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USEPA Manual of Methods for Virology Chapter 2 xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
The following is an electronic text form of CHAPTER 2 of the
USEPA MANUAL OF METHODS FOR VIROLOGY - EPA publication
EPA/600/4-84/013 (R-2). The hardcopy form of this chapter can be
obtained by contacting:
Dr. Robert S. Safferman
National Exposure Research Laboratory
U.S. Environmental Protection Agency
26 West M.L. King Dr.
Cincinnati OH 45268 USA
e-mail: safferman.robert@epa.gov
Date of Publication (in Revised form): May 1991
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CHAPTER 2
CLEANSING LABORATORY WARE AND EQUIPMENT
1. INTRODUCTION
Laboratory ware (laboratory glassware and plasticware) and
equipment must be carefully cleaned to avoid trace chemical
contaminants that may introduce toxic substances. When
laboratory ware is not chemically clean, laboratories often
experience considerable losses in personnel time and supplies.
These losses may temporarily suspend research activities with
down time occurring when experiments clearly have been
adversely affected. The failure to ensure the absence of
residues on laboratory glassware, plasticware or equipment also
may result in fluctuations in data that are often attributed to
experimental error.
Deionized distilled water should be used as the final rinse in
the cleansing process. However, distilled water may be used in
place of deionized distilled water for rinsing whenever quality
control tests show that distilled water is adequate.
Chemical contaminants that adversely affect experimental
results can be difficult to detect. The problem of improper
washing is usually worse in large laboratories with central
preparation facilities that are staffed with personnel
ineffectively trained in processing virological laboratory ware
and equipment. The key to an effective preparation facility
lies in the careful training of hands-on personnel who must
understand that a preparation facility is not a kitchen. It is, of course, imperative that the supervisor of the
preparation facility understands and appreciates the need to
meet established specifications for chemically clean laboratory
ware. Competent supervisors who understand the need can
achieve the necessary quality of cleanliness, even with
personnel who do not grasp the concepts involved. High
performance standards in the cleansing of laboratory ware and
equipment are essential to assure the validity of the product
in all subsequent laboratory operations.
All personnel within their particular laboratories must
exercise independent judgment to determine the adequacy of the
safety activities currently in practice. Consequently, they
must be made aware of the hazards associated with the
monitoring for waterborne viruses and of each worker's
responsibility for his or her own safety.
1.1 Precautions.
1.1.1 Sterilize contaminated laboratory ware and equipment
before cleansing them (see Chapter 3).
1.1.2 Soiled laboratory ware and equipment are difficult to
clean when allowed to dry.
1.1.3 During the washing process, do not allow laboratory ware
or equipment to dry until after the final rinse in deionized
distilled water. Detergent that has dried on laboratory ware
or equipment is difficult to remove.
1.1.4 Monitor routinely for proper operation of the distilled
and deionized water sources. See Chapter 4 for servicing and
monitoring of the distilled and deionized water sources.
1.1.5 Select detergent which is compatible with area water and
leaves behind no undesirable residues on the cleansed
laboratory ware and equipment. See Section 1.1.8 for testing
for undesirable residues.
1.1.6 Transport strong acids only in appropriate safety
carriers.
1.1.7 Once detergent solution or acid used to clean a vessel
has been rinsed away, do not touch lip or inside of vessel
with hands. Detergent or acid on hands or gloves and oil even
from clean skin are sources of contamination.
1.1.8 Check cleansed laboratory ware and equipment for acid
residues in accordance with procedures given in Section 2.5.
Detergents used in washing may contain inhibitory substances. As necessary, test for the presence of inhibitory residues
(e.g. a new supply of detergent). For appropriate procedure,
see Standard Methods for the Examination of Water and
Wastewater, 17th Edition, American Public Health Association,
Washington, D.C., 1989, p. 9-8.
1.1.9 Use nontoxic stainless steel, nontoxic glass, nontoxic
nonbreakable plastic, or other nontoxic materials for plumbing
that carries deionized distilled water. Do not use copper
plumbing. Do not use plumbing that contains any ions that may
be toxic.
