ASTM D5245-92(1998)
(Practice)Standard Practice for Cleaning Laboratory Glassware, Plasticware, and Equipment Used in Microbiological Analyses
Standard Practice for Cleaning Laboratory Glassware, Plasticware, and Equipment Used in Microbiological Analyses
SCOPE
1.1 In microbiology, clean glassware is crucial to ensure valid results. Previously used or new glassware must be thoroughly cleaned. Laboratory ware and equipment that are not chemically clean are responsible for considerable losses in personnel time and supplies in many laboratories. These losses may occur as down time when experiments clearly have been adversely affected and as invalid data that are often attributed to experimental error. Chemical contaminants that adversely affect experimental results are not always easily detected. This practice describes the procedures for producing chemically clean glassware.
1.2 The values stated in SI units are to be regarded as the standard.
1.3 This practice does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. For specific precautions, see Section 5, 7.3.1, and Notes 1 and 2.
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An American National Standard
Designation:D5245–92(Reapproved1998)
Standard Practice for
Cleaning Laboratory Glassware, Plasticware, and Equipment
,
1 2
Used in MicrobiologicalAnalyses
This standard is issued under the fixed designation D 5245; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 4. Reagents
1.1 In microbiology, clean glassware is crucial to ensure 4.1 Purity of Reagents—Reagent grade chemicals shall be
valid results. Previously used or new glassware must be used in all tests. Unless otherwise indicated, it is intended that
thoroughly cleaned. Laboratory ware and equipment that are all reagents conform to the specifications of the Committee on
not chemically clean are responsible for considerable losses in Analytical Reagents of the American Chemical Society where
personnel time and supplies in many laboratories. These losses such specifications are available. Other grades may be used,
may occur as down time when experiments clearly have been provided it is first ascertained that the reagent is of sufficiently
adversely affected and as invalid data that are often attributed high purity to permit its use without lessening the accuracy of
to experimental error. Chemical contaminants that adversely the determination.
affect experimental results are not always easily detected. This 4.2 Purity of Water— Unless otherwise indicated, refer-
practice describes the procedures for producing chemically ences to water shall be understood to mean Type IV of
clean glassware. Specification D 1193.
1.2 The values stated in SI units are to be regarded as the 4.3 Detergent Solution, for machine-washing glassware and
standard. equipment. Use according to manufacturer’s instructions.
1.3 This practice does not purport to address all of the 4.4 Detergent Powder, for hand-washing glassware and
safety problems, if any, associated with its use. It is the equipment. Use them according to manufacturer’s instructions.
responsibility of the user of this standard to establish appro- There now are a number of effective biogradable detergent
priate safety and health practices and determine the applica- products available that allow the laboratory to avoid acid
bility of regulatory limitations prior to use. For specific cleaning of most if not all glassware.
precautions, see Section 5, 7.3.1, and Note 1 and Note 2. 4.5 NitricAcid(1+9)—Pour100mLofconcentratedHNO
3 slowly into 900 mL of water. To avoid dangerous splatters,
2. Referenced Documents
never pour water into concentrated acid.
2.1 ASTM Standards: 4.6 Chromic Acid Solution—Chromic acid replacement is
D 1193 Specification for Reagent Water
applicable.
3. Significance and Use 5. Hazards
3.1 This practice provides uniform guidance for cleaning
5.1 The analyst/technician must know and observe normal
the laboratory glassware, plasticware, and equipment used in good laboratory practices and safety procedures required in a
routine microbiological analyses. However, tests that are ex-
microbiology laboratory in preparing, using, and disposing of
tremely sensitive to toxic agents (such as virus assays) may cultures, reagents, and materials, and while operating steriliza-
require more stringent cleaning practices.
tion and other equipment and instrumentation.
5.2 Sterilize contaminated laboratory ware and equipment
before cleaning.
This practice is under the jurisdiction ofASTM Committee D-19 on Water and
is the direct responsibility of Subcommittee D19.24 on Water Microbiology. “Reagent Chemicals, American Chemical Society Specifications, Am. Chemi-
Current edition approved May 15, 1992. Published September 1992. cal Soc., Washington, DC. For suggestions on the testing of reagents not listed by
A significant portion of this practice was taken from: Berg, G., Safferman, R. theAmerican Chemical Society, see “Analar Standards for Laboratory Chemicals,”
S., Dahling, D. R., Berman, D., and Hurst, C. J., USEPA Manual of Methods for BDH Ltd. Poole Dorset, U.K. and the “United States Pharmacopeia.”
Virology, EPA-600/4-84-013, Chapt. 2, “Cleansing Laboratory Ware and Equip- MIR-A-KOL, available from Diversey Corp., 255 East Fifth St., Cincinnati,
ment, Environmental Monitoring and Support Laboratory—Cincinnati,” USEPA, OH 45202, or equivalent.
