EUROPEAN PHARMACOPOEIA 9.2 5.1.6.

Alternative methods for control of microbiological quality

For example, biological indicators available for dry heat 5-1. TEST MICRO-ORGANISMS
sterilisation have D160 °C-values within a range of 1 to 5 min. Spores of Bacillus pumilus (e.g. ATCC 27142, NCTC 10327,
When exposed to the reference cycle of 2 h at 160 °C, a NCIMB 10692 or CIP 77.25) or other strains of
biological indicator with a D160 °C-value of 2.5 min would micro-organisms having demonstrated equivalent or better
be inactivated by 48 log10 scales. For dry-heat sterilisation performance are recommended.
processes, z-values of about 20 °C are typically assumed
in calculations of equivalence of cycle effectiveness 6. MICROBIAL PREPARATIONS FOR STERILISATION
(FH-calculations). FH is the equivalent time in minutes at a GRADE FILTRATION
temperature of 160 °C delivered by the sterilisation process to As stated in general chapter 5.1.1, certain products that
the product in its final container. For a biological indicator cannot be sterilised in their final container may be sterilised
with a D160 °C-value of 5 min, the D150 °C-value would be by a filtration process. In contrast to the biological indicators
about 16 min, and inactivation in the reference cycle would discussed in the previous sections, which assess kill-based
be 7.5 log10 scales. The use of a sterilisation process at a sterilisation, the biological challenge assesses the retention of
temperature reduced from the target temperature by 10 °C micro-organisms by the filters.
would give an expected 1 in 30 biological indicator units To validate the sterilisation process, it must be demonstrated
having surviving micro-organisms. that the filtration process (usually in a scaled-down model) is
3-3-1. Test micro-organisms capable of completely retaining a microbial challenge of at
least 107 CFU per square centimetre of effective filter surface
Spores of Bacillus atrophaeus (e.g. ATCC 9372, NCIMB 8058, using a suitable test micro-organism. This test should mimic
NRRL B-4418, or CIP 77.18) have been found to be suitable the actual filtration process as closely as possible. Where
for use as biological indicators for dry heat sterilisation feasible, the test is carried out in the product using the
processes performed at temperatures between 160 °C and specified filtration conditions. If this is not possible, e.g. due
180 °C. Where a test micro-organism other than Bacillus to the antimicrobial properties of the product, a medium as
atrophaeus is used, to ensure its suitability, the resistance similar as possible to the product must be used in the test.
of the test micro-organism for the sterilisation process is
evaluated as described in section 3-1-2. 6-1. TEST MICRO-ORGANISMS
For processes using a filtration system with a nominal pore
size not greater than 0.22 μm, a suspension of Brevundimonas
4. BIOLOGICAL INDICATORS FOR GAS STERILISATION diminuta (ATCC 19146, NCIMB 11091 or CIP 103020) is
recommended. The Brevundimonas diminuta suspension
The use of biological indicators is necessary for the must be prepared in order to achieve predominantly single
development, validation and monitoring of all gaseous cells of the smallest possible size. Other micro-organisms, for
sterilisation processes. Gas sterilisation is a multi-factorial example natural flora isolated from the product or process in
process : gas concentration, humidity, temperature, time, question, may be used if presenting a stronger challenge to
surface characteristics interact in a complex manner. A the sterile filtration system than Brevundimonas diminuta.
number of gas sterilisation processes are currently used, For filtration systems with a nominal pore size of 0.1 μm or
including ethylene oxide, hydrogen peroxide and peracetic less, a suspension of Acholeplasma laidlawii (ATCC 23206)
acid or combinations of the latter. may be used.
Gas surface disinfection is widely used for medical devices,
isolators, chambers, etc. Use for such purposes is outside
the scope of the European Pharmacopoeia but the use of 07/2017:50106
biological indicators as described in this general chapter may
assist in the validation of such disinfection processes.
4-1. TEST MICRO-ORGANISMS
4-1-1. Ethylene oxide sterilisation
5.1.6. ALTERNATIVE METHODS FOR
The use of spores of Bacillus atrophaeus (e.g. ATCC 9372,
NCIMB 8058, NRRL B-4418, or CIP 77.18), or other CONTROL OF MICROBIOLOGICAL
strains of micro-organism having demonstrated equivalent QUALITY
performance, is recommended for ethylene oxide sterilisation.
The number of viable spores is greater than or equal to 106 per The following chapter is published for information.
carrier. Test micro-organisms shall have D-values relevant to
1. GENERAL INTRODUCTION
the process to be validated. These biological indicators are
used routinely during each sterilisation cycle thus allowing the The objective of this chapter is to facilitate the implementation
effectiveness of the process to be checked. and use of alternative microbiological methods where this
can lead to efficient microbiological control and improved
4-1-2. Other processes assurance for the quality of pharmaceutical products.
It is the responsibility of the user to define the sterilisation The microbiological methods described in the European
cycle and the suitability of any biological indicator used. Pharmacopoeia have been used for over a century and
Geobacillus stearothermophilus has been found suitable for these methods for detecting, enumerating and identifying
vaporised hydrogen peroxide processes. micro-organisms still serve microbiologists well. Over the
years, these methods have been invaluable for the production
of microbiologically safe pharmaceutical products. However,
5. BIOLOGICAL INDICATORS FOR IONISING these microbiological methods are slow, and in the case of
RADIATION STERILISATION sterility tests, results are not available before an incubation
Unless otherwise indicated, biological indicators are not period of 14 days. Consequently, the results from these
generally considered necessary for validation of the sterilising methods seldom enable proactive corrective action to be taken.
dose for radiation sterilisation. The use of biological Alternative methods for the control of microbiological quality
indicators may however be required for the development and have shown potential for real-time or near real-time results
validation of ionising radiation sterilisation e.g. of tissues, with the possibility of earlier corrective action. These new
cell preparations or other specific cases (e.g. products with a methods, if validated and adapted for routine use, can also
potential for spore protection). offer significant improvements in the quality of testing.

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5.1.6. Alternative methods for control of microbiological quality EUROPEAN PHARMACOPOEIA 9.2

