ENVIRONMENTAL MONITORING ANALYSIS

Project thesis submitted for partial fulfilment of the requirements for the
award of the degree of

MASTER OF SCIENCE

IN

MICROBIOLOGY

SUBMITTED BY

MS. MEDIDI. ANANDINI

(REG.NO:2267934016)

Under the supervision of

U.SRINIVAS
( MANGING DIRECTOR)
BIOCHEMLABS
VISAKHAPATNAM

DEPARTMENT OF THE MICROBIOLOGY

DR B.R. AMBEDKAR UNIVERSITY
ETCHERLA, SRIKALULAM-532410
2024
 ENVIRONMENTAL MONITORING ANALYSIS
Project thesis submitted for partial fulfilment of the requirements for the
award of the degree of

MASTER OF SCIENCE

IN

MICROBIOLOGY

SUBMITTED BY

MS. MEDIDI.ANANDINI

(REG.NO:2267934016)

Under the supervision of

U.SRINIVAS
(MANGING DIRECTOR)
BIOCHEMLABS
VISAKHAPATNAM
 DEPARTMENT OF BIOTECHNOLOGY
DR.B.R. AMBEDKAR UNIVERSITY ETCHERLA, SRIKALULAM

CERTIFICATE

This is certify that, Ms. MEDIDI.ANANDINI, M.sc Microbiology has

successfully completed the project entitled “ENVIRONMENTAL
MONITORING ANALYSIS” is a bonafied work carried out under the guidance

of U.SRINIVAS, Managing Director ,Biochemlabs ,Visakhapatnam from January

2024-February 2024, for the partial fulfilment of MSc Microbiology course Dr.
B.R. Ambedkar University, 2022-2024.

EXAMINARS
1.
2.
Professor & HOD
(Department of Biotechnology)

ACKNOWLEDGEMENTS
I sincerely thank U.Srinivas, my project supervisor, managing director of
Biochem labs, Visakhapatnam I wholeheartedly thank all the Juniorette
faculty at Biochem labs, Visakhapatnam for the encouragement and support
provided to me towards the successful completion of my project work at
Biochem Labs Visakhapatnam.

I express our sincere gratitude to entire faculty DR.P.SUJATHA,

DR.S.UDAYBHASKAR,DR.K.SWAPNAVAHINI,DR.N.LOKESHWARI And
P.MADHAV RAO for this transcendent suggestions during the process of
work .

I convey my highest regards to my parents and friends who helped me

through their support towards the successful completion of my project.

MEDIDI ANANDINI

INDEX

1. Content
2. Abstract
3. Introduction
4. Materials and Methodology
Air Monitoring
Personnel Monitoring
 Surface monitoring
5. Results and Discussion
6. Discussion and Conclusion
7. Reference

ABSTRACT

Environmental monitoring is a scrutinizing program for microbial and particulate

contamination of clean rooms and associated clean zones for aseptic processing.
The current study was therefore, designed to evaluate the quality of clean rooms
environment of local Coronary drug eluting stents and PTCA ballon catheters
manufacturing unit in innvolution health care private limited with the international
cleanroom standards proposed by the World Health Organization(WHO) for the
production of quality biological products.

In the manufacturing unit of Innovolution Healthcare, coronary drug-eluting stents

and PTCA balloon catheters are produced with meticulous attention to quality and
patient safety. This is achieved through the implementation of comprehensive
monitoring methods including air monitoring, personnel monitoring, and surface
monitoring. Air monitoring ensures the continuous assessment of airborne
contaminants to identify and mitigate potential sources of contamination.
Personnel monitoring focuses on ensuring strict adherence to hygiene protocols
and the proper use of protective gear among workers to minimize the risk of
personnel-induced contamination. Surface monitoring involves regular inspection
and cleaning of surfaces to prevent the accumulation of contaminants on
equipment and workstations. These monitoring methods collectively contribute to
maintaining a clean and sterile manufacturing environment, thereby ensuring the
production of high-quality medical devices free from contamination.

INTRODUCTION

Environmental monitoring is a designed scrutiny system for microbial

contamination of cleanrooms and other close processing environments. It is a
cascade of procedures which gives monitoring, testing and responses to the
microbial concentration in aseptic processing environments. Environmental
monitoring defines the procedures and actions that require occurring to
characterize and scrutinize the quality of the environment. The prime goal of
environmental monitoring of cleanrooms or isolators are to regulate the numbers of
airborne viable and Non-viable particles within defined limits, predict the risk to
the environment and regularly estimate the efficacy of cleaning and disinfecting
processes in sterile areas.

In microbiology environmental monitoring (EM) is a process that determines the

quality of a controlled environment via microbial data collection. Data comes from
samples of air, surfaces, and personnel in a clean space. Environmental monitoring
plays a critical role in developing sterile and non-sterile.

