1、Designation: E2564 13 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 oforigi
2、nal 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 countingmethod (DMC
3、) for the enumeration of the acid fast stainedmycobacteria population in metalworking fluids. It can be usedto detect levels of total mycobacteria population, includingculturable as well as non-culturable (possibly dead or mori-bund ) bacterial cells. This practice is recommended for allwater-based
4、metalworking fluids.1.2 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to
5、 use. For additionalsafety information, see Laboratory Safety: Principle andPractices, 4th Edition22. Referenced Documents2.1 ASTM Standards:3D2881 Classification for Metalworking Fluids and RelatedMaterials3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 acid-fast bacteria, na
6、 distinctive staining propertyof Mycobacteria due to their lipid-rich cell walls.3.1.1.1 DiscussionOnce stained, mycobacterium resist de-colorization when exposed to acidified organic solvents, andare therefore, informally designated acid-fast.3.1.2 non-tuberculous Mycobacteria (NTM)environmental my
7、cobacteria, not associated with tuberculosis.3.1.3 microscopic factor (MF), na calibrated conversionfactor for calculating the Mycobacterium count per mLsample.3.1.3.1 DiscussionThe average number of mycobacteriumcells per one microscopic field (or oil field, OIF) is multipliedby the MF to give the
8、concentration of mycobacterium per mLof sample.3.1.4 oil immersion field (OIF), nthe circular area of amicroscopic field visible in the eye piece of the microscopeusing oil immersion objective.4. Summary of Practice4.1 The practice describes a semi quantitative test forenumerating acid fast stained
9、environmental mycobacterium(AFB) from metal working fluids by direct microscopic count-ing (DMC) method.4It is used to determine total mycobacte-rium counts including culturable and possibly dead or mori-bund cells in the sample. This practice cannot be used todetermine the total viable mycobacteriu
10、m population in thesample. A known sample volume (centrifuged or direct) isspread over a known area (1 cm2or similar) on a microscopeslide (marked by frosted or painted circles). Following differ-ential acid-fast staining,5the acid-fast cells are counted inseveral microscopic fields over the designa
11、ted area. Thecalculation is based on using a calibrated microscope with aknown Microscopic Factor (MF). The MF is determined by themicroscopic area over which a known amount of sample wasspread, the number of microscopic fields in the marked circle,and the volume of sample examined. The number of ac
12、id faststained mycobacterium cells per microscopic field multipliedby the MF gives the mycobacterium number per mLof sample.5. Significance and Use5.1 During the past decade, it has become increasinglyapparent that non-tuberculous mycobacteria are common mem-bers of the indigenous MWF bacterial popu
13、lation. Measure-ment of mycobacterial cell count densities is an important step1This practice is under the jurisdiction of ASTM Committee E34 on Occupa-tional Health and Safety and is the direct responsibility of Subcommittee E34.50 onHealth and Safety Standards for Metal Working Fluids.Current edit
14、ion approved July 1, 2013. Published July 2013. Originally approvedin 2007. Last previous edition approved in 2011 as E2564 - 11. DOI: 10.1520/E2564-13.2Mary J. R. Gilchrist: Biosafety Precautions for Airborne Pathogens, inLaboratory Safety Principles and Practices, pp. 67-76, 1995, ASM Press3For re
15、ferenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.4Standard Methods for the Examination of Dairy Products, Chapter: 10
16、: DirectMicroscopic Methods for Bacteria or Somatic Cells, 16th ed.America Public HealthAssociation, Inc., Washington, DC, 1978.5Ebersole L.L.: Acid-fast stain procedures, pp. 3.5.13.5.11. In Clinical Micro-biology Procedures Handbook, Vol. 1. American Society for Microbiology, 1994,Washington, D.C.
