ASTM D5245-92(2005)
(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 standard does not purport to address all of the safety concerns, 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 Note 1 and Note 2.
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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: D5245 − 92 (Reapproved 2005)
StandardPractice for
Cleaning Laboratory Glassware, Plasticware, and Equipment
1,2
Used in Microbiological Analyses
This standard is issued under the fixed designation D5245; 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 (´) indicates an editorial change since the last revision or reapproval.
1. Scope tremely sensitive to toxic agents (such as virus assays) may
require more stringent cleaning practices.
1.1 In microbiology, clean glassware is crucial to ensure
valid results. Previously used or new glassware must be
4. Reagents
thoroughly cleaned. Laboratory ware and equipment that are
4.1 Purity of Reagents—Reagent grade chemicals shall be
not chemically clean are responsible for considerable losses in
used in all tests. Unless otherwise indicated, it is intended that
personnel time and supplies in many laboratories. These losses
all reagents conform to the specifications of the Committee on
may occur as down time when experiments clearly have been
Analytical Reagents of the American Chemical Society where
adversely affected and as invalid data that are often attributed
such specifications are available. Other grades may be used,
to experimental error. Chemical contaminants that adversely
provided it is first ascertained that the reagent is of sufficiently
affect experimental results are not always easily detected. This
high purity to permit its use without lessening the accuracy of
practice describes the procedures for producing chemically
the determination.
clean glassware.
4.2 PurityofWater—Unlessotherwiseindicated,references
1.2 The values stated in SI units are to be regarded as the
to water shall be understood to mean Type IV of Specification
standard.
D1193.
1.3 This standard does not purport to address all of the
4.3 Detergent Solution, for machine-washing glassware and
safety concerns, if any, associated with its use. It is the
equipment. Use according to manufacturer’s instructions.
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica-
4.4 Detergent Powder, for hand-washing glassware and
bility of regulatory limitations prior to use. For specific
equipment. Use them according to manufacturer’s instructions.
precautions, see Section 5, 7.3.1, and Note 1 and Note 2.
There now are a number of effective biogradable detergent
products available that allow the laboratory to avoid acid
2. Referenced Documents
cleaning of most if not all glassware.
2.1 ASTM Standards:
4.5 Nitric Acid (1+9)—Pour 100 mL of concentrated
D1193 Specification for Reagent Water
HNO slowly into 900 mL of water. To avoid dangerous
splatters, never pour water into concentrated acid.
3. Significance and Use
4.6 Chromic Acid Solution—Chromic acid replacement is
3.1 This practice provides uniform guidance for cleaning
applicable.
the laboratory glassware, plasticware, and equipment used in
routine microbiological analyses. However, tests that are ex-
5. Hazards
5.1 The analyst/technician must know and observe normal
This practice is under the jurisdiction of ASTM Committee D19 on Water and
good laboratory practices and safety procedures required in a
is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
microbiology laboratory in preparing, using, and disposing of
Current edition approved Oct. 1, 2005. Published February 2006. Originally
approved in 1992. Last previous edition approved in 1998 as D5245 – 92 (1998).
DOI: 10.1520/D5245-92R05.
2 4
A significant portion of this practice was taken from: Berg, G., Safferman, R. “Reagent Chemicals, American Chemical Society Specifications, Am. Chemi-
S., Dahling, D. R., Berman, D., and Hurst, C. J., USEPA Manual of Methods for cal Soc., Washington, DC. For suggestions on the testing of reagents not listed by
Virology, EPA-600/4-84-013, Chapt. 2, “Cleansing Laboratory Ware and theAmerican Chemical Society, see “Analar Standards for Laboratory Chemicals,”
Equipment, Environmental Monitoring and Support Laboratory—Cincinnati,” BDH Ltd. Poole Dorset, U.K. and the “United States Pharmacopeia.”
USEPA, Cincinnati, OH. The sole source of supply of the apparatus known to the committee at this time
For referenced ASTM standards, visit the ASTM website, www.astm.org, or is Monostat Corp., 519 Eighth St., New York, NY 10018. If you are aware of
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM alternative suppliers, please provide this information to ASTM International
Standards volume information, refer to the standard’s Document Summary page on Headquarters.Your comments will receive careful consideration at a meeting of the
the ASTM website. responsible technical committee, which you may attend.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5245 − 92 (Reapproved 2005)
cultures, reagents, and materials, and while operating steriliza- laboratory ware and equipment for residues in accordance with
tion and other equipment and instrumentation. proceduregivenin7.2.7.Thisprocedureissimilartothatgiven
in Footnote 7.
5.2 Sterilize contaminated laboratory ware and equipment
before cleaning.
7.2 Manual Washing Procedure:
5.3 Transport hazardous acids only in appropriate safety 7.2.1 Immerse vessels in detergent solution, and soak ves-
sels overnight. Use fresh detergent solution daily. Solutions
carriers.
thataresavedmaybecomeheavilycontaminatedwithbacteria.
5.4 See 7.3 and 7.4 for details on proper cleaning with acids
7.2.2 Brush-wash vessels with hot (50 to 60°C) detergent
and alkalies.
solution. Hot tap water that exceeds 50°C is adequate for
6. Cleaning Rules
preparing detergent solution.
