Work Item
ASTM WK67672

Revision of E2966-14(2019) Standard Test Method for Quantitative Assessment of Sanitizing Solutions for Carpet


5.3 This test method provides for efficient recovery of surviving bacteria and fungi from inoculated carpets. 6. Apparatus 6.1 For broadloom-type carpets (typically a 3.7-m roll carpet), a 2 cm thick plywood cut (40 by 40 cm) with the same dimension tempered hardboard attached is used to mount the carpet. Brass brads are used to secure the carpet sample to the tempered hardboard. 6.2 For carpet tile with a dimensionally stable backing, no mounting board is required. 6.3 Cutting the carpet into samples can be accomplished with a traditional carpet knife or the use of mechanical cutting dies and a hydraulic press. One mechanical cutting die, 20 cm by 30 cm, and another mechanical cutting die, 5 cm by 5 cm, are used. 6.4 One-sided adhesive tabs are used to temporarily secure the precut 5-cm by 5-cm carpet carriers in plane with the remaining carpet during the scrubbing procedure. Alternately, the 90 corners of the 20 cm by 30 cm section may be nailed or tacked to the mounting board. 6.5 Spray DeviceA spray unit is used to atomize the carpet sanitizer. NOTE 1An atomizer may also be used. Aerosol formulated spray products may be directly sprayed onto the carpet. 6.6 Scrub Brushes, surgical hand brush. 6.7 Extraction Bottles, wide-mouth round 500-mL polypropylene bottles with screw caps. NOTE 2For the procedure, each bottle will contain 100 mL of sterile neutralizer broth. 6.8 Ultrasonic Bath. 6.9 Wrist-action-shaker. 6.10 Autoclave unit capable of achieving 121C 15 psi pressure. 7. Reagents and Media 7.1 Sanitizer Solutions: 7.1.1 Test a single lot of the candidate carpet sanitizer. Consult the appropriate regulatory 101 guidelines for lot replication requirements for registration purposes (for example, US EPA 810 102 Guidelines). 7.1.2 If the product is to be used as a One-step cleaner-sanitizer, a 5% soil load (for example, animal sera) may be added to the inoculum. 7.1.3 Recommended application rates (volume per unit area, that is, ml/m2) are extrapolated and reported from the proposed carpet application rate to the sample size used for this test method. 7.2 Carpet Specifications: 7.2.1 Two carpet types are to be tested. For example, carpet with nylon face fiber and carpet white polypropylene face fiber may be used for this test. If the candidate product is to be used on wool carpet, a wool carpet sample shall be included in the test. 7.2.2 The test report should indicate: face fiber composition, weight of the pile fiber (kg/m), pile density, and pile height. 7.3 Media: 7.3.1 Phosphate-buffered saline. 7.3.2 Nutrient agar. 7.3.3 Nutrient broth. 7.3.4 Potato dextrose agar or Saubarouds agar (Emmons Modified). 7.3.5 Appropriate neutralization media / technique must be selected that allows for immediate neutralization at the completion of the contact time. For example double strength neutralizer broth (Letheen broth + 0.7-g lecithin and 5-g polysorbate 80 per litre) may be used for quaternary actives. Sodium thioglycolate 0.1 %, and 0.01 % iso-octyl-phenoxy-poly ethoxyethanol, are suggested for heavy metal and halogen-based actives. Neutralizer efficacy can be confirmed using Test Method E1054. EPA 810.2000 also outlines neutralization requirements. 7.3.6 Neutralizing agar (Letheen agar) or Dey-Engley neutralizing agar. 7.3.7 Sterile deionized water. 8. Microorganisms 8.1 Staphylococcus aureus ATCC 6538. 8.2 Enterobacter aerogenes ATCC 13048. 8.3 Pseudomonas aeruginosa ATCC 15442. 8.4 Aspergillus brasiliensis ATCC 9642 or ATCC 16404. 8.5 Maintain bacterial stocks on nutrient agar slants or as frozen stocks. Bacterial stocks should be purchased from the supplier every 18 months. Stock cultures should be transferred monthly. Transfers from lyophilized stocks are limited to six before replacement is required. 8.6 Maintain mold on potato dextrose agar or as suspensions of conidia. Mold stocks should be purchased from the supplier every 18 months. Stock cultures should be transferred monthly. Transfers from lyophilized stocks are limited to six before replacement is required. 9. Inoculum Preparation 9.1 Bacteria: 9.1.1 Transfer one 4 mm loop scraping of Staphylococcus, Enterobacter and Pseudomonas bacteria from stock slants to separate 9.0 ml tubes of sterile nutrient broth. Additional test organisms may require different media or growth conditions. 