USEPA Manual of Methods for Virology: Revised May 1991, Chapter 2


&EPA
United States      Office of Research and   EPA/600/4-84/013(R-2)
Environmental Protection  Development      May 1991
Agency         Washington, DC 20460   Revision

USEPA Manual of
Methods for Virology

Chapter 2
Revised May  1991

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May 1991
Chapter 2
Cleansing Laboratory Ware and Equipment
1. Introduction

Laboratory ware* 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 fluctua-
tions 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 estab-
lished 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.
'Laboratory ware comprises laboratory glassware
and plasticware.
All personnel within their particular
laboratories must exercise indepen-
dent 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 labora-
tory 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 deion-
ized water sources.
See Chapter 4 for servicing and
monitoring of the distilled and deion-
ized 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 equip-
ment.
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

2-1
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 proce-
dure, 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.
Printed on Recycled Paper

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May 1991
  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 prob-
lems, 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 de-
scribed herein are adequate for most
laboratory situations. These proce-
dures 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
(Secthn 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 unsatisfac-
tory for propagating cell monolayer or
render them unsuitable due to difficul-
ties removing residues that are toxic to
celts.

 1.2.5  When contaminants refractory
to detergent procedures are encoun-
tered on acid-resistant laboratory ware
or equipment, nitric acid or, if neces-.
sary, 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 man-
agement guidelines for its environmen-
tally safe disposal.
  Nitric add 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%.
  To prepare 10% 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%.
  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% chromic acid,
dissolve 25 g of sodium dichromate
(Na2CrzO7) 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.

2.5.7  Select several pieces of clean
glassware or plasticware for testing.

2.5.2  Add a few drops of 0.04%
bromthymol blue into each of the
selected pieces.
  Prepare 0.04% bromthymol blue
indicator by adding 16 mL of a 0.01 M
solution ofNaOH to 0.1 g of
bromthymol blue and dilute to 250 mL
with distilled deionized water.

2.5.3   Observe color reaction.
  Color reaction should be blue-green
in the neutral pH range. A yellow color
reaction identifies the presence of an
acid residue. The presence of an
alkaline residue is distinguished by a
blue color reaction.

3.   Procedures for
Cleansing  Laboratory Ware
and  Equipment
                            •
  All laboratory ware and equipment
must be decontaminated before
cleansing. A summary of decontamina-
tion procedures is given in Section 5.
See Chapter 3 for the detailed steps to
decontaminate laboratory ware and
equipment.
  Laboratory ware.and equipment may
be cleansed in several ways. Those
used for cell cultures may require
special care (see Section 1.2.4).

3.1  Cleansing with Detergent.

3.1.1   General Laboratory Ware and
Washable Equipment.

(a)  Mechanical washing procedure.
  Lightly soiled laboratory ware and
washable equipment may not require
Steps (a. 1)  and (a.2)
  Equip washing machine so as to
deliver four deionized distilled water
rinses. Additional deionized distilled
water rinses may require machine
operation using manual cycle.
   The water jets in some washing
machines are not strong enough to
reach all walls inside tall vessels. This
results in poor washing and rinsing.
The water jets in other washing
machines are too strong for test tubes
and similar vessels and for many other
narrow-necked vessels. Jets that are
too powerful hold detergent and rinse
water in place and do not allow such
vessels to drain properly. If washing
machine is unable to wash or rinse
adequately, use manual washing
procedure described in Section 3.1.1,
Step (b).

(a. 1)   Immerse decontaminated
laboratory ware and washable
equipment in detergent solution and
soak them overnight.
  If any laboratory item is too large to
immerse, fill it to brim with detergent
solution and soak it overnight.
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May 1991
(a.2) Brush-wash soaked items with
hot detergent solution.
Hot tap water that exceeds 50° C is
adequate for preparing detergent
solution.

(a.3) Machine-wash vessels.
Follow manufacturer's instructions
carefully. Add four deionized, distilled
water rinses if not included in
manufacturer's instructions.
The added four deionized distilled
water rinses may require machine
operation using manual cycle.

(a.4) Drain and dry washed items at
ambient temperature or dry in hot-air
chamber.

(a.5) Sterilize the cleansed item by
appropriate method.
See Chapter 3 for sterilization
procedure.

