6.3.04 (1) Synthetic broth (SB).
—Use for (10 mL) daily transfers and
AOAC Official Method 961.02 (10 mL) test cultures for S. enterica, S. aureus, and P. aeruginosa.
Germicidal Spray Products Solution A.—Dissolve 0.05 g L-cystine, 0.37 g DL-methionine, 0.4 g
as Disinfectants L-arginine×CHCl, 0.3 g DL-histidine×HCl, 0.85 g L-lysine×HCl,
First Action 1961 0.21 g L-tyrosine, 0.5 g DL-threonine, 1.0 g DL-valine, 0.8 g
Final Action 1964 L-leucine, 0.44 g DL-isoleucine, 0.06 g glycine, 0.61 g DL-serine,
Revised First Action 2009 0.43 g DL-alanine, 1.3 g L-glutamic acid×HCl, 0.45 g L-aspartic acid,
Revised First Action 2012 0.26 g DL-phenylalanine, 0.05 g DL-tryptophan, and 0.5 g L-proline
(Applicable for testing spray and pressurized spray disinfectants in 500 mL H2O containing 18 mL 1 N NaOH.
to determine effectiveness as disinfectants for contaminated hard
Solution B.—Dissolve 3.0 g NaCl, 0.2 g KCl, 0.1 g MgSO4×7 H2O,
non p o r o u s, i n a n i mate e n v iro n men tal s u rface s . The s e
1.5 g KH2PO4, 4.0 g Na2HPO4, 0.01 g thiamine×HCl, and 0.01 g
microbiological methods are technique-sensitive in which careful
niacinamide in 500 mL H2O.
adherence to the method with identified critical control points, good
microbiological techniques, and quality control is required for Mix Solutions A and B, final pH should be 7.1 ± 0.1, dispense in
proficiency and validity of results.) 10 mL portions in 20 ´ 150 mm tubes, and steam sterilize 20 min at
Notes: (1) All manipulations of the test organism are required to 121°C. Before using for daily transfers or test cultures, aseptically
be performed in accordance with appropriate biosafety practices add 0.1 mL sterile 10% glucose (dextrose) solution per tube.
stipulated in the institutional biosafety regulations. Use the (2) Nutrient broth.—Alternatively, use for (10 mL) daily
equipment and facilities indicated for the test organism. For transfers and (10 mL) test cultures for P. aeruginosa. Boil 5 g beef
recommendations on safe handling of microorganisms, refer to the extract (Difco; paste or powder), 5 g NaCl, and 10 g peptone
CDC/NIH Biosafety in Microbiological and Biomedical (Anatone, peptic hydrolysate of pork tissues, manufactured by
Laboratories Manual. American Laboratories, Inc., Omaha, NE, USA) in 1 L H2O 20 min
(2) Disinfectants may contain a number of different active and dilute to volume with H2O; adjust to pH 6.8 ± 0.1. (If
ingredients, such as heavy metals, aldehydes, peroxides, and colorimetric method is used, adjust broth to give dark green with
phenol. Personal protective clothing or devices are recommended bromothymol blue.) Filter through paper (Whatman No. 4, or
during the handling of these items for purposes of activation, or equivalent), place 10 mL portions in 20 ´ 150 mm test tubes, and
efficacy testing. A chemical fume hood or other containment steam sterilize 20 min at 121°C.
equipment may be employed when appropriate during performing
tasks with concentrated products. The study analyst may wish to (3) Subculture media.—Use (i) or (ii).
consult the Materials Safety Data Sheet for the specific (i) Fluid thioglycolate medium USP.—Mix 0.5 g L-cystine,
product/active ingredient to determine best course of action. 0.75 g agar, 2.5 g NaCl, 5.5 g glucose×H2O, 5.0 g H2O soluble yeast
(3) References to water (H2O) mean reagent grade, except where extract, and 15.0 g pancreatic digest of casein with 1 L H2O. Heat in
otherwise specified. Use reagent grade water. Reagent grade water water bath to dissolve, add 0.5 g Na thioglycolate or 0.3 g
should be free of substances that interfere with analytical methods. thioglycolic acid, and adjust with 1 N NaOH to pH 7.1 ± 0.2. If
Any method of preparation of reagent grade water is acceptable filtration is necessary, reheat without boiling and filter hot through
provided that the requisite quality can be met. Reverse osmosis, filter paper moistened with sterile water. Add 1.0 mL freshly
distillation, and deionization in various combinations all can prepared 0.1% Na resazurin solution, transfer 20 mL portions to 38 ´
produce reagent grade water when used in the proper arrangement. 200 or 38 ´ 100 mm tubes, and steam sterilize 20 min at 121°C. Cool
See Standard Methods for the Examination of Water and Wastewater at once to 25°C and store at 20–30°C, protected from light.
