An Introduction to

Microbiological

Environmental Monitoring

Laura B. Guardi

An Introduction to EM

Common sampling tools

The limitations of EM

Tools for interpreting EM data

EM risk assessments

Limitations of EM

Just because you

don’t catch anything,

doesn’t mean there’s

nothing to catch

Looking in the

wrong place

Not looking for

long enough

Using the wrong

tools

Poor technique

Sampling Tools – Settle Plates (Passive Air)

So called as organism

settle ono the media

Airflows/currents will

impact the rate of

sedimentation

Enables continuous

monitoring – up to a point

Also used for “finger dabs”

Sampling Tools – Air Sampling (Active)

Sample a given volume

of air

Beware that different

samplers will have

different efficiencies –

compare apples with

apples

Recovery ~50%

Sampling Tools – Contact Plates

Used for sampling surfaces

& personnel garments

AKA Rodac plates (Replicate

Organism Detection And

Counting)

Should contain neutralisers

to inactivate disinfectant

surface residues

Can be used with an

applicator to ensure constant

pressure

~50% recovery

Swabs

Lower recovery than contact plates

Plain swabs ~10-20% recovery

Flocked swabs 40-60% recovery

Good for uneven/shaped surfaces and difficult to access locations

Good for qualitative rather than quantitative assessment

Precise technique should be followed

© FSL 7

EM – what it is, what it is not…

EM is one of a number of tools designed to perform a health

check of your clean rooms

It is not a stand alone system that defines drug product quality

It is a tool that tells you how other cleanroom controls are

functioning

HVAC

Gowning

Behaviours

Bioburden control of consumables, raw materials, and utlties

Cleaning and disinfection

Long term trending tool AND a snap shot of a time and place

It doesn’t control the environment; but it can show the

environment is in control

The Limitations of EM – ability to culture

70-90% of microbes will not grow on standard growth media

Is this an issue?

Not if you get it right – EM is a monitoring tool, not a sterility

test.

The media you use must be able to grow that small % of bugs that

can cope with the media used

Good supplier with a reproducible manufacturing method

Correct handling of the media – dehydration

Demonstrating the media can grow standard and ‘local’

isolates.

Real-time monitoring solutions that can identify viable organisms

exist, but have not yet been widely adopted

Limitations of EM – sample size

Sampling of a table by contact plate

Contact plate has a 55mm diameter > area of 2376mm2

A clean table 2 meters long by 1 meter wide – area of 2 million mm2

The contact plate samples 0.12% of the table.

What does a result of 3cfu on a contact plate tell you about the status

of the table?

An air sample?

1m3 sampled

Volume of air in a clean room? 5 meters, by 5 meters by 2.5 meters.

That is a volume of 62.5m3

With 20 air changes an hour that is 1250m3 of air an hour

30,000 m3 a day

210,000 m3 a week.

A single weekly sample is 0.000005% of that total airflow

Interpretation of Results

It is important to understand what a result of 0cfu means

Sterile

Viable non-culturable (the 90%)

Poor media control/quality

Poor recovery (recovery of a contact plate is ~50%)

Interference from disinfectant residues

But what does 1 CFU mean?

Often a CFU is not a single bacterium

A colony could arise from one cell or several.

Issue can occur through:

Poor sample mixing e.g. bacteria clumping together,

Settle plate picking up skin detritus.

Limitations of EM

The samples are very small.

Can you take more samples on a more frequent basis

to make up for the size (without having a negative

impact)?

Can you look at the data over a bigger period of time?

Trending.

Location of the sample is critical. It must represent the

activities in the room.

This does not necessarily mean the location most

likely to be the dirtiest – e.g. floor, but a location

that indicates the microbial load associated with the

activity being performed

Trending EM Data

Is this a problem?

0

5

10

15

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35

Micro Count

32 CFU (Human skin

commensals)

Action Limit of 50 CFU

Trending EM Data

Is it a problem now?

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Period

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Microbial Count

Trending EM Data

How about now?

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p1 p6 p11 p16 p21 p26 p31

MicrobialCount

Trending EM Data

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Trending EM Data

Are you OK with this data?

