ASTM E2563-23

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: E2563 − 18 E2563 − 23 An American National Standard
Standard Practice for
Enumeration of Non-Tuberculosis Mycobacteria in Aqueous
Metalworking Fluids by Plate Count Method
This standard is issued under the fixed designation E2563; 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope Scope*
1.1 This practice covers the detection and enumeration of viable and culturable rapidly growing Mycobacteria (RGM), or
non-tuberculosis Mycobacteria (NTM) in aqueous metalworking fluids (MWF) in the presence of high non-mycobacterial
background population using standard microbiological culture methods.
1.2 The detection limit is one colony forming unit (CFU)/mL metalworking fluid.
1.3 This practice involves culture of organisms classified as Level 2 pathogens, and should be undertaken by a trained
microbiologist in an appropriately equipped facility. The microbiologist should also be capable of distinguishing the diverse
colonies of Mycobacteria from other microorganism colonies on a Petri dish and capable of confirming Mycobacteria by acid-fast
staining method.
1.4 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.
1.5 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.
2. Referenced Documents
2.1 ASTM Standards:
D5465 Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
E1326 Guide for Evaluating Non-culture Microbiological Tests
E2523 Terminology for Metalworking Fluids and Operations
2.2 Other Document:
Kinyoun Acid-Fast Staining Procedure
3. Terminology
3.1 For definitions of terms used in this standard, refer to Terminology E2523.
This practice is under the jurisdiction of ASTM Committee E34 on Occupational Health and Safety and is the direct responsibility of Subcommittee E34.50 on Health
and Safety Standards for Metal Working Fluids.
Current edition approved Oct. 1, 2018Oct. 1, 2023. Published October 2018October 2023. Originally approved in 2007. Last previous edition approved in 20132018 as
E2563 – 13. DOI: 10.1520/E2563-18.18. DOI: 10.1520/E2563-23.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Public Health Microbiology: A Guide for the Level III Laboratory, Centers for Disease Control, U.S. Department of Health and Human Services, Atlanta, GA, 1985.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2563 − 23
3.2 Definitions:
3.2.1 rapidly growing mycobacteria (RGM)—non-tuberculous Mycobacteria that grow and produce visible colonies in four to
seven days.
4. Summary of Practice
4.1 For recovery and enumeration of viable and culturable Mycobacteria population in metalworking fluid field samples, selective
culture medium containing antimicrobial agents to suppress bacterial and fungal contamination is recommended. (See Section 8.)
Standard microbiological spread and droplet plating techniques are used for the enumeration of Mycobacteria (see Practices
D5465). After a minimum of 14 days incubation at 30 °C, the Mycobacteria colonies are counted and confirmed by acid-fast
staining technique specific for Mycobacteria.
5. Significance and Use
5.1 This practice allows for the recovery and enumeration of viable and culturable, non-tuberculosis, rapidly growing
Mycobacteria (M. immunogenum,M. chelonae,M. absessus,M. fortuitum, and M. smegmatis) in the presence of high Gram-negative
background populations in metalworking fluid field samples. During the past decade, it has become increasingly apparent that
non-tuberculous Mycobacteria are common members of the indigenous MWF bacterial population. This population is
predominantly comprised of Gram-negative bacteria and fungi. Mycobacterial contamination of metalworking fluids has been
putatively associated with hypersensitivity pneumonitis (HP) amongst metalgrinding machinists. The detection and enumeration
of these organisms will aid in better understanding of occupational health-related problems and a better assessment of antimicrobial
pesticide efficacy.
5.2 The measurement of viable and culturable mycobacterial densities (Guide E1326), combined with the total mycobacterial
counts (including viable culturable (VC), viable nonculturable (VNC) and nonviable (NV) counts), is usually the first step in
establishing any possible relationship between Mycobacteria and occupational health concerns (for example, HP).
5.3 The practice can be employed in survey studies to characterize the viable-culturable mycobacterial population densities of
metalworking fluid field samples.
5.4 This practice is also applicable for establishing the mycobacterial resistance of metalworking fluid formulations by
determining mycobacterium survival by means of plate count technique.
5.5 This practice can also be used to evaluate the relative efficacy of microbicides against Mycobacteria in metalworking fluids.
6. Interferences
6.1 In some metalworking fluid samples, very high (>10 /mL) microbial background population levels, mainly Gram-negative
pseudomonads and fungi, can interfere with the enumeration of Mycobacteria by “overgrowth” on the agar surface.
6.2 Sample dilution or smaller sample size can be used to minimize interference of non-target bacterial and fungal densities.
Replicates of sample dilutions could be also plated and the results combined.
6.3 In some metalworking fluid samples, chemicals (antimicrobial pesticides, functional additives, and other components) can
interfere with the culturability of total viable Mycobacteria count in the sample. If interference by chemicals is suspected, sample
dilution may also overcome this interference but will reduce sensitivity.
7. Apparatus
7.1 Laboratory Incubator, 30 6 2 °C.
7.2 Microscope, with oil immersion lens, magnification 1000×.
7.3 Staining Tray or Sink, with running water and drying rack.
E2563 − 23
8. Reagents and Materials
8.1 Test Tubes,
...