1、Designation: D5245 92 (Reapproved 2012)Standard Practice forCleaning Laboratory Glassware, Plasticware, and EquipmentUsed in Microbiological Analyses1, 2This standard is issued under the fixed designation D5245; the number immediately following the designation indicates the year oforiginal adoption
2、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 In microbiology, clean glassware is crucial to ensurevalid results. Previousl
3、y used or new glassware must bethoroughly cleaned. Laboratory ware and equipment that arenot chemically clean are responsible for considerable losses inpersonnel time and supplies in many laboratories. These lossesmay occur as down time when experiments clearly have beenadversely affected and as inv
4、alid data that are often attributedto experimental error. Chemical contaminants that adverselyaffect experimental results are not always easily detected. Thispractice describes the procedures for producing chemicallyclean glassware.1.2 The values stated in SI units are to be regarded as thestandard.
5、1.3 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 use. For specificpr
6、ecautions, see Section 5, 7.3.1, and Note 1 and Note 2.2. Referenced Documents2.1 ASTM Standards:3D1193 Specification for Reagent Water3. Significance and Use3.1 This practice provides uniform guidance for cleaningthe laboratory glassware, plasticware, and equipment used inroutine microbiological an
7、alyses. However, tests that are ex-tremely sensitive to toxic agents (such as virus assays) mayrequire more stringent cleaning practices.24. Reagents4.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the spe
8、cifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.4Other grades may be used,provided it is first ascertained that the reagent is of sufficientlyhigh purity to permit its use without lessening the accuracy ofthe determination.4.
9、2 Purity of Water Unless otherwise indicated, refer-ences to water shall be understood to mean Type IV ofSpecification D1193.4.3 Detergent Solution, for machine-washing glassware andequipment. Use according to manufacturers instructions.4.4 Detergent Powder, for hand-washing glassware andequipment.
10、Use them according to manufacturers instructions.There now are a number of effective biogradable detergentproducts available that allow the laboratory to avoid acidcleaning of most if not all glassware.4.5 Nitric Acid (1 + 9)Pour 100 mL of concentrated HNO3 slowly into 900 mL of water. To avoid dang
11、erous splatters,never pour water into concentrated acid.4.6 Chromic Acid SolutionChromic acid replacement5isapplicable.5. Hazards5.1 The analyst/technician must know and observe normalgood laboratory practices and safety procedures required in amicrobiology laboratory in preparing, using, and dispos
12、ing ofcultures, reagents, and materials, and while operating steriliza-tion and other equipment and instrumentation.1This practice is under the jurisdiction of ASTM Committee D19 on Water andis the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved June 1, 20
13、12. Published August 2012. Originallyapproved in 1992. Last previous edition approved in 2005 as D5245 92 (2005).DOI: 10.1520/D5245-92R12.2A significant portion of this practice was taken from: Berg, G., Safferman, R.S., Dahling, D. R., Berman, D., and Hurst, C. J., USEPA Manual of Methods forVirolo
14、gy, EPA-600/4-84-013, Chapt. 2, “Cleansing Laboratory Ware and Equip-ment, Environmental Monitoring and Support LaboratoryCincinnati,” USEPA,Cincinnati, OH.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of AST
15、MStandards volume information, refer to the standards Document Summary page onthe ASTM website.4“Reagent Chemicals, American Chemical Society Specifications, Am. Chemi-cal Soc., Washington, DC. For suggestions on the testing of reagents not listed bythe American Chemical Society, see “Analar Standar
16、ds for Laboratory Chemicals,”BDH Ltd. Poole Dorset, U.K. and the “United States Pharmacopeia.”5The sole source of supply of the apparatus known to the committee at this timeis Monostat Corp., 519 Eighth St., New York, NY 10018. If you are aware ofalternative suppliers, please provide this informatio
17、n to ASTM InternationalHeadquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee,1which you may attend.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.2 Sterilize contaminated
18、 laboratory ware and equipmentbefore cleaning.5.3 Transport hazardous acids only in appropriate safetycarriers.5.4 See 7.3 and 7.4 for details on proper cleaning with acidsand alkalies.6. Cleaning Rules6.1 Once detergent solution or acid used to clean a vesselhas been rinsed away, do not touch lip o
