Carpet Sanitizers
DIS/TSS-8 / April 18, 1981
EFFICACY DATA REQUIREMENTS
Carpet Sanitizers
Products bearing label claims for effectiveness as sanitizers for pre-cleaned
carpeting must be tested by a protocol incorporating the basic elements
of the attached recommended method. If the product is intended to be represented
in labeling as a "one-step" cleaner-sanitizer, the method must
be modified by including an appropriate soil with the bacterial inoculum.
(a) Test requirements.
(1) Three product samples representing 3 separate batches, one of which
is at least 60 days old, must be tested against Staphylococcusaureus
ATCC 6538 and Enterobacteraerogenes ATCC 13048 with 2 different types
of representative synthetic carpeting, such as acrylic and polypropylene
tufted-loop types. If the application is intended for hospitals or medical
institutions, the product must also be tested against Pseudomonasaeruginosa
ATCC 15442. If the product is also intended for use on wool carpeting,
an additional representative sample of wool carpet must be tested; otherwise,
the label must bear a disclaimer for use on wool. All carpet samples
tested must be fully identified by the pile fiber type, pile yarn weight
of finished carpet, pile density, and tuft height. Adequate controls
must demonstrate that bacteriostatic agents in the carpet pile or backing
do not yield false-negative data which interfere with the test results.
(2) The amount of solution applied to the sample carpeting in the tests
must be determined and extrapolated to obtain the amount of the solution
of product to be applied to carpeting (volume per unit area) as stated
on the label.
(b) Performance requirements. A 99.9% reduction of test
bacteria over the scrubbed control count must be demonstrated.
Note: If the product is intended for use in hospitals or medical
institutions, a wet vacuum pickup must be specified in the label directions
for use. In no case will the so-called "dry shampoo treatment"
be considered for use in such areas.
Sanitizers - Carpets
(Proposed method prepared by Registration Division,
Office of Pesticide Programs, EPA, 1976; revised 1981)
a. Special equipment and materials.
1. Carpet mounting board. Mount a piece of l/8-in. (0.3 cm)
tempered hardboard, tempered surface up, on a 16 x 16-in. (40.6 x 40.6
cm) base of 3/4-in. (1.8 cm) thick marine plywood, with 3/4-in. (1.8
cm) brads.
2. Cutting equipment. 2 x 2-in. (5.1 x 5.1 cm) squares of 1/4-in.
(0.6 cm) acrylic plastic with 3/32-in. (0.24 cm) holes in the center
as templates, and a sharp knife with replaceable blade.
3. Scrub brushes. 1 1/4 x 3 1/2-in. (4.2 x 8.9 cm) surgical
hand brush with 5/8-in. (0.6 cm) nylon bristles.
4. Extraction bottles. 8-oz. (236.6 ml), widemouth, round, polypropylene
bottles with screw caps (Nalgene 2105 or equivalent) containing 10 stainless
steel penicylinders and 100 ml of appropriate neutralizer broth. Similar
style glass bottles may be used but care must be taken to prevent breakage
during shaking.
5. Spray device. Adjustable spray atomizer modified to feed from a
calibrated test tube or bottle. A Model 15 DeVilbiss atomizer on a 2-oz.
(59.2 ml) bottle graduated with 10-ml marks may be used.
6. Carpet. If the product is intended for use on commercial grade carpeting,
2 representative carpets, such as acrylic and polypropylene tufted-loop
type must be tested. No carpeting is available to serve as a standard.
If the product is intended for use on wool carpeting, a representative
wool sample must additionally be tested. All carpet samples tested must
be fully identified, and the pile fiber type, pile yarn weight of finished
carpet, pile density and tuft height must be reported. Adequate controls
must demonstrate that bacteriostatic agents in the carpet pile or backing
do not interfere with the test results.
b. Test cultures and media.
1. Test bacteria. Use Staphylococcusaureus (ATCC 6538) and Enterobacteraerogenes
(ATCC 13048). If the product is intended for use in hospitals, Pseudomonasaeruginosa
PRD-10 (ATCC 15442) must additionally be tested.
2. NutrientagarB. AOAC Methods, sec. 4.023 (a) (2).
3. Phosphate buffer dilution water. AOAC Methods, sec. 4.023 (f).
4. Double strength neutralizer broth. For phenolic based products,
Letheen broth [AOAC Methods, sec. 4.001 (d) (3)] plus an additional
0.7 g lecithin (Azolectin) and 5 g polysorbate 80 (Tween 80) per liter
may be used; or a defoaming neutralizer consisting of nutrient broth
[AOAC Methods, sec. 4.001 (a)] plus 1.0 % Pluronic 25R2 (Meroxapol 252)
has been suggested. In the case of halogen or heavy metal based products,
0.1% sodium thioglycollate and 0.01% isooctylphenoxypolyethoxyethanol
(Triton X-100) in phosphate buffer (pH 7.2) may be used.
5. Neutralizerplatecountagar. Tryptone glucose extract agar
[AOAC Methods, sec. 4.037 (a)] plus 0.7 g lecithin (Azolectin) and 5
g polysorbate 80 (Tween 80) per liter.
c. Bacterial inoculum. Prepare French square culture bottles with
nutrient agar B and test bacteria (AOAC Methods, sec. 4.026). Prepare
standardized bacterial stock suspensions by washing growth from bottles
and adjust to a density of 10 x 10 bacteria per ml with phosphate buffer
dilution water (AOAC Methods, sec. 4.026).
d. Procedure.
1. Cut the carpet into 8 x 12-in. (20.3 x 30.5 cm) pieces. With the
aid of the 2 x 2-in. (5.1 x 5.1 cm) template, cut six 2 x 2-in. squares
(2 rows of 3 squares per row) from the backing side of the carpet, leaving
at least 4 in. (10.2 cm) between the center of each square. The preferred
method is to leave about 1/8 in. (0.32 cm) of backing intact at each
corner of each cut square so that the entire piece of carpeting can
be sterilized and inoculated without separation. Mark the pile surface
in the center of each test square with a waterproof marking pen with
the aid of the hole in the center of the template. Cover the pile surface
of the carpeting with aluminum foil and fold over edges to secure. Steam
sterilize and dry. Only carpet that has been determined to be free of
residual bacteriostatic activity on the pile or backing, following autoclaving,
shall be used. A seeded agar plate overlay technique should be used
for this determination.
2. Dilute the standardized bacterial stock suspensions, prepared as
in (c) above, with phosphate buffer dilution water containing 0.01%
isooctylphenoxypolyethoxyethanol to a concentration 10 x 10 bacteria
per ml. Inoculate the previously marked center of each cut square with
0.1 ml of the bacterial suspension. (Retain the bacterial suspensions
for determination of inoculation numbers.) Dry inoculated carpet in
an incubator at 35-37°C for 60 min. with the foil wrap loosely
in place.
3. Condition brushes by immersing the bristles in separate containers
(15- cm glass petri dishes or equivalent) of diluted test solution and
a control solution without the active antimicrobial ingredient(s) for
15 min. (If such a control solution is not available, use sterile distilled
water containing 0.01% isooctylphenoxypolyethoxyethanol.) Fasten 2 pieces
of inoculated carpet (each containing 6 test squares) onto the carpet
mounting board by nailing each corner with upholstery tacks, and with
the foil wrapping positioned so as to protect the controls during spraying
and scrubbing with the test solution. Place the board in a biological
hood or glove box. A simple safety chamber can be constructed from a
large plastic bag.
4. Determine the amount of test solution intended to be applied to
one piece of the carpeting containing 6 spots of dried bacterial inoculum
[96 sq. in. or 2/3 sq. ft. (244 sq. cm)] and subtract approximately
15 ml which will be applied later in the brushing procedure. Apply the
pre-determined amount of diluted test solution at room temperature uniformly
by metered spray to one piece of the test carpet. Shake excess test
solution from a conditioned brush and transfer to a fresh dish containing
100 ml of test solution at room temperature. Dip bristles of brush and
transfer the retained test solution to an inoculated spot on the sprayed
carpet. Scrub the spot for 30 sec. using 30 circular clockwise strokes
and 30 circular counterclockwise strokes. A circular area of pile approximately
3 in. (7.6 cm) in diameter around each spot must be covered by this
treatment. Moderate to heavy pressure should be applied downward on
the brush to work the solution to the base of the pile. Repeat dipping
of brush into test solution and scrubbing procedure until each of the
6 spots is treated. The brush dipped into the solution no more than
6 times will deliver about 15 ml of solution to the carpet. Do not exceed
this amount. Record the total volume of solution applied by spray and
brush. Allow the treated carpet piece to remain at room temperature
for 60 min. for partial drying of the treated areas.
5. While the piece of carpet treated with the test solution is drying,
spray the non-active control solution at room temperature onto half
of the other (control) piece of carpet so as to cover 3 of the 6 spots
of dried inoculum. Position the aluminum foil over the remainder. Spray
an amount equivalent to half of the amount of sprayed test solution.
Scrub the 3 wet spots in the same manner as the test carpet. The remaining
3 spots are unscrubbed controls to determine the numbers of bacteria
which survived drying of the inoculum. Care must be taken not to wet
or scrub over the unscrubbed control area. Allow the scrubbed and unscrubbed
controls to remain at room temperature for 60 min. as with the test
piece.
6. Following the 60-min. drying periods, cut each 2 x 2-in. test square
free with flamed forceps and knife. Transfer each square of carpet to
a separate extraction bottle of neutralizer broth. Shake each extraction
bottle vigorously for at least 1 min. to free the bacteria from the
carpet fibers. Determine the number of viable bacteria in each sample
bottle by plating duplicate dilutions in neutralizer plate count agar.
Similarly determine the number of viable bacteria in 0.1 ml of the suspension
used for inoculating the carpet. Also incubate all broth extraction
bottles to determine whether neutralization of the test sample was achieved.
7. Determine the percent reduction of viable bacteria by the test solution
by comparing the number of survivors from each treated test square against
the average viable count from the scrubbed control squares. An average
viable count of at least 1.0 x 106 bacteria from the extracted
unscrubbed control squares is necessary for a valid test.
Also see:
Horowitz, William, ed. 1975. Official Methods of Analysis of the Association
of Official Analytical Chemists, 12th ed. Association of Official Analytical
Chemists, Washington, D.C.
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