Sterilization by Radiation

Sterilization by radiations is also known as “cold sterilization” because no heat is used in this method. The
microorganisms are very susceptible to the lethal effects of ionizing radiations. The exact mechanism by
which the microorganisms are destroyed is not clear. According to one theory alteration of chemicals
presents in microorganisms takes place with the formation of new compounds which destroy the
microorganisms. According to another theory the vital structure of cells, such as nucleoproteins are
destroyed by which the microorganisms are killed.

Radiations can be divided into two groups

1. Electromagnetic waves, e.g. infra-red radiation, ultra-violet light, X-rays and gamma-rays.

2. Streams of minute particles of matter, e.g. alpha and beta radiations.

Among these infra-red radiation, ultra-violet light, gamma-rays and high velocity electron (a type of beta
radiation) are used for sterilization.

X-rays, alpha and other types of beta radiations are unsuitable for sterilization for their certain harmful
effects.

Excitation & ionization:

An atom consists of a small heavy nucleus surrounded by planetary electrons. As ultra-violet light, gamma
rays & high-speed electrons pass through matter they give energy to the electrons of the constituent atoms,
which can give one of the following effects:

 The electron may acquire sufficient energy to tear themselves free from the atoms--- this is called
ionization.

 The acquired energy may not be sufficient to permit electrons to escape but it raises the atoms to
an excessively energetic state--- this is known as excitation.

Although both excitation & ionization of the atoms of essential molecules in microorganisms can cause
death, ionization is far more effective.

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- UV light is of relatively low energy and rarely causes ionization, it destroys by excitation.

- Gamma rays & high velocities electrons are far more energetic, and therefore produce ionization; they
are called ionizing radiations and much more efficient sterilizing agents than UV light.

Radiation energies:

Although UV light & gamma rays are wave motions and have no mass, they behave in contact with matter
as if they consisted of small packets of energy; these are called quanta or photons.

The energies of electromagnetic and particulate radiations are expressed by the energies of their individual
quanta or particles respectively. The energy unit used is electron-volt (eV).

A particle or quantum has an energy of 1 eV if it has the same kinetic energy as an electron accelerated
through a potential difference of 1 volt.

 The quantum energy of the most effective ultra-violet wavelength for sterilization purposes is only
about 5 eV.

 The quanta of the two important gamma rays emitted from 60Co (a radioactive isotope) have
energies of 1.3 & 1.17MeV.

 A typical energy for the high velocity electrons, used for the same purpose is 4 MeV.

Ultra-Violet Light (UV-Radiation)

 Direct sunlight can destroy microorganisms which is largely due to ultra-violet rays of comparatively
long wavelength.
 Shorter wavelengths are far more damaging to living cells are emitted by the sun but fortunately are
absorbed by the earth's atmosphere.
 Only a narrow range of wavelengths is suitable for sterilisation because above 310 and below 200 nm
antimicrobial activity is very low.

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 Wavelength (nm) Effectiveness (%)
220 25
253.7 97
265 100
300 10
320 0.4

 Quantum energy increases as wavelength decreases and therefore, the fall in efficiency below 265 nm
indicates that the antibacterial activity must be influenced by another factor.
 To kill microorganisms, the radiation must be absorbed by an essential cell component.
 The degree of absorption - at any particular wavelength depends on the chemical structure of the
component.
 The wavelength of 265 nm and adjacent wavelengths are strongly absorbed by nucleoproteins, e.g. the
Deoxyribonucleic acid protein of the cell nucleus.
 Disruption of these vital compounds would be expected to cause serious damage to an organism i.e.
death, cessation of cell division and mutations.
 Wavelengths absorbed by less essential substances are relatively harmless.

Sources of Ultra-violet Light:

 The main method of generating ultra-violet light for sterilisation is by passing a low current at high
voltage through mercury vapour in an evacuated glass tube.
 The electrons of the outer orbit of the mercury atoms become excited and discharge their excess
energy as ultra-violet radiation.
 About 90 per cent of this is at 253.7 nm, which although not the wavelength of maximum efficiency, is
highly microbicidal.
 Ordinary glass tubes may contain impurities such as iron, sulphur, iodine and titanium which can
cause them to absorb antimicrobial wavelengths (253.7 nm).
 Therefore, the tubes are quartz or Vycor (a quartz like substance derived from boro-silicate glass).
 The popular size of the tube for air treatment has an effective length of 610 mm.

