FINAL TECHNICAL REPORT
DISINFECTANT TEST METHOD UPDATE
COOPERATIVE AGREEMENT CR 811204
THE UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF PESTICIDE PROGRAMS
WASHINGTON, D.C. 20460
AND
THE UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILL
DIVISION OF INFECTIOUS DISEASES
CHAPEL HILL, N.C. 27514
OCTOBER 23, 1983-XTOBER 23, 1985
U.S. Environmental Protection Agency
library, Room 2f04 FH-211-A
401 M Street, S.W.
Washington, DC 20460
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Table of Contents
Page
I. BACKGROUND AND OBJECTIVES 4
II. RECOMMENDATIONS 5
A. Test Organisms 5
B. Culture Broth 5
C. Carrier 6
D. Neutralization/Recovery .. 6
3. Phenol Resistance 7
F. Good Laboratory Practices 7
G. Pass/Fail Criteria 8
III. PROPOSED REVISED USE-DILUTION METHOD 9
IV. LABORATORY EXAMINATIONS OF DEFICIENCIES 19
A. Evaluation of Initial Broth Inoculum 20
B. Removal of Bacteria from Penicylinders 23
C. Growth Curves and Inoculation of Penicylinders .... 26
D. Bacterial Attachment and Surface Texture 29
E. Penicylinder Drying Conditions . 45
F. Wash-Off from Penicylinders 48
G. Stainless Steel Penicylinders 51
H. Consistency of S&L Penicylinders 59
IP Pseudomonas Pellicle Removal .....67
J. Pseudomonas Pellicle Reduction 71
K. Electron Microscopy of Pseudomonas Pellicle 75
L. Standardization of Bacterial Numbers 76
M. Twenty-four Hour Broth Cultures 87
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Page
N. Evaluation of Asparagine 101
0. Analysis of Test Bacteria 116
P. Evaluation of Letheen Broth (Difco) 128
Q. Phenol Resistance Testing 134
R. Use-Dilution Method Statistics 139
S. Use-Dilution Collaborative Study 144
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Principal Investigators and Key Personnel
William A. Rutala, Ph.D, M.P.H.
Research Associate Professor
Division of Infectious Diseases
University of North Carolina
School of Medicine
Chapel Hill, NC 27514
Phone: (919) 966-2536
Eugene C. Cole, Dr.P.M.
Research Associate
Division of Infectious Diseases
The University of North Carolina
School of Medicine
Chapef Hi I I, NC 27514
Phone: (919) 966-2536
Edith M. Alfano, M.S.P.H.
Research Assistant
Division of Infectious Diseases
The University of North Carolina
School of Medicine
Chapel H? I I, NC 27514
Phone: (919) 966-2536
Gregory P. Samsa, M.S.
Biostati stictan
Division of Infectious Diseases
The University of North Carolina
School of Medicine
Chapef Hill, NC 27514
Phone: (919) 966-2536
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Background
The Association of Official Analytical Chemists (AOAC) Use-Dilution
Method has been the official methodology for evaluating the germicidal
activity of hospital disinfectants. For many years the Environmental
Protection Agency (EPA) performed intramural pre- and post-registration
efficacy testing of chemical disinfectants. In 1982 this was curtailed,
presumably for budgetary reasons. Thus, manufacturers presently do not
need verification of efficacy claims by the EPA or an independent
testing laboratory when registering a disinfectant.
In the past five years, there have been many criticisms of the test
method and several federal, commercial, state and university
laboratories performing the test have been unable to substantiate the
manufacturers' bactericidal claims. While this may be attributed to
intrinsic deficiencies of some disinfectants, there is concern that the
p rob I err may be test methodology. At least seventeen preseumptive
inadequacies of deficiencies have been identified in the Use-Dilution
Method.
Object i ves
This investigation was undertaken to investigate (a) presumptive
deficiencies in the AOAC Use-Dilution Method, and (b) variability among
disinfectant testing laboratories and EPA registered disinfectants using
the current methodology.
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Recommendations
The following recommendations for the revision of the AOAC Use-Dilution
Method are based primarily on the results of laboratory investigation
conducted under Cooperative Agreement CR 811204 between the United
States Environmental Protection Agency and the University of North
Carolina at Chapel Hi If. Recommendations of the Steering Committee of
the AOAC Use-Dilution Task Force were also considered.
Test Organisms
Purchase test bacteria directly from the American Type Culture
Collection (ATCC) on an annual basis.
Use ATCC method of storing reconstituted bacteria, i.e., pellet 48h
broth culture, resuspend in fresh broth, add equal amount of 2Q%
glycerol, aliquot, and store at -70.0 degrees C.
Prior to using broth cultures, streak on plates of tryptic soy agar
with 5% sheep blood, incubate overnight at 35-37 degrees C, and
examine colonial morphology for _S_. choteraesuis ATCC 10708 and P_.
aeruginosa, ATCC 15442 and pigmentation for S_. aureus ATCC 6538. If
the gram-negative organisms display both colonial variants (rough
and smooth), it may be necessary to use only one type for
disinfectant testing. Yellow pigment colonies of S_. aureus are
preferable for use.
No quantitated inoculum
culture broths.
is necessary for the initial inocluation of
5. Organism counts on inoculated carriers should not be required,
Culture Broth
1. Use 22-26 broth cultures of each test strain. No other
standardization is necessary.
2. Use only cultures having undergone 3-12 daily transfers beyond
initial broth inoculation.
3. Nutrient broth (AOAC 4.001a) is recommended for S^ choteraesuis and
P. aeruginosa, while Synthetic Broth (Dtfco) is recommended for ^.
aureus.
4. Remove Pseudomonas pellicle by vacuum suction with steri(e Pasteur
pipet, then decant broth into another tube for penicylinder
inocuI at ion.
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Carrier
2.
3.
Only one brand of stainless stee! pen icy Under should be used (S & L
Metal Products, Corp., Maspeth, NY). It is recommended however that
other carrier types or brands may be approved for use following
comparative inter laboratory studies with at least three
laboratories. It is recommended that the newly developed Colgate
planchet be so evaluated.
After carrier inoculation, drying time should be 60 minutes.
Carriers that are visibly imperfect (gouged, dented, etc.) must not
be used.
4. Biologically screen carriers before use. Perform Use-Dilution
testing with S. aureus ATCC 6538 and 500 ppm a Ikyl/dimethyl/ammoniurn
choloride, alkyl chain distribution of C14, 50%; C12, 40£; C16, 10$
(e.g., Onyx 8TC-835). Do not use those carriers giving positive
resu Its.
5. Include sonic cleaning (at least 10 minutes) as part of carrier
prepart ion.
6. Substitute sterile, glass distilled water for asparagine.
7. For carrier inocufation, remove distilled water from tube of sterile
carriers and aseptically transfer carriers to another sterile tube
to which the correct amount of thoroughly mixed broth culture is
added.
Neutra t i zat ion/Recovery
1. Letheen Broth (Difco) should be included as the subculture medium
for quaternary ammonium and phenolic disinfectants.
2. Eliminate secondary subcultures for quaternary ammonium and phenolic
d i sinfectants.
3. For classes of disinfectants other than phenolic and quaternary
ammonium compounds, tests should be included in the revised method
to confirm effective neutralization and the absence of
bacteriostasis.
4. Continue research to identify suitable subculture media for use with
the other classes of disinfectants.
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Phenol Resistance
Retain the loop method, but require three replicates, at least one
of which must yield the acceptable pattern of resistance in order to
validate final use-dilution results.
Use only rough colonies for testing S^. choIeraesuis and P_.
aerugi nosa.
Good Laboratory Practices
1. Retain phenol as the resistance standard.
2. Require a one-year expiration date for all degradeable test
components (media, reagents, organisms).
3. Require the use of glass distilled water (or equivalent) for all
aspects of testing (media and reagent preparation, disinfectant
d i tut ton, etc.).
4. Require glassware cleaning with non-ionic detergent followed by a
rinsing scheme that meets or exceeds APHA requirements (6-12
successive rinsings to remove detergent residue and insure freedom
from residual bacteriostatic action) in Standard Methods for the
Examination of Water and Wastewater, 15th ed.
5. Allow variability in test temperature: _+1.0 degrees C (incubation),
and +0.5 degrees C (testing).
6. Require all thermometers be calibrated with an NBS certified
thermometer.
7. Confirm identification of growth from penicylinder tubes by agar
plating.
8. Require dilution of test disinfectants in Class A volumetric flasks
on I y.
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Pass/FaiI Criteria
For statistical and economic reasons, a sequential testing scheme with
revised pass/fail criteria is recommended following standardization of
the method and after product and laboratory variability have been
control led.
Method Revision
A revision of the AOAC Use-Dilution Method (attached) is currently being
evaluated by the AOAC Use-Dilution Task Force. This revision
incorporates the aforementioned test recommendations and data.
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Use-Dilution Methods
Testing Disinfectants Against
Salmonella choleraesuis
Use-Dilution Method
Interim Official First Action
(Applicable to testing disinfectants miscible with H-0 to determine the
effectiveness of a given bactericidal concentration using standard test
strains under controlled conditions. Test results may not necessarily reflect
a product's efficacy as used on a variety of inanimate surfaces or within
specified environments).
4.007
Culture Media
(a) Nutrient broth. Dissolve 5g beef extract (Difco, 1 Ib. jar only), 5g
NaCl, and lOg peptone (Anatone, peptic hydrolysate of pork tissue,
manufactured by American Laboratories, Inc., 4410 South 102nd St., Omaha,
NB 68127) in 1L glass distilled H20 and adjust to pH 6.8. Place 10 mL
portions in 20 x 150mm tubes and sterilize in autoclave 15 min. at 121°.
Use this broth for daily transfers of test cultures. Use as subculture
medium when testing alcohols, aldehydes, iodophors and hypochlorites.
(b) Letheen broth. See 4.001(d)3 or Letheen Broth (Difco). Prepare according
to manufacturer's directions, dispense in 10 mL amounts into 20 x 150mm
test tubes and sterilize in autoclave 15 min. at 121°. Use for
subcultures when testing quaternary ammonium, phenolic, and pine
disinfectants.
(c) Fluid thioglycolate medium. See 4.001(d)2 or Bacto Fluid Thioglycolate
Medium (Difco). Prepare according to manufacterer's Instructions,
transfer lOmL portions to 20 x 150mm tubes, and autoclave 15 min at 121°.
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10
Cool to room temperature and store at 20-30°, protected from light. Use
for subcultures when testing mercurials.
(d) Pour plate agar. Plate Count Agar (Difco). Prepare according to
manufacturer's directions in bottles or tubes. Sterilize at 121° for 15
miri. , place in 43-45 environment and allow to come to temperature before
use. Alternatively, 20 ml may be poured into 15 x 100 mm sterile petri
dishes for spread plate method.
4.008
Test Organism
Salmonella choleraesuis ATCC 10708. Obtain directly from American Type
Culture Collection, 12301 Parklawn Dr., Rockville, MD 20852. Reconstitute
using Nutrient: broth. After overnite incubation, streak on plate of
Trypticase Soy Agar with 5% sheep blood, incubate overnite at 35-37° and
examine for rough and smooth colonies. If both types are observed, select the
rough variant for storage and testing. To prepare for storage, make 3
successive 24hr transfers using 4mm id loop. Spin broth tubes to pellet
cells, pour off supernatant, replace with fresh nutrient broth, resuspend
cells, add equal amount 20% glycerol, aliquot into sterile vials (or inoculate
sterile glass beads) and freeze at 70 or in liquid nitrogen. Thaw vials at
room temperature when used. From frozen stock culture inoculate tube of
nutrient broth and make at least 3 subsequent consecutive daily transfers (<
12), incubating at 36°+1.0°, before using culture for testing. Use 48hr +3
culture of organism in test. Swirl or vortex before using.
4.009
Reagents
(a) Phenol solution 5%. See 4.002(f). Make fresh or retiter annually. Store
in well stoppered amber bottle protected from light, at 20-25°.
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11
(b) Glass distilled water. For reagent and media preparation. Reagent water
Type II as defined by the American Society for Testing and Materials
(ASTM): Type II grade of reagent water shall be prepared by distillation
using a still designed to produce a distillate having a conductivity of
less than 1,0 umho/cm at 298K(25°). Ion exchange distillation or reverse
osmosis may be required as an initial treatment prior to distillation if
the purity cannot be attained by single distillation. Measure
conductivity after H.,0 is freshly prepared (^4hr).For sterile glass dist
H_0 prep stock supply by placing freshly prepared H-O in capped bottles or
flasks and sterilize 20 rain at 121°. Use to prepare dilns of germicides
and to cover carriers.
(c) Sodium hydroxide solution. Approximately 1N(4%). For cleaning metal
carriers before use.
(d) Phenolphthalein solution. See 11.047(d).
(e) Phosphate buffer dilution water (PBDW). See A.020(f).
4.010
Apparatus
(a) Glassware. Serological pipets or volumetric pipets, reusable or
disposable; 250, 500, or 1000 ml vol flask (class A) for germicide
dilution; 20 x 150mm, 25 x 150mm borosilicate tubes; 15 or 20 x 100mm
glass (Pyrex) petri dishes; Pasteur pipets, 15cm. Sterilize by dry heat
at 170 for 60 min (loose glassware), 170° for 2 hr (for glassware in
metal containers), or steam (121 20 min. with dry cycle). Clean
glassware manually or automatically with suitable laboratory detergent and
hot H20, followed by hot HjO rinsing then a distilled H.O rinse. Perform
APHA test for inhibitory residues on glassware using the 3 Use-Dilution
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12
Method test strains, initially and every 6 mos thereafter, or when
cleaning agents change.
(b) Water baths. Constant temperature, capable of uniformly maintaining
20°+0.5° and holding rack with at least 20 holes for 25 x 150mm tubes.
(c) Racks. Any convenient style, to hold 20 x 150mm and 25 x 150mm tubes.
(d) Wire loop., For broth culture transfer and phenol resistance testing. 95%
platinum, 3.5% rhodium alloy, 4mm id loop; B&S 0.19mm dia., 75mm shank.
(Obtain from American Scientific Products).
(e) Wire hook,. For carrier transfer. Make 3-5mm bend (approx 60 ) at end of
suitable nichrome wire in appropriate holder.
(f) Petri dishes. (1) See A.OlO(a). Prep for use in drying carriers. Place
2 layers of Whatman No. 2, 9cm filter paper in ea dish. Autoclave 20 min.
at 121° with dry cycle. (2) Polystyrene, 15 X 100mm, sterile.
(g) Thermometer. Subdivided to 1/10°, with manufacturer's certification of
traceability to NBS. Use for calibration of thermometers used to monitor
laboratory equipment.
(h) Carriers. Polished stainless steel penicylinders, length 10mm, 6mra id,
8mm od type 304 stainless steel. (Obtain from S&L Metal Products Corp.,
58-29 57th Dr., Maspeth, NY 11378). Discard carriers that are visibly
unacceptable (chipped, dented, gouged, etc.) Biologically screen those
remaining. See 4.011(b).
4.011
Operating Technique
(a) Carrier preparation. Place new or used penicylinders one layer deep in
distilled H-° °r detergent solution in sonic cleanser bath for at least 10
min, rinse 2 X in distilled HO, and place overnight in IN NaOH. Rinse
with tap H20 until rinse H20 is neutral to phthln, then rinse 2 X with
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13
distilled HO. Place cleaned carriers (22 or 33) in tubes or flasks,
cover with glass distilled H-O and sterilize 20 min at 121 . Let cool and
store at room temperature.
(b) Carrier screening. Test visibly acceptable new or previously unscreened
old penicylinders by performing use-dilution method with S. aureus ATCC
6538 and 500 ppm alkyl/dimethyl/ammonium chloride, alkyl chain
distribution C14, 50%; C12, 40%,; C16, 10% (e.g. BTC-835, Onyx Chemical
Co., Jersey City, NJ 07302). Do not use carriers giving initial positive
results. Postive carriers may be re—screened at any time and used for
testing if found negative. Discard carriers that consistently (3-4x)
fail.
(c) Carrier inoculation. Decant sterile H^O from previously screened and
prepared oenicylinders, remove remainder on bottom with pipet. Using
pipet, add equivalent no. of mL of thoroughly mixed culture broth as no.
of penicylinders. Cap tubes or bottles, and allow to remain at room
temperature for 15 min. Using flamed, then cooled hook remove each
carrier from broth, taking care to not allow carriers to touch sides of
tube, and place on end on filter paper in previously prepared dishes,
4.010(f). Insure that carriers are well drained with no bubble or pool at
base of cylinder. Insert flamed needle through center of cylinder to
disperse bubble. Each dish must contain 11 carriers spaced to avoid
touching. Cover dishes and transport to incubator taking care to not
allow carriers to tip over. Carriers that fall over may not be used for
testing. Dry in incubator (without humidification system) at 36°+1.0 for
40 min. The llth cylinder in each plate serves as an extra if needed.
(d) Germicide preparation. Make stock solution of germicide to be tested in
sterile, class A vol flask. Prepare appropriate concentration according
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14
to manufacturer's recommendation by dilution in sterile glass distilled
H20, 4.009(c). From this solution dispense lOmL amounts into 20 or 30, 20
x 150mm tubes. Place tubes in H?0 bath at 20°+0.5 and let come to
temperature (at least 5 min.).
(e) Carrier exposure. Add one contaminated dried cylinder to germicide tube
every 30 seconds if testing 20 carriers and every 20 seconds if testing
30. Donot swirl or shake tubes after adding carrier. Start timer when
first carrier is placed into solution. At exactly 10 min begin extracting
carriers (every 20 or 30 sec) in the order exposed using one or more
flamed hooks. Place each carrier into a tube of appropriate subculture
broth. Shake well and incubate all subculture tubes at 36°+1.0 for at
least 48hr and observe for growth or no growth. Confirm growth as test
organism by culture or gram stain.
(f) Secondary subculture. When desired, and particularly when testing
germicides with coned acids and alkalies, vortex primary subculture tube
with carrier 15 sec then with sterile pipet transfer 1.0 ml to secondary
subculture tube within 60 min of initial transfer and incubate both
primary and secondary subculture tubes at 36°+1.0 for at least 48hr.
(g) Neutralization confirmation. The absence of residual effects of a test
germicide in the subculture medium must be assured. Randomly select one
negative tube for each 10 tubes tested and to each tube add the
appropriate amount of 18-48hr test strain broth culture in diluted PBDW to
deliver 5-100 cells per tube. Incubate at 36°+1.0 Confirm number of
cells added by pour plate method (inoculate 15 x 100mm sterile petri
dishes and overlay with 15-20ml Pour Plate Agar) or use spread plate
technique with glass rod. Incubate at 36°+1.0 for at least 48hr. Count
colonies on plates to determine size of inoculum (at least 5 cells on each
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15
plate for valid results), and examine tubes for growth. Growth in all
tubes indicates effective germicide neutralization. Absence of growth in
one or more tubes indicates presence of residual germicide and
use-dilution test results are invalid. When using secondary subcultures,
only the secondary tubes must be tested for adequate neutralization. If
same lot of disinfectant is tested repeatedly, only one neutralization
confirmation is necessary.