Medical equipment contaminated by microbes can cause immediate and long-term

harm to patients treated with these products. Microorganisms can also impact the
products efficacy. microbiological testing ensures that a medical equipment is safe
and effective.

In a manufacturing facility, key sources of contamination are the air, equipment,

surfaces, or even the person performing the activity. And to avoid these
contaminations, the manufacturing facility should regularly monitor the air,
equipment, surfaces, or personnel wearables.

Environmental monitoring is not the same as environmental control.

Environmental control is concerned with the design measures necessary to
maintain environments within the required operating parameters. Such parameters
include: temperature, relative humidity, air velocity, unidirectional air flow, HEPA
filtration, and pressure differentials between rooms of different classification.
Environmental monitoring does, however, relate to environmental control in that
monitoring can indicate a failure of a control and it is sensible to, from a risk-based
perspective, target monitoring where control is weakest. The implementation of
environmental monitoring in an organization should be through the construction of
a planned environmental monitoring programme. The requirement to perform
monitoring is described in the following standards and guidelines:
• USP <1116>
• ISO 14698 Part 1

The essential factors of any well-designed environmental monitoring programme

are similar. However, the way in which different factors are weighted and the
decisions made by facilities will vary in terms of how programmes are carried out.

The objectives of an environmental monitoring programme are:

• To monitor cleanrooms, collect data and to examine trends to show the state of
microbiological control of an environment. Monitoring is more meaningful when
the environment is assessed under representative conditions (normally when
cleanrooms are occupied, and processing is taking place)

 Data collection may relate to either numbers of microorganisms or to the

incidence of detection, or to both (using pre-defined monitoring limits). In
addition, some of the microorganisms recovered should be characterized and
trended

 To show that contamination levels do not increase through manufacturing as

the process is designed to become cleaner.

 To assess the risk to the environment and most importantly to the product.
This is achieved by selecting monitoring locations which are meaningful and
by monitoring at frequencies will allow the trend.

 When action levels are exceeded or adverse trends are detected appropriate
investigations must be performed using documented procedures to determine
the contamination source, the impact upon the product and to set corrective
or preventative actions. For this risk base methodologies can be deployed.

 To allow the effectiveness of the cleaning and sanitization programme to be

assessed.

 To understand the performance of the people and equipment, and the

suitability of operating protocols.

 To provide information about environmental control in constructing an

environmental monitoring programme, it is important to be aware of the
limitations of monitoring.

The methods deployed, for example, are highly variable in terms of collection
efficiency Furthermore, the culture media selected and the incubation parameters
chosen will only detect those microorganisms which will grow under the set of
conditions adopted. A further limitation is the time of sampling. Monitoring only
provides a ‘snapshot’ of one moment in time and this may or may not reflect
conditions throughout the process. It follows that individual environmental
monitoring results are rarely significant (with the exception of the continual
monitoring of aseptic processing batches where some samples can relate to specific
events). Thus, the most important aspect of environmental monitoring is the
examination of trends over time.

The environments in which processing occurs are either classified (to a cleanroom
standard, such as ISO 14644) or controlled. Cleanrooms and clean zones are
typically classified according to their use (the main activity within each room or
zone), controlled through the physical operation of HVAC (Heating Ventilation
and Air Conditioning), with the classification confirmed by the cleanliness of the
air by the measurement of particles. In addition, recommended limits are applied
for microorganisms. With control of cleanliness, the pharmaceutical manufacturing
environment is based around a series of rooms with specially controlled
environments. These are termed ‘cleanrooms’. A cleanroom, on one level, is
simply a room that is clean. The key aspect, however, is that the level of
cleanliness is controlled. The definition of cleanliness. A room with control of
particulates and set environmental parameters. Construction and use of the room is
in a manner to minimize the generation and retention of particles. The
classification is set by the cleanliness of the air While control of airborne particles
is important, the ISO 14644 standard does not differentiate whether these particles
are inert or biologic. Reference to biocontamination in relation to cleanrooms is
detailed in ISO 14698, although this latter standard is not, in this author’s
experience, widely followed in drug manufacture (although it does contain some
useful advice relating to the qualification of environmental monitoring methods).A
controlled environment is similar, in that airborne particulate and microorganism
levels are controlled, but the rooms are not classified against a national or
international standard (Whyte, 2001). These are sometimes referred to as
‘controlled but not classified’ (CNC) areas.