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Penetrating Power:
The most serious disadvantage of UV light as a sterilising agent is its poor penetrating power; this is a
result of strong absorption by many substances. Therefore,

 Although it rapidly destroys naked or droplet enclosed micro-organisms it is far less effective against
organisms within or on the unexposed sides of dust particles or dried mucus.
 When used for the sterilisation of the air of asepsis rooms - it should be combined with strict dust
control measures and a method, e.g. filtration or electrostatic precipitation that removes atmospheric
dust efficiently.
 It is not applicable to the sterilisation of packed pharmaceuticals.
 Its use for the sterilisation of thermolabile medicaments and preparations, prior to aseptic packing, is
severely limited.
 It is less effective against organisms in the atmosphere or on surfaces if the relative humidity is high.
A relative humidity less 50% is desirable.

Hazards:

 Bactericidal ultra-violet light causes acute conjunctivitis and erythema of the skin.

 Exposure of the eyes and skin to direct or high-intensity reflected radiation must be prevented.

Applications:

1. Irradiation of incoming or internal air of the sterile filling areas of antibiotic plants:

UV radiation can provide air of adequate bacteriological quality when combined with a method for
removing dust. The workers need to protect themselves by wearing eye shields, hoods, gowns with
long sleeves & rubber gloves.

2. As an aid to asepsis: (https://www.youtube.com/watch?v=cTK7QnrKe1M)

(a) In manufacturing houses: Over the working area during the filling and closing of containers of
sterile thermolabile products.

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 (b) In hospitals: Inside a screen during aseptic processing.

3. To prevent cross-infection in hospitals, schools etc:

(a) Indirect Method:

Lamps are mounted above the heads of occupants, on the walls, or ceiling, and with the radiation
directed upwards by suitable reflectors.

The result is an irradiated zone across the top of the room through which the air is encouraged to pass.

(b) Direct Method:

Lamps are mounted over the doors of rooms and cubicles containing infectious patients to provide an
intense bactericidal curtain down to the floor.

4. Sterilization of thermolabile substances:

Poor penetration, unreliability and the possibility of photochemical damage have largely prevented the
application of this method to the sterilization of thermolabile pharmaceuticals. It has been used--

(a) To destroy the virus of homologous serum jaundice in blood plasma: It is difficult to ensure
adequate penetration & complete destruction of the virus without damage to plasma protein.

(b) To inactivate viruses and bacteria in vaccine production: UV light has been successful in a few
cases where heat & chemical treatment seriously affect antigenicity.

5. Improvement of bacteriological quality of water used to manufacture non-sterile

pharmaceuticals:

Since complete freedom from microorganisms is not essential for non-sterile pharmaceutical products,
ultra-violet radiation is suitable for controlling bacteriological quality of water especially when large
volume is considered.

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 Ionizing radiations

Ionizing energy can be obtained from machines or radioactive isotopes.

 Machine-generated radiations include X-rays, electrons, protons and alpha-particles.
 Radioactive isotopes provide sources of alpha-particles, beta particle and gamma-ray.
All ionizing radiations, in adequate dosage, can destroy micro-organisms, but most are unsuitable for the
sterilization of pharmaceutical and medical materials because they fail to satisfy one or more of the
following requirements:

1. Good Penetrating Power:

 X ray and gamma radiations: penetrate well because they are
-uncharged,
-massless and
-have highly energetic quanta.
 Alpha and beta particles have charge & mass and their passage through matter is impeded by
interaction with atomic fields and collisions with atomic particles. These penetrations are
inadequate for routine sterilization.

2. High Sterilizing Efficiency:

 Alpha-particles and protons:
- produce dense tracks of ions
-which cause far more ionizations than necessary
-are less efficient than radiations, such as electrons, X-rays and gamma-rays
 Electrons, X-rays and gamma-rays:
-produce ionizations which are more sparsely distributed
-are more efficient sterilizer

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 3. Minimum Damage to Irradiated Materials:
 Exposure to ionizing radiations can cause adverse chemical and physical changes.
 The risk is greater with particles that produce dense ionizations (Alpha-particles and protons).

4. Satisfactory Production Efficiency:

 X-radiation suitable for sterilization can be produced by bombardment of a heavy metal target
with a beam of high-speed electrons.
 But only a small proportion of beam energy is converted to X-rays.
 Which makes it economically unsuitable than the alternatives.

The only radiations that adequately fulfill these four requirements are-
 High speed electrons from machines and
 Gamma rays from radioactive isotopes.

