Industrial Microbiology
Definition:
Industrial Microbiology is a branch of applied microbiology in which microorganisms are used
for the production of important substances, such as antibiotics, food products, enzymes, amino
acids, vaccines, and fine chemicals.
The main features of industrial microbiology:
● It uses microbial sciences to derive industrial products.
● It involves manipulation of microorganisms to increase yield.
● Introduction to mutagens causes mutations in organisms.
● Real world applications include enzymes, antibiotics, hormones, amino acids.
● It involves processes like fermentation, enzymatic reactions, etc.
Difference between Industrial Microbiology and Microbiology:
Industrial microbiology includes food processing and manufacture of high value products like
drugs, chemicals,fuels, all using microorganisms. While microbiology deals with detailed
information about microorganisms, immunology, virology, etc.
Importance of Industrial Microbiology:
In large-scale industrial processes, microbes are widely used to synthesize a number of products
valuable to human beings. There are numerous industrial products that are derived from
microbes such as:
● Food additives.
● Alcoholic and non-alcoholic beverages.
● Biofuels, metabolites, and biofertilizers.
● Few Chemicals, Enzymes and other Bioactive Molecules.
● Vaccines and other Antibiotics to kill or retard the growth of disease-causing microbes.
STERILIZATION
DEFINITION:
Sterilization is a term referring to any process that eliminates (removes) or kills all
forms of microbial life, including transmissible agents (such as fungi, bacteria, viruses spore
forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound such as
biological culture media.
It is a process of destroying all forms of microbial life. A sterile object in microbiological
sense is free of living micro-organisms. The aim of sterilization is the reduction of initially
present microorganisms or other potential pathogens
INTRODUCTION:
Products to be sterilized include bacteriological contaminated glassware and Petri dishes,
dressings, sutures, ligatures, surgical instruments, etc., as well as certain raw materials and forms
of pharmaceutical dosage. It is considered necessary to sterilize all of these as they could
constitute a potential health hazard to patients.
Sterilization is the process of killing or removing microorganisms. A sterile material is one that
contains no living organisms at all and the term sterile is therefore an absolute one.
However, with all articles to be sterilized there is the chance that the sterilizing treatment
will have a detrimental effect. This is particularly true of pharmaceutical dosage forms where it
is important that the chosen process should not cause changes in the formulation, which would
reduce its therapeutic efficacy or patient acceptability. For this reason, with the design of all
sterilization processes a balance has to be achieved between the maximum acceptable risk of
failing to achieve sterility and the maximum permissible concomitant damage caused to the
treated articles.
APPLICATIONS:
➢ Foods:
One of the first steps toward sterilization is made by Nicolas Appert.
He learned that thorough cooking (applying a suitable amount of heat over a suitable period of
time) slowed the decay of foods and various liquids, preserving them for safe consumption for a
longer time than was typical.
Canning of foods is an extension of the same principle, and has helped to reduce food borne
illness ("food poisoning").
➢ Medicine and surgery
Joseph Lister was a pioneer of antiseptic surgery
In general, surgical instruments and medications that enter an already aseptic part of the body
(such as the bloodstream, or penetrating the skin) must be sterilized to a high sterility assurance
level. Examples of such instruments include scalpels, hypodermic needles and artificial
pacemakers. This is also essential in the manufacture of parenteral pharmaceuticals.
Preparation of injectable medications and intravenous solutions for fluid replacement therapy
requires not only a high sterility assurance level, but also well-designed containers to prevent
entry of adventitious agents after initial product sterilization.
METHODS:
European pharmacopeia recognized Following methods for sterilization of pharmaceutical
products.
Physical methods
• Steam or moist heat sterilization
• Dry heat sterilization
• Ionizing radiation sterilization
Chemical methods
• Gas sterilization
Mechanical methods
• Filtration
1) STEAM OR MOIST HEAT STERILIZATION:
It is recognized as efficient biocidal agents from early stage of bacteriology, when it was
principally developed for sterilization of culture media.