Once a room has been assigned a classification, certain environmental parameters

(physical and microbiological) are to be met on a routine basis. This is assigned by
collecting data and examining the results of monitoring against pre-set criteria. Part
of this assessment is through a microbiological environmental monitoring
programme. The extent of monitoring and the monitoring limits assigned will
relate to the class of cleanroom, the activities taking place and the relative risks.
Thus, an important question to answer before launching a monitoring programme
is “what cleanliness levels are expected.
Sources of microbial contamination

There are different sources of microbiological contamination within clean

environments: water, air, surfaces (both within the room and from equipment) and
personnel. These hazards should be evaluated by the microbiologist in terms of the
relative risks to the product, and the environmental monitoring programme should
be orientated towards the points of greatest risk. The sampling methods should be
appropriate in relation to the types of contamination sources (and a comprehensive
monitoring programme should assess each of the main contamination sources). The
greatest risks are those which could lead to product contamination. This is
illustrated in the diagram below:

The primary sources of contamination are people and water. This is because both
are vectors of contamination. People are the most significant source of
contamination, although they are a highly variable and unpredictable source.
Microorganisms are shed from hair, skin, eyes and mucous membranes.
Microorganisms are either deposited into the air stream or can spread through
contact. Water is a common feature in processing (as an ingredient, a cleaning
agent, a diluent for disinfectants, steam supply, and so on).
The concern with water in cleanrooms is that it not only provides a means for
microorganisms to survive, it provides the opportunity for the numbers of
microorganisms to increase and microorganisms are invariably found in all
residues of water (some bacteria, especially Gram-negative rods, can grow and
multiply in low nutrient states). The secondary sources of contamination are air
and surfaces. The air in most areas contains microorganisms. However, the number
of microorganisms will vary according to the cleanroom grade. Air is a secondary
contamination source because air is a vector for microorganisms, but it is not a
nutritive environment and whilst some bacteria can survive in air streams they
cannot multiply. Generally Gram-positive bacteria are more commonly found in air
[typically Bacillus species, Staphylococcus species, and Micrococcus species] .
Bacteria in air are normally in association with dust particles or skin flakes, rather
than as individual microorganisms (for which the term ‘microbial carrying particle’
is sometimes used). This makes the microorganisms heavier and more prone to
gravitational settling. Therefore, what often matters most is not the
microorganisms in the air but their potential for settling. A well-designed
cleanroom will filter air (to dilute the number of microorganisms) and have a
pressure cascade to prevent re-contamination of a clean area from a less clean area
(since microorganisms cannot move against an air current). The other secondary
contamination source is materials and surfaces. Here, the key risks are the transfer
of items in and out of a clean area, where materials are more at risk if they are of a
design that cannot be easily cleaned or disinfected; and from personnel touching
surfaces. Another risk is the contamination of surfaces through deposition (such as
settling from the air) . Based on these contamination sources certain factors will
lead to contamination risks being more likely. These factors include:

• Poorly designed cleanrooms.

• Water remaining on surfaces for prolonged periods.

• Inadequate cleaning and sanitization.

• Inadequate personnel gowning.

• Poor aseptic practices such as direct surface-to-

surface transfer (such as by personnel directly
touching the product or contaminated water
entering the process)

• Airborne transfer, often arising from personnel

shedding microorganisms. Shedding increases with
https://www.innvolution.in//insignia/
https://www.innvolution.in//eternia/

https://www.innvolution.in/scoreflex/
https://www.innvolution.in/sapphire-ii/

Contamination in the manufacturing process of coronary drug-eluting stents

and PTCA balloon catheters can lead to various adverse effects on patients.
Contaminants can introduce foreign particles or substances into the devices,
potentially causing allergic reactions, infections, or even device malfunction.
These effects could manifest as increased risk of thrombosis, inflammation at the
implantation site, or compromised efficacy of the devices in dilating arteries or
delivering drugs. Proper quality control measures are crucial to minimize
contamination and ensure patient safety in the production of these medical devices.

MATERIALS AND METHODOLOGY

MEDIA

● Soyabean casein digest agar

REAGENTS

● Sodium chloride
● Peptone

GLASSWARE

● Sterile petri plates

● Conical flask
● Beakers
● Test tubes
● Glotted tube

INSTRUMENTS

● Laminar air flow

● Autoclave
● Incubator
● Active air sampler

PREPARATION OF MEDIUM
SOYABEAN CASEIN DIGEST AGAR

• Dissolve 20 grams of soyabean casein digest agar in 50 ml of distilled water

up to 500 ml of distilled water and sterile by steam sterilization at 1210c for 15-20
minutes.

PEPTONE WATER

Dissolve 1.5 grams of peptone in 100ml of distilled water and sterile by steam
sterilization at 1210c for15- 20 minutes.

SODIUM CHLORIDE PREPARATION

Dissolve 2 grams of NaCl in 250 ml de-ionized or distilled water in a clean

container mix it properly without any lumps in the container.

PREPARATION OF SWAB STICKS

Use the disposable swab sticks or prepared the swab sticks by non absorbent cotton
fold on swab sticks and steam sterilization it at 121°c for 15-20 minutes.