-1

1

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p1 p4 p7 p10 p13 p16 p19

MicrobialCount

Trending EM Data

Does this demonstrate a good state of control?

33

33.5

34

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35.5

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p1 p4 p7 p10 p13 p16 p19

MicrobialCounts

Trending EM Data

Same data, different scale

0

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p1 p4 p7 p10 p13 p16 p19

Microbial Counts

What is Typically Recovered?

Product

Adjacent areas

Supply air

Cleanroom air

Machine and

ancillaries

Room surfaces

People

Materials

Water sources

What is Typically Recovered?

Very few studies of pharmaceutical cleanroom

microflora published

Sandle, T. (2011): ‘A Review of Cleanroom Microflora:

Types, Trends, and Patterns’, PDA Journal of

Pharmaceutical Science and Technology, Vol. 65, No.

4, July–August 2011, pp392-403

A Review of Cleanroom Microflora

Pharmaceutical facility in south-east England

The cleanrooms examined represented:

40 Grade B rooms (of which five had Grade A

cleanzones)

35 Grade C cleanrooms

20 Grade D cleanrooms

EM regime used TSA and incubation regime:

20-25oC ≤5 days / 30-35oC ≤2 days

Time period: 2001 - 2010

A Review of Cleanroom Microflora

EU GMP Grade A and B clean areas = 6,729 isolates

A Review of Cleanroom Microflora

Grade A and B major

species:

Skin related microflora

represent the most common

genera isolated, with the

family Micrococcaceae (the

genera Micrococci and

Staphylococci) representing

>90% of the isolates

Order of detection:

Micrococcus luteus

Micrococcus lylae

Staphylococcus spp

Micrococcus spp

Staphylococcus epidermidis

Staphylococcus capitis

Staphylococcus hominis

Bacillus spp

Staphylococcus haemolyticus

A Review of Cleanroom Microflora

EU GMP Grade C and D

clean areas = 2, 500

isolates

More diversity due to

nature of operations and

presence of water

Order of detection:

Micrococcus luteus

Bacillus spp.

Micrococcus lylae

Micrococcus spp.

Staphylococcus spp.

Bacillus spp

Bacillus cereus

Pseudomonads

Corynebacterium spp

A Review of Cleanroom Microflora

Association between the microorganisms commonly

found in cleanrooms and those which are transient to

(short-term or long term-residents on) human skin .

Low incidents of Bacillus spp.,

Possible transfer into the cleanrooms via

personnel, dust, and material transfer .

Occasional, low-level incidences of microorganisms

resident within the human body.

Where there is a water source, some microorganisms

associated with water detected.

A Review of Cleanroom Microflora

Little variation over time - variation signalled that something

had gone wrong e.g. cleaning techniques, changes to

personnel, HVAC failure

Sample types:

Personnel samples: Gram-positive cocci occur most

frequently

Air samples: Gram-positive cocci occur very frequently

(people shedding)

Surfaces: Gram-positive cocci occur quite frequently.

Higher levels of Gram-positive rods (possible equipment

transfer link)

Where Gram-negative rods occur, this is from water on

surfaces

A Review of Cleanroom Microflora

Majority mesophilic aerobic or

facultatively aerobic bacteria.

Where specialist gases are

used some anaerobic found.

Thermophiles and

extremotolerant bacteria very

rare.

Common fungi in cleanrooms

are: Aspergillus, Penicillium

and Trychophyton.

I keep six honest serving men

(They taught me all I knew);

Their names are What and Why and When

And How and Where and Who.

Rudyard Kipling

Kipling’s Questions

What – surfaces, air, materials, utilities?

Where – which locations, where does the

action happen?

Why – is the action important, what’s the risk

to the process/product?

When – in operator, at rest, frequency?

How – contact plates, swabs, active

air/passive air?

Who – which people, if any?

Environmental Monitoring Workshop

Two scenarios to review:

Grade B filling room, with filling line under Grade A

Grade C ultrafiltration room, with a closed and

automated process

In your groups decide what you would monitor, how

you would monitor it and why (i.e. what the sample

tells you)