Alternative methods may be used for in-process samples of and Raman spectroscopy, restriction endonuclease banding
pharmaceutical products, particularly for the application of patterns and the use of genome sequencing methods such as
Process Analytical Technology (PAT), for environmental 16S rRNA gene sequence analysis for prokaryotes.
monitoring and for industrial utilities (e.g. production and Traditional biochemical and phenotypic techniques have been
distribution of water, steam etc.), thereby contributing to the shown to be less accurate and precise than genotypic methods.
quality control of these products.
Pure cultures are required for a precise identification and such
In this chapter, alternative microbiological methods cultures must be fresh and cultivated in appropriate media.
for pharmaceutical application are described. For each Databases are part of the systems and are included in the
method, the basic principle is stated and the advantages and primary validation. As identification methods depend on the
disadvantages of the method are discussed along with any use of databases, the extent of coverage of the database with
critical aspects to be considered. Potential uses that may be respect to the range of micro-organisms of interest must be
envisaged based on the principles of the method concerned are taken into account during validation. Appropriate software
given, but it is not intended to suggest that such applications allows customisation of the database, thereby allowing the
have been realised or that the list provided is exhaustive. user to add micro-organisms not previously included. This
It is not the intention of this chapter to recommend one possibility must be considered during the validation.
method over another, nor is it the intention to provide 2. GENERAL PRINCIPLES OF ALTERNATIVE METHODS
an exclusive or exhaustive list of alternative methods that
can be used for pharmaceutical microbiological control. Alternative microbiological methods employ direct and
The information herein may be used, however, in the indirect methods of detection ; in some instances amplification
process of choosing an alternative microbiological method of the signal is achieved by enrichment methods. In
as a supplement or as an alternative to pharmacopoeial recognition of these differences, and for convenience
microbiological methods and to give guidance on validation within this chapter, alternative methods for the control of
of the chosen method. If a suitable method is described in the microbiological quality are divided into 3 categories :
Pharmacopoeia, this method is the reference method. In this – growth-based methods, where a detectable signal is usually
rapidly developing field, other methods are likely to appear achieved by a period of culture ;
and the guidance offered herein may be equally applicable – direct measurement, where individual cells are
in these cases. differentiated and/or imaged ;
There are 3 major types of determination specific to – cell component analysis, where the expression of specific
microbiological tests : cell components offers an indirect measure of microbial
– qualitative tests for the presence or absence of presence and identification of micro-organisms.
micro-organisms ; In some instances, these distinctions are artificial, but enable a
working classification to be created.
– quantitative tests for enumeration of micro-organisms ;
2-1. GROWTH-BASED METHODS
– identification tests.
2-1-1. General critical aspects of methods based on early
1-1. QUALITATIVE TESTS FOR THE PRESENCE OR detection of growth
ABSENCE OF MICRO-ORGANISMS Such methods are critically dependent on microbial growth
In conventional microbiological analysis, this type of test is in order to provide an indication of the presence and/or
characterised by the use of turbidity or other growth-related number of micro-organisms. For the typically low levels of
changes in a culture medium as evidence of the presence of microbial contamination seen in pharmaceutical products,
viable micro-organisms in the test sample. The most common detection may take 24 h or longer. Increased sensitivity
example of this test is the test for sterility (2.6.1). Other can be achieved with filtered products. In this case, after
examples include those tests designed to evaluate the presence filtration, the membrane filter is incubated in or on the
or absence of a particular type of viable micro-organism in a medium and the result is expressed as presence or absence
sample. The conventional sterility test may be replaced by, in the quantity corresponding to the filtered volume. These
for example, tests based on bioluminescence or solid phase systems, if they use an incubation step in liquid media, do
cytometry, gas detection or autofluorescence. Nucleic acid not offer quantitative information, but a presence/absence
amplification techniques (NAT) (2.6.21) may also be used for determination in the quantity analysed. Analysis of more
the detection of mycoplasmas (2.6.7). than one sample quantity may offer a semi-quantitative
1-2. QUANTITATIVE TESTS FOR ENUMERATION OF estimation (limit test). The major benefit of early detection
MICRO-ORGANISMS methods compared to classical methods is often the capacity
Membrane filtration and plate count methods are to simultaneously process a large number of samples and the
conventional methods used to estimate the number of potential to obtain a result in a shorter time.
viable micro-organisms present in a sample. The Most The methods described below can be used for quantitative,
Probable Number (MPN) method is another example of such semi-quantitative or qualitative analyses. They are also
methods and was developed as a means of estimating the non-destructive, therefore subsequent identification of the
number of viable micro-organisms present in a sample not micro-organism is possible.
amenable to direct plating. Examples of alternative methods 2-1-2. Electrochemical methods
for enumeration include autofluorescence, flow cytometry,
direct epifluorescent filter technique (DEFT) and solid phase Principles of measurement. Micro-organisms multiplying and
cytometry. metabolising in appropriate growth media produce highly
charged ionic metabolites from weakly charged organic
1-3. IDENTIFICATION TESTS nutrients leading to the modification of electrical properties
Biochemical and morphological characterisation of an in such media. These changes in impedance (measured by
unknown micro-organism is the classical approach to conductance or capacitance) are monitored with electrodes
identification. Recently developed methods have streamlined included in the culture vessels and in contact with the culture
and automated aspects of this identification, especially in the medium. The measurable end-point is the time taken to detect
areas of data handling, analysis and storage. Several alternative a predetermined impedance change ; for particular types of
approaches that have been integrated into these methods micro-organisms, the detection time is inversely proportional
include biochemical reactions, carbon substrate utilisation, to the initial inoculum size. For yeasts and moulds, which
characterisation of fatty acid composition, mass spectroscopy only produce small changes in electrical impedance, an

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EUROPEAN PHARMACOPOEIA 9.2 5.1.6. Alternative methods for control of microbiological quality

indirect measurement of conductance can be used. Direct generally a destructive process which should be considered
measurement of capacitance can also be carried out. with respect to any subsequent need for identification of
Critical aspects. There is no direct relationship between the detected micro-organisms.
original microbial level and the detectable end-point. Potential uses. Presence/absence testing of filterable or
non-filterable samples (e.g. final drug products, in-process
Potential uses. Microbiological assay of antibiotics, efficacy of control samples, media fill), total aerobic microbial count
antimicrobial preservation and presence/absence testing. (TAMC), environmental and water monitoring, testing for
2-1-3. Measurement of consumption or production of gas efficacy of antimicrobial preservation.
Principles of measurement. Appropriate growth media 2-1-5. Turbidimetry
is utilised by actively multiplying and metabolising Principles of measurement. Microbial growth leads to
micro-organisms, leading to the production of metabolites or detectable changes in medium opacity, which can be accurately
the elimination of specific nutrients. These methods detect quantified by optical density measurements at a specified
microbial growth either by changes in the electrical properties wavelength. In its simplest form, such measurements are
of a sensor in response to a change in gas composition performed using a standard spectrophotometer, generally over
or by colorimetric changes of a sensor in response to a wavelength range of 420-615 nm. Alternative automated
physicochemical changes in the growth medium in contact systems employ microtitre plate readers offering a continuous
with that sensor. The systems are based on non-destructive readout with early detection of optical density change.
techniques which enable subsequent identification or strain Critical aspects. Attempts have been made to extrapolate the
typing of the micro-organisms. Bacteria and/or fungi may be initial microbial contamination from the time to detection, but
grown in closed containers and continuous monitoring can this is limited to healthy micro-organisms with reproducible
be performed using automated instruments that measure gas growth characteristics.
evolution (e.g. CO2) or consumption (e.g. O2) as surrogate
markers of microbial growth. Furthermore, the production of Potential uses. By means of calibration graphs, determination
metabolites or elimination of nutrients can lead to changes in of the inoculum size of microbial suspensions for use in
pH or redox potential. All of these changes can be measured pharmacopoeial tests. In automated mode, microbiological
either directly or indirectly as changes in colorimetric markers assay of antibiotics and testing for efficacy of antimicrobial
in the growth medium. preservation.
Critical aspects. There is no direct relationship between the 2-1-6. Growth detection using selective and/or indicative
original microbial level and the detectable end-point. The media
incubation temperature, the physiological state and type of Principles of measurement. The ability to detect the presence
micro-organism, the initial load and the algorithm for data of specific enzymes using suitable chromogenic substrates has
processing can significantly affect the results or the time to led to the development of a large number of methods for the
detection. identification of micro-organisms employing either manual or
automated techniques. The incorporation of such substrates
Potential uses. Presence/absence testing of filterable or into a selective or non-selective primary isolation medium
non-filterable samples (e.g. final drug products, in-process can eliminate the need for further subculture and biochemical
control samples, media fill or container closure integrity testing for the identification of certain micro-organisms.
testing). Consequently, chromogenic liquid or solid culture media are
2-1-4. Bioluminescence designed to reveal specific enzymatic activities for detection
Principles of measurement. Adenosine triphosphate (ATP) is and differentiation of micro-organisms. In these particular
a well-documented marker of cell viability. In this method, media, defined substrates are introduced into the formulation
ATP first needs to be released from the micro-organisms and are metabolised by the specific cell enzyme of a given
using an appropriate extractant, followed by an assay using bacterium or fungus during growth. These substrates, which
the luciferin/luciferase enzyme system, which emits light in are linked to coloured indicators, are chosen according to
proportion to the ATP present. The signal-to-noise ratio can the diagnostic enzymatic activity sought. Furthermore,
be increased by addition of ADP and converting this ADP chromogenic broth can be used for early or improved
into released ATP. detection of contamination (e.g. in media fill or broth-based
detection methods).
Qualitative method : micro-organisms are cultivated in
The use of innovative media presents several advantages,
liquid medium. The emitted light is measured with a
namely improved discrimination of colonies in a mixed
bioluminometer and is expressed in relative light units (RLU)
culture, ease of use and ease of interpretation. In addition,
(e.g. bioluminescence in a tube or a well of a microtitre
response times are shorter as the growth and identification of
plate). The RLU obtained from the sample is compared with a
the micro-organism are simultaneous.
pre-determined threshold value. The result is positive if the
RLU obtained with the analysed sample exceeds the threshold Critical aspects. Validation of the media must be undertaken
value. carefully to ensure a combination of specificity, selectivity and
robustness. The quality of the signal is based not only on the
Quantitative method : micro-organisms are captured on a careful choice of the enzymes or indicators used as the basis
membrane and cultivated by incubation on agar medium. of detection (as these enzymes may be present in different
Using a charge coupled device (CCD) camera, the ATP micro-organism genera), but also on the physico-chemical
released from microcolonies can be detected by light emission characteristics of the medium, e.g. pH.
and a quantitative determination is possible.
Potential uses. Detection of specified micro-organisms and
Critical aspects. If the sample has a high level of bacterial qualitative testing (e.g. media fill and container closure
contamination, the detection is rapid. For low levels of integrity testing) and quantitative testing (e.g. water testing).
contamination, it is necessary to increase the number
2-2. DIRECT MEASUREMENT
of micro-organisms using an incubation step in culture
media (liquid or solid). The yield of ATP varies from 2-2-1. Solid phase cytometry
one micro-organism to another and can depend on Principles of measurement. Micro-organisms are stained for
several factors including the species, the growth phase viability by exposure to a conjugated, initially non-fluorogenic,
of the cell, the nutritional status, the cellular stress or fluorophore. An intact cellular membrane is required to
the cellular age. Additional factors such as turbidity, retain and accumulate the fluorophore within the cytoplasm.
sample colour or product matrix effects can also influence Inside metabolically-active microbial cells, the conjugate is
bioluminescence measurements. Extraction of ATP is enzymatically cleaved and the fluorescent derivative is released

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intracellularly. Micro-organisms are collected on a membrane surfaces), subsequent staining on the sheet itself, followed by
filter either before or after viability staining. direct observation using an epifluorescence microscope.
Membrane surfaces retaining vital-stained cells are then Critical aspects. The distribution of micro-organisms on
scanned by a laser beam and epifluorescent excitation allows the membrane affects method robustness. The intensity of
the detection of single, viable fluorescent micro-organisms. fluorescence can be influenced by the staining process and the
Appropriate software allows differentiation of viable metabolic status of the micro-organisms. Fluorescence is not
micro-organisms from autofluorescent particles. The high necessarily an indicator of viability. A brief period of culture
sensitivity and rapidity of the method permit detection of on the filter surface prior to staining allows microcolony
microbial contaminants within a few hours. Total cell counts formation ; these microcolonies stain readily, can be easily
(viable and non-viable) can be obtained using fluorescent enumerated and are demonstrable evidence of viability.
staining. Potential uses. DEFT is generally limited to low viscosity
Critical aspects. Metabolically active, fastidious and viable fluids, although pre-dilution or pre-filtration has occasionally
non-culturable micro-organisms can all be detected. This been applied to viscous or particulate products. Monitoring
may result in reappraisal of the microbial limits established of microbial contamination has been successfully applied to
for the samples under evaluation. Spores require initiation of aqueous pharmaceuticals.
germination to enable detection. Single cell detection may be 2-2-4. Autofluorescence
achievable, but identification of isolates might not be possible.
False positives may occur due to autofluorescent particles that Principles of measurement. The presence of endogenous
can be difficult to differentiate from micro-organisms. Signal autofluorescent molecules and metabolites (e.g. NADPH,
discrimination and enhancement can be aided by microcolony flavoproteins) within micro-organisms allows the early
growth. detection and quantitative enumeration of microcolonies
or single cells. For direct measurements, the laser-induced
Potential uses. Rapid and sensitive method for the non-specific
autofluorescence of a single micro-organism is captured
evaluation of microbial contamination.
by a detector, while for growth-based systems, automated
2-2-2. Flow cytometry sequential imaging of the membrane surface on agar medium
Principles of measurement. Fluorophore-labelled over the incubation period is employed and image overlay
micro-organisms can be detected in suspension as they allows differentiation of growing microcolonies from
pass through a flow cytometer. Viable micro-organisms fluorescent particulates. The emitted light is detected by a
can be differentiated from non-viable particles by use of CCD camera. Non-destructive detection allows identification
a viability-indicating fluorophore (see 2-2-1). The cell of contaminants at the end of the incubation period.
suspension stream is dispersed into a narrow channel Critical aspects. For a non-growth based measurement,
and exposed to a laser which excites the fluorophore. viable, but non-culturable, micro-organisms might be
Micro-organisms and particles are then counted in different detected. It may be difficult to distinguish between culturable
channels depending on whether or not they contain a micro-organisms, viable but non-culturable micro-organisms
fluorescent cell. and/or other particles.
Critical aspects. Direct flow cytometry may be applied to the Potential uses. Environmental monitoring, filterable in-process
microbiological analysis of both filterable and non-filterable samples, water testing and product release for both sterile and
materials, and after possible enrichment in the case of the non-sterile applications.
low contamination levels. It gives near real-time detection,
but is not as sensitive as solid phase cytometry. To increase 2-3. CELL COMPONENT ANALYSIS
sensitivity for use in the pharmaceutical field, it is often 2-3-1. Phenotypic techniques
necessary to add an incubation step in culture media, in which 2-3-1-1. Immunological methods
case the method becomes a combination of a growth-based
Principles of measurement. Antibody-antigen reactions
method and a direct detection method. Particle size and
can be employed to detect unique cellular determinants of
number may have a significant effect on performance, and
specific micro-organisms. These reactions can be linked to
samples may require serial dilution. With the exception of
agglutination phenomena and colorimetric or fluorimetric
filterability, similar considerations to those in solid phase
end-points, which offer both quantitative and qualitative
cytometry apply. Clumping of bacteria can be a problem (e.g.
detection. Enzyme-linked immunosorbent assays (ELISA)
Staphylococcus aureus).
offer simple solid-phase methodologies.
Potential uses. In contrast to solid phase cytometry, this
method offers the potential to detect and enumerate microbial Critical aspects. Immunological detection methods
contamination in materials containing particulate matter and depend on the unique expression of specific identifiers,
if the material cannot be filtered. If a pre-incubation step is but do not necessarily demonstrate the presence of viable
needed, the method becomes a qualitative determination. micro-organisms.
Potential uses. Detection and identification of specified
2-2-3. Direct epifluorescent filtration technique (DEFT) micro-organisms.
Principles of measurement. This technique may be considered
2-3-1-2. Fatty acid profiles
a forerunner of solid phase cytometry. Micro-organisms,
concentrated by filtration of the sample, are stained with a Principles of measurement. The fatty acid composition of
fluorescent dye (formerly acridine orange and now more micro-organisms is stable, well conserved and shows a high
commonly 4,6-diamidino-2-phenylindole (DAPI)), that can degree of homogeneity within different taxonomic groups.
be detected by epifluorescent illumination. Fluorescent vital The isolate is grown on a standard medium and harvested.
staining techniques, as employed in solid phase cytometry The fatty acids are saponified, methylated and extracted,
(see 2-2-1), are amenable to DEFT, and fluorescent redox dyes and the occurrence and amount of the resulting fatty acid
such as 5-cyano-2,3-ditolyltetrazolium chloride (CTC) can be methyl esters are measured using high-resolution gas
used to highlight respiring cells. Coupled with microscopy, chromatography. The fatty acid composition of an unknown
the method allows rapid detection of micro-organisms with isolate is compared with a database of known isolates for a
an absolute sensitivity that is dependent on the volume of possible match and identification.
product filtered and the number of fields of view examined. Critical aspects. The use of fatty acid profiles for microbial
Semi-automated auto-focusing systems coupled to image identification requires a high degree of standardisation. It is
analysis have served to improve the utility of this method. critical for the fatty acid composition of microbial cells that
A modification of the principle involves sampling using isolates are grown using standardised media and standard
an adhesive sheet (which permits collection of cells from incubation conditions. Standard conditions for operation of

