1Hojlyng,
N.,
Holten­
Andersen,
W.,
and
S.
Jepsen.
1987.
Cryptosporidiosis:
a
case
of
airborne
transmission.
Lancet.
2:
271­
272.
Blagburn,
B.
L.,
and
W.
L.
Current.
1983.
Accidental
infection
of
a
researcher
with
human
Cryptosporidium.
J.
Infect.
Dis.
142:
772­
773.

Draft
June
2003
1
BIOSAFETY
GUIDELINES
FOR
LABORATORIES
ANALYZING
ENVIRONMENTAL
SAMPLES
FOR
CRYPTOSPORIDIUM
1.0
Introduction
Laboratory­
related
infections
with
Cryptosporidium
can
occur
if
personnel
are
not
properly
trained
in
biosafety
techniques.
In
addition
to
waterborne,
fecal­
oral,
person
to
person
transmission,
and
animal
to
person,
circumstantial
evidence
suggests
that
airborne
transmission
of
oocysts
may
occur1.
Adoption
of
a
biosafety
policy
by
laboratory
management
that
includes
commitment
to
technician
safety,
training
and
supervision,
as
well
as,
rigid
adherence
to
biosafety
guidelines
will
prevent
the
occurrence
of
Cryptosporidium
infection
(
cryptosporidiosis)
in
laboratory
personnel.

Symptoms
associated
with
cryptosporidiosis
may
include:
watery
diarrhea,
abdominal
cramps,
nausea,
low­
grade
fever,
dehydration,
weight
loss,
and
loss
of
appetite.
Symptoms
may
develop
within
2
to
10
days
after
infection.
There
are
no
antibiotics
or
drug
treatments
that
will
cure
cryptosporidiosis.
For
additional
information
:
www.
cdc.
gov/
ncidod/
dpd/
parasites/
cryptosporidiosis/
default.
htm
Biosafety
Level
2
(
BSL
2)
practices
and
facilities
are
recommended
for
activities
with
infective
stages
of
Cryptosporidium.
A
BSL
2
facility
is
appropriate
for
agents
known
to
cause
disease
in
humans.

Biosafety
Level
2
practices
and
facilities
include
the
following
requirements:

°
Laboratory
personnel
have
specific
training
in
handling
pathogenic
agents
and
are
directed
by
competent
scientists
°
Access
to
the
laboratory
is
limited
when
work
is
being
conducted
°
Certain
procedures
in
which
infectious
aerosols
or
splashes
may
be
created
are
conducted
in
biological
safety
II
cabinets
2.0
Scope
and
Application
The
biosafety
guidelines
described
in
this
document
are
adapted
from
Laboratory
Safety:
Principles
and
Practices,
Second
Edition
(
Reference
8.1)
and
Biosafety
in
Microbiological
and
Biomedical
Laboratories
(
BMBL)
Fourth
Edition
(
Reference
8.2).
A
readily
available
laboratory­
specific
biosafety
manual
should
be
developed
,
and
maintained,
to
address
the
safety,
handling,
and
laboratory
practices
described
below.
The
manual
should
be
distributed
to
all
employees
and
available
at
all
times.
It
should
be
reviewed
annually,
or
as
recommended,
by
the
laboratory
safety
officer.
Personnel
are
required
to
read
and
sign
off
on
the
document
on
a
regular
basis,
as
determined
by
the
laboratory
safety
officer.

3.0
General
Safety
Practices
3.1
Basic
Laboratory
Safety
3.1.1
Closed­
toed
shoes
shall
be
worn
in
the
laboratory.

3.1.2
All
work
surfaces
and
floors
should
be
cleaned
regularly
and
free
of
clutter.

3.1.3
All
emergency
numbers
should
be
posted
in
the
laboratory.
LT2
Biosafety
Guidance
Draft
June
2003
2
3.1.4
All
employees
should
be
trained
in
the
use
and
location
of
all
safety/
emergency
equipment
in
each
work
area.

3.1.5
Biological
safety
II
cabinets
must
be
tested
and
certified
annually.

3.1.6
All
laboratory
personnel
should
be
trained
in
the
proper
procedures
to
clean
up
biological
spills.

4.0
Microbiological
Practices
4.1
Access
to
the
laboratory
is
limited
or
restricted
at
the
discretion
of
the
laboratory
director
or
laboratory
safety
officer
when
experiments
are
in
progress.

4.2
Persons
wash
their
hands
after
they
handle
viable
materials
and
animals,
after
removing
gloves,
and
before
leaving
the
laboratory.

