1、Designation: E 2564 07An American National StandardStandard Test Method forEnumeration of Mycobacteria in Metalworking Fluids by DirectMicroscopic Counting (DMC) Method1This standard is issued under the fixed designation E 2564; the number immediately following the designation indicates the year ofo
2、riginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method describes a direct microscopic count-ing m
3、ethod (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 al
4、lwater-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 limitat
5、ions prior to use. For additionalsafety information, see Laboratory Safety: Principle and Prac-tices, 4th Edition22. Referenced Documents2.1 ASTM Standards:3D 2881 Classification for Metal Working Fluids and RelatedMaterials3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 acid-
6、fast 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)e
7、nvironmental 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 th
8、e MF 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 forenumeratin
9、g acid 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 t
10、otal 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 microscopi
11、c fields 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 e
12、xamined. 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 ind
13、igenous MWF bacterial population. Measure-ment of mycobacterial cell count densities is an important stepin establishing a possible relationship between mycobacteriaand occupational health related allergic responses, for example,Hypersensitivity Pneumonitis (HP) in persons exposed toaerosols of meta
14、lworking fluids. It is known that the viablemycobacteria count underestimates the total mycobacterial1This 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
15、ing Fluids.Current edition approved April 15, 2007. Published May 2007.2Mary J. R. Gilchrist: Biosafety Precautions for Airborne Pathogens, inLaboratory Safety Principles and Practices, pp. 67-76, 1995, ASM Press3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Cus
16、tomer 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: DirectMicroscopic Methods for Bacteria or Somatic Cells, 16th ed.America Public
17、 HealthAssociation, Inc., Washington, DC, 1978.5Ebersole L.L.: Acid-fast stain procedures, pp. 3.5.1-3.5.11. In Clinical Micro-biology Procedures Handbook, Vol. 1. American Society for Microbiology, 1994 ,Washington ,D.C.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshoh
18、ocken, PA 19428-2959, United States.levels by not counting the non-culturable, possibly dead ormoribund population that is potentially equally important in theinvestigation of occupational health related problems. TheDirect Microscopic Counting Method (DMC) described heregives a quantitative assessm
19、ent 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 volumeover a known area. Although other microbesparticularly theActinomycetesalso stain acid
20、 fast, they are differentiatedfrom the mycobacteria because of their morphology and size.Non-mycobacteria, acid-fast microbes are 50-100 times largerthan mycobacteria. The method provides quantitative informa-tion on the total (culturable and non-culturable viable, andnon-viable) mycobacteria popula
21、tions. 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 techniqueis a semi- quantitative method with a relatively fast turnaroundtime.5.3 The DMC method can also be employed in field surveystudies to charac
22、terize 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 or centri-fuged, etc.) examined.6. Interferences6.1 Some metalworking fluid formulations fail
23、 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 difficult to interpret as heat fixing may notprovide full adherence. These samples should be re
24、-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 detectionlimit (1 cell/mL) than the DMC method.7. Apparatus7.1 Centrifuge, (“microfuge”) 14,
25、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, 10 L,1.0 mL, 5 mL.7.4 Microscope slides, with 100 mm2or similar areasmarked by frosted or
26、 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,103 magnification, equippedwith a net micrometer (10 mm by 10 mm) or similar.7.8 Slide drying apparatus, (box) 50-60C w
27、ith 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 8.1).7.13 Analytical balance8. Reagents and Materials8.1 Staining Reagents for Acid-Fast Staining Procedure forStaining Mycobacteria by the Kinyoun (C
28、old) 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: 970 mL8.1.3 TB Quick Stain Methylene Blue Reagent B: MethyleneBlue (alcoholic) 2.0 g, acid-
29、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 good laboratorypractices and safety procedures required in the microbiologylaboratory in p
30、reparing, using and disposing of cultures,reagents and materials.10. Sampling, Test Specimens, and Test Units10.1 Use sterile screw-capped plastic containers (100-200mL) for microbiological sampling of metalworking fluids. Thesample should be a random representative portion of 50-100mL that is from
31、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 documentation procedure inaccordance with good laboratory practices.11. Procedure11.1 Gently agitate samp
32、le 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 relative gravities for 30minutes at 22C.11.3 Remove supernatant gently using a disposable micropi
33、-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 micropipet 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
34、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 longerdrying time. These samples can be dried as long as overnighton the drying box. The slides that remain oily even after theextended drying time are usually
35、 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. The heat-fixed slide shouldE2564072be warm, not hot after flaming. In order to avoid overheatingthe slides th
36、e 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 staining kit:for example, Modified Kinyoun Staining Kit, although otheracid-fast staining methods can also be used.11.8 If the Modified Kinyoun Acid F
37、ast 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 rinsing on thefrosted part of the slide, not directly on the sample. Gentlyremove excess water.NOTE 1The stain is viscous and will not completely clea
38、r11.8.3 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 with TB Quick Stain MethyleneBlue / Reagent B for 4-5 minutes. (Staining with BrilliantGreen for 30 second
39、s 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 for the Oil Field (OIF) area,which is a single microscopic field that can be seen by the eyepiece.11.10.1 Us
40、e 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 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.
41、 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 factor has to be considered for calculation of 1-mLsample.)11.10.5 Place slide under low power dry (103) objec
42、tiveand 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 diameter 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.
43、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 (50/OIF) count only 5 OIFs. Forsamples with very low count (1/OIF) continue to count thefull diameter of t
44、he 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 may be prepared. A handtally or an electrical counter can be used to keep track with the number ofOIFs and
45、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 is multiplied by the calculated MF for the volume ofsample examined. Use the following equation to calcul
46、ate thetotal mycobacteria count per mL of metal working fluidsample:Total mycobacteria per mL 5Sum of AFB counts in all OIFs examinedNumber of OIFs counted 3 MFWhere MF is the Microscopic Factor calculated for 1-mLsample volume. If less than 1 mL of sample is examined (forexample, 10 L) dilution fac
47、tor of 1003 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 Bias14.1 PrecisionThe repeatability standard deviation hasbeen determined to be 0.99 (P 0.0001). Reproducibil
48、ity ofthe test method has not been determined yet.14.2 BiasNo information can be presented on the biasprocedure in this test method for measuring direct microscopicmycobacteria counts because no material having an acceptedreference value is available.15. Keywords15.1 acid-fast Bacteria (AFB); acid-f
49、ast staining; directmicroscopic count (DMC); hypersensitivity pneumonitis (HP);metalworking fluid (MWF); microscopic factor (MF); non-tuberculous mycobacterium (NTM)E2564073ASTM 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 rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the respon