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EUROPEAN PHARMACOPOEIA 9.2 5.1.6. Alternative methods for control of microbiological quality

the gas chromatograph must also be employed, with frequent an antimicrobial agent. The results are based on measurable
runs of calibration standards and known isolates being very changes (e.g. turbidity, chromogenic or fluorogenic reaction)
important. due to the growth or inhibition of the micro-organism
Potential uses. Identification or characterisation of under investigation. Comparison of the metabolic and/or
environmental and product microbial contamination antimicrobial resistance profile with a database allows for
(for contaminant tracing and detection of specified identification of the culture. These methods can be performed
micro-organisms). manually or by semi- or fully automated instruments.
2-3-1-3. Fourier transform infrared (FTIR) spectroscopy Complementary tests can be performed in cases of poor
discrimination. Subcultures can help in cases of indeterminate
Principles of measurement. A Fourier transformation of the results.
infrared spectrum of whole micro-organisms gives a stable,
Critical aspects. A fresh physiological culture is required. The
recognisable pattern typical of the taxonomic groups of
performance of the system is also dependent on the selected
micro-organisms. The analysis of the FTIR pattern can be
phenotypic parameters, which must be stable, significant and
performed with commercially available instruments. The
in sufficient number.
isolate is grown on a standard medium and harvested. Cell
mass is transferred to a carrier, and the infrared spectrum is Potential uses. Identification or characterisation of
recorded. The Fourier transformation is calculated and the environmental and product microbial contamination
pattern is compared with a database of known isolates for a (for contaminant tracing and detection of specified
possible match and identification. micro-organisms).
Critical aspects. The use of FTIR patterns for microbial 2-3-2. Genotypic techniques
identification requires a high degree of standardisation. It is Identification and detection of micro-organisms as well as
critical for the FTIR pattern of microbial cells that isolates characterisation of strains belonging to the same species may
are grown using standardised media and standard incubation be achieved by direct detection of nucleotide target sequences
conditions. The cells must be in the same state of the growth that are unique for a particular microbial species or microbial
cycle when analysed, and particular attention must be paid to group, and are targets of the genotypic (DNA or RNA-based)
this in the validation process. detection techniques. These detection techniques may be
Potential uses. Identification or characterisation of separated into 3 broad categories : direct hybridisation, nucleic
environmental and product microbial contamination acid amplification and genetic fingerprinting.
(for contaminant tracing and detection of specified 2-3-2-1. Direct hybridisation
micro-organisms). General principles of measurement. DNA probes are short,
2-3-1-4. Mass spectrometry labelled, single-strand segments of DNA that hybridise with a
Principles of measurement. Ionised particles released by complementary region of microbial DNA or RNA. The probe
exposing microbial isolates to a laser in a vacuum can be or target DNA is usually labelled with either radioactive,
analysed by mass spectrometry, providing characteristic fluorescent or chromogenic molecules in order to provide a
spectra. Similarly, intact microbial cells, when subject to hybridisation signal. Hybridisation assays include fluorescence
intense ionisation under matrix-assisted laser desorption in situ hybridisation (FISH) and microarray-based techniques.
ionisation-time of flight (MALDI-TOF) mass spectrometry, General critical aspects. Hybridisation generally requires a
release a distinctive pattern of charged species. Such spectra large amount of the target DNA for analysis, which may result
can be compared with known profiles. in lower detection sensitivity. The availability of suitable
Critical aspects. The isolates must be cultured under probes may be limited.
standardised conditions prior to analysis. Potential uses. Due to the high specificity of the
Potential uses. Identification or characterisation of sequence-based hybridisation reaction, this method may be
environmental and product microbial contaminants used for both detection and identification of micro-organisms.
(for contaminant tracing and detection of specified 2-3-2-2. Nucleic acid amplification techniques (NAT)
micro-organisms). General principles of measurement. NAT relies on the
2-3-1-5. Biochemical assays based on physiological reactions reiteration of the DNA polymerisation process, leading to
an exponential increase of a specific nucleic acid fragment.
Principles of measurement. Systems capable of performing The polymerase chain reaction (PCR) is the most widely
biochemical assays based on physiological reactions are used method for target DNA amplification. In this cyclic
used for the identification of micro-organisms. In the
process, a specific DNA fragment is copied by a thermostable
presence of a pure colony, the five basic steps for these DNA polymerase enzyme in the presence of nucleotides and
assays are preparation, inoculation, incubation, readings oligonucleotide primers, previously designed to flank the
and interpretation. These steps are usually preceded by a
target sequence and to hybridise with it (see also general
description of the colony morphology, a differentiation test chapter 2.6.21). After PCR, the amplified nucleic acid targets
(e.g. Gram stain), a description of the cellular morphology can be analysed using several methods of post-amplification
and/or other early biochemical differentiation tests (e.g.
analysis : fragment size analysis in gel electrophoresis, DNA
oxidase, catalase, coagulase) in order to determine the sequencing or specific detection by hybridisation with
appropriate testing protocol. a fluorescent-labelled probe. Real-time PCR eliminates
The Gram stain is often a key characteristic upon which the need for further post-amplification processing and
further testing is based. Alternatives to the traditional staining offers the additional advantage that the likelihood of
method include the potassium hydroxide (KOH) string cross-contamination is minimised. An important advantage of
test, the aminopeptidase test, a fluorescent staining method real-time PCR is the ability to quantify the starting amount of
and a limulus amoebocyte lysate (LAL) based assay. Test the DNA target sequence in the original sample, in contrast to
kits are available for the latter 3 methods. The fluorescent conventional PCR techniques, which are based on end-point
staining method requires a fluorescence microscope or a flow detection. Since the amount of PCR product detected at
cytometer. the beginning of the exponential phase of the amplification
Microbial cell suspensions are tested using biochemical reaction correlates with the initial starting amount of the
(assimilation or susceptibility) test kits (plates or strips). DNA target, modern real-time PCR techniques have been
Anaerobic and aerobic micro-organisms develop characteristic developed to measure this exponential phase of the reaction.
reactions to selected biochemical substances. They are also Automated real-time PCR systems are commercially available.
known to utilise specific carbon, nitrogen, phosphorus and For identification of species, either species-specific probes or
sulfur sources or to be inhibited by a specific concentration of primers can be used.