PRINCIPLE OF DESTRUCTION OF MICRO-ORGANISM BY MOIST HEAT:
Bacterial death by moist heat is due to denaturation and coagulation of essential protein
molecules (enzymes) and cell constituents. When heat is applied in the presence of sufficient
water, disulphide bonds and hydrogen bonds between proteins can be broken. New linkages are
formed resulting in denaturation of proteins.
Conditions:
The USP and BP 1988 recommended the following condition:
· Pressure: 15 lb. / square inch
· Temperature: 121 0C
· Time: 15 minutes
Biological indicator:
Spores of Bacillus stearothermophilus
Spores of Clostridium sporogenes.
Temperature-Holding Time Cycle:
The most commonly applied standard Temperature-Holding Time Cycles are:
115-118 0C - 30 mins
121-124 0C - 15 mins
134-138 0C - 03 mins
121 0C for 15 mins cycle is most commonly used. 115 0C for 30 mins cycle is considered
as alternative to this cycle. But now it is no longer considered sufficient to give desired sterility
assurance level for products which may contain significant concentration of spores.
EQUIPMENTS:
Portable sterilizer:
Steam sterilizers or autoclave are stainless steel vessels designed to withstand the steam pressure
employed in steam sterilization. Portable sterilizers are used for small pilot scale or laboratory
scale sterilization and for treatment of instruments and utensils.
A portable or bench autoclave is very similar to domestic pressure cookers which consist of an
upright aluminium or stainless-steel vessels with capacity of 15 liters. Steam for sterilization can
be generated within the sterilizer.
Large scale sterilizers:
These are used for routine hospital and industrial uses. These are cylindrical or rectangular
chambers with the capacity of 400-800 liters either a single door or doors at both ends. Steam
can be provided from a separate boiler. In large sterilizer steam jackets surrounding the chamber
is used to heat chamber. The same jacket can also be filled with water at the end of the cycle to
facilitate cooling and thus reduce the overall cycle time. During sterilization the doors are held
closed by a locking mechanism which prevents opening when the chamber is under pressure and
until the chamber has cooled to a pre-set temperature, typically 80°C.
STAGES OF OPERATION OF STERILIZER :
1. Air removal and steam addition:
i. Air is removed by downward displacement of steam. Heavier cool air is forced
out from discharge chamber by incoming hot steam.
ii. By application of vacuum pump.
iii. Combinations of both methods.
2. Heating and sterilization stage:
When sterilizer reaches its operating temperature and pressure, sterilization stage
begins. The duration of exposure may include heating up time and holding time.
3. Drying stage:
Surgical dressings or porous load may become damp during sterilization process. It must
be dried before removal from chamber which is achieved by steam exhaust by application of
vacuum and is often assisted by heat from steam filled jacket. After drying, atmospheric pressure
is restored by administration of sterilized filter air.
4. Cooling stage:
For bottle fluids final stage of sterilization process is cooling which is achieved by
circulating water through jacket that surrounds the chamber.
APPLICATION OF AUTOCLAVE :
It is used to sterilize anything, which is not injured by steam and high temperature of
sterilization. These includes:
1. Aqueous parenteral solutions e.g. distilled water, saline solutions.
2. Aqueous liquid media e.g. liquid media with or without carbohydrate and gelatin.
3. Surgical dressings and fabrics.
4. Plastic and rubber closures.
5. Metal instruments.
6. Glass apparatus and containers.
7. Ophthalmic preparations (eye drops, ointments).
8. Irrigation fluids (fluids that are used to wash body cavities).
2) DRY HEAT STERILIZATION:
Principle
In dry-heat processes, the primary lethal process is considered to be oxidation of cell
constituents.
Introduction
Dry-heat sterilization requires a higher temperature than moist heat and a longer exposure time in
the range 160–180°C and requires exposure times of up to 2hours depending upon the
temperature employed. The method is, therefore, more convenient for heat-stable, non-aqueous
materials that cannot be sterilized by steam because of its deleterious effects or failure to
penetrate. Such materials include glassware, powders, oils, and some oil-based injectables.