(b) Manual washing procedure.
» Use fresh detergent solution daily.
Solutions that are saved may become
heavily contaminated with bacteria.

(b.1) Immerse decontaminated
laboratory ware and washable
equipment in detergent solution.
Soak lightly soiled laboratory ware
and washable equipment for 1 hour
and heavily soiled items overnight.

(b.2) Brush-wash soaked items with
hot detergent solution.
Hot tap water that exceeds 50° C is
adequate for preparing detergent
solution.

(b.3) Swish-rinse washed laboratory
ware and washable equipment ten
times with cold tap water.
To swish-rinse, pour into the vessel
or apparatus a volume of tap water
equal to about 10% of its volume and
swish water around entire surface with
each rinse.

(b.4) Swish-rinse vessel or appara-
tus five times with deionized distilled
water.

(b.5) Drain and dry rinsed items at
ambient temperature or dry in hot-air
chamber.

(b.6) Sterilize the cleansed item by
appropriate method.
See Chapters for sterilization
procedure.
3.1.2 Test Tubes.
Test tubes may be washed by
procedure described in Section 3.1.1,
Step (a) unless a washing machine is
unavailable or washing machine jets
are so powerful they do not allow
adequate evacuation of tubes and thus
interfere with washing and rinsing. In
either event, the procedure that follows
may be used instead of the washing
machine procedure.

(a) Place decontaminated test tubes
open end up into covered wire basket,
place basket into stainless steel or
plastic vessel sufficient in size to allow
complete immersion of tubes, and fill
vessel with hot detergent solution (50-
60°C).

(b) Brush-wash test tubes by hand or
machine.
Heavily soiled test tubes should be
soaked overnight before brush
washing.

(c) Empty tubes and flush with cold
tap water.
To achieve thorough rinsing,
introduce tap water into bottom of
vessel with a hose connected to tap.

(d) Fill and empty tubes ten times
with cold tap water.

(e) Fill and empty tubes five times
with deionized distilled water.
Run hose from deionized distilled
water line to bottom of vessel to
achieve thorough rinsing.

(f) Drain and dry tubes at ambient
temperature or dry in hot-air chamber.

(g) Sterilize screw-cap or cotton-
plugged test tubes.

(g. 1) Place screw-cap or cotton-
plugged tubes in test tube racks.

(g.2) Sterilize test tubes in dry heat
oven for one hour.
Oven temperature is maintained at
170°C.

(h) Sterilize test tubes with caps or
semipermeable plastic inserts.

(h. 1) Protect mouths of tubes with
caps or with semipermeable plastic
inserts.

(h.2) Place tubes with caps or
plastic inserts in test tube racks.

2-3
(h.3) Sterilize tubes by autoclaving
at 121°C for 30 minutes.

3.7.3 Pipettes.

(a) Remove cotton plugs from
decontaminated pipettes.
ff necessary, remove cotton plugs by
forcing a jet of air or water through
delivery tips of pipettes.

(b) Place pipettes, with tips up, into
pipette holder.

(c) Place pipette holder into a pipette
jar and fill jar with hot detergent
solution.
Hot tap water that exceeds 50° C is
adequate for preparing detergent
solution. Pipettes must be completely
immersed. If air bubbles are present in
pipettes, raise and tower pipette holder
several times to remove bubbles.

(d) Soak pipettes in detergent
solution overnight.
Raise and lower pipette holder five
or six times during this time period to
agitate detergent solution and thus
help remove soil and debris from
pipettes.
Pipettes that are lightly soiled may
not require soaking overnight in
detergent solution.

(e) Place pipette holder into auto-
matic pipette washer and rinse
pipettes through ten cycles of cold tap
water.

(f) Rinse pipettes through five cycles
of deionized distilled water.

(g) Remove pipettes from automatic
pipette washer and allow pipettes to
drain and dry at ambient temperature.

(h) Plug pipettes with cotton.

(i) Place pipettes in pipette canisters
and sterilize in dry heat oven for one
hour.
Oven temperature is maintained at
170°C.

