Static vs. Dynamic Environments
Copyright ©2009 by Gary Fromert, M.S.
PETRI Project, Northampton Community College
After completing this exercise, you should be able to:
1.
2.
3.
4.
Explain what is meant by a static environment.
Explain what is meant by a dynamic environment.
Explain what is meant by the settling plate method.
Demonstrate an understanding for the necessity to monitor a controlled environment
for viable microorganisms.
Environmental monitoring for particulate and microorganisms is a critical process
utilized to assess clean areas and other controlled environments within the
pharmaceutical, electronics, and biotechnology industries. It serves as an important proactive tool for quality assurance programs in the manufacture and control of sterile and
non-sterile products and support areas. Clean area control parameters should be supported
by microbiological and particle data obtained during qualification studies. Initial
cleanroom qualification includes an assessment of air quality under as-built, static
conditions. It is important for area qualification and classification to place most emphasis
on data generated under dynamic conditions with personnel present, equipment in place,
and operations ongoing. An adequate aseptic processing facility monitoring program also
will assess conformance with specified clean area classifications under dynamic
conditions on a routine basis.
Environmental monitoring evaluates HVAC systems, personnel, cleaning, and
sanitization activities. It monitors both viable and non-viable particles. Viable particles
would include microorganisms such as bacteria, yeast, and molds. Microorganisms such
as bacteria and fungi as well as bacterial spores and fungal spores are almost always
present in air. The purpose of monitoring viable particulate is to determine environmental
bioburden which is the total number of microorganisms that can be detected. Nonviable
particles would be inanimate air particulates of various sizes. The numbers of particles,
whether viable or nonviable, vary by site and environmental conditions.
Sampling sites are typically chosen by how critical that stage of the process is to the
product at that particular location in the processing stream. Ideally, environmental
monitoring should occur under dynamic conditions during regular work activity.
Monitoring is best conducted at the end of the work day when the environment has been
most stressed as the viable and nonviable particles should be at there highest levels.
In this laboratory you will take samples to determine the number of viable
microorganisms using the settling plate method. Gravitational or settling plates contain
sterile growth media in which air can be monitored for viable microorganisms. The plates
are passively exposed to an environment typically for 60 minutes allowing airborne
microorganisms to settle on to the surface of the media over the total elapsed time period.
Viable microorganisms that settle on to the media surface, due to gravity, will grow when
54
the plates are incubated. Settling plates provide minimal interruption to the environment
allowing them to be easily used in dynamic controlled environments as well as being
used under static conditions. Sampling areas for this laboratory exercise will include both
static and dynamic environments. Environmental monitoring will be accomplished by
exposing sterile uncovered nutrient agar plates and Sabouraund's agar plates to the
surrounding air for an extended period of time. After a specified exposure period and
incubation period, the total number of microorganisms will be counted, recorded and the
results from the two environments will be compared. Under these two different
environmental conditions it should be expected to see different levels of microbial
contamination.
Materials (per student)
3 Nutrient Agar or Trypticase Soy Agar (TSA) Plates
3 Sabouraund's Agar Plates
Timer
China Marker or Sharpie
Procedure 1
Environmental monitoring of a static and a dynamic environment
Note: You will be instructed as to the rooms or locations that will be designated as
static and dynamic environments.
Settling Plate Method
1. Mark the bottom of one of the nutrient agar plates "Control", include your initials and
the date, and then set aside. Do the same for one of the Sabouraud's agar plates.
2. Mark the bottom of one of the remaining nutrient agar plates "Static 60 min"; include
your initials and the date. Do the same for one of the remaining Sabourand's agar plates.
3. Mark the bottom of the remaining nutrient agar plate "Dynamic 60 min"; include your
initials and the date. Do the same for the remaining Sabourand's agar plate.
4. Expose one each of the nutrient agar plates and one each of the Sabourand's agar
plates marked "Static 60 min" simultaneously to a static environment by removing the
cover plate and placing it face down next to its respective plate. Allow the exposure time
to elapse then cover each plate with its respective cover.
5. Expose one each of the nutrient agar plates and one each of the Sabourand's agar
plates marked "Dynamic 60 min" simultaneously to a dynamic environment by removing
the cover plate and placing it face down next to its respective plate. Allow the exposure
time to elapse then cover each plate with its respective cover.
6. Incubate all three of the nutrient agar plates at 37°C for 24 to 48 hours. Incubate all
three of the Sabourand's agar plates at room temperature for 3 to 5 days.
7. Observe all the plates for colonies of viable microorganisms that have grown on the
respective media. Count the colonies and record.
55
Results and Observations
After examining your plates, count the colonies and record your results in the following
table. For all plates, quantify your results as follows:
No growth
1 to 200 colonies
> 200 colonies
0
Record the actual colony counts
TNTC (Too numerous to count)
Settling Plate Method
Nutrient Plate (Total Count)
Sabourand's Plate (Total Count)
Control
Static 60 min
Dynamic 60 min
Laboratory Review
1. Explain what is meant by static conditions in regard to a controlled environment.
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
2. Explain what is meant by dynamic conditions in regard to a controlled environment.
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
3. Explain what is meant by a settling plate and its usage.
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
4. What is the purpose of monitoring a controlled environment for viable
microorganisms?
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
5. Compare your results for the nutrient agar plates of the static environment and the
dynamic environment and speculate as to difference in the number of colonies that grew.
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
56