Vous êtes sur la page 1sur 10

D-value (microbiology)

From Wikipedia, the free encyclopedia

In microbiology, D-value refers to decimal reduction time (or decimal reduction dose) and is
the time (or dose) required at a given condition (e.g. temperature), or set of conditions, to kill
90% (or 1 log) of the exposed microorganisms. The term originated from assessing microbial
thermal resistance and thermal death time analysis; however, it has analogous uses in other
microbial resistance and death rate applications, such as (but not limited to) those of ethylene
oxide and radiation processing.

Thus after a colony is reduced by 1 D, only 10% of the original organisms remain, i.e., the
population number has been reduced by one decimal place in the counting scheme. Generally,
each lot of a sterilization-resistant organism is given a unique D-value. When referring to D-
values, for the purpose of thermal analysis, it is proper to give the temperature as a subscript of
the "D". For example, given a hypothetical organism which is reduced by 90% after exposure to
temperatures of 150 C for 20 minutes, the D-value would be written as D150C = 20 minutes. If
generally describing D-value for any temperature, a common abbreviation is DT (where T is
temperature) until a value for T is relevant to express, specifically. Another more general
abbreviated expression of D-value is D10 (as to denote 10% reduction).

D-value determination is often carried out to measure a disinfectant's efficiency to reduce the
number of microbes present in a given environment.[1]

Determining D-Value: The Importance of Standardized Measurements

Posted on: 10 January 2014

By: REVOX Steriliza...

Many factors affect sterilization; concentration of the sterilizing agent, temperature, pH, relative
humidity, cleanliness of product, whether organic or inorganic matter, innate resistance &
population of microorganisms, plus duration of exposure or time.

With so many variables at hand, there is a need to define an industry-wide accepted methodology
that characterize sterilization in terms of correlation between duration that achieves sterilization
under a standardized set of conditions.

Such duration may be addressed by calculating a D-value decimal reduction time, which is the
time required (usually in minutes) to kill 90% of the organisms being tested. Sterility is
controlled by allowing for an SAL (sterility assurance level) usually at 10-3 or 10-6; this means a
one in a thousand and one in a million chance respectively that a device would not be organism
free. In order to prove this, one must show at least a 3 or 6 log reduction.
Most biological indicators (BIs) have a population of 106, or six logs, of colony forming units
(CFU) that need to be completely killed in a half cycle for the sterilization validations success
when dealing with medical devices.

In the overkill method, an assumption is made that some level of microorganisms (bioburden) is
present on the device before sterilization. Therefore, a 12 spore log reduction (SLR) must be
shown for safety and obtaining the needed SAL. One method of determining a D-value is the
Stumbo/Murphy/Cochran that uses the formula D = U/ log No log Nu ; where D is the D-value,
U = exposure time, No = population of BIs used, Nu = ln (n/r) where n = total number of BIs
used and r = number of negative BIs after exposure time. For the most accurate results, use 20
BIs in the run and choose a time point where approximately half your BIs are negative.

This cycle is known as a fractional or sub lethal cycle and will be much shorter than the half
cycle, it will be an estimate for the cycle duration but results are acceptable if one has either 20%
survival to 20% kill. Total kill or no kill renders this formula unusable.

Once a D-value is calculated, an SAL can be determined for the device with the understanding
that spore kill is log linear. A two minute D-value means a 12 SLR in 24 minutes of exposure.
This level provides safety for bioburden on devices while still covering the SAL of 10-6.

The use of D-value is widely accepted in the field of industrial microbiology, which includes the
food industry, medical device and pharmaceutical industries. In the case of industrial
sterilization, as product characteristics are developed during cycle development, it is imperative
to understand the D-value of the product sterilization cycle, so one may know the effectiveness
of such cycle when compared with other sterilization methods.

Kinetics of microrganism growth and inactivation

Thermal inactivation

The decrease of a microbial population, under thermal processing, also follows a first order
kinetics and all the relationships deduced for chemical kinetics can be applied. However, in
dealing with microrganisms different parameters are used and here we will describe the
relationship existing between the parameters used in chemical kinetics and those used in thermal
processing of foods.