for details on reagent grade water.
(ii) Letheen broth.—Dissolve 0.7 g lecithin (American Lecithin
(4) Commercial media made to conform to the specified recipes
Co., Oxford, CT, USA) and 5.0 g polysorbate 80 (Tween 80, or
may be substituted.
equivalent) in 400 mL hot water, and boil until clear. Add 600 mL
(5) Revised Salmonella nomenclature: ATCC currently lists solution of 5.0 g beef extract (Difco), 10 g peptone (Anatone),
ATCC 10708 as Salmonella enterica and cites Tindall et al. (2005) (a)(1), and 5 g NaCl in H2O and boil 10 min. Adjust with 1 N NaOH
Nomenclature and Taxonomy of the Genus Salmonella, Int. J. Syst. and/or 1 N HCl to pH 7.0 ± 0.2 and filter through coarse paper;
Evol. Microbiol. 55, 521–524, to support this revision.
transfer 20 mL portions to 38 ´ 200 or 38 ´ 100 mm tubes, and steam
(6) Methods for preparing stock cultures and test cultures of
sterilize 20 min at 121°C.
Salmonella enterica, Staphylococcus aureus, and Pseudomonas
aeruginosa are derived from the AOAC use-dilution methods: (iii) Other subculture media.—Use (2)(i) with 0.7 g lecithin
955.14 (S. enterica) (see 6.2.01), 955.15 (S. aureus) (see 6.2.04), (Alcolec Granules, American Lecithin Co.) and 5.0 g polysorbate 80
and 964.02 (P. aeruginosa) (see 6.2.06). Methods for preparing the (Tween 80, or equivalent) added; or suspend 29.8 g dehydrated fluid
stock spore suspensions and the standardized test conidial thioglycolate medium (Difco), 0.7 g lecithin, and 5.0 g polysorbate
suspension for Trichophyton mentagtrophytes are derived from 80 in 1 L H2O, and boil until solution is clear. Cool, dispense in
955.17 (see 6.3.02). 20 mL portions in 38 ´ 200 or 38 ´ 100 mm tubes, and steam sterilize
(7) Alternate organism preparation procedures may be used for 20 min at 121°C. Store at 20–30°C. Protect from light. Adjusted
test organisms not mentioned herein. concentrations of listed neutralizing constituents and/or alternate
ingredients (e.g., sodium thiosulfate) may be added to the base
A. Reagents subculture media or alternate subculture media (e.g., Dey/Engley
(a) Culture media for stock and test cultures.—All media should broth) may be used to allow for adequate test substance
be subjected to appropriate quality control practices. neutralization, as necessary.
© 2013 AOAC INTERNATIONAL
(iv) Tryptic Soy Broth (TSB).—For use in rehydrating added per plate for resuspending with subsequent aspiration. Mix
lyophilized vegetative culture of test organism. Prepare according to the pooled contents of the vessel thoroughly. Immediately after
manufacturer’s instructions. mixing, pipet approximately 1.0 mL quantities of the diluted
(v) Tryptic Soy Agar (TSA).—For use in propagation of the test suspension into cryovials (e.g., 1.5 mL). Place and store cryovials in
organism to generate frozen cultures. Prepare according to –70°C or below freezer; these are the frozen stock cultures. Each
manufacturer’s instructions. Alternatively, TSA with 5% sheep cryovial is considered as single use only. Store stock cultures up to
blood may be used. 18 months. Reinitiate stocks using a new lyophilized culture.