3.1.4 Automatic-Pipettor (Brewer-
type).
To ensure continual proper opera-
tion of the automatic pipettor, fill
reservoir with tap water immediately
after pipettor has been used and
carefully pump sufficient water through
the system to remove cellular debris
and other materials that might adhere
to apparatus. Water pumped through

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May 1991
automatic pipettor is returned to
reservoir. Decontaminate automatic
plpettor and reservoir as a unit.
Cleanse  apparatus according to
Instructhns given in Steps (a) through
(d). To prevent contamination of cell
cultures,  do not use detergent or acid
to dean plpettor.

(a) Disassemble decontaminated
automatic plpettor from  reservoir.
   Cleanse automatic pipettor as a
unit—syringe, tubing, valve, and
cannula (see Step (d) for cleansing
reservoir).

(b) Force  250 to 500 mL of deionized
distilled water through automatic
pipettor.

(c) Allow pipettor to drain and dry at
ambient temperature.

(d) Reservoir.

(d. 1)   Fill reservoir with hot detergent
solution.
   Hot tap  water that exceeds 50°C is
adequate for preparing detergent
solution.

(d.2)   Brush-wash reservoir with hot
detergent solution.
   If reservoir remains soiled, rinse
with cold tap water and treat with 10%
nitric add or if necessary with 1%
chromic  add (see Section 3.2.1).
   Take care to avoid exposure to acid
(see precautions noted under Section
3.2).

(d.3)   Swish-rinse reservoir ten times
with cold tap water.
   To swish-rinse, pour into the vessel
a volume of tap water equal to about
10% of the volume of the vessel, and
swish water around entire surface with
each rinse.

(d.4)   Machine-wash reservoir.
   Follow manufacturer's instructions
carefully. Add four deionized distilled
water rinses if not induded in
manufacturer's instructions. The
added four deionized distilled water
rinses may require machine operation
using manual cycle.
   If reservoir cannot be mechanically
washed, proceed to swish-rinse vessel
an addittonal five times with cold tap
water and five times with  deionized
distilled  water.
   Drain and dry reservoir at ambient
temperature or dry in hot air chamber.
(e)  Pour 10 to 20 mL of deionized
distilled water into reservoir.

(f)  Cover opening of reservoir with
aluminum foil or Kraft paper.

(g)  Protect cannula with glass tube
cover, and wrap syringe, interconnect-
ing tubing and protected cannula in
cloth or Kraft paper.

(h)  Reconnect tubing to reservoir.

(i)  Autoclave assembled  apparatus
at 121° C for 60 minutes.
  Use slow exhaust.

(j)  Allow apparatus to cool and store
in closed cabinet  until needed.

3.1.5  Automatic Syringe  (Cornwall-
type).
  To ensure continual proper opera-
tion of the automatic syringe,  fill
reservoir with tap water immediately
after pipettor has been used and
carefully pump sufficient water through
the system to remove cellular debris
and other materials that might adhere
to apparatus. Water pumped through
automatic syringe is  returned to
reservoir.
  To prevent chemical contamination
of cell culture preparations, do not use
detergent or acid to clean pipettor.

(a)  Disassemble decontaminated
automatic syringe from reservoir.
  Reservoir is generally a flask or
bottle which is cleaned as regular
glassware by the method described in
Section 3.1.1.
  Cleanse decontaminated automatic
syringe as a unit,  syringe, tubing,
•valve, and cannula. If syringe has not
been delivering properly, check inlet
and outlet valves. Replace either
valve, or both valves, if damaged or
hardened.

(b)   Force 250 to 500 mL of deionized
distilled water through automatic
syringe.

(c)  Allow syringe to drain and dry at
ambient temperature.

(d)   Protect cannula with glass tube
cover, and wrap syringe, interconnect-
ing tubing and  protected cannula in
cloth or Kraft paper.

(e)   Autoclave  apparatus at 121°C for
30 minutes.
  Use slow exhaust.

(f)  Allow apparatus to cool and store
in closed cabinet until needed.

3.1.6  Disc Filter Holder.

(a)  Disassemble decontaminated
filter holder and discard membrane.

(b)  Rinse filter holder components
with 2 to 5 liters of cold tap water.
If debris remains after tap water rinse,
brush-wash filter holder with hot (50-
60° C) detergent solution. Remove
refractory debris with  nonabrasive
scrubber. Use fine grade steel wool
only if absolutely necessary. Rinse
again with 2 to 5 liters of cold tap
water.

(c)  Rinse filter holder components
with 1 to 2 liters of deionized distilled
water.

