Procedures
for Collecting Surface Environmental Samples for Culturing Bacillus
anthracis
Preface
The
decision to collect environmental samples for culturing Bacillus
anthracis should be made by medical, environmental, and industrial
hygiene professionals familiar with the organism and with the
environmental sampling methodologies described in this document.
This decision should be based on the nature and location of the
suspected contamination, the medical diagnoses and opinions, the
potential for the contaminant to migrate, and the activity for
which the facility is used, following a pre-planned sampling strategy.
Representatives from local, state, and federal agencies should
be consulted during the decision-making process.
Environmental
sampling can be used to help determine the extent and degree of
contamination, to support decisions regarding the need for cleanup,
and to provide guidance regarding when cleanup is adequate to
permit re-entry into an area. The use of experienced investigators
to conduct the environmental sampling will provide the best probability
of locating and identifying B. anthracis spores if present.
Currently,
no environmental exposure standards exist for B. anthracis spores.
Investigators who review and interpret the results of environmental
sampling for such spores must consider these uncertainties and
use professional judgment in interpreting any positive or negative
findings.
Before
sampling is begun, the building's engineer should be consulted
to determine airflow patterns and the design of the heating, ventilating,
and air-conditioning system(s). Since most building ventilation
systems recirculate air to other locations in the building, the
ventilation system serving the contaminated area should be shut
off to prevent further airborne spread of any B. anthracis.
Depending on the size of the area involved, the types of surfaces
potentially contaminated, and the extent of contamination, it
may be necessary to isolate and control access to the contaminated
area to prevent the spread of contamination through the movement
of people or equipment:
-
If the
contaminated area is small, discrete, and only lightly contaminated,
cordoning off the area may provide adequate protection.
-
If
the contaminated area is large, the affected area should
be sealed off using an interim dust barrier made from impervious
lightweight plastic (e.g., 6 mil polypropylene) sheeting.
Tight seals should be maintained at the full perimeter of
temporary walls and sealed by tape at ceiling height in
the same way that areas are sealed off for asbestos abatement
or dust control during building renovation. Air vents in
the area should also be sealed with plastic sheeting and
tape to control the risk of dust dispersal and recirculation.
The sampling method and number of samples collected will
be influenced by the nature, circumstances, and setting
of the potential contamination. The methods used may include
bulk or surface sampling strategies. Since the extraction
efficiency of spores from the various building materials
is not known, the results of bulk or surface sample analyses
are qualitative. A sufficient number of samples must be
taken to increase the probability that the sampling is representative.
Obtaining samples from additional locations may provide
more specific information on the source of the contamination.
For each sample collected, the usual, non-forensic chain-of-custody
procedures should be followed and documented as designated
by the local state health laboratory reporting requirements.
Taking photographs of the location where the samples are
collected is often helpful.
The first priority should be to collect samples in locations
that are near suspected release source(s). Samples should
be collected by moving inward in concentric circles toward
the suspected release source, following the path over which
spores may have dispersed. If the aerosol containing B.
anthracis spores has an aerodynamic size of less than 10
microns, the particles will remain suspended in the air
for extended periods of time. In such cases, the spores
can quickly spread throughout an air space and into adjacent
areas. Spores can also be carried if they attach to clothing,
shoes, or other objects; thus, more distant sampling may
be needed. (Personnel who enter the contaminated area must
follow a safety and infection control plan developed for
the particular site).
Bulk
sampling
Bulk samples can help investigators characterize the presence
of contamination on building materials such as carpets, office
equipment, and supplies. However, because extracting spores from
bulk samples can pose exposure concerns for laboratory personnel,
appropriate precautions (such as double-bagging of samples) should
be taken.
Surface
sampling
Surface samples are collected by wiping non-porous surfaces with
an absorptive medium from which spores can be extracted in the
laboratory. The absorptive media, wetting agent, and bags used
to transport samples should be selected with input from the laboratory
personnel who will be analyzing the samples so that collection
procedures will be compatible with the laboratory's analytical
procedures.
There
are several absorptive media available, but non-cotton swabs are
preferred. These swabs must be sterile and used with a sterile
wetting agent such as sterile water, a sterile saline solution,
or a sterile phosphate-buffer solution.
Samples
collected by vacuuming
Although collecting samples by vacuuming offers the advantages
of covering large surfaces and collecting material from porous
areas such as carpets, only high-efficiency particulate air (HEPA)
vacuum cleaners must be used.