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 for the
time and at the temperature given in the table below. Other conditions may be necessary for
different preparations to ensure the effective elimination of all undesirable microorganisms.
Temperature Minimum sterilization time
(°C) (min)
160 180
170 60
180 30
Specific conditions of temperature and time for certain preparations are stated in individual
monographs.
Sterilizer design
Dry heat sterilization is usually carried out in a hot air oven which comprises an insulated
polished stainless-steel chamber, with a usual capacity of up to 250 litres, surrounded by an outer
case containing electric heaters located in positions to prevent cool spots developing inside the
chamber. A fan is fitted to the rear of the oven to provide circulating air, thus ensuring more
rapid equilibration of temperature. Shelves within the chamber are perforated to allow good
airflow. Thermocouples can be used to monitor the temperature of both the oven air and articles
contained within it.
Sterilizer operation
Articles to be sterilized must be wrapped or enclosed in containers of sufficient strength and
integrity to provide good post-sterilization protection against contamination. Suitable materials
are paper, cardboard tubes or aluminium containers. Container shape and design must be such
that heat penetration is encouraged in order to shorten the heating-up stage. In a hot-air oven,
heat is delivered to articles principally by radiation and convection; thus, they must be carefully
arranged within the chamber to avoid obscuring centrally placed articles from wall radiation or
impending air flow. Heating-up times, which may be as long as 4 hours for articles with poor
heat-conducting properties, can be reduced by preheating the oven before loading. Following
sterilization, the chamber temperature is usually allowed to fall to around 40°C before removal
of sterilized articles; this can be accelerated by the use of forced cooling with filtered air.
The bioindicator strain proposed for validation of the sterilization process is spores of Bacillus
subtilis for which the D-value is 5-10 minutes at 160 °C using about 106 spores per indicator.
OTHER METHODS OF DRY HEAT STERILIZATION:
Red heat: Articles such as bacteriological loops, straight wires, tips of forceps and searing
spatulas are sterilized by holding them in Bunsen flame till they become red hot. This is a simple
method for effective sterilization of such articles, but is limited to those articles that can be
heated to redness in flame
Flaming: This is a method of passing the article over a Bunsen flame, but not heating it to
redness. Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips are
passed through the flame a few times. Even though most vegetative cells are killed, there is no
guarantee that spores too would die on such short exposure.
This method too is limited to those articles that can be exposed to flame. Cracking of the
glassware may occur.
Incineration: Using a direct flame can incinerate microbes very rapidly. For example: The flame
of the Bunsen burner is employed for a few seconds to sterilize the bacteriological loop before
removing a sample from a culture tube. Disposable hospital gowns and certain plastic apparatus
are examples of materials that may be incinerated. In past centuries, the bodies of disease victims
were burned to prevent spread of the plague. It still is common practice to incinerate the
carcasses of cattle that have died of anthrax and to put the contaminated field to the torch
because anthrax spores cannot be destroyed adequately by other means.
Pasteurization: This process was originally employed by Louis Pasteur. Currently this
procedure is employed in food and dairy industry. There are two methods of pasteurization, the
holder method (heated at 63oC for 30 minutes) and flash method (heated at 72oC for 15 seconds)
followed by quickly cooling to 13oC. Other pasteurization methods include Ultra-High
temperature (UHT), 140oC for 15 sec and 149oC for 0.5 sec. This method is suitable to destroy
most milk borne pathogens like Salmonella, Mycobacterium, Streptococci, Staphylococci and
Brucella.
3) 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
• UV light
Principle
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
a) Gamma-rays
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.
Gamma-ray sterilizers
Sterilizer design and operation
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 on disintegration emits radiation at two
energy levels of 1.33 and 1.17MeV. The isotope is held as pellets packed in metal rods, each rod
carefully arranged within the source and containing up to 20kCi of activity; these rods are
replaced or re-arranged as the activity of the source either drops or becomes unevenly
distributed. Articles being sterilized are passed through the irradiation chamber on a conveyor
belt.
b) 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
wall or ceiling-mounted for air disinfection or fixed to vessels for water treatment.