(d)  Allow filter holder components to
drain and dry at ambient temperature.
  Check gaskets for distortion
(flattened areas), and replace gaskets
if necessary. If filter holder is to be
used for the VIRADEL disc filter
procedure in Chapters, proceed to
Step (e). If filter holder is to be used in
the preparation of media for cell
culture, proceed to Step (i).

(e)  Reassemble filter holder.

(f)  Cover the ports of filter holder
with aluminum foil or  Kraft paper.

(g)  Autoclave filter holder at 121 °C
for  30 minutes.

(h)  Allow filter holder to cool and
store in closed cabinet until needed.

(i)  Attach tube to outlet port of filter
holder.

(j)  Attach glass filling bell to outlet
port tubing.

(k)   Place filter support on base of
filter holder.

(I)  Add 10 to 20 mL of deionized
distilled water to base of filter holder.

(m) Place membrane filter or filters on
filter support.

(n)  Reassemble filter holder.
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                                                                                                            May 1991
   Loosely tighten down top of filter
 holder.

 (o)  Add 10 to 20 mL of deionized
 distilled water through inlet port of
 filter holder.

 (p)  Cover filling  bell and inlet port
 with aluminum foil or Kraft paper.

 (q)  Autoclave filter and filter holder at
 121 °C for 30 minutes.
   Use slow exhaust.

 (r)   Allow filter holder to cool.

 (s)  Loosely secure top of  holder and
 store in closed cabinet until needed.
   If bolts are kept tightly fastened
 during storage, filter may subsequently
 crack.

 3.1.7  Dispensing Pressure Vessel.

 (a)  Remove lid from decontaminated
 dispensing pressure vessel.

 (b)  Rinse pressure vessel and lid
 with 4 to 5 liters of cold tap water.
  If debris remains after tap water
 rinse, brush-wash vessel and lid with
 hot (50-60° C)detergent solution.
 Remove refractory debris with
 nonabrasive scrubber. Use fine grade
 steel wool only if absolutely necessary.
 Rinse again with 4 to 5 liters of cold
 tap water.

 (c)   Swish-rinse pressure vessel and
 lid five times with deionized distilled
 water.
  To swish-rinse, pour into the vessel
 a volume of water equal to about 10%
 of the volume of the vessel, and swish
 water around entire surface with each
 rinse.

 (d)  Allow vessel and lid to drain and
 dry at ambient temperature.

 (e)  Wrap lid and cover vessel
 opening and inlet and outlet ports with
aluminum foil or Kraft paper.
  Be certain vent/relief valve on vessel
 is open.

 (f)  Autoclave vessel and lid at 121°C
for 30 minutes.

 (g)  Close vent/relief valve on vessel.

 (h)  Store in closed cabinet until
needed.
 3.1.8   Plastic Screw-Cape..

 (a)  Pour water from vessel containing
 decontaminated caps and rinse caps
 with 5 to 10 liters of cold ttap water.
   Run hose from tap to bottom of
 vessel to achieve thorough rinsing.

 (b)  Add deionized distilled water to
 vessel containing rinsed caps and
 autoclave at  121°C for 15 minutes.

 (c)  Rinse caps with 4 to 5 liters  of
 deionized distilled water.
   Run hose from deionized distilled
 water line to bottom of vessel to
 achieve thorough rinsing.

 (d)  Place caps with opening posi-
 tioned downward on towel and allow
 caps to drain and dry at ambient
 temperature.

 (e)  Place caps with opening posi-
 tioned downward in glass petri plates.

 (f)   Place petri plates in petri plate
 canister.

 (g)  Autoclave caps at 121°C for 45
 minutes.
   Use fast exhaust and a 15 minute
 dry cycle.
   Leave top off canister during
 autoclaving to allow penetration of
 steam.

 (h)  Secure cover on canister, allow
 canister and contents to cool, and
 store in closed cabinet until needed.

 3.1.9  Cartridge Filters and Filter
 Holders.
                          §
 (a)  Filter and filter  holder from
 VIRADEL cartridge filter procedures.
   The VIRADEL cartridge filter
procedures are described in Chapter
 6.

 (a. 1)  Remove filter from cartridge
filter holder and decontaminate filter
and filter holder.
   Cartridge filters are decontaminated
by steam sterilization. Filter holders
 are decontaminated chemically with
 either ethylene oxide or chlorine.