(vi) Glucose agar slants.—Glucose 2%, Neopeptone (BD (d) Preparation of stock culture for
Sciences, Codified Cat. No. 211681) prepared as a 1% solution, agar T. mentagtrophytes.—Initiate stock culture of fungus on glucose
2%, adjusted to pH 6.1–6.3. Sterilize at 121°C for 15 min and slant. agar slants or SDA slants; incubate 10 days at 25–30°C and store at
(vii) Sabouraud dextrose agar (SDA) slants.—Suspend 65 g 2–5°C. At intervals £3 months, transfer to fresh agar slants, incubate
Difco SDA in 1 L H2O. Heat agar media with frequent agitation and 10 days at 25–30°C, and store at 2–5°C. Do not use culture that has
boil for 1 min to completely dissolve the powder. Avoid overheating been kept at or above room temperature >10 days as source of
which could cause a softer medium. Transfer 10 mL portions to 20 ´ inoculum for culture. (Cultures may be kept at room temperature to
150 mm tubes, steam sterilize at 121°C for 15 min, and slant to cool. preserve the strain and to inoculate cultures if transferred at intervals
(viii) Glucose agar plates.—Glucose 2%, Neopeptone (BD £10 days.) Use same culture medium to prepare cultures for
Sciences, Codified Cat. No. 211681) prepared as a 1% solution, agar obtaining conidial suspension, and use fluid medium of same
2%. Steam sterilize at 121°C for 15 min. nutrient composition (without agar) to test survival and viability of
(ix) SDA plates.—Suspend 65 g Difco SDA in 1 L H2O. Heat agar conidia after exposure to fungicide.
media with frequent agitation and boil for 1 min to completely B. Apparatus
dissolve the powder. Avoid overheating which could cause a softer (a) Pipets and glassware.—(1) Volumetric pipets and
medium. Steam sterilize at 121°C for 15 min. Pour into sterile Petri volumetric flasks of various volumes for disinfectant preparation.
dishes and cool. (2) Positive displacement pipets with corresponding sterile tips
(x) Glucose broth.—Glucose 2%, Neopeptone (BD Sciences, or fixed volume pipets able to deliver 0.01 mL.
Codified Cat. No. 211681) prepared as a 1% solution, adjusted to (3) Test tubes.—For neutralizer and subculture, autoclavable 38
pH 6.1–6.3. Steam sterilize at 121°C for 15 min. ´ 100 or 38 ´ 200 mm (Bellco Glass Inc., Vineland, NJ, USA);
(xi) Sabouraud dextrose broth.—Suspend 30 g Difco Sabouraud reusable or disposable 20 ´ 150 mm (for cultures). Cap with closures
dextrose broth in 1 L H2O. Heat agar media with frequent agitation before sterilizing. Sterilize all glassware 2 h in hot air oven at 180°C
and boil for 1 min to completely dissolve the powder. Transfer or steam sterilize for a minimum of 20 min at 121°C with drying
10 mL portions to 20 ´ 150 mm tubes, steam sterilize at 121°C for cycle. Alternate, appropriate sized, autoclavable vessels with caps
15 min. may be used.
(xii) 3MTM PetrifilmTM Aerobic Count Plate.—3M Food Safety (b) Racks or other tube holding device.—Any convenient style.
(St. Paul, MN, USA; Cat. No. 6400). (c) Transfer loops or equivalent.—(1) Transfer loop.—Make
(b) Test organisms.—Obtain directly from a reputable supplier 4 mm id single loop at end of 50–75 mm (2–3 in.) Pt or Pt alloy wire
(ATCC, or equivalent). No. 23 B&S gage or 4 mm loop fused on 75 mm (3 in.) shaft
(1) Salmonella enterica.—subsp. enterica serovar Choleraesuis (available from Johnson Matthey, West Chester, PA, USA). Fit other
(ATCC 10708). end in suitable holder. Bend loop at 30° angle with stem.
(2) Staphylococcus aureus.—ATCC 6538. (2) Calibrated volumetric transfer devices may be used instead of
(3) Pseudomonas aeruginosa.—PRD 10 ATCC 15442. transfer loops.
