Incubation conditions for Environmental Monitoring -

Justification
Introduction
Recently, a number of different documents have been making statements similar
to incubation conditions and media used must be justified. Many companies
were able to cite USP <1116> as a rationale for using two different incubation
conditions including 20C-25C and 30C-35C. However, the most recent
revision to USP states: Time and incubation temperatures are set once the
appropriate media have been selected. Typically, for general microbiological
growth media such as SCDM, incubation temperatures in the ranges of
approximately 20C35C have been used with an incubation time of not less
than 72 hours. [1]. With a 15C range in temperature, it is not likely that a
single study will provide adequate data to justify the incubation conditions use.
As such, companies need to generate data to support the incubation conditions
utilized.
Background
For many years, pharmaceutical companies have used two different media for
conducting monitoring of the environment. One of the media was designed for
the recovery of bacteria and the other was chosen to isolate fungi (mold and
yeast). Typically the bacterial media was incubated at 30C-35C for a set
number of days and the fungal media was incubated at 20C-25C for a specified
number of days.
Marshall, et al. published an article entitled Comparative Mold and Yeast
Recovery Analysis (The Effect of Differing Incubation Temperature Ranges and
Growth Media). This article described the studies performed to support the use
of a single type of media to isolate both bacteria and fungi under a specified set
of conditions that included two different temperature ranges. [2]
In the process of evaluating a novel sterility test method, Kielpinski, et al. studied
the appropriate incubation temperature to use. Their data indicated that an
incubation temperature of 32C provided improved detection of the
microorganisms in the sterility test method over the values obtained using the
incubation conditions in the compendia. [3]
In 2012, the USP revised monograph <1116> from Microbiological Evaluation of
Clean Rooms and Other Environments to a chapter entitled Microbiological
Control and Monitoring of Aseptic Processing Environments. As part of the
change, this indicates that when using two-temperature incubation of
environmental monitoring plates, if one chooses to incubate at the lower
temperature first, the recovery of Gram-positive cocci may be compromised.
These are important as they are associated with humans, i.e., typical human
contaminants. [4]
In addition to the other variables associated with monitoring, the media used
may contain neutralizing agents, like polysorbate 80 (Tween 80) and/or lecithin
to improve the recovery of microorganisms from areas that may have been
exposure to sanitizers or other antimicrobial substances.
Other types of concerns with environmental monitoring include: whether or not
anaerobic monitoring should be conducted routinely, whether different culture
media should be utilized, whether the incubation conditions selected can recover
slow-growing microorganisms, and so forth.
This article describes a testing methodology that can be used to evaluate the
environmental monitoring procedures utilized to justify the methods used. In
most cases, a company would find the use of a single medium and a single set of
incubation conditions the most cost effective approach to use if it were
acceptable.
A two-phase approach to the protocol was developed to justify the environmental
monitoring conditions. In this approach, Phase 1 involves testing a variety of
samples in the laboratory to select an appropriate media and set of incubation
conditions to evaluate in situ. Phase 2 is a comparative side-by-side study in situ
looking at the existing environment monitoring conditions used and the proposed
monitoring method selected in Phase 1.
Testing Scheme Phase 1 Laboratory Testing to Select the Proposed Test
Conditions
During this phase of testing, consideration should be taken to determine the
various types of organisms and media conditions to be evaluated. Test organisms
should include both laboratory stock cultures and environmental isolates from
the facility. A rationale should be prepared to identify how organisms were
selected, e.g., at least one each of a Gram-positive cocci and bacillus, a Gram
negative cocci and bacillus, a mold and yeast. Additionally, prominent organisms
previously isolated in environmental monitoring should be utilized. It is important
to include a slow-growing microorganism like P. acnes in the test scheme. Some
companies choose to include more organisms of a specific type that they
routinely find in their environment, or have been associated with adverse trends
or sterility failures.
If you have a program currently established, you will likely want to include those
conditions routinely used in the test environment, especially if you intend to
have acceptance criteria that compares to that same data.
Another consideration for the testing is the types of media to evaluate. It is
common to look at a common nutrient media, like Trypticase Soy Agar. Fungal
media such as Sabouraud Dextrose Agar or Potato Dextrose Agar can also be
used. Some companies like to evaluate the performance of media with lower
levels of nutrients, like R2A. There are companies that prefer Brain Heart Infusion
broth/agar for recovery. Each of the media selected can be tested both with and
without neutralizing agents, if desired. One might also try use of a designated
anaerobic media (or regular media and an anaerobic system) to evaluate the
difference in counts using anaerobic incubation.