19、r inside of vessel withhands. Detergent or acid on hands or gloves and even oil fromclean skin are sources of contamination.6.2 Do not allow soiled laboratory ware and equipment todry. Soak glassware if cleaning is delayed.6.3 Use only cold water for tap water rinsing. Hot watermay contain grease or
20、 oil removed from plumbing. Use onlycold water to wash laboratory ware heavily contaminated withproteinaceous material. Hot water may coagulate such mate-rial.6.4 Inspect washed laboratory ware and equipment forcleanliness. Reclean by appropriate procedures. Check labora-tory ware and equipment for
21、cracks, chips, or other damage andreplace.6.5 Use nontoxic stainless steel, glass, nonbreakable plastic,or other nontoxic materials for plumbing that carries water. Donot use copper plumbing.6.6 Use disposable glass and plasticware for pathogenicwork and test conditions that severely soil or etch gl
22、assware.7. Cleaning Procedures7.1 Machine Washing Equip washing machine with capa-bility for delivering four water rinses. The water jets in somewashing machines are not strong enough to reach all walls intall vessels. This results in poor washing and rinsing. The waterjets in other washing machines
23、 are too strong for test tubes andsimilar vessels and for many other narrow-necked vessels. Jetsthat are too powerful hold detergent and rinse water in placeand do not allow them to drain properly. If washing machine isunable to wash or rinse adequately, use procedure described in7.2.7.1.1 Immerse w
24、ashable vessels in detergent solution, andsoak them overnight. If vessels are too large to immerse, fillthem to brim with detergent solution, and soak them overnight.7.1.2 Brush-wash vessels with hot (50 to 60C) detergentsolution. Hot tap water that exceeds 50C is adequate forpreparing detergent sol
25、ution.7.1.3 Machine-wash vessels. Follow manufacturers in-structions carefully. Add four water rinses if not included inmanufacturers instructions.7.1.4 Drain and air dry vessels, or dry vessels in dryingchamber.7.1.5 Detergents used in washing may contain inhibitorysubstances. As necessary, test fo
26、r the presence of inhibitoryresidues (for example, a new supply of detergent). Check cleanlaboratory ware and equipment for residues in accordance withprocedure given in 7.2.7. This procedure is similar to that givenin Footnote 7.67.2 Manual Washing Procedure:7.2.1 Immerse vessels in detergent solut
27、ion, and soak ves-sels overnight. Use fresh detergent solution daily. Solutionsthat are saved may become heavily contaminated with bacteria.7.2.2 Brush-wash vessels with hot (50 to 60C) detergentsolution. Hot tap water that exceeds 50C is adequate forpreparing detergent solution.7.2.3 Swirl-rinse ve
28、ssels ten times with cold tap water. Toswirl-rinse, pour into the vessel a volume of tap water equal toabout 10 % of the volume of the vessel, and swirl water aroundentire surface with each rinse. Swirl-rinse vessels five timeswith water.7.2.4 Drain and air dry vessels, or dry vessels in dryingchamb
29、er.7.2.5 Test TubesTest tubes may be washed by the proce-dure described in 7.1, unless a washing machine is unavailableor washing machine jets are so powerful they do not allowadequate evacuation of tubes and thus interfere with washingand rinsing or by the following procedure.7.2.5.1 Remove marking
30、s from tubes with solvent beforewashing.7.2.5.2 Place test tubes open end up into covered wirebasket, place basket into stainless steel or plastic vesselsufficient in size to allow complete immersion of tubes, and fillvessel with hot detergent solution.7.2.5.3 Steam autoclave (100C) immersed tubes f
31、or 30min.7.2.5.4 Empty vessel and tubes, and run cold tap water in toflush out detergent solution. Introduce tap water into bottom ofvessel with a hose connected to tap. Wax pencil and other scumwill wash over rim of vessel.7.2.5.5 Fill and empty tubes in vessel ten times with cold tapwater. Fill an
32、d empty tubes in vessel five times with water.7.2.5.6 Drain and air dry tubes, or dry tubes in dryingchamber.7.2.5.7 Inspect, rewash if not clean, and use alternatecleaning method if appropriate. If glassware still does not meetrequirements, discard.7.2.6 Pipets:7.2.6.1 Remove cotton plugs from pipe
33、ts. If necessary,remove cotton plugs by forcing a jet of air or water throughdelivery tips of pipets.7.2.6.2 Place pipets, with tips up, into pipet holder.7.2.6.3 Place pipet holder into a pipet jar, and fill jar withhot (50 to 60C) detergent solution. Hot tap water that exceeds50C is adequate for p
34、reparing detergent solution. Pipets mustbe completely immersed. If air bubbles are present in pipets,raise and lower pipet holder several times to remove bubbles.7.2.6.4 Soak pipets in detergent solution for 24 h. Raise andlower pipet holder five or six times during the 24-h period toagitate deterge