A first order kinetics is described by the following equations:

c kt kt co = the initial concentration


log ---- = - ------ log c - log co = - ----
co 2.3 2.3 c = concentration at time t

t = time
k = kinetic constant.

In chemical kinetics, a reaction is generally characterized by its half-life, i.e. the time needed to
halve the initial concentration. In other words the time at which c = co/2. By substituting c with
co/2 and rearranging, we get an expression for half-life (t1/2):

kt1/2 kt1/2
log (co/2) - log co = - ------ log co - log 2 - log co = - -------
2.3 2.3

kt1/2 log 2 (2.3) 0.693


- log 2 = - -------- t1/2 = ------------ = --------
2.3 k k
From this equation, it should be evident that the time to halve the initial concentration (t1/2) is
independent from the initial concentration.

Decimal reduction time

In thermal processing, a different parameter is used to characterize the rate of reduction of a


microbial population. This is the decimal reduction time (generally indicated as D) which is
defined as the time necessary to destroy 90% of the initial population.

Suppose we have 1000 microrganisms, after a 90% reduction 100 microrganisms will survive
and thus the initial population is reduced by a factor of 10 (100 = 1000/10). By indicating with
D the time necessary for 90% reduction and substituting c with co/10 in the first order equation,
we can obtain an expression for D:

kD kD
log (co/10) - log co = - ------ log co - log 10 - log co = - ------
2.3 2.3

kD 2.3
- log 10 = -1 = - ------ D = ------
2.3 k
It is worth to note that D as t1/2 is independent from the initial concentration.

The equation - log (c/co) = kt/2.3 (first order kinetic) represents a straight line with slope = k/2.3:

Remembering that D = 2.3/k, i.e., the inverse of the slope, we can easily determine the decimal
reduction time. From the graph we have that
slope = (y/x) = 0.5/2.1

D = x/y = 2.1/0.5 = 4.2 seconds

Normally D values are given with a subscript indicating the temperature at which it refers, as an
example D60 indicates the decimal reduction time at 60 C.

As seen above D = x/y and thus D = t / (log co - log c). In other words, D can be defined as
the time needed to have a unit variation of log co - log c. A unit variation on a decimal logarithm
graph is referred to as a one log cycle, thus D is the time corresponding to one log cycle. As
shown above, for a 90% reduction it results that

log co - log 10 - log co = - log 10 = -1.

Therefore one log cycle corresponds to 90% reduction of microbial population. It is worth noting
that if we take y = 1 (one log cycle) we can directly read, from the graph, the value of D. As an
example, in going from 6 to 5 (one log cycle) we can read a value of D of 4.2 seconds.

To Assess or Determine? Unraveling the D-


Value
Posted by mfontanazza on March 4, 2010
Understanding the difference between assessing and determining
a D-value is important when testing biological indicators.
Share

There is a misconception that U.S. Pharmacopeia (USP) requires


the end-users of biological indicators (BIs), except under specific
conditions, to perform a D-value determination on incoming lots
of BIs prior to acceptance and use. Assessing the D-value is much
easier and much less costly than determining it. This article
clarifies the difference between assessing and determining the D-
value, and discusses what USP requires to perform these
evaluations. For either practice, it is essential to be aware of the
associated pitfalls.

BIs and the D-Value


Each year, numerous end-users send incoming BIs out to third-
party labs for assessment or verification of the D-value or a
population assay of the BIs prior to acceptance for use. A D-value
is the amount of time (or dose) required to reduce the population
of a BI by 90% or one log of the population under specific Certified BIER vessels must
document the temperature, time, and
pressure during cycle operation.
exposure conditions. For example, when using a steam autoclave, a BI with a D-value of 1.7
minutes would mean that when exposing the BI to a temperature of 121C for 1.7 minutes, the
population of the BI would be reduced by one log or 90%. This is an important BI characteristic
for the end-user to be aware of during cycle validation or cycle development work.