1. High Speed Electrons:

 Electrons are generated from a suitable source in a machine known as a Van de Graaff accelerator.
 Electrons are then accelerated along a highly evacuated tube by a tremendous potential difference
between the ends.
 The particles in the emergent beam are travelling at near to the speed of light
 They have energies from 1 to 4 MeV.
 The beam, which is narrow and intense, is used to irradiate articles on a conveyor belt

2. Gamma Rays:
 Radiation from the radioactive isotope of Cobalt, 60Co, is used.
 Almost all of the unstable atoms of this isotope emit, when they disintegrate, two gamma-rays in
series.
 These have energies of 1.33 and 1.17 MeV, and therefore products are treated with gamma
radiation with a mean energy of 1.25 MeV.

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  The half-life of 60Co is 5.25 years.
 The unit of radioactivity is the curie (Ci).
 This is the amount of any radioactive substance that undergoes 3.7x1010 disintegrations/second.

Mode of Action:
 Ionizing radiations cause both excitations and ionizations and their absorption is not affected by
the structure of the molecules.
 Harmful effects on microorganisms and organic compounds may be the result of -
1. Direct action (Target) Theory
2. Indirect action (Chemical) Theory

1. Direct Action (Target) Theory:

 This proposes that every micro-organism and living cell contains a target region so highly sensitive to
radiation damage that a single ionization inside it causes inactivation or death.
 In very small organisms, such as bacteriophages & small viruses nucleic acid is the sensitive material.
 In larger organisms, radiation sensitive genes are thought as target.

2. Indirect Action (Chemical) Theory:

 Absorption of radiation by the water, within or surrounding living cells, produces free radicals.
 These are powerful oxidizing and reducing agents capable of damaging essential molecules and,
therefore, causing death.

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 The oxidizing radicals OH and HO2 and H2O2 are more destructive than the reducing radical.

Sterilizing Dose:
The dose unit is the rad (radian)- the amount of ionizing radiation from any source that delivers 0.01 J of
energy/kg of absorbing material.
A report on the sterilization of pharmaceuticals published by the Association of British Pharmaceutical
Industry concludes that,

 Gram negative organisms appear more sensitive than Gram positive organisms.

 Spore producers are the most resistant.

 A dose of 2.5 x 1O6 rads appears adequate to ensure sterility.

Sterilization Time:
 The intense beams or pulses of electrons from accelerators can deliver a sterilization dose in a
fraction of a second to a few seconds depending on the size and density of the material or article
being irradiated.

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  With isotope sources, because of the diffuse and penetrating nature of their emissions, the dose rate
is much less and, therefore, the sterilizing dose has to be accumulated over several days (3 to 3.5).

Penetration:
 Gamma radiation (isotope source) has great penetrating power.
 The intensity of the radiation decreases exponentially with the thickness of the absorber.

Deleterious Effects:
Radiation sterilization appears an attractive method for thermolabile medicaments and equipment because
the rise in temperature caused by a sterilizing dose is very small-about 4°C. But they have some
deleterious effects:
 Undesirable effects include decomposition, immediately or after storage, and alterations in colour,
texture and solubility.
 Potency changes range from almost nil or nil (e.g. certain antibiotics and steroid hormones) to
serious loss (eg. insulin, posterior pituitary hormones and cyanocobalamin).
 Destruction is often greater when substances are irradiated in solution (e.g. heparin).

Reduction of Undesirable Effects:

Methods of reducing unwanted radiation damage particularly in connection with the sterilization of foods:

1. Irradiation in the Frozen State:

Freezing slows down the diffusion of free radicals and, therefore, fewer reach the molecules of
sensitive substances.

2. Addition of Chemical Protectants:

-Examples: ascorbic acid, cysteine, thiourea

-Cysteine: protects healthy cells of the human body from damage by X-rays.

3. Removal of Oxygen:

Decreases oxidative effects by causing a reduction in the number of oxidizing free radicals.

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 4. Use of Pulsed Radiation:

Discontinuous electron beam.

Applications:
 The method is firmly established for disposable surgical materials and equipment.
 Articles regularly treated on a commercial scale include plastic syringes & catheters, hypodermic
needles and scalpel blades, adhesive dressings, single-application capsules of eye ointment etc.
 Popular packaging materials include plastic films and aluminium foil.

Advantages:
1. The temperature rise is insignificant.
2. The processes can be continuous because exposure is so short (machines) or a large amount of
material can be treated at once (isotopes).
3. There is no aseptic handling since sterilization can be performed after packing in the final
containers.
4. The methods are reliable and can be very accurately controlled.
5. Dry, moist and, with electrons, frozen materials can be treated.
6. Some bacterial and viral vaccines can be sterilized without loss of antigenicity.

Disadvantages:
1. Capital and replacement costs are high.
2. Elaborate & expensive precautions must be taken to protect operators from the harmful effects of
ionizing radiations.
3. Deleterious changes are produced in many medicaments, fats and foods.

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