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ASTM E1533-00(2006)

(Practice)

Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4`-6-Diamidino-2-2 Phenylindole (DAPI) Staining (Withdrawn 2014)

Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4`-6-Diamidino-2-2 Phenylindole (DAPI) Staining (Withdrawn 2014)

SIGNIFICANCE AND USE
Mycoplasma contamination of cell cultures is a common problem that can affect the growth, metabolism, and function of cultured animal cells. The ability to detect mycoplasma in cell cultures provides an opportunity to ensure that cells are free of contamination, and to replace those that are not. For additional information, see Practices E 1531, E 1532, and E 1536. Strict adherence to established, well-tested procedures is necessary. This practice was developed by Task Group E48.01.02 to assist in developing and maintaining an established regimen for mycoplasma detection by indirect 4′-6-Diamidino-2-Phenylindole (DAPI) fluorochrome staining.
This practice is intended for use in examining cultured animal cells for the presence of mycoplasma contamination.
This practice is not intended for use in the detection of mycoplasma contamination in serum, culture media, or systems other than cultures of animal cells.
All cell cultures to be examined for mycoplasma should undergo a minimum of two passages in antibiotic-free tissue culture medium before testing.
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.
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 and health practices and determine the applicability of regulatory limitations prior to use.
WITHDRAWN RATIONALE
This practice covers procedures used for the detection of mycoplasma contamination by indirect DNA staining.
Formerly under the jurisdiction of Committee E55 on Manufacture of Pharmaceutical Products, this practice was withdrawn in August 2014. This standard was withdrawn without replacement due to its limited use by the industry.

General Information

Status
Withdrawn
Publication Date
31-Oct-2006
Withdrawal Date
04-Aug-2014
ICS
07.100.10 - Medical microbiology
Technical Committee
E55 - Manufacture of Pharmaceutical and Biopharmaceutical Products
Drafting Committee
E55.04 - General Biopharmaceutical Standards
Current Stage
Ref Project

Relations

Replaces
ASTM E1533-00 - Standard Practice for Indirect Detection of Mycoplasma in Cell Culture by 4'-6-Diamidino-2-2 Phenylindole (DAPI) Staining
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
01-Nov-2006

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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)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)
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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)
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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: E1533 − 00(Reapproved 2006)
Standard Practice for
Indirect Detection of Mycoplasma in Cell Culture by 4*-6-
Diamidino-2-2 Phenylindole (DAPI) Staining
This standard is issued under the fixed designation E1533; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3.1.2 direct detection of mycoplasma—detection of myco-
plasma by cultivation in culture media.
1.1 Thispracticecoversproceduresusedforthedetectionof
mycoplasma contamination by indirect DNA staining.
3.1.3 indirect detection of mycoplasma—detection of myco-
plasma by DNAstaining or any method other than cultivation.
1.2 This practice does not cover direct methods for the
detection of mycoplasma or other indirect methods such as
3.1.4 mycoplasma—the smallest prokaryotes capable of liv-
enzymatical detection or DNA probes.
ing freely, lacking a cell wall, having a circular double-
1.3 This practice does not cover methods for the identifica-
stranded DNA relatively rich in adenine and thymine, and
tion of mycoplasma organisms.
containing16sand23sribosomalRNAs.Theycanbefoundas
contaminants in cell cultures.
1.4 The values stated in SI units are to be regarded as the
standard. The values given in parentheses are for information
only. 4. Significance and Use
1.5 This standard does not purport to address all of the
4.1 Mycoplasmacontaminationofcellculturesisacommon
safety concerns, if any, associated with its use. It is the
problem that can affect the growth, metabolism, and function
responsibility of the user of this standard to establish appro-
of cultured animal cells. The ability to detect mycoplasma in
priate safety and health practices and determine the applica-
cell cultures provides an opportunity to ensure that cells are
bility of regulatory limitations prior to use.
free of contamination, and to replace those that are not. For
additional information, see Practices E1531, E1532, and
2. Referenced Documents
E1536. Strict adherence to established, well-tested procedures
2.1 ASTM Standards:
is necessary. This practice was developed by Task Group
E1531PracticeforDetectionofMycoplasmaContamination
E48.01.02 to assist in developing and maintaining an estab-
of Cell Cultures by Growth on Agarose Medium
lished regimen for mycoplasma detection by indirect 4`-6-
E1532PracticeforDetectionofMycoplasmaContamination
Diamidino-2-Phenylindole (DAPI) fluorochrome staining.
of Cell Cultures by Use of Bisbenzamide DNA-Binding
Fluorochrome
4.2 This practice is intended for use in examining cultured
E1536PracticeforDetectionofMycoplasmaContamination
animal cells for the presence of mycoplasma contamination.
of Bovine Serum by Large Volume Method
4.3 This practice is not intended for use in the detection of
3. Terminology
mycoplasma contamination in serum, culture media, or sys-
tems other than cultures of animal cells.
3.1 Definitions:
3.1.1 DAPI staining—staining of DNA in particular by
4.4 All cell cultures to be examined for mycoplasma should
using DAPI fluorochrome stain.
undergo a minimum of two passages in antibiotic-free tissue
culture medium before testing.
This practice is under the jurisdiction of ASTM Committee E55 on Manufac-
5. Quality Control
ture of Pharmaceutical Products and is the direct responsibility of Subcommittee
E55.04 on General Biopharmaceutical Standards.
5.1 Visually examine the DAPI stain concentrate routinely
Current edition approved Nov. 1, 2006. Published December 2006. Originally
approved in 1993. Last previous edition approved in 2000 as E1533–00. DOI:
forcontamination.Freshstockshouldbepreparedperiodically.
10.1520/E1533-00R06.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
5.2 Indicator cells:
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
5.2.1 Indicator cells support the growth of mycoplasma
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. species and provide positive and negative controls.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1533 − 00 (2006)
5.2.2 Use continuous cell lines such as the African green 6.5.1 Add 2 mL DAPI staining solution to each tube.
monkey kidney cell line,Vero,AmericanType Culture Collec- 6.5.2 Incubate for 30 min at 37°C.
tion (ATCC CCL81) as indicator cells as described in this 6.5.3 Wash twice with 2 mL PBS pH 7.2.
practice; 3T6 mouse fibroblast (ATCC CCL 96) may also be
6.6 Mounting of Cover Slips:
used.
6.6.1 Remove the cover slip from the Leighton tube and
5.2.3 Do not use transformed cells as indicators since they
placeitonaglassslide.Themonolayermustbedirectedtothe
produce large amounts of extra nuclear fluorescence.
glass slide. Do not allow the culture to dry.
6.6.2 Label each slide to identify the specimen being tested.
6. Procedure
6.7 Observation and Recording of Results:
6.1 Preparation of DAPI Stain Concentrate:
6.7.1 Observe each specimen, including both the positive
6.1.1 Add 1.0 mg DAPI stai
...

