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ASTM E1533-00

(Practice)

Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4'-6-Diamidino-2-2 Phenylindole (DAPI) Staining

Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4'-6-Diamidino-2-2 Phenylindole (DAPI) Staining

SCOPE
1.1 This practice covers procedures used for the detection of mycoplasma contamination by indirect DNA staining.
1.2 This practice does not cover direct methods for the detection of mycoplasma or other indirect methods such as enzymatical detection or DNA probes.
1.3 This practice does not cover methods for the identification of mycoplasma organisms.
1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.
1.5 This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

General Information

Status
Historical
Publication Date
09-May-2000
ICS
07.100.10 - Medical microbiology
Technical Committee
E48 - Bioenergy and Industrial Chemicals from Biomass
Current Stage
Ref Project

Relations

Replaces
ASTM E1533-93 - Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4`-6-Diamidino-2-2 Phenylindole (DAPI) Staining
Effective Date
10-May-2000
Replaced By
ASTM E1533-00(2006) - Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4`-6-Diamidino-2-2 Phenylindole (DAPI) Staining (Withdrawn 2014)
Effective Date
01-Nov-2006
Referred By
ASTM E2363-23 - Standard Terminology Relating to Manufacturing of Pharmaceutical and Biopharmaceutical Products in the Pharmaceutical and Biopharmaceutical Industry
Effective Date
10-May-2000

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ASTM E1533-00 - Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4'-6-Diamidino-2-2 Phenylindole (DAPI) StainingASTM E1533-00 - Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4'-6-Diamidino-2-2 Phenylindole (DAPI) Staining
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ASTM E1533-00 - Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4'-6-Diamidino-2-2 Phenylindole (DAPI) Staining
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E 1533 – 00
Standard Practice for
Indirect Detection of Mycoplasma in Cell Culture by 4*-6-
1
Diamidino-2-2 Phenylindole (DAPI) Staining
This standard is issued under the fixed designation E 1533; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 3.1.3 indirect detection of mycoplasma—detection of my-
coplasma by DNA staining or any method other than cultiva-
1.1 Thispracticecoversproceduresusedforthedetectionof
tion.
mycoplasma contamination by indirect DNA staining.
3.1.4 mycoplasma—the smallest prokaryotes capable of
1.2 This practice does not cover direct methods for the
living freely, lacking a cell wall, having a circular double-
detection of mycoplasma or other indirect methods such as
stranded DNA relatively rich in adenine and thymine, and
enzymatical detection or DNA probes.
containing 16s and 23s ribosomal RNAs.They can be found as
1.3 This practice does not cover methods for the identifica-
contaminants in cell cultures.
tion of mycoplasma organisms.
1.4 The values stated in SI units are to be regarded as the
4. Significance and Use
standard. The values given in parentheses are for information
4.1 Mycoplasmacontaminationofcellculturesisacommon
only.
problem that can affect the growth, metabolism, and function
1.5 This standard does not purport to address all of the
of cultured animal cells. The ability to detect mycoplasma in
safety problems, if any, associated with its use. It is the
cell cultures provides an opportunity to ensure that cells are
responsibility of the user of this standard to establish appro-
free of contamination, and to replace those that are not. For
priate safety and health practices and determine the applica-
additional information, see Practices E 1531, E 1532, and
bility of regulatory limitations prior to use.
E 1536. Strict adherence to established, well-tested procedures
2. Referenced Documents is necessary. This practice was developed by Task Group
2
E48.01.02 to assist in developing and maintaining an estab-
2.1 ASTM Standards:
lished regimen for mycoplasma detection by indirect 48-6-
E 1531 Practice for Detection of Mycoplasma Contamina-
Diamidino-2-Phenylindole (DAPI) fluorochrome staining.
tion of Cell Cultures by Growth on Agrose Medium
4.2 This practice is intended for use in examining cultured
E 1532 Practice for Detection of Mycoplasma Contamina-
animal cells for the presence of mycoplasma contamination.
tion of Cell Cultures by Use of the Bisbenzamide DNA-
4.3 This practice is not intended for use in the detection of
Binding Flurorochrome
mycoplasma contamination in serum, culture media, or sys-
E 1536 Practice for Detection of Mycoplasma Contamina-
tems other than cultures of animal cells.
tion of Bovine Serum by the Large Volume Method
4.4 All cell cultures to be examined for mycoplasma should
3. Terminology undergo a minimum of two passages in antibiotic-free tissue
culture medium before testing.
3.1 Definitions:
3.1.1 DAPI staining—staining of DNA in particular by
5. Quality Control
using DAPI fluorochrome stain.
5.1 Visually examine the DAPI stain concentrate routinely
3.1.2 direct detection of mycoplasma—detection of myco-
forcontamination.Freshstockshouldbepreparedperiodically.
plasma by cultivation in culture media.
5.2 Indicator cells:
5.2.1 Indicator cells support the growth of mycoplasma
1
This practice is under the jurisdiction of ASTM Committee E48 on Biotech-
species and provide positive and negative controls.
nology and is the direct responsibility of Subcommittee E48.02 on Characterization
5.2.2 Use continuous cell lines such as the African green
and Identification of Biological Systems.
monkey kidney cell line, Vero,American Type Culture Collec-
Current edition approved May 10, 2000. Published July 2000.
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
tion (ATCC CCL81) as indicator cells as described in this
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
practice; 3T6 mouse fibroblast (ATCC CCL 96) may also be
Standards volume information, refer to the standard’s Document Summary page on
used.
the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
1

