ASTM E2564-07
(Test Method)Standard Test Method for Enumeration of Mycobacteria in Metalworking Fluids by Direct Microscopic Counting (DMC) Method
Standard Test Method for Enumeration of <i>Mycobacteria</i> in Metalworking Fluids by Direct Microscopic Counting (DMC) Method
SIGNIFICANCE AND USE
During the past decade, it has become increasingly apparent that non-tuberculous mycobacteria are common members of the indigenous MWF bacterial population. Measurement of mycobacterial cell count densities is an important step in establishing a possible relationship between mycobacteria and occupational health related allergic responses, for example, Hypersensitivity Pneumonitis (HP) in persons exposed to aerosols of metalworking fluids. It is known that the viable mycobacteria count underestimates the total mycobacterial levels by not counting the non-culturable, possibly dead or moribund population that is potentially equally important in the investigation of occupational health related problems. The Direct Microscopic Counting Method (DMC) described here gives a quantitative assessment of the total numbers of acid-fast bacilli. It involves using acid-fast staining to selectively identify mycobacteria from other bacteria, followed by enumeration or direct microscopic counting of a known volume over a known area. Although other microbes—particularly the Actinomycetes—also stain acid fast, they are differentiated from the mycobacteria because of their morphology and size. Non-mycobacteria, acid-fast microbes are 50-100 times larger than mycobacteria. The method provides quantitative information on the total (culturable and non-culturable viable, and non-viable) mycobacteria populations. The results are expressed quantitatively as mycobacteria per mL of metalworking fluid sample.
The DMC method using the acid-fast staining technique is a semi- quantitative method with a relatively fast turnaround time.
The DMC method can also be employed in field survey studies to characterize the changes in total mycobacteria densities of metalworking fluid systems over a long period of time.
The sensitivity detection limit of the DMC method depends on the MF and the sample volume (direct or centrifuged, etc.) examined.
SCOPE
1.1 This test method describes a direct microscopic counting method (DMC) for the enumeration of the acid fast stained mycobacteria population in metalworking fluids. It can be used to detect levels of total mycobacteria population, including culturable as well as non-culturable (possibly dead or moribund ) bacterial cells. This test method is recommended for all water-based metalworking fluids.
1.2 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. For additional safety information, see Laboratory Safety: Principle and Practices, 4th Edition
General Information
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Standards Content (Sample)
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An American National Standard
Designation:E2564–07
Standard Test Method for
Enumeration of Mycobacteria in Metalworking Fluids by Direct
1
Microscopic Counting (DMC) Method
This standard is issued under the fixed designation E2564; 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 3.1.3.1 Discussion—The average number of mycobacte-
rium cells per one microscopic field (or oil field, OIF) is
1.1 This test method describes a direct microscopic count-
multiplied by the MF to give the concentration of mycobacte-
ing method (DMC) for the enumeration of the acid fast stained
rium per mL of sample.
mycobacteria population in metalworking fluids. It can be used
3.1.4 oil immersion field (OIF), n—the circular area of a
to detect levels of total mycobacteria population, including
microscopic field visible in the eye piece of the microscope
culturable as well as non-culturable (possibly dead or mori-
using oil immersion objective.
bund ) bacterial cells. This test method is recommended for all
water-based metalworking fluids.
4. Summary of Test Method
1.2 This standard does not purport to address all of the
4.1 The method describes a semi quantitative test for
safety concerns, if any, associated with its use. It is the
enumerating acid fast stained environmental mycobacterium
responsibility of the user of this standard to establish appro-
(AFB) from metal working fluids by direct microscopic count-
priate safety and health practices and determine the applica-
4
ing (DMC) method . It is used to determine total mycobacte-
bility of regulatory limitations prior to use. For additional
rium counts including culturable and possibly dead or mori-
safety information, see Laboratory Safety: Principle and Prac-
2 bund cells in the sample. This test method cannot be used to
tices, 4th Edition
determine the total viable mycobacterium population in the
2. Referenced Documents sample. A known sample volume (centrifuged or direct) is
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spread over a known area (1 cm or similar) on a microscope
2.1 ASTM Standards:
slide (marked by frosted or painted circles). Following differ-
D2881 Classification for Metal Working Fluids and Related
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ential acid-fast staining , the acid-fast cells are counted in
Materials
several microscopic fields over the designated area. The
3. Terminology calculation is based on using a calibrated microscope with a
known Microscopic Factor (MF).The MF is determined by the
3.1 Definitions of Terms Specific to This Standard:
microscopic area over which a known amount of sample was
3.1.1 acid-fast bacteria, n—a distinctive staining property
spread, the number of microscopic fields in the marked circle,
of Mycobacteria due to their lipid-rich cell walls.
and the volume of sample examined. The number of acid fast
3.1.1.1 Discussion—Once stained, mycobacterium resist
stained mycobacterium cells per microscopic field multiplied
decolorization when exposed to acidified organic solvents, and
bytheMFgivesthemycobacteriumnumberpermLofsample.
are therefore, informally designated acid-fast.
3.1.2 non-tuberculous Mycobacteria (NTM)—
5. Significance and Use
environmental mycobacteria, not associated with tuberculosis.
5.1 During the past decade, it has become increasingly
3.1.3 microscopic factor (MF), n—a calibrated conversion
apparentthatnon-tuberculousmycobacteriaarecommonmem-
factorforcalculatingtheMycobacteriumcountpermLsample.
bers of the indigenous MWF bacterial population. Measure-
ment of mycobacterial cell count densities is an important step
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This test method is under the jurisdiction of ASTM Committee E34 on
in establishing a possible relationship between mycobacteria
Occupational Health and Safety and is the direct responsibility of Subcommittee
andoccupationalhealthrelatedallergicresponses,forexample,
E34.50 on Health and Safety Standards for Metal Working Fluids.
Current edition approved April 15, 2007. Published May 2007. DOI: 10.1520/
E2564-07.
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Mary J. R. Gilchrist: Biosafety Precautions for Airborne Pathogens, in Standard Methods for the Examination of Dairy Products, Chapter: 10: Direct
Laboratory Safety Principles and Practices, pp. 67-76, 1995, ASM Press MicroscopicMethodsforBacteriaorSomaticCells,16thed.AmericaPublicHealth
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For referenced ASTM standards, visit the ASTM website, www.astm.org, or Association, Inc., Washington, DC, 1978.
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contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Ebersole L.L.: Acid-fast stain procedures, pp. 3.5.1-3.5.11. In Clinical Micro-
Standards volume information, refer to the standard’s Document Summary page on biology Procedures Handbook,
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