Precautions
Operators present in an irradiated room should wear appropriate protective clothing and eye
shields because of eye and skin damaging effects of these radiations.
The UV lamp is kept on for 20 minutes prior to commencement of lab. work.
Applications.
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
• For the treatment of manufacturing-grade water (aqueous oral preparations)
c) Accelerated Electrons
Concentrated beam of high energy electrons about 7Mev are produced by electrostatic
accelerators or by microwave linear accelerators. These electrons have low penetration power
than gamma rays, so electron irradiation is confined to sterilization of small items such as
individual dressing and sutures spread in a thin layer.
4) GASEOUS STERILIZATION
The chemically reactive gases ethylene oxide (CH2)2O and formaldehyde possess broad-
spectrum biocidal activity, and have found applications
• In the sterilization of re-usable surgical instruments,
• Certain medical, diagnostic and electrical equipment,
• The surface sterilization of powders.
Sterilization processes using ethylene oxide sterilization are far more commonly used on an
international basis than those employing formaldehyde.
Principle of destruction of microorganism by gas sterilization
The mechanism of antimicrobial action of the two gases is assumed to be through alkylation of
sulfhydryl, amino, hydroxyl and carboxyl groups on proteins and imino groups of nucleic acids.
As alkylating agents, both gases are potentially mutagenic and carcinogenic; they also produce
symptoms of acute toxicity including irritation of the skin, conjunctiva, and nasal mucosa.
Consequently, strict control of their atmospheric concentrations is necessary and safe working
protocols are required to protect personnel.
a) Ethylene oxide
Ethylene oxide gas is highly explosive in order to reduce this explosion hazard it is usually
supplied for sterilization purposes as a 10% mix with carbon dioxide, or as an 8.6% mixture with
HFC 124 (2-chloro-1,1,1,2 tetrafluoroethane), which has replaced fluorinated hydrocarbons
(freons). Alternatively, pure ethylene oxide gas can be used below atmospheric pressure in
sterilizer chambers from which all air has been removed.
Organisms are more resistant to ethylene oxide treatment in a dried state, as are those protected
from the gas by inclusion in crystalline or dried organic deposits. Thus, a further condition to be
satisfied in ethylene oxide sterilization is attainment of a minimum level of moisture in the
immediate product environment. This requires a sterilizer humidity of 30–70% and frequently a
preconditioning of the load at relative humidities of >50%.
Sterilizer design and operation
An ethylene oxide sterilizer consists of a leak-proof and explosion-proof steel chamber, normally
of 100–300-litre capacity, which can be surrounded by a hot-water jacket to provide a uniform
chamber Temperature.
Successful operation of the sterilizer requires
• removal of air from the chamber by evacuation
• humidification
• Conditioning of the load by passage of sub-atmospheric pressure steam followed by a
further evacuation period
• The admission of preheated vaporized ethylene oxide from external pressurized canisters
or single-charge cartridges.
• Forced gas circulation is often employed to minimize variations in conditions throughout
the sterilizer chamber.
• After treatment, the gases are evacuated either directly to the outside atmosphere or
through a special exhaust system
b) Formaldehyde
Formaldehyde gas for use in sterilization is produced by heating formalin (37% w/v aqueous
solution of formaldehyde) to a temperature of 70–75°C with steam. Heating of formaldehyde
produces its fumes which serve the purpose of sterilization.
Disadvantages
• Formaldehyde has a similar toxicity to ethylene oxide
• A major disadvantage of formaldehyde is low penetrating power. So, can only be used for
surface sterilization.
• Irritant and Pungent
5) FILTRATION STERILIZATION (Mechanical method)
It is unique in sense. It removes rather than destroy microorganism. It prevents passage of both,
viable (living) or non-viable (non-living) microorganisms. Sterilization by passage through a
bacteria proof filter is used for thermolabile solutions and gases including air. It is used for
clarification and sterilization of liquids and gases.