(4) Trichophyton mentagtrophytes.—ATCC 9533. Used for (3) Purchased calibrated disposable inoculating loops may also
measuring fungicidal activity; prepare 12 slides using 0.01 mL of a be used.
standardized spore suspension, spraying and subculturing as (d) Microscope slides (carriers).—Noncorrosive, 25 ´ 25, 18 ´
described below. 36, or 25 ´ 75 mm glass slides.
(c) Preparation of frozen stock cultures for S. enterica, S. aureus, (e) Timer.—Any certified timer that can display time in seconds.
and P. aeruginosa.—Using a tube containing 5–6 mL TSB (for (f) Petri dishes.—Matted with 2 layers of S&S No. 597 or
S. aureus and P. aeruginosa) or NB (for S. enterica), aseptically Whatman No. 2, 9 cm filter paper.
withdraw 0.5 to 1.0 mL and rehydrate the lyophilized culture. (g) Tissue grinder.—Thomas Scientific, No. 3431E20, Size B, or
Aseptically transfer the entire rehydrated pellet back into the equivalent.
original tube of broth. Mix well. Incubate for 24 ± 2 h at 36 ± 1°C. (h) Bacteriological culture tubes.—Pyrex, 32 ´ 200 or 38 ×
Using a sterile spreader, inoculate a sufficient number of TSA plates 100 mm (Bellco Glass, Inc.).
(e.g., 5 to 10 plates per organism) with 100 µL each of the culture. (i) Metal forceps.—Sharp points, straight, 115 mm long, or other
Incubate plates at 36 ± 1ºC for 24 ± 2 h. Following incubation, add appropriate size.
5 mL cryoprotectant solution (TSB with 15% v/v glycerol) to the
(j) Electronic Plate Scanning Device.—3MTM PetrifilmTM Plate
surface of each agar plate. Resuspend the cells in this solution using
Reader (3M Food Safety; Cat. No. 6499, or equivalent).
a sterile spreader or a sterile swab and aspirate the cell suspension
from the surface of the agar. Transfer suspension into a sterile vessel. C. Operating Technique
Repeat by adding another 5 mL cryoprotectant to the agar plates, (a) Carrier preparation.—Visually screen carriers. Discard
resuspend the cells, aspirate suspension and pool with the initial cell carriers that are visibly damaged (scratched, chipped, or nicked).
suspension. Alternately, 10 mL cryoprotectant solution may be Prior to sterilization, carriers should be cleaned to remove oil and
© 2013 AOAC INTERNATIONAL
film. (Example: Rinse in 95% ethanol followed by a rinse with macerate with 25 mL sterile physiological saline solution (0.85%
deionized water to remove oil and film on the slides.) Place NaCl) or 0.85% saline with 0.05% Triton X, or to heat-sterilized
individual carriers in Petri dish matted with 2 pieces of 9 cm filter Erlenmeyer flask containing 25 mL sterile saline solution with glass
paper (Whatman No. 2, or equivalent). Sterilize in hot air oven 2 h at beads and shake thoroughly. Filter suspension through sterile
180°C, or steam sterilize for a minimum of 20 min at 121°C with a absorbent cotton or equivalent to remove hyphal elements. Estimate
drying cycle. density of the conidial suspension by counting in a hemacytometer
(b) Test culture preparation.—For S. aureus and S. enterica, or by direct plate count using glucose agar or SDA. Store suspension
defrost a single cryovial at room temperature and briefly vortex to at 2–10°C. This represents the stock spore suspension; it should
mix. Each cryovial should be single use only. Add 10 µL of the contain approximately 1 ´ 108 conidia/mL. Use for up to 4 weeks for
thawed frozen stock to a tube containing 10 mL synthetic broth and preparing test suspensions of conidia. Standardize test conidial
then vortex to mix. Incubate at 36 ± 1°C for 24 ± 2 h. Briefly vortex suspension as needed by diluting (using sterile saline solution) or
the 24 h culture prior to transfer. For this final subculture step, concentrating the stock spore suspension so that it contains a
inoculate a sufficient number of 20 ´ 150 mm tubes containing minimum of 5 ´ 106 conidia/mL. Add 0.02 mL Triton X-100/10 mL
10 mL synthetic broth with 10 mL per tube of the 24 h synthetic broth suspension to facilitate spreading, if previously not incorporated.