(h) Phenol resistance. From 5% phenol soln (1:20) 4.002(f) make 1:90 and
1:100 dilns. Place 5mL of each dilution into 25 x 100mm tubes. Place
tubes in 20°+0.5 H20 bath and let come to temp (at least 5 rain.). Add 0.5
mL of test: culture suspension to 1:90 diln; after 20 or 30 sec interval,
add 0.5 mL to 1:100 diln. After adding culture, swirl tubes to distribute
bacteria, and replace in bath; 5 min after seeding first tube, aseptically
transfer one loopful (4mm id loop) of mixture from that tube to a
corresponding subculture tube. After 30 sec transfer loopful from 1:100
tube to another subculture tube; 5 min after making first set of transfers
begin second set of transfers for 10 min period. Repeat for 15 min
period. Incubate subculture tubes at 36 +1.0 for 48h and observe for
growth or no growth. S_. choleraesuis ATCC 10708 must demonstrate at least
the following resistance to phenol in at least one of 3 replicate
determinations on the day of testing in order to validate results.
Phenol
1:90
1: 100
5 min.
+ or 0
10 min.
+ or 0
15 min.
0
+ or 0
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4.012
Testing Disinfectant against
Staphylococcus aureus
Use-Dilution Method
Interim Official First Action
Determination
(a) Test organism. Staphylococcus aureus ATCC 6538. Obtain directly from
American Type Culture Collection. Reconstitute with synthetic broth.
Proceed as in 4.008 using synthetic broth, 4.012(b). Following
reconstitution of original lyophilized sample, streak on plate of
Trypticase Soy Agar with 5% sheep blood or other suitable medium and
incubate at 35-37° for 18hr. Select yellow pigraented colonies only for
transfer, storage and subsequent testing.
(b) Synthetic broth. Bacto Synthetic Broth AOAC (Difco). Prepare according
to directions dispensing exactly lOmL amts into 20 x 150mm tubes prior to
sterilization.
(c) Operating technique. Before using broth culture to inoculate
penicylinders, decant into clean, sterile tubes or bottles to eliminate
residue on bottom. Swirl or vortex before using. Proceed as in 4.011
except for phenol dilutions and minimum resistance requirements which are
as follows:
Phenol
1:60
1:70
5 rain.
+ or 0
10 rain.
+ or 0
15 rain.
0
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17
Testing Disinfectants Against
Pseudomonas aeruginosa
Use-Dilution Method
Interim Official First Action
(a) Test organism. Pseudomonas aeruginosa ATCC 15442. Obtain directly from
American Type Culture Collection. Reconstitute with nutrient broth
(4.007a). After overnite incubation, streak on plate of Trypticase Soy
Agar with 5% sheep blood or other suitable medium and incubate overnite at
35-37 and examine for rough and smooth colonies. If both types are
observed, select the smooth variant for storage and testing. Proceed as in
4.008, taking care to not disturb pellicle. When making daily transfers
tilt tube to retract pellicle before inserting loop. When preparing final
test broth, remove pellicle using sterile Pasteur pipet attached to vacuum
source, then decant into other, clear, sterile tube or bottle; swirl or
vortex before using.
(b) Operating technique. Proceed as in 4.011 except for phenol dilutions and
minimum resistance requirements which are as follows:
Phenol
1:80
1:90
5 min.
+ or 0
10 min.
+ or 0
15 min.
0
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18
Changes IncIuded In Draft of Rev1sed Use-DlIutIon Method
1. Specified broth medium for each bacterial test strain.
2. Subculture media specified for disinfectant classes. Letheen Broth
(Dlfco) nay be used. Incubation of subculture broths for "at least" 48h.
3. No more than 15 consecutive dally broth transfers.
4. Required use of Beef Extract (Dlfco) In I Ib. jar only.
5. Test strains to be acquired directly from ATCC.
6. Use 18-24h broth cultures rather than 48-54 h.
7. Use of Nutrient agar for Pseudomonas stock cultures.
8. Transfer cultures dally by using 4mm Id loop or lOul pipette.
9. Substitution of asparaglne with distilled water for storage of carriers.
10. Distilled water must be "glass" distilled and meet specifications for ASTM
Type II Reagent water. Water may be lab distilled or commercially
prepared.
11. Use of class A volumetric flasks for germicide dilution on v/v basis.
12. Inclusion of glassware cleaning method and test for detergent residue.
13. Identification of organism In positive tubes.
14. Only one carrier authorized (S&L).
15. Elimination of swirling tubes after carrier exposure to disinfectant.
16. Visible screening of carriers before use.
17. Biological screening of test carriers required.
18. Only one brand and type of filter paper specified for use In drying
carriers.
19. Drying of carriers In non-humidified Incubator for 40 mln.
20. Test required to confirm neutralization.
21. Two determinations confirming minimum resistance to phenol required during
life of broth cultures (_<15 days).
22. Clumpy J5. jujreus cultures may not be used.
23. Removal of Pseudomonas pellicle by suction method.
24. Sonic cleaning of carriers required.
25. Calibration of thermometers with NBS traceable one.
26. Methods for preparing phenolphthaleln solution, phosphate buffer dilution
water, and pour ptate agar Included.
27. Phenol solution (5%) - prepare fresh or retlter annually.
28. Carriers may be tested In batches of 20 or 30.
29. SARC device permitted.
30. Use of boroslllcate glass tubes.
31. New secondary subculture procedures.
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19
Laboratory Examinations of Presumed Deficiencies
Included in this Report
1. Evaluation of initial broth inoculum.
2. Removal of attached test bacteria from stainless steel
penicylinders.
3. Bacterial growth curves and the inoculation of stainless steel
penicylinders.
4. Evaluation of bacterial challenge and surface texture of
penicylinders.
5. Penicylinder drying conditions.
6. Bacterial wash-off from stainless steel penicylinders.
7. Tn-use comparison of stainless steel penicylinders.
8. Consistency of manufacture of S & L penicylinders.
9. Comparison of Pseudomonas pellicle removal methods.
10. Alteration of broth medium to effect reduction or inhibition of
Pseudomonas pellicle.
11. Electron microscopy of Pseudomonas pellicle.
12. Standardization of bacterial numbers on disinfectant test
penicylinders: Interlaboratory Study.
13. Bacterial numbers on penicylinders: Use of 24-hour standardized
broth cultures.
14. Evaluation of asparagine.
15. Analysis of AOAC recommended test bacteria.
16. Evaluation of commercial letheen broth for inactivation of hospital
disinfectants.
17. Phenol resistance testing: Loop vs. micropipette.
IB. Use-Dilution Method statistics.
19. Collaborative study of the AOAC Use-Dilution Method to assess
interlaboratory variability of results and set specifications for
pass/fai1.
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Evaluation of Initial Broth Inoculum
In the Use-Dilution Method
BACKGROUND: The current Use-Dilution Method does not require a
quantitated inoculum when transferring organisms from the
stock culture slants to the culture broth.
PURPOSE: To determine whether variations >n initial inoculum will
eventually be reflected in the numbers of organisms fn the
broth used to inoculate test pentcyIinders.
METHODOLOGY: 1,
For each of the 3 AOAC Use-Dilution test strains, 2
tubes (20 x 150mm) of nutrient broth AOAC 4.001) are
inoculated — 1 lightly (inoculum gathered by barely
touching edge of loop to slant growth) and 1 heavily
(inoculum gathered by raking loop through slant growth
several times). Incubate tubes at 37°C.
2. Transfer growth 6 times at 24 hour Intervals using 4mm
id loop and incubating at 37°C.
3. Remove five 1 ml aliquots from both lightly and
heavily inoculated broth for serial dilutions at 24
hours and 4 aliquots at 48 hours. Perform dilutions
in phosphate buffer dilution water {AOAC 4.020).
Plate 10 dilution in duplicate using pour plate
method and Difco Plate Count Agar. Incubate plates at
37°C for 48 hours.
4. Count plates with colony range between 30 and 300.
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RESULTS:
21
Salmonella choferaesuis 10708
24 hour
Light
6.2 x 10
8
6.7 x 10
8
6.4 x 10
8
7.2 x 10
8
6.0 x 10
8
x=6.5 x 10
8
Heav\
7.4 x 10
8
8.6 x 10
8
7.3 x 10
8
6.9 x 10
8
7.6 x 10
8
7.6 x 10
8
Light
7.6 x 10
8
7.1 x 10
8
6.6 x 10
8
6.5 x 10
8
7.0 x 10
8
48 hour
HeavN
6.4 x 10
8
6.4 x 10
8
6.3 x 10
8
7.5 x 10
8
6.7 x 10
8
P s e udomon a s aer iig j nosa 15442
24 hour
Light
9.9 x 10
8
8
9.6 x 10
1.0 x 10"
1.1 x 10'
1.1 x 10'
x=1.0 x 10'
Heav\
1.0 x 10'
1.0 x 10'
1.0 x 10'
9.6 x 10
8
9.2 x 10
8
9.8 x 10
8
Light
1.3 x 10'
1.2 x 10'
1.1 x 10'
1.3 x 10'
1.2 x 10'
48 hour
Heavv
1.2 x 10'
1.3 x 10'
1.3 x 10'
1.2 x 10'
1.3 x 10'
-------
22
Staphylococcus aureus 6538
24 hour
Light
3.9 x 13
8
3.2 x 10C
3.6 x 10
8
3.5 x ID
8
3.6 x 10
8
x=3.6 x 10
8
Heavv
3.3 x 10
3.5 x 10Q
8
2.6 x 10
8
2.5 x 10
8
2.6 x 10
8
2.9 x 10
8
Light
5.6 x 10
8
7.0 x 10
8
6.5 x 10
8
6.3 x 10
8
6.4 x 10
8
48 hour
Heavv
7.8 x 10
8
6.2 x 10
8
6.2 x 10
8
7.1 x 10
8
6.8 x 10
8
CONCLUSION: Comparison of mean values indicates no significant
differences. AOAC text does not need to specify
quantltated inoculum for initial broth transfer
-------
23
Removal of Attached Test Bacteria from
Stainless Steel PenicyIinders
Background: No procedure has been described to effectively quantitate
test bacteria attached to stainless steel penicylindens
used in the Use-Dilution Method. Our original removal
procedure consisted of vortexing penicyItnders
individually in 10ml of phosphate buffer dilution water
(PBDW) in 20 x 150mm tubes at a Vortex-Genie setting of #4
for 30 seconds. Scanning electron microscopy confirmed
the efficiency of this procedure.
Purpose: To determine the efficiency of a revised vortex ing method
of removing bacteria attached to stainless steel
pen icy Iinders.
Methodology: 1.
Transfer stock cultures of each AOAC Use-Dilution
tester strain to 10ml nutrient broth (AOAC 4.001) in
20 x 150mm tubes.
caps loose.
Incubate overnight at 37 C with
2. Make at least three consecutive 24hr broth transfers
using 4mm id loop with incubation at 37°C.
3. Transfer 10ml aliquots of 48-54hr inoculated broths to
25 x 150mm tubes.
4.
5.
For each organism, place 10 previously prepared (AOAC
4.009) S&L pen icy Iinders into each broth culture tube
for 15min at room temperature, followed by drying
(AOAC 4.009) for 40min at
37°C.
Each carrier is placed in a 20 x 150mm screw-capped
tube containing 10ml (PBDW) AOAC 4.020 (f).
Five tubes are individually spun on a Vortex-Genie,
setting #4, for one minute. The other 5 tubes are
simultaneously placed in an operating sonic cleaner
bath for one minute, then individually spun on the
Vortex-Genie, setting #6-7, for 2-4 min.
Serial dilutions using PBDW are made from each tube,
with 1.0ml aliquot from and 10-4 dilutions prepared as
pour plates in duplicate using Difco Plate and Count
agar.
Incubate plates for 48 hours at 37°C and count plates
showing colonies between 30 and 300.
-------
24
Results:
Qrcjanism
_S. aureus
6538
4.7 x 10
3.6 x 10
3.2 x 10
2.8 x 10
2.1 x 10
x=3.3 x 10
^. choleraesuls
10708
1.8 x 10
1.3 x 10
1.1 x 10
7.3 x 10'
6.0 x 10
5.0 x 10
4.2 x 10
3.8 x
3.4 x 10
4.5 x 10
1.2 x 10
1.2 x 10
1.2 x 10
1.2 x 10
8.8 x 10-
6
P. aerucjinosa
15442
x=1.2 x
1.2 x
4.5 x
3.3 x
2.8 x
2.5 x
x=5.0 x
io6
107
fi
10
f.
i r\
106
106
6
10°
1. 1 x
5.7 x
3.4 x
3.0 x
2.8 x
2.7 x
3.5 x
106
io6
10
g
10
106
io6
6
10
A = Vortex #4, 1 min
B = Sonicat ion 1 min, vortex #6-7, 2-4 min
P values for t-test on log-transformed responses:
^. aureus = .087
S_. cnoleraesuis = .920
P_. aerug i nosa = 5.31
-------
Conclusion: No significant differences between removal methods for S^.
choleraesu i s and P_. aerugjnosa. Although the ^. aureus
comparison is suggestive of statistical significance, the
difference (if any), between mean values appears to be
sIi ght.
-------
26
Bacterial Growth Curves and the Inoculation
of Stainfess Steel Pen icy Iinders
Background:
Purpose:
Methodology:
Disinfectant testing methodolgies of other nations utilize
24hr broth cultures. The AOAC Use-Dilution Method
requires the use of a 48-54hr broth culture, which may not
provide an optimum number of cells for the inoculation of
pen i cyIinders.
To exami ne
test strat
Salmons I(a
the growth kinetics of two AOAC Use-Dilution
s, Pseudomonas aerug i nosa ATCC 15442 and
choteraesuis ATCC 10708; to examine carrier
loads when seeded according to growth cycles; to assess
the degree of variability in cell numbers that may exist
bewteen a 48 and 54 hour broth culture; and ultimately to
utilize this information in attempts to standardize
bacterial numbers on penicyItnders.
1. Simultaneously inoculate seven 10m! tubes of nutrient
broth (AOAC 4.001) with 4mm id loop from nutrient
broth previously transferred at least 4 times at 24hr
intervals. Incubation at a 11 times is 37°C.
2. At desired intervals (6h, 12h, 18h, etc.), remove two
1.0ml aliquots from a single broth tube, perform
serial dilutions in phosphate buffered dilution water
(PBDW) and plate in duplicate using pour plate method
and Difco Plate Count Agar.
3. At desired intervals (18h, 24h, etc.), after removing
aliquots for broth counts, place 5ml of broth culture
into sterile 25 x 150mm tubes for the inoculation of 5
S&L carriers (prepared as in 4.009).
4. Allow penicyIinders to remain undisturbed in broth
culture for 15min at room temperature. Remove and dry
for 40min at 37°C as in AOAC 4.009.
5. Place each of three carriers into individual 10ml
tubes of PBDW, spin on Vortex-Genie at setting #4 for
1 min, make serial dilutions in PBDW and plate 1ml
aliquots in duplicate using pour plate method and
Difco Plate Count Agar.
6. Incubate plates for 48hr at 37°C and count plates with
colonies between 30 and 300.
-------
27
Results:
Salmonella choleraesuis 10708
Mean Numbers of Organisms
Broth Pen icy 1 inders % Attached
6 hr
12 hr
18 hr
24 hr
36 hr
48 hr
54 hr
1.6 x
3.8 x
7.7 x
5.9 x
6.3 x
5.9 x
3.9 x
108
108
108
to8
108
108
108
3. 1 x
2.2 x
2.4 x
1.9 x
1.8 x
106
106
106
106
106
.37$
.37$
.38$
.32$
.46$
Conelus ions:
1. Greater numbers of organisms will attach to
pen icy I inders at the peak of the growth curve.
2. Utilizing an 18 or 24hr culture to increase bacterial
attachment to penicytinders seems plausible.
3. While broth count decreases significantly from 48 to
54hrs the effect on numbers of bacteria attaching to
penicyIinders appears negligible.
-------
28
Results:
Pseudomonas aerug inosa 15442
Mean Numbers of Organisms
Broth
6
12
18
24
36
48
54
hr
hr
hr
hr
hr
hr
hr
5
5
8
1
1
1
5
.1 x
.5 x
.4 x
.1 x
.2 x
.1 x
.2 x
10
10
10
10
10
10
10
Pen i cy 1 inders
8
8
8 7
8 3.2 x 10'
9 3.3 x 107
9 2.7 x 10?
9 2.8 x 107
8 9.3 x 106
% Attached
3.8*
3.0?
2.3*
2.6%
1.8*
Cone I us ions:
1. Pseudomonas broth counts are significantly higher than
those of Sa tmoneI la.
2. Pseudononas exhibits a greater ability to attach to
carrier surfaces than SaImoneI I a.
3. As the broth count decreases significantly from 48 to
54 hrs, the number of organisms attaching to
pentcyIinders similarly decreases.
-------
29
Evaluation of Penicylinders Used in Disinfectant Testing:
Bacterial Attachment and Surface Texture
Eugene C. Cole1*, William A. Rutala1, and
Johnny L. Carson^
Department of Medicine, Division of Infectious Diseases1 and Department
of Pediatrics, Division of Infectious Diseases2, The University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina 27514
*Correspcnding author
Presented in part at the 85th Annual Meeting of the American Society for
Microbiology, Las Vegas, Nevada, March, 1985.
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30
ABSTRACT
The AOAC Use-Dilution Method for EPA registration of liquid
disinfectants has several presumed deficiencies which include physical
disparity between the available brands of penicylinders and variability
of bacterial numbers on penicylinders depending upon test strain and
penicylinder surface texture. Two brands of stainless steel (SS)
penicylinders, one brand of porcelain and one brand of glass were
investigated by scanning electron microscopy to assess textural
differences. The outer surfaces of the AOAC recommended S&L SS
penicylinders were principally smooth but exhibited grooves where
bacteria resided. The inner surfaces of the S&L and the outer/inner
surfaces of the Fisher SS penicylinders showed deep grooves and marked
pitting. Porcelain penicylinders were extremely irregular and the glass
were very smooth. Vortexing (30 sec at #4) the penicylinders after
using the AOAC method of bacterial inoculation and drying (40 min at
37oC) allowed assessment of bacterial numbers attached to a
penicylinder. Utilizing this methodology the three AOAC recommended
bacterial test strains attached to stainless steel carriers
differentially; approximataely 10^ for Pseudomo_nas aeruginosa, 5 x 10^
for Staphylococcus aureus, and 106 for Salmonella choleraesuis^ Glass
(moist at. 40 min) retained lO^-lO7 organisms of all three test strains,
whereas the porcelain retained about 106-107 S_. aureus but 105-106 P_.
aeruginosa and lO3-^ _S. choleraesuis. These data suggest that
disinfectants are not similarly challenged with the different AOAC test
bacteria and that alternate methodology should be considered to ensure
comparable numbers of bacteria on the penicylinders.
