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II. Microbiological Testing II. Basic Microbiological Tests

Medical devices contaminated with pathogens
may be a source of infection for humans. There-
fore, according to the Medical Device Directive,
devices must be designed and manufactured in
such a way as to eliminate or reduce the risk of
infection to the patient, user or third parties as
far as possible.
Consequently, medical device manufacturers, in
particular if they manufacture sterile devices,
must produce their products under adequate and
microbiologically monitored conditions involving
a clear zone concept. Furthermore, the final pro-
ducts, and if necessary the starting and packaging
materials, must be subjected to bioburden deter-
minations in order to support the later steriliza-
tion success.
The microbiological basic tests of mdt inform
the manufacturer about the microbiological sta-
tus of their products, determine antiinfective
properties of materials and disinfectants and help
to control manufacturing processes and steriliza-
tion procedures.
II. Basic Microbiological Tests
1. BIOBURDEN DETERMINATIONS
The bioburden of a product is determined in order to
assess the number of viable microorganisms present on
a medical device or its packaging material during or at
the end of the manufacturing process.
As the recovery rates of bacteria and molds, which
adhere to a medical device or its package, strong-
ly depend on the respective material properties, any
bioburden determination procedure needs to be vali-
dated according to EN ISO 11737-1. The determined
recovery rate is then applied to the measured biobur-
den value in order to evaluate the real microbiological
status of a product. For the validation of the biobur-
den determination two different guidelines can be used:
First, EN ISO 11737-1 can be used. This standard
specifies the use of the following three microorganisms:
Staphylococcus aureus, Fusarium solani and Bacillus
subtilis. Alternatively, the validation can also be per-
formed using the harmonized Pharmacopeia (Ph. Eur.,
USP, and JP). According to these three harmonized
Pharmacopeia a total of five microorganisms must be
used: Staphylococcus aureus, Pseudomonas aerugino-
sa, Bacillus subtilis, Candida albicans and Aspergillus
brasiliensis.
1.1. Validation of the Bioburden Determination
A. Procedure according to EN ISO 11737-1 and
EN ISO 11137-2
Inoculation of sterile test items with the following
microorganisms: Staphylococcus aureus, Fusarium
solani and Bacillus subtilis
Detachment of inoculated microorganisms using a
rinsing solution
Membrane filtration, dilution series or spread
plates
Determination of recovery rates
Incubation time up 1 5 days
II. Basic Microbiological Tests
B. Procedure in analogy to harmonized Pharmacopeia
(Ph. Eur., USP and JP)
Inoculation of sterile test items with the following
microorganisms: Staphylococcus aureus, Pseu-
domonas aeruginosa, Bacillus subtilis, Candida
albicans and Aspergillus brasiliensis
Detachment of inoculated microorganisms using
a rinsing solution
Membrane filtration, dilution series or spread
plates
Determination of recovery rates
Incubation time 1 5 days
1.2. Bioburden Determination
Procedure according to Ph.Eur., USP, or
EN ISO 11737-1
Membrane filtration, dilution series or spread
plates
Application of 12 media
Incubation time 7 days
Applicable for products and packaging materials
2. STERILITY TESTING
Sterility tests confirm that a product does not contain
any viable microorganisms. Sterility tests, however,
can not be applied to prove the success of a steriliza-
tion procedure. These procedures have to be validated
separately. CHAPTER III
2.1. Validation of the Sterility Test
Procedure according to Ph.Eur.and USP (confirmation
of the absence of antimicrobial activity of product)
Application of 12 media
Comparison of growth of microorganisms (< 100
CFU) in the absence and presence of product
Test of up to 6 different microorganisms
Incubation time 14 days
Applicable for products and packaging materials
II. Basic Microbiological Tests
2.2. Direct Transfer to Culture Medium
Procedure according to EN ISO 11737-2 or in
analogy to Ph.Eur and USP
Application of 12 media
Positive controls
Incubation time 14 days
Applicable for products and packaging materials
2.3. Membrane Filtration Method
Procedure according to EN ISO 11737-2 or in
analogy to Ph.Eur and USP
Rinsing of samples in saline solution
Application of 12 media
Positive controls
Incubation time 14 days
Applicable for products and packaging materials
3. DIFFERENTIATION AND DETECTION OF PATHO-
GENIC MICROORGANISMS
Procedure according to Ph.Eur. or USP
Determination of bacteria, molds and yeasts
Microscopic differentiation, gram-staining
Differentiation using culture techniques
Differentiation using biochemical tests
Detection / exclusion of pathogenic microorganisms
by amplification in selective nutrient media, e. g.