culture; incubate 48–54 h at 36 ± 1°C. Using a Vortex-style mixer, (e) Carrier inoculation.—Using a sterile capillary pipet, a
mix synthetic broth test cultures 3–4 s and let stand 10 min at room calibrated positive displacement pipet, or a 4.0 mm loop, transfer
temperature before continuing. Remove the upper portion of each 0.01 mL of the test culture or spore suspension onto approximately
culture, leaving behind any debris or clumps, and transfer to a sterile 1 sq. in. of the sterile test carrier in the Petri dish. (Note: Vortex mix
flask; pool cultures in the flask and swirl to mix. Aliquot a sufficient the inoculum periodically during inoculation of the carriers.)
volume of culture into a sterile test tube. Immediately spread the inoculum uniformly over the majority of the
(c) Test culture preparation.—For P. aeruginosa, defrost a single carrier surface using a sterile loop. Do not touch the edges of the
cryovial at room temperature and briefly vortex to mix. Each carrier. Cover dish immediately and repeat operation until a
cryovial should be single use only. Add 10 µL of the thawed frozen sufficient number of carriers have been prepared for the test,
stock to a tube containing 10 mL broth (synthetic or nutrient broth) viability controls and quantification of microbe on carriers. Once all
and then vortex to mix. Incubate at 36 ± 1ºC for 24 ± 2 h. Do not of the carriers have been inoculated, place in incubator at 36 ± 1°C
vortex the 24 h culture prior to transfer. For this final subculture step, and let dry 30–40 min. Use only visually dry carriers for testing.
inoculate a sufficient number of 20 ´ 150 mm tubes containing Notes: Use inoculated carriers for determining carrier counts, (j),
and performing efficacy testing. Use inoculated carriers within 2 h
10 mL broth (synthetic or nutrient) with 10 mL per tube of the 24 h
of drying. For general planning purposes, 60 inoculated carriers plus
broth culture; incubate 48–54 h at 36 ± 1°C. Do not shake 48–54 h
those for viability and microbe enumeration are necessary for
test culture. The pellicle from the 48–54 h cultures must be removed
determining bactericidal efficacy against one microbe and a single
from the broth either by decanting the liquid aseptically into a sterile
lot of product; 10 carriers plus those for viability and microbe
tube, by gently aspirating the broth away from the pellicle using a
enumeration are necessary for determining fungicidal efficacy of a
pipet, or by vacuum removal. Avoid harvesting pellicle from the
single lot of product.
bottom of the tube.
(f) Disinfectant sample preparation.—Ready-to-use products
Note: Any disruption of the pellicle resulting in dropping, or are tested as received; no dilution is required. For products that
breaking up of the pellicle in culture before or during its removal require dilution prior to spray application, prepare the disinfectant
renders that culture unusable in the Germicidal Spray Products as within 3 h of performing the assay unless test parameters specify
Disinfectants test. This is extremely critical because any pellicle otherwise.
fragment remaining will result in uneven clumping and layering of Aseptically prepare dilution as directed. Prepare all dilutions with
organism, allowing unfair exposure to disinfectant and causing
sterile standardized volumetric glassware. Use ³1.0 mL or 1.0 g of
false-positive results.
sample disinfectant to prepare the dilution to be tested. Use v/v
Pool the test culture from each tube and visually inspect culture dilutions for liquid products and w/v dilutions for solids. Round to 2
for pellicle fragments. Presence of pellicle in the final culture makes decimal places toward a stronger concentration. Aseptically fill
it unusable for test. spray container, then insert spray device (pump) and tighten closure.
