THE CONTROL OF MICROBIAL GROWTH

"Control of growth", means to prevent growth of microorganisms. This

control is affected in two basic ways: (1) by killing microorganisms or (2)
by inhibiting the growth of microorganisms.

Control of growth usually involves the use of physical or chemical

agents, which either kill or prevent the growth of microorganisms.
Agents, which kill cells, are called cidal agents; agents which inhibit
the growth of cells (without killing them) are referred to as static
agents. Thus the term bactericidal refers to killing bacteria and
bacteriostatic refers to inhibiting the growth of bacterial cells. A
bactericide kills bacteria; a fungicide kills fungi, and so on.

Sterilization can be defined as any process that effectively kills or

eliminates transmissible agents (such as fungi, bacteria, viruses and prions)
from a surface, equipment, foods, medications, or biological culture
medium.

Methods of Sterilization
Physical Methods of Microbial Growth:
Heat (Moist Heat, Dry Heat)
Low Temperatures
Filtration
Radiation
Gaseous
Chemical Methods
Alcohols
Aldehydes
Biguanides:
Halogens: Hypochlorites
Halogens: Iodine
Phenol & Phenolics
Quaternary Compounds

Heat: Heat Kills microorganisms by denaturing their enzymes and other proteins.
Thermal Death Point (TDP): Lowest temperature at which all of the microbes in a
liquid suspension will be killed in ten minutes.
Thermal death time (TDT): Shortest time needed to kill all organisms in a
suspension at a specified temperature under specific conditions.
Decimal reduction time (D value): The time required to reduce a population of
microbes by 90% at a specified temperature and specified conditions.
Z-value: it is defined as number of degrees temperature change required to achieve
a ten fold change in D-value. e.g, if D-value for Bacillus stearothermophilus spores at
110oC is 20 minutes and they have Z-value of 9oC, this means that at 119oC D-value
would be 2 minutes and at 128oC the D-value would be 0.2 minutes.

Moist Heat
The mechanism of killing by moist heat is a combination of protein/nucleic acid
denaturation and membrane disruption. Effectiveness mainly dependent on type of
cells present as well as environmental conditions (type of medium or substrate).
Bacterial spores much more difficult to kill than vegetative cells.
Methods of Moist Heat:
Boiling at 100C: Vegetative cells are killed almost immediately at 90-100 oC, but
the sporing bacteria require prolonged periods of boiling. It is generally used to
disinfect drinking water but unsuitable method for heat sensitive chemicals & many
foods
Autoclaving/pressure canning:
Temperatures above 100C achieved by steam under pressure.
The principle of the autoclave or steam sterilizer is that water boils when its vapour
pressure equals to the surrounding atmosphere. Hence when pressure inside a closed
vessel increases, the temperature at which water boils also increases.
Saturated steam has penetrative power. When steam comes in to contact with a
cooler surface it condenses to water and gives up its latent heat to that surface (1600
ml steam at 100 oC and at atmospheric pressure condenses in to water 1 ml of water
at 100 oC and releases 518 calories of heat). Most procedures use 121.1C, achieved
at approx. 15-psi pressure, with 15 - 30 min autoclave time to ensure sterilization.

Pressure in psi (pound per Temperature oC

square inch)
15
121 to 124
20
126 to 129
30
134 to 138

Holding time
(minutes)
15
10
03

Autoclaving is used to sterilize culture media, instruments, dressing, intravenous

equipments, solutions, syringes, transfusion equipments and numerous other items
that can withstand high temperatures and pressures. Large industrial autoclaves
are called retorts.

Disadvantages:
1. During autoclaving, the pH of aqueous solutions changes due to water loss.
2. Oils do not get sterilized in autoclave as they are hydrophobic in nature and do
not allow the steam to penetrate it.

Autoclaves,
or
steam
sterilizers
essentially consist of following:
1.A cylindrical or rectangular chamber,
with capacities ranging from 400 to
800 liters.
2.Water heating system or steam
generating system
3.Steam outlet and inlet valves
4.Single or double doors with locking
mechanism.
5.Thermometer or temperature gauge
6.Pressure gauges
Operation
For porous loads (dressings) sterilizers
are generally operated at a minimum
temperature of 1340C, and for bottled
fluid, sterilizers employing a minimum
temperature of 1210C are used. Ensure
that there should be sufficient water in
the autoclave to produce the steam.
The stages of operation of autoclaves
include air removal, steam admission
and sterilization cycle (includes heating
up, holding/exposure, and cooling
stages).