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SCOPE
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5.1 This guide should be used by producers and potential producers of non-culture tests to determine the accuracy, selectivity, specificity, and precision of the tests, as defined in Practice E691. Results of such studies should identify the limitations and indicate the utility or applicability of the non-culture test, or both, for use on different types of samples. Guide E1488 recommends other statistical tools for evaluating the suitability and applicability of proposed new test methods.  
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SCOPE
1.1 The purpose of this guide is to assist users and producers of non-culture microbiological tests in determining the applicability of the test for processing different types of samples and evaluating the accuracy of the results. Culture test procedures such as the Heterotrophic (Standard) Plate Count, the Most Probable Number (MPN) method and the Spread Plate Count are widely cited and accepted for the enumeration of microorganisms. However, these methods have their limitations, such as performance time. Moreover, any given culture test method typically recovers only a portion of the total viable microbes present in a sample. It is these limitations that have recently led to the marketing of a variety of non-culture procedures, test kits and instruments.  
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SCOPE
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1.3 This is one of a series of reference test methods for the assessment of cytotoxic potential, employing different techniques.  
1.4 Assessment of cytotoxicity is one of several tests employed in determining the biological response to a material, as recommended in Practice F748.  
1.5 The L-929 cell line was chosen because it has a significant history of use in assays of this type. This is not intended to imply that its use is preferred; only that the L-929 is a well characterized, readily available, established cell line that has demonstrated reproducible results in several laboratories.  
1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.7 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.  
1.8 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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ASTM
ASTM E1968-19(Practice)
Standard Practice for Microcrystal Testing in Forensic Analysis for Cocaine

SIGNIFICANCE AND USE
5.1 This technique involves a chemical-precipitation reaction between cocaine and the precipitating reagent. The habit and the aggregation of the crystals formed could be used to distinguish cocaine from other drugs (6).  
5.2 This technique can be utilized on cocaine present in either the salt or free base form.  
5.3 This technique does not distinguish between the salt and free base forms.
SCOPE
1.1 This practice describes procedures applicable to the analysis of cocaine using multiple microcrystal tests (1-6).2  
1.2 These procedures are applicable to cocaine, which is present in solid form or an injectable liquid form. They are not typically applicable to the analysis of cocaine in biological samples.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 These procedures could generate observations indicating a positive test for cocaine or its enantiomers which could be incorporated into the analytical scheme as defined by the laboratory.  
1.5 This standard cannot replace knowledge, skills, or abilities acquired through appropriate education, training, and experience (see Practice E2326) and is to be used in conjunction with professional judgment by individuals with such discipline-specific knowledge, skills, and abilities.  
1.6 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.  
1.7 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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ASTM
ASTM E2125-19(Practice)
Standard Practice for Microcrystal Testing in Forensic Analysis for Phencyclidine and Its Analogues

SIGNIFICANCE AND USE
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  
1.2 These procedures are applicable to phencyclidine and its analogues which are present in solid form or in a liquid form. They are not typically applicable to the analysis of phencyclidine and its analogues in biological samples.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 These procedures could generate observations indicating a positive test for phencyclidine and its analogues which could be incorporated into the analytical scheme as defined by the laboratory.  
1.5 This standard cannot replace knowledge, skills, or abilities acquired through appropriate education, training, and experience (see Practice E2326) and is to be used in conjunction with sound professional judgment by individuals with such discipline-specific knowledge, skills, and abilities.  
1.6 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.  
1.7 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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