Using a Vortex-style mixer, mix test cultures 3–4 s and let stand (g) Test procedure.—After the required drying time, the slides
10 min at room temperature before continuing. Remove the upper are sequentially sprayed in a horizontal position for a specified time
portion of each culture, leaving behind any debris or clumps, and and distance and number of pumps. Use a certified timer to time the
transfer to a sterile flask; pool cultures in the flask and swirl to mix. spray interval. Modify intervals to accommodate exposure times
Aliquot a sufficient volume of culture into a sterile test tube. other than 10 min. After spraying, maintain carriers in a horizontal
Note: For each bacterium, one daily transfer is required prior to position. Treated carriers should be kept undisturbed during the
the inoculation of a final test culture. Daily cultures may be contact time. After the carriers have been exposed to the
subcultured for up to 5 days; each daily may be used to generate a disinfectant, and the exposure time is complete, the carriers are then
test culture. transferred in a sequentially timed fashion into the subculture tubes
(d) Test culture preparation.—For T. mentagrophytes, prepare containing the appropriate neutralizer (20 mL in 38 ´ 200 or 38 ´
Petri dish cultures (5 plates) by planting inoculum from a stock 100 mm tubes). The carriers are removed from the Petri dish with
culture at the center of the glucose agar or SDA plate and incubating sterile forceps. The excess disinfectant is drained and the carrier
culture at 25–30°C for 10 to 15 days. Remove mycelial mats from transferred into the subculture tube. Carriers should be drained
surface of the 5 agar plate cultures, using a sterile spatula or similar without touching the Petri dish or filter paper. Flame forceps after
device. Transfer growth to a heat-sterilized glass tissue grinder and each carrier transfer. Alternatively, autoclaved forceps may be used.
© 2013 AOAC INTERNATIONAL
The remaining carriers are moved into their corresponding Maximum dilution of germicide which kills test organism on 10
subculture tubes at the appropriate time. As with the spray time, carriers in 10 min interval represents presumed maximum safe
transfers into subculture tubes should be within ±5 s of the actual germicidal spray product for practical disinfection.
transfer. For products with a £1 min contact time, the transfers Note: While killing in 10 of 10 replicates specified provides
should be made within ±3 s. reasonably reliable index in most cases, killing in 59 of 60 replicates
After the carrier is deposited in the subculture tube, recap the is necessary for confidence level of 95%.
subculture tube and shake culture thoroughly. Incubate tubes into 36
(j) Enumeration of viable bacteria from carriers (carrier
± 1°C for 48 ± 2 h for bacteria and 25–30°C for 10–15 days for counts).—After the carriers have dried, assay carriers in two sets of
fungus. The subculture medium (primary subculture tube) should three carriers, one set prior to conducting the tests and one set
serve as a suitable neutralizer for the test substance as well as an following the test. Place each of the inoculated, dried carriers in a 38
adequate growth medium which must be confirmed in advance or
´ 100 mm culture tube, sterile 50 mL polypropylene conical tube, or
concurrently with the germicidal spray products test.
other appropriate vessel containing 20 mL letheen broth or
Note: If a secondary subculture tube is deemed necessary to
appropriate neutralizing subculture broth as used in testing. Vortex
achieve neutralization and support growth, then transfer carrier
immediately, 60 ± 5 s for P. aeruginosa or 120 ± 5 s for S. aureus and
from the primary tube to a secondary tube of sterile medium after a
S. enterica. After vortexing, make serial 10-fold dilutions in 9 mL
minimum of 30 ± 5 min from the end of the initial transfer. Within
phosphate-buffered dilution water. If the serial dilutions are not
25–60 min of the initial transfer, transfer the carriers using sterile
made and plated immediately, keep the vortexed tubes at 2–5°C until
forceps to a second subculture tube containing 20 mL of the
this step can be done; however, dilution and plating should be
appropriate subculture medium which may contain a suitable
performed within 2 h of vortexing. Alternatively, the letheen broth
neutralizer. Move the carriers in order but the movements do not
tubes may be pooled after vortexing for each set of 3 carriers. An
have to be timed. Thoroughly shake the subculture tubes after all of
aliquot of the pooled media (60 mL) will be serially diluted and
the carriers have been transferred. Incubate both the primary and
plated, and the average carrier count per set will be calculated.