Pasteurization: Used to reduce microbial numbers in milk and other beverages

while retaining flavor and food quality of the beverage. This method kills most
vegetative bacterial cells including pathogens such as streptococci, staphylococci
and Mycobacterium tuberculosis.
Application:
1.Pasteurization is most often employed to prevent the transmission of milk borne
diseases from infected cows. The primary target of pasteurization is the control of
non-spore forming pathogens.
2.To kill unwanted contaminants in beer and wine.
Limitations:
It does not destruct thermoduric organisms and their spore.
Pasteurization can be done by one of the following methods.
Classic Method of Pasteurization (Low temperature holding method or
LTH): Milk exposed to 63 oC for 30 minutes.
High Temperature Short Time Pasteurization (HTST): Most milk
pasteurization today uses higher temperature at least 72 oC but only for 15
seconds. This treatment, known as High Temperature Short Time Pasteurization.
Ultra High Temperature Pasteurization (UHT): Milk is treated at 140oC for 3
seconds and then cooled very quickly in a vacuum chamber. Advantage: Milk can
be stored at room temperature for several months.

Dry Heat: practically the temperature of dry heat ranges from 160oC to several
thousand degrees Celsius. Microbes are generally more resistant to dry heat than
moist heat. This is because of less penetration and diffusion of dry heat compared
to moist heat. Dry heats kill microbes by oxidation effects.
Direct Flaming: Used to sterilize inoculating loops and needles. Heat metal until it
has a red glow.
Incineration: Effective way to sterilize disposable items (paper cups, dressings)
and biological waste.
Hot Air Sterilization: Hot air oven is equipment for dry heat sterilization by hot
air under atmospheric pressure. Hot air is circulated within compartment making
contacts with the items and transferring the heat. Dry heat is transfers heat less
effectively to a cool body, than moist heat.
The longer period and higher
temperature are required because the heat in water is more readily transferred to a
cool body than is the heat in air.
The most common conditions used for sterilization using dry heat as follows:
Temperature
160 oC
170 oC
180 oC

Holding time (min)

120
60
30

Hot-air oven
Dry heat sterilization is usually carried out in a hot air oven, which consists of the
following:
1.An insulated chamber surrounded by an outer case containing electric heaters.
2.A fan
3.Shelves
4.Thermocouples
5.Temperature sensor
6.Door locking controls.
Operation
7.Articles to be sterilized are first wrapped or enclosed in containers of cardboard,
paper or aluminum.
8.Then, the materials are arranged to ensure uninterrupted air flow.
9.Oven may be pre-heated for materials with poor heat conductivity.
10.The temperature is allowed to fall to 400C, prior to removal of sterilized
material.
Application of hot air sterilization:
.To sterilize needles, porcelain and metal equipments.
.To sterilize oils and fats, including oily injections.
.Various powders can be sterilized by this method eg: talc
Disadvantages:
.Not suitable for aqueous solutions, plastic, papers and cloths.
.It takes longer time compared to autoclaving.

Radiation: Three types of radiation kill microbes:

1. Ionizing Radiation: Gamma rays, X rays, electron beams, or higher energy
rays. They have short wavelengths (less than 1 nanometer). Ionic radiations
dislodge electrons from atoms and form ions. One of the most sensitive targets of
ionizing radiation is DNA molecule. These radiation cause mutations in DNA and
produce peroxides.
Application:
Used to sterilize pharmaceuticals and disposable medical supplies. Food like meat
can also sterilized by ionizing radiation.
Disadvantages: Penetrates human tissues. May cause genetic mutations in
humans.
2. Ultraviolet light (Nonionizing Radiation): Wavelength is longer than 1
nanometer. UV rays have wavelength ranges from approximately 100nm- 400nm.
Mercury lamp is commonly used source of UV rays. DNA absorbs ultraviolet
radiation at 260 nm wavelengths. This causes damage to DNA in the form of
thymine dimer mutations.
Application: Used to disinfect operating rooms, nurseries, and cafeterias.
Disadvantages: Damages skin, eyes. Does not penetrate paper, glass, and cloth.
3. Microwave Radiation: Wavelength ranges from 1 millimeter to 1 meter.
Microwaves as such do not kill microbes directly. But they can destroy microbes
because of heat they generated. Water molecules present in microbial cells absorb
heat. Microwaves may kill vegetative cells in moist foods. Bacterial endospores,
which do not contain water, are not damaged by microwave radiation. Microwaves
unevenly penetrate solid foods.