7.2.3 Swirl-rinse vessels ten times with cold tap water. To
6.1 Once detergent solution or acid used to clean a vessel
swirl-rinse, pour into the vessel a volume of tap water equal to
has been rinsed away, do not touch lip or inside of vessel with
about 10 % of the volume of the vessel, and swirl water around
hands. Detergent or acid on hands or gloves and even oil from
entire surface with each rinse. Swirl-rinse vessels five times
clean skin are sources of contamination.
with water.
6.2 Do not allow soiled laboratory ware and equipment to
7.2.4 Drain and air dry vessels, or dry vessels in drying
dry. Soak glassware if cleaning is delayed.
chamber.
6.3 Use only cold water for tap water rinsing. Hot water
7.2.5 Test Tubes—Test tubes may be washed by the proce-
may contain grease or oil removed from plumbing. Use only
dure described in 7.1, unless a washing machine is unavailable
cold water to wash laboratory ware heavily contaminated with
or washing machine jets are so powerful they do not allow
proteinaceous material. Hot water may coagulate such mate-
adequate evacuation of tubes and thus interfere with washing
rial.
and rinsing or by the following procedure.
6.4 Inspect washed laboratory ware and equipment for
7.2.5.1 Remove markings from tubes with solvent before
cleanliness. Reclean by appropriate procedures. Check labora-
washing.
torywareandequipmentforcracks,chips,orotherdamageand
7.2.5.2 Place test tubes open end up into covered wire
replace.
basket, place basket into stainless steel or plastic vessel
6.5 Use nontoxic stainless steel, glass, nonbreakable plastic, sufficient in size to allow complete immersion of tubes, and fill
or other nontoxic materials for plumbing that carries water. Do
vessel with hot detergent solution.
not use copper plumbing.
7.2.5.3 Steam autoclave (100°C) immersed tubes for 30
min.
6.6 Use disposable glass and plasticware for pathogenic
work and test conditions that severely soil or etch glassware. 7.2.5.4 Empty vessel and tubes, and run cold tap water in to
flush out detergent solution. Introduce tap water into bottom of
7. Cleaning Procedures
vesselwithahoseconnectedtotap.Waxpencilandotherscum
7.1 Machine Washing— Equip washing machine with capa- will wash over rim of vessel.
bility for delivering four water rinses. The water jets in some
7.2.5.5 Fillandemptytubesinvesseltentimeswithcoldtap
washing machines are not strong enough to reach all walls in
water. Fill and empty tubes in vessel five times with water.
tallvessels.Thisresultsinpoorwashingandrinsing.Thewater
7.2.5.6 Drain and air dry tubes, or dry tubes in drying
jets in other washing machines are too strong for test tubes and
chamber.
similar vessels and for many other narrow-necked vessels. Jets
7.2.5.7 Inspect, rewash if not clean, and use alternate
that are too powerful hold detergent and rinse water in place
cleaning method if appropriate. If glassware still does not meet
and do not allow them to drain properly. If washing machine is
requirements, discard.
unable to wash or rinse adequately, use procedure described in
7.2.6 Pipets:
7.2.
7.2.6.1 Remove cotton plugs from pipets. If necessary,
7.1.1 Immerse washable vessels in detergent solution, and
remove cotton plugs by forcing a jet of air or water through
soak them overnight. If vessels are too large to immerse, fill
delivery tips of pipets.
them to brim with detergent solution, and soak them overnight.
7.2.6.2 Place pipets, with tips up, into pipet holder.
7.1.2 Brush-wash vessels with hot (50 to 60°C) detergent
solution. Hot tap water that exceeds 50°C is adequate for 7.2.6.3 Place pipet holder into a pipet jar, and fill jar with
preparing detergent solution.
hot (50 to 60°C) detergent solution. Hot tap water that exceeds
7.1.3 Machine-wash vessels. Follow manufacturer’s in- 50°C is adequate for preparing detergent solution. Pipets must
structions carefully. Add four water rinses if not included in
be completely immersed. If air bubbles are present in pipets,
manufacturer’s instructions. raise and lower pipet holder several times to remove bubbles.
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 (Reapproved 2005)
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
tions are used) of a water sample yielding 30 to 300 colonies
producing burns even when used in relatively dilute solutions. When
working with these or with other acids, avoid inhalation of fumes. Protect
to triplicate petri dishes from each group (A, B, and C).
eyes with safety goggles or with full-face mask. Protect clothing with
Proceed according to the heterotrophic plate count method.
acid-resistant laboratory coat or apron. If eyes are accidently exposed to
7.2.7.6 Differences in bacterial counts of less than 15 %
acid, immediately wash them with copious quantities of tap water for at
among all groups indicate the detergent has no toxicity or
least 15 min. Consult a physician immediately thereafter. If other parts of
inhibitory effect. Differences in bacterial counts of 15 % or the body are exposed to acid, immediately remove clothing over exposed
areas and flood with large volumes of tap water. Consult a physician
more between Groups A and B demonstrate that inhibitory
immediately if affected area is large or if exposure has been lengthy.
residues are left on glassware after the normal washing
Subsequently, wash exposed areas of clothing with copious quantities of
procedure used. Disagreement in averages of less than 15 %
tap water. To avoid dangerous splatters, always add acid to water, not the
between Groups A and B, and greater than 15 % between
reverse (see also 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 deli
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