-------
31
INTRODUCTION
The Association of Official Analytical Chemists (AOAC) Use-Dilution
Method (1) has been the official methodology for evaluating the
germicidal activity of hospital disinfectants. For many years the
Environmental Protection Agency (EPA) performed intramural pre- and
post-registration efficacy testing of chemical disinfectants. In 1982
this was curtailed, presumably for budgetary reasons. Thus,
manufacturers presently do not need verification of efficacy claims by
the EPA or an independent testing laboratory when registering a
disinfectant.
In the past five years, there have been many criticisms of the test
method and several federal, commercial, state and university
laboratories performing the test have been unable to substantiate the
bactericidal claims of some manufacturers (2,3). This may be attributed
to intrinsic deficiencies of the disinfectants or problems with the test
methodology.
This investigation was undertaken to examine two of the deficiencies
that have been proposed to account for the AOAC Use-Dilution test
variability: physical disparity between the available brands of
penicylinders and its relation to the adherence of bacteria. The data
suggest that disinfectants are not similarly challenged with the AOAC
test bacteria and that alternate methodology should be considered to
ensure comparable numbers of bacteria on the stainless steel
penicylinders.
-------
32
MATERIALS AND METHODS
Test Organisms. The three recommended AOAC Use-Dilution Method strains
were used in this investigation, they are: Pseudomonas aeruginosa PRD10
(ATCC 15442), Staphylococcus aureus FDA209 (ATCC 6538), and Salmonella
cholerae;mis (ATCC 10708). Stock cultures of test organisms were
maintained on agar slants as in AOAC 4.001 (1) with monthly transfers.
Broth medium. Nutrient broth was prepared as in AOAC 4.001a (1), with
10 ml portions placed in 20 x 15mm screw-capped, flint glass tubes, and
sterilized at 121°C, tor 15 rain. Broths were inoculated from the stock
culture slants and incubated overnight at 37oC. At least three
consecutive 24 hour broth transfers using 4 mm id platinum/rhodium loop
(American Scientific Products, McGraw Park, 1L) were made before
inoculated broths were used for testing at 49 hours.
Penicylinders. Four types of penicylinders were evaluated: S&L
stainless steel (type 304, S&L Metal Products Corp, Maspeth, NY), Fisher
SS (Fisher Scientific, Pittsburgh, PA), porcelain (Fisher Scientific),
and glass (University Research Glassware, Carrboro, NC). All had the
same dimensions: 8jHmm od., 6^1mm id., and length lOjvlmm.
Penicylinder preparation. New penicylinders were boiled in distilled
water for at least 10 minutes to remove oil residues. After the
distilled water was poured off, the penicylinders were sonicated in
distilled water for at least 5 min to remove additional debris. Used
penicylinders were steam sterilized for 15 min at 121°C prior to
sonication. After sonic cleaning and rinsing in distilled water, all
penicylinders were placed in IN NaOH overnight as specified in AOAC
-------
33
4.009 (1). The penicyllnders were then rinsed in tap water until
neutral to phenolphthalein, rinsed twice in distilled water, drained,
and placed 10 to a tube (20 x 150mm, screwcap, flint glass).
Penicylinders were then covered with fresh 0.1% asparagine solution,
sterilized at 121°C (15 psi) for 15 min and held at room temperature
until ready for use. During preparation, any cylinders with noticeable
imperfections (scratches, chips, etc.) were discarded.
Penicylinder inoculation. Test penicylinders were aseptically placed in
swirled broth cultures with the volume of broth directly proportional to
the number of penicylinders (i.e., 1 ml broth per penicylinder). When
simultaneously inoculating more than one type of penicylinder, their
placement; into the tubes was alternated. If necessary, tubes were
shaken to rearrange the cylinders in the broth so that all were covered
by the broth culture. Tubes remained undisturbed at room temperature
for 15 min.
Penicylinder drying. Following inoculation, the penicylinders were
removed from the broth using a flamed wire hook and placed on end in
sterile glass petri dishes (no more than 10 per dish) matted with two
layers of Whatman No. 2, 9 cm filter paper. The dishes were covered and
placed in an incubator at 37°C, and dried for 40 min at 25-35% relative
humidity.
Evaluation of cell removal methods. Two methods of mechanically
removing attached bacteria from penicylinders using a vortex device were
compared. For each of the three bacteria tested, 9 penicylinders were
inoculated as described above. Six penicylinders were randomly selected
and each placed Into a 20 x 150mm screw-capped, flint glass tube
-------
34
containing 10ml of sterile phosphate buffer dilution water (PBDW), AOAC
4.020f (1). Method A involved the use of a Vortex-Genie (Fisher
Scientific) set at number 4 for 30 sec, while method B employed a
Vortex-Genie set at number 8 for 3 min. After each vortexing procedure,
dilutions of 10""^ and 10~5 in PBDW were prepared for S_. aureus and _P.
aeruginosa, and dilutions of 10-3 and 10-4 were prepared for S_.
chpleraesuls. For each dilution, 1.0ml was inoculated into each of two
15 x lOOrim sterile petri dishes to which 15-17ml Plate Count Agar (Difco
Laboratories, Detroit, MI) at 42-45°C was added. When solidified, all
plates were inverted and incubated for 48hr at 37oC. Colonies on all
plates were enumerated using a colony counter, and those with counts
between 30 and 300 were used for calculations.
Bacterial removal from penicylinders. After drying, each of 5
penicylinders was aseptically placed into a 20 x 15mm screw-capped,
flint glass tube containing 10ml of sterile PBDW. Organisms were
removed from the dried penicylinders by placing the tube on a
Vortex-Genie (Fisher Scientific) set at number 4 for 30 seconds.
Dilutions of 10-3, 10-4 and 10-5 were prepared in PBDW using sample
aliquots of 1.0 ml. At each of the three dilutions 1.0ml was plated in
duplicate by the pour plate method, described above. When solidified,
all plates were inverted and incubated for 48hr at 37°C. Following
incubation, colonies were enumerated using a colony counter, and those
with counts between 30 and 300 used for calculations.
Scanning Electron Microscopy. Sterile penicylinders for surface texture
examination were prepared as described above. They were aseptically
removed from the sterile 0.1% asparagine solution and placed into
-------
35
sterile glass petrl dishes matted with two layers of Whatman No. 2
filter paper and allowed to dry at 37oC for at least 30 rain.
Penicylinders with adherent bactria were inoculated and dried as
described above. All penicylinders were immediately fixed in a mixture
of 2% glutaraldehyde/2% paraformaldehyde (pH 7.2) and subsequently
rinsed in phosphate buffer. They were post-fixed in a buffered 1%
osmium tetroxide solution, dehydrated in a graded ethanol series, passed
through graded solutions into a transition fluid of Freon 113, and dried
by the critical point technique using Freon 13. The penicylinders were
mounted on SEM specimen stubs with silver paste, and spatter coated with
gold or gold/palladium. The specimens were examined in an ETEC Autoscan
scanning electron microscope at an accelerating voltage of 20kV. Prior
to photographing a surface, at least 20 typical fields were examined.
RESULTS
Surface texture of penicylinders types. Examinations were performed to
determine^ if physical differences exist between the four types of
available penicylinders: stainless steel (both S&L and Fisher),
porcelain, and glass (Fig. 1). As can be seen in Fig. 2, significant
differences in surface texture were noted among the penicylinders when
examined by scanning electron microscopy (SEM). The two brands of
stainless steel penicylinders were markedly different, with the outer
surfaces of the AOAC recommended S&L brand principally smooth (Fig. 2A)
but exhibiting some grooves where bacteria may reside. The inner
surface of the S&L penicylinder (Fig. 2B) and the outer/inner surfaces
of the occasionally used Fisher penicylinder (Fig. 2C,D) showed deep
grooves and significant pitting. Porcelain penicylinders were extremely
-------
36
irregular (Fig. 2E) and the glass penicylinders were very smooth (Fig.
2F).
Cell removal from penicylinders. For _S. aureus, both methods A and B
revealed identical mean values of 1.3 x 107 organisms (p>0.1).
Examination of a randomly selected penicylinder by SEM revealed no cells
attached to the penicylinder following processing according to method A.
Mean values for j^. choleraesuis were 9.7 x 10^ for method A, and 1.2 x
106 for method B (p<0.05). P.aeruginosa showed a mean value of 6.3 x
10^ cells using method A, and 1.5 x 107 cells using method B (p<0.05).
Since the number of cells per penicylinder did not differ by more than
0.5 log when employing the two removal methods and method B often
resulted in tube breakage, method A was employed.
.^^ Cell attachment to penicylinder types. When the adherence of $_• aureus
^^ to the different penicylinder types was evaluated, it was found that
mean numbers of organisms on S&L and Fisher SS were directly comparable
(about 5 x 106) as were glass and porcelain (Table 1). There were no
significant differences among the four penicylinder types. A comparison
of mean numbers of ]?. aeruginosa attached to the four types, again shows
S&L and Fisher SS penicylinders directly comparable, while differences
were observed between porcelain (low cell numbers) and the other
penicylinders (Table 1). An SEM of a porcelain penicylinder inoculated
with P. aeruginosa, however, showed cell attachment in large numbers,
most likely indicating difficulty in removal of the cells from the rough
surface. No significant difference was observed between glass and
stainless steel. A comparison of mean numbers of S. choleraesuis on
different penicylinders can also be seen in Table 1. Again, the S&L and
-------
37
Fisher SS are directly comparable with about 106 per penicylinder, while
the porcelain and glass both differed significantly from the stainless
steel.
In assessing numbers of the three AOAC test bacteria adhering to the
recommended S&L penicylinder (Table 1), P_, aeruginosa appeared most
adherent, with a mean number of organisms approaching 1.4 x 10^', §_•
aureus somewhat less attached at approximately 6.0 x 106 and j>_.
choleraesuis attaching the least at 8.2 x 10^. These findings were
visually corroborated by SEM when inoculated penicylinders were examined
(Fig. 3).
-------
38
DISCUSSION
The surface texture of available penicylinders that might be used in the
AOAC Use-Dilution Method of disinfectant testing varies significantly.
The methodology recommends only the S&L brand of stainless steel
penicylinder (S&L); however, in 1982, S&L Metal Products Corporation
suspended the manufacture of their penicylinders for 14 months. At that
time many laboratories were forced to purchase stainless steel
penicylinders from the other major manufacturer, Fisher Scientific.
The Fisher penicylinders display the same measurement (8mm od x 6mm x
10mm length) as the S&L, but the Fisher penicylinder has ends beveled to
the inside. A difference in the degree of polish on both brands of
stainless steel penicylinders used in this study was visibly noticeable.
The Fisher brand of penicylinder appeared less polished than the S&L,
although both companies have manufacturing specifications that are
nearly identical. S&L Metal Products Corporation manufactures their
penicylinders using 304 grade stainless steel, with a maximum degree of
polish of 32 urn on the outside and 36 urn on the inside. Fisher
Scientific also uses 304 grade stainless steel but specifies a maximum
degree of polish of 32 urn on the outside and 32 urn on the inside. A
measurement of 32 urn indicates that the distance from the lowest to the
highest point of a depression on the polished surface can be no greater
than 32 urn. Scanning electron microscopy corroborated the marked
textural difference between the two brands. The three bacterial test
strains, however, exhibited similar adherence to both brands of
penicylinders.
-------
39
Porcelain penicylinders are required by the EPA to be used as carriers
in the Use-Dilution Method if a germicidal claim is made for a porous
surface. Fisher Scientific is the sole manufacturer and supplier of
this penicylinder which is identical to the Fisher stainless steel
penicylinder in terms of size and design. The porcelain surface was
found microscopically to be extremely rough. Bacterial adherence to the
porcelain varied significantly among the test bacteria, although values
for P_. aeruginosa may be falsely low due to difficulty in removing cells
from the matrix of the porcelain cylinder by the vortex method. In
addition, Lhe porcelain is susceptible to cracking and chipping. For
these reasons it cannot be recommended as a substitute for stainless
steel in use-dilution testing.
Glass penicylinders were included in this study because of their
extremely smooth inner and outer surfaces. These were custom-made from
Pyrex tubing (with fire polished ends) to the exact dimensions of the
S&L stainless steel penicylinders. Glass demonstrated the most similar
bacterial adherence (lO^-lO?) for all of the test strains, when compared
with steel or porcelain penicylinders. The glass penicylinders,
however, dried very poorly and it is possible that the bacterial
attachment is not entirely representative of organisms dried onto the
glass surface, but rather represents organisms carried over in the
remaining droplet of undried broth.
The most significant finding of the study was that the three AOAC test
bacteria attached to stainless steel penicylinders differently with more
than lO^for _P. aeruginosa, approximately 5 x 10^ for ^. aureus and 10^
or less for S. choleraesuis. Thus, there could be a 1.5 log difference
-------
40
or more among the test organisms. This might not be unexpected
considering differing numbers of cells in broth culture as well as other
morphological and physiological differences among the test strains which
may affect bacterial attachment to penicylinders. This is the first
demonstration that disinfectants claiming efficacy against various
bacteria are not similarly challenged in the AOAC Use-Dilution Method.
In conclusion, there are textural differences among the types of
available penicylinders (porcelain, glass and two brands of stainless
steel) that may affect the attachment of bacteria to them. Both brands
of stainless steel demonstrated comparable numbers of test bacteria for
all three test strains. We recommend that stainless steel be retained
as the required carrier in future revision of the Use-Dilution Method.
We also recommend a comparative use-dilution study of S&L and Fisher
brands of stainless steel penicylinders. Since the three AOAC
recommended test bacteria attached to stainless steel penicylinders
differently for each organism, with more than 107 for ]>. aeruginosa,
approximately 5 x 106 for S_. aureus, and 106 or less for j>.
choleraesuis, we suggest that alternate methodologies be considered to
ensure comparable numbers of challenge bacteria.
ACKNOWLEDGEMENT
This work was funded by the US Environmental Protection Agency under.
assistance agreement CR811204-01-0 to the University of North Carolina
at Chapel Hill.
-------
40a
LITERATURE CITED
1. Association of Official Analytical Chemists. 1984. Official
methods of analysis, 14th ed. Association of Official Analytical
Chemists, Arlington, VA.
2. Rutala, W.A., M.M. Stiegel, F.A. Sarubhi. 1982. Ineffectiveness of
disinfectants against hospital strains of bacteria. Abs. Annual
Meeting Am. Soc. Microbiol. Q138:233.
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41
Table 1. Mean (n=5) numbers of organisms (xlO^) on penicylinders seeded
with AOAC recommended test bacteria.
Penicvlinder
S. aureus
(ATCC 6538)
P. aeruginosa S.choleraesuis
(ATCC 15442)
(ATCC 10708)
S&L
Fisher S3
Porcelain
Glass
5.98
5.38
7.04
7.94
13.70
18.92
.97a,c,d
13.22
.82d
.99
l.70c»d
an=4
bStainless steel
cSignifleant difference between penicylinder types, p<0.05
dSignlfleant difference between test strains, p<0.05
-------
42
FIG. 1. Types of available penicyllnders for the Use-Dilution Method,
From left to right; porcelain (Fisher Scientific); stainless steel
(Fisher Scientific); stainless steel (S&L Metal Products Corp.); and
glass (University Research Glassware).
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43
FIG. 2. Scanning electron photomicrographs of penicylinder surface
textures. S&L stainless steel, outside (A) and inside (B); Fisher
stainless steel, outside (C) and inside (D); porcelain, outside (E) and
glass, outside (F). Bar, 1 urn.
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44
FIG. 3. Comparison of numbers of Use-Dilution test bacteria seeded on
recommended S&L stainless steel penicylinders: (A) _P. aeruginosa, (C)
J3. aureus, and (D) _S. choleraesuis. (B) shows JP. aeruginosa residing in
penicylinder (S&L) grooves. Bars 1 um.
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45
Test Protocol
Penicyllnder Drying Conditions
Background:
Current Use-Dilution methodology calls for the drying
of inoculated penicylinders for 20 to 60 minutes.
Realizing the extent of variability inherent in this
range, agreement among testing laboratories has been to
use a 40 min drying time. Penicylinders are not
completely dry within this time period, however, and a
longer drying time may be necessary.
Purpose:
To assess numbers of bacteria adhered to penicylinders
undergoing increased drying times beyond and compared
to the current 40 minute period.
Methodology:
1. Transfer each of the AOAC Use-Dilution bacterial
stock cultures to 20 ml Nutrient Broth (AOAC
4.001) in 20 x 150 mm tube and incubate overnight
at 37C.
2. Make at least 3 consecutive 21* hr broth transfers
using a 4 mm id loop with incubation at 37C.
3. Prepare S&L penicylinders as in AOAC 4.009 and
aseptically place 12 in sterile 25 x 150 mm tubes,
after withdrawing asparagine solution with pipet.
4. Cover penicylinders with 12 ml of vortexed broth
cultures and allow to sit undisturbed for 15 min
at room temperature.
5. Aseptically remove carriers from broth and place
alternatively in 3 sterile glass petri dishes
matted with 2 layers of Whatman No. 2, 9 cm filter
paper. There will be 4 carriers per dish.
6. For each organism,label the three dishes 40, 60
and 80 min, and place in incubator at 37C.
7. After 40 min remove plate from incubator. Place
each carrier into 10 ml phosphate buffered
dilution water (PBDW) {AOAC 4.020f) in 20 x 150 mm
tube and agitate for one minute on Vortex-Genie,
setting #4.
-------
46
8.
9.
Results:
Prepare and plate dilutions (10**3 fOr c
£.ll£i££§S3.y.i.S» 10 for *Lt._aiiC£Jlfi» 10" for £A
a£r_U£.illQ.fi.a) in triplicate""with 1.0 ml inoculum for
pour plate method using Difco Plate Count Agar.
Continue similarly with 60 and 80 minute plates.
Incubate plates 48 hr at 37C and count plates with
colonies between 30 and 300.
Cylinder Drying Conditions
StaE.bylfic.£2.e.U£_ayxe_iis. 6538
lo__ain
6.4
6.4
7.1
x = 67F
x =
10°
1°6
106
10°
To6
1 .0 x 107
1.7 x 107
2.2 x 107
2.6 x 107
x = 1 .9 x 10
n7
•J.