METHODS

ENVIRONMENTAL
MONITORING

SURFACE
AIR MONITORING PERSONNEL
MONITORING
MONITORING

ACTIVE AIR PASSIVE AIR CONTACT FINGER DAB SWAB TEST

SAMPLING SAMPLING

● AIR MONITORING
I. Active air sampling
II. Passive air sampling

● PERSONNEL MONITORING
I. Contact Plate
II. Finger Dab Plate

● SURFACE MONITORING
I. Swab test
PREOPERATIONAL CHECKS FOR ENIRONMENTAL MONITORNG

 Two days pre incubated plates shall be used.

 Check the plates for any microbial contamination and physical integrity of
solid media.
 Outer surface of the plates should be sanitized with a filtered sporicidal
agent.
 Check the expiry and use before date of media.
 Check the cleaning status of the area where the plate has to exposed.
 Ensure the temperature humidity and differential pressure of the area within
the limit or not.

AIR MONITORING

Air Monitoring is an assessment of air quality determined by the measurement of

pollutants and particulates in air. It is used in industrial environments to protect
workers and prevent environmental and product contamination. When measuring
air contaminants, investigators must consider not only the sampling method but
also the phase and identity of the pollutant or particulate of interest.

ACTIVE AIR-SAMPLING

Active (volumetric or bio aerosol) air-samplers are a slightly different measure of

air than settle plates. The settle plate indicates the number of micro-organisms that
may deposit onto a surface the active air-sampler indicates the number of micro-
organisms present in a given volume of air.

within the range of the air-sampler. Both of these approaches have merits and any
comprehensive programme will use both active air-samples and settle plates. The
volume of air sampled is normally 1m³ of air. Therefore, like the settle plate, the
data can be quantified. Within a Grade A (ISO 5) clean room environment the
number of micro-organisms present would be expected to be < 1 Cfu/m3.
Therefore, the efficiency of the sampler is of great importance Variations are
experienced with all types of active air-sampling.

These include:
• The non-random distribution of micro-organisms in the environment.
• Imprecision in the various sampling techniques
• The rate of sampling at lower velocities, there is a danger that micro-
organisms will not be deposited onto the agar medium whereas at higher
velocities there is a danger of desiccation of the culture medium.
These variations must be considered when choosing between different sampler
types and models.

TYPES OF ACTIVE AIR-SAMPLER

There are three main types of active air-sampler:

• Impaction
• Centrifugal
• Filtration

However, other types of sampler are available. In assessing the three main types,
an impaction air-sampler functions by accelerating air at an angle of 90 ⁰ through
holes in the head of an air sampler (often a 'sieve-like' design) and impacting any
micro-organisms onto an agar strip or plate. A centrifugal air-sampler draws air
into the sampler head through a rotating vane mechanism. The vane causes micro-
organisms to be thrown out of the air and into the agar through the effect of the
centrifugal force. During filtration, air-samplers air is sucked through a filter and
any micro-organisms are captured onto a membrane filter, which is then
transferred to the surface of a culture medium and incubated.

Effective air-samplers must be able to precipitate particle sizes of at least 2mm. In

relation to most clean rooms, particles sized 5mm or over have greater significance
most airborne micro-organisms are typically between 5-15mm and increase to 15-
18mm for naturally- occurring airborne particles such as skin flakes, which contain
bacteria (1,2). There are also differences in the sampling volumes of different
model samplers, with most models capable of sampling at least 1,000 liters.

There is considerable debate as to which type of air-sampler is the most efficient.

The most common types of air sampler and the methods of operation are:
Slit-to-Agar Air Sampler (STA)

The air is drawn by a self-contained vacuum pump through a standardized slit

below which is placed a slowly revolving Petri dish containing agar. Particles in
the air that have sufficient mass impact on the agar surface. The agar plates are
incubated and viable organisms are allowed to grow out.

Sieve Impactor

The apparatus consists of a container designed to accommodate a petri dish

containing agar. The cover of the unit is perforated with the perforations of a pre-
determined size. A vacuum pump draws a known volume of air through the cover
and the particles in the air containing micro-organisms impact on the agar medium
in the petri dish Some samplers are available with a cascaded series of containers
containing perforations of decreasing size.

The above two methods allow determination of the distribution of the size ranges
of particulates containing viable micro-organisms, based on which size
perforations admit the particles onto the agar plates. Other air-sampler types
include:

Centrifugal Sampler

The unit consists of a propeller or turbine that pulls a calculated volume of air into
the unit and then propels the air outward to impact on a tangentially-placed nutrient
agar strip set on a flexible plastic base.

Sterilizable Microbiological Atrium

The unit is a variant of the single-stage sieve impactor. The unit's cover contains
uniformly spaced orifices. The base of the unit accommodates one petri dish
containing a nutrient agar. Air is drawn through the unit by vacuum and allowed to
impact on the agar surface.
Surface Air System Sampler

This integrated unit consists of an entry section that accommodate an agar contact
plate. Immediately behind the contact plate is a motor and turbine that pulls air
through the unit's perforated cover over the agar contact plate and beyond the
motor, where it is exhausted.