---------------------- Page: 1 ----------------------
E1533–00
5.2.3 Do not use transformed cells as indicators since they 6.6 Mounting of Cover Slips:
produce large amounts of extra nuclear fluorescence. 6.6.1 Remove the cover slip from the Leighton tube and
place it on a glass slide.The monolayer must be directed to the
6. Procedure
glass s
...

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5.1 The presence of cell growth medium complicates a direct analysis of cells with SIMS. Attempts to wash out the nutrient medium results in the exposure of cells to unphysiological reagents that may also alter their chemical composition. This obstacle is overcome by using a sandwich freeze-fracture method (1). This cryogenic method has provided a unique way of sampling individual cells in their native state for SIMS analysis.  
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1.1 This practice covers a reference method of direct contact cell culture testing which may be used in evaluating the cytotoxic potential of materials for use in the construction of medical materials and devices.  
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1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
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5.1 This technique involves a chemical-precipitation reaction between the phencyclidine or its analogues and the precipitating reagent. The habit and the aggregation of the crystals formed could be used to distinguish phencyclidine or its analogues from other drugs.  
5.2 This technique can be utilized on phencyclidine or its analogues present in either the salt or free base form.  
5.3 This technique does not distinguish between salt and free base forms.
SCOPE
1.1 This practice describes procedures applicable to the analysis of phencyclidine and its analogues using microcrystal tests (1-8).2  
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1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
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SIGNIFICANCE AND USE
5.1 The propensity of a material to stimulate delayed contact hypersensitivity must be assessed before clinical application of devices containing this material. Delayed hypersensitivity may occur anywhere in the body. Systemic delayed hypersensitivity may have a complex set of reactions and consequences depending on the actual tissue/organ site of reaction. Although the reactions are seldom life-threatening, severe tissue and organ damage my result over time. Skin is the usual test site to determine the propensity of a material to cause delayed hypersensitivity.  
5.2 The standard historical test methods have involved the use of guinea pigs with a cutaneous application and observation of the reaction site. The use of the murine local lymph node assay results in a numerical quantitation of stimulation, rather than subjective evaluation and could be used to determine dose responses.  
5.3 This practice may not be predictive of events occurring during all types of implant applications. The user is cautioned to consider the appropriateness of the method in view of the materials being tested, their potential applications, and the recommendations contained in Practice F748.
SCOPE
1.1 This practice provides a methodology to use a combination of in vivio and in situ procedures for the evaluation of delayed contact hypersensitivity reactions.  
1.2 This practice is intended to provide an alternative to the use of guinea pigs for evaluation of the ability of a device material to stimulate delayed contact hypersensitivity reactions. This alternative is particularly applicable for materials used in devices that contact only intact skin. However, the guinea pig maximization test is still the recommended method when assessing the delayed hypersensitivity response to metals or when testing substances that do not penetrate the skin but are used in devices that contact deep tissues or breached surfaces. This practice may be used for testing metals, with the exception of nickel-containing metals, unless the unique physicochemical properties of the materials may interfere with the ability of LLNA to detect sensitizing substances.  
1.3 This practice consists of a protocol for assessing an increase in lymphocyte proliferation in the lymph nodes draining the site of test article administration on the ears of mice.  
1.4 The LLNA has been validated only for low-molecular-weight chemicals that can penetrate the skin. The absorbed chemical or metabolite must bind to macromolecules, such as proteins, to form immunogenic conjugates.  
1.5 This practice is one of several developed for the assessment of the biocompatibility of materials. Practice F748 may provide guidance for the selection of appropriate methods for testing materials for a specific application.  
1.6 Identification of a supplier of materials or reagents is for the convenience of the user and does not imply a single source. Appropriate materials and reagents may be obtained from many commercial supply houses.  
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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  • Standard
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    English language
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