Conventional home or industrial vacuum cleaners should not be
used for sample collection because these vacuum cleaners will
further disperse spores. A hose or diffuser can be retrofitted
to the vacuum cleaner exhaust so that the HEPA-filtered exhaust
can be vented outside the contaminated zone to prevent reaerosolization
of spores within the contaminated area.
There
are several methods for collecting vacuum samples. One option
is to connect a filtering Alsock® (dust collection trap manufactured
by Healthy Home Air or equivalent)* to the inlet nozzle of a HEPA
vacuum cleaner. A second option is to collect a sample on a 37-millimeter
(mm) diameter mixed cellulose ester (MCE) filter contained in
a plastic sampling cassette. The analytical results from this
type of sampling are qualitative. Finally, when selecting sampling
equipment, consideration should be given to whether and how it
can be decontaminated.
Collecting
Bulk Samples
For most laboratories, bulk samples are not acceptable. Therefore,
the receiving laboratory should be contacted before bulk samples
are collected to determine whether such samples will be accepted.
Bulk
samples may include items such as sections of carpet, office equipment,
supplies, or vials of dust.
1. Don sterile, non-powdered nitrile or vinyl examination gloves
over the gloves that are part of standard PPE and clothing.
2. Collect and bag the item; seal the bag.
3. Clean the outside of the sealed bag with a 0.5-0.6% (5,000-6,000
ppm) sodium hypochlorite solution just prior to leaving the contaminated
area. Typical household bleach sold in the United States contains
approximately 5.25-6% (52,500-60,000 ppm) sodium hypochlorite.
The disinfection solution is made by adding 1 part household bleach
to 9 parts water (a 1:10 dilution).Final solutions should be in
a pH range of ~6-8. Clorox® bleach* diluted 1:10 meets these requirements.
When using other brands, one should confirm the buffering capacity
and sodium hypochlorite concentrations.
4. Place the cleaned, sealed bag in another unused, self-sealing
bag, and prepare for shipping according to CDC guidelines (http://www.cdc.gov/od/ohs/biosfty/shipregs.htm
or http://www.bt.cdc.gov/LabIssues/PackagingInfo.pdf
).
5. Record the following items:
* Measured size of the area sampled
* Type of sample
* Time and date of sample
* Name of person collecting sample To collect another sample,
change gloves to prevent cross-contamination and repeat steps
1-5.
6. Submit the samples to the laboratory for culture.
7. Transport samples to the laboratory at ambient temperature.
8. Maintain appropriate chain-of-custody documentation and procedure.
Collecting
Sterile Swab Samples
The
following steps are used to collect samples for laboratory culture
from small non-porous surfaces or objects.
1. Don sterile, non-powdered nitrile or vinyl examination gloves
over the gloves that are part of standard PPE and clothing.
2. Remove a sterile, non-cotton swab from the package.
3. Moisten the swab with 100-200µl (or 1-2 drops) of a sterile
water, sterile saline, or sterile phosphate-buffered saline (PBS)
solution. This can be done by using a disposable Pasteur pipette
and aseptic technique. Note: Check with the laboratory that will
do the analysis to determine which type of swab or solution is
preferred.
4. Wipe the surface. Recommended wipe area is 100 cm2. Avoid letting
the swab dry completely.
5. Place the sampled swab into a sterile conical vial, and cap
the vial.
6. Record the following items: * Measured size of the area sampled
* Type of sample * Time and date of sample * Name of person collecting
sample
7. Label the vial, and place it in a self-sealing bag (such as
a Ziploc® bag or Whirlpak®).*
8. Clean the outside of the sealed bag with a 0.5-0.6% (5,000-6,000
ppm) sodium hypochlorite solution just prior to leaving the contaminated
area. Typical household bleach sold in the United States contains
about 5.25-6% (52,500-60,000 ppm) sodium hypochlorite. The disinfection
solution is made by adding 1 part household bleach to 9 parts
water (a 1:10 dilution). Final solutions should be in a pH.range
of ~6-8. Clorox® bleach diluted 1:10 meets these requirements.
When using other brands, one should confirm the buffering capacity
and sodium hypochlorite concentrations.
9. Place the cleaned, sealed bag in another unused similar bag.
To collect another sample, change gloves to prevent cross-contamination
and repeat steps 1-9.
10. Prepare samples for shipping according to CDC guidelines (http://www.cdc.gov/od/ohs/biosfty/shipregs.htm
(pdf) or http://www.bt.cdc.gov/LabIssues/PackagingInfo.pdf
) and submit the
samples to the laboratory for analysis.