2
8
1
2
1
1
1
1
1
.3
.8
.1
.9
.3
.0
.1
.1
.9
U.*L*
X
X
X
X
x
x
X
X
X
4
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
5
5
6
6
6
6
6
6
x =
x =
x = 1.3 x 10'
x =
5,
5,
5,
5.
at T* r
«.£.*
6.C
1 .
1 .
1 .
2.
1 .
1 a •
JL£<
6.Q
9.
1 .
1 .
1 .
1 .
1 .
1 .
1 .
1 .
1 .
.1
.2
,6
.2
is.
u
,0
2
2
0
4
ca
. i
8
2
3
6
r
0
1
3
3
3
x
x
X
X
Ins
ait
x
X
X
X
X
.££J
uln
X
X
X
X
X
X
X
X
X
X
1
li
L
1
1
1
1
1
L
1
1
1
1
1
1
1
1
1
1
1C
1C
1C
1C
IS
10
10
0
0
0
4 ,
ij
o
0
0
0
0
0
0
0
0
0
'.'
I6
L
,7
J
7
7
7
a
5
g
6
6
6
6
g
V
6
x =
15442
10708
x =
4.4
5.0
5.2
5.9
5.1
£
9
9
1
1
1
SL
9
1
1
2
1
1
1
1
1
.0
.0
.7
.8
.3
.6
.3
.8
.0
.5
.1
.2
.3
.3
x
x
X
X
X
aij
X
X
X
X
X
ail
X
X
X
X
X
X
X
X
X
10fi
106
106
106
To6
X6
10;
10!
10
i
10
10
10
To
10
10
10
10
)<
T
5
6
6
6
S
6
6
6
6
1 .3 x 10'
-------
47
Conclusions:
1 . Increased numbers of Sjt_aiiEfiU£ and Ej._afi.EUS.iacfi.a
at 40 min may possibly result from incomplete
drying. Their 60 and 80 min counts are comparable
and probably reflect a more accurate assessment of
the dried bacterial loads.
2. £j._C.h_D_le.cae.£iii£. appears to dry sufficiently at ^0
min, since numbers of viable organisms at all
three dyring times are comparable.
Recommendations:
In order to effect a more consistent, dried carrier
load, consideration should be given to changing the
current 40 rain drying period to 60 minutes.
No specific relative humidity needs to be recommended,
as the relative humidity inside the covered petri dish
will be quite high.
-------
48
Bacterial Wash-Off from Stainless Steel Pen icy Iinders
Background: The AOAC Use-Dilution Method of disinfectant efficacy
testing uses, as a bacterial challenge, organisms dried to
the surface of stainless steet cylinders. When these
carriers are immersed (and swirled) in test disinfectants,
particularly those that are detergent/cleaners, some or
all of the organisms are washed off. If all of the
bacteria wash-off into suspension, then the carrier thus
becomes a very Inaccurate sampling device transferring
approximately 0.01 ml of bacteria/disinfectant suspension
into subculture broth. Data relating to the extent of
such wash-off are lacking.
Purpose: The purpose of this study is to approximate the bacterial
wash-off when stainless steel pentcyIinders inoculated
with the 3 Use-Dilution Method bacteria are exposed to a
fluid environment as in Use-Dilution testing.
Methodology: 1. AOAC Nutrient Broths (AOAC 4.001a) are Inoculated with
the three Use-Dilution test strains and incubated and
transferred as described in the Method.
2. For each test organism, 11 S&L penicyIinders are
inoculated In 11 ml broth, cultured and allowed to
remain undisturbed at room temperature for 15 mins
They are then removed and dried for 40 mlns at 37°C as
described in the Method (AOAC 4.009).
-------
49
3. From the 11 penicyUnders, 4 are selected and placed
individually into 10ml tubes of Phosphate Buffer
Dilution Water (PBDW), AOAC 4.020f for 10 min at 20°C
as per use-dilution testing.
4. At 10 min, a 1 ml aliquot ts removed from the PBDW,
diluted 10~3, 10~4, 10~5 with each dilution plated in
duplicate using 1 ml aliquot and Difco Plate Count
Agar. Also at 10 min the carrier !s transferred to a
20 x 150mm tube with 10ml PBDW, vortexed at No. 4 for
1 min and diluted to 10~2, 10~3, 10~4. AM dilutions
are likewise plated using 1 ml allquots and pour plate
method. Incubation of all plates is for 48 hours at
37 C, after which colonies are counted.
5. The subcultured PBDW Into which the carrier is placed
contains bacteria washed off from the cylinders'
surfaces. Organisms in the PBDW after vortex ing
represent cells adhered to the pen icy Iinders in
addition to those carried over in the subculture
process. The combination of the two figures should
represent the original number of organisms seeded on
the carriers. Thus a percentage of cells washed off
can be calculated.
-------
4»
50
Results:
Table 1.
Evaluation of bacterial wash-off from stainless steel
pen icy Iinders inoculated with Use-Dtlution Method Strains
Organism Washed-0ffa
S. aureus
P. aeruginosa
S. choleraesuis
2
5
2
.20 x
.21 x
.23 x
IO6
IO6
IO6
Transferred
3.
5.
2.
46 x
47 x
35 x
106
106
to5
5
1
2
Total
.66 x
.10 x
.47 x
1
106
io7
io6
Wash-Off6
39
48
90
.2%
.6%
.1*
Mean value based on 4 replicates.
Discussion: While the results show considerable wash-off for each test
organism, the numbers of bacteria transferred appear
adequate (at least 10 ) for each strain. It must be
realized, however, that the inoculated penicyIInders were
exposed to PBDW and not disinfectant/detergents containing
numerous surfactants as would occur in efficacy testing.
It is thus presumed that wash-off under such conditions
would be greater than that demonstrated here, perhaps
resulting in a decreased probability of recovering viable
organisms and raises the question of whether the carrier is
an acceptable sampling device.
Since varying degrees of bacterial wash-off have been
demonstrated for each of the Use-Dilution test bacteria and
these may ultimately influence final test results, it Is
recommended that a quantitative suspension test be
developed as alternative to the carrier method.
-------
51
Comparison of Two Brands of Stainless Steel PenicyIinders
Used for Disinfectant Testing
Eugene C. Cote,* William A. Rutala
and Edith M. Alfano
Department of Medicine, Division of Infectious Diseases
The University of North Carolina at Chapel HiM
Chapel Hill, NC 27514
Corresponding Author
-------
52
Abstract
Two brands of stainless steel pen icy Ifnders (S&L Metal Products and
Fisher Scientific) were tested to determine if they provide comparable
results when used in the AOAC Use-Dilution Method of disinfectant
testing. Two tots of each brand were evaluated. One lot of S&L and
one lot of Fisher pen icy Iinders were macroscopicaIly smooth and
polished, while another lot of each brand was dull. Using the
Use-Dilution Method, the two brands of penicyUnders were evaluated with
the three AOAC bacteria (StaphyIococcus ajjreus ATCC 6538, Salmonella
choleraesuis ATCC 10708, and Pseudomonas aeruglnosa ATCC 15442), and two
hospital disinfectants. Results showed consistently more positive tubes
for the Fisher brand of penicylinders than for the S&L, regardless of
the test cylinders' surface finish. These data suggest that the use of
highly polished S&L stainless steel pen!cylinders can standardize the
disinfectant efficacy test by eliminating inter laboratory variation in
results due to penicylinder brand.
-------
53
The AOAC Use-Dilution Method of disinfectant efficacy testing
specifies the use of type 304 stainless steel penicyIinders manufactured
by S&L Metal Products, Inc., Maspeth, N.Y. These cylinders are
inoculated with three specific test bacteria which serve as disinfectant
challenges. When S&L suspended the manufacture of their penicyIinders
for 14 months, many laboratories purchased stainless steel penicyIinders
from Fisher Scientific, Pittsburgh, PA. Fisher's penicyIinders are also
304 stainless steel, but unlike S&L's model, Fisher's penicy1inders are
manufactured with beveled ends. Microscopic examination of both brands
of penicyIinders reveals defects in the surface of both pen I cylinders
where bacteria can reside and perhaps be protected from disinfectant
2
exposure . In addition, the degree of polish of both brands of
pen icy Iinders has varied considerably in the past two years.
A previous study has shown that both types of stainless steel
pen icy Iinders are inoculated with approximately the same number of test
2
bacteria . The purpose of this study was to simultaneously test S&L and
Fisher stainless steel pen icy Iinders using the AOAC Use-Dilution Method
to determine if the brands yield comparable test results.
When the three AOAC test bacteria were Inoculated onto both brands
of penicyItnders and exposed to E.P.A. registered hostpital-grade
phenolic and quaternary ammonium disinfectant-detergents, the results
demonstrated (with only one exception), a significantly greater number
of positive Fisher brand pen icy Iinders. Moreover, since there were
significantly fewer positive S&L polished pen icy Iinders compared to dull
pen Icy Iinders of the same brand, it appears that the quality of the
surface finish may also effect the results.
-------
54
The three recommended AOAC Use-Dilution strains used in this
investigation were Salmonella choleraesuis 10708, StapnyIococcu s aureu s
6538 and Pseudomonas aeruginosa ATCC 15442 (American Culture Collection,
Rockville, MD). Stock cultures of test organisms were maintained on
nutrient and cystine trypticase agar slants as specified in AOAC
4.001c . Tubes of nutrient or Synthetic Broth AOAC {Difco Laboratories,
Detroit, Ml) were inoculated from the stock culture slants and incubated
overnight at 37°C. At least four consecutive broth transfers were made
before inoculating the broths to be used for testing.
Pen icy Iinders were prepared by steam sterilization (15 min at
121°C), rinsing in tap water, sonicating tn distilled water for at least
5 min, rinsing in distilled water and draining. The cylinders were then
placed in tubes (20 per tube), covered with 1.0N NaOH and further
prepared as In AOAC 4.009. Prior to testing, asparagine was removed
from the tubes of pen icy Iinders and the penicyfinders were aseptically
transferred to 25 x 150mm sterile tubes. The appropriate number of
48-54h 10ml broth cultures used to inoculate 120 pen icy 1inders for each
day's testing was pooled and mixed. Twenty ml of broth culture was
added to the tube containing the 20 pen icy Iinders to be tested and
allowed to remain undisturbed at room temperature for 15 min. Upon
removal as specified in the Method, penicyfinders were dried at 37°C for
40 min.
The disinfectants were prepared according to the manufacturers'
recommended use-dilutions in sterMe, distilled water (Steri le Water for
Irrigation, USP, Travenof Laboratories, Deerfield, ID. Formulations
for the quaternary ammonium product and the phenolic were as follows:
-------
55
Didecyl dimethyl ammonium chloride 7.50/E
N-alkyl dimethyl benzyl ammonium chloride 5.00?
Tetrasodtum ethylene dfamine tetraacetate 1.69?
Inert ingredients 85.81?
0-8enzyf-p-chlorophenoI 7.24?
I sopropy I alcohol 4.04?
0-phenyI phenol 2.23?
Inert ingredients 86.49?
In batches of twenty, sixty pen Icy 11nders of each brand were tested
on the same day with the same broth culture against the same
disinfectant. The test brand of pent cylinder was alternated for each
batch (S&L, Fisher, S&L, Fisher, etc.), with the order varied on each
day of the experiment. Prior to testing each group of sixty
pen icy Iinders, the test disinfectant was freshly prepared in a sterile
1L volumetric flask; then, using a 10ml volumetric pipet, the
disinfectant was dispensed into 25 x 150mm tubes in a 20 C circulating
waterbath. The Use-Difution Method was then performed . Letheen Broth
(Dlfco) was used for neutralization and subculture.
The S&L polished and the Fisher dull pen icy IInders were compared
first. At the time of testing, these were the pen icy Ifnders that were
available for purchase from the manufacturers. Tested against the 1:256
quaternary ammonium disinfectant, the Fisher penicy I inders yielded 7/60
and 13/60 positive for ^. choleraesuis and P_, aeruglnosa respectively,
as opposed to the S&L penicyIinders, which remained negative (Table 1).
Tested against the 1:128 phenolic disinfectant, Fisher pen Icy Iinders
again produced more positive results than the S&L, with 5/60 vs. 0/60
-------
56
for £. choleraesuts, 14/60 vs. 4/60 for S_. aureus and 2/60 vs. 0/60 for
H* aeruginosa (Table 2). Several months later, S&L penicyIinders were
produced with a dull finish, while the Fisher brand was highly polished.
Another simultaneous use-dilution comparison was thus performed using
the same quaternary ammonium and phenolic disinfectants previously
employed in the experiment. Again, the Fisher penicyItnders yielded
more positive tubes than S&L when tested against the 1:256 quaternary
ammonium disinfectant (Table 1). Further testing of the penfcyIinders
against the 1:128 phenolic again resulted in more positives with the
Fisher penicy I inders than the S&L (Table 2). WhMe the data comparing
S&L dull vs. S&L polished are not significant (using Fisher's exact
test, 2-sided), they are significant with respect to the Use-Dilution
Method pass/fail criterion (> 1/60 designates product failure). As
shown Jn Table 1, S&L dull penlcyHnders failed the quaternary ammonium
disinfectant for each of the three test bacteria, while S&L polished
penicyIinders yielded results that confirmed the product's efficacy
against those same strains.
As these results suggest, it appears that the use of highly
polished S&L stainless steel penicyIinders can help standardize the AOAC
Use-Dilution Method by eliminating variability in test results due to
penicylfnder brand.
-------
57
This work was supported by the U.S. Environmental Protection
Agency, Office of Pesticide Programs under Cooperative Agreement
CR8II204-02 to the University of North Carolina at Chapel HIM.
LITERATURE CITED
Association of Official Analytical Chemists. 1984. Official
methods of analysis, 14th ed. Association of Official Analytical
Chemists, Arlington, VA.
Cole, E.G., W.A. Rutala and J.L. Carson. 1985. AOAC disinfectant
testing: evaluation of penicyUnder surface texture and bacterial
load. Abs. Ann. Meeting Am. Soc. Microbiol., Q41, p. 264.
-------
58
Table 1. Positive use-dilution tubes for Sil and Fisher brands of stainless
steel pen icy Iinders exposed to 1:256 quaternary ammonium
disinfectant.
Organism
S. choleraesuis
S. aureus
P. aeruqinosa
S&L3
(pol Ished)
0/60
1/58
0/60
Fisher3
(dull)
7/60*
1/58
13/60*
S&Lb
(dull)
4/60
5/60
3/60
Fisher
(pol f shed)
15/60
6/60
7/60
a,b
Simultaneously tested for each organism.
* p-vaIue < 0.05 as generated from Fisher's exact test (2-slded)
Table 2. Positive use-dilution tubes for SiL and Fisher brands of stainless
steel penicyIinders exposed to 1:128 phenolic disinfectant.
Organism S&La Fishera S&Lb
(polished) (dull) (dull)
S. choleraesuis 0/60 5/60 0/60
S. aureus 4/60 14/60* 7/60
P. aeruginosa 0/60 2/60 0/60
Fisher
(pol Ished)
3/60
11/60
1/60
' Simultaneously tested for each organism.
*p-value < 0.05 as generated from Fisher's exact test (2-sided).
-------
59
CONSISTENCY OF MANUFACTURE OF S & L PENICYLINDERS
Most disinfectant testing laboratories use stainless steel
penicylinders manufactured by the S & L Metal Products
Corporation of Maspeth, New York. This brand of
penicylinder is mentioned in the AOAC Use-Dilution Method,
but is not required. Inconsistencies in manufacture have
been noted over the past three years. These deficiencies
were brought to the attention of the President of S & L (see
attached correspondence) and the problem is apparently
corrected.
If S & L Metal Products can consistently maintain a high-
quality product, then it is recommended that S & L stainless
steel penicylinders be specified and required for use in the
AOAC Use-Dilution Method.
-------
60
THE UNIVERSITY OF NORTH CAROLINA
AT
CHAPEL HILL
School of Medicine
Department of Medicine
Division of Infectious Diseases
919-966-25 J6
The University of North Carolina «t Chapel Hill
?47 Clinical Sciences Building 229 H
Chapel Hill. N.C. 27514
February 6, 1984
Mr. Judah Klein
President
S & L Metal Products Corp.
58-29 57th Drive
Maspeth, NY 11378
Dear Mr. Klein:
I should like to request some information regarding the stainless steel
penicylinders that your corporation manufacturers.
I am currently conducting research, supported by the Environmental Protection
Agency, aimed at defining deficiencies in the current Use-Dilution Method of
the Association of Official Analytical Chemists (A.O.A.C.), which has been used
by the E.P.A. to confirm disinfectant efficacy prior to registration. As you
are probably aware, the A.O.A.C. procedure has specified for many years that the
stainless steel penicylinders be obtained from S & L. When your corporation
recently suspended penicylinder manufacture for about 14 months, many laboratories
went to purchasing cylinders from Fisher Scientific. There were in the past and
still are now, significant differences between the two brands. At the present
time, the design and finish of each is significantly different. S & L penicylinder:
are now more highly polished than Fisher's. This is actually more desirable, as
a highly polished finish should provide fewer microscopic cracks and crevices
wherein bacteria coated onto the cylinders might be protected from disinfectant
exposure. Scanning electron microscopy conducted here last year confirmed some
microscopic differences between older S & L and Fisher cylinders. Significant
differences were also noted when the penicylinders were compared during actual
disinfectant efficacy testing.
Comments from the disinfectant testing community included questions as to the
exact composition of Fisher's penicylinders, and, if S & L did resume production,
would it be permanent?
The E.P.A. has recently suspended its disinfectant testing program for an indefinite
period. There is the possibility that if the agency does not resume its own
testing, it may require the individual states to set up their own disinfectant
testing laboratories. In the meantime, it is expected that within the next three
years, the official A.O.A.C. methodology will be significantly revised. The
quality and availability of the stainless steel penicylinders that are finally
-------
Mr. Judah Klein
Page 2
February 6, 1984
61
recommended will have to be consistent. In this regard, would you kindly
provide me with information concerning the composition of the penicylinders,
the consistency from lot to lot in terms of composition and degree of polish,
and whether or not you plan to continue production of them on a permanent
basis.
I thank you for your assistance in this matter.
Sincerely,
Eugene C. Cole, Dr.P.H.
Research Associate
ECC/kac
-------
62
AEROSPACE PRODUCTS
SgfL METAL PRODUCTS CORPORATION
58-29 57th DRIVE, MASPETH, NEW YORK 1 1378 • (212) 894-4042 • TWX 710-582-2461
The, University of i!orth Caroline
Livision of infectious Liseases
r;l+7 Clinical Science 31c£. - 229 H
Chapel Hill, h.C. 2?;H
ATT: kr. Gene Cole.