Gelatine Filter Sampler

The unit of a vacuum pump with an extension hose terminating in a filter holder
that can be remotely located in the critical space. The filter consists of random
fibers of gelatin capable of retaining airborne micro-organisms microorganisms.
After a specified exposure time ,the filter is aseptically removed, dissolved in an
appropriate diluent and then plated on an appropriate agar medium to estimate its
microbial content . the filter may also be plated directly on an agar surface.

In addition to the wide choice of various sampler types, different models of air
sampler vary in their efficiency. Some devices themselves can generate non viable
particle counts, and this should be considered in the design qualification of such
devices. Other design considerations include the suitability of the sampler to any
sanitization procedure for equipment to enter the clean room and the samplers
operations as an isokinetic sampler (that is to match the airflow speed within the
UDAF).

TIIE PRACTICAL USE OF AIR-SAMPLERS

There are a number of important issues to consider when using active air-samplers,
especially in critical zones. There are arguments against and in favor of active air-
samples in the rade A/ISO Class 5 environment. This debate centers on the
disruption of the air-flow caused by the operation of the air-sampler. The number
of active air-samples taken during a session should be considered. The effect of the
air-sampler can be examined through air-flow visualizations studies where the
disruption of the air-flow can be visualized by smoke studies (at a time when the
clean room is decommissioned).
Furthermore, the act of placing and removing an active air-sample is an
intervention into the environment and this must be carefully practiced during
media simulation trials or during practice sterility testing session. Due to this,
where isolators are being monitored, many users have fitted the active air-sampler
outside of the isolator environment. This is achieved by feeding a length of tubing
into the isolator, so that air can be drawn out and into the sampler. This is a similar
concept to the placement of many particle counters and reduces the risks associated
with intervention. The user Inay also wish to consider the ease of cleaning air-
samplers and the reaction of the air-sampler material to different disinfectants.
Some organizations have performed studies in order to determine if the active air
sampler itself generates particle counts in addition to the disruption of the airflow.
Most models of active air-sampler will generate some level of particles. The key
concern is if these are at a level that could be detected by discrete particle counters,
and therefore generate 'false' count data when processes are being monitored.

A further area of consideration is the effect of dehydration on the culture media

used in the active air-sampler. This is an area that organizations may need to
examine for themselves because the rate of moisture loss will relate to the type of
culture media used (and the fill volume). It will also depend upon the incubation
conditions, times and temperatures. Such studies should be performed post-
incubation and use a range of micro-organisms with which the growth promoting
properties of the culture medium should be assessed. It is common to include
isolates from the manufacturing facility in such studies4.4

● Prepare the Soybean casein digest agar plates as we required "Procedure for
Storage and Preparation of Microbiological Culture Media" Use the prepared
plates after 48 hours of pre-incubation.

● Transfer the required number of media cassette/plate, and accessories into

pass box

● Enter the innovulation health care private limited.

● Aseptically check the pre-incubated plates for any evidence of

microbiological contamination

● Discard, if the plates having any microbiological growth.

● Sanitize the external surface of the media cassette/ plate with the filtered
sporicidal agent.

● Mark the Petri-plates at the base with the following details with a marker,
For negative control plate For all the other plates Media lot Number Name of the
location Date of use Date of Sampling Used by Sampled by

● Perform the air sampling as mentioned

● Place the media cassette / Plate on the Air sampler and operate the
instrument about 600 seconds for air sampler.

● Take the instrument to the locations, where the air is to be sampled, hold it
in such a way plate facing the airflow at the required working height, and collect
1000liters of air which passes through the air sampler.

● After completion of sampling collect all the plates and incubate in an

inverted position in the incubator at 22.5 ± 2.5⁰C for 3 days and 32.5 ± 2.5 ⁰C for 2
days.

● After incubation observe and count the number of colonies on the colony
counter or in the light source with the help of a marker.

● Note down the observation in the format.

● If the counts obtained are above the limits specified below investigate the
results and take necessary actions.

Frequency of Monitoring

Class A: Once in a day

Class B: Once in a day
Class C: Once in a day
Acceptance criteria:

Alert limit Action limit

Class A: 1 CFU/ plate Class A: 1 CFU/ plate
Class B: 7 CFU / plate Class B: 10 CFU / plate
Class C: 75 CFU / plate Class C: 100 CPU / plate

PASSIVE AIR SAMPLING

Passive sampling is performed with settle plates. These are standard Petri dishes
filled with prepared culture media, and left open to allow colonies of bacteria to
settle and develop. The results can then be counted, enabling measurement of the
viable biological particles in the air and microbial contaminants.