Filtration: Removal of microbes by passage of a liquid or gas through a screen like

material with small pores. This is a mechanical way of sterilization and is generally
used for heat sensitive fluids. Following filters are most commonly used in
sterilization:
Seitz filter: It is asbestos filter. The disk of pressed asbestos is tightly clamped
between two smooth metal rings by screw. The liquid to be filtered is poured in to a
funnel like attachment and drawn through the asbestos disk by vacuum.
Membrane filter: membrane filters are prepared from a variety of polymeric
material like polycarbonate and polyesters. Membrane filters are only used for
sterilization, but also for separation of microorganisms and for collecting microbial
sample. Different sizes of membrane filters are:
0.22 and 0.45m Pores: Used to filter most bacteria. Dont retain spirochetes,
mycoplasmas and viruses.
0.01 m Pores: Retain all viruses and some large proteins.
HEPA filter: (High efficiency particulate air): These filters are widely used for
getting sterile air. HEPA filters are made up of cellulose acetate and are available in
various pore sizes. A generally available size of HEPA filter pore is 0.3 . It provides
99.9% efficiency. The HEPA filters are generally equipped with laminar airflow,
which is very important instrument for providing aseptic condition. The laminar
airflow widely used to carry out sterility testing, drug packing, and ampoule fillings.
Laminar airflow is equipped with UV lamp.

Low Temperature: The effect of low temperature on microorganisms depends on

particular microbe and intensity of treatment applied.
Refrigeration: Temperatures from 0 to 7oC, reduces metabolic rate of most
microbes so they cannot reproduce or produce toxins. In other words low
temperature has a bacteriostatic effect.
Freezing: Temperatures below 0oC.
Flash Freezing: Does not kill most microbes.
Slow Freezing: More harmful because ice crystals forms disrupt cell structure. Most
parasites are killed by a few days of freezing.
Desiccation: In the absence of water, microbes cannot grow or reproduce, but some
may remain viable for years. After water becomes available, they start growing
again. Susceptibility to desiccation varies widely:
Neisseria gonnorrhea: Only survives about one hour.
Mycobacterium tuberculosis: May survive several months.
Viruses are fairly resistant to desiccation.
Clostridium spp. and Bacillus spp.: May survive decades.
Osmotic Pressure: The use of high concentrations of salts and sugars in foods is
used to increase the osmotic pressure and create a hypertonic environment, which
causes water to leave the microbial cell, resulting in the plasmolysis. The principle of
osmotic pressure used in the preservation of foods. For example, concentrated salt
solutions are used to cure meats, and thick sugar solutions are used to preserve
fruits. As a general rule, molds and yeasts are much more capable than bacteria of
growing in materials with low moisture or high osmotic pressures. This property of
molds, sometimes combined with their ability to grow under acidic conditions, is the
reason fruits and grains are spoiled by molds rather than by bacteria.

Gaseous Sterilization:
Ethylene oxide (ETO) is the most commonly used form of chemical sterilization.
Due to its low boiling point of 10.4C at atmospheric pressure, EtO behaves as a gas
at room temperature. EtO chemically reacts with amino acids, proteins, and DNA to
prevent microbial reproduction. The sterilization process is carried out in a
specialized gas chamber. After sterilization, products are transferred to an aeration
cell, where they remain until the gas disperses and the product is safe to handle.
Ethylene oxide can be used with a wide range of plastics (e.g. petri dishes, pipettes,
syringes, medical devices, etc.) and other materials without affecting their integrity.
Ozone sterilization has been recently approved for use in the U.S. It uses oxygen
that is subjected to an intense electrical field that separates oxygen molecules into
atomic oxygen, which then combines with other oxygen molecules to form ozone.
Ozone is used as a disinfectant for water and food. It is used in both gas and liquid
forms as an antimicrobial agent in the treatment, storage and processing of foods,
including meat, poultry and eggs. Many municipalities use ozone technology to purify
their water and sewage. Ozone is used to disinfect swimming pools, and some
companies selling bottled water use ozonated water to sterilize containers.
Low Temperature Gas Plasma (LTGP) is used as an alternative to ethylene oxide.
It uses a small amount of liquid hydrogen peroxide (H2O2), which is energized with
radio frequency waves into gas plasma. This leads to the generation of free radicals
and other chemical species, which destroy organisms.