9.1.2 Grow broth cultures overnight (18-24 h) at 372C. Incubate Enterobacter aerogenes at 25-302C. 9.1.3 Adjust cell density to 1-5 109 CFU/mL in sterile phosphate-buffered saline. This may be based on hemacytometer, McFarland standards, spectrophotometer or historical culture counts, or a combination thereof. Note that concentration by centrifugation may be required to achieve the above titer. 9.1.4 Add 5 % horse or fetal bovine serum to mimic soil load (if one-step cleaner and sanitizer claim is to be made) 9.2 Mold: 9.2.1 Grow Aspergillus brasiliensis for 7-14 d at 302C on potato dextrose or Sabaroud dextrose agar (modified) until mature conidia are present. Multiple plates of mature Aspergillus growth may be required along with pooled conidia suspensions from these plates to achieve the specified spore densities. 9.2.2 Harvest mature conidia using phosphate-buffered saline and gently scraping the surface with a bent glass rod. 9.2.3 Filter hyphal fragments through sterile funnels fitted with sterile glass wool or sterile gauze. 9.2.4 Standardize condia solution in phosphate buffered saline to 1-5 109 CFU/ml using a hemocytometer. 10. Procedure 10.1 Carpet Preparation: 10.1.1 Use a utility knife or mechanical die to cut the carpet into 20 cm by 30 cm samples. Two of these carpet samples should be prepared for each test substance and carpet type tested, one carpet piece for evaluating the test substance lot and one 20 cm by 30 cm carpet piece for the scrubbed and unscrubbed control solution. 10.1.2 Use a utility knife or mechanical die to cut 5 cm by 5 cm square carriers (two rows with three carrier squares per row) within the 20 cm by 30 cm rectangular piece of carpet. Space the cuts leaving approximately 10 cm between the centers of each carrier square. The test substance is evaluated on 6 carriers from a single 20 cm by 30 cm carpet piece. From a separate 20 by 30 cm carpet section, three 5 cm by 5 cm carriers are used as scrubbed controls and three 5 cm by 5 cm carriers are used as un-scrubbed controls for the inert solution. 10.1.3 Wrap the carpet in aluminum foil, autoclave for 15 min at 121C; 15 lbs/psi, and then air dry. Make sure the foil seam is at the top of the carpet for access to the samples. 10.1.4 After autoclaving, apply the adhesive tabs to the backside of the carpet to secure the smaller square carpet carriers within the larger rectangular carpet. Use a marking pen to mark the center of each small carpet carrier (carpet pile side). 10.1.5 Only carpets with no antimicrobial activity can be used. Test Method E2471 (seeded agar overlay test) may be used to document no inherent inhibitory activity. 10.2 Inoculation of Carpet: 10.2.1 Place 0.1 mL of the standardized bacterial innoculum onto the center (previously marked) of each 5 cm by 5 cm carpet square. 10.2.2 Allow inoculated carpet to dry for 1 h 2 min in a 35-37 2C incubator with foil cover loosely in place. 10.2.3 Inoculate the control solution carpet sample in the same manner as described in 10.2.1. 10.2.4 Determine 0-hr cell density by performing serial dilution and plating of the inoculum. 10.3 Application of Sanitizer: 10.3.1 Calculate the intended application rate of the carpet sanitizer per square meter and extrapolate the amount of material to be applied to a 600 cm2 test piece of carpet. 10.3.2 Use the spray device unit to apply the sanitizer uniformly to the inoculated carpet. Alternately, glass chromatography spayers may be used to apply the product in a manner consistent with intended application and dosing rate. 10.3.3 Dip the surgical hand brush into fresh sanitizer and scrub the first inoculated square of carpet with a clockwise motion for 30 s making 30 clockwise and 30 counter-clockwise passes with moderate pressure (slight bend of brush bristles). Repeat this process until all six inoculated samples have been scrubbed. Report the actual total sanitizer volume applied to the carpet by calculating the liquid holding capacity of the scrub brush. This can be accomplished by dipping and tapping the brush (five replicates) into a weigh boat and calculating based on mass of the liquid. 10.3.4 Allow the carpet with applied sanitizer to remain at room temperature for 60 min. 10.4 Control Carpet: 10.4.1 The following steps can be performed during the 60-min contact period for the sanitizer-treated carpet. 10.4.2 Apply the inert control solution (sterile phosphate-buffered saline) to three of the six inoculated squares on the control carpet. Use half the volume calculated for the treated samples. Scrub three of the six inoculated control carrier samples as described in 10.3.3 but with brush dipped into the inert control solution. Do not scrub three of the carriers designated as unscrubbed population controls. 