filters by blower fans, and prefilters are
used to remove larger particles to extend the lifetime of the HEPA filter.
The operational efficiency and integrity of a HEPA filter can be monitored by pressure
differential and airflow rate measurements, and dioctylphthalate smoke particle
penetration tests.
Other applications of filters include sterilization of venting or displacement air in tissue
and microbiological culture (carbon filters and hydrophobic membrane filters);
decontamination of air in mechanical ventilators (glass fibre filters); treatment of
exhausted air from microbiological safety cabinets (HEPA filters); and the clarification and
sterilization of medical gases (glass wool depth filters and hydrophobic membrane filters).

4th semester 10