 (a.2)  Discard filter.

 (a.3)  Rinse filter holder components
with 10 to 15 liters of cold tap water.
  If debris remains after tap water
rinse, brush-wash filter holder with hot
 (50-60°C) detergent solution. Remove
 refractory debris with nonabrasive
 scrubber. Rinse again with 10 to 15
 liters of cold tap water. '

 (a.4)   Rinse filter holder components
 with 5 to 10 liters  of deionized distilled
 water.

 (a.5)   Allow filter holder components
 to drain and dry at ambient tempera-
 ture.

 (a.6)   Reassemble filter holder.

 (a. 7)   Cover the  ports of filter holder
 with Kraft paper if holder is to be
 chemically sterilized with ethylene
 oxide.
   Proper aeration after gas steriliza-
 tion is essential.
   Do not cover ports if holder is to be
 treated with chlorine. After chlorine
 treatment cover ports with sterile
 aluminum foil or sterile Kraft paper.

 (a.8)   Chemically sterilize .filter holder
 according to procedure given in
 Chapter 3.

 (b)  Prefilter and filter holder from
 media preparation.
   Media preparation is described in
 Chapter 9.
   Until clogged, the prefliter may be
 reused for media preparation. Dispose
 of clogged pref liters in plastic-lined
 trash can.

 (b. 1)  Backwash  cartridge prefilter
 with 100 liters of deionized distilled
 water.

 (b.2)  Disassemble filter holder.

 (b.3)  Remove prefilter from cartridge
 holder and drain and dry prefilter at
 ambient temperature.

 (b.4)  Resterilize  prefilter according
to manufacturer's instructions.

 (b.5)  Swish-rinse filter holder ten
times with cold tap water.
   To swish-rinse, pour into apparatus
 a volume of tap water equal to about
 10%   of its volume and swish water
around entire surface with each rinse.

 (b.6)  Swish-rinse filter holder five
times with deionized distilled  water.
                                                       2-5

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May 1991
(b.7)   Allow filter holder components
to drain and dry at ambient tempera-
ture.

(b.8)   Reassemble filter holder.
       Cover the ports of filter holder
with Kraft paper if holder is to be
chemically sterilized with ethylene
oxWe.
  Proper aeration after gas steriliza-
tion is essential.
  Sterilization of filter holder with
ethylane oxide is an optional step. If
gas sterilizer is not available, distilled
water rinse and air drying is sufficient
treatment for filter holder used for
housing ofprefilter.

3.2  Cleansing with Acid.

  Either 10% nitric acid or, if neces-
sary, 1% chromic acid may be used to
cleanse glassware. Cleansing with
nitric add necessitates longer contact
time than is required with chromic
acid, but residual nitric acid is not as
likely to be toxic to cell cultures as
residual chromic acid. Also spent
chromic add Is not disposed of by
conventional means.
  Replacement products for chromic
acid have been reported. Examples of
such products are given in Section
2A. Do not expose metals or other
materials to acids unless certain that
those substances are acid-resistant.
  Acids may react  violently with
organhs or other oxidizable sub-
stances. Take care to avoid such
reactions.
   Cleanse laboratory ware with
detergent solutions before  cleansing
them with acids.
   Nitric and chromic acids are strong
acids capable of producing severe
bums even when used in relatively
dilute solutions. Safety procedures are
described In part 1090 of the 17th
edition of Standard Methods for the
Examination of Water and  Wastewater
and in Material Safety Data Sheets
provided by chemical manufacturers in
compliance with OSHA's Hazard
 Communication Standard.  When
 working with these or with other strong
 acids, avoid inhalation of fumes.
 Protect eyes with safety goggles or
 with full face mask. Protect clothing
 w'stfi acid-resistant laboratory coat or
 apron. If eyes are accidentally ex-
posed to acid, immediately flush them
 with copious quantities of water for at
toast 15 minutes. Consult a physician
Immediately thereafter. If other parts of
the body are exposed to acid, immedi-
ately remove clothing overexposed
areas and wash exposed areas with
copious quantities of water. Consult a
physician if affected area is large or if
exposure has been lengthy.

3.2.1   General Acid-resistant Labora-
tory Ware and Equipment.

(a) Nitric acid procedure.