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RNA can also be amplified by both conventional and Real-time PCR is needed for quantitative or semi-quantitative
real-time PCR after transcription into cDNA using a reverse analysis of the target. Besides quantitative determinations,
transcriptase enzyme. This technique is known as reverse the real-time PCR technique allows simultaneous detection
transcriptase PCR (RT-PCR) and it enables detection of multiple targets in a single sample, as long as appropriate
and identification of RNA viruses or viable organisms. primers and probes that allow for multiplexing are
Alternatively, specific RNA-based amplification techniques, employed. The sequencing of different genes (e.g.16S rDNA,
for example nucleic acid sequence-based amplification or 23S rDNA, rpoB, Gyr) is best applied to the identification of
transcription-mediated amplification, are available. Both micro-organisms.
techniques produce RNA amplicons, in contrast to PCR which 2-3-2-3. Genetic fingerprinting
only produces DNA amplicons, even when starting from an Principles of measurement. Genetic fingerprinting is the
RNA target. identification of a strain on the basis of its DNA profile
Types of target to be amplified. Regardless of the type of NAT (or RNA for RNA viruses). Individual DNA profiles may
used, the specificity of the test is determined by the target DNA be different due to genetic diversity between strains of the
sequence under evaluation. For identification/characterisation same species, and the aim of the fingerprinting methods is
purposes, the 16S or 23S ribosomal RNA genes may be used to discriminate between these strains. The classical genetic
as targets. The 16S rRNA gene is an evolutionary-conserved fingerprinting technique characterises micro-organisms using
gene present in all bacterial species, and is a broad range restriction fragments of chromosomal DNA from bacterial
target as it is a universal marker for bacterial detection. The and fungal genomes.
23S rRNA gene is not widely used as a single target, but the Different strains from the same species may exhibit different
16S-23S rRNA transcribed intergenic spacer regions can be patterns and these differences are referred to as restriction
employed to distinguish between certain closely related species fragment length polymorphisms (RFLPs). As cutting the
and/or to identify subtypes. Alternative broad-range targets chromosomal DNA with restriction enzymes generates
include the groEL and tuf genes. Apart from broad-range too many fragment bands to be efficiently and accurately
targets, species-specific sequences can be used as targets for compared, several modifications of the conventional
micro-organism identification. Depending on the species, RFLP-based method have been developed. Examples of
either specific surface antigens, virulence factors or genes the kind of technologies used are ribotyping, pulsed-field
which code toxins may be amplified to detect and identify gel electrophoresis (PFGE) and amplified fragment length
micro-organisms. polymorphism (AFLP). Several other fingerprinting methods
General critical aspects : use PCR to selectively amplify defined subsets of DNA
– the target and the primers chosen must be specific for a restriction fragments from the entire genome, for example
particular micro-organism or group of micro-organisms ; random amplified polymorphic DNA (RAPD) and variable
number tandem repeats (VNTR).
– the sensitivity of the methods is highly dependent on
the efficiency of the lysis protocol and how successfully Critical aspects. All fingerprinting techniques require that
the DNA targets can be purified and concentrated in the the micro-organism is present as a pure culture. Depending
sample ; on the method, a preliminary enrichment cultivation step
may be necessary if a defined quantity or a specific DNA
– the presence of inhibitors of the enzymatic process results preparation is required for the test, e.g. AFLP and PFGE.
in false negative reactions ; The discriminatory power, the reproducibility, the expertise
– the procedures are prone to cross-contamination from needed and the labour-burden vary among techniques.
background DNA resulting in false positive results. The major criticism of conventional RFLP analysis is the
Depending on the aim, a choice must be made between complexity of the banding patterns. The discriminatory power
amplification of either a DNA or an RNA target, as this target of ribotyping (based on patterns of rRNA genes) is less than
choice affects the correlation with viability. DNA targets that of PFGE (based on patterns of the whole genomic DNA)
are generally more widely used for identification purposes, or some PCR-based methods, but it has the advantage that it
but the use of DNA as a marker has the disadvantage that can be a highly automated system. Although PFGE is one of
dead micro-organisms can also be detected. As mRNA is the most highly discriminatory fingerprinting methods, it is
rapidly degraded in dead cells, it is considered a marker for time-consuming and technically demanding in the laboratory
viability. Furthermore, mRNA is the obligatory target for the as it is not automated. It also requires the use of standardised
identification of RNA viruses. protocols. AFLP has high reproducibility, but requires
Critical aspects of (semi-) quantitative detection by real-time technical expertise and the interpretation of results may need
PCR. Quantification of the target requires generation of automated computer analysis. The reproducibility of RAPD
appropriate standards and the use of standardised procedures. may be poor, so it must be performed in a standardised way.
Potential uses. Genetic fingerprinting methods are mainly
Critical aspects of RT-PCR. RNA is less stable compared used for strain discrimination (characterisation below species
to DNA, so it requires more attention during processing. level). They are a powerful tool for investigating and tracing
Depending on the quality of the RNA isolation, the efficiency the source and the spread of microbial contamination.
of the cDNA synthesis can vary. RT-PCR can be used to
specifically detect RNA if DNA contamination of the RNA 3. VALIDATION OF ALTERNATIVE MICROBIOLOGICAL
sample is low. METHODS
Critical aspects of using the 16S or 23S rRNA gene as a target 3-1. INTRODUCTION
for species identification. 16S rRNA gene sequencing is a Validation, whilst subject to a variety of context-specific
valuable method for identification of bacteria provided that definitions, can be generally defined as a method to establish
appropriate universal primers from databases are selected. Its documented evidence that a process will consistently achieve
discriminatory power depends on the variability and the length its intended goal. Therefore, to validate an alternative
of the 16S rRNA gene within a certain species. Regarding microbiological method, it is essential to understand and
the use of assays targeting the 16S-23S rRNA intergenic define what the procedure is intended to achieve.
spacer regions, the choice of appropriate species-specific Typically, pharmaceutical microbiological methods use
primers/probes is of critical importance due to the potential specific characteristics of micro-organisms as indicators or
polymorphism of such regions. detection principles in order to determine microbiological
Potential uses of NAT. Due to the high sensitivity and quality. The information generally sought is presence/absence,
specificity of amplification techniques, they are suitable number, viability and/or identity of micro-organisms in
for both detection and identification of micro-organisms. a given product or environment. Any given method will