17、Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of Inter
18、national Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1in establishing a possible relationship between mycobacteriaand occupational health related allergic responses, for example,Hypersensitivity Pneumonitis (HP) in persons
19、exposed toaerosols of metalworking fluids. It is known that the viablemycobacteria count underestimates the total mycobacteriallevels by not counting the non-culturable, possibly dead ormoribund population that is potentially equally important in theinvestigation of occupational health related probl
20、ems. TheDirect Microscopic Counting Method (DMC) described heregives a quantitative assessment of the total numbers of acid-fast bacilli. It involves using acid-fast staining to selectivelyidentify mycobacteria from other bacteria, followed by enu-meration or direct microscopic counting of a known v
21、olumeover a known area. Although other microbesparticularly theActinomycetesalso stain acid fast, they are differentiatedfrom the mycobacteria because of their morphology and size.Non-mycobacteria, acid-fast microbes are 50 to 100 timeslarger than mycobacteria. The practice provides quantitativeinfo
22、rmation on the total (culturable and non-culturable viable,and non-viable) mycobacteria populations. The results areexpressed quantitatively as mycobacteria per mL of metal-working fluid sample.5.2 The DMC method using the acid-fast staining techniqueis a semi- quantitative method with a relatively
23、fast turnaroundtime.5.3 The DMC method can also be employed in field surveystudies to characterize the changes in total mycobacteriadensities of metalworking fluid systems over a long period oftime.5.4 The sensitivity detection limit of the DMC methoddepends on the MF and the sample volume (direct o
24、rcentrifuged, etc.) examined.6. Interferences6.1 Some metalworking fluid formulations fail to com-pletely dry or provide an uneven film on the microscope slide(for example, synthetic fluids and metalworking fluids withhigh trap tramp oil content and debris). For these samples theresults can be diffi
25、cult to interpret as heat fixing may notprovide full adherence. These samples should be re-stained ora new slide may be prepared.6.2 A negative acid fast staining reaction does not necessar-ily indicate that a sample will be culturally negative forMycobacteria since the culture method has a lower de
26、tectionlimit (1 cell/mL) than the DMC method.7. Apparatus7.1 Centrifuge, (“microfuge”) 14,000 relative gravities.7.2 Centrifuge tubes with caps, disposable, 1 mL-2 mLcapacity, such as Eppendorf SafeLock Tube or any othersuitable centrifuge tubes.7.3 Calibrated variable pipet, with sterile tips: 5 L,
27、 10 L,1.0 mL, 5 mL.7.4 Microscope slides, with 100 mm2or similar areasmarked by frosted or painted circles and frosted labeling ends.7.5 Calibrated stage micrometer, 0.01 mm or similar divi-sions.7.6 Compound microscope, with oil immersion lens.7.7 Microscope eye pieces, 10 magnification, equippedwi
28、th a net micrometer (10 by 10 mm) or similar.7.8 Slide drying apparatus, (box) 50 to 60C with leveldrying rack.7.9 Staining hood7.10 Staining rack and running water7.11 Hand tally or electrical counter7.12 Kinyoun Acid-Fast Stain Kit, (see 8.1).7.13 Analytical balance8. Reagents and Materials8.1 Sta
29、ining Reagents for Acid-Fast Staining Procedure forStaining Mycobacteria by the Kinyoun (Cold) Acid-Fast Pro-cedure:8.1.1 TB Quick Stain Carbol-Fuchsin, Reagent A: BasicFuchsin (alcoholic) 17.0g, Aqueous Phenol 1000.0 mL8.1.2 TB-Decolorizer: Hydrochloric Acid, 30.0 mL, Dena-tured Ethanol/Methanol: 9
30、70 mL8.1.3 TB Quick Stain Methylene Blue Reagent B: MethyleneBlue (alcoholic) 2.0 g, acid-alcohol 1000.0 mL; (acid-alcohol:30 mLHCl 970 mL, 90-95 % Ethanol) or Brilliant Green Stain:Brilliant Green 2.0 g, Sodium Hydroxide 0.02 g, DistilledWater 1000 mL9. Hazards9.1 The analyst must know and observe
31、good laboratorypractices and safety procedures required in the microbiologylaboratory in preparing, using and disposing of cultures,reagents and materials.10. Sampling, Test Specimens, and Test Units10.1 Use sterile screw-capped plastic containers (100 to 200mL) for microbiological sampling of metal
32、working fluids. Thesample should be a random representative portion of 50 to 100mL that is from the circulating tank opposed to a pooled,spillover or stagnant hose contents. Refrigerate samples untilanalyzed. Maximum sample storage time is 24 h at refrigera-tion temperatures. Follow sample documenta
33、tion procedure inaccordance with good laboratory practices.11. Procedure11.1 Gently agitate sample to re-suspend any sediment.Dispense 1 mL directly into the centrifuge tube. In the case ofvery viscous fluids, a 1-g sample should be weighed on ananalytical balance.11.2 Centrifuge samples at 13.000 r
