ASTM E2563-07
(Test Method)Standard Test Method for Enumeration of Non-Tuberculosis Mycobacteria in Aqueous Metalworking Fluids by Plate Count Method
Standard Test Method for Enumeration of Non-Tuberculosis Mycobacteria in Aqueous Metalworking Fluids by Plate Count Method
SIGNIFICANCE AND USE
This method 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 metal grinding 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.
The measurement of viable and culturable mycobacterial densities combined with the total mycobacterial counts (including viable culturable (VC), viable-non culturable (VNC) and non viable (NV) counts) is usually the first step in establishing any possible relationship between Mycobacteria and occupational health concerns (for example, HP).
The method can be employed in survey studies to characterize the viable-culturable mycobacterial population densities of metal working fluid field samples.
This method is also applicable for establishing the mycobacterial resistance of metalworking fluid formulations by determining mycobacterium survival by means of plate count technique.
This method can also be used to evaluate the relative efficacy of microbicides against Mycobacteria in metalworking fluids.
SCOPE
1.1 This test method 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 test method 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
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.
General Information
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: E2563 − 07 AnAmerican National Standard
Standard Test Method 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.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. Terminology
1.1 This test method covers the detection and enumeration 3.1 Definitions:
ofviableandculturablerapidlygrowing Mycobacteria(RGM), 3.1.1 rapidly growing mycobacteria (RGM)—non-
or non-tuberculosis Mycobacteria (NTM) in aqueous metal- tuberculous Mycobacteria that grow and produce visible colo-
working fluids (MWF) in the presence of high non- nies in four to seven days.
mycobacterial background population using standard micro-
4. Summary of Test Method
biological culture methods.
4.1 For recovery and enumeration of viable and culturable
1.2 The detection limit is one colony forming unit
Mycobacteria population in metalworking fluid field samples
(CFU)/mL metalworking fluid.
selective culture medium containing antimicrobial agents to
1.3 This test method involves culture of organisms classi-
suppress bacterial and fungal contamination is recommended.
fied as Level 2 pathogens, and should be undertaken by a
(See Section 8). Standard microbiological spread and droplet
trained microbiologist in an appropriately equipped facility.
plating techniques are used for the enumeration of Mycobac-
Themicrobiologistshouldalsobecapableofdistinguishingthe
teria. After a minimum of 14 days incubation at 30°C, the
diverse colonies of Mycobacteria from other microorganism
Mycobacteria colonies are counted and confirmed by acid-fast
colonies on a Petri dish and capable of confirming Mycobac-
staining technique specific for Mycobacteria.
teria by acid fast staining method
5. Significance and Use
1.4 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the 5.1 Thismethodallowsfortherecoveryandenumerationof
responsibility of the user of this standard to establish appro- viable and culturable, non-tuberculosis, rapidly growing My-
priate safety and health practices and determine the applica- cobacteria (M.immunogenum, M.chelonae, M. absessus, M.
bility of regulatory limitations prior to use.
fortuitum, and M.smegmatis) in the presence of high gram
negative background populations in metalworking fluid field
2. Referenced Documents
samples. During the past decade it has become increasingly
apparent that non-tuberculous Mycobacteria are common
2.1 ASTM Standards:
members of the indigenous MWF bacterial population. This
D5465Practice for Determining Microbial Colony Counts
population is predominantly comprised of gram negative
from Waters Analyzed by Plating Methods
bacteria and fungi. Mycobacterial contamination of metal-
E1326GuideforEvaluatingNonconventionalMicrobiologi-
working fluids has been putatively associated with hypersen-
cal Tests Used for Enumerating Bacteria
sitivity pneumonitis (HP) amongst metal grinding machinists.
2.2 Other Documents:
The detection and enumeration of these organisms will aid in
Kinyuon Acid-Fast Staining Procedure
better understanding of occupational health related problems
and a better assessment of antimicrobial pesticide efficacy.
This test method is under the jurisdiction of ASTM Committee E34 on
5.2 The measurement of viable and culturable mycobacte-
Occupational Health and Safety and is the direct responsibility of Subcommittee
rial densities combined with the total mycobacterial counts
E34.50 on Health and Safety Standards for Metal Working Fluids.
(including viable culturable (VC), viable-non culturable
Current edition approved Oct. 1, 2007. Published October 2007. DOI: 10.1520/
E2563-07.
(VNC) and non viable (NV) counts) is usually the first step in
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
establishing any possible relationship between Mycobacteria
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
and occupational health concerns (for example, HP).
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
5.3 The method can be employed in survey studies to
Public Heatlth Microbiology:AGuidefortheLevelIIILaboratory.Centersfor
characterize the viable-culturable mycobacterial population
Disease Control, U.S. Department of Health and Human Services, Atlanta, GA,
1985. densities of metal working fluid field samples.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2563 − 07
NOTE 1—Brilliant Green stain can be used instead of the Methylene
5.4 This method is also applicable for establishing the
Blue stain (Brilliant Green 2.0 g, Sodium Hydroxide 0.02 g, Distilled
mycobacterial resistance of metalworking fluid formulations
water 1000 mL).
by determining mycobacterium survival by means of plate
count technique.
9. Hazards
5.5 This method can also be used to evaluate the relative
9.1 The analyst must know and observe good laboratory
efficacyofmicrobicidesagainst Mycobacteriainmetalworking
practices and safety procedures required in the microbiology
fluids.
laboratory in preparing, using and disposing of cultures,
reagents and materials.
6. Interferences
6.1 In some metal working fluid samples very high (>10 /
10. Sampling, Test Specimens, and Test Units
mL) microbial background population levels; mainly gram
10.1 Usesterilescrew-capped,non-breakableplasticscrew-
negative pseudomonads and fungi can interfere the enumera-
capped containers (100-200 mL) for metalworking fluid sam-
tion of Mycobacteria by “overgrowth” on the agar surface.
pling for microbiological analysis. The sample should be a
6.2 Sample dilution or smaller sample size can be used to
random representative portion that is from the circulating tank
minimize interference of non-target bacterial and fungal den-
as opposed to a pooled spillover or stagnant hose content.
sities. Replicates of sample dilutions could be also plated and
Collect approximately 100–mLsize samples.Analyze samples
the results combined.
immediately if possible, or refrigerate until analyzed. Maxi-
6.3 In some metalworking fluid samples chemicals (antimi-
mumsamplestoragetimeis24hatrefrigerationtemperatures.
crobial pesticides, functional additives, and other components)
Follow sample docu
...
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