To test a sterilization cycles effectiveness, BIs may be routinely used to monitor the cycle by being
placed into the autoclave chamber along with the items to be sterilized. After the cycles
completion, the BIs are removed and processed. With a successful delivery of a sterilization cycle,
BIs placed into the chamber should also be killed or sterilized. This is a very important way to
check cycle lethality delivered. If the BIs show growth after a cycles completion, this is an
indication that a problem existed with the cycle and that all pertinent cycle parameters were not
met. It also means that the sterilized or processed load needs to be held and an investigation
conducted before the release of any goods from that particular cycle. As a result, end-users must
have accurate D-value information for the BIs that are being used to prevent false positives.
If one is using a certified log 6 BI that has a D-value of 2 minutes at 121C, then it should die in a
sterilization cycle as soon as the temperature has reached 121C for an exposure time of two
minutes for each log of population or approximately 12+ minutes (2-minute D-value 6 logs of
population = 12 minutes). Thus, to check the accuracy of the BI manufacturers certified D-value
of a given BI, manufacturers may look to the USP for guidance. This is where some
misconceptions originate.

Assessing the D-Value


USP 31, Users Responsibility, states:

The user should establish in-house acceptance standards for biological indicator lots and consider
rejection in the event the biological indicator lot does not meet the established in-house
performance standards. A certificate of performance should be obtained for each lot of indicators,
and the user should routinely perform audits of the manufacturers facilities and procedures.1

USP is recommending that end-users establish in-house acceptance criteria for BI performance.
This may pertain to a users acceptable population or D-value range for the cycles they use. In
doing so, a company may set a performance standard that states it will not accept a BI for use
unless the certificate of analysis shows the D-value to be within a range of 1.52.0 minutes, for
example, and a population between 1.0 and 3.0 of the desired log. It is further recommended that
the end-user obtain a certificate of performance for each lot of BIs obtained, and routinely perform
audits of the BI manufacturers site and the procedures that are used.

USP 31 also states:

Upon initial receipt of the biological indicator from a commercial


supplier, the user should verify the purity and morphology of the
purchased biological indicator organisms. Verification of at least
the proper genus is desirable. Also, a microbial count to determine
the mean count per biological indicator unit should be conducted.
The manufacturers comments relative to D-value range, storage
conditions, expiration dating, and stability of the biological

Step 1. A group of BIs ready to be


placed into a BIER unit.
indicator should be observed and noted. The user may consider conducting a D-value assessment
before acceptance of the lot.

After reading these USP excerpts, many facilities have instituted in-house acceptance criteria for
incoming BIs prior to allowing them to be used. As per USP 31, a microbial count should be
conducted, and a large number of end-users are performing population counts in-house as a result. If
they cannot perform the population assays themselves, they send them out for third-party population
verification.

Test Methods
Where D-value is concerned, most facilities do not have the proper equipment to do a D-value
assessment, so they send BIs off to a third party for testing. As USP states in the above excerpt,
The user may consider conducting a D-value assessment before acceptance of the lot. One should
note that may consider is not a must or should consider. If one chooses to consider a D-value
assessment or the facility protocol requires a D-value assessment prior to use, then a qualified third-
party testing lab is usually needed. When the test BIs are sent off to the third-party testing lab, the
end-user should request a D-value assessment.
This is not the same as a D-value determination. If requesting such, the same D-value testing
methods used initially by the BI manufacturer to determine and certify the D-value for that
particular lot of BIs must be used. D-value determination requires that two methods be used. Also,
the ISO 11138 series of documents pertain to BI manufacturers for initial resistance testing. ISO
allows the use of any two of the following methods:

Fraction negative method.

Survivor curve method.

Survive/kill method.

In a fraction negative method such as the Spearman/Karber


method, various partial or sublethal exposure times are run in a BI
evaluator resistometer (BIER) vessel with several groups of BIs
from the same lot. This ensures that one exposure results in killing
all of the BIs exposed and another exposure keeps alive all of the
BIs exposed. Several exposures that run in between these two
exposure times will result in exposures where a fraction of the BIs
are positive for growth and a fraction are negative for growth, and
is thus called a fraction negative method.

In a survivor curve method, various groups of BIs are run in a


BIER vessel to sublethal exposure times such as 1.5- or 2-minute Step 2. A group of BIs are
intervals between exposure times. The BIs from each exposure are placed into the exposure
chamber of a BIER unit.
processed and quantified for remaining viable organisms. This is
done as per USP, and the serial dilutions are plated out on trypticase soy agar and incubated.2 Upon
incubation, the colony-forming units on each plate are counted, and the resulting surviving
population can be enumerated. As a result, the amount of exposure time to reduce the population
by one log can be determined.