Methods
Heat sterilization

Mechanism
Destroys
bacterial
endotoxins

Merits
Most widely used
and reliable method
of
sterilization,
involving
destruction
of
enzymes and other
essential
cell
constituents.

Gaseous sterilization

Alkylation

Penetrating ability of
gases

Radiation sterilization

Ionization
of It
is
a
useful
nucleic acids
method
for
the
industrial
sterilization of heat
sensitive products.

Filtration sterilization

Does
not
destroy
but
removes
the
microorganisms

It is used for both

the clarification and
sterilization
of
liquids and gases as
it is capable of
preventing
the
passage
of
both
viable
and
non
viable particles.

Demerits
Can be applied only to
the
thermostable
products

Applications
Dry heat is applicable for
sterilizing
glassware
and
metal surgical instruments
and moist heat is the most
dependable
method
for
decontamination
of
laboratory waste and the
sterilization
of
laboratory
glassware,
media,
and
reagents.
Gases being alkylating Ethylene oxide gas has been
agents are potentially used widely to process heatmutagenic
and sensitive devices.
carcinogenic
Undesirable
changes Radiation
sterilization
is
occur
in
irradiated generally applied to articles in
products, an example the dry state; including
is
aqueous
solution surgical instruments, sutures,
where
radiolysis
of prostheses,
unit
dose
water occurs.
ointments, plastics
Does not differentiate This method is Sterilizing
between viable and grade filters are used in the
non viable particles
treatment of heat sensitive
injections and ophthalmic
solutions, biological products
and air and other gases for
supply to aseptic areas.

Chemical Methods of Microbial Control:

Disinfection: Any substance or agent that opposes sepsis or inhibits the growth and activity
of microorganism is called antiseptic. It is used on skin or other tissues. Sometimes,
antiseptics are diluted form of disinfectants (applied directly on inanimate objects), but not all
the disinfectants are a concentrated form of an antiseptic.
Factors affecting efficacy of Disinfectants:
The efficacy of disinfection is affected by various factors, including temperature, pH, and
concentration of the substances, time of action nature of organisms and the presence of
organic matter.
Temperature: Temperature is a determinant factor in the action of disinfectants. At high
temperatures, the disinfecting action is faster as long as the decomposition of the disinfectant
does not occur. At low temperatures the biocidal efficacy of most disinfectants decreases.
pH: pH also affects the action of disinfectants. Many disinfectants have an optimum pH
range/level, and product choice should depend on the pH of the diluent (water). For example,
quaternary ammonia is more efficient at alkaline pH while iodine and iodophores are more
efficient at neutral or acid pH.
Concentration: Concentration of the disinfectants may be -static at certain concentrations
and -cidal at higher concentrations
Types of organisms: The initial choice of a chemical disinfectant depends upon the
resistance of the microorganisms of concern. The most susceptible are vegetative bacteria,
fungi and enveloped viruses. Mycobacteria and nonenveloped viruses are less susceptible;
bacterial spores and protozoan cysts are generally the most resistance.
Presence of organic materials: The presence of organic material and greasy substances
may significantly reduce the efficacy of a disinfectant. Therefore, surfaces should be cleaned
thoroughly before applying disinfectants. Special attention should be paid to organic matters
material and greasy substances that can significantly reduce the efficacy of the disinfectant.
Therefore, surfaces should be It is recommended to cleaned thoroughly the surfaces to be
disinfected before applying disinfectants, as their actions can drastically decrease due to the
presence of these elements.

Disinfectant
Category
Sample
Trade Names

Alcohols

Aldehydes

Biguanides:

Halogens:
Hypochlorites
Bleach

Halogens:
Iodine
Betadyne
Providone

Ethyl alcohol
Isopropyl alcohol

Formaldehyde
Glutaraldehyde

Chlorhexidine

Mechanism
of Action

Precipitates
proteins
Denatures lipids

Denatures
proteins
Alkylates
nucleic acids

Advantages

Fast acting
Leaves no
residue

Disadvantages

Phenol &
Phenolics
Chlorocresol,
triclosan,
Hexachlorophane
Denatures
proteins
Alters cell wall
permeability