This method of EM and its test results are not quantitative, in that it cannot capture
and detect smaller particles suspended in the air, or sample specific volumes of air.
Depending on the level of contamination, settle plates do not always provide
accurate data to drive improvements and solutions. Yet they are cost-effective, and
require only minimal additional equipment, such as settle plate stands or plate
carriers.

Passive air sampling is a method primarily used to sample for gases and vapors. In
this case, passive air sampling is more accurately termed "diffusive sampling"
because it relies on the natural process of diffusion. This method may be deemed
"passive" because, unlike active sampling, it does not involve pumping air to
collect it.

Diffusion samplers come in various types, but they all have the same basic design.
At one end of the container is the diffusive surface, where gaseous and vapor
molecules enter. At the other end is the sorbent medium, where the molecules are
collected. Each analyte — that is, a substance being measured — has its own
diffusion coefficient, or uptake rate. With this information combined with the
amount of exposure time, you can determine a time-averaged sample concentration
for the analyte. Diffusive sampling works especially well for long-term testing.

Passive air sampling Settle plate method

● Prepare the Soyabean casein digest agar plates as required for storage and
preparation of microbial media. Use the prepared plates after 48 hours of
preincubation.
● Transfer the required number of pre-incubated Petri plate into pass box
(LAL test room to Incubator).
● Enter the innovolution health care private limited.

● Aseptically check the pre-incubated plates for any evidence of

microbiological contamination.

● Discard the plates having microbiological growth.

● Decontaminate the external surface of the Petri plates with the help of a
sterile mop soaked in a filtered sporicidal agent.

● Mark the Petri plates at the base with the following details with a marker.

● Place the Petri plates on the plate exposure stands and remove the upper lid
of the Petri plate and keep it in an inverted position on the resting stand. (Note:
Inclined Petri plate stand in front of the riser and straight Petri plates stand at all
other location if there is no stand available expose the plates on the floor at the
specified location not less than 4 hours.

● Expose the media plates at all the locations for 4 hours.

● After 4 hours of exposure, close the Petri plate with lid. Note: collect the
plates in the same sequence in which the plates were exposed.

● Collect all petri plates and transfer the plates into the incubator.

● Incubate all exposed Petri plates in an inverted position in the incubator at

22.5 ±2.5 ⁰C for 3 days and 32.5 ± 2.5⁰ C for 2 days. (Keep each set not more than
10 plates in Stack)

● Once in a month (preferably first week of the month) expose the separate
set of plates inside the RABS and incubate an aerobically at 32.5 ± 2.5 0 C for 3
days,

Note: For anaerobic incubation the plates shall be pre-incubated under anaerobic
condition for 2 days

● After incubation observe and count the number of colonies on the colony
counter or in the light source with the help of a marker.

● Note down the observation in the format.

● If the counts obtained are above the limits specified below investigate the
results and take necessary actions.

Frequency of Monitoring

Class A: Once in a day where there is activity

Class B: Once in a day
Class C: Once in a day class
Class :Twice in a week

Note: For the above classes, frequency of monitoring can be done on demand basis
or whenever applicable.
Whenever a sterility test is to be performed in a day monitoring shall be done at the
same time.
 Alert limit
Class A: 1 CFU / plate
Class B: 3 CFL] / plate
Class C: 35 CFU / plate
Class D: 75 CFU / plate
Action limit
Class A: 1 CFU / plate
Class B: 5 CFU / plate
Class C: 50 CFU / plate
Class D: 100 CFU / plate

Note: For transferring the plates into sterility testing area place the plates in pass
box (Buffer room to LAL test room)

PERSONNEL MONITORING

Humans are the biggest source of contaminants in an aseptic environment Even

with hygiene practices and proper PPE (gowns, gloves, goggles, face shields, and
N95 masks), humans are the most significant risk of source of contaminants to an
aseptic environment.

Personnel Monitoring is done in a highly controlled area where the surrounding

environment is of class B and the working environment is of class A.

The person who is entering the classified section should be qualified for gowning.
He or she should know and follow the gowning procedure. It is necessary because
each individual can bring particulate and microbial contaminants into the area.

Individuals can easily perform personnel testing on themselves in one of two ways:
swabbing the forehead or elbows with a cotton swab dipped in Soyabean casein
digest agar or direct contact with a Soyabean casein digest agar plate and the
fingertips. Plates will then be covered and incubated for about 18 hours before
checking if microbes are present.

CONTACT PLATE :
● Prepare the soyabean casein digest agar media plates as required.

● Transfer the required number of media cassette plate and accessories into
pass box.

● Enter the innovolution health care private limited.

● Aseptically check the pre-incubated plates for any evidence of

microbiological contamination.

● Discard the plates having microbiological growth.

● Sanitize the external surface of the media cassette/plate with the filtered
sporicidal agent

● Mark the petri plates at the base with following details with a marker for
negative control plate for all other plates media, lot number, name of the
location ,date of sampling used by sampled by.