35、nt solution and help remove soil and debris frompipets.6Standard Methods for the Examination of Water and Wastewater, 17th Ed.,American Public Health Association, Washington, DC, Section 9020B, 3.a, 2, 1989,pp. 98.D5245 92 (2012)27.2.6.5 Place pipet holder into automatic pipet washer, andrinse pipet
36、s through ten cycles of cold tap water.7.2.6.6 Rinse pipets through five cycles of water.7.2.6.7 Remove pipets from automatic pipet washer, andallow them to drain and air dry.7.2.6.8 Plug pipets with cotton.7.2.7 Test Procedure for Suitability of Detergent Used inWashing:7.2.7.1 Wash and rinse six p
37、etri dishes in the usual manner.These are Group A.7.2.7.2 After normal washing, rinse a second group of sixpetri dishes twelve times with successive portions of water.These are Group B.7.2.7.3 Wash six petri dishes with the detergent wash waterusing detergent concentrations normally employed, and dr
38、ywithout rinsing. These are Group C.7.2.7.4 Sterilize dishes in the usual manner.7.2.7.5 Add the proper dilution (usually two different dilu-tions are used) of a water sample yielding 30 to 300 coloniesto triplicate petri dishes from each group (A, B, and C).Proceed according to the heterotrophic pl
39、ate count method.7.2.7.6 Differences in bacterial counts of less than 15 %among all groups indicate the detergent has no toxicity orinhibitory effect. Differences in bacterial counts of 15 % ormore between Groups A and B demonstrate that inhibitoryresidues are left on glassware after the normal wash
40、ingprocedure used. Disagreement in averages of less than 15 %between Groups A and B, and greater than 15 % betweenGroups A and C indicates that detergent used has inhibitoryproperties that are eliminated during routine washing.7.2.8 Automatic Pipetor (Brewer-Type):7.2.8.1 Immediately after pipettor
41、has been used, fill reser-voir with tap water and carefully pump sufficient water throughthe system to remove cellular debris and other materials thatmight adhere to apparatus. Determine whether syringe deliversproperly without cannula connected.7.2.8.2 Remove tubing from reservoir, and remove syrin
42、gefrom pipettor; autoclave valve, tubing, reservoir, and syringe at121C for 60 min.7.2.8.3 Disassemble syringe, and remove cannula.7.2.8.4 Cleanse syringe. Rinse plunger and barrel of syringewith copious quantities of cold tap water. Soak tubing over-night in water. Allow tubing to drain and air dry
43、.7.2.8.5 Fill reservoir with hot (50 to 60C) detergent solu-tion, and soak reservoir overnight. Hot tap water that exceeds50C is adequate for preparing detergent solution. Brush-washreservoir with hot (50 to 60C) detergent solution. If reservoirdoes not come clean, rinse it with tap water, and soak
44、itovernight in HNO3(1 + 9) or in chromic acid (1 + 9). Thenrinse reservoir ten times with cold tap water, swirl-rinse fivetimes with water, and allow to drain and air dry.7.2.8.6 ValveIf syringe has been delivering properly withthe cannula removed, no further attention to valve is needed. Ifsyringe
45、has not been delivering properly with the cannularemoved, remove valve from apparatus. Soak valve overnightin (1 + 9) HNO3or in chromic acid (1 + 9). Rinse copiouslywith cold tap water and reagent water. Allow to drain and airdry and return to apparatus.7.2.8.7 Connect cannula to a clean syringe and
46、 forcethrough 50 mL of water.7.2.8.8 Rinse tubing copiously with cold tap water. If tubingdoes not come clean, place it in hot (50 to 60C) detergentsolution, remove air bubbles, and allow tubing to soak for 24h.7.3 Cleaning With Acid:7.3.1 Use acid cleaning only when there is no alternative.Consider
47、 disposable glassware as a possible alternative. Chro-mic acid or HNO3(1 + 9) may be used to clean glassware. Tenpercent HNO3requires longer contact (24 h) with tubes thanchromic acid requires, but residual HNO3is not as likely to betoxic to microorganisms.NOTE 1Warning: Do not expose metals or othe
48、r materials to acidsunless certain that those substances are acid-resistant. Chromic acidcleaning solutions5and other acids may react violently with organics orother oxidizable substances. Take care to avoid such reactions.NOTE 2Warning: Chromic acid and nitric acid are capable of pro-ducing burns e
49、ven when used in relatively dilute solutions. When workingwith these or with other acids, avoid inhalation of fumes. Protect eyes withsafety goggles or with full-face mask. Protect clothing with acid-resistantlaboratory coat or apron. If eyes are accidently exposed to acid, immedi-ately wash them with copious quantities of tap water for at least 15 min.Consult a physician immediately thereafter. If other parts of the body areexposed to acid, immediately remove clothing over exposed areas andflood with large volumes of tap water. Consult a physician immedia
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