Incubation conditions (temperature and time) should also be specified, e.g.,
20C-25C for three days followed by 30-35C for an additional three days or
continue to incubate for up to seven, 30C-35C for three days followed by 20-
25C for an additional three days, 20C-25C for up to seven days (final
incubation time to be determined at the end of phase 1 testing), 30C- 35C for
up to seven days (final incubation time to be determined at the end of phase 1
testing), and so forth.
Following selection of the test organisms, test media, and incubation conditions,
a series of studies are set up where each test organism is tested on each media
for each of the indicated incubation conditions. For the extended incubation
periods, such as up to seven days, it is worthwhile to record the counts daily to
determine the optimal time for recovery of all specified organisms. While this is
an extensive test, it provides a wealth of information for the facility.
The number of replicates to run for each test condition should also be specified.
A higher number of replicates can be useful if using statistical analysis for the
data.
Following collection of the data for all the test conditions, the results are
analyzed to determine which test condition provides the optimal results. In most
cases, it is costbeneficial to use a single media and a single incubation
temperature. Some companies may choose to use different test conditions for
different areas of the facility, e.g., use of media with neutralizers in areas where
recovery may be stifled without the use of neutralizers.
Detailed instructions should be provided in the protocol to address how to select
the desired test conditions, e.g., what type of recovery level is acceptable, if the
media should be required to recover all of the test organisms, should
slowgrowing organisms be recovered in the test, do anaerobes need to be
recovered, and so forth. Levels of recovery should also be addressed, e.g., if the
counts must be the same or better than an existing method being used. There
are different criteria that can be used for recovery levels, e.g., within 70% of a
reference level (perhaps an existing methodology), within 0.5 log, or other
method of choice. An important concern in this discussion is what to do if none of
the media tested is successful in recovery for all of the identified acceptance
criteria. It is important to seriously consider the criteria you will use for test
condition acceptance, as you could design a study that precludes use of any of
the test conditions identified. In some cases, you may also find that a variety of
methods provide acceptable recovery and it becomes important to identify how
to select the method to use.
Phase 2 in situ testing: Comparative study
For a facility already conducting environmental monitoring, the new method
selected (optimal method) will be compared to the existing methods (reference
method) being used in the site, e.g., a biphasic, two media method. The purpose
of this phase is to assess the appropriateness of the selected medium and
incubation conditions in situ.
Environmental monitoring is conducted in accordance with the site monitoring
procedures, however at every selected monitoring point, test samples are
collected (using the optimum method selected in Phase 1) in addition to the
required samples in the site standard operating procedure (SOP). The samples
are incubated and evaluated per the appropriate methodology, i.e., the existing
method plates are incubated as specified in the SOP while the test plates, using
the conditions specified in Phase 1, are incubated as tested in Phase 1. A
minimum of 20 separate monitoring locations should be identified for evaluation.
For these types of comparisons, it is easier to include rooms where levels of
recovery are expected, e.g., ISO 7 and 8 areas. This will aid in reducing variability
if very low levels of microorganisms are isolated. The choice of sampling sites
may be based upon previous recovery data. If no data exists, sampling sites may
include lower level classified areas, hallways, or unclassified areas. Following
incubation, the average recovered counts, in colony forming units (cfu) are
determined. A comparison of the counts using the test method and the reference
method will be conducted. Acceptance criteria for the comparison of data should
be established. In most cases, the test method should give results that are
equivalent and/or better than the existing method used. It is useful to maintain
all the samples (refrigerated) until the end of the analysis, so that plates can be
recounted or analyzed if necessary.
If a reference method is not available for comparison, the test method should be
used in situ for a specified number of days much like one might do with an initial
performance qualification of an environmental monitoring program.
Conclusion
This testing scheme involves a good deal of work but provides conclusive data on
the appropriateness of the environmental monitoring conditions utilized. When
audited, it provides documented evidence to support the methods selected and
addresses the concerns of some regulators on which incubation condition should
be initiated first. Lastly, in many cases companies can end up with a set of
conditions that are more cost effective than the current methods.