Cincinnati, OH. Chromerge, a registered trademark of Monostat Corp., 519 Eighth St., New
Annual Book of ASTM Standards, Vol 11.01. York, NY 10018 has been found suitable for this purpose.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D5245–92 (1998)
5.3 Transport hazardous acids only in appropriate safety laboratory ware and equipment for residues in accordance with
carriers. proceduregivenin7.2.7.Thisprocedureissimilartothatgiven
in Footnote 7.
5.4 See 7.3 and 7.4 for details on proper cleaning with acids
and alkalies. 7.2 Manual Washing Procedure:
7.2.1 Immerse vessels in detergent solution, and soak ves-
sels overnight. Use fresh detergent solution daily. Solutions
6. Cleaning Rules
thataresavedmaybecomeheavilycontaminatedwithbacteria.
6.1 Once detergent solution or acid used to clean a vessel
7.2.2 Brush-wash vessels with hot (50 to 60°C) detergent
has been rinsed away, do not touch lip or inside of vessel with
solution. Hot tap water that exceeds 50°C is adequate for
hands. Detergent or acid on hands or gloves and even oil from
preparing detergent solution.
clean skin are sources of contamination.
7.2.3 Swirl-rinse vessels ten times with cold tap water. To
6.2 Do not allow soiled laboratory ware and equipment to
swirl-rinse, pour into the vessel a volume of tap water equal to
dry. Soak glassware if cleaning is delayed.
about 10 % of the volume of the vessel, and swirl water around
6.3 Use only cold water for tap water rinsing. Hot water
entire surface with each rinse. Swirl-rinse vessels five times
may contain grease or oil removed from plumbing. Use only
with water.
cold water to wash laboratory ware heavily contaminated with
7.2.4 Drain and air dry vessels, or dry vessels in drying
proteinaceous material. Hot water may coagulate such mate-
chamber.
rial.
7.2.5 Test Tubes—Test tubes may be washed by the proce-
6.4 Inspect washed laboratory ware and equipment for
dure described in 7.1, unless a washing machine is unavailable
cleanliness. Reclean by appropriate procedures. Check labora-
or washing machine jets are so powerful they do not allow
torywareandequipmentforcracks,chips,orotherdamageand
adequate evacuation of tubes and thus interfere with washing
replace.
and rinsing or by the following procedure.
6.5 Use nontoxic stainless steel, glass, nonbreakable plastic,
7.2.5.1 Remove markings from tubes with solvent before
or other nontoxic materials for plumbing that carries water. Do
washing.
not use copper plumbing.
7.2.5.2 Place test tubes open end up into covered wire
6.6 Use disposable glass and plasticware for pathogenic
basket, place basket into stainless steel or plastic vessel
work and test conditions that severely soil or etch glassware.
sufficient in size to allow complete immersion of tubes, and fill
vessel with hot detergent solution.
7. Cleaning Procedures
7.2.5.3 Steam autoclave (100°C) immersed tubes for 30
7.1 Machine Washing— Equip washing machine with capa- min.
bility for delivering four water rinses. The water jets in some 7.2.5.4 Empty vessel and tubes, and run cold tap water in to
washing machines are not strong enough to reach all walls in flush out detergent solution. Introduce tap water into bottom of
tallvessels.Thisresultsinpoorwashingandrinsing.Thewater
vesselwithahoseconnectedtotap.Waxpencilandotherscum
jets in other washing machines are too strong for test tubes and will wash over rim of vessel.
similar vessels and for many other narrow-necked vessels. Jets 7.2.5.5 Fillandemptytubesinvesseltentimeswithcoldtap
that are too powerful hold detergent and rinse water in place water. Fill and empty tubes in vessel five times with water.
and do not allow them to drain properly. If washing machine is 7.2.5.6 Drain and air dry tubes, or dry tubes in drying
unable to wash or rinse adequately, use procedure described in chamber.
7.2. 7.2.5.7 Inspect, rewash if not clean, and use alternate
7.1.1 Immerse washable vessels in detergent solution, and cleaning method if appropriate. If glassware still does not meet
requirements, discard.
soak them overnight. If vessels are too large to immerse, fill
them to brim with detergent solution, and soak them overnight. 7.2.6 Pipets:
7.2.6.1 Remove cotton plugs from pipets. If necessary,
7.1.2 Brush-wash vessels with hot (50 to 60°C) detergent
remove cotton plugs by forcing a jet of air or water through
solution. Hot tap water that exceeds 50°C is adequate for
delivery tips of pipets.
preparing detergent solution.
7.2.6.2 Place pipets, with tips up, into pipet holder.
7.1.3 Machine-wash vessels. Follow manufacturer’s in-
7.2.6.3 Place pipet holder into a pipet jar, and fill jar with
structions carefully. Add four water rinses if not included in
hot (50 to 60°C) detergent solution. Hot tap water that exceeds
manufacturer’s instructions.
50°C is adequate for preparing detergent solution. Pipets must
7.1.4 Drain and air dry vessels, or dry vessels in drying
chamber.