Se?r I--r. Cole:
As per our telephone conversation, v:e v:ish to advise
that our pcnicylinderu ^.re mace vat;, a 30k ^r^-le of
stainless c.tcol. The naxinu-T. decree of polish is
32 micro on the outride £ no 36 micro on the inside.
r.7e are enclosing our p«'n;p:;let as v;ell as our price-
list fcr any otner information you rr.ay neec.
Yours very truly
Jiicvh L. Klein. President
-------
63
THE UNIVERSITY OF NORTH CAROLINA
AT
CHAPEL HILL
School of Medicine
Department of Medicine
Division of Infectious Diseases
919-966-2536
The L'im««it>p of North Carolina at Chape! Hill ]
547 Clinical Sciences Building 229 H
Chapel Hill. N.C. 27514
August 2, 1985
Mr. Jut-dab L. Klein, President
S & L Metal Products Corp.
58-29 57th Drive
Maspoth, New York 11378
Dear Mr. Klein:
I would like to address what appears to be inconsistency in the
manufacture and/or polish of your stainless steel penicylinders.
I currently serve as an Associate Referee For the Association of
Official Analytical Chemists (AOAC) with responsibility to assist in
revising the Official Use-Dilution Method of disinfectant efficacy
testing. As I am sure you are aware, S & L penicylinders have been
specified for use in that method for the past 25 years. Recently
however, there have been obvious problems concerning your product. Some
three years ago penicylinder manufacture was stopped for about 14
months. Testing laboratories were then forced to purchase an
alternative brand which, based on research that I have conducted,
appears to introduce significant variability into the test. Since
manufacture was resumed, S & L penicylinders have appeared to exhibit
the consistency required in an official method. This assessment is
based on examination with the electron microscope and in-use evaluations
with a variety of disinfectants.
Very recent problems however have been brought to my attention. I
have enclosed two S & L penicylinders for your examination. The more
highly polished one was purchased by myself in November of 1983 while
the other was received in an order of 1000 by the Florida Department of
Agriculture laboratories in June of this year. I instructed them not to
use those penicylinders since the lack of polish may produce aberrant
results when they are inoculated with bacteria and then exposed to
various disinfectant solutions. They subsequently contacted your
offices and after exploring the situation were told to return them for
replacement. What they received were apparently the same penicylinders
that had been repolished on the outer surface, while the interior and
ends remained dull. There were also deep grooves on the outer surface
that were not present before. Thus, these also cannot be used for the
standardized testing for which they are required. The Florida State Lab
will thus need further replacements or a refund if production problems
continue.
-------
64
If you anticipate a long-term problem with consistent penicylinder
quality, then I will need to advise the more than 60 laboratories
throughout the country that perform AOAC disinfectant testing to refrain
from the further purchase of S & L penicylinders. If such were the
case, an alternative device that consistently maintained its
specifications as to composition and polish would be needed. In fact,
one has been developed through Colgate-Palmolive. It is a Flat, 304
type stainless steel planchet with little interior surface and a number
7 polish. Consistency is guaranteed, although cost is high at $3.00
each.
At the end of 1984 I received a letter from you {copy enclosed)
stating the specifications for the S & L penicylinder. With the
difficulties I've outlined it's apparent that these specifications are
not being met consistently. Hopefully these problems can be resolved to
everyone's satisfaction.
I would appreciate your assistance in this matter.
Sincerely,
Eugene C. Cole, Dr. P.M.
Associate Referee
Use-Dilution Method
ECC/rct
Enclosure
-------
65
AEROSPACE PRODUCTS
ScrL METAL PRODUCTS CORPORATION
58-29 57th DRIVE, MASPETH, NEW YORK 1 1378 • (54^.894-4042 • TWX 710-582-2461
The University of North Carolina
Division of Infectious Diseases
547 Clinical Science Bldg. - 229H
Chapel Hill, N.C. 27514
ATTENTION: Dr. Eugene C. Cole
September 18, 1985
Dear Dr. Cole,
As per our telephone conversation of this date I am
enclosing 10 Penicylinders for your inspection and
comments .
Thank you for your attention in this matter.
Sincerely yours,
S & L METAL PRODUCTS CORPORATION
JLK/ad
End.
dah L. Klein
esident
-------
66
THE UNIVERSITY OF NORTH CAROLINA
AT
CHAPEL HILL
School of Medicine
Department of Medicine
DKision of Infmious Disease*
919-966-2536
Stanley M. Lemon. M.D.. Division Chief
Myron S. Cohen, M.D.
Janet 1 Fischer. M.D.
Joseph S. Kigano. M.D.
M. Lynn Smiley. M.D.
P. Frederick Sparling, M.D.
David J Weber. M.D.. M.P.H.
Hospital Fpt«l?mioti>jO/lnf«ction Control
David J Wcbcr. M.D.. M.P.H.
William A. Rinala. Ph.D.
The University of North Carolina ai Chape! Mil ]
547 Clinical Sciences Building 229 H
Chapel Hill. N.C. 275)4
November 5, 1985
Mr. Judah L. Klein, President
S&L Metal Products Corporation
58-29 57th Drive
Maspeth, NY 11378
Dear Mr. Klein:
I've appreciated the several opportunities we've had to discuss
problems relating to the stainless steel penicylinders. I'm grateful for
the attention that you've given to this matter.
I received the representative sample of penicylinders that you sent
me and have examined them both macroscopically and microscopically(partic-
ularly in regard to finish). These are acceptable for the standard AOAC
Use-Dilution Method of disinfectant efficacy testing. If consistency of
penicylinder manufacture can be assured in regard to this quality, then I
will recommend that S&L stainless steel penicylinders be specified in the
Official Methods of Analysis of the AOAC.
I thank you again for your cooperation in resolving this problem.
Sincerely,
^
Eugene C. Cole,Dr.P.M.
Research Associate
Associate Referee, Use-Dilution Method
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67
Comparison of PseudomQna.s Pellicle Removal Methods
Background:
Pseudomonas aeruginosa ATCC 15442 when grown in a liquid medium produces
a dense mat of growth, or pellicle, at the broth/air interface. Clumps
from the pellicle can introduce significant variability into the Use-
Dilution test. The effect of the pellicle on disinfectant efficacy has
been aptly demonstrated using the Use-Dilution Method and four
quaternary ammonium compounds (Rutala, unpublished data).
The official AOAC methodology currently specifies decanting of the
Pseudomonss broth culture to another tube so as to leave the pellicle
behind. This method, as well as pellicle removal by sterile vacuum
pipet is currently used by testing laboratories. Broth filtration has
been suggested as another alternative removal method.
Purpose
The purpose of this study Is to compare three methods for the effective
removal of the pellicle from Pseudononas broth cultures: suction,
filtration and decanting. The evaluation of broth variability after
pellicle-removal is based on an assessment of phenol resistance,
bacterial load on penicylinders, and use-dilution testing.
Media and Reagents:
1. Nutrient Broth (AOAC 4.001a) using Anatone and Beef Extract
(Difco) as specified. Prepare without boiling, using glass
distilled water. Adjust pH to 6.8 prior to sterilization.
2. Letheen Broth (Difco) . Prepare as directed using glass distilled
water.
3- Quaternary ammonium disinfectant. Prepare at recommended use-
dilution concentration of 1:256, using glass distilled water.
4. Glass distilled water. Sterile Water for Irrigation, USP
(Travenol).
Methodology:
1. Transfer stock culture of Pseudomonas aeruginosa ATCC 15442 to 10
ml nutrient broth in 20x150 mm screw capped tube. Incubate
overnight at 37°C with cap loose.
2. Make at least three consecutive 24 h broth transfers using 4mm id
loop with incubation at 37°C. Inoculate sixteen 10 ml tubes for
one day's testing.
3. Prepare S&L SS penicylinders by autoclaving, sonication (for at
least 5 min), and continue processing as in AOAC 4.009.
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68
U. On each day of testing 80 penicylinders undergo use-dilution
testing - 20 for each of the three removal methods and 20 that are
inoculated in broth containing the pellicle. For each removal
method 3-4 tubes of 48-5*111 culture broth are pooled after pellicle
removal to be used for penicylinder seeding. Decanting involves
pouring the broth slowly into another sterile tube, thus leaving
the intact pellicle on the wall of the tube. Suction requires a
side-arm flask vacuum system with a sterile Pasteur pipette for
pellicle removal. Broths are then aseptically decanted into a
sterile tube. Filtration requires that culture broths be
carefully decanted through 4 layers of sterile cotton gauze (4in x
4in) into a sterile tube. As a positive control or worst case
situation, culture broths with pellicles are vortexed for 10 sec
on a Vortex-Genie at setting No. U. After broths have been
prepared, they undergo use-dilution methodology, determination of
resultant bacterial load on carriers, and phenol resistance
testing.
5. Tubes containing sterile penicylinders are drained of asparagine
and 22 carriers are aseptically transferred to a clean, sterile
25x150 mm tube. Twenty-two ml of prepared broth is added and
cylinders are allowed to remain for 15 min at room temperature,
after which they are aseptically removed, placed on end in a
sterile glass petri dish (11 per dish), matted with 2 layers of
Whatman No. 2 filter paper and dried at 37°C for 10 min.
6. Twenty dried penicylinders undergo use-dilution testing, while two
are used for bacterial load determination. To determine bacterial
load, each penicylinder is placed in a 20 x 150 nun screw-capped
tube containing 10 ml of phosphate buffered dilution water (AOAC
4.020f) and vortexed for one minute at No. 4 setting. Buffer is
then further diluted to 10-4 and one ml aliquots are plated in
duplicate using pour plate technique and Difco Plate Count Agar.
Plates are incubated at 37°C for 48h and those plates are counted
showing colonies between 30 and 300. Phenol resistance is
performed using the loop method (AOAC 4.009).
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69
Results:
Kse-Dilution
No. of Positives
Total
Phenol RealBt.aprf
Eecant
P_ecani
0/80
000
denotes failure
Suction
Filtep
1/80
1/80
1:90 Dilution
Suction
++0*
+00*
+++
+00*
Decant
Suction
Filtration
Mean
5.745
5.816
5.867
Variance
(.073)
(,010)
(.025)
HoJeooval
0/20
0/20
0/20
0/20
0/20
1/20
0/20
0/20
1/20
0/20
0/20
0/20
20/20
17/20
1/20
10/20
48/80
Filtao
+++
000*
+00*
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70
Discussion:
Comparison of pellicle removal methods shows no significant differences
based upon use-dilution results, phenol resistance testing, and
bacterial load data. The effect of retaining the pellicle in use-
dilution testing has been aptly demonstrated.
The suction and filtration methods of pellicle removal are in fact
refinements of the decant procedure since decanting is still performed
after pellicle removal by each of the two methods.
There are no significant differences among the three removal methods
based upon the limited use-dilution testing with one disinfectant in
this study.
Phenol resistance testing did reveal failures with the 1:90 dilution for
each of the three removal methods. The suction method exhibited 3
failures out of the H determinations; filtration showed 2 out of 4; and
the decant procedure failed only once out of the four tests. The
significance of these results is questionable, however, due
to recognized error of loop sampling and a limited number of
replicates.
Recommendations:
Based upon these study results, and considering ease and practicability
of the removal methods, it is recommended that sterile pipet suctioning
followed by decantation, be the approved method of pellicle removal in
the Use-Dilution test.
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71
Alteration of Broth Medium to Effect Reduction or Inhibition
of Pseudomonas Pellicle
Background: Pseudomonas aeruginosa 15442, when grown in a liquid
medium, produces a dense mat of growth, or pellicle, at
the broth/air interface. Clumps from the pellicle can
introduce significant variability into the Use-Dilution
test. The effect of the pellicle on disinfectant
efficacy has been aptly demonstrated using the
Use-Dilution method and four quaternary ammonium
compounds (Rutala, unpublished data).
Purpose:
To investigate a number of broths with varying pH,
differing concentrations of sucrose or dextrose, and the
presence or absence of a surfactant, in order to achieve
an inhibition or reduction of the Pseudomonas pellicle.
Media and
Reagents:
1. Nutrient broth AOAC 4.001, pH 6.82
2. Nutrient broth AOAC 4.001, pH 8.22
3. Disinfectant Test (DT) Broth (Difco), pH 6.87
4. FDA Broth (BBL), pH 6.79
5. Synthetic Broth AOAC (Difco), pH 8.32
6. Synthetic Broth AOAC (Difco), pH 7.11
7. ]Q% sucrose/10^ dextrose, used with Synthetic Broth
AOAC, 0.1 ml/tube
8. Tween 80 (J.T. Baker Chemical Company)
9. Dextrose (BBL); Sucrose (Fisher)
10. Potassium gluconate broth (substituting dextrose for
potassium gluconate):
tryptone 1.5g
yeast extract 1.Og
potassium phosphate, dibasic 1.0g
dextrose, 40.Og
distiI led water, 1L
adjust pH to 7.0 and Filter sterilize
11. Distilled Water (Sterile Water for Irrigation, USP,
TravenoI)
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72
Methodology A: 1.
Transfer stock culture of IP. aeruginosa 15442 to
10ml of each broths No. 1-6, in 20 x 150mm new,
screw-capped, flint glass tubes, and incubate
overnight at 37 C. (Synthetic Broths are prepared
with sucrose and dextrose).
2. Make at least 4 consecutive broth transfers using
4mm id loop before ffna! inoculation of 2 tubes of
each of the 9 media.
3. To each of the duplicate broth tubes, one drop of
Tween 80 is carefully added to the surface of the
broth. Incubation fs upright at 37 C for 48 hours.
Cultures are examined at 24 and 48 hours in
comparison to the Nutrient Broth AOAC, pH 6.8, with
no tween added, which serves as a control.
Results:
Broth
Nutrient,
pH 6.8,
pH 6.8,
pH 8.2,
pH 8.2,
DT Broth:
pH 6.8,
pH 6.8,
FDA Broth;
pH 6.8,
pH 6.8,
Synthetic
pH 7.1,
PH 7.1,
pH 8.3,
pH 8.3,
pH 7.1,
pH 7.1,
pH 8.3,
pH 8.3,
AOAC:
tween
no tween
tween
no tween
tween
no tween
tween
no tween
dextose, tween
no tween
dextrose, tween
no tween
sucrose, tween
no tween
sucrose, tween
no tween
Pellicle Formation (48hr)
Stringy, surface ring, fragmenting
NormaI
No pel Iicle
NormaI
Reduced, stringy, fragmenting
Thinner than control
Reduced, unattached, fragmenting
Thinner than control
Extensive, droopy, fragmenting
Normal (extensive)
Reduced, droopy, fragmenting
NormaI
Small, unattached, stringy,
fragmenting
NormaI
Reduced, unattached, nonfragment ing
NormaI
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73
Conclusions:
1. Greatest pellicle reduction was in Nutrient Broth,
pH 8.2, with tween. At 24hr there was no pellicle
formed, only a sMght ring of growth around the
tube. At 48hr, pellicle had formed but was small,
with no glass attachment. This pellicle did not
fragment when the tube was shaken. Easily removable
with pi pet.
2. Synthetic broth, pH 8.3 with tween and sucrose,
exhibited a reduced pellicle that formed a single,
droopy, unattached strand that was not very
fragmenting and appeared easily removable with a
plpet.
3. In any consideration of adopting these or other
altered media into the standard methodology,
attention must be given to retaining the organisms'
proper disinfectant resistance.
Methodology B:
Filter sterilize Nutrient Broth AOAC 4.001 with
final sucrose concentrations of 6%, B% and 10? and
place 10ml allquots into 20 x 150mm tubes. Do the
same with dextrose concentrations of 4?, 6%, 8% and
10*.
Inoculate standard Nutrient Broth AOAC 4.001 from
stock slant, making three additional 24hr transfers.
Using 4mm id loop, inoculate 2 tubes of nutrient
broth for each of the 3 sucrose and 4 dextrose
concentrations from the original broth.
Incubate all tubes at 37 C, observing pellicle
formation at 24 and 48 hours.
Results:
1. After 24hr incubation, there was no inhibition of
pellicle formation observed with any of the media
regardless of the concentrations of dextrose and
sucrose uttI I zed.
2. After 48hr incubation, there was no reduction of
pellicles observed with either of the media at any
of the concentrations of dextrose and sucrose
utiIized.
Conclusion: Although sucrose and dextrose have been reported to
inhibit slime production in Pseudomonas aeruginosa in
low concentrations (4#), there was no observed pellicle
inhibition or reduction in this study which utilized
several concentrations.
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Methodology C: 1.
74
Filter sterilize potassium gluconate broth (Haynes,
J. Gen. Micro 5, 939-950, 1951) substituting dextose
for potassium gluconate as recommended to Inhibit
slime formation.
2. Inoculate 10ml aliquot of this modified broth from
stock slant, making consecutive 24hr transfers for 5
days.
3. Examine dai ly the 24hr and 48hr broths.
Results:
1. At 24 hours there is a slight reduction of pellicle
growth as compared with Nutrient Broth AOAC. The
pellicle appears thin, but it does cover the entire
broth surface, with some growth on the glass above.
2. At 48 hours there is no significant pellicle
reduction. The pellicle easily fragments when the
tube is disturbed.
Conclusion: Hayne's medium does not effectively prevent or reduce
the formation of a pellicle at the broth surface.
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75
Electron Microscopy of Pseudomonas Pellicle
A.
B.
D.
Intact pellicle.
High magnification of pellicle showing an interlacing network of
cells. Differences in contrast among the organisms may indicate
viable and non-viable cells.
Pellicle fragment attached to a stainless steel penicylinder showing
some densely packed cells.
Large and extremely dense pellicle fragment attached to a stainless
steel penicylinder. Germicide exposure of all cells may be
compromised, leading to false-positive results.
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76
Standardization of Bacterial Numbers on Disinfectant Test Penicylinders:
Interlaboratory Study
EUGENE C. COLE, WILLIAM A. RUTALA, AND GREGORY P. SAMSA.
The University of North Carolina at Chapel Hill, Department of Medicine,
Division of Infectious Diseases, Chapel Hill, NC 27514
An interlaboratory study was conducted to evaluate a method of standardizing
bacterial numbers on disinfectant test penicylinders used in the AOAC
Use-Dilution Method (A.007-4.015) of disinfectant testing. Eight
participating laboratories followed a standardization method using their media
and scock cultures of Staphylococcus aureus ATCC 6538, Salmonella choleraesuis
ATCC 10709, and Pseudomonas aeruginosa ATCC 15442. The culture broths that
were used to inoculate the penicylinders were incubated for 48h at 37°C after
several (4-6) 24h passages. McFarland turbidity standards of 1.0 and 0.5 were
used to visually standardize the cultures of S. aureus and P. aeruginosa
respectively. j>. choleraesuis was used undiluted. The results showed
significant variability in numbers of test bacteria which adhered to the
penicylinders, with mean values of 1.6 x 10 for £. choleraesuis, 3.5 x 106
for J3. aureus and 8.2 x 10 for £. aeruginosa. The results from collaborating
laboratories attempting standardization of bacterial numbers on penicylinders
demonstrated significant interlaboratory and cylinder variation for all three
test organisms.