34、elative gravities for 30minutes at 22C.11.3 Remove supernatant gently using a disposable micropi-pet end.E2564 13211.4 Remove oily residues completely from the tube using asterile cotton swab. Gently remove the whole pellet with asterile loop or a micropipet end without disturbing the sedi-ment.11.5
35、 Transfer the whole amount of sediment to the 1-cm2designated 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 60C forminimum of one hour. Some fluid formulations require longerdrying time. These samples can be dried as lo
36、ng as overnighton the drying box. The slides that remain oily even after theextended drying time are usually the result of a poorly decantedtube and for these samples the slide preparation must berepeated.11.7 Heat-fix the dried slides by gently passing the slidethrough a flame fast once or twice. T
37、he heat-fixed slide shouldbe warm, not hot after flaming. In order to avoid overheatingthe slides the flaming can be substituted by a standardtemperature heat block at 75C for 10 to 20 minutes. Afterheat-fixing the slides, stain them using an acid-fast staining kit:for example, Modified Kinyoun Stai
38、ning Kit, although otheracid-fast staining methods can also be used.11.8 If the Modified Kinyoun Acid Fast Staining Method isused:11.8.1 Flood slide with TB Quick Stain Carbol-FuchsinReagent A for 4 to 5 minutes.11.8.2 Rinse slide gently with water. Start rinsing on thefrosted part of the slide, not
39、 directly on the sample. Gentlyremove excess water.NOTE 1The stain is viscous and will not completely clear11.8.3 Flood slide with TB Acid Alcohol Decolorizer for15-30 seconds.11.8.4 Rinse slide gently with water until rinse water ismostly clear. Gently remove excess water.11.8.5 Counterstain slide
40、with TB Quick Stain MethyleneBlue / Reagent B for 4 to 5 minutes. (Staining with BrilliantGreen for 30 seconds 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 completely11.9 Direct Microscopic Counting11.10 Calibrate the microscope
41、 for the Oil Field (OIF) area,which is a single microscopic field that can be seen by the eyepiece.11.10.1 Use a stage micrometer slide with 0.1 and 0.01-mmdivisions to determine the diameter of one field under the oilimmersion lens. Make the reading to the third decimal point.11.10.2 Determine the
42、area of the OIF (r2)inmm2.11.10.3 Convert the OIF area to cm2.11.10.4 Determine the number of OIFs in 1 cm2. Thisnumber will provide the Microscopic Factor (MF) for 1 mL ofsample if the whole sediment is examined. (If less than 1-mLsample volume (for example, 10 L or 5 L) is examined, thedilution fa
43、ctor has to be considered for calculation of 1-mLsample.)11.10.5 Place slide under low power dry (10) objective andscan the slide for evenness of the sample distribution. If thesample appears to be even, move the slide so that the frostededge of the widest diameter is centered in your field of view.
44、Place oil over the sample and change to oil immersionobjective (100) at the edge of the frosted circle.11.10.6 Start examination by slowly rolling and focusing fora full diameter of the designated 1-cm2area counting thenumber of mycobacteria in each field as you go. If themycobacteria count is high
45、(50/OIF) count only 5 OIFs. Forsamples with very low count (1/OIF) continue to count thefull diameter of the 1-cm2area (50-55 OIFs) For samples with(30/OIF) AFB count 20-25 OIFs.NOTE 2Slides that have oil or debris may be difficult to count. In thiscase the slide could be re-stained or a new slide m
46、ay be prepared. A handtally or an electrical counter can be used to keep track with the number ofOIFs and the total number of AFBs counted.12. Calculation or Interpretation of Results12.1 To obtain estimates of mycobacteria per mL of metal-working fluid, the average number of mycobacteria per oneOIF
47、 is multiplied by the calculated MF for the volume ofsample examined. Use the following equation to calculate thetotal mycobacteria count per mL of metal working fluidsample:Total mycobacteria per mL5Sum of AFB counts in all OIFs examined 3 MFNumber of OIFs countedWhere MF is the Microscopic Factor
48、calculated for 1-mLsample volume. If less than 1 mL of sample is examined (forexample, 10 L) dilution factor of 100 has to be taken intoconsideration when calculating the concentration for 1-mLsample.13. Report13.1 Report mycobacterium count per mL of metalworkingfluid sample.14. Precision and Bias1
49、4.1 PrecisionSince precision will depend on the fluids,challenge microbes, and microbicide treatments used to per-form individual investigations, no statement on precision ismade.14.2 BiasSince there is no accepted reference materialsuitable for the bias in this practice, no statement on bias ismade.15. Keywords15.1 acid-fast Bacteria (AFB); acid-fast staining; directmicroscopic count (DMC); hypersensitivity pneumonitis (HP); metalworking fluid (MWF); microscopic factor (MF); non-tuberculous mycobacterium (NTM)E2564 133ASTM International takes no position