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EUROPEAN PHARMACOPOEIA 9.2 5.1.6. Alternative methods for control of microbiological quality

usually provide an indirect and conditional measure of 3-2-3. Primary validation

microbiological quality. For example, the total number and The supplier, using a panel of test micro-organisms appropriate
viability of micro-organisms can be indicated by the number for the intended use, must characterise the principle of
of colonies appearing under a certain set of conditions of detection. Depending on the type of alternative method,
sample preparation, cultivation and incubation ; reproduction relevant validation criteria shall be selected from those listed
in classical microbiology is hence taken as the general below :
indicator for viability. There are other parameters, however,
that can be used as a viability measure, such as the level of – prerequisite treatment of sample or micro-organisms ;
ATP or the accumulation or metabolism of substrates in living – type of response ;
cells. The results from different viability-indicating methods
– specificity ;
may not always be identical ; micro-organisms may not be able
to reproduce on a given medium, but may still accumulate and – detection limit ;
metabolise a substrate. Conversely, micro-organisms may be – quantitation limit ;
unable, at a given state of damage, to accumulate a substrate,
but may still be able to recover and reproduce. – range ;
– linearity ;
Similar considerations arise with the multiplicity of methods
used for identification of micro-organisms. Therefore, – accuracy and precision ;
while characterisation of the pattern of metabolic activity is – robustness of the method in a model system.
frequently used for species identification, alternative methods
3-2-4. Validation for the intended use
also exist. Again, the outcomes obtained may not be fully
consistent for the different identification methods, as one Validation for the intended use should encompass the
answer may be appropriate for the construction of a correct entire process, from the decision to change any aspects of a
phylogenetic correlation tree, while another may be more microbiological testing programme to on-going routine use. It
useful in the context of pathogenicity or other property of the should consist of the following phases :
differentiated micro-organisms. – user requirement specification (URS) ;
3-2. VALIDATION PROCESS – design qualification (DQ) ;
Two levels of validation must be envisaged for the application – installation qualification (IQ) ;
of alternative microbiological methods, namely primary
validation and validation for the intended use. The supplier – operational qualification (OQ) ;
of the alternative technology typically performs primary – performance qualification (PQ).
validation of a method, whereas validation for the intended
use, which is a verification of the suitability or applicability The supplier and user have different tasks to perform with
of the method in a given situation, must be seen as the regard to the validation and implementation of an alternative
responsibility of the user. method. These tasks are summarised in Table 5.1.6.-1.
Where specific equipment is critical for the application of a Table 5.1.6.-1 – Tasks to be undertaken during the validation
method, the equipment, including computer hardware and process
software, must be fully qualified.
Normally carried out by
3-2-1. Description of the technique Activity
Supplier User
In order to characterise a specific microbiological method,
Primary validation + -(1)
the principle of detection must be clearly described by the
supplier. Through primary validation, the method must be URS (instrument, application) - +
fully detailed with respect to the conditions required for
Description of the technique + -(2)
application, the materials and equipment needed and the
expected signal. The user shall critically review the available Risk benefit analysis -(3) +
information.
Design qualification (DQ) - +
3-2-2. Risk-benefit analysis (4)
Installation qualification (IQ) - +
For validation of specific alternative microbiological methods, (4)
Operational qualification (OQ) - +
it is critical that the purpose of the quality assurance procedure
is precisely outlined, as this defines the type and depth of Performance qualification (PQ) :
information needed. The information obtained by, and the
- verification of primary validation data - +
limitations of, the pharmacopoeial method and the alternative
given by the supplier ;
method must be considered and compared in a risk-benefit
analysis. - verification for the intended use (e.g. - +
sterility testing, TAMC/TYMC, …) ;
The risk level in adopting an alternative method varies
- method suitability test - +
depending on the technology considered, the methodology it
replaces, the nature of the measurements taken (qualitative, (1) The user performs primary validation if they employ the alternative
quantitative or identification), the particular product method for a use other than that defined by the supplier.
or process attribute being evaluated, the location of the (2) The user shall critically review information provided by the
measurement in the manufacturing process chain and various supplier.
other factors. (3) As part of commercialisation, the supplier may list advantages of
the alternative method over pharmacopoeial techniques.
Risk analysis tools may be utilised in order to determine (4) IQ/OQ for complex equipment, IQ/OQ is often outsourced to
which alternative method is to be implemented, to assist in supplier.
the justification of its implementation or to better understand
the impact of implementation on production and/or product 3-2-4-1. User requirement specification (URS)
quality. An alternative method can be justified for use if the The URS describes the functions that the method must be
information obtained gives a scientifically sound measure of capable of performing and will form the basis of the method
microbiological quality, and if the limitations of the method selection process. It is an essential document, as acceptance
are not more severe than those of the pharmacopoeial method. testing will be based on the requirements detailed therein. It

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5.1.6. Alternative methods for control of microbiological quality EUROPEAN PHARMACOPOEIA 9.2