4.3
Eating,
drinking,
smoking,
handling
contact
lenses,
and
applying
cosmetics
are
not
permitted
in
the
work
areas.
Persons
who
wear
contact
lenses
in
laboratories
should
also
wear
goggles
or
a
face
shield.
Food
is
stored
outside
the
work
area
in
cabinets
or
refrigerators
designated
for
this
purpose
only.

4.4
Mouth
pipetting
is
prohibited.

4.5
All
procedures
are
performed
carefully
to
minimize
the
creation
of
splashes
or
aerosols.
Ensure
that
lids
are
used
during
all
centrifugation
and
vortexing.
Any
procedures
with
open
containers
should
be
performed
inside
a
BSL
2
hood.

4.6
Work
surfaces
are
decontaminated
before
and
after
each
use
and
after
any
spill
of
viable
material.

4.7
All
cultures,
stocks,
and
other
regulated
wastes
are
decontaminated
before
disposal
by
an
approved
decontamination
method
such
as
autoclaving.

4.7.1
Materials
to
be
decontaminated
outside
of
the
immediate
laboratory
are
to
be
placed
in
a
durable,
leakproof
container
and
closed
for
transport
from
the
laboratory.

4.7.2
Materials
to
be
decontaminated
off­
site
from
the
laboratory
are
packaged
in
accordance
with
applicable
local,
state,
and
federal
regulations
before
removal
from
the
facility.

5.0
Special
Practices
when
Processing
Cryptosporidium
Samples
5.1
In
general,
persons
who
are
at
increased
risk
of
acquiring
infection
or
for
whom
infection
may
be
unusually
hazardous
are
not
allowed
in
the
laboratory
or
animal
rooms.
For
example,
persons
who
are
immunocompromised
may
be
at
risk
of
acquiring
infections.

5.2
The
laboratory
safety
officer
has
the
final
responsibility
for
assessing
each
circumstance
and
determining
who
may
enter
or
work
in
the
laboratory.
The
laboratory
safety
officer
will
report
to
the
laboratory
director
on
a
regular
basis
on
the
status
of
safety
in
the
laboratory,
conduct
training
and
maintain
outside
professional
contacts
to
exchange
safety
information
relevant
to
laboratory
operations.

5.3
The
laboratory
director
or
laboratory
safety
officer,
establishes
policies
and
procedures,
whereby
only
persons
who
have
been
advised
of
the
potential
hazard
and
meet
specific
entry
requirements,
enter
the
laboratory
or
animal
rooms.

5.4
When
the
infectious
agent(
s)
in
use
in
the
laboratory
require
special
provisions
or
special
training
for
entry,
a
hazard
warning
sign
incorporating
the
universal
biohazard
symbol
is
posted
on
the
access
door
to
the
laboratory
work
area.
The
hazard
warning
sign
identifies
the
infectious
agent,
lists
the
name
and
telephone
number
of
the
laboratory
director
or
other
responsible
person(
s),
and
indicates
the
special
requirement(
s)
for
entering
the
laboratory.
LT2
Biosafety
Guidance
Draft
June
2003
3
5.5
Laboratory
personnel
receive
appropriate
training
on
the
potential
hazards
associated
with
the
work
involved,
the
necessary
precautions
to
prevent
exposures,
and
the
exposure
evaluation
procedures.
Personnel
receive
annual
updates,
or
additional
training
as
necessary
for
procedural
or
policy
changes.

5.6
Materials
containing
Cryptosporidium
are
placed
in
a
container
that
prevents
leakage
during
collection,
handling,
processing
storage,
transport,
or
shipping.

5.7
Laboratory
equipment
should
be
decontaminated
with
an
appropriate
disinfectant
before
and
after
the
equipment
is
used,
and
especially
after
overt
spills,
splashes,
or
other
contamination
by
infectious
materials.
Contaminated
equipment
must
be
decontaminated
according
to
any
local,
state,
or
federal
regulations
before
it
is
sent
for
repair
or
maintenance
or
packaged
for
transport
in
accordance
with
applicable
local,
state,
or
federal
regulations
before
removal
from
the
facility.

5.8
Spills
and
accidents
which
result
in
overt
exposures
to
infectious
materials
are
immediately
reported
to
the
laboratory
director
or
laboratory
safety
officer.
Medical
evaluation,
surveillance,
and
treatment
are
provided
as
appropriate
and
written
records
are
maintained.

