ASTM D5245-19(2024)
(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
4.1 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.2
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 standard. No other units of measurement are included in this 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use. For specific precautions, see Section 6, 5.7.3.1, and 8.3.1.
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
General Information
Relations
Standards Content (Sample)
This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D5245 − 19 (Reapproved 2024)
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:
valid results. Previously used or new glassware must be D1129 Terminology Relating to Water
thoroughly cleaned. Laboratory ware and equipment that are D1193 Specification for Reagent Water
not chemically clean are responsible for considerable losses in
personnel time and supplies in many laboratories. These losses 3. Terminology
may occur as down time when experiments clearly have been
3.1 Definitions:
adversely affected and as invalid data that are often attributed
3.1.1 For definitions of terms used in this standard, refer to
to experimental error. Chemical contaminants that adversely
Terminology D1129.
affect experimental results are not always easily detected. This
practice describes the procedures for producing chemically
4. Significance and Use
clean glassware.
4.1 This practice provides uniform guidance for cleaning
1.2 The values stated in SI units are to be regarded as
the laboratory glassware, plasticware, and equipment used in
standard. No other units of measurement are included in this
routine microbiological analyses. However, tests that are ex-
standard.
tremely sensitive to toxic agents (such as virus assays) may
1.3 This standard does not purport to address all of the require more stringent cleaning practices.
safety concerns, if any, associated with its use. It is the
5. Reagents
responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter-
5.1 Purity of Reagents—Reagent grade chemicals shall be
mine the applicability of regulatory limitations prior to use.
used in all tests. Unless otherwise indicated, it is intended that
For specific precautions, see Section 6, 5.7.3.1, and 8.3.1.
all reagents conform to the specifications of the Committee on
1.4 This international standard was developed in accor-
Analytical Reagents of the American Chemical Society where
dance with internationally recognized principles on standard-
such specifications are available. Other grades may be used,
ization established in the Decision on Principles for the
provided it is first ascertained that the reagent is of sufficiently
Development of International Standards, Guides and Recom-
high purity to permit its use without lessening the accuracy of
mendations issued by the World Trade Organization Technical
the determination.
Barriers to Trade (TBT) Committee.
1 3
This practice is under the jurisdiction of ASTM Committee D19 on Water and For referenced ASTM standards, visit the ASTM website, www.astm.org, or
is the direct responsibility of Subcommittee D19.24 on Water Microbiology. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Current edition approved April 1, 2024. Published April 2024. Originally Standards volume information, refer to the standard’s Document Summary page on
approved in 1992. Last previous edition approved in 2019 as D5245 – 19. DOI: the ASTM website.
10.1520/D5245-19R24. ACS Reagent Chemicals, Specifications and Procedures for Reagents and
A significant portion of this practice was taken from: Berg, G., Safferman, R. Standard-Grade Reference Materials, American Chemical Society, Washington,
S., Dahling, D. R., Berman, D., and Hurst, C. J., USEPA Manual of Methods for DC. For suggestions on the testing of reagents not listed by the American Chemical
Virology, EPA-600/4-84-013, Chapter 2, “Cleansing Laboratory Ware and Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset,
Equipment, Environmental Monitoring and Support Laboratory—Cincinnati,” U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharma-
USEPA, Cincinnati, OH. copeial Convention, Inc. (USPC), Rockville, MD.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D5245 − 19 (2024)
5.2 Purity of Water—Unless otherwise indicated, references 6.2 Sterilize contaminated laboratory ware and equipment
to water shall be understood to mean Type IV of Specification before cleaning.
D1193.
6.3 Transport hazardous acids only in appropriate safety
5.3 Bromothymol blue, 0.4 %. carriers.
5.3.1 Bromothymol blue.
6.4 See 8.3 and 8.4 for details on proper cleaning with acids
5.3.2 Sodium hydroxide solution, 240 g/L.
and alkalis.
5.3.3 Adding 16 mL of a NaOH solution (240 g/L) to 0.1 g
of bromothymol blue.
7. Cleaning Rules
5.3.4 Dilute to 250 mL with Type III reagent water of
7.1 Once detergent solution or acid used to clean a vessel
Specification D1193.
has been rinsed away, do not touch lip or inside of vessel with
5.4 Detergent Solution, for machine-washing glassware and
hands. Detergent or acid on hands or gloves and even oil from
equipment. Use according to manufacturer’s instructions.
clean skin are sources of contamination.