Membrane filters with pore sizes between 0.2-0.45 μm are commonly used to remove particles
from solutions that can't be autoclaved
The process of sterilization consists of three main steps: -
1-Passage of Solution: - Passage of solution to be sterilized through a previously sterilized filter
unit.
2-Aseptic transference of filtrate: - Aseptic transference of filtrate to sterile container is carried
out which are then sealed aseptically.
3-Test for sterility: Test on sterility is carried out on filtered product.
Advantages of Filtration Sterilization:
The advantages of filtration sterilization are: -
• It is ideal for thermolabile substances.
• It removes all bacteria, fungi and often clarifies solutions.
• It is useful for sterilization for large volume solutions.
• It is useful for eye drops.
Disadvantages of Filtration Sterilization:
The disadvantages of filtration sterilization are: -
• As it is aseptic technique. So highly trained staff and trained technicians are
needed.
• Viruses and certain bacterial products like toxins and pyrogens are not removed.
• Adsorption can occur with some filters.
• Some filters shed fibers.
• It cannot sterilize suspensions.
Types of Filtration Units:
There are two types of filtration units: -
• Positive Pressure Unit:
In this case, solution is forced to filter by compressed air.
• Negative Pressure Unit:
In this case, solution is sucked through filter. It is also known as vacuum type unit
Applications of Filtration Sterilization in Medical and Pharmaceutical Fields:
Filtration sterilization has following applications: -
• Treatment of heat sensitive injections and ophthalmic solutions.
• Certain biological products, air and other gases for supply to aseptic area.
• These filters are part of fermenter, centrifuge, autoclave and freeze drier
(lyophilizer)
STERILITY TESTING
“A test which accesses whether a sterilized medical or pharmaceutical product is free from
contaminated microorganisms”
There are three methods which are commonly used: -
1-Direct Inoculation Method: -
In this method, test sample is directly introduced into nutrient media. European Pharmacopeia
recommended two media:
The first one is Fluid Mercaptoacetate Media or Fluid Thioglycolate Media suitable for
growth of anaerobes and incubation temperature is 30-35 degree Celsius.
The second one is Soya bean Casein Digest Media. It supports the growth of both aerobes
and fungi. In case of bacteria, media incubation temperature is 30-35 degree Celsius and for
fungi is 20-25 degree Celsius.
2-Membrane Filtration Method: -
This method is recommended by most pharmacopeias. It involved filtration of fluid through a
sterile membrane filter with a pore size of 0.4um. The microorganisms present will be retained
on membrane filter. Filter is then divided aseptically, and portions are transferred to suitable
culture media and are then incubated.
3-Sensitive Method: -
A sensitive method is used for detecting low level of contamination. In this method, culture
media is transferred to fluid in its original container. So, sampling of entire volume is obtained.
Validation and in-process monitoring of sterilization procedures:
There are several definitions of ‘validation’ but, in simple terms, the word means demonstrating
that a process will consistently produce the results that it is intended to. Thus, with respect to
sterile products, validation would be necessary for each of the individual aspects of the
manufacturing process, e.g. environmental monitoring, raw materials quality assessment, the
sterilization process itself and the sterility testing procedure. Of these, it is the sterilization
process that is likely to be subject to the most detailed and complex validation procedures, and
these will be used to exemplify the factors to be considered. A typical validation procedure for a
steam sterilization process is likely to incorporate most, or all, of the following features:
• The calibration and testing of all the physical instruments used to monitor the process, e.g.,
thermocouples, pressure gauges and timers.
• Production of evidence that the steam is of the desired quality (e.g., that the chamber
temperature is that expected for pure steam at the measured pressure).
• The conduct of leak tests and steam penetration tests using both an empty chamber and a
chamber filled with the product to be sterilized in the intended load conformation.
• The use of biological indicators either alone or in combination with bioburden organisms to
demonstrate that the sterilization cycle is capable of producing an acceptable level of sterility
assurance under ‘worst case’ conditions.
• The production of data to demonstrate repeatability of the above (typically for three runs).