is important to consider data management capabilities at this make it possible to satisfy the criteria described by the supplier
stage, particularly within a regulatory context. The URS shall of the method in the model system used for the primary
at least address the following items : validation.
– application of the instrument : Verification of primary validation data given by the supplier
– the type of analysis to be performed (e.g. quantitative, (see 3-2-3). The method is verified using the panel of test
semi-quantitative, qualitative or identification). micro-organisms given by the corresponding pharmacopoeial
– detection limit or quantitation limit (sensitivity): chapter. The alternative method must be applied according to
the specified procedure of the supplier, without the samples
– the detection limit may be linked to time to detection to be analysed under the responsibility of the user, and must
(TTD) ; be shown to give comparable results as characterised in the
– the required level of sensitivity, which will depend on model system used by the supplier.
the current specification, the dilution regime and the Verification for the intended use (e.g. sterility testing, total
test sample size for the existing test method under aerobic microbial count (TAMC)/total combined yeasts/moulds
replacement. count (TYMC), etc). The following points, where applicable,
– specificity : should be addressed :
– the ability of the alternative test method to – compatibility of the response with the sample preparation
selectively detect the micro-organisms or classes of that the user normally performs for product testing
micro-organisms ; this should be based on historical (method suitability testing) ;
data generated from the pharmacopoeial test method
– limit and range of detection of the method with regard to
and complemented by information from the supplier sample size and sample availability ;
of the alternative method ;
– specificity of the response with regard to the influence of
– the ability to detect only the required viable
the product ingredients ;
micro-organisms ;
– for identification methods, the extent of coverage of the – linearity of the response with regard to the types of samples
database with respect to the range of micro-organisms to be analysed ;
of interest. – accuracy and precision of the response with regard to the
– number and type of samples : types of samples to be analysed.
– the nature of samples to be tested and the manufacturing Acceptance criteria for the method will need to be defined as
output per batch or work-shift. a function of the application and the validation data.
– time to detection (TTD) or time to result (TTR) : 3-3. TYPES OF MICROBIOLOGICAL TESTS
– the TTD or TTR is an important attribute for alternative Validation of a microbiological method is the process
microbiological methods ; for monitoring purposes, a whereby it is experimentally established by the user that
relatively short TTD (e.g. a few hours) allows corrective the performance characteristics of the method meet the
actions to be taken at an early stage ; for quality control requirements of the intended application. As microbiological
purposes, a short TTD may be less critical. tests have 3 basic applications (qualitative, quantitative
and identification), 3 separate sets of validation criteria are
– data management capabilities : required. These criteria are described below and summarised
– the new instrumentation may need to have laboratory in Table 5.1.6.-2.
information management system (LIMS) interface
capability and external server compatibility, and the Table 5.1.6.-2 – Validation criteria for qualitative, quantitative
data management tools should be defined ; evidence of and identification tests
software validation and functional testing reports will Criterion
Qualitative Quantitative Identification
be required to support each part of the software and test test test
firmware functions. Accuracy +(1) + +
3-2-4-2. Design qualification (DQ) Precision - + -
The DQ provides documented evidence that the design of Specificity + + +
any associated equipment is suitable for correct performance
(2)
of the method. Most alternative method systems are Detection limit + - -
based on commercial off-the-shelf equipment. The DQ Quantitation limit - + -
is most suitably performed, therefore, by the instrument
developer/manufacturer. Nevertheless, the user shall verify Linearity - + -
that the equipment meets the specifications laid down in the Range - + -
URS for the intended application.
3-2-4-3. Installation qualification (IQ) Robustness + + +

The IQ provides documented evidence that the equipment Suitability testing + + -

has been provided and installed in accordance with its + + -
Equivalence testing
specifications.
(1) Performing an accuracy test of the alternate method with respect to
3-2-4-4. Operational qualification (OQ) the pharmacopoeial method can be used instead of the validation of
The OQ provides documented evidence that the installed the limit of detection test.
equipment operates within predetermined limits when used (2) May be needed in some cases.
in accordance with its operational procedures.
3-2-4-5. Performance qualification (PQ) 3-3-1. Validation of alternative qualitative tests for the
presence or absence of micro-organisms
The PQ provides documented evidence that the method,
with the equipment installed and operated according to 3-3-1-1. Specificity
operational procedures, consistently performs in accordance The specificity of an alternative qualitative method is its ability
with predetermined criteria and thereby yields correct to detect only the required micro-organisms, i.e. does not
results for the method. This is typically done with a panel of generate false positive results. This can be demonstrated using
micro-organisms (e.g. pharmacopoeial test strains, in-house a panel of appropriate micro-organisms. Where relevant for
isolates or stressed/slow-growing micro-organisms). This the purpose of the test, mixtures of micro-organisms are used
assures that the conditions employed by the user laboratory during validation. For qualitative methods that rely on growth

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EUROPEAN PHARMACOPOEIA 9.2 5.1.6. Alternative methods for control of microbiological quality

to demonstrate presence or absence of micro-organisms, Accuracy may be shown by preparing and testing a suspension
specificity is adequately addressed by demonstrating the of micro-organisms at the upper end of the test range and
growth promotion properties of the media. For those methods serially diluting to the lower end of the test range. For
that do not require growth as an indicator of microbial example, if the alternative method is meant to replace the
presence, the specificity assures that extraneous matter in the pharmacopoeial plate count method for viable counts, then
test system does not interfere with the test. a reasonable range might be 100-106 CFU/mL. If instead, it
3-3-1-2. Detection Limit is a replacement for the MPN method, a much narrower
range may be used. At least 1 suspension for each test
The detection limit of an alternative qualitative method is
micro-organism dilution must be analysed.
the lowest number of micro-organisms in a sample that
can be detected under the stated analytical conditions. A The alternative method should be shown to recover at least as
microbiological limit test determines the presence or absence many micro-organisms as the pharmacopoeial method using
of micro-organisms in a defined quantity of the sample under appropriate statistical analysis.
test. Due to the nature of microbiological tests, the detection The protocol used to check the linearity of the method
limit reflects the number of micro-organisms present in (see 3-3-2-5) may also be used to check the accuracy.
the original sample before any dilution or incubation steps. The suspensions of micro-organisms prepared for the
The detection limit of the alternative method must not be a alternative method are counted at the same time using the
number greater than that of the pharmacopoeial method. pharmacopoeial method.
It is essential that the detection limit is determined using a 3-3-2-2. Precision
sufficient number of replicates and a number of independent The precision of an alternative quantitative method is the
determinations. degree of agreement between individual test results when
3-3-1-3. Robustness the procedure is applied repeatedly to multiple samplings
The robustness of an alternative qualitative method is a of homogeneous suspensions of micro-organisms under
measure of its capacity to remain unaffected by small but the prescribed conditions. Precision should be split into
deliberate variations in method parameters (e.g. incubation repeatability and intermediate precision under normal or
period or incubation temperature range). Robustness is a routine operating conditions. Repeatability (also referred to as
validation parameter best suited to determination by the within-run variability) refers to the use of the microbiological
supplier of the method. Nevertheless, if the user modifies method with the same sample (replicate) in the same
critical parameters, any effect on robustness must be evaluated. laboratory over a short period of time with the same analyst
Robustness of a qualitative method is judged by its ability to and the same equipment. It gives the minimum variability
detect the test micro-organisms after deliberate variations to of the method. Intermediate precision (includes run-to-run
the method parameters. variability and within-run variability) refers to the use of
the microbiological method applied to different sample
3-3-1-4. Suitability testing preparations of the product under test in the same laboratory
The alternative method must be applied according to the with different analysts, equipment and/or on different days. It
specified procedure and with the samples to be analysed under gives the maximum variability of the method. The precision
the responsibility of the user. It must be shown that the test of a microbiological method is usually expressed as the
sample does not interfere with the system’s detection capacity standard deviation or relative standard deviation (coefficient
or microbial recovery. Specific points to be addressed are : of variation). At least 1 suspension in the middle of the
– the ability of the test to detect micro-organisms in the test range is analysed. The number of replicates is chosen
presence of the sample matrix ; so that the entire test can be carried out during the same
– verifying if the sample matrix interferes with the alternative working session, i.e. under the same operating conditions and
system (e.g. background signal or inhibiting chemical without any change in the suspension of micro-organisms.
reactions). For intermediate precision, other working sessions are
then carried out under conditions of maximum variability
Acceptance criteria for the method in routine use will need to (different reagents, operators and/or days, etc.). The variance
be defined as a function of the application and the validation in the results observed in each of the working sessions is
data. calculated. If the variances are homogeneous, the variance of
3-3-1-5. Equivalence testing the repeatability can be calculated. The inter-group variance
Equivalence testing of 2 qualitative methods can be conducted of the results is also calculated and the resultant variance
directly on the validation parameters. This approach of the intermediate precision is given as the sum of the
requires an adequate comparison experiment at low levels of variance of the repeatability and the inter-group variance.
inoculation (e.g. less than 5 CFU) with sufficient numbers The coefficient of variation is then calculated. Alternative
of replicates for relevant strains of test micro-organisms. methods must demonstrate precision comparable to that of
Alternatively, and in some cases additionally, equivalence the pharmacopoeial methods.
testing can be carried out by the parallel testing of a predefined 3-3-2-3. Specificity
number of samples or for a predefined period of time. This The specificity of an alternative quantitative method is its
parallel testing can be justified based on a risk assessment. The ability to quantify only the required micro-organisms, i.e. does
alternative method must enable an unequivocal decision as to not generate false positive results. This may be demonstrated
whether compliance with the standards of the monographs using a panel of appropriate micro-organisms. Where relevant
would be achieved if the official method was used. for the purpose of the test, mixtures of micro-organisms are
3-3-2. Validation of alternative quantitative tests for used during validation. For those methods that do not require
enumeration of micro-organisms growth as an indicator of microbial presence, the specificity
3-3-2-1. Accuracy assures that extraneous matter in the test system does not
interfere with the test.
The accuracy of an alternative quantitative method is the
closeness of the test results obtained by the alternative method 3-3-2-4. Quantitation limit
to those obtained by the pharmacopoeial method. Accuracy The quantitation limit of an alternative quantitative method
must be demonstrated across the practical range of the is the lowest number of CFUs in a sample which can be
test. It is usually expressed as the percentage recovery of quantitatively determined with suitable precision and accuracy.
micro-organisms by the alternative method compared to the It is essential that the quantitation limit is determined from a
percentage recovery using the pharmacopoeial method, taking number of replicates. The results of the linearity and accuracy
into account statistical analysis. studies can also be used. In this case, the lowest concentration