Alters
membrane
permeability

Denatures
proteins

Denatures
proteins

Broad
spectrum

Broad
spectrum

Broad spectrum
Short contact
time
Inexpensive

Stable in
storage
Relatively
safe

Rapid
evaporation
Flammable

Carcinogenic
Mucous
membranes and
tissue irritation
Only use in
well ventilated
areas

Only
functions in
limited pH
range
(57)

Inactivated
Can cause skin
by QACs
and
Requires
eye irritation
frequent
application
Corrosive
Stains clothes
and treated
surfaces

Vegetative
Bacteria

Effective

Effective

Effective

Inactivated by
sunlight
Requires
frequent
application
Corrodes
metals
Mucous
membrane
and tissue
irritation
Effective

Effective

Effective

Mycobacteria
Enveloped
Viruses
Non-enveloped
Viruses
Spores Variable
Fungi
Effi cacy with
Organic Matter
Effi cacy with
Hard Water
Effi cacy
with Soap/
Detergents

Effective
Effective

Effective
Effective

Variable
Limited

Effective
Effective

Limited
Effective

Variable
Effective

YESGram Positive
LimitedGram
Negative
Variable
Variable

Variable

Effective

Limited

Effective

Limited

Variable

Not Effective

Not Effective
Effective
Reduced

Effective
Effective
Reduced

Not Effective
Limited
?

Variable
Effective
Rapidly reduced

Not Effective
Variable
Effective

Not Effective
Variable
Inactivated

Reduced

Effectiv

Limited
Effective
Rapidly
reduced
?

Effective

Inactivated

Reduced

Inactivated

Inactivated

Effective

Effective

Inactivated

Good effi cacy

with organic
material
Non-corrosive
Stable in storage

Quaternary
Compounds
Bezylkonium
Chloride
Denatures
proteins
Binds
phospholipids
of cell membrane
Stable in storage
Non-irritating to
skin
Effective at high
temperatures and
high pH (9-10)

Evaluating a Disinfectant:
Disinfectants are evaluated for its efficacy. Most commonly used methods for the
evaluation of disinfectants are:
Phenol Coefficient test
The filter paper method
Phenol Coefficient test: Water soluble disinfectants are tested using phenol as a
standard. The concept was originated by Rideal and Walker in 1903 and now being
used as a mean to evaluate water soluble disinfectants having similar nature as that
of phenol. It expresses the disinfection ability of a given substances as compared to
the activity of pure phenol.
Test Organism: Salmonella typhi, Staphylococcus aureus or Bacillus subtilis can also
be used.
A series of dilutions of phenol and the experimental disinfectant are prepared in
sterile tubes.
Viable test bacteria (Staphylococcus aureus or Salmonella typhi )are inoculated into
each tube and incubated at 37 C.
After exposure for 5, 10, and 15 minutes, samples of inoculated tubes are transferred
into fresh culture medium or plated on agar plate that do not have disinfectant and
incubated for 48 hours.
The broth culture tubes are evaluated for growth by turbidity.
The dilutions of the test disinfectant that show growth in 5 min but not in 10 min and
the dilution of phenol having the same effect is to be considered for calculating the
phenol coefficient.
Record the presence (+) or absence (-) of growth of the microorganisms in
subcultures after an interval of 5, 10 and 15 minutes in a tabular form:

Disinfectant

1:10 1:70
1:80
Phenol
1:90

Presence of growth
in subcultures
i.e., nutrient broth
Exposure
time
(min)
5
10
15
+
+

1:100
1:400

Test
+
1:450
Example:Suppose
tets chemical +dilution +
of 1:450 - showed no
growth at 10 minutes
but growth at 5 minutes; and phenol of 1:90
1:500

showed no growth at 10 minutes but growth at 5 minutes. Then the

phenol coefficient of test chemical would be:
=1: 450/1:90 = 5.
Agents with a phenol coefficient > 1 are more effective than
phenol.
The higher the phenol coefficient value, the more effective the test
antimicrobial is in killing bacteria under the test conditions. Note
that phenol coefficient tests are only valid for bactericidal (not
bacteriostatic) disinfectants, since cells inhibited (but not killed) by
bacreriostatic would resume growth when transferred to fresh
medium

Filter Paper Method:

