1、Designation: E2564 13E2564 18 An American National StandardStandard Practice forEnumeration of Mycobacteria in Metalworking Fluids byDirect Microscopic Counting (DMC) Method1This standard is issued under the fixed designation E2564; the number immediately following the designation indicates the year
2、 oforiginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice describes a direct microscopic counting me
3、thod (DMC) for the enumeration of the acid fast acid-fast stainedmycobacteria population in metalworking fluids. It can be used to detect levels of total mycobacteria population, includingculturable as well as non-culturable (possibly dead or moribund ) moribund) bacterial cells. This practice is re
4、commended for allwater-based metalworking fluids.fluids (Classification D2881).1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety and health practices and dete
5、rmine the applicability of regulatorylimitations prior to use. For additional safety information, see Laboratory Safety: Principle and Practices, 4th Edition.21.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the use
6、r of this standard to establish appropriate safety, health, and environmental practices and determine the applicability ofregulatory limitations prior to use. For additional safety information, see Laboratory Safety: Principle and Practices, 4th Edition.21.3 This international standard was developed
7、 in accordance with internationally recognized principles on standardizationestablished in the Decision on Principles for the Development of International Standards, Guides and Recommendations issuedby the World Trade Organization Technical Barriers to Trade (TBT) Committee.2. Referenced Documents2.
8、1 ASTM Standards:3D2881 Classification for Metalworking Fluids and Related MaterialsE2523 Terminology for Metalworking Fluids and Operations3. Terminology3.1 For definitions of terms used in this standard, refer to Terminology E2523.3.2 Definitions of Terms Specific to This Standard:3.2.1 acid-fast
9、bacteria, na distinctive staining property of Mycobacteria due to their lipid-rich cell walls.3.2.1.1 DiscussionOnce stained, mycobacteriummycobacteria resist decolorization when exposed to acidified organic solvents, and are therefore,therefore informally designated acid-fast.3.2.2 non-tuberculous
10、Mycobacteria (NTM)environmental mycobacteria,mycobacteria not associated with tuberculosis.3.2.3 microscopic factor (MF), na calibrated conversion factor for calculating the Mycobacteriummycobacterium count permL sample.1 This practice is under the jurisdiction of ASTM Committee E34 on Occupational
11、Health and Safety and is the direct responsibility of Subcommittee E34.50 on Healthand Safety Standards for Metal Working Fluids.Current edition approved July 1, 2013Oct. 1, 2018. Published July 2013October 2018. Originally approved in 2007. Last previous edition approved in 20112013 asE2564 - 11.E2
12、564 13. DOI: 10.1520/E2564-13.10.1520/E2564-18.2 Gilchrist, Mary J. R. Gilchrist, R., “Biosafety Precautions for Airborne Pathogens,” in Laboratory SafetySafety: Principles and Practices, pp. 6776, 1995, ASMPress.ASM Press, 1995, pp. 6776.3 For referencedASTM standards, visit theASTM website, www.as
13、tm.org, or contactASTM Customer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what c
14、hanges have been made to the previous version. Becauseit 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 versionof the standard as published by ASTM is to be considered the of
15、ficial document.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.2.3.1 DiscussionThe average number of mycobacterium cells per one microscopic field (or oil field, OIF) is multiplied by the MF to give theconcentration of mycobacteriu
16、mmycobacteria per mL of sample.3.2.4 oil immersion field (OIF), nthe circular area of a microscopic field visible in the eye piece eyepiece of the microscopeusing oil immersion objective.4. Summary of Practice4.1 TheThis practice describes a semi quantitative semi-quantitative test for enumerating a
17、cid fast acid-fast stainedenvironmental mycobacteriummycobacteria (AFB) from metal working metalworking fluids by direct microscopic counting(DMC) method.4 It is used to determine total mycobacterium counts, including culturable and possibly dead or moribund cells inthe sample. This practice cannot
18、be used to determine the total viable mycobacterium population in the sample. A known samplevolume (centrifuged or direct) is spread over a known area (1 cm2 or similar) on a microscope slide (marked by frosted or paintedcircles). Following differential acid-fast staining,5 the acid-fast cells are c