10.4.3 Allow the scrubbed and unscrubbed control carriers to stand at room temperature for 60 1 min. 10.5 Recovery of Viable Cells from Carpet: 10.5.1 After the 601 min contact period, use sterile forceps or hemostats or both to lift the small carpet carrier samples from the larger carpet field. 10.5.2 Transfer each carpet carrier sample to separate extraction bottles containing 100 ml of neutralizing broth. 10.5.3 Place the bottles with neutralizing solution and carpet into a sonic bath for 1 min 10 s. The fluid level in the sonic bath should equal the fluid level in the recovery bottle when immersed. 10.5.4 After sonication, pat bottles dry with paper towels and mount them onto a wrist-action shaker. Shake the bottles at maximum setting for 1 min 10 seconds. Alternately, manually shake the bottles for the same time period. 10.5.5 Perform serial dilutions from each neutralizing solution bottle. Plate each serial dilution in duplicate (pour plates or spread plates for bacteria, spread plates for mold) and incubate as described in sections 9.1.2 or 9.2.1. 11. Controls 11.1 Sterility controlsIncubate alongside the test as least one tube or plate of each type of broth or agar media used in the study. Where soil load was used, add 1 ml of soil to 20 ml of growth media and incubate alongside the test. Where a neutralization solution was used add 1 mL it to 20 mL of growth media and incubate alongside the test. These sterility controls should not demonstrate growth. 11.2 Neutralization ControlTo confirm neutralization of the test substance, conduct the test using sterile carpet carriers. Use sufficient carriers to evaluate each lot of test substance and each test organism. After the contact time has elapsed, transfer the carriers to the neutralizer/growth media as in the test. Inoculate the neutralizer carrier tubes with 100 CFU/ml media. To ensure the target level of bacteria/media volume is achieved, it may be necessary to test several dilutions of a test organism. Mix the tubes, plate 1 mL aliquots in duplicate from each tube, and incubate alongside the test. Plate 1 mL of each inocula level to confirm the inoculation density of each organism or dilution of organism evaluated, or both. Neutralization has been achieved where the neutralized test substance plate counts fall within 0.51 log of the inoculum density. 12. Calculation 12.1 Use the following formula to determine the log reduction of the microbial population from the sanitized carpet when compared to the set of scrubbed control carpet samples. 12.1.1 Log Reduction Formula: Determine log (x * 10a ) of control samples. Determine log (x * 10a ) of treated samples Determine geometric mean of control samples: (1) Log values of control samples: b1, b2, b3, bn. (2) Mean = (b1 + b2 + b3bn)/n. Determine geometric mean of treated samples: (1) Log values of treated samples: c1, c2, c3, cn. (2) Mean = (c1 + c2 + c3 +cn)/n. where: x=Value of samples, a=Exponent value, b=Log value of control samples, c=Log value of treated samples, and n=Number of log values in set. 13. Report 13.1 Report the intended application rate of the carpet sanitizer (mL/m2 ). 13.2 Report the volume (sprayed plus brush volume) and application method of sanitizer applied. 13.3 Report the presence or absence of organic load in the inoculum. 13.4 Report the log reduction of the sanitized versus scrubbed control carpet. 14. Precision and Bias 14.1 PrecisionThe repeatability standard deviation from a single operator has been determined to be 0.14 (0.0943 for 95 % confidence limit) for scrubbed control carpet samples, 0.22 (0.178 for 95 % confidence limit) for unscrubbed control carpet samples, and 0.23 (0.152 for 95 % confidence limit) for the sanitizer treated carpet samples. An interlaboratory study of this test method is being planned and a complete precision statement is expected to be available on or before Dec. 31, 2021. 14.2 BiasNo information can be presented on the bias of the procedure in Test Method E2966 for measuring the bacterial log reduction of the sanitized versus control carpet because no material having an accepted reference value is available.


Developed by Subcommittee: E35.15

Committee: E35

Staff Manager: Brian Milewski

Work Item Status

Date Initiated: 04-02-2019

Technical Contact: Daniel Price

Item: 001

Ballot: E35.15 (19-02)

Status: Will Reballot Item

Item: 004

Ballot: E35 (19-03)

Status: Will Reballot Item

Item: 003

Ballot: E35 (20-02)

Status: Will Reballot Item

Item: 004

Ballot: E35 (20-06)

Status: Will Reballot Item