Escherichia coli
Pseudomonas aeruginosa
Staphylococcus aureus
Salmonella spec.
4. ANTIMICROBIAL ACTIVITY TESTING
Some medical devices can be applied repeatedly or can
be used up by patients over a longer period of time. For
such products effective disinfectants, respectively effec-
tive preservative agents, are required. In the following,
some tests are presented which are applied to investi-
gate such antimicrobial properties.
II. Basic Microbiological Tests
4.1. Suspension Test to Determine Bactericidal
Effects of Disinfectants
Procedure according to EN 1040; further procedures
according to EN 1276, EN 13697 or EN 13727
Test of final disinfectants or active ingredients
Validation of inactivation of the disinfecting agent
Inoculation for example with Pseudomonas
aeruginosa and Staphylococcus aureus
Determination of time-dependent killing rates
Evaluation of success: reduction by 10
5
after
60 min at 20 C
4.2. Suspension Test to Determine Fungicidal Effects
of Disinfectants
Procedure according to EN 1275; further procedures
according to EN 1650, EN 13697 or EN 13624
Test of final disinfectants or active ingredients
Validation of inactivation of the disinfecting agent
Inoculation with Candida albicans and Aspergillus
niger
Determination of time-dependent killing rates
Evaluation of success: reduction by 10
4
after
60 min at 20 C
4.3. Preservative Efficacy Test (PET)
Procedure according to Ph.Eur. or USP
Freshly prepared samples or samples after
simulated use
Validation of inactivation of the preservative agent
Inoculation of test solution with Pseudomonas
aeruginosa, Staphylococcus aureus, Candida
albicans and Aspergillus brasiliensis
(USP: Escherichia coli in addition)
Determination of bioburden at various times
intervals
Evaluation of results (fulfillment of criteria A and B)
II. Basic Microbiological Tests
4.4. Oligodynamic Activity of Metals and Plastic
Materials
Modified Preservative Efficacy Test according to
Ph.Eur.
Equilibration of test items in physiological saline
solution
In case of strong antimicrobial materials: parallel
testing in CASO Bouillon
Validation of inactivation of the preservative
materials effect
Inoculation with suitable microorganisms
Inoculation with 100 to 1000 cfu per ml
Bioburden determination at relevant time intervals
Growth controls in a parallel time schedule
Determination of reduction rates in comparison
to controls
5. MICROBIOLOGICAL MONITORING OF
PRODUCTION
According to the MDD devices which are supposed to
be sterilized must be manufactured under appropriate
and controlled conditions. In this regard the European
GMP Guideline offers helpful suggestions as how the
practical performance concerning the production area,
and the personnel working in this area, can be realized.
In cooperation with the manufacturer, mdt develops
a suitable sampling plan which specifies the time regi-
men and sampling positions in the production area
and which determines action and alert limits for each
sampling position. Thereby the microbiological status
of the controlled environment can be documented and
evaluated for the following test systems:
Microorganisms on surfaces: Total colony count
(e. g., Tryptic Soy Agar with Neutralizer)
Airborne microorganisms: Sedimentation test
(e. g., CASO-, SAB-Agar Plates)
Microorganisms in water supplies: Plating of
samples (e. g., CASO-, SAB-Agar Plates)
Furthermore, mdt offers hygiene training courses for
employees working in a cleanroom environment.