ASTM D5245-92(2012)
(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
SIGNIFICANCE AND USE
This practice provides uniform guidance for cleaning the laboratory glassware, plasticware, and equipment used in routine microbiological analyses. However, tests that are extremely sensitive to toxic agents (such as virus assays) may require more stringent cleaning practices.
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.
General Information
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D5245 − 92 (Reapproved 2012)
Standard Practice 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 2. Referenced Documents
1.1 In microbiology, clean glassware is crucial to ensure 2.1 ASTM Standards:
D1193 Specification for Reagent Water
valid results. Previously used or new glassware must be
thoroughly cleaned. Laboratory ware and equipment that are
3. Significance and Use
not chemically clean are responsible for considerable losses in
3.1 This practice provides uniform guidance for cleaning
personnel time and supplies in many laboratories. These losses
the laboratory glassware, plasticware, and equipment used in
may occur as down time when experiments clearly have been
routine microbiological analyses. However, tests that are ex-
adversely affected and as invalid data that are often attributed
tremely sensitive to toxic agents (such as virus assays) may
to experimental error. Chemical contaminants that adversely
require more stringent cleaning practices.
affect experimental results are not always easily detected. This
practice describes the procedures for producing chemically
4. Reagents
clean glassware.
4.1 Purity of Reagents—Reagent grade chemicals shall be
1.2 The values stated in SI units are to be regarded as the
used in all tests. Unless otherwise indicated, it is intended that
standard.
all reagents conform to the specifications of the Committee on
1.3 This standard does not purport to address all of the Analytical Reagents of the American Chemical Society where
safety concerns, if any, associated with its use. It is the such specifications are available. Other grades may be used,
responsibility of the user of this standard to establish appro- provided it is first ascertained that the reagent is of sufficiently
priate safety, health, and environmental practices and deter- high purity to permit its use without lessening the accuracy of
mine the applicability of regulatory limitations prior to use.
the determination.
For specific precautions, see Section 5, 7.3.1, and Note 1and
4.2 PurityofWater—Unlessotherwiseindicated,references
Note 2.
to water shall be understood to mean Type IV of Specification
1.4 This international standard was developed in accor-
D1193.
dance with internationally recognized principles on standard-
4.3 Detergent Solution, for machine-washing glassware and
ization established in the Decision on Principles for the
equipment. Use according to manufacturer’s instructions.
Development of International Standards, Guides and Recom-
4.4 Detergent Powder, for hand-washing glassware and
mendations issued by the World Trade Organization Technical
equipment. Use them according to manufacturer’s instructions.
Barriers to Trade (TBT) Committee.
There now are a number of effective biogradable detergent
products available that allow the laboratory to avoid acid
cleaning of most if not all glassware.
This practice is under the jurisdiction of ASTM Committee D19 on Water and
is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved June 1, 2012. Published August 2012. Originally For referenced ASTM standards, visit the ASTM website, www.astm.org, or
approved in 1992. Last previous edition approved in 2005 as D5245 – 92 (2005). contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
DOI: 10.1520/D5245-92R12. Standards volume information, refer to the standard’s Document Summary page on
A significant portion of this practice was taken from: Berg, G., Safferman, R. the ASTM website.
S., Dahling, D. R., Berman, D., and Hurst, C. J., USEPA Manual of Methods for “Reagent Chemicals, American Chemical Society Specifications, Am. Chemi-
Virology, EPA-600/4-84-013, Chapt. 2, “Cleansing Laboratory Ware and cal Soc., Washington, DC. For suggestions on the testing of reagents not listed by
Equipment, Environmental Monitoring and Support Laboratory—Cincinnati,” theAmerican Chemical Society, see “Analar Standards for Laboratory Chemicals,”
USEPA, Cincinnati, OH. BDH Ltd. Poole Dorset, U.K. and the “United States Pharmacopeia.”
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5245 − 92 (Reapproved 2012)
4.5 Nitric Acid (1+9)—Pour 100 mL of concentrated and do not allow them to drain properly. If washing machine is
HNO slowly into 900 mL of water. To avoid dangerous unable to wash or rinse adequately, use procedure described in
splatters, never pour water into concentrated acid. 7.2.