5.5 Detergent Powder, for hand-washing glassware and
7.2 Do not allow soiled laboratory ware and equipment to
equipment.
dry. Soak glassware if cleaning is delayed.
NOTE 1—There now are effective biodegradable detergent products
7.3 Use only cold water for tap water rinsing. Hot water
available that allow the laboratory to avoid acid cleaning of most if not all
may contain grease or oil removed from plumbing. Use only
glassware.
cold water to wash laboratory ware heavily contaminated with
5.6 Nitric Acid (1 + 9)—Pour 100 mL of concentrated
proteinaceous material. Hot water may coagulate such mate-
HNO slowly into 900 mL of water.
rial.
NOTE 2—To avoid dangerous splatters, always pour concentrated acid
7.4 Inspect washed laboratory ware and equipment for
into water.
cleanliness. Reclean by appropriate procedures. Check labora-
5.7 Chromic Acid Solution:
tory ware and equipment for cracks, chips, or other damage and
5.7.1 Sodium dichromate (K Cr O ) or potassium dichro-
2 2 7
replace.
mate (Na Cr O ), 25 g.
2 2 7
7.5 Use nontoxic stainless steel, glass, nonbreakable plastic,
5.7.2 Sulfuric acid, concentrated (36.8 N), 2.5 L.
or other nontoxic materials for plumbing that carries water. Do
5.7.3 To prepare chromic acid (1 + 9), dissolve 25 g of
not use copper plumbing.
sodium dichromate or potassium dichromate in 2.5 L of con-
centrated sulfuric acid.
7.6 Use disposable glass and plasticware for pathogenic
5.7.3.1 Warning—Chromic acid and nitric acid are capable
work and test conditions that severely soil or etch glassware.
of producing burns even when used in relatively dilute solu-
tions. When working with these or with other acids, avoid
8. Cleaning Procedures
inhalation of fumes. Protect eyes with safety goggles or with
8.1 Machine Washing—Equip washing machine with capa-
full-face mask. Protect clothing with acid-resistant laboratory
bility for delivering four water rinses. The water jets in some
coat or apron. If eyes are accidently exposed to acid, immedi-
washing machines are not strong enough to reach all walls in
ately wash them with copious quantities of tap water for at least
tall vessels. This results in poor washing and rinsing. The water
15 min. Consult a physician immediately thereafter. If other
jets in other washing machines are too strong for test tubes and
parts of the body are exposed to acid, immediately remove
similar vessels and for many other narrow-necked vessels. Jets
clothing over exposed areas and flood with large volumes of
that are too powerful hold detergent and rinse water in place
tap water. Consult a physician immediately if affected area is
and do not allow them to drain properly. If washing machine is
large or if exposure has been lengthy. Subsequently, wash
unable to wash or rinse adequately, use procedure described in
exposed areas of clothing with copious quantities of tap water.
8.2.
To avoid dangerous splatters, always add acid to water, not the
8.1.1 Immerse washable vessels in detergent solution, and
reverse (see also precautions noted under Section 6).
soak them overnight. If vessels are too large to immerse, fill
NOTE 3—Chromic acid replacement is applicable.
them to brim with detergent solution, and soak them overnight.
8.1.2 Brush-wash vessels with hot (50 °C to 60 °C) deter-
6. Hazards
gent solution. Hot tap water that exceeds 50 °C is adequate for
6.1 The analyst/technician must know and observe normal
preparing detergent solution.
good laboratory practices and safety procedures required in a
8.1.3 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.
8.1.4 Drain and air dry vessels, or dry vessels in drying
chamber.
The sole source of supply of the apparatus known to the committee at this time
8.1.5 Detergents used in washing may contain inhibitory
is Monostat Corp., 519 Eighth St., New York, NY 10018. If you are aware of
substances. As necessary, test for the presence of inhibitory
alternative suppliers, please provide this information to ASTM International
residues (for example, a new supply of detergent). Check clean
Headquarters. Your comments will receive careful consideration at a meeting of the
responsible technical committee, which you may attend. laboratory ware and equipment for residues in accordance with
D5245 − 19 (2024)
procedure given in 8.2.7. This procedure is similar to that given 8.2.6.4 Soak pipets in detergent solution for 24 h. Raise and
in Standard Methods for the Examination of Water and lower pipet holder five or six times during the 24 h period to
Wastewater. agitate detergent solution and help remove soil and debris from
pipets.