7.1.5 Detergents used in washing may contain inhibitory
Standard Methods for the Examination of Water and Wastewater, 17th Ed.,
substances. As necessary, test for the presence of inhibitory
American Public HealthAssociation,Washington, DC, Section 9020B, 3.a, 2, 1989,
residues (for example, a new supply of detergent). Check clean pp. 9–8.
D5245–92 (1998)
be completely immersed. If air bubbles are present in pipets, 7.2.8.6 Valve—If syringe has been delivering properly with
raise and lower pipet holder several times to remove bubbles. the cannula removed, no further attention to valve is needed. If
syringe has not been delivering properly with the cannula
7.2.6.4 Soak pipets in detergent solution for 24 h. Raise and
removed, remove valve from apparatus. Soak valve overnight
lower pipet holder five or six times during the 24-h period to
in (1 + 9) HNO or in chromic acid (1 + 9). Rinse copiously
agitate detergent solution and help remove soil and debris from
with cold tap water and reagent water. Allow to drain and air
pipets.
dry and return to apparatus.
7.2.6.5 Place pipet holder into automatic pipet washer, and
7.2.8.7 Connect cannula to a clean syringe and force
rinse pipets through ten cycles of cold tap water.
through 50 mL of water.
7.2.6.6 Rinse pipets through five cycles of water.
7.2.8.8 Rinse tubing copiously with cold tap water. If tubing
7.2.6.7 Remove pipets from automatic pipet washer, and
does not come clean, place it in hot (50 to 60°C) detergent
allow them to drain and air dry.
solution, remove air bubbles, and allow tubing to soak for 24
7.2.6.8 Plug pipets with cotton.
h.
7.2.7 Test Procedure for Suitability of Detergent Used in
7.3 Cleaning With Acid:
Washing:
7.3.1 Use acid cleaning only when there is no alternative.
7.2.7.1 Wash and rinse six petri dishes in the usual manner.
Consider disposable glassware as a possible alternative. Chro-
These are Group A.
mic acid or HNO (1 + 9) may be used to clean glassware. Ten
7.2.7.2 After normal washing, rinse a second group of six
percent HNO requires longer contact (24 h) with tubes than
petri dishes twelve times with successive portions of water.
chromic acid requires, but residual HNO is not as likely to be
These are Group B.
toxic to microorganisms.
7.2.7.3 Wash six petri dishes with the detergent wash water
NOTE 1—Warning: Do not expose metals or other materials to acids
using detergent concentrations normally employed, and dry
unless certain that those substances are acid-resistant. Chromic acid
without rinsing. These are Group C.
cleaning solutions and other acids may react violently with organics or
7.2.7.4 Sterilize dishes in the usual manner.
other oxidizable substances. Take care to avoid such reactions.
7.2.7.5 Add the proper dilution (usually two different dilu-
NOTE 2—Warning: Chromic acid and nitric acid are capable of pro-
ducing burns even when used in relatively dilute solutions.When working
tions are used) of a water sample yielding 30 to 300 colonies
withtheseorwithotheracids,avoidinhalationoffumes.Protecteyeswith
to triplicate petri dishes from each group (A, B, and C).
safety goggles or with full-face mask. Protect clothing with acid-resistant
Proceed according to the heterotrophic plate count method.
laboratory coat or apron. If eyes are accidently exposed to acid, immedi-
7.2.7.6 Differences in bacterial counts of less than 15 %
ately wash them with copious quantities of tap water for at least 15 min.
among all groups indicate the detergent has no toxicity or
Consult a physician immediately thereafter. If other parts of the body are
inhibitory effect. Differences in bacterial counts of 15 % or
exposed to acid, immediately remove clothing over exposed areas and
flood with large volumes of tap water. Consult a physician immediately if
more between Groups A and B demonstrate that inhibitory
affected area is large or if exposure has been lengthy. Subsequently, wash
residues are left on glassware after the normal washing
exposed areas of clothing with copious quantities of tap water. To avoid
procedure used. Disagreement in averages of less than 15 %
dangerous splatters, always add acid to water, not the reverse (see also
between Groups A and B, and greater than 15 % between
precautions noted under Section 5).
Groups A and C indicates that detergent used has inhibitory
7.3.2 Chromic Acid Cleaning:
properties that are eliminated during routine washing.
7.3.2.1 Chromic acid should be used only when stubborn
7.2.8 Automatic Pipetor (Brewer-Type):
contaminants are not effectively removed by other cleaning
7.2.8.1 Immediately after pipettor has been used, fill reser-
reagents.Replacementproductsforchromicacidareofferedby
voirwithtapwaterandcarefullypumpsufficientwaterthrough
several manufacturers.
the system to remove cellular debris and other materials that
7.3.2.2 To prepare chromic acid (1 + 9), dissolve 25 g of
might adhere to apparatus. Determine whether syringe delivers
sodium dichromate (Na Cr O ) or potassium dichromate
properly without cannula connected. 2 2 7
(K Cr O ) in 2.5 L of concentrated sulfuric acid. Follow
2 2 7
7.2.8.2 Remove tubing from reservoir, and remove syringe
instructions in Note 2.
from pipettor; autoclave val
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