Environmental monitoring of clean room

Purposes of environmental monitoring

1. To estimate the microbial and particulate
content of room air and surfaces.
2. An effective environmental monitoring
program can alert some one regarding
conditions
contributing
to
excessive
microbial levels arise due to ineffective
cleaning,
sanitation
or
other
personnel/equipment issues.

3. To provide crucial information on the quality of

the aseptic processing environment during
manufacturing.

4. To minimize the potential for inadvertent

product contamination.
5. To prevent the release of potentially
contaminated batch if appropriate standards
are not fulfilled.

6. Prevents future contamination by detecting

adverse trends.

Environmental Monitoring Components

Airborne nonviable particulate monitoring.
Airborne viable (microbial) contaminant
monitoring.
Viable contaminant monitoring of surfaces.
Viable contaminant monitoring of
personnel.
Temperature and humidity monitoring.
Pressure differential monitoring.
Water monitoring.

Airborne particulate monitoring

Electronic air particle counters.
Count all particles in the environment.
Air particle counters are useful in
determining the number of particles per
cubic foot (or per cubic meter) to classify the
cleanness of a clean room area i.e. Class 100
(A/B), Class (C) 10,000
Class (D) 100,000.

Particle counter

HEPA Filter Integrity

Ensure that HEPA filter fabric/seals are not
damaged/broken .
Test HEPA filter with smoke of known
particle size upstream of the filter .

Scan for smoke penetration using ,

Aerosol photometer and particulate counter.

Hepa filter

Air velocity
Velocity at various points in the clean room
measured .
Hand-held velometer checks the velocity of
the airflow along the entire filter surface
area.
LAF 90 FPM (0.45 m/s) for horizontal.
Velocity of <70 FPM indicates filter clogging,
filter failure and needs replacement.

Air velocity counter

Microbial Monitoring

(viable)

Non-volumetric air sampling.

Settling plate.
Volumetric air sampling.
Slit-to-Agar air sampler.
Liquid impinger .
Centrifugal air sampler.
Surface sampling.
Replicate Organism Detection and
Counting(RODAC) plate.
Swab-rinse test.
Personnel monitoring.
Gloves finger dabs.

Settling plates
Simplest
means
of
evaluating
the
microbiological quality of air.
Petri dishes containing agar medium are
exposed at sampling locations for a certain
period (for 4 hrs) to collect airborne
particles settling by gravity.
Plates are incubated at 32C for 48 hours .
Disadvantage: volume of air sampled is
unknown (non-volumetric) .

Settling plates

Volumetric air sampling

Known volume of air is drawn from the
environment and contaminants in the
air are impinged/ impacted on to a
suitable microbial growth medium.
Growth medium is incubated.
Colonies of microbial growth are
counted and expressed as CFU per cubic
meter.

Slit-to-Agar Air Sampler

Sterile agar plate (150 mm) is placed on a
circular plate and the cover containing the
slit is secured above the agar plate. The plate
rotates under the inlet slit.
Air is drawn through the slit, accelerated and
impacted on the agar plate.
Rotation of plate is made constant by a clock
mechanism.
Colony counts is related to time of collection.

Liquid Impinger
Collects viable microbial contamination
within a given cubic foot of air.
Uses a vacuum source that draws air at
high velocity through an isotonic fluid.
Fluid is filtered through a membrane
filter.
Filter is incubated on agar plate.
Disadvantage: microbial counts may be
underestimated because the high
velocity of impingement kills some
microorganisms upon impact

Liquid Impinger

Centrifugal Impaction
It utilizes a rotor device in the head of the
air sampler to draw air and microorganisms
in and on to a special strip containing
growth medium.
The centrifugal force causes particles and
microorganisms to impact the medium at a
rate dependent on the size of the particle.

Reuter centrifugal sampler (RCS)

The principle of collection is centrifugation.
Air is drawn into the sampler, accelerated
toward the inner wall of the drum.
Applied centrifugal force (~ 4000 rpm)
causes airborne particles to impact upon a
strip of agar medium that lines the inner wall
.
Strips are incubated .

Surface monitoring

RODAC Plate method.

Swab method.
Surface Rinse method.
ATP Bioluminescence Hygiene Monitoring
Method.
ATP: Adenosine triphosphate.

 RODAC plate Method

(Replicate Organism Detection and Counting)
Made by pouring suitable agar media into
the petri dishes till a convex surface extends
above the rim of the base plate.
When the molten agar has solidified, the
agar surface have to be gently pressed
against a selected surface, e.g. LAF
workbench.
Plates are incubated (commonly 48 hours at
32C) to reveal growth of microorganisms .
RODAC plates contain Trypticase Soy Agar with
Lechitin and Polysorbate 80.

RODAC plate

Sterile swabs
Moistened cotton swab tips are used to swab
the surfaces that are difficult for RODAC
plates to reach .

Swabs are then streaked onto solid growth

media in plates .
Plates are incubated to reveal microbial
growth .

Personnel monitoring
Finger tips of gloved hands are pressed onto
surface of solid growth media .

Plates are incubated .

Adenosine triphosphate (ATP) is an enzyme

that is present in all living cells, and an ATP
monitoring system can detect the amount of
organic matter that remains after cleaning
an environmental surface, a medical device
or a surgical instrument.
ATP Sample Testing Device contains a
natural enzyme found in fireflies. This
enzyme, called luciferase, produces a simple
bioluminescence (light-producing) reaction
when it comes into contact with ATP.

 Luminometer can measure extremely low

levels of ATP. The bioluminescence reaction
is immediate so results can be processed
in seconds, at the testing site, by inserting
the swab device into the system.
Results are then expressed numerically on
the luminometers screen as Relative Light
Units (RLU).

Media & Incubation Conditions

Routine monitoring should be conducted
using Soybean - Casein Digest Agar
incubated at 30-35C for 48 hours and/or
20-25C for 72 hours.
Periodic monitoring only should be
conducted using Sabourauds Agar
incubated at 20-25C for up to 5 days.