1.1.10 If a washing machine is used, ensure that jets are
strong enough to reach all parts of deep vessels. Ensure also
that jets are not so powerful they fill narrow-necked vessels
and prevent draining during the time that water is being
delivered. Check daily that jets and drains are not clogged.
Ensure that washing machine operates properly. Check weekly
timing of wash and rinse cycles. Descale lime deposits with
descaler when necessary.
1.1.11 Use only cold water for tap water rinsing. Hot water
may contain grease or oil removed from plumbing.
1.1.12 Use only cold water to wash laboratory ware heavily
contaminated with proteinaceous material. Hot water may
coagulate such material.
1.1.13 Inspect washed laboratory ware and equipment for
cleanliness. Recleanse unclean laboratory ware. Overnight
soaking or brush-washing with hot detergent solution may be
required prior to machine washing.
1.1.14 Check laboratory ware and equipment for damage.
Laboratory ware and equipment that is chipped, cracked,
excessively scratched or otherwise damaged beyond repair should
be replaced.
1.1.15 In a multipurpose laboratory in which different levels
of cleanliness are required, user must code all laboratory ware
and equipment to cleansing specifications required for
laboratory studies. Cleansing problems, breakage, and damage
must be reported to user.
1.2 Alternate Procedures.
1.2.1 Disposable laboratory ware may be used when available.
1.2.2 Cleansing procedures described herein are adequate for
most laboratory situations. These procedures focus specifically at rendering laboratory ware and equipment
suitable for cell culture preparation.
1.2.3 Less rigorous procedures may be used when quality
control tests show they are adequate for laboratory's needs.
When cleansing with detergents (Section 3.1), all vessels may
not require overnight soaking or brush-washing with hot
detergent solution prior to machine washing.
1.2.4 Some critical laboratory ware are washed without
detergent or acid to avoid the possibility of retaining
undesirable residues. Cleaning reagents may render the surface
of laboratory ware unsatisfactory for propagating cell
monolayer or render them unsuitable due to difficulties
removing residues that are toxic to cells.
1.2.5 When contaminants refractory to detergent procedures are
encountered on acid-resistant laboratory ware or equipment,
nitric acid or, if necessary, chromic acid may be used to
cleanse them. Spent chromic acid, being a hazardous waste, is
not disposed of by conventional means. Contact safety or waste
control officer for waste management guidelines for its
environmentally safe disposal. Nitric acid must be neutralized
before disposal into the sewage system. Neutralize by adding
acid to a large volume of an ice water solution of sodium
hydroxide. For concentrated acid use 6 M sodium hydroxide.
2. PREPARATION OF CLEANSING COMPOUNDS AND REAGENTS
2.1 Liquid Detergent Compound for Machine-Washing Laboratory
Ware and Equipment (MIR-A-KOL, product no. 03030, Du Bois
Chemical Co., or equivalent). Use according to manufacturer's
instructions.
2.2 Detergent Powder for Hand-Washing Laboratory Ware and
Equipment (Buell Cleaner, product no. 222, Polychem Corp., or
equivalent). Use according to manufacturer's instructions.
2.3 Nitric Acid, 10 percent. To prepare 10 percent nitric acid,
pour 100 mL of concentrated nitric acid slowly into 900 mL of
cold deionized distilled water. To avoid dangerous splatters,
never pour water into concentrated acid (see also precautions
noted under Section 3.2).
2.4 Chromic Acid (dichromate solution), 1 percent. Chromic
acid should be used only when stubborn contaminates are not
effectively removed by other cleaning reagents. Replacement
products for chromic acid have been reported by several
manufacturers (examples of such products are: Nochromix, Godax
Laboratories; PCC-54 concentrate or RBS-35 concentrate, Pierce Chemical Company; Isoclean concentrate, Isolab Incorporated).
To prepare 1 percent chromic acid, dissolve 25 g of sodium
dichromate (Na2Cr2O7) or potassium dichromate (K2Cr2O7) in 2.5
liters of concentrated sulfuric acid. Potassium and sodium
dichromate are strong oxidizing agents and must be handled
cautiously. Take care to avoid exposure to acid (see
precautions noted under Section 3.2).
2.5 Test for Acid Residues on Laboratory Ware.
2.5.1 Select severa