9040 WASHING AND STERILIZATION*
Clean glassware† is essential in microbiology laboratories to
ensure reliability in analytical tests. Clean before first use. All
contaminated laboratory ware must be sterilized before cleaning.
Wash labware after use. If material has dried on glassware, a
pre-soaking may be needed. Consistently follow the same wash-
ing procedures, whether using an automated system or washing
by hand. Where possible, use mechanical glassware washers. Do
not use units designed for home use or with copper plumbing.
Instead, use stainless steel or other nontoxic material for influent,
distribution, and rinse-water systems. Ensure that the water jets’
spray reaches all parts of the vessels, including deep vessels.
Cleanse all glassware thoroughly with warm to hot water and
a suitable laboratory-grade detergent containing no phosphates.
To remove all traces of residual washing compound, rinse five to
ten times with cold water after bubbles/foam are gone. Then,
rinse two to three times with reagent-grade water. Alternatively,
any washing method in a commercial glass washer can be used.
Perform the bromothymol blue pH check on each batch (all
items washed at the same time) and type of glassware to dem-
onstrate that no alkali or acid residues are present. If residues are
found, rewash glassware. If this test is done on each batch of
washed glassware, run the glassware inhibitory residue test be-
fore initial use of a washing compound and whenever a new
formulation or washing procedure is used. If the bromothymol
blue test is not done consistently, also run the toxicity test
annually. NOTE: Glass items washed and sterilized in the labo-
ratory may contain toxic detergent residues not detected by the
bromothymol blue pH test. These items must be checked peri-
odically for toxic detergent residues. Review the information in
Section 9020B.5a2). Perform the bromothymol blue pH check
[Section 9020B.5a1)] on each batch and type of glassware to
* Approved by Standard Methods Committee, 2013.
Joint Task Group: Margo E. Hunt (chair), Gil Dichter, Nancy H. Hall, Robin K.
Oshiro.
† The term glassware refers to both borosilicate glass and heat-resistant plastic
materials.
https://doi.org/10.2105/SMWW.2882.182
demonstrate that no alkali or acid residues are present. If residues
are found, rewash glassware. NOTE: Glassware used for some
analyses [e.g., biochemical oxygen demand (BOD) and assimil-
able organic carbon (AOC)] must be organic-free. Use a furnace
set at 550°C for ⱖ4 h.
Protect clean glassware and materials against dust during
storage, in conditions that will maintain its cleanliness.
To sterilize glassware via dry heat, use a hot-air oven set at
ⱖ170°C for 2 h or longer. Alternatively, sterilize glassware by
autoclaving at 121°C for at least 30 min. For all bottles, loosen
caps before autoclaving. If desired after autoclaving, remove
moisture present in empty sterile containers by placing items in
a drying oven. For glass pipets in metal containers, sterilize
using a hot-air oven set at ⱖ170°C for at least 2 h.
As long as the bromothymol blue pH test is being done on
each batch of washed glassware, only run the glassware inhibi-
tory residue test [Section 9020B.5a2)] before initial use of a
washing compound and whenever a new formulation or washing
procedure is used. If the bromothymol blue test is not done
consistently, run the glassware inhibitory residue test on a per-lot
or annual basis, whichever is more frequent. Review Sections
9020B.4h and 5d for quality-control checks on autoclaves and
sterilized bottles.
Bibliography
BORDNER, R.H., J.A. WINTER & P.V. SCARPINO, eds. 1978. Microbiolog-
ical Methods for Monitoring the Environment, Water and Wastes;
EPA-600/8-78-017. U.S. Environmental Protection Agency, Envi-
ronmental Monitoring & Support Lab., Cincinnati, Ohio.
U.S. ENVIRONMENTAL PROTECTION AGENCY. 2005. Manual for the Certi-
fication of Laboratories Analyzing Drinking Water, 5th ed.; EPA
805-R-05-004. Cincinnati, Ohio.
AMERICAN SOCIETY FOR TESTING & MATERIALS. 2012. Standard Practices
for Cleaning Laboratory Glassware, Plasticware, and Equipment
Used in Microbiological Analyses; D5245-92 (Reapproved 2012).
West Conshohocken, Pa.
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