1、Designation: E2564 11An American National StandardStandard Test Method forEnumeration of Mycobacteria in Metalworking Fluids by DirectMicroscopic Counting (DMC) Method1This standard is issued under the fixed designation E2564; the number immediately following the designation indicates the year ofori
2、ginal 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 test method describes a direct microscopic count-ing meth
3、od (DMC) 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 test method is recommended for allwa
4、ter-based 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 limitation
5、s prior to use. For additionalsafety information, see Laboratory Safety: Principle and Prac-tices, 4th Edition22. Referenced Documents2.1 ASTM Standards:3D2881 Classification for Metal Working Fluids and RelatedMaterials3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 acid-fast
6、 bacteria, na distinctive staining propertyof Mycobacteria due to their lipid-rich cell walls.3.1.1.1 DiscussionOnce stained, mycobacterium resistdecolorization when exposed to acidified organic solvents, andare therefore, informally designated acid-fast.3.1.2 non-tuberculous Mycobacteria (NTM)envir
7、onmental mycobacteria, 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 mycobacte-rium cells per one microscopic field (or oil field, OIF) ismultiplied by the MF
8、 to give the concentration of mycobacte-rium per mL of 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 Test Method4.1 The method describes a semi quantitative test forenumerating ac
9、id fast stained environmental mycobacterium(AFB) from metal working fluids by direct microscopic count-ing (DMC) method4. It is used to determine total mycobacte-rium counts including culturable and possibly dead or mori-bund cells in the sample. This test method cannot be used todetermine the total
10、 viable mycobacterium 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 staining5, the acid-fast cells are counted inseveral microscopic fi
11、elds over the designated 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 exami
12、ned. The number of acid faststained mycobacterium cells per microscopic field multipliedby the MF gives the mycobacterium number per mL of sample.5. Significance and Use5.1 During the past decade, it has become increasinglyapparent that non-tuberculous mycobacteria are common mem-bers of the indigen
13、ous MWF bacterial population. Measure-ment of mycobacterial cell count densities is an important step1This test method is under the jurisdiction of ASTM Committee E34 onOccupational Health and Safety and is the direct responsibility of SubcommitteeE34.50 on Health and Safety Standards for Metal Work
14、ing Fluids.Current edition approved Jan. 1, 2011. Published January 2011. Originallyapproved in 2007. Last previous edition approved in 2007 as E2564 - 07. DOI:10.1520/E2564-11.Current edition approved . Published May 2007.2Mary J. R. Gilchrist: Biosafety Precautions for Airborne Pathogens, inLabora
15、tory Safety Principles and Practices, pp. 67-76, 1995, ASM Press3For referenced 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 websi
16、te.4Standard Methods for the Examination of Dairy Products, Chapter: 10: DirectMicroscopic Methods for Bacteria or Somatic Cells, 16th ed.America Public HealthAssociation, Inc., Washington, DC, 1978.5Ebersole L.L.: Acid-fast stain procedures, pp. 3.5.1-3.5.11. In Clinical Micro-biology Procedures Ha
17、ndbook, Vol. 1. American Society for Microbiology, 1994 ,Washington ,D.C.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.in establishing a possible relationship between mycobacteriaand occupational health related allergic responses,
18、for example,Hypersensitivity Pneumonitis (HP) in persons 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
19、 in theinvestigation of occupational health related problems. 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
20、 enu-meration or direct microscopic counting of a known volumeover 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 50-100 times largertha
21、n mycobacteria. The method provides quantitative informa-tion on the total (culturable and non-culturable viable, andnon-viable) mycobacteria populations. The results are ex-pressed quantitatively as mycobacteria per mL of metalwork-ing fluid sample.5.2 The DMC method using the acid-fast staining te
22、chniqueis a semi- quantitative method with a relatively 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
23、 methoddepends on the MF and the sample volume (direct or centri-fuged, 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 cont
24、ent and debris). For these samples theresults can be difficult 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 negati
25、ve forMycobacteria since the culture method has a lower detectionlimit (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 tu
26、bes.7.3 Calibrated variable pipet, with sterile tips: 5 L, 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 le
27、ns.7.7 Microscope eye pieces,103 magnification, equippedwith a net micrometer (10 mm by 10 mm) or similar.7.8 Slide drying apparatus, (box) 50-60C with level dryingrack.7.9 Staining hood7.10 Staining rack and running water7.11 Hand tally or electrical counter7.12 Kinyoun Acid-Fast Stain Kit, (see 8.
28、1).7.13 Analytical balance8. Reagents and Materials8.1 Staining 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:
29、Hydrochloric Acid, 30.0 mL, Dena-tured Ethanol/Methanol: 970 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, DistilledWat
30、er 1000 mL9. Hazards9.1 The analyst must know and observe 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 conta
31、iners (100-200mL) for microbiological sampling of metalworking fluids. Thesample should be a random representative portion of 50-100mL 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
32、refrigera-tion temperatures. Follow sample documentation 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 anan
33、alytical balance.11.2 Centrifuge samples at 13.000 relative gravities for 30minutes at 22C.11.3 Remove supernatant gently using a disposable micropi-pet end.11.4 Remove oily residues completely from the tube using asterile cotton swab. Gently remove the whole pellet with asterile loop or a micropipe
34、t end without disturbing the sedi-ment.11.5 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 50-60C forminimum of one hour. Some fluid formulations require longerdryin
35、g time. These samples can be dried as long as overnightE2564 112on 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 passi
36、ng the slidethrough a flame fast once or twice. The 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 10-20 minutes. Afterheat-fixing the slides, stain them using an acid-fast s
37、taining kit:for example, Modified Kinyoun Staining 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 4-5 minutes.11.8.2 Rinse slide gently with water. Start r
38、insing on thefrosted part of the slide, not 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 rem
39、ove excess water.11.8.5 Counterstain slide with TB Quick Stain MethyleneBlue / Reagent B for 4-5 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 Microscopi
40、c Counting11.10 Calibrate the microscope 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 th
41、ird decimal point.11.10.2 Determine the area of the OIF (r2p)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,
42、 10 L or 5 L) is examined, thedilution factor has to be considered for calculation of 1-mLsample.)11.10.5 Place slide under low power dry (103) objectiveand scan the slide for evenness of the sample distribution. If thesample appears to be even, move the slide so that the frostededge of the widest d
43、iameter is centered in your field of view.Place oil over the sample and change to oil immersionobjective (1003) 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 a
44、s you go. If themycobacteria count is high (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
45、 slide could be re-stained or a new slide may 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, th
46、e average number of mycobacteria per oneOIF 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 mL 5Sum of AFB counts in all OIFs examined 3 MFNumber of OIF
47、s countedWhere MF is the Microscopic Factor calculated for 1-mLsample volume. If less than 1 mL of sample is examined (forexample, 10 L) dilution factor of 1003 has to be taken intoconsideration when calculating the concentration for 1-mLsample.13. Report13.1 Report mycobacterium count per mL of met
48、alworkingfluid sample.14. Precision and Bias14.1 PrecisionThe repeatability standard deviation hasbeen determined to be 0.99 (P 0.0001). Reproducibility ofthe test method has not been determined yet.14.2 BiasNo information can be presented on the biasprocedure in this test method for measuring direc
49、t microscopicmycobacteria counts because no material having an acceptedreference value is available.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 113ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent