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77
INTRODUCTION
Previous investigations have demonstrated significant differences in the
numbers of bacteria dried onto the surfaces of stainless steel penicylinders
(1) used in the AOAC Use-Dilution Method (2). The numbers of the three AOAC
recommended test bacteria which adhered to the stainless steel penicylinders
varied widely, with mean values of more than 10 for £. aeruginosa,
approximately 5 x 10 for S. aureus and 10 or less for S. choleraesuis.
Presumably, this variability among the three test bacteria is due to unique
morphological and physiological characteristics, which affect adherence as
well as bacterial numbers In broth. These data suggest that disinfectants are
not similarly challenged by the AOAC test bacteria, and alternative
methodologies should be considered to ensure comparable bacterial numbers on
penicylinders.
Studies were conducted to standardize bacterial numbers on disinfectant
test penicylinders. Preliminary investigations demonstrated that comparable
numbers of bacteria may be obtained using various McFarland turbidity
standards. An Interlaboratory study was thus undertaken to: (1) assess
laboratory to laboratory variation of bacterial numbers on penicylinders when
a standardized culture broth is employed, and (2) determine if more equitable
numbers of bacteria on penicylinders were attainable using the three test
organisms.
Interlaboratory Study
Those participating in this study included 4 industrial research laboratories,
3 laboratories from state government and one academic research laboratory.
Participants were sent a protocol detailing the method to be used with their
penicylinders and Use-Dilution bacteria and were also instructed to complete a
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4»
79
Apparatus
(a) Vortex mixer. Vortex-Genie (Fisher Scientific, Pittsburgh, PA
15219).
(b) Colony counter. (New Brunswick Scientific, New Brunswick, NJ).
(c) Test tubes. 25 x 150mm; 20 x 150mra screw-capped (SC).
(d) Pe^tri dishes. (1) glass, 20 x 100mm; (2) polystyrene, 15
xlOOmm.
Organism and Penicylinder Preparation
From a stock culture slant make from 4 to 6 consecutive 24hr broth
transfers of test organisms in 10ml of nutrient broths. Keep caps loose and
incubate at 37 C. Final broth for penicylinder inoculation must be in 25 x
150mm tube. Incubate broth culture at 37°C for 48hr. Prepare stainless steel
penicylinders as in AOAC 4.009.
Standardization of Culture Broths
For the inoculation of penicylinders use 48hr broths. Use S_.
choleraesuis broth undiluted; adjust turbidity of S. aureus to that comparable
with a 1.0 McFarland standard; adjust turbidity of P. aeruginosa to that
comparable with a 0.5 McFarland. For Pseudomonas , an aliquot of broth is
removed for turbidity adjustment by tilting the tube and guiding a pipet under
the pellicle. Alternatively, a sterile Pasteur pipet with a vacuum source may
be used to remove the pellicle. The turbidity of broth cultures is adjusted
with sterile phosphate buffer dilution water (PBDW) to obtain a turbidity
visually comparable to that of the turbidity standard. The turbidity standard
and broth tube are agitated on a vortex mixer immediately prior to use. For
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jflt
80
turbidity adjustment, simultaneously view a white background and contrasting
black line with an adequate light source through both tubes.
Inoculation of Penicylinders
Transfer 10ml of adjusted broth to a 25 x 150mm sterile tube. Pour off
asparagine from prepared penicylinders, using a sterile pipet to withdraw any
remaining solution from the bottom of the tube. Briefly vortex (1.0 sec) the
broth In the tube to be used for inoculation, and using a flamed wire hook,
aseptically transfer 10 penicylinders to the broth. The tube may be shaken to
rearrange the penicylinders so all penicylinders are submerged. Let tube sit
undisturbed at room temperature for 15 minutes. Aseptically remove
penicylinders from tube and place on end In sterile glass petri dish matted
with two layers of Whatman No. 2, 9 cm filter paper. Incubate covered dish at
37°C for 40 min.
Removal and Quantitation of Organisms
Place each of 5 randomly-selected penicylinders into a 20 x 15mm SC tube
containing 10ml sterile PBDW. Spin each tube on a vortex mixer, setting #4,
for one minute. Using 1.0ml transfer aliquots, prepare serial dilutions in
PBDW to 10~ . Using a pour plate method, plate 10~3 and 10~4 dilutions in
duplicate using 1.0ml samples and 15- 17ml of plate count agar at 42-4 5°C.
Incubate plates for 48hr at 37°C and count those with colonies between 30 and
300.
Replicates
Using the above procedures, process 5 carriers per day on 3 consecutive
days, for each of the three test organisms.
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81
Statistical Analysis
For each organism, a nested random effects analysis of variance model was
used to estimate components of variance and variance proportions (Table 1).
This was accomplished as follows. For each cylinder the amount of variation
in numbers of organsisms between separate replicates was calculated. This
variation is then averaged over all cylinders to obtain the component of
variance for "replicates within cylinders." This result measures the typical
variability between replicates after accounting for all other sources of
variation (i.e., cylinders, days and laboratories). Similar calculations are
made to obtain the components of variance for "cylinders within days," "days
within laboratories" and "laboratories," using as input the number of
organisms per cylinder, the average number of organisms per day and the
average number of organisms per laboratory, respectively. Variance
proportions simply place the components of variance on a relative, rather than
an absolute scale, and each variance proportion is reported "per 100% of total
variability in numbers of organisms."
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82
Results and Discussion
The data on bacterial counts from inoculated penlcylinders for all
participating laboratories are presented in Figure 1. Table 1 analyzes the
variability of these data. Interlaboratory variability was high for all three
test bacteria but particularly with £. aureus and P. aeruginosa. Day to day
variability was high with JP. aeruginosa, while cylinder variation (within a
batch) was extremely high, with variation of 70.70%, 47.352 and 28.24% for S.
choleraesuis, £. aureus and JP. aeruginosa, respectively. These data
demonstrate that most of the variability in organism numbers observed may be
attributed to differences between cylinders. These data show that even when a
standardized method for adjusting broth cultures is used there remains a
significant difference in mean bacterial numbers on stainless steel
penicylinders with 1.6 x 10 for £. choleraesuis, 3.5 x 106 for £. aureus and
8.2 x 10 for JP. aeurginosa. In addition, although diluted cultures of
Pseudomonas and Staphylococcus provided fewer organisms per penicylinder than
previously reported data, dilution did not provide more equitable bacterial
numbers on penicylinders among the three test strains (Figure 1). Further
dilution of Pseudomonas and Staphylococcus broth cultures may be attempted to
achieve equitable bacterial numbers on penicylinders.
Although this study utilized a standard procedure for the preparation of
broth cultures used to inoculate penicylinders, the methodology employed in
each participating laboratory was not identical. For example, the
penicylinders used varied in brand and age. Questionnaire responses revealed
that 4 laboratories used the recommended S&L peniclyinder, one laboratory used
Fisher penicylinders (Fisher Scientific) and another used a combination of
both brands. Decontamination and treatment of penicylinders prior to re-use
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83
also varied. Treatment methods included rinsing in water, exposure to
sonication and immersion in detergent solution. In addition, oae laboratory
(No. 6) reported difficulty when visually standardizing broth cultures.
Conclusions
An interlaboratory study to assess a method of standardizing bacterial
numbers on disinfectant test penicylinders revealed an inability to attain
comparable cell numbers. The results also demonstrated significant
interlaboratory and cylinder variation for all three test bacteria. It is
recommended that alternative methodologies (e.g., suspension tests) be
evaluated to ensure a comparable bacterial challenge when performing
disinfectant efficacy testing.
Acknowledgement s
The authors express thanks to the following study participants: A. Bittle and
S. Dahiya, North Carolina Department of Agriculture, Raleigh, NC; A. Buzin and
G. Fischler, Colgate-Palmolive Company, Piscataway, NJ; W. De Castro, Airwick
Industries, Inc., Secaucus, NJ; D. Fredell, Economics Laboratory, Inc.,
Mendota Heights, MN; F. Porter and B. Woodward, Florida Department of
Agriculture, Tallahassee, FL.
This work was funded by the U.S. Environmental Protection Agency, Office of
Pesticide Programs, under cooperative agreement CR811204-01-0 to the
Univeristy of North Carolina at Chapel Hill.
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84
References
(1) Cole, E.G., Rutala, W.A., & Carson, J.L. (1985) Abs. Ann. Meeting Am.
Soc. Microbiol., Q41, p. 264.
(2) Official Methods of Analysis (1984) 14th ed., AOAC, Arlington, VA,
Chapter 4, Disinfectants,
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85
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456
LABORATORY
Figure 1. The vertical lines for each laboratory represent daily mean numbers
of bacteria on penicylinders using visually adjusted broths of S.
aureus aad P. aeruginosa and an unadjusted broth of S. ~
choleraesuis. Interlaboratory, day to day, and cylinder
variability of bacterial numbers on penicylinders among
laboratories is also shown.
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86
Table 1. Variability (%) of bacterial numbers on penicylinders
Variation
Inter laboratory3
Day to day
Cylinders0
Replicates
S. choleraesuis
16.85
10.40
70.20
2.55
S. aureus
44.59
6.05
47.35
2.01
P. aeruginosa
40.10
22.40
28.24
9.26
Percent of total variability from laboratory to laboratory considering
all data.
Percent of varibility within a typical laboratory on a daily basis.
°Percent of variability within a typical batch (5 cylinders per organism
per day).
Percent of variability for a given penicyliader.
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87
Bacterial Numbers on PenicyIinders Used In Disinfectant Testing: Use of
Twenty-four Hour Standardized Broth Cultures
EUGENE C. COLE AND WILLIAM A. RUTALA.
The University of North Carolina at Chapel Hill, Department of Medicine,
Division of Infectious Diseases
Chapel H» I 1, NC 27514
The current AOAC Use-Dilution Method of disinfectant efficacy testing
requires the use of 48hr non-standardized broth cultures of Salmonella
choleraesuis, Staphylococcus aureus and Pseudomonas aeruginosa for the
inoculation of stainless steel penicyIInders. This results in
non-comparable numbers of organisms on pen icy Iinders among the test
strains. In an atttempt to ensure more equitable bacterial numbers on
the penicyIinders, 24hr Nutrient and Synthetic broth cultures were
utilized. ^. choleraesuts broth was used undiluted
-------
be given to the use of 24hr broth cultures for the tnoculat'on of
penlcy I inders in the Use-DHution Method.
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89
INTRODUCTION
The AOAC Use-Dilution Method (1) requires the use of 48-54hr
bacterial broth cultures for the inoculation of stainless steel
penicyfinders used in disinfectant testing. Previous investigations
have demonstrated significant differences in the numbers of bacteria
dried onto the surfaces of these stainless steel penicyIinders (2). The
three AOAC test bacteria adhered to the penicyIinders differently, with
more than 10 for P_. aeruglnosa, approximately 5 x 10 for j^. aureus and
10 or less for ^. choleraesuis. Presumably this is due to different
morphological and physiological differences among the three test
organisms which affect adherence as well as bacterial numbers in broth.
Thus, these data suggest that disinfectants are not similarly challenged
with the AOAC test bacteria.
In an attempt to assure more comparable numbers of cells on the
penicyIinders, among the three test strains, the use of 24hr broth
cultures was evaluated. Several disinfectant test methodologies of
other countries utilize 24hr broth cultures, as do five of the other
AOAC disinfectant test procedures (1). It is also recognized that in
general, for disinfectant test purposes, 18-24hr broth cultures have
been found to be the most suitable (3). Presumably such cultures
represent the range of maximum cell density per ml. Growth studies have
been initiated using ^. choleraesuis and P_. aeruginosa for the
determination of cell numbers in broth at timed intervals, along with
corresponding numbers of cells attaching to the penicyIinders.
Results showed that the use of the 24hr broth cultures for all
three test strains along with standardized adjustment of S. aureus and
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90
F\ aeruginosa broths contributes to more comparable test challenges In
the Use-Dilution Method.
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91
METHODS
Media and Reagents
(a) Nutrient Broth.—AOAC 4.001(a), 10m! in 20 x 150 mm tubes using
Anatone (American Laboratories, Inc., Omaha, NE 68127), and Beef Extract
(Difco Laboratories, Detroit, Ml 48232) as specified. Prepare according
to directions using glass distMled water. Adjust the pH to 6.8 prior
to ster IIizatton.
(b) Synthetic broth. —-Bacto Synthetic Broth AOAC (Dlfco Laboratories)
(c) Pour plate agar.—Bacto Plate Count Agar (Dlfco Laboratories).
(d) DlstiI led water.--Sterile Water for Irrigation, USP (Travenol
Laboratories, Inc., Deerfield, IL 60015).
(e) Phosphate buffer dilution water.—AOAC 4.020(f).
(f) BaHum chloHde.—Crystals, reagents grade.
(g) Suituric acid.—ACS reagent grade.
(h) McFarland standards.—Prepare 1^ (w/v) aqueous sulfuric acid
solutions using distilled water. For a 1.0 standard, add 1.0 ml of the
barium chloride solution to 9.9 ml of the "\% sulfuric acid. This will
Q
approximate a bacterial density of 3.0 x 10 cells/ml. For a 0.5
standard add 0.05 ml of the barium chloride solution to 9.95 ml of the
\% sulfuric acid. This will approximate a bacterial density of 1.5 x
Q
10 cells/ml. Prepare fresh standards each week and protect from light.
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92
Organ isms
Stajphry lococcus aureus 6538, Sal none I la choleraesuis 10708, P^seudompnaj
aeruglnosa 15442 (American Type Culture Collection, RockviIle, MD
20852>.
Apparatus
(a) Vortex mixer.—Vortex-Gen'e (Fisher Scientific, Pittsburgh,
PA 15219).
(b) Colony counter.—New Brunswick Cjolony Counter (New Brunswick
Scientific, New Brunswick, NJ), ore equivalent.
(c) Pen Icy Iinders.—S&L Metal Products Corp., Maspeth, NY 11378.
(d) Sonic cleaner.—E/MC model 450 (RAI Research Corp.,
Hauppauge, NY 11787), or equivalent.
(e) Inoculating loop.—4mm Id p latlnum/rhod'urn loop (American
Scientific Products, McGraw Park, ID
Culture Broth Preparation
From stock culture slant make at least 4 consecutive broth
transfers (using 4mm id loop), of test organisms in 10ml broths. Keep
caps loose and incubate broths at 37°C. Final broths for penicylinder
inoculation must be in 25 x 150mm tubes. Incubate broths at 37°C for 24
hr.
Penicylinder Preparation
Previously used S&L penlcyllnders are decontaminated by steam
sterilization, followed by sonlcatlon for at least 5 mln, rinsed with
distilled water, and processed as in AOAC 4.009. Cleaned penlcyllnders
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93
are then placed in groups of 10 into 20 x 150mm screw-capped tubes and
covered with 0.1$ asparagine. Tubes are then sterilized at 121 C for 15
min and allowed to cool.
Standardization of Culture Broths
For the inoculation of penicylinders use 24hr broths. Use j^.
choleraesuls broth undiluted; adjust turbidity of ^. aureus to that
visually comparable with a No. 1 McFarfand standard; adjust turbidity of
P_. aeruginosa to that visually comparable with a 0.5 McFarland. For
Pseudomonas, remove an aliquot of broth to be adjusted by tilting the
tube and guiding a pipet under the pellicle. Alternatively, a sterile
Pasteur pipet with a vacuum source may be used to remove the pellicle.
The turbidity of broth cultures are adjusted with PBDW to obtain a
turbidity visually comparable to that of the turbidity standard. The
turbidity standard and broth tube are agitated on a vortex mixer
immediately prior to use. For turbidity adjustment, simultaneously view
a white background and contrasting black line with an adequate light
source through both tubes.
Inoculation of Pen icy Iinders
Transfer 10ml of test broths to 25 x 150mm sterile tubes. Pour off
asparaglne from prepared carriers, using sterile pipet to withdraw any
remaining solution in bottom of tube. Briefly vortex (1.0 sec) broth in
tube to be used for inoculation, and using a flamed wired hook,
aseptically transfer 6 pen icy Iinders to the broth. The tube may be
shaken to rearrange the cylinders so all are submerged. Let tube sit
undisturbed at room temperature for 15 minutes. Aseptically remove
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94
carriers from tube and place on end in sterl le glass petrl dish matted
with two layers of Whatman No. 2, 9cm filter paper. Incubate covered
dish at 37°C for 40 min.
Removal and Quant I tatI on of Organisms
Place each of the 6 Inoculated pentcyfinders Into a 20 x 150 mm
screw-capped tube containing 10ml sterile PBDW. Spin each tube on a
vortex mixer, setting #4, for one minute. The efficiency of this
removal method has been demonstrated (unpublished results). Using 1.0ml
transfer allquots, prepare serial dilutions In PBDW to 10~ . Using a
pour plate method, plate 10*" and 10~ dilutions In duplicate usfng
1.0ml samples and 15-17 ml of plate count agar at 42-45°C. Incubate
plates for 48hr at 37°C and count those with colonies between 30 and
300.
Cell Attachment According to Growth Curves
Since preliminary data Indicate the greatest difference In numbers
of bacteria attaching to penlcyIInders to be between S_. choleraesuls and
P_. aeruglnosa, growth studies of both organisms are conducted. Seven
10ml tubes of nutrient broth for each organism are Inoculated and
transferred as described above. At specific Intervals (6hr, 12hr, 18hr,
etc.), two I.Omf aliquots from a single broth tube are removed, serially
diluted In PBDW, and duplicate pour plates are prepared. The plates are
incubated and qunatltated as described above. At Intervals of 18, 24,
36, 48 and 54 hrs after removal of aliquots, 5.0 ml of broth culture of
each organism is placed Into sterile 25 x 150mm tubes for the
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95
inoculation of S&L pentcylindens and subsequent cell removal as
described above.
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96
Results and Discussion
As presented in Table 1, growth studies of S^. choleraesuls showed
maximum numbers In broth and attachment to penicylinders at 18hrs, with
a mean of 7.7 x 10 cells/ml of broth, and a mean of 3.1 x 10
eelIs/cyUnder. At 24hrs only 0,37% of S_. choleraesuls cells in broth
culture attached to the penicylinders as opposed to 3.0$ for P^.
aerugInosa (Table 2).
This study showed that numbers of ^. choleraesuls attaching to
stainless steel penlcyIInders inoculated with undiluted 24hr culture
broths were consistently above 10 . Several replicates approached or
exceeded 5 x 10 cells per pen icy Under. Results of a 11 three test
bacteria attaching to penicyllnders in both Nutrient and Synthetic
broths are presented In Table 1. ^. aureus, with turbidtty visually
adjusted to that of a 1.0 McFarland standard, demonstrated mean numbers
of organisms on penicylinders comparable to those of _S. choleraesuts.