19、ounted in several microscopic fields over the designatedarea. The calculation is based on using a calibrated microscope with a known Microscopic Factormicroscopic factor (MF). TheMF is determined by the microscopic area over which a known amount of sample was spread, the number of microscopic fields
20、in the marked circle, and the volume of sample examined. The number of acid fast acid-fast stained mycobacterium cells permicroscopic field multiplied by the MF gives the mycobacterium number per mL of sample.5. Significance and Use5.1 During the past decade, it has become increasingly apparent that
21、 non-tuberculous mycobacteria are common members ofthe indigenous MWF bacterial population. Measurement of mycobacterial cell count densities is an important step in establishinga possible relationship between mycobacteria and occupational health related health-related allergic responses, for exampl
22、e,Hypersensitivity Pneumonitishypersensitivity pneumonitis (HP) in persons exposed to aerosols of metalworking fluids. It is knownthat the viable mycobacteria count underestimates the total mycobacterial levels by not counting the non-culturable, possibly deador moribund population that is potential
23、ly equally important in the investigation of occupational health related health-relatedproblems. The Direct Microscopic Counting Methoddirect microscopic counting method (DMC) described here gives aquantitative assessment of the total numbers of acid-fast bacilli. It involves using acid-fast stainin
24、g to selectively identifymycobacteria from other bacteria, followed by enumeration or direct microscopic counting of a known volume over a known area.Although other microbesparticularly the Actinomycetesalso stain acid fast, acid-fast, they are differentiated from themycobacteria because of their mo
25、rphology and size. Non-mycobacteria, acid-fast microbes are 50 to 100 times larger thanmycobacteria. TheThis practice provides quantitative information on the total (culturable and non-culturable viable, andnon-viable) mycobacteria populations. The results are expressed quantitatively as mycobacteri
26、a per mL of metalworking fluidsample.5.2 The DMC method using the acid-fast staining technique is a semi-quantitative method with a relatively fast turnaround time.5.3 The DMC method can also be employed in field survey studies to characterize the changes in total mycobacteria densitiesof metalworki
27、ng fluid systems over a long period of time.5.4 The sensitivity detection limit of the DMC method depends on the MF and the sample volume (direct or centrifuged, etc.)examined.6. Interferences6.1 Some metalworking fluid formulations fail to completely dry or provide an uneven film on the microscope
28、slide (forexample, synthetic fluids and metalworking fluids with high trap tramp oil content and debris). For these samples, the results canbe difficult to interpret, as heat fixing may not provide full adherence. These samples should be re-stained or a new slide may beprepared.6.2 A negative acid f
29、ast acid-fast staining reaction does not necessarily indicate that a sample will be culturally negative forMycobacteria, since the culture method has a lower detection limit (1 cell/mL) than the DMC method.7. Apparatus7.1 Centrifuge, (“microfuge”) 14,000(“microfuge”), 14 000 relative gravities.4 Sta
30、ndard Methods for the Examination of Dairy Products, Chapter: 10: Direct Microscopic Methods for Bacteria or Somatic Cells, 16th ed.Americaed.,American PublicHealth Association, Inc., Washington, DC, 1978.5 Ebersole L. L., “Acid-Fast Stain Procedures,” pp. 3.5.13.5.11. In3.5.13.5.11, in Clinical Mic
31、robiology Procedures Handbook, Vol. 1.Vol 1, American Society forMicrobiology, 1994, Washington, DC.DC, 1994.E2564 1827.2 Centrifuge Tubes with Caps, disposable, 1 mL-2 mL1 mL to 2 mL capacity, such as Eppendorf SafeLock TubeSafe-Locktube or any other suitable centrifuge tubes.7.3 Calibrated Variabl
32、e Pipet, with sterile tips: 5 L, 10 L, 1.0 mL, 5 mL.7.4 Microscope Slides, with 100 mm100 mm2 or similar areas, marked by frosted or painted circles and frosted labeling ends.7.5 Calibrated Stage Micrometer, 0.01 mm 0.01 mm or similar divisions.7.6 Compound Microscope, with oil immersion lens.7.7 Mi
33、croscope Eye Pieces, 10 magnification, equipped with a net micrometer (10 by 10 mm) or similar.7.8 Slide Drying Apparatus, (box) 50 to 60C60 C, with level drying rack.7.9 Staining Hood.7.10 Staining Rack and Running Water.7.11 Hand Tally or Electrical Counter.7.12 Kinyoun Acid-Fast Stain Kit, (see 8
34、.1).7.13 Analytical Balance.8. Reagents and Materials8.1 Staining Reagents for Acid-Fast Staining Procedure for Staining Mycobacteria by the Kinyoun (Cold) Acid-Fast Procedure:8.1.1 TB Quick Stain Carbol-Fuchsin,Carbolfuchsin, Reagent ABasic Fuchsin (alcoholic) 17.0g, Aqueous Phenol 1000.0mLfuchsin
