Microorganisms and test methods
Use test organisms such as Salmonella choleraesuis (ATCC 10708),
Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442), and
Enterococcus faecalis (ATCC 29212). These organisms would be passaged on
tryptic soy agar and incubated aerobically at 37°C. Stock cultures can be
maintained frozen in skim milk at –70°C.
Test soil and inoculation methods
To provide an organic and inorganic challenge that mimics what medical
devices would be exposed to in the body, an artificial test soil (ATS) is
used. The
composition of
ATS is based on the worst-case levels of protein, carbohydrate, endotoxin,
and hemoglobin detected from patient-used flexible endoscopes. The test
organisms are suspended in the ATS to provide a final concentration of 10
8
cfu/mL. The inoculation of the test carrier is performed by the use of a
micropipette to place 10 or 50μL of the test suspension on the inner surface
of a PVC test carrier. This provides an inoculum of 10
6 cfu/carrier.
The test carrier consists of a 1-cm piece of PVC tubing with an inner
diameter of 3 mm. Once inoculated, the test carrier is placed in a petri
dish inside a biosafety cabinet and allowed to dry overnight at room
temperature (RT). These inoculated dried test carriers are then used to
assess the killing efficacy of various detergent formulations, which
represents a “
worst-case”
challenge to the detergent.
Test method and quantitation
Lumen test carrier.
The dried inoculated test carriers are exposed to the detergent when it was
placed into a test tube that contains 1 mL of the detergent to be evaluated.
The test carrier is exposed for the detergent manufacturer’s recommended
time and temperature. Quantitation is based on the viable counts that are
performed from serial dilutions of the original sample (limit of detection,
10 cfu/carrier) on the spread plate and filtration for quantitation of the
original sample (limit of detection, 1 cfu/carrier). All inoculated plates
are incubated aerobically at 35°C for 24 to 48 hours before the number of
colonies are counted. All results may be presented as the number of viable
organisms per test carrier. To assess the cleaning efficacy of the various
detergents, the lumen test carrier method is used to assess soil parameters
before and after treatment. The inoculation and processing method are the
same as described
above. To prevent loss of
soil caused by exposure to fluid, the dried inoculum on the lumen carrier
may be fixed by immersing in 1% glutaraldehyde for 1 minute at RT. The final
sample is assayed to determine the level of hemoglobin, carbohydrate,
protein, and endotoxin. The methods for quantitation of each of these soil
parameters is as described by
Alfa et al.
Control
To ensure that “wash-off” from fluid exposure is separated from the
microbial killing ability, all experiments should include controls that
consist of quantitative testing of the recoverable bioburden after exposure
of the inoculated/dried carrier to phosphate buffered saline solution for
the time and temperature equivalent to the detergent exposure conditions.
A study utilizing this
method