• Comprehensive documentation of all of these aspects
INDICATORS FOR STERILIZATION ASSURANCES:
There are three types of indicators for sterilization assurances;
Physical indicators
In heat sterilization processes, a temperature record chart is made of each sterilization cycle with
both dry and moist heat (i.e., autoclave) sterilizers; this chart forms part of the batch
documentation and is compared against a master temperature record (MTR). It is recommended
that the temperature be taken at the coolest part of the loaded sterilizer. Further information on
heat distribution and penetration within a sterilizer can be gained by the use of thermocouples
placed at selected sites in the chamber or inserted directly into test packs or bottles. For gaseous
sterilization procedures, elevated temperatures are monitored for each sterilization cycle by
temperature probes, and routine leak tests are performed to ensure gas-tight seals. Pressure and
humidity measurements are recorded. In radiation sterilization, a plastic dosimeter which
gradually darkens in proportion to the radiation absorbed gives an accurate measure of the
radiation dose and is considered to be the best technique currently available for following the
radio sterilization process.
Sterilizing filters are subject to a bubble point pressure test, which is a technique employed for
determining the pore size of filters, and may also be used to check the integrity of certain types
of filter device (membrane and sintered glass) immediately after use. The principle of the test is
that the wetted filter, in its assembled unit, is subjected to an increasing air or nitrogen gas
pressure differential. The pressure difference recorded when the first bubble of gas breaks away
from the filter is related to the maximum pore size. When the gas pressure is further increased
slowly, there is a general eruption of bubbles over the entire surface. The pressure difference
here is related to the mean pore size. A pressure differential below the expected value would
signify a damaged or faulty filter.
Chemical indicators
Chemical monitoring of a sterilization process is based on the ability of heat, steam, sterilant
gases and ionizing radiation to alter the chemical and/or physical characteristics of a variety of
chemical substances. Ideally, this change should take place only when satisfactory conditions for
sterilization prevail, thus confirming that the sterilization cycle has been successfully completed.
Chemical indicators generally undergo melting or color changes the relationship of this change
to the sterilization process is being influenced by the design of the test device. It must be
remembered, however, that the changes recorded do not necessarily correspond to
microbiological sterility and consequently the devices should never be employed as sole
indicators in a sterilization process. Nevertheless, when included in strategically placed
containers or packages, chemical indicators are valuable monitors of the conditions prevailing at
the coolest or most inaccessible parts of a sterilizer.
Biological indicators
Biological indicators (BIs) for use in thermal, chemical or radiation sterilization processes
consist of standardized bacterial spore preparations which are usually in the form either of
suspensions in water or culture medium or of spores dried on paper, aluminum or plastic carriers.
As with chemical indicators, they are usually placed in dummy packs located at strategic sites in
the sterilizer.
After the sterilization process, the aqueous suspensions or spores on carriers are aseptically
transferred to an appropriate nutrient medium, which is then incubated and periodically
examined for signs of growth. Spores of Bacillus stearothermophilus in sealed ampoules of
culture medium are used for steam sterilization monitoring, and these may be incubated directly
at 55°C; this eliminates the need for an aseptic transfer. The bacterial species to be used in a BI
must be selected carefully, as it must be non-pathogenic and should possess above-average
resistance to the particular sterilization process.
While certainly offering the most direct method of monitoring sterilization processes, it should
be realized that BIs may be less reliable monitors than physical methods and are not
recommended for routine use, except in the case of gaseous sterilization.
Definitions.
Thermal death time is the minimum time required to kill a suspension of organisms at a
predetermined temperature in a specified environment
Expressions of resistance
D-value
The resistance of an organism to a sterilizing agent can be described by means of the D-value.
For heat and radiation treatments, respectively, this is defined as the time taken at a fixed
temperature or the radiation dose required to achieve a 90% reduction in viable cells.
Z-value
For heat treatment, a D-value only refers to the resistance of a microorganism at a particular
temperature. In order to assess the influence of temperature changes on thermal resistance a
relationship between temperature and log D-value can be developed, leading to the expression of
a z-value, which represents the increase in temperature needed to reduce the D-value of an
organism by 90%.