secondary subculture tubes at the appropriate time and temperature
as in the test. Record the results from both tubes (a carrier set) after For bacterial organisms, plate 0.1 mL aliquots of appropriate
this time. dilutions in duplicate on TSA or TSA with 5% sheep blood using
Report results as + (growth) or – (no growth) as determined by pour- or surface-spread plating; dilutions of 10–1 through 10–3
presence or absence of turbidity. For bacteria, growth in tubes should result in plates with a countable range of colonies. Briefly
should be checked by Gram stain to ensure that no contamination is mix each serial dilution tube on a vortex mixer prior to plating. For
present. pour-plating, add molten TSA tempered to approximately 45°C to
Note: Specialized neutralizer/subculture media such as each plate. Swirl the pour-plates to distribute cells evenly and allow
Dey/Engley broth will not show turbidity; rather, a color change to agar to solidify. Incubate plates (inverted) concurrently with the
the medium (yellow for growth of S. aureus and S. enterica) or the efficacy subculture tubes at 36 ± 1°C for up to 48 ± 2 h.
presence of pellicle at the surface of the medium (for P. aeruginosa) Alternatively, Petrifilm may be used for enumeration of bacterial
must be used to assess the results as a positive or negative outcome. organisms. Follow manufacturer’s instructions for preparation and
Use viability controls, (h) Sterility and Viability controls, for incubation of Petrifilm cards. Note: A culture purity check should be
comparative determination of a positive tube. If the product passes conducted on one dilution of one carrier. For Trichophyton
the performance standard, a minimum of 20% of the negative tubes mentagrophytes, plate 0.1 mL aliquots of appropriate dilutions in
should be assayed for the presence of the test microbe using isolation duplicate on SDA using pour- or surface-spread plating. Incubate
streaks on TSA or TSA with 5% sheep blood. carrier count plates for 44–76 h at 25–30°C. Incubate subculture
tubes for 10 days at 25–30°C.
Once the results are recorded, it is important that the carriers be
reprocessed, including visual screening, before use in another study. Count the colonies by hand or with a colony counter. Use
(h) Sterility and viability controls.—On the day of testing, place dilutions yielding counts up to 300 for enumeration; plate counts of
a sterile uninoculated carrier into a tube of the neutralizing 0 are to be included in the calculations.
subculture broth and one (or two) dried inoculated carrier(s) into Calculate the log10 density (LD) for each carrier by taking the
separate tubes of the neutralizing subculture broth (if primary and log10 of the density (per carrier). The mean LD across carriers is the
secondary media are different). Incubate tubes for 48 ± 2 h at 36 ± mean LD for the test. The mean LD must be at least 5.0
1°C. In order to validate the test system, no growth must be (corresponding to a geometric mean density of 1.0 × 105) and not
associated with the uninoculated carrier and growth must occur for above 6.5 (corresponding to a geometric mean density of 3.2 × 106)
the inoculated carrier(s). for P. aeruginosa and S. aureus; a mean LD below 5.0 or above 6.5
(i) Verification of positive carriers.—Positive carriers are invalidates the test (see retesting guidance below). For S. enterica,
examined for test organism by inoculating onto the appropriate the mean LD must be at least 4.0 (corresponding to a geometric
medium (e.g., TSA, TSA with 5% sheep blood, glucose agar, SDA mean density of 1.0 × 104) and not above 5.5 (corresponding to a
and selective media) for the test microbe. Incubate inoculated media geometric mean density of 3.2 × 105); a mean LD below 4.0 or above
as in the test. Examine plates for colonial morphology characteristic 5.5 invalidates the test (see retesting guidance below). Note: If the
to the test organism (conforming to the morphology in Bergeys Germicidal Spray Products as Disinfectants method is strictly
Manual). Bacterial growth from subculture media should be followed, mean log densities of at least 5.0 (for P. aeruginosa and
checked by Gram stain. In addition, any suitable S. aureus) and 4.0 (for S. enterica) are expected; values lower than
confirmation/identification may be done. This may include, but not these levels may be indicative of a dilution error, poor media quality,
be limited to, selected biochemical testing, manual and/or interference by environmental parameters (e.g., carrier drying and
automated identification (e.g., VITEK). culture incubation conditions), or lack of adherence to the method.