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in the linear range is considered to be the quantitation limit of If the result of the alternative method cannot be expressed
the method. The quantitation limit of the alternative method as the number of CFUs, equivalence testing is performed
must not be greater than that of the pharmacopoeial method. using suitable parameters, followed by statistical analysis to
3-3-2-5. Linearity demonstrate that the results of the alternative method enable
an unequivocal decision as to whether compliance with the
The linearity of an alternative quantitative method is its ability standards of the monographs would be achieved if the official
(within a given range) to produce results that are proportional method was used.
to the concentration of micro-organisms present in the sample.
The linearity must be determined over a reasonable range 3-3-3. Validation of alternative identification tests
(e.g. 100-106 CFU/mL) so as to correspond to the purpose There is a large body of evidence that different methods vary
of the alternative method. One approach would be to select considerably in their ability to identify micro-organisms. It
different concentrations of each test micro-organism and test must be accepted that a method of identification needs to
several replicates. For each concentration, an appropriate be internally consistent, but may differ from others in its
number of replicates is chosen to confirm linearity. The identification of micro-organisms.
number of replicates is chosen so that the entire test can be 3-3-3-1. Accuracy
carried out during the same working session. After checking The accuracy of an alternative identification method is
the homogeneity of the variances of the results obtained for its ability to identify the desired micro-organism to the
each concentration, the regression line is calculated. Linearity required taxonomic level. It must be demonstrated using
is demonstrated if the estimated slope is significant and if the well-characterised reference micro-organisms, e.g. type
test for deviation from linearity is non-significant (see general strains. Accuracy of the identification method is usually
chapter 5.3). expressed as the number of correct identifications divided by
3-3-2-6. Range the total number of identifications.
The range of an alternative quantitative method is the interval 3-3-3-2. Specificity
between the upper and lower levels of micro-organisms as The specificity of an alternative identification method is its
determined from the related studies of precision, accuracy ability to discriminate micro-organisms actually present
and linearity using the specified method ; it is dependent on from interfering factors that cause false identification
the intended application. results. Such factors include chemical substances and
3-3-2-7. Robustness mixtures of micro-organisms, which cause the test to identify
The robustness of an alternative quantitative method is a micro-organisms not actually present in the sample material
measure of its capacity to remain unaffected by small but (e.g. the presence of mixtures of DNA material from 2
deliberate variations in method parameters (e.g. incubation micro-organisms in a sequencing test leading to the false
period or incubation temperature range). Robustness is a identification of a third micro-organism).
validation parameter best suited to determination by the 3-3-3-3. Robustness
supplier of the method. Nevertheless, if the user modifies The robustness of an alternative identification method is a
critical parameters, the effects on robustness must be measure of its capacity to remain unaffected by small but
evaluated. Robustness of an alternative quantitative method deliberate variations in method parameters (e.g. incubation
is judged by its ability to accurately enumerate the test period or incubation temperature range). Robustness is a
micro-organisms after deliberate variations to the method validation parameter best suited to determination by the
parameters. supplier of the method. Nevertheless, if the user modifies
3-3-2-8. Suitability testing critical parameters, the effects on robustness have to be
evaluated. Robustness of an identification method is judged
The alternative method must be applied according to the
by its ability to correctly identify the test micro-organisms
specified procedure and with the samples to be analysed
after deliberate variations to the method parameters.
under the responsibility of the user. It must be shown that the
test sample does not interfere with the system’s enumeration
capacity or microbial recovery. Specific points to be addressed 07/2017:50111
are :
– the ability of the test to detect micro-organisms in the
presence of the sample matrix ;
– verifying if the sample matrix interferes with the alternative 5.1.11. DETERMINATION OF
system (e.g. background signal or inhibiting chemical
reactions). BACTERICIDAL, FUNGICIDAL
Acceptance criteria for the method are defined as a function OR YEASTICIDAL ACTIVITY OF
of the application and of the validation data. ANTISEPTIC MEDICINAL PRODUCTS
3-3-2-9. Equivalence testing This general chapter describes a test that can be used for
Equivalence testing of 2 quantitative methods can be the determination of antimicrobial activity in antiseptic
conducted directly on the validation parameters. This medicinal products that are miscible with water and intended
approach requires an adequate comparison experiment for administration by direct contact with the skin or mucous
at low levels of inoculation (e.g. less than 5 CFU) with membranes. The extent of testing is dependent on the declared
sufficient numbers of replicates for relevant strains of antimicrobial activity of the product.
test micro-organisms. Alternatively, and in some cases The test determines whether a product exhibits bactericidal,
additionally, equivalence testing can be carried out by the fungicidal or yeasticidal activity and complies with an
parallel testing of a predefined number of samples or for a established specification for such activity.
predefined period of time. This parallel testing can be justified This test cannot replace or confirm the assessment of the clinical
based on a risk assessment. efficacy of such preparations.
If the result of the alternative method can be expressed as
the number of CFUs per weight or per volume, statistical 1. PRINCIPLE
analysis of the results shall demonstrate that the results of Antimicrobial activity is determined by adding test
the alternative method enable an unequivocal decision as to suspensions of micro-organisms (bacteria, fungi or yeasts,
whether compliance with the standards of the monographs separately) to the sample antiseptic product. The mixture
would be achieved if the official method was used. is maintained at 33 ± 1 °C for contact times of 5 min for

4348 See the information section on general monographs (cover pages)