ISO/TR 4752:2025
(Main)Biotechnology — Inventory of methods for detection of microbiological contamination in mammalian cell culture
Biotechnology — Inventory of methods for detection of microbiological contamination in mammalian cell culture
This document provides an inventory of methods for the detection of microbiological contamination in mammalian cell culture. This document includes considerations for the selection of methods to test the presence of common contaminants such as bacteria, fungi, viruses and mycoplasma. This document is not applicable to prions and protists. This document is intended for use by biomedical researchers, biobank operators and others performing mammalian cell culture.
Biotechnologie — Inventaire des méthodes pour la détection de la contamination microbiologique dans la culture cellulaire de mammifères
General Information
Standards Content (Sample)
Technical
Report
ISO/TR 4752
First edition
Biotechnology — Inventory
2025-06
of methods for detection of
microbiological contamination in
mammalian cell culture
Biotechnologie — Inventaire des méthodes pour la détection de
la contamination microbiologique dans la culture cellulaire de
mammifères
Reference number
© ISO 2025
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General concepts . 3
4.1 Introduction .3
4.2 Detection process.3
5 Contamination test information . 4
6 Critical control points in detection . 5
6.1 Samples .5
6.2 Reagents and Equipment .5
6.3 Operation .6
6.4 Personnel .6
6.5 Environment .6
Annex A (informative) Available and exemplary testing methods for bacteria and fungi . 7
Annex B (informative) Available and exemplary testing methods for Viruses .12
Annex C (informative) Available and exemplary testing methods for mycoplasma .15
Bibliography .18
iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee
has been established has the right to be represented on that committee. International organizations,
governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely
with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
The document was prepared by Technical Committee ISO/TC 276, Biotechnology, Subcommittee SC 1,
Analytical methods.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
Introduction
Cell culture plays an extremely important role in biotechnology and life science research. Therefore, the
reliability of scientific data from cell culture is essential. One of the most common problems in cell culture
is microbiological contamination. This usually results in catastrophic cell death or contamination sustained
at low levels (sometimes due to the use of antibiotics), and thus impacts the reliability of experimental data
derived from cell culture. Nonetheless, despite its prevalence and importance, the lack of open discussion
discourages development and learning of best practices to avoid microbial contamination in cell culture.
Microbial contamination is an economic issue as it affects the reproducibility of scientific data, the efficiency
of in vitro cell culture work and most importantly, the safety of laboratory operators. It is therefore
necessary to monitor microbial contamination through a full understanding of the source of contamination
and employ good testing techniques before and during cell culture.
When cultured cells have been contaminated, first the various aspects of the contamination are identified,
including the nature of contamination, the time of contamination and the operating environment.
Contaminants can include bacteria, fungi, mycoplasma, viruses and other types of organisms. In order to
demonstrate the absence of microbiological contamination in cell culture, it can be necessary to conduct a
series of tests for likely organisms and such an approach will benefit from risk assessment of the cell type,
cell origin and reagents used for their culture.
Environmental monitoring in areas where cells are cultured and stored can reduce the risk of microbial
contamination from the processing environment.
This document provides an inventory of methods for the detection of microbiological contamination in
mammalian cell culture in order to provide users with an overview that also includes information on pro-
and contra indications for the listed methods in relation to the appropriate sample type.
v
Technical Report ISO/TR 4752:2025(en)
Biotechnology — Inventory of methods for detection of
microbiological contamination in mammalian cell culture
1 Scope
This document provides an inventory of methods for the detection of microbiological contamination in
mammalian cell culture. This document includes considerations for the selection of methods to test the
presence of common contaminants such as bacteria, fungi, viruses and mycoplasma. This document is not
applicable to prions and protists.
This document is intended for use by biomedical researchers, biobank operators and others performing
mammalian cell culture.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
accuracy
closeness of agreement between a measured quantity value and the true quantity value of a measurand
[SOURCE: ISO/IEC Guide 99:2007, 2.13, modified — Notes to entry deleted.]
3.2
digital PCR
dPCR
procedure in which nucleic acid templates are distributed across multiple partitions of nominally equivalent
volume, such that some partitions contain template and others do not, followed by PCR amplification of
target sequences and detection of specific PCR products, providing a count of the number of partitions with
a positive and negative signal for the target template
Note 1 to entry: Nucleic acid target sequences are assumed to be randomly and independently distributed across the
partitions during the partitioning process.
Note 2 to entry: The count of positive and negative partitions is normally based on end point detection of PCR products
following thermal cycling, however real-time qPCR monitoring of PCR product accumulation is additionally possible
for some dPCR platforms.
[SOURCE: ISO 20395:2019, 3.10]
3.3
intended use
intended purpose
use for which a product, process, or service is intended according to the specifications, instructions or
information or multiple of them provided by the manufacturer or user
[SOURCE: ISO 23033:2021, 3.26]
3.4
microbial contamination
presence of unintended bacteria, fungi, mycoplasma, or viruses
[SOURCE: ISO 11139:2018, 3.171]
3.5
next generation sequencing
NGS
non-Sanger-based high-throughput nucleic acid sequencing
Note 1 to entry: Millions or billions of nucleic acid strands can be sequenced in parallel, yielding substantially more
throughput.
Note 2 to entry: NGS (next-generation-sequencing) is also well recognized as MPS (massively parallel sequencing) in
the ISO 20397 series.
Note 3 to entry: MPS or NGS covers long read sequencing and short read sequencing.
[SOURCE: ISO/DIS 20397-3:2024, 3.16]
3.6
PCR assay
qPCR or dPCR (3.2) measurement method with specified oligonucleotide primers (and, in some cases, a
probe or probes) that is used to identify or quantify a nucleic acid target
[SOURCE: ISO 20395:2019, 3.23]
3.7
reagent
substance used in chemical/biochemical analysis or other reaction
[SOURCE: ISO 20391-1:2018, 3.19]
3.8
resolution
smallest change in a quantity being measured that causes a perceptible change in the corresponding
indication
Note 1 to entry: Resolution can depend on, for example, ratio of signal and noise (internal or external). It can also
depend on the value of a quantity being measured.
[SOURCE: JCGM 200:2012, 4.14, modified — Friction not mentioned in Note 1 to entry.]
3.9
ribosomal RNA
rRNA
non-coding ribonucleic acid contained in ribosomes
Note 1 to entry: The nucleotide sequence of rRNA subunits can be used to detect microorganisms (e.g. 16S rRNA, 18S rRNA).
3.10
sample
one or more parts taken from a system
[SOURCE: ISO 23033:2021, 3.46]
3.11
sensitivity
quotient of the change in an indication of a measuring system and the corresponding change in a value of a
quantity being measured
Note 1 to entry: Sensitivity of a measuring system can depend on the value of the quantity being measured.
Note 2 to entry: The change considered in a value of a quantity being measured must be large compared with the
resolution.
[SOURCE: ISO/IEC Guide 99:2007, 4.12, modified — "sensitivity" is given as the only preferred term.]
3.12
test sample
small aliquot of the sample that is prepared for measurement in the method of interest
Note 1 to entry: Generally, test samples are representative of the sample they are prepared from and are sometimes
referred to as “representative test sample(s)”.
[SOURCE: ISO 20391-2:2019, 3.36]
3.13
validation
confirma
...
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