In the survive/kill method as described in USP, the USP-calculated survive exposure time and kill
time are used.3 Two groups of 20 BIs each are exposed to the survive and kill times in a BIER
vessel. All BIs in the survive time must survive and show growth, and all BIs within the kill time
group must show no growth for the requirements of the test to be met and thus verify reliability of
the D-value being tested.

The methods initially used by the BI manufacturer should be stated on the certificate of analysis
provided by the manufacturer with the BI lot. It is important to remember that in a D-value
assessment or verification test, you are getting an assessment from the third-party lab, which
isnt the same as a determination. To obtain a D-value assessment, one could use the USP
survive/kill resistance performance test for D-value verification. The third-party testing lab
would only need to run the two USP-calculated cycles (one for survive time and one for kill
time) based on the certified D-value of the BI in question. If the requirements of the test are met,
the D-value has been assessed and verified, and the BI can be used.

Even if a manufacturer is requesting verification with use of a BIER vessel from a third-party lab,
it is strongly recommended to audit the third-party facility first to ensure that it is following ISO
and USP testing procedures. A very important question to ask during the audit would be whether
the third-party facility that is using a BIER vessel is ISO/AAMI compliant. In preparation for the
audit, the auditor needs to become familiar with ANSI/AAMI/ISO requirements for BIER vessel
performance.4 The survive/kill test seems like a very straightforward method to perform, but the
use of a BIER vessel is still critical.

To accomplish successful and accurate testing of a BI with regard to resistance, exposure intervals
must be as accurate in duration as possible. A 15-minute exposure time needs to be as close to 15
minutes (plus or minus 610 seconds) as possible. It cannot be the usual 25-minute come-up time,
along with a typical 2-minute comedown time at cycle end. If a BIER vessel is used, a 15-minute
cycle will typically involve a total time of around 15 minutes and 12 seconds. A BIER vessels
chamber is very small and responds very quickly to temperature increases. It can maintain a target
temperature accurately, within 0.5C of the target temperature throughout the entire exposure
phase. As with the Spearman/Karber method, a 4.5-minute exposure must be 4.5 minutes. Thus,
when auditing a third-party testing lab prior to contracting work, the auditor should review a few
BIER vessel run printouts and note whether the test equipment performance requirements are being
met. Does the chamber reach target temperature within 10 seconds or less? Is the target
temperature stable, and does it remain within 0.5C for the entire exposure cycle? Are the BIs
removed quickly from the unit following a fast comedown phase? Is the vessel itself capable of
recording time, temperature, and pressure as specified within such regulatory standards as
ANSI/AAMI/ISO 18472:2006?

To be a certified or compliant BIER vessel, the unit must independently document the temperature,
time, and pressure occurring during the cycle operation. Some units claim to be ISO and AAMI
compliant, yet they need to use several outside measuring devices to record and document the
events that are occurring within the BIER vessel chamber. This does not meet regulatory
requirements. Needing a separate instrument other than the actual BIER vessel to record the
temperature or pressure will add variables to an already delicate and precise testing process.
Knowing what D-value test methods are allowed by ISO or USP, the methods specific steps, and
having the equipment performance documentation should all be part of prequalification process
in selecting a lab or facility to conduct third-party testing.

If a testing facility is following ISO or USP procedure, obtaining


the protocols to review prior to testing should not be an issue,
because the procedures are not proprietary. If one is paying for a
standard service, it should be privy to the procedure to be used
prior to contracting the service.

If a lab follows USP or ISO guidance on D-value testing without


variation and uses compliant equipment and suitable recovery
media, D-value assessment or determination by a third-party
testing lab can be successful and repeatable. When selecting a
third-party lab to use for testing results, a quality assurance
department can work closely with the selected lab to ensure that
Step 3. Following the incubation of
test BIs, six groups of 10 ampoules
all testing parameters are followed and that the third-party testing
each are exposed to different labs testing methods and media are the same as those used by the
exposure times. The yellow color BI manufacturer during initial D-value testing. BIER vessels
indicates growth. should be checked for current calibrations and to verify that they
are operating properly.