(a. 1)   Fill vessel to brim with 10%
nitric acid.
   Small laboratory ware and equip-
ment components may be immersed
directly in a vat of acid.
   Take care to avoid splatter and do
not allow acid to contact skin.  When
necessary, wear acid-resistant gloves.
Gloves must possess good gripping
qualities, because acid makes vessels
slippery.

(a.2)   Allow 10% nitric acid to remain
overnight in contact with vessel
surface.

(a.3)   Pour neutralized nitric acid
down sewer drain and flush with tap
water.
   Neutralize by adding acid to a large
volume of an ice water solution of
sodium hydroxide. For concentrated
acid use 6 M sodium hydroxide.

(a.4)   Fill and empty acid-washed
vessel ten times with cold tap water.
   Be certain that all acid is removed
from outside of vessel and that the
initial rinse is neutralized before
disposal down sewer drain.

(a.5)   Swish-rinse vessel five times
with deionized distilled water.
   To swish-rinse, pour into the vessel
a volume of water equal to about 10%
of the volume of the vessel, and swish
water around entire surface with each
rinse.

(a.6)   Drain and dry vessel at
ambient temperature or dry in hot-air
chamber.

(a. 7)   Sterilize vessel by appropriate
method (see Chapter 3).

(b)  Chromic acid procedure.
   Chromic acid should be used only
 when other cleansing procedures fail.
 Glassware and other acid-resistant
laboratory ware cleansed with chromic
acid may retain some chromium ions
even after extensive rinsing. For some
 work, these ions maybe undesirable.
Chromic acid may be toxic to cells.
Glassware and other laboratory ware   :
used for cell culture work, if washed
with chromic acid, may subsequently
need to be washed with detergent
solution to remove chromium ions (see
Section 3.1).

(b. 1)   From an acid reservoir pour
into vessel a volume of 1% chromic
acid equal to about 10% of the
capacity of the vessel.
  Small laboratory ware and equip-
ment components may be immersed
in a vat of acid.
  Take care to avoid splatter. Do not
allow acid to contact eyes or skin.
Wear eye protection and an acid-
resistant laboratory coat or an acid-
resistant apron. Wear acid-resistant    -.
gloves. Gloves must possess good
gripping qualities, because acid makes
vessels slippery.

(b.2)   Rotate vessel so that acid
covers entire inside area of vessel.

(b.3)   Allow chromic acid to remain
in contact with vessel for about five
minutes.

(b.4)   Pour acid from vessel into acid
reservoir.
  Chromic acid is reusable until
oxidized (green). If chromic acid is
oxidized, dispose of it safely as with
other hazardous wastes. Contact
safety or waste control officer for waste
management guidelines for its
environmentally safe disposal.

(b.5)    Fill and empty acid-washed
vessel ten times with cold tap water.
  Be certain that all acid is removed
from outside of vessel and that the
initial rinse water is disposed of safely.

(b.6)    Swish-rinse vessel five times
with deionized distilled water.
   To swish-rinse, pour into the vessel
a volume of water equal to about 10%
of the  volume of the vessel, and swish
water around entire surface with each
rinse.

(b.7)    Drain and dry vessel at
ambient temperature or dry in hot-air
chamber.

(b.8)    Sterilize  vessel by appropriate
method (see Chapter 3).
                                                       2-6

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May 1991
3.2.2 Test Tubes.
(a) Place tubes open end up into
covered acid-resistant basket and
place basket into acid-resistant
vessel.

(b) Fill vessel with 10% nitric acid or,
if necessary, with 1% chromic acid.
Take care to avoid splatter. Do not
allow acid to contact eyes or skin.
Wear eye protection and an acid-
resistant laboratory coat or an acid-
resistant apron. Wear acid-resistant
gloves. Gloves must possess good
gripping qualities, because acid makes
tubes slippery.
If 10% nitric acid is used, allow acid
to remain overnight in contact with
tubes.
If1% chromic acid is used, allow
acid to remain in contact with tubes for
about five minutes.

(c) Pour acid from tubes.
Chromic acid is reusable until
oxidized (green) and may be poured
back into reservoir. If chromic acid is
oxidized, dispose of it safely as with
other hazardous wastes. Contact
safety or waste control officer for
waste management guidelines for its
environmentally safe disposal.
Pour neutralized nitric acid down
sewer drain and flush with tap water.
Neutralize by adding acid to a large
volume of an toe water solution of
sodium hydroxide. For concentrated
acid use 6 M sodium hydroxide.