© 2013 AOAC INTERNATIONAL
The prescribed minimum count also accounts for the addition of 5% Health and Human Services, Public Health Service,
OSL to the inoculum. Centers for Disease Control and Prevention and
Note: For the purpose of achieving the carrier count range, National Institutes of Health.
dilution of the final test culture may be performed using the sterile Official Methods of Analysis (2012) 19th Ed., AOAC
culture medium used to generate the final test culture (synthetic or INTERNATIONAL, Gaithersburg, MD, Methods
nutrient broth). Dilution of the final test culture (e.g., one part 955.14 (see 6.2.01), 955.15 (see 6.2.04), 964.02 (see
culture plus one part sterile broth) should be made prior to the 6.2.06), 955.17 (see 6.3.02).
addition of the OSL to the inoculum. Concentration of the final test
culture may be necessary in the event the bacterial titer in the final J. AOAC Int. 96, 567(2013)
test cultures is too low. Concentration may be achieved using DOI: 10.5740/jaoacint.12-406
centrifugation (e.g., 5000 g for 20 min) and resuspending the pellet Additional Guidance
in the appropriate volume of the sterile final test culture medium The information provided in this section is not considered a
necessary to meet the carrier count range. In addition, the use of a component of the official test; rather it serves as procedural
spectrophotometer to measure optical density (OD at 650 nm) is guidance to augment germicidal spray product testing of specific
recommended to provide a tool (i.e., development of a standard antimicrobial products and specific test conditions as the need
curve) for assessing the need to concentrate or dilute the final test arises.
culture. Sterile broth medium should always be used to calibrate the
A. Neutralization Confirmation
spectrophotometer.
A neutralization confirmation test must be performed in advance
Retesting guidance.—For tests where the product passes and the
or in conjunction with the germicidal spray products test. Historical
TestLD value is above 6.5 for S. aureus and P. aeruginosa or above
use of neutralizer media for specific active ingredients may also be
5.5 for S. enterica, no retesting is necessary. For a test where the
taken into consideration. A neutralization confirmation procedure
product fails and the TestLD is below 5.0 for S. aureus and
must demonstrate the recovery of a low level (e.g., 10–100 CFU) of
P. aeruginosa or below 4.0 for S. enterica, no retesting is necessary.
the test organism in the subculture media. For example:
For tests where the product fails and the mean TestLD is above 6.5
(a) In a separate assay to simulate actual test conditions, expose a
for S. aureus and P. aeruginosa or above 5.5 for S. enterica, retesting
sterile carrier to the test material and transfer to subculture medium
may be conducted.
(or both primary and secondary tubes if used in the efficacy test) as
References: J. Bacteriol. 49, 526(1945). in the test procedure. Immediately following the transfer, inoculate
Am. J. Vet. Res. 9, 104(1948). the tube(s) with 10–100 CFU/tube of the specified culture and
incubate as in the test. Confirm number of cells in the suspension in
JAOAC 36, 466(1953); 44, 422(1961); duplicate by pour plate or spread plates. Count colonies on plates to
50, 763(1967); 70, 318(1987); 71, 117(1988); determine inoculum level. Examine tubes for growth. Growth in
72, 116(1989). tubes indicates effective neutralization.
Soap Chem. Spec. 38(2), 69(1962); 61, 400(1978). (b) See also Method ASTM E 1054.
ASTM International Method E 1054–Standard Test B. Hard Water
Methods for Evaluation of Inactivators of For spray product requiring hard water, see 960.09E (see 6.3.03).
Antimicrobial Agents.
C. Organic Burden
American Public Health Association, Washington, For one-step cleaner disinfectants, an organic burden is
DC, USA. incorporated into the test culture prior to carrier inoculation. Vortex
Standard Methods for the Examination of Water and mix the 48–54 h test culture. Allow culture to stand for ³10 min
Wastewater (2005) 21st Ed., American Public Health before using, and proceed as in Operating Technique, C(b)–(c). For
Association, Washington, DC, USA. a 5% preparation, pipet 19 mL culture and 1 mL organic soil/serum
Biosafety in Microbiological and Biomedical into a 25 ´ 150 mm test tube and mix.
Laboratories (2007) 5th Ed., U.S. Department of Posted: March 2013
© 2013 AOAC INTERNATIONAL