The filter paper method is used in teaching laboratories to evaluate the
efficacy of a chemical agent. A disk of filter paper is soaked with a chemical
and placed on an agar plate that has been previously inoculated with test
organism. After incubation, if the chemical is effective, a clear zone
representing inhibition of growth can be seen around the disk.
Dynamics of disinfection:
Chick's Law: The death of microorganisms is first order with respect to time. Thus,
the remaining number of viable microorganisms in water, N, decreases with time, t,
according to:
Where k is an empirical constant (Chick's constant) descriptive of
microorganism, pH and disinfectant used.
Integrating with respect to time, and replacing limits (N = No at t = 0) yields:
Removing the natural logarithm:
The equation 2 is linear equation; the value of k can be
calculated by plotting the graph between ln (N/No) and time.
The time required to reduce the concentration of viable microrganisms will be;

the

Validation of sterilization methods & equipments

The goal of validation is to demonstrate that a process, when operated within
established limits, produces a product of consistent and specified quality.
Biological indicators:
Biological indicators are characterized and standardized preparations of microorganisms having known stable high resistance to one or more sterilization procedures.
A biological indicator is used to:
1.Assist in the qualification of the physical operation of a sterilizer,
2.Develop and establish a validated sterilization process for a particular article and for
the sterilization of equipment, materials and packaging components for aseptic
processing,
The following factors govern the choice of indicator organisms:
3.The test strain should be stable and non-pathogenic;
4.The resistance of the test strain to the particular sterilization process should be great
compared with the resistance of all species of micro-organisms likely to contaminate
the product, where possible, the ambient flora in the production environment;
5.The recovery of the test strain should be reproducible when cultivated under carefully
standardized conditions.

A biological indicator is characterized by a strain of test organism, the total viable

spore count per carrier, the D-value (Decimal Reduction Value), the Z-value and the
expiry date.
The D-value is a measure of the resistance of a micro-organism to a particular type
of sterilization process. It is the time (in minutes) required the microbial population
by 90%, at a specific temperature. In the case of steam sterilization, the D-value is
expressed by the time in minutes at a defined temperature, e.g. D121, D170 indicate
the temperature of sterilization. In the case of radiation sterilization, the D-value is
expressed by the absorbed dose and subscripts are frequently used to show the log
system used, e.g. D10.
The Z-value relates the heat resistance of a micro-organism to changes in
temperature. The Z-value is the change in temperature required to alter the d value
by a factor of 10.
Steam sterilization (autoclaving)
Spores of Bacillus stearothermophilus or Clostridium sporogenes are recommended
for steam sterilization. The number of viable spores should be more than 105 per
carrier. The D-value at 121 0C for spores of Bacillus stearothermophilus is
approximately 1.5 minuites and for Clostridium sporogenes is approximately is 0.8
minutes but for quantitative work these values should be checked in the fluid in
which the indicator is to be added.

Dry Heat Sterilization

Spores of Bacillus subtilis is recommended for dry heat sterilization. The
number of viable spores should be more than 105 per carrier and the D-value at
1600C should be approximately 5 to 10 minutes.
Gas Sterilization:
The gas generally employed in gaseous sterilization is ethylene oxide of acceptable sterilizing
quality. The process is difficult to control, the gas itself being toxic and, when mixed with
certain proportions with air, explosive. The sterilizing efficiency of ethylene oxide is related to
the concentration of gas, the humidity, the time of exposure, the temperature and the nature
of the load. Knowledge of the performance of the process is routine use should be gained by
monitoring and suitably recording the temperature, relative humidity and the concentration of
ethylene oxide achieved within the load in the sterilizer. The effectiveness of each sterilization
cycle should be demonstrated by the use of suitable biological indicators distributed
throughout the load
Spores of Bacillus subtilis is recommended for gas sterilization. The stock suspension should
contain predominantly non-germinating spores that have been held in a non-nutritive liquid.
Radiation sterilization:
Sterilization of drug substances, dosages forms and medical devices may be achieved by
exposure in the final containers or packages to ionizing radiation in the form of gamma
radiation. For gamma radiation sterilization, an effective sterilizing dose, which is tolerated
without damaging effect, is about 25kGy (2.5 Mrads). If the doses less than 25 kGy are used,
additional microbiological monitoring of the product before irradiation will be necessary. In
order to validate the efficacy, particularly of the lower exposure levels, it is necessary to
determine the magnitude (number and / or degree) of the natural radiation resistance of the
microbial population of the product. Specific product loading patterns must be established and
absorbed minimum and maximum dosages distribution must be determined.
Spores of Bacillus pumilus may be used for a minimum dose of 25kGy. They should contain 10 7
to 108 viable spores per carrier and the D-value should be approximately 3kGy.