(d) Run cold tap water into vessel to
flush acid from tubes.

(e) Fill and empty tubes in vessel ten
times with cold tap water.
Be certain that the initial rinse water
is disposed of safely.

(f) Fill and empty tubes in vessel five
times with deionized distilled water.
Tubes for cell culture work that have
been cleansed with chromic acid must
be cleansed with detergent solution.
For such tubes, proceed to Section
3.1.2.

(g) Drain and dry tubes at ambient
temperature or dry in hot-air chamber.

(h) Sterilize screw-cap or cotton-
plugged tubes.

(h.1) Place screw-cap and cotton-
plugged tubes in test tube racks.
(h.2) Sterilize tubes in dry heat oven
for one hour.
Oven temperature is maintained at
170°C.
See Section 3.1.8 for processing
plastic screw caps.

(i) Sterilize tubes with caps or
semipermeable plastic inserts.

(i. 1) Protect mouths of tubes with
caps or with semipermeable plastic
inserts.

(i.2) Place tubes with caps or
plastic inserts in test tube tacks.

(1.3) Sterilize tubes by autoclaving
at 121° C for 30 minutes.

3.2.3 Pipettes.

(a) Remove cotton plugs from
decontaminated pipettes.
H necessary, remove cotton plugs by
forcing a jet of air or water through
delivery tips of the pipettes,

(b) Place pipettes, with tips up, into
an acid-resistant plastic pipette holder.

(c) Carefully place pipette holder into
pipette jar filled with 10% nitric acid.
Take care to avoid acid splatter. Do
not allow acid to contact eyes or skin.
Wear eye protection and an acid-
resistant laboratory coat or an acid-
resistant apron. Wear acid-resistant
gloves. Gloves must possess good
gripping qualities, because acid makes
pipettes and pipette holders slippery.

(d) Carefully raise and lower pipette
holder several times to force air
bubbles from pipettes.

(e) Soak pipettes overnight in acid.
Carefully raise and lower pipette
holder five or six times during this time
period to agitate acid and thus help
remove contaminants and debris from
pipettes.

(i) Carefully remove pipette holder
from pipette jar and place holder and
pipettes in automatic pipette washer.
Ta!
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May 1991
(c) Decontaminate filled plastic bags
or sharp waste containers.
Decontamination procedures are
summarized In Section 5. See
Chapters for the detailed steps to
decontaminate waste materials.
Use care while handling bags so as
not to puncture them. Containers
should be closed tightly to prevent
loss of contents while handling them.
If bags or containers must be moved
from laboratory area for decontamina-
tion, they must be securely sealed to
prevent leakage and transported in
rigid, sturdy receptacles that are taped
chsed. The receptacles must be
properly labelled with the bhhazard
symbol and the location and name of
the person disposing of the infectious
waste.

(d) After decontamination, label
plastic bags or containers "non-
hazardous".

(e) Minimize waste production by
recycling waste materials whenever
applicable; otherwise dispose of waste
in appropriate trash receptacle.

4.1.2 Disposal of Agar Media.

{a) Use autoclavable, leakage- and
tear-resistant plastic bags imprinted
with biohazard symbol.

(b) Place plastic bags in a durable
ieakproof receptacle.
Containers to be recycled may be
placed In a metal pan. After rendering
the waste nonfnfecthus, spent agar
madia from containers may be flushed
with tap water into sewer system.

Decontamination procedures are
Summarized in Section 5. See
Chapter 3 for the detailed steps to
carry out decontamination of the agar
media.
Use care while handling the bag so
as not to puncture the bag.
If bags must be moved from
laboratory area for decontamination,
they must be securely sealed to
prevent leakage and transported in
rigid, sturdy receptacles that are taped
chsed. The receptacles must be
properly labelled with the biohazard
symbol and the location and name of
the person disposing of the infectious
waste.

(d) After decontamination, label
plastic bags nonhazardous.
(e) Dispose of waste in appropriate
trash receptacle.

4.1.3 Disposal of Liquid Media.

(a) Decontaminate liquid media.