6.0
Safety
Equipment
(
Primary
Barriers)

6.1
Properly
maintained
biological
safety
II
cabinets,
and
other
appropriate
personal
protective
equipment
or
physical
containment
devices
are
used
whenever:

6.1.1
Procedures
with
potential
for
creating
infectious
aerosols
or
splashes
are
conducted.
These
may
include
centrifuging,
grinding,
blending,
vigorous
shaking
or
mixing,
sonic
disruption,
or
opening
containers
of
infectious
materials
whose
internal
pressures
may
be
different
from
ambient
pressures.

6.1.2
High
concentrations
or
large
volumes
of
infectious
agents
are
used.
Such
materials
may
be
centrifuged
in
the
open
laboratory
if
sealed
rotor
heads
or
centrifuge
safety
cups
are
used,
and
if
these
rotors
or
safety
cups
are
opened
only
in
a
biological
safety
cabinet.

6.2
Face
protection
(
goggles,
mask,
faceshield,
or
other
splatter
guards)
is
used
for
anticipated
splashes
or
sprays
of
infectious
or
other
hazardous
materials
to
the
face,
when
the
microorganisms
must
be
manipulated
outside
the
biological
safety
cabinet.

6.3
Protective
laboratory
coats,
gowns,
smocks,
or
uniforms
designated
for
lab
use
are
worn
while
in
the
laboratory.
This
protective
clothing
is
removed
and
left
in
the
laboratory
before
leaving
for
non­
laboratory
areas
(
e.
g.,
cafeteria,
library,
administrative
offices).
All
protective
clothing
is
either
disposed
of
in
the
laboratory
or
laundered
by
the
institution;
it
should
never
be
taken
home
by
personnel.

6.4
Gloves
are
worn
when
handling
infected
animals
and
when
hands
may
contact
infectious
materials,
contaminated
surfaces,
or
equipment.

6.4.1
Wearing
two
pairs
of
gloves
may
be
appropriate;
if
a
spill
or
splatter
occurs,
the
hand
will
be
protected
after
the
contaminated
glove
is
removed.

6.4.2
Gloves
are
disposed
of
when
contaminated,
removed
when
work
with
infectious
materials
is
complete,
and
are
not
worn
outside
the
laboratory.

6.4.3
Disposable
gloves
are
not
washed
or
reused.
LT2
Biosafety
Guidance
Draft
June
2003
4
7.0
Laboratory
Facilities
(
Secondary
Barriers)

7.1
Each
laboratory
contains
a
sink
for
handwashing.

7.2
The
laboratory
is
designed
so
that
it
can
be
easily
cleaned.
Rugs
in
laboratories
are
not
appropriate,
and
should
not
be
used
because
proper
decontamination
following
a
spill
is
extremely
difficult
to
achieve.

7.3
Bench
tops
are
impervious
to
water
and
resistant
to
acids,
alkalis,
organic
solvents,
and
moderate
heat.

7.4
Laboratory
furniture
is
sturdy,
and
spaces
between
benches,
cabinets,
and
equipment
are
accessible
for
cleaning.

7.5
If
the
laboratory
has
windows
that
open,
they
are
fitted
with
fly
screens.

7.6
A
method
for
decontamination
of
infectious
or
regulated
laboratory
wastes
is
available
(
e.
g.,
autoclave,
chemical
disinfection,
incinerator,
or
other
approved
decontamination
system).

7.7
An
eyewash
facility
is
readily
available.

7.8
The
laboratory
facilities
should
be
clean,
temperature
and
humidity
controlled,
and
have
adequate
lighting
at
bench
tops.

8.0
References
8.1
D.
O.
Fleming,
J.
H.
Richardson,
J.
J.
Tulis,
and
D.
Vesley.
Laboratory
Safety:
Principles
and
Practices.
1995.
Second
Edition.
American
Society
for
Microbiology,
Washnigton,
D.
C.,
publisher.

8.2
US
Department
of
Health
and
Human
Services.
Biosafety
in
Microbiological
and
Biomedical
Laboratories.
1999.
Fourth
Edition.
U.
S.
Department
of
Health
and
Human
Services,
Centers
for
Disease
Control
and
Prevention,
and
National
Institutes
of
Health,
US
Government
Printing
Office,
Washington,
D.
C.,
publisher.

8.3
Fayer,
Ronald,
Ed.
1997.
Cryptosporidium
and
Cryptosporidiosis.
CRC
Press,
Inc.,
Boca
Raton.