● Now the take the contact plates open the lid of contact plate and gently
pressing the right elbow and left elbow and forehead on media plate smoothly on
the surface which has to be monitor, after sampling, close the lid of the plate.

● After sampling carries out the contact plates for incubation.

● Incubate all exposed contact plates in an inverted position in the incubator at

22.5 ±2.5 ⁰C for 3 days and 32.5 ± 2.5⁰ C for 2 days.

● After incubation observe the number of colonies on the colony counter in the
light source with the help of marker.

● Note down the observation.

FINGERDAB PLATE:

● Prepare the soyabean casein digest agar media plates.

● Transfer the required number of media cassette plate and accessories into
pass box.

● Enter the innovolution health care private limited.

● Aseptically check the pre-incubated plates for any evidence of

microbiological contamination.

● Discard the plates having microbiological growth.

● Sanitize the external surface of the media cassette/plate with the filtered
sporicidal agent

● Mark the petri plates at the base with following details with a marker for
negative control plate for all other plates media, lot number, name of the location ,
date of sampling used by sampled by.

● Open the lid of soyabean casein digest agar media plates gently pressing the
four fingers and thumb of right hand on media surface follow the same plate of
soyabean casein digest agar media.

● After collecting the sample discard the glove and wear the new gloves

● After sampling close the lid of plates and sanitize the gloves with 70%IPA.

● Then carries out the contact plates for incubation.

● Incubate all exposed contact plates in an inverted position in the incubator at

22.5 ±2.5 ⁰C for 3 days and 32.5 ± 2.5⁰ C for 2 days.

● After incubation observe the number of colonies on the colony counter in the
light source with the help of marker.

● Note down the observation.

SURFACE MONITORING

No matter how thoroughly a lab or cleanroom has been sanitized, microorganisms

can quickly transfer from one surface to another by touching the surface with
something that is contaminated.

The surface monitoring procedure is performed to monitor the contaminants which

stick to the surface of the working machine, laminar air flows, conveyor belts, door
surface, floor surface, window glass wall surface.In these surface monitoring swab
method is used.
The swab method is suitable for small areas and areas with cracks and crevices.
Swabs can cover larger areas compared to contact plate.

Surface swab test:

• Dissolve 2 grams NaCl (mw 58.44) in 250 ml de-ionized or distilled water in

clean container mix it properly without any lumps in the container, and add water
to bring total solution volume to 1000 ml.

• Make 10 ml aliquots in sterile 15 ml culture tubes.

• Prepare saline peptone solution and dispense 10 ml quantity in a test tube

and put one cotton swab in it and sterilize in the autoclave at 15 lbs pressure and
121 o c for 15 minutes.

• Sanitize the outer surface of the saline tube with a filtered sporicidal agent
and transfer the tube into cRABS through pass box.
• Remove the excess solution from swab by pressing on side of the tube. Swab
5 X 5 cm2 area using parallel overlapping stroke with slow rotation of swab.
Repeat the by the stroke at the right angle to original stroke. After taking the swab
put the swab stick back into saline or peptone solution. The sample according to
the sampling plan. After taking swab write the location of swab and date of swab
with a market.

• Bring the tubes to quality control laboratory and vortex for 1 minute and
perform the bio burden according to membrane filtration method as mentioned
below.

• 0.1% Peptone Salt Solution is used as diluents for different test method.
Suspend 9.50 grams in 1000 ml distilled water. Heat if necessary to dissolve the
medium completely. Sterilize by autoclave at 15 lbs pressure (121 ⁰C) for 15
minutes i.e. validated cycle .
• Clean the syringe or the filter instrument 2-3 time or 40-60ml of the peptone
water.

• Incubate the Petri dishes at 22.5 ± 2.50 C for 3 days followed by 32.5 ± 2.50
C for 2 days in an upright position.

• After incubation count and observe the number of colonies on colony

counter or under a light source with the help of a marker. Record the results.

• Acceptance criteria

CLASS ACTION LIMIT

A 1CFU/plate
B 1CFU/plate

RESULT :
By performing environmental monitoring in innovulation health care
private limited the given results are observed.

ACTIVE AIR SAMPLING

SNO SAMPLE GRADE RESULTS

 LOCATION CFU/m3
TBC TFC TAMC
1 Left corner Grade A Air 0 0 0
[Laminar air flow] supply
2 Left front[Laminar Grade A Air 0 0 0
air flow] supply
3 Right Grade A AIR 0 0 0
corner[Laminar air Supply
flow]
4 Right Grade A Air 0 0 0
front[Laminar air supply
flow]
5 Center[Laminar air Grade C 01 01 01
flow]
6 [Near]BCM HEPA Grade C 02 02 02