Decontamination procedures are
summarized in Section 5. See Chapter
3 for the detailed steps to decontami-
nate liquid media.

(b) Flush with tap water decontami-
nated spent liquid media into sewer
system.

4.2 Disposal of Nonhazardous Waste.

4.2.1 Glassware.

(a) Place broken or disposable
glassware in plastic-lined puncture-
resistant container.

(b) Minimize waste production by
recycling waste glassware whenever
possible; otherwise dispose of waste
in appropriate trash receptacle.

4.2.2 Plasticware.

(a) Place disposable plastic labora-
tory ware in plastic-lined trash can.

(b) Minimize waste production by
recycling disposable plastic laboratory
ware whenever possible; otherwise
dispose of waste in appropriate trash
receptacle.

4.3 Disposal of Chemical Wastes.

4.3.1 Hazardous Chemicals.

(a) Collect chemical in a suitable
container.
Container must be sturdy,
nonleaking and sealable.

(b) Label container with the name of
the chemical waste, its hazardous
properties (e.g. corrosive, caustic,
toxic, flammable, irritant), quantity,
date of disposal, and location and
name of person disposing of the
waste.

(c) Transfer chemical to hazardous
waste storage facility for ultimate
disposition by safety or waste control
officer.
2-8
4.3.2 Acids.

(a) Add ice water solution of 6 M
sodium hydroxide until pH is above 6.

(b) Flush neutralized solution into
sewer system with tap water.

5. Summary of
Decontamination Procedures
See Figures 1 and 2 for an overview
of procedures that are used for
packaging, decontaminating and
disposing of infectious agents.

5.1 Decontamination Procedures.

5.1.1 Decontamination by autoclav-
ing at 121°C for 30 minutes.

(a) glass and plastic laboratory ware.

(b) towels, lab coats, and gloves.

(d) filters, and autoclavable equip-
ment.

5.1.2 Chemical Decontamination
Procedures.

(a) Ethylane oxide.
Decontaminate by gas sterilizing
with ethylene oxide for 4 hours and
then aerating for 4 hours.

(a. 1) Reusable filters.

(a.2) Filter housings.

(a.3) Hoses.

(a.4) Nonautoclavable equipment.

(b) Chlorine.
Decontaminate with 10 to 15 mg of
chlorine per liter by holding in chlorine
solution for 30 minutes or by
recirculating solution through equip-
ment for 30 minutes.

(b.1) Hoses.

(b.2) Filter housings.

(b.3) Heat-sensitive equipment (e.g.
pumps).

(b.4) Large vessels (e.g. drums).

(b.5) pH electrodes.
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May 1991
6. Bibliography

Collins, C. H. Laboratory-Acquired
Infections, 2nd Edition,
Butterworths, London, 1988.
Furr, Keith A. CRC Handbook of
Laboratory Safety, 3rd Edition, CRC
Press, Boca Raton, Florida, 1990.
Paul, John. Cell and Tissue Culture,
5th Edition, Churchill Livingstone,
New York, 1975.
Standard Methods for the Examination
of Water and Wastewater, 17th
Edition, American Public Health
Association, Washington, D.C.,
1989.
Figure 2-1. Overview of temperature decontamination and disposal procedures of
infectious microbiological wastes.
Glassware
Small Equipment
Towels
Labcoats
Spent Media
Sewage
Sludge
QB V0

o
Steam Autoclave

Prep Room
Broken/Discard Glass
Disposable Plastic Items
Filters i\
Paper V
Cloth
Small or Wet Disposable Items
C
SB o0oo
o
Steam Autoclave

(Open Autoclavable
Bag in Metal Pan)
Laundry
Glass Disposal Box

"'• " ^
Plastic Bag
Lined Trash Can
Steam Autoclave
Trash Pick-up
2-9
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May 1991

Figure 2-2. Overview of chemical decontamination and disposal procedures of infectious
microbiological wastes.
Fiftor Housings

Hoses

Non-autoclavabte
Equipment
Ethylene Oxide
Gas Sterilizer
O O
Aerator
O O
Prep Room
Hosos

Fitter Housings

Pumps, Apparatus f\

Heat-Sensitive Containers
Prep Room
10-15 mg Chlorine per Liter
*US GOVERNMENT PRINTING Of FICE. 1991-548-187/25616
2-10
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