35、(alcoholic) 17.0 g, aqueous phenol 1000.0 mL.8.1.2 TB-DecolorizerHydrochloric Acid, 30.0 mL, Denatured Ethanol/Methanol: 970 mLacid, 30.0 mL; denatured ethanol/methanol, 970 mL.8.1.3 TB Quick Stain Methylene Blue Reagent BMethylene Blue (alcoholic) 2.0 g, acid-alcohol 1000.0 mL; (acid-alcohol: 30mL
36、HCl 970 mL, 90-95 % Ethanol) 90 to 95 % ethanol) or Brilliant Green Stain:stain: Brilliant Green 2.0 g, SodiumHydroxidesodium hydroxide 0.02 g, Distilled Waterdistilled water 1000 mLmL.9. Hazards9.1 The analyst must know and observe good laboratory practices and safety procedures required in the mic
37、robiology laboratoryin preparing, using, and disposing of cultures, reagents, and materials.10. Sampling, Test Specimens, and Test Units10.1 Use sterile screw-cappedsterile, screw-capped, plastic containers (100 to 200 mL) 200 mL) for microbiological samplingof metalworking fluids. The sample should
38、 be a random representative portion of 50 to 100 mL100 mLthat is from the circulatingtank tank, as opposed to a pooled,pooled spillover or stagnant hose contents. Refrigerate samples until analyzed. Maximum samplestorage time is 24 h at refrigeration temperatures. Follow sample documentation procedu
39、re in accordance with good laboratorypractices.11. Procedure11.1 Gently agitate sample to re-suspend any sediment. Dispense 1 mL directly into the centrifuge tube. In the case of veryviscous fluids, a 1-g1 g sample should be weighed on an analytical balance.11.2 Centrifuge samples at 13.00013 000 re
40、lative gravities for 30 minutes 30 min at 22C.22 C.11.3 Remove supernatant gently using a disposable micropipet end.11.4 Remove oily residues completely from the tube using a sterile cotton swab. Gently remove the whole pellet with a sterileloop or a micropipet end without disturbing the sediment.11
41、.5 Transfer the whole amount of sediment to the 1-cm1 cm2 designated area on the microscope slide and spread it evenlyusing a disposable pipet end.11.6 Dry slides over a level drying box at 50 to 60C60 C for a minimum of one hour. 1 h. Some fluid formulations requirelonger drying time. These samples
42、 can be dried as long as overnight on the drying box. The slides that remain oily even after theextended drying time are usually the result of a poorly decanted tube and, for these samples, the slide preparation must be repeated.11.7 Heat-fix the dried slides by gently passing the slide through a fl
43、ame fast once or twice.The heat-fixed slide should be warm,not hot after flaming. In order to avoid overheating the slides, the flaming can be substituted by a standard temperature heat blockat 75C75 C for 10 to 20 minutes.min. After heat-fixing heat fixing the slides, stain them using an acid-fast
44、staining kit:kit, forexample, Modified Kinyoun Staining Kit,the modified Kinyoun staining kit, although other acid-fast staining methods can also beused.E2564 18311.8 If the Modified Kinyoun Acid Fast Staining Method modified Kinyoun acid-fast staining method is used:11.8.1 Flood slide with TB Quick
45、 Stain Carbol-Fuchsin Reagentquick stain carbolfuchsin reagent A for 4 to 5 minutes.min.11.8.2 Rinse slide gently with water. Start rinsing on the frosted part of the slide, not directly on the sample. Gently removeexcess water.NOTE 1The stain is viscous and will not completely clearclear.11.8.3 Flo
46、od slide with TB Acid Alcohol Decolorizer for 15-30 seconds.acid alcohol decolorizer for 15 to 30 s.11.8.4 Rinse slide gently with water until rinse water is mostly clear. Gently remove excess water.11.8.5 CounterstainCounter-stain slide with TB Quick Stain Methylene Blue / Reagent quick stain Methy
47、lene Blue/reagent Bfor 4 to 5 minutes.min. (Staining with Brilliant Green for 30 secondss can replace Methylene Blue.)11.8.6 Rinse slide under running water, g.11.8.7 Place slide on a drying rack and dry it completelycompletely.11.9 Direct Microscopic Counting:11.10 Calibrate the microscope for the
48、Oil Fieldoil field (OIF) area, which is a single microscopic field that can be seen by theeye piece.eyepiece.11.10.1 Use a stage micrometer slide with 0.1 and 0.01-mm0.01 mm divisions to determine the diameter of one field under theoil immersion lens. Make the reading to the third decimal point.11.1
49、0.2 Determine the area of the OIF (r2pi) in mm2.11.10.3 Convert the OIF area to cm2.11.10.4 Determine the number of OIFs in 1 cm2. This number will provide the Microscopic Factormicroscopic factor (MF) for1 mL of sample if the whole sediment is examined. (If less than 1-mL1 mL sample volume (for example, 10 L or 5 L) isexamined, the dilution factor has to be considered for calculation of 1-mL1 mL sample.)11.10.5 Place slide under low power low-power dry (10) objective and scan the slide for evenness of the sample distribution.If the sample appears