7 AL3 RIS Grade D 03 03 03

8 Negative control
No Growth

PASSIVE AIR SAMPLING

SNO SAMPLING GRADE RESULTS

LOCATION CFU/Plate
TBC TFC TVMC
1 Left corner[Laminar Grade A Air supply 0 0
air flow]

2 Left front[Laminar Grade A Air supply 0 0

air flow]
3 Right Grade A Air supply 0 0
corner[Laminar air
flow]
4 Right front[Laminar Grade A Air supply 0 0
air flow]

5 Center[Laminar air Grade A Air supply 0 0

flow ]
6 BCM RIS Grade C 01 01

7 Ballon forming Grade C 01 01

8 Neck down Grade C 01 01

9 AL4 RIS Grade D 02 02

10 Negative control No Growth

CONTACT:

NAME OF THE PERSON RESULTS CFU/CONTACT PLATES

SNO Left elbow Right elbow
1 Person 1 0 0
2 Person 2 0 0
3 Person 3 0 0
4 Person 4 01 01
5 Person 5 01 01
6 Person 6 0 0
7 Person 7 02 02
8 Person 8 02 02
9 Person 9 02 02
10 Negative control No Growth

FINGER DAB:
 NAME OF THE PERSON RESULTS CFU/FINGER DAB
SNO
Left hand Right hand

1 Person 1 0 0
2 Person 2 0 0
3 Person 3 0 0
4 Person 4 01 01
5 Person 5 01 01
6 Person 6 0 0
7 Person 7 02 02
8 Person 8 02 02
9 Person 9 02 02
10 Negative control No Growth

SWAB TEST:

SNO SAMPLE LOCATION GRADE RESULT

CFU/SWAB

1 Laminar air flow 1 Grade A 00

2 Laminar air flow 2 Grade A 00
3 Leak test machine Grade C 00
4 Flat tester machine Grade C 01
5 Flat form Grade C 00
6 Ballon wrapping machine Grade C 02
7 Hot jaw bonder [right] Grade C 00
8 Hot jaw bonder[left] Grade C 01
9 Pass box Grade C 01
10 Annealing oven Grade C 01
 11 Marker swaging Grade C 02
12 Negative control No growth

DISSCUSSION :

Implementing air monitoring, personnel monitoring, and surface monitoring

methods in the manufacturing unit of coronary drug-eluting stents and PTCA
balloon catheters is crucial for ensuring product quality and patient safety.

Air monitoring involves the continuous assessment of air quality in the

manufacturing environment to detect airborne contaminants such as dust,
microbes, or volatile organic compounds. This method helps in identifying
potential sources of contamination and allows for corrective actions to be taken to
prevent the introduction of contaminants into the manufacturing process.

Personnel monitoring involves monitoring the workers involved in the

manufacturing process to ensure that they adhere to proper hygiene practices and
wear appropriate protective gear. This helps in minimizing the risk of personnel-
induced contamination, such as shedding of skin cells or hair, which can
potentially contaminate the medical devices being manufactured.

Surface monitoring involves regular inspection and cleaning of surfaces within the
manufacturing facility to prevent the accumulation of contaminants on equipment,
workstations, and other surfaces. This method helps in maintaining a clean and
sterile environment, which is essential for producing high-quality medical devices
free from contamination.

CONCLUSION:

By implementing air monitoring, personnel monitoring, and surface monitoring

methods, Innovolution healthcare manufacturing unit can effectively control and
mitigate the risk of contamination in the production of coronary drug-eluting stents
and PTCA balloon catheters. These proactive measures not only ensure product
quality but also uphold patient safety by reducing the likelihood of adverse effects
associated with contaminated medical devices. Continuous monitoring and
adherence to strict hygiene protocols are essential for maintaining the integrity of
the manufacturing process and delivering safe and effective medical devices to
patients

It is concluded that all sections of coronary heart stent manufacturing unit are
comply with world health organization standards in each sampling sites of all clean
zones shows viable and non viable counts within the limits set by local and
international clean room standards in risk assessment study.it is established that
personnel and external environment .differential positive air pressure is a barrier to
the mixing the external air with clean room environment.it counter the air pressure
of adjacent lower clean zone that separate the clean room environment from lower
clean zone.
REFERENCE:

https://www.cherwell-labs.co.uk/cherwell-labs-post/microbiological-air-samplers-
and-their-role-in-effective-em-programs

https://www.sigmaaldrich.com/IN/en/applications/environmental-testing-and-
industrial-hygiene/air-testing

https://www.tmmedia.in/environmental-monitoring-in-pharmaceutical-industries/

https://pharmaguidesline.com/sop-for-environmental-monitoring-by-swab-
personal-monitoring/

https://www.researchgate.net/publication/
332333561_A_Brief_Overview_on_Active_Air_Sampling_Procedure_for_Enviro
nment_Monitoring

https://www.viroxylabs.com/microbiological-testing-services/environmental-
monitoring-service/surface-monitoring-with-swab-method/