11. Transport samples to the laboratory at ambient temperature.
12. Maintain appropriate chain-of-custody documentation and procedure.
LRN
Level A Protocol for Rule-Out Testing of Bacillus anthracis
1. Process low-risk (non-powder) environmental samples taken and
transported according to above procedure in a CLIA-certified laboratory
using biosafety level (BSL) 2 facilities and BSL-3 safety practices.
2. Follow standardized Laboratory Response Network (LRN) Level
A testing protocol (http://www.asmusa.org/pcsrc/ban.asm.la.cp.102401f.pdf
) with modification
for elution and plating as follows (per LRN Level B):
a. Place each sample swab in 3 ml of sample-processing solution
(0.3% Tween in PBS).
b. Vortex for approximately 1 minute.
c. Transfer 1.5 ml of buffer to a tube labeled "C." Label the
remainder of the sample "A."
d. Heat-shock the "C" tube in the 65*C water bath for 10 minutes.
e. Inoculate the "A" and "C" samples onto 3 blood agar plates
each using 0.1 ml inoculation volumes.
f. Streak the plates for isolation.
g. Incubate the plates at 35-37*C for 18-24 hours and examine
for suspicious colonies. Identify suspicious colonies using the
LRN Level A Bacillus anthracis methods referred to above.
OR
a. Remove the swab from the transport container and place into
1.5 ml of sterile saline or a nutrient broth such as trypticase
soy broth, brain heart infusion broth, or equivalent. Vigorously
twist the swab, and recap the tube.
b. Leave the swab in the tube. Place the broth suspension into
a 65*C water bath for 30 minutes.
c. Plate 0.1-0.2 ml of broth on 5% sheep blood agar plate and
incubate at 35-37*C for 18-24 hours. Many B. anthracis will have
visible growth in 12-18 hours; observe for characteristics of
B. anthracis.
All
culture isolates that cannot be ruled out and are therefore presumptively
positive should be referred to an LRN State Public Health Laboratory
for confirmatory testing by the LRN Level B protocol and standardized
algorithm for identification of Bacillus anthracis.
Collecting
Samples with a HEPA Vacuum Cleaner
The
following steps should be used to collect samples for laboratory
culture from large porous or non-porous surfaces such as carpeting,
ceiling tiles, ventilation systems, and papers.
1.
Don sterile non-powdered nitrile or vinyl examination gloves over
the gloves that are part of the standard PPE and clothing.
2. Insert a cone-shaped filtering Alsock® (dust collection trap
manufactured by Healthy Home Air or equivalent)* into the vacuum
cleaner nozzle.
3. Fold the plastic sleeve over the outside of the nozzle, and
secure it with tape or an elastic band.
4. HEPA-vacuum the surface. Note: 1-2 tablespoons of vacuumed
debris are needed. Technique: Make one pass of the entire sampling
area at a slow rate (12 inches per 5 seconds).
5. Record the following items:
* Measured size of the area sampled
* Type of sample
* Time and date of sample
* Name of person collecting sample
6. After collecting the sample, remove the tape or elastic band
and discard these items as contaminated waste.
7. Remove the cone-shaped dust collection trap, and place it in
a self-sealing bag (such as a Ziploc® bag or Whirlpak®),* or roll
the filter and place it in a sterile conical vial.
8. Place the sample in a clean self-sealing bag and label it.
9. Clean the outside of the sealed bag with a 0.5-0.6% (5,000-6,000
ppm) sodium hypochlorite solution just prior to leaving the contaminated
area. Typical household bleach sold in the United States contains
about 5.25-6% (52,500-60,000 ppm) sodium hypochlorite. The disinfection
solution is made by adding 1 part household bleach to 9 parts
water (a 1:10 dilution). Final solutions should be in a pH range
of ~6-8. Clorox® bleach diluted 1:10 meets these requirements.
When using other brands, one should confirm the buffering capacity
and sodium hypochlorite concentrations.
10. Place the cleaned sealed bag in another unused self-sealing
bag. To collect another sample, change gloves and clean the nozzle
with the bleach solution followed by alcohol to prevent cross-contamination,
and repeat steps 1-10.
11. Prepare samples for shipping according to CDC guidelines (http://www.cdc.gov/od/ohs/biosfty/shipregs.htm
or http://www.bt.cdc.gov/LabIssues/PackagingInfo.pdf
) and submit the
samples to the laboratory for analysis.
12. Transport samples to the laboratory at ambient temperature.
13. Maintain appropriate chain-of-custody documentation and procedure.
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