Numbers of P_. aerug t nosa attaching to pen icy Unders, after broth culture
dilution comparable with 0.5 McFarland, remained significantly higher
than the other two test strains.
It has been demonstrated that the use of 24hr broth cultures can
Increase the numbers of _S. choleraesuls consistently above 10 , thus
producing a bacterial challenge to germicide testing that Is comparable
to ^. aureus. P^. aerug I nosa broth cultures would require further
dilution beyond that employed In this study in order to achieve
comparability with the other two test bacteria.
It »s recommended that the use of 24hr broth cultures be considered
in the future revision of the Use-Dilution Method.
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97
AcknowIedgement
This work was funded by the U.S. Environmental Protection Agency,
Office of Pesticide Programs under Cooperative Agreement CR811204-02 to
the University of North CaroHna at Chapel Hill.
REFERENCES
(1) Official Methods of Analysis (1984) 14th ed., AOAC, Arlington, VA,
Chapter 4, Disinfectants.
(2) Cole, E.G., Rutala, W.A., & Carson, J.L. (1985) Abs. Ann. Meeting
Am. Soc. Mtcrobiol., Q41, p. 264.
(3) Crenriieux, A., & Fleurette, J. (1983) in Disinfection,
Sterilization and Preservation, S.S. Block (ed), Lea & Febiger,
Philadelphia, PA, p. 921.
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98
Table 1. Mean numbers of S_. choleraesuts ATCC 10708 In broth and on
stainless steel pen* cylinders
Urne Broth Penlcy 1 Inders
6
12
18
24
36
48
54
1.6 x
3.8 x
7.7 x
5.9 x
6.3 x
5.9 x
3.9 x
108
108
108 3.1 x 106
108 2.2 x 106
108 2.4 x 106
108 1.9 x 106
108 1.8 x 106
% Attached
.37
.37
.38
.32
.46
Mean vaIues of 2 rep I!cates
Mean values of 3 repMcates
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99
Table 2. Mean numbers of P. aerugInosa ATCC 15442 'n broth and on
stainless steel pen(cylinders
Time (h)
6
12
18
24
36
48
54
5
5
8
1
1
1
5
Broth
.1 X
.5 x
.4 x
.1 x
.2 x
.1 x
.2 x
10
10
10
10
10
10
10
a
8
8
8
Q
y
9
9
8
Pen
3.
3.
2.
2.
9.
Icyl
2 x
3 x
7 x
8 x
3 x
!indersb
10
7
7
10'
10
10
10
7
7
6
£ Attached
3
3
2
2
1
.8
.0
.3
.6
.8
Mean values of 2 replicates
Mean values of 3 repMcates
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100
Table 3. Mean (n=6) numbers of bacteria on S&L penicyItnders following
Inoculation with standardized3 24hr broths
Organisms
NutHent Broth
Synthetic Broth
S. aureus
S. choleraesuis
3.4 x 10
2.4 x 10
3.8 x 10
6b
3.8 x 10
6c
P. aeruginosa
1.4 x 10
8.4 x 10
' S_. aureus broth adjusted to turbfdtty of 1.0 McFarland standard; P_.
aerugjnosa broth adjusted to turbidity 0.5 McFarland standard; S_.
choleraesuls broth undiluted.
Mean value of twelve replicates.
"Mean value of fWe repMcates.
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Evaluation of Asparagine in the AOAC Use-Dilution Method
EUGENE C. COLE AND WILLIAM A. RUTALA
Department of Medicine, Division of Infectious Diseases
The University of North Carolina at Chapel Hill
Chapel Hill, NC 27514
An asparagine solution (0.1$) is required in the AOAC Use-Dilution Method to
cover stainless steel carriers during sterilization and storage. The
rationale of an asparagine soak is unclear. It has been proposed that
asparagine enhances bacterial adherence to carriers or inhibits corrosion of
the metal carriers or both. The former theory was assessed in this study by
comparing bacterial adherence on seeded carriers stored in 0.1/f and 1$
asparagine solution to those stored dry or in distilled water. The anti-
corrosive properties of asparagine on stainless steel carriers was
microscopically evaluated after a 9 month period. The results demonstrate
that asparagine neither enhanced bacterial adherence to carriers nor is
necessary to prevent corrosion.
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Asparagine solution 0.1? (AOAC Jj.OOTe) is required in the Association of
Official Analytical Chemists (AOAC) Use-Dilution Method (1) to "cover metal
carriers for sterilization and storage". The asparagine soak takes place
prior to transferring the carriers to a broth culture for inoculation with one
of three recommended test bacteria. The Use-Dilution Method however, does not
describe a specific function for the asparagine. Two theories have been
proposed for the inclusion of asparagine in the methodology: (1) to enhance
bacterial adherence to the stainless steel carrier surface; and (2) to inhibit
corrosion of the stainless steel carriers.
Asparagine is the beta-amide of aspartic acid. It is a non-essential amino
acid, and in 1806 was the first amino acid to be isolated. The Bacto-
Asparaglne (Difco Laboratories, Detroit, MI 48232) specified in the Method
(AOAC 4.007e) is L-asparagine of exceptional purity (2). It is occasionally
used as an ingredient of culture media where it serves as a source of organic
nitrogen (2).
This paper compares numbers of test bacteria seeded on penicylinders stored in
asparagine with those stored dry or in distilled water, and also examines the
effect of asparagine on inhibiting corrosion of stainless steel carriers. The
data demonstrate that asparagine neither promotes bacterial adherence to
stainless steel carriers nor is necessary to prevent corrosion of the carrier.
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Experimental
Media and Reagents
(a) Nutrient broth.—(AOAC l4.001a) using Anatone (American Laboratories,
Inc., Omaha, NE 68127), and Beef Extract (Difco Laboratories, Detroit,
HI 48232) as specified. Prepare according to directions using
distilled water. Adjust the pH to 6.8 prior to sterilization.
(b) Pour Plate Agar..—Plate Count Agar (Difco)-Bacto Tryptone 5.0g, Bacto
Yeast Extract 2.5g, Bacto Dextrose 1.0g, Bacto Agar 15.Og, per liter.
(c) Glass distilled water.— Sterile water for irrigation, USP (Travenol
Laboratories, Inc., Deerfield, IL 60015).
(d) Laboratory distilled (non-glass) water.—Less than 1.5 megohm
resistance.
(e) Asparagine.— Eacto-Asparagine (Difco) 0.1$ and 1.0$ (w/v) aqueous
solutions.
(f) Ethanol.—(1)v/v. 95$ for concentrations of 25$, 50$, 75$, 95$. (2)
Absolute alcohol.
(g) Freon 13.—(E.I. duPont de Nemours and Co., Wilmington, DE).
Freon 113.—(Polysciences Inc., Warrington, PA),
Fixative.—2$ glutaraldehyde/2$ paraformaldehyde. Glutaraldehyde 50$
(Ted Pella, Inc., Tustin, CA). Paraformaldehyde, reagent grade (Fisher
Scientific, Pittsburgh, PA 15219).
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(j) Sgrenson's Phosphate buffer. 0.2M.—To 363.5ml distilled water add
10.325g sodium phosphate, dibasic, (Fisher Scientific) dissolve and add
136.5ml distilled water and 3«7l5g potassium phosphate, monobasic.
Adjust pH to 7.3.
(k) Sorenson's phosphate buffer. 0.1M.—Dilute Sorenson's 0.2M buffer 1:2
with distilled water.
(1) Osmium tetroxide, I.Ot.—Dissolve 1.Og osmium (Electron Microscopy
Sciences, Ft. Washington, PA) in 50.0ml distilled water and dilute 1:2
with Sorenson's phosphate buffer 0.2M.
(»»> Dilution water.—-Phosphate buffer dilution water, AOAC *J.020(f).
Organisms
Staphylococcus aureus e6538, Salmonella choJeraesuis 10708, Pseudotnonas
aeruginosa 15^2 (American Type Culture Collection, Rockville, MD 20852).
Apparatus
(a) Stainless steel penicylinders.—S&L Metal Products Corp., Maspeth, NY
11378; and Fisher Scientific, Pittsburgh, PA 15219 (purchased 12/83,
used V8U-8/8K).
(b) Vortex mixer.—Vortex Genie (Fisher Scientific).
(c) Colony Counter.—New Brunswick Colony Counter (New Brunswick Scientific,
New Brunswick, NJ).
(d) Dissecting microscope.—Bausch and Lomb, Rochester, NY 14602.
(e) Screw-capped (SO tubes.—Flint glass, 20x150mm (Fisher Scientific).
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105
(f) Petri dishes.—Polystyrene, 100x15mm (Fisher Scientific).
(g) Scanning electron microscope.—ETEC Autoscan (Perkin-Elmer Electron Beam
Technology, Hayward, CA 94545).
(h) Sonicator.—Ultrasonic Cleaner model 450 (RAI Research Corp., Hauppauge,
NY 11787).
Procedure
(a) Asparagine versus distilled water: Comparison of bacterial numbers.
Transfer stock culture of S. aureus to 10 ml nutrient broth in a 20x150 mm
screw-capped (SC) tube. Incubate the nutrient broth overnight at 37°C with
cap loose. Make at least 3 consecutive 24h broth transfers using a 4 mm i.d.
loop (AOAC 4.002d).
Prepare S&L penicylinders by steam sterilization, followed by sonication in
laboratory distilled water for 5-15 min with processing continued as in AOAC
4.009. Cleaned carriers are then placed in 20x150 mm SC tubes and covered
with 0.1% asparagine solution or laboratory distilled water. The same
distilled water is used for covering carriers as for preparing the asparagine.
Tubes with carriers are then steam sterilized at 121OC for 15 min and allowed
to cool.
Gently vortex (1.0 sec) 48-54h culture broth, withdraw 9.0 ml of §. aureus
with sterile pipet and place 4.5 ml into each of two sterile 20x150 mm SC
tubes. Aseptically transfer 4 asparagine coated carriers to one tube and 4
distilled water coated carriers to the other. Ensure that all carriers are
covered by the broth and allow to stand undisturbed for 15 min at room
temperature. As in AOAC 4.009, remove carriers from broth and place on end in
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106
sterile glass petri dish matted with 2 layers of Whatman No. 2, 9 cm filter
paper and dry at 3?oc for 40 rain. Place each carrier into 10 ml sterile
Phosphate buffer dilution water in 20x150 mm SC tube and vortex for 30 sec at
setting #14. Prepare and plate 10-3, 10-1*, and 10-5 dilutions in duplicate
with 1.0ml inoculum into petri dishes, adding 16.5ml Plate Count Agar.
Incubate 48 hr at 37 DC and count colonies from plates with between 30 and 300
colonies. Repeat procedure with S. choleraes^jg and £. aeruginosa.
(b) Asparagine concentrations with wet vs. dry carriers. - Glass distilled
water is used to prepare asparagine solutions and_S_. choleraesuis
only is used in this experiment. Broth inoculation and transfers, as well as
carrier preparation are the same as described in procedure (a). Four carriers
are then placed in each of seven 20x150 mm SC tubes. The carriers in 5 tubes
are covered with 0.1$ asparagine, and those in the remaining 2 tubes with 1.0$
asparagine. Caps are tightened and tubes are sterilized for 15 min at 121OC
and allowed to cool. Four dry carriers are placed in an eighth tube with cap
loose, and sterilized as above to include a 10 min drying cycle.
On each of 4 days, carriers prepared in the recommended 0.156 asparagine prior
to bacterial inoculation are processed for bacterial adherence and compared to
one of % variables - 1.0% asparagine, carriers used wet (Day 1), 1.0$
asparagine, carriers used dry (Day 2), 0.1$ asparagine, carriers used dry (Day
3) and no asparagine, carriers used dry (Day 4).
For each of the comparisons to be made, gently vortex (1 .Osec) 49h culture
broth, withdraw 9-0 ml with sterile pipet and place 4.5 ml into 2 sterile
25x150 mm tubes. Wet carriers to be evaluated are placed directly into the
broth culture tubes (4 per tube) and allowed to sit undisturbed for 15 min at
room temperature. Asparagine coated carriers to be used dry are placed on end
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107
in sterile glass petri dishes matted with 2 layers of Whatman No. 2, 9 cm
filter paper and allowed to remain at 37OC for HO min prior to seeding.
Carrier seeding and drying, as well as bacterial removal, dilution
preparation, and pour plate method are as described under procedure (a).
(c) Inhibition of corrosion. The ability of asparagine to inhibit corrosion
of stainless steel penicylinders is investigated. Prepare two brands of
penicylinders (Fisher Scientific and S&L Metal Products Corp.) as previously
described in procedure (a). After a distilled watar rinse, carriers are
allowed to air dry. After the carriers are visually inspected and found
devoid of imperfections (e.g. chips, cracks, gouges, or corrosion), eight of
each carrier brand are placed into four 20x150 mm SC tubes, two carriers per
tube. One tube of each brand is left uncapped, thus allowing the dry carriers
a constant air exposure. Two other tubes (one for each brand) are capped
tightly to prevent air exposure. Asparagine solution 0.1% is added to another
tube of each brand so that carriers are completely covered and caps are
tightened. Distilled water is added to the last two tubes to cover the
carriers and the caps are then tightened. All of the tubes are placed in a
rack to support them vertically and sterilized at 121 OG for 15 min. After 9
months at room temperature all carriers were examined using a dissecting
microscope.
(d) Scanning electron microscopy - Sterile penicylinders are prepared,
seeded, and dried as previously described in (a), using carriers prepared in
asparagine (0.1$) and those prepared in distilled water. After seeding and
drying, all penicylinders are immediately fixed in a mixture of 2%
glutaraldehyde/25E paraformaldehyde (pH 7.2) and subsequently rinsed in
Sorenson's phosphate buffer 0.1M. They are post-fixed in a buffered 1$ osmium
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108
tetroxide solution, dehydrated in a graded ethanol series, passed through
graded solutions into a transition fluid of Freon 113t and dried by the
critical point technique using Freon 13. The penicylinders are mounted on SEM
specimen stubs with silver paste, and spatter coated with gold or
gold/palladium. The specimens are examined in an ETEC Autoscan scanning
electron microscope at an accelerating voltage of 20 kv.
Results and Discussion
Procedure (a) was done to determine if the use of 0.1$ asparagine solution
enhances bacterial adherence of stainless steel carriers (Table 1). £. aureus
and S. choleraesuis show no significant difference when bacterial numbers per
penicyllnder are compared. For P.. aeruginosa. the carriers stored in
distilled water had significantly more organisms than those coated with
asparagine.
Procedure (b) was performed to determine if adherence of £.. choleraesuis to
carriers could be improved by increasing the asparagine concentration from
0.1$ to 1.0$ and transferring asparagine soaked carriers either dry or wet (as
recommended) to the broth culture of the test organisms for inoculation.
Carriers in 0.1$ asparagine were also dried before bacterial inoculation to
determine what effect this would have on bacterial numbers. The last
alternative used sterile, dried carriers, with no exposure to asparagine. S.
choleraesuis was used because it is the test bacterium that consistently
exhibits the lowest bacterial numbers per carrier (3). Table 2 demonstrates
that the use of 1.0? asparagine, either wet or dry, did not increase the
attachment of £. choleraesuj.s to the penicylinders. Likewise, drying the
carriers coated with 0.1$ asparagine did not promote bacterial adherence to
the stainless steel surface.
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109
The inability of asparagine to promote bacterial adherence was visually
demonstrated by scanning electron microscopy. Figure 1 compares
representative surface areas of asparagine coated and distilled water coated
Penicylinders inoculated with £.. aerueinosa. Similar comparisons with S.
aureus and §. choleraesulg also indicated no visible differences in regard
to bacterial adherence.
Procedure (c) involved examination by light microscopy of 16 stainless steel
carriers (eight of each brand) stored for 9 months: (a) in distilled water,
(b) in 0.1$ asparagine;(c) dry with air exposure, (d) dry without air
exposure. No surface changes were evident on any of the test carriers, as
might be expected from stainless steel's property of corrosion resistance.
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no
Recommendation
Since asparagine neither enhances bacterial adherence to stainleas steel
penicylinders nor is necessary to inhibit corrosion, it is recommended that
the use of asparagine be eliminated from the Use-Dilution Method. Two
alternatives for consideration are: (1) substituting distilled water for 0.1?
asparagine; or (2) using dried carriers, by placing prepared penicylinders
into clean, capped Erlenmeyer flasks and dry heat sterilizing at 170oC for
at least one hour.
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in
Acknowledgements
The authors wish to thank Dr. Johnny L. Carson for his expertise in conducting
the scanning electron microscopy.
This work was funded by the U.S. Environmental Protection Agency Office of
Pesticide Programs, under cooperative agreement CR81120U-01-0 to the
University of North Carolina at Chapel Hill.
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112
References
1. Official Methods of Analysis (198*0 14th ed., AOAC, Arlington, VA,
Chapter 4, Disinfectants
2. Difco Manual (1953) 9th Ed., Difco Laboratories Inc., Detroit, MI, p.
268
3. Cole, E.G., Rutala, W.A., & Carson, J.L. (1985) Abs. Ann Meeting Am.
Soc. Microbiol., QHl, p. 261
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113
Table 1. Comparison of bacterial numbers (x106) from stainless steel
penicylinders (n=4) stored in 0.15E asparagine or distilled water.
Asparagine Distilled Water
S. aureus ATCC 6538 5.08 5.03
£. aeruginosa ATCC 15442 21.18 25.78*
S. choJeraesuis ATCC 10708 1.04 1.28
•Significant difference between asparagine and distilled water using Students
t test of independent means, p<0.05.
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114
Table 2. Comparison of numbers of S. choleraesuis (x1C)6) on stainless steel
penicylinders (n=4) stored in asparagine (0.1? or 1.0$) and used wet or
dry .a,b
Asparagine 0.1$ Asparagine (concentration,
(wet)a carrier state)
Day 1
Day 2
Day 3
Day 4
1.39 1.32 (1.0$, wet)
3.19 3.24 (1.0$, dry)
1-6U 1.37 (0.1$, dry)
I*10 1.16 (no asparagine,
dry)
aWet or dry refers to the stainless steel penicylinder state before
inoculation.
bNo significant difference (p>0.1) between comparison of values found on
individual days using Students t test of independent means.
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115
FIG. 1. Comparison of Pseudotnonas aeruginosa seeded on penicylinders
prepared in asparagine (A) and those prepared in distilled water (B).
Bars, 1 urn.