5 7.1.1 Immerse washable vessels in detergent solution, and
4.6 Chromic Acid Solution—Chromic acid replacement is
soak them overnight. If vessels are too large to immerse, fill
applicable.
them to brim with detergent solution, and soak them overnight.
7.1.2 Brush-wash vessels with hot (50 to 60°C) detergent
5. Hazards
solution. Hot tap water that exceeds 50°C is adequate for
5.1 The analyst/technician must know and observe normal
preparing detergent solution.
good laboratory practices and safety procedures required in a
7.1.3 Machine-wash vessels. Follow manufacturer’s in-
microbiology laboratory in preparing, using, and disposing of
structions carefully. Add four water rinses if not included in
cultures, reagents, and materials, and while operating steriliza-
manufacturer’s instructions.
tion and other equipment and instrumentation.
7.1.4 Drain and air dry vessels, or dry vessels in drying
5.2 Sterilize contaminated laboratory ware and equipment
chamber.
before cleaning.
7.1.5 Detergents used in washing may contain inhibitory
substances. As necessary, test for the presence of inhibitory
5.3 Transport hazardous acids only in appropriate safety
residues (for example, a new supply of detergent). Check clean
carriers.
laboratory ware and equipment for residues in accordance with
5.4 See 7.3 and 7.4 for details on proper cleaning with acids
proceduregivenin7.2.7.Thisprocedureissimilartothatgiven
and alkalies.
in Footnote 7.
6. Cleaning Rules
7.2 Manual Washing Procedure:
7.2.1 Immerse vessels in detergent solution, and soak ves-
6.1 Once detergent solution or acid used to clean a vessel
sels overnight. Use fresh detergent solution daily. Solutions
has been rinsed away, do not touch lip or inside of vessel with
thataresavedmaybecomeheavilycontaminatedwithbacteria.
hands. Detergent or acid on hands or gloves and even oil from
7.2.2 Brush-wash vessels with hot (50 to 60°C) detergent
clean skin are sources of contamination.
solution. Hot tap water that exceeds 50°C is adequate for
6.2 Do not allow soiled laboratory ware and equipment to
preparing detergent solution.
dry. Soak glassware if cleaning is delayed.
7.2.3 Swirl-rinse vessels ten times with cold tap water. To
6.3 Use only cold water for tap water rinsing. Hot water
swirl-rinse, pour into the vessel a volume of tap water equal to
may contain grease or oil removed from plumbing. Use only
about 10 % of the volume of the vessel, and swirl water around
cold water to wash laboratory ware heavily contaminated with
entire surface with each rinse. Swirl-rinse vessels five times
proteinaceous material. Hot water may coagulate such mate-
with water.
rial.
7.2.4 Drain and air dry vessels, or dry vessels in drying
chamber.
6.4 Inspect washed laboratory ware and equipment for
7.2.5 Test Tubes—Test tubes may be washed by the proce-
cleanliness. Reclean by appropriate procedures. Check labora-
dure described in 7.1, unless a washing machine is unavailable
torywareandequipmentforcracks,chips,orotherdamageand
or washing machine jets are so powerful they do not allow
replace.
adequate evacuation of tubes and thus interfere with washing
6.5 Use nontoxic stainless steel, glass, nonbreakable plastic,
and rinsing or by the following procedure.
or other nontoxic materials for plumbing that carries water. Do
7.2.5.1 Remove markings from tubes with solvent before
not use copper plumbing.
washing.
6.6 Use disposable glass and plasticware for pathogenic
7.2.5.2 Place test tubes open end up into covered wire
work and test conditions that severely soil or etch glassware.
basket, place basket into stainless steel or plastic vessel
sufficient in size to allow complete immersion of tubes, and fill
7. Cleaning Procedures
vessel with hot detergent solution.
7.1 Machine Washing— Equip washing machine with capa- 7.2.5.3 Steam autoclave (100°C) immersed tubes for 30
bility for delivering four water rinses. The water jets in some
min.
washing machines are not strong enough to reach all walls in 7.2.5.4 Empty vessel and tubes, and run cold tap water in to
tallvessels.Thisresultsinpoorwashingandrinsing.Thewater
flush out detergent solution. Introduce tap water into bottom of
jets in other washing machines are too strong for test tubes and vesselwithahoseconnectedtotap.Waxpencilandotherscum
similar vessels and for many other narrow-necked vessels. Jets
will wash over rim of vessel.
that are too powerful hold detergent and rinse water in place
7.2.5.5 Fillandemptytubesinvesseltentimeswithcoldtap
water. Fill and empty tubes in vessel five times with water.
The sole source of supply of the apparatus known to the committee at this time
is Monostat Corp., 519 Eighth St., New York, NY 10018. If you are aware of
alternative suppliers, please provide this information to ASTM International Standard Methods for the Examination of Water and Wastewater, 17th Ed.,
Headquarters.Your comments will receive careful consideration at a meeting of the American Public HealthAssociation,Washington, DC, Section 9020B, 3.a, 2, 1989,
responsible technical committee, which you may attend. pp. 9–8.
D5245 − 92 (Reapproved 2012)
7.2.5.6 Drain and air dry tubes, or dry tubes in drying 7.2.8.3 Disassemble syringe, and remove cannula.
chamber. 7.2.8.4 Cleanse syringe. Rinse plunger and barrel of syringe
7.2.5.7 Inspect, rewash if not clean, and use alternate with copious quantities of cold tap water. Soak tubing over-
cleaning method if appropriate. If glassware still does not meet night in water. Allow tubing to drain and air dry.
requirements, discard. 7.2.8.5 Fill reservoir with hot (50 to 60°C) detergent
7.2.6 Pipets: solution, and soak reservoir overnight. Hot tap water that
7.2.6.1 Remove cotton plugs from pipets. If necessary, exceeds 50°C is adequate for preparing detergent solution.
Brush-wash reservoir with hot (50 to 60°C) detergent solution.
remove cotton plugs by forcing a jet of air or water through
delivery tips of pipets. If reservoir does not come clean, rinse it with tap water, and
soak it overnight in HNO (1 + 9) or in chromic acid (1 + 9).
7.2.6.2 Place pipets, with tips up, into pipet holder.
Then rinse reservoir ten times with cold tap water, swirl-rinse
7.2.6.3 Place pipet holder into a pipet jar, and fill jar with
five times with water, and allow to drain and air dry.
hot (50 to 60°C) detergent solution. Hot tap water that exceeds
7.2.8.6 Valve—If syringe has been delivering properly with
50°C is adequate for preparing detergent solution. Pipets must
the cannula removed, no further attention to valve is needed. If
be completely immersed. If air bubbles are present in pipets,
syringe has not been delivering properly with the cannula
raise and lower pipet holder several times to remove bubbles.
removed, remove valve from apparatus. Soak valve overnight
7.2.6.4 Soak pipets in detergent solution for 24 h. Raise and
in (1 + 9) HNO or in chromic acid (1 + 9). Rinse copiously
lower pipet holder five or six times during the 24-h period to 3
with cold tap water and reagent water. Allow to drain and air
agitate detergent solution and help remove soil and debris from
dry and return to apparatus.
pipets.
7.2.8.7 Connect cannula to a clean syringe and force
7.2.6.5 Place pipet holder into automatic pipet washer, and
through 50 mL of water.
rinse pipets through ten cycles of cold tap water.
7.2.8.8 Rinse tubing copiously with cold tap water. If tubing
7.2.6.6 Rinse pipets through five cycles of water.
does not come clean, place it in hot (50 to 60°C) detergent
7.2.6.7 Remove pipets from automatic pipet washer, and
solution, remove air bubbles, and allow tubing to soak for 24
allow them to drain and air dry.
h.
7.2.6.8 Plug pipets with cotton.
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.
NOTE 2—Warning: Chromic acid and nitric acid are capable of
7.2.7.5 Add the proper dilution (usually two different dilu-
producing burns even when used in relatively dilute solutions. When
tions are used) of a water sample yielding 30 to 300 colonies
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
acid, immediately wash them with copious quantities of tap water for at
7.2.7.6 Differences in bacterial counts of less than 15 %
least 15 min. Consult a physician immediately thereafter. If other parts of
among all groups indicate the detergent has no toxicity or
the body are exposed to acid, immediately remove clothing over exposed
inhibitory effect. Differences in bacterial counts of 15 % or
areas a
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