8.2 Manual Washing Procedure:
8.2.6.5 Place pipet holder into automatic pipet washer, and
8.2.1 Immerse vessels in detergent solution, and soak ves-
rinse pipets through ten cycles of cold tap water.
sels overnight. Use fresh detergent solution daily. Solutions
8.2.6.6 Rinse pipets through five cycles of water.
that are saved may become heavily contaminated with bacteria.
8.2.6.7 Remove pipets from automatic pipet washer, and
8.2.2 Brush-wash vessels with hot (50 °C to 60 °C) deter-
allow them to drain and air dry.
gent solution. Hot tap water that exceeds 50 °C is adequate for
8.2.6.8 Plug pipets with cotton.
preparing detergent solution.
8.2.7 Test Procedure for Suitability of Detergent Used in
8.2.3 Swirl-rinse vessels ten times with cold tap water. To
Washing:
swirl-rinse, pour into the vessel a volume of tap water equal to
8.2.7.1 Wash and rinse six petri dishes in the usual manner.
about 10 % of the volume of the vessel, and swirl water around
These are Group A.
entire surface with each rinse. Swirl-rinse vessels five times
8.2.7.2 After normal washing, rinse a second group of six
with water.
petri dishes twelve times with successive portions of water.
8.2.4 Drain and air dry vessels, or dry vessels in drying
These are Group B.
chamber.
8.2.7.3 Wash six petri dishes with the detergent wash water
8.2.5 Test Tubes—Test tubes may be washed by the proce-
using detergent concentrations normally employed, and dry
dure described in 8.1, unless a washing machine is unavailable
without rinsing. These are Group C.
or washing machine jets are so powerful they do not allow
8.2.7.4 Sterilize dishes in the usual manner.
adequate evacuation of tubes and thus interfere with washing
8.2.7.5 Add the proper dilution (usually two different dilu-
and rinsing or by the following procedure.
tions are used) of a water sample yielding 30 to 300 colonies
8.2.5.1 Remove markings from tubes with solvent before
to triplicate petri dishes from each group (A, B, and C).
washing.
Proceed according to the heterotrophic plate count method.
8.2.5.2 Place test tubes open end up into covered wire
8.2.7.6 Differences in bacterial counts of less than 15 %
basket, place basket into stainless steel or plastic vessel
among all groups indicate the detergent has no toxicity or
sufficient in size to allow complete immersion of tubes, and fill
inhibitory effect. Differences in bacterial counts of 15 % or
vessel with hot detergent solution.
more between Groups A and B demonstrate that inhibitory
8.2.5.3 Steam autoclave (100 °C) immersed tubes for
residues are left on glassware after the normal washing
30 min.
procedure used. Disagreement in averages of less than 15 %
8.2.5.4 Empty vessel and tubes, and run cold tap water in to
between Groups A and B, and greater than 15 % between
flush out detergent solution. Introduce tap water into bottom of
Groups A and C indicates that detergent used has inhibitory
vessel with a hose connected to tap. Wax pencil and other scum
properties that are eliminated during routine washing.
will wash over rim of vessel.
8.2.8 Automatic Pipetor (Brewer-Type):
8.2.5.5 Fill and empty tubes in vessel ten times with cold tap
8.2.8.1 Immediately after pipettor has been used, fill reser-
water. Fill and empty tubes in vessel five times with water.
voir with tap water and carefully pump sufficient water through
8.2.5.6 Drain and air dry tubes, or dry tubes in drying
the system to remove cellular debris and other materials that
chamber.
might adhere to apparatus. Determine whether syringe delivers
8.2.5.7 Inspect, rewash if not clean, and use alternate
properly without cannula connected.
cleaning method if appropriate. If glassware still does not meet
8.2.8.2 Remove tubing from reservoir, and remove syringe
requirements, discard.
from pipettor; autoclave valve, tubing, reservoir, and syringe at
8.2.6 Pipets:
121 °C for 60 min.
8.2.6.1 Remove cotton plugs from pipets. If necessary, 8.2.8.3 Disassemble syringe, and remove cannula.
remove cotton plugs by forcing a jet of air or water through
8.2.8.4 Cleanse syringe. Rinse plunger and barrel of syringe
delivery tips of pipets. with copious quantities of cold tap water. Soak tubing over-
8.2.6.2 Place pipets, with tips up, into pipet holder. night in water. Allow tubing to drain and air dry.
8.2.8.5 Fill reservoir
...
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