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116
Analysis of Use-Dilution Method Bacteria
Background;
Stock culture maintenance of Use-Dilution test bacteria varies from
laboratory to laboratory. Some purchase fresh ATCC cultures
periodically whi le others do not. Many use the recommended maintenance
media and refrigeration method of storage, while some laboratories use
alternative media and various methods of freezing. Recognizing the high
inter laboratory variability in collaborative study results, as well as
between Industry and enforcement testing data, the biotype, antibiotic
susceptibility and resistance to phenol of the Use-Dilution Method
bacteria were Investigated. Those organisms transferred monthly for
several years might be expected to have genotyplcally and phenotypical!y
changed, perhaps to an extent that affects their intrinsic resistance to
germicides. Several laboratories were requested to forward their AOAC
Usp-DMution bacteria to our laboratory for analysis.
Purpose;
To assess the degree of variability of organisms used in the
Use-Dilution test from several different laboratories. The following
character!sites were examined: biochemical test patterns; colonial
morphology, hemolysis and pigment production; antibiotic susceptibility
and resistance to phenol.
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117
Method;
(A) A request for test bacteria (attached) was mailed to laboratories
routinely performing the use-dilution test. As organisms were received
in the laboratory, they were immediately stored at 2-5°C. For analysis,
the bacteria were removed from refrigeration and the type of maintenance
media was noted. The bacteria were transferred to 10ml of nutrient
broth (AOAC 4.001a) with overnight incubation at 35-37°C. All broths
were then subcultured to plates of Tryptlease Soy Agar (ISA) with 5%
sheep blood (BBL, Cockeysville, MD), streaked for isolation, and
incubated for 24h at 35-37°C.
(B) Isolated colonies were examined for morphological variation (rough
vs. smoooth) pigmentation, and beta-hemolysis if ^. aureus. AH
isolates were prepared for biotyping and antibiotic susceptibility
testing by reiso I at ion on ISA sheep blood plates with overnight
incubation at 35-37°C.
(C) Biotyping was done using commercial identification systems
manufactured by API Analytab Products, Plainview, NY. These included
the API 20 E system for j^. choleraesuis, the dms Rapid NFT system for P_.
aeruginosa, and the Staph I dent and Staphase III systems for ^. aureus.
Organisrr test suspensions were prepared, test strips inoculated, and
reactions interpreted, according to the manufacturer's instructions for
each system. In addition, j>_. choleraesuis was tested for motiIity and
sulfide production by the inoculation of Sulfide Indole Motility (SIM)
medium. Incubation was at 37 C and room temperature.
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118
(D) Antiobiotic susceptibility testing was performed by disk diffusion
according to procedure described by Barry and Thornsberry in Manual of
Clinical Microbiology, 4th ed., American Society of Microbiology, 1985.
From ISA 5% sheep blood plates, well isolated colonies were inoculated
to trypticase soy broth and incubated at 37°C until a slight turbidity
appeared that was equivalent to a freshly prepared 0.5 McFarfand
standard. Broths too visibly turbid were adjusted with 0.9^ sterile
saline. Mue!ler-Hinton Agar plates (approx 4mm deep) 15 x 150mm were
inoculated by using a sterile, nontoxic swab on an applicator stick
which was dipped Into the standardized suspension, and pressed to the
side of the tube above the culture to express excess broth. The swab
was then streaked over the ptate eventy in three directions. A final
sweep of the agar rim with the swab was made. The plate was allowed to
dry for 3-5 min before application of the antibiotic disks (BBL).
Plates were incubated at 35-37 C for 16-18h, after which zone sizes were
measured. The method and its materials were quality controlled on a
daily basis using standard recommended strains—Bactrol Disks Set A
(Difco): Escherichia coli ATCC 25922, StaphyIococcus aureus ATCC 25923,
Pseudomonas aeruginosa ATCC 27853. The antibiotics tested against ^.
choleraesuis and P_. aeruginosa are listed in Table 1, while those
antibiotics tested against Staphylococcus aureus are listed in Table 2.
(E) Phenol resistance testing was performed according to the
methodology in 4.009 of the Use-Dilution Method, with resistance
requirements for each of the test organisms as outlined in the Phenol
Coefficient Method (4.001-4.006).
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119
Results:
Twelve laboratories submitted the three Use-Dilution test bacteria
on the media that each normally uses for maintenance and storage. One
laboratory submitted S. aureus only, and one isolate of each of the test
strains (each from a different lab) was not suitable for analysis. With
the inclusion of our own strains, a total of 13 samples of J>. aureus, 12
of ^. c ho Ie raes u t s, and 12 of P. aerugrnosa were analyzed.
jL- choleraesuis
Of the 12 samples examined, 6 (50$) showed both rough and smooth
colonial variants; thus, 18 isolates were biotyped (11 smooth, 7 rough).
Using the API 20E system, there was complete agreement among the 18
isolates for all reactions with the exception of lysine decarboxylase (1
negative, rough) and cttrate utilization (8 positive, 10 negative). One
rough variant did not decarboxylate lysine, whereas the smooth variant
from the same culture did. The negative lysine decarboxylase isolate
also gave weak fermentation reactions with glucose and melibiose. Four
isolates (mostly rough variants) were strongly citrate positive, while 4
others (mostly smooth variants) were weakly positive.
Two (1 rough, 1 smooth) of the 18 isolates (11?) exhibited motility
}r, SIM medium, both at 37 C and at room temperature. Ability to produce
hydrogen sulfide was measured after 5 days incubation at 37°C. Twelve
(9 smooth) of 18 (67%) showed evidence of H_S production. In 5 (4
smooth) of the 12 (41.6#), H S was not detectable at 72h.
The eighteen rough and smooth isolates of S. choIeraesuis were
evaluated for antiobiotic susceptibility. All fslofates (100$) were
susceptible to the 10 antibiotics tested (Table 1).
-------
120
Results of replicate comparative phenol resistance testing for a
rough and smooth variant from the same laboratory indicated consistently
greater resistance for the rough variant (Table 3).
^. aureus
Of the 13 laboratory samples examined, only one showed 2 pigmented
colonies (yellow and white). One other isolate did not produce yellow
pigment at 24h and had an irregular colony shape. Of the 14 isolates
tested, 10 (71.4?) produced the expected beta-hemo lysis after 24h, while
4 gave weak reactions. AM 14 isolates were in agreement with 9 of the
10 biochemical reactions studied. Three isolates (21.4$) produced
positive urease reactions while the other 11 were negative.
Fourteen isolates of _S. aureus were evaluated for antibiotic
susceptibility. Twelve of the 14 (85.7$) were uniformly susceptible to
all eleven antibiotics (Table 2). Of the 2 remaining isolates, one
demonstrated resistance to penicillin, ampicillin, oxacillfn,
tetracycl ine, and erythromyci n. The other showed resistance to
oxaci I I in on ly .
Results of comparative phenol resistance testing showed that all 13
isolates tested met the minimum resistance requirements at 1:60 and 1:70
as currently specified in the method.
•
Of 12 isolates submitted and examined, 9 (75$) showed both rough
and smooth variants. Of the remaining 3, 2 were rough variants and one
was smooth. Thus 19 Isolates were biotyped. There was complete
agreement among the 19 isolates for 16 of the 20 biochemical reactions
-------
121
tested. Eight (4 rough, 4 smooth) isolates (42/S) were argfnfne
dihydrolase negative, 1 fsolate (5%) was urease positive, 4 (3 rough, 1
smooth) isolates (2\%) were gelatinase negative, and 5 (3 rough, 2
smooth) isolates (26%} were weakly positive for the assimilation of
N-AcetyI-gIucosam i ne.
The 19 isolates (12 rough, 7 smooth) were evaluated for
susceptibility to 10 antibiotics (Table 1). All isolates were uniformly
susceptible or resistant to 8 of the 10 drugs. Three (2 rough, 1
smooth) isolates demonstrated resistance to Carbenici I I in while three (2
rough, 1 smooth) were resistant to Mezlocillin. It was also noted that
two (\]%) of the isolates did not produce green pigmentation on the
Mueller-Hinton agar.
Replicate comparative phenol resistance testing was performed for
rough and smooth variants of 2 isolates from different laboratories.
Taking into account the extreme variability that p_. aeruginosa is known
to demonstrate in phenol resistance testing, the comparisons did show
slightly greater resistance for the smooth variants than for the rough
ones.
Discussion;
These data suggest the presence of different biotypes for each of
the Use-Dilution bacteria, paratlcu lar ly ^. aureus and F^. aeruginosa.
The emergence of differing biotypes could be related to the
procurement, maintenance, and storage of the organisms. Most
laboratories submitting their test strains indicated that they follow
AOAC procedure and store the strains on the designated media at 2-5°C.
Some laboratories use other freezing techniques and/or use alternative
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122
maintenance media. One lab stores the bacteria on commercially prepared
nutrient agar (FDA, BBL), three labs maintain F^. aeruginpsa on AOAC
Nutrient agar instead of the specified CTA, and one laboratory submitted
.§.* cho leraesuf s In CTA. Some laboratories purchase new cultures each
year, while others maintain the same organisms indefinitely.
I* is not known if different biotypes would effect the outcome of
phenol resistance testing or final Use-Dilution results. Consistent
with standard quality assurance practices, however, it is recommended
that the revised Use-Dilution Method contain specific requirements for
the procurement, maintenance and storage of test bacteria, such that the
probability for genetic change is virtually eliminated.
It is further recommended that procurement of organisms be directly
from the American Type Culture Collection, Rockvllle, MD.
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123
Table 1. Antiobiotic disks used to test JS. choleraesuis 10708 and
IP. aeruginosa 15442
Gentamictn (10 ug)
CarbeniciI I in (100 ug)
Ceftoxime (30 ug)
ChloramphenicoI (30 ug)
AmpiciI I in (10 ug)
Tetracyciine (30 ug)
MezlociI)in (15 ug)
Amikazin (30 ug)
Tobramycin (10 ug)
Trimethoprim-suIfamethoxazole
(1.25/23.75 ug)
Table 2. Antibiotic disks used to test S. aureus 6538
Ampici I I in (10 ug)
ChloramphenicoI (30 ug)
Cefotaxime (30 ug)
Erythromycin (15 ug)
Cefoxitin (30 ug)
Gentamicin (10 ug)
OxaclI I in (1 ug)
Penici11 in G (10 ug)
Tetracyciine (30 ug)
Varcomycin (30 ug)
Trimethoprim-Suifamethoxazole (1.25/23.75 ug)
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124
Table 3. Phenol resistance of rough and smooth variants of a single
S. choleraesuis isolate
Variant
Di lution
Day No. 1
Replicates (5, 10, 15 min)
Rough
Smooth
Rough
Smooth
1:90
1:100
1:90
1:100
1:90
1:100
1:90
1:100
++0 +00 +00 +++ +00
+++
000 +00 +00 +00 000
++0 ++0 ++0 ++0 ++0
Day No. 2
+++ „+ w +++ +++
+++ +++ +++ +++ +++
++0 ++0 ++0 ++0 ++0
+++ +4.+ +++ +++ +++
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125
THE UNIVERSITY OF NORTH CAROLINA
AT
CHAPEL HILL
School of Medicine
Ilepanmein of Medicine
Division of Infectious Diseases
9(9-966-2536
The University of North Carolina ai Chapel Hill
547 Clinical Sciences Building 229 H
Chapel Mil!, N.C. 27514
MEMORANDUM
TO: Participants in Collaborative Studies of the
AGAC Use-Dilution Task Group
FROM: Eugene C. Cole, Dr. P. H,
Co-Associate Referee
AOAC Use-Dilution Method
DATE: June 17, 1985
RE: Analysis of Use-Dilution Test Bacteria
The current Use-Dilution Method does not specify periodic
purchase of the recommended ATCC test strains. Some
laboratories- purchase new cultures annually while most
apparently do not. Some laboratories store frozen aliquots
of freshly reconstituted bacteria for routine use, while
others maintain the organisms on refrigerated slants,
passing them indefinitely at the required monthly interval.
« f
The AOAC Use-Dilution Task Group recognizes that differences
do exist among laboratories in regard to storage and
maintenance of the test bacteria. In addition, questions
have also been raised concerning the integrity of ATCC
lyophilized cultures. One laboratory reports that it
reconstituted a newly purchased vial of Salmone ^ 1 a
choloraesu is ATCC 10708 and found it to be hydrogen-sulfide
positive. Perhaps most alarming are the different phenol
resistance values encountered from laboratory to laboratory.
The recent Collaborative Study by Karen Q'Donnell
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126
examination o-f colonial variants, antibiotic susceptibility
patterns, and phenol resistance testing. In this regard you
are invited to submit slants o-f th 3 AOAC Use-Dilution
bacteria 'that you routinely use in your testing. To insure
survival, particularly in hot weather, shipment via Federal
Express or similar carrier is recommended. Please include
with your organisms in-format ion regarding the type o-f media
on which they are growing, when they were orginally
reconstituted -from the lyophilized state
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127
ANALYSIS OF USE-DILUTION TEST BACTERIA
Please ship cultures of the three AOAC Use-Dilution test
bacteria, you are currently using, and this completed form to
the following address:
Eugene C. Cole, Dr. P.M.
The University of North Carolina
Division of Infectious Diseases
547 Clinical Sciences Bldg. 229H
Chapel Hill, NC 27514
Laboratory
Name :
Address:
Phone :
OrQan i sms
S. choloraesuis ATCC 10708:
Type of media
Date reconstituted
(wi th ATCC batch tt
Method of storage
f r om original
i f possi ble)
cu1ture
S. aureus ATCC 6538:
Type of media u ._ ,
Date reconstituted
(wi th ATCC batch tt
Method of storage
from original culture
if possible)
IP. aeruoinosa ATCC 15442:
Type of media
Date reconstituted
Cwi th ATCC batch tt
Method of storage
from orjginal
i f poss i bl e _
cu1ture
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128
Evaluation of Commercial Letheen Broth for Inactivation
Of Hospital Disinfectants in the AOAC Use-Dilution Method
Background:
To prevent an inhibitory concentration of a disinfectant
from being transferred to the recovery medium, it is
essential that the activity of the disinfectant be
neutralized. Normally, the neutralizing agent is included
in the recovery medium,
Letheen broth has long been recommended for the inactivation
of phenolic and quaternary ammonium disinfectants.
Phenolics are effectively neutralized by a non-ionic
surfactant, such as polysorbate 80 (Tween); while quaternary
ammonium compounds are inactivated by lecithin, and usually
a combination of lecthin and Tween 80. These two
inactivators are included in letheen broth (AOAC 4.001d3)
and Bacto Letheen Broth (Difco Laboratories, Detroit, MI).
Such subculture media must not only effectively neutralize
carryover disinfectant, but must support the recovery and
growth of injured microorganisms.
Purpose:
To evaluate Letheen Broth (Difco) for the effective
neutralization of EPA registered phenolic and quaternary
ammonium hospital disinfectants.
Media and
Reagents:
Nutrient broth (AOAC A.OOla) using Anatone and Beef Extract
(Difco) as specified. Prepare according to directions using
glass distilled water. Adjust pH to 6.8 prior to
sterilizaation.
Letheen Broth (Difco). Prepare as directed using glass
distilled water.
Glass distilled water. Sterile Water for Irrigation, USP
(Travenol Laboratories, Deerfield, IL).
Phosphate buffer dilution water (PBDW). AOAC 4.020(f).
Pour plate agar. Plate Count Agar (Difco). Prepare as
directed.
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129
Apparatus:
Vortex mixer. Vortex-Genie (Fisher Scientific, Pittsburgh,
PA).
Colony counter.
Test tubes. 20 x 150 mm, borosilicate glass, with slip-on
caps.
Petri dishes. Polystyrene, 15 x 100 mm.
Organisms:
Disinfectants:
Phenolic
Products:
Staphylococcus aureus 6538, Salmonella choberaesius 10708,
Pseudomonas aeruginosa 15442 (American Type Culture
Collection, Rockville, MD).
Quaternary ammonium products (with recommended
use-dilution):
Tergiquat (1:64), Lehn & Fink Products Group, Montvale, NJ.
Quat-128 (1:128), The Davies-Young Company, Maryland
Heights, MO.
Virex (1:128), S.C. Johnson & Son, Inc., Racine, WI.
Organic-Quat (1:128), Lehn & Fink Products Group.
Tor _(1:64), Huntington Laboratories, Inc., Huntington, IN.
Lysol (1:00), Lehn & Fink Products Group.
Beaucoup (1:128), Huntington Laboratories, Inc.
0-syl (1:128), Lehn & Fink Products Group.
Expose (1:128), S.C. Johnson & Son, Inc.
EDC (1:128), The Davies-Young Company.
-------
Method:
130
From stock culture slants make six consecutive 24h broth
transfers of test organisms in 10 ml of nutrient broth. Keep
caps loose and incubate at 37
incubated for 48 h.
C. Final broth must be
Eleven tubes of Letheen Broth are used for each disinfectant
and test organism. To tubes 1-10 a disinfectant at the
recommended use-dilution is added in increasing volumes from
0.1 to 1.0 ml per tube. After the addition of disinfectant,
each tube is mixed for 1-2 sec on the vortex mixer, setting
#3. The eleventh tube serves as a control.
A 10-7 serial dilution in PBDW is made for each challenge
organism. For £. aeruginosa, 0.1 ml is added to each of the
eleven tubes. For j^. aureus and S, choleraesuis , 0.2 ml is
added to each tube. All tubes are gently swirled after the
addition of organisms. Using this inoculation scheme
approximately 10 cells will be added to each tube. To
determine the precise number of cells added, a single petri
dish is inoculated, after each of the 11 tubes, with the
appropriate amount of diluted broth culture, and overlaid with
15-17 ml of 43 degres C Plate Count Agar. Both tubes and
plates are incubated at 37° C for 48h after which the tubes
are examined for growth and colonies on plates are counted.
The 5 plates from each group of quats and phenolics are
averaged to determine the mean number of bacterial cells added
to each tube.
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131
Results and Discussion
Quaternary Ammonium Disinfectants
Table 1 shows the degree of neutralization of the five quaternary ammonium
disinfectants tested with the 3 Use-Dilution Method bacteria. As might be
expected, S. aureus was inhibited as the amount of disinfectant added to the
Three disinfectants (Organic Quat, Quat-128 and
Tergiquat showed inactivation
While
leethen broth was increased.
Virex) were not neutralized beyond 0.2 ml.
through 0.3 ml, while Tor was effectively neutralized through 0.7 ml.
recovery was not measured using damaged cells, the mean numbers of organisms
added to the test broths were: ^. aureus, 11.0; J5. choleraesuis, 4.0; and j?.
aeruginosa, 5.6. Since the amount of disinfectant carryover on test
penicylilnders in the Use-Dilution Method has been approximated at 0.01 ml, it
is felt that the Letheen Broth (Difco) is adequate for the neutralization and
probable recovery of organisms