In Table I, 15 different lots of both ampoule and spore strip BIs were sent out to a third-party lab
for D-value testing. Listed is the initial certified D-value found for the BI by the BI manufacturer
and the third-party labs determined D-value for the lot. In most cases, the Spearman/Karber
fraction negative method was initially used by the third-party lab. Once a D-value was
determined by fraction negative method, the USP survive/kill method was run.

If verification results pass USP requirements, the D-value can be used as certified on the
certificate of analysis and should be accepted as a lot of BIs to be used. The certified D-value has
been assessed, substantiated, supported, or confirmed. However, it cannot replace the
manufacturers determined, certified D-value. Unfortunately, this is happening in far too many
cases. When this happens, the end-user uses the third-party testing labs result for D-value (or
population) as the new certified D-value. Or, if the testing doesnt pass requirements, the BIs are
classified as noncompliant with performance requirements.

Unless two methods have been used and are the same as those used by the manufacturer, the testing
result is not an ISO-compliant determination result. Such relabeling cannot be done. A D-value
testing assessment cannot replace or be used to relabel the certified D-value determined by the
manufacturer. To initially determine and certify the manufacturers stated D-value, methods allowed
by ISO and USP were used. As stated earlier in ISO 11138 series, to obtain a label claim or certify a
determined D-value, one must use two of the three methods outlined in ISO and USP.3,5 A third-
party assessment is not a determination. In almost all cases, only one method was used and may not
in any way be used to relabel a BI resistance characteristic that
was certified by the manufacturer.

Population Verification
When sending out a BI for a population assay, a similar situation
exists. For the BI to pass the population verification set out in
USP, the population result needs to be within +300% and 50%
of the labeled population. If the population being verified falls
within this range, the requirements of the test are met, and the
populationhas been verified. However, the verified population is
Table I. The initial and the third-
not the new certified population to be used in further studies or party D-values are all within an
validations done with this lot of BIs. In most situations, BI acceptable +/-20% range of
manufacturers are much more familiar, have validated, and have difference.
extensive experience in performing population assays on their
particular BIs. Having a third-party labs assay result replace a manufacturers certified
population, even if only slightly different than the certificate of analysis listed population, makes
little sense.

The bottom line is that third-party verifications are not intended to replace a manufacturers
certified BI characteristic such as
D-value or population. Some testing labs are performing recertifications and some end-users are
using the third-party verification to replace the certificate of analysis-labeled values. If
verification criteria are met, the certified characteristics on the manufacturers certificate of
analysis should be used.

Conclusion
If a manufacturer cannot do a D-value assessment or verification in-house due to the lack of a
compliant BIER vessel and wants to obtain a verification, it should have a contract lab run the
USP resistance test, the survive/kill method. This will involve two BIER runs and be much less
expensive (around $600$800) than conducting a full determination involving two methods and
costing upwards of $6000$7000.

When dealing with any third-party lab, before putting any money out or contracting work,
perform an audit of the equipment and methods usedeven if it has to be a paper audit.
Qualifying a lab prior to having work done can save money and numerous needless problems
that can cause work delays and frustration. Remember, to consider an assessment is not the same
as to determine. To determine a BIs resistance is very costly, is usually done by the BI
manufacturer, and involves at least two test methods. FDA or an auditor might not look favorably
upon relabeled or possibly adulterated BI usage.

References
1. USP 31, Biological Indicators for Sterilization (Rockville, MD: U.S. Pharmacopeia), 401.
2. USP 28, Total Viable Spore Count (Rockville, MD: U.S. Pharmacopeia), 244.
3. USP 31, Resistance Performance Tests, (Rockville, MD: U.S. Pharmacopeia), 1532.
4. ANSI/AAMI/ISO 18472:2006, Sterilization of Health Care Products, Biological and
Chemical IndicatorsTest Equipment, (Arlington, VA: AAMI, 2006).
5. ISO 11138-1:2006, Sterilization of Health Care ProductsBiological IndicatorsPart 1
(Geneva: International Organization for Standardization, 2006).

Russ Nyberg is director of retail sales and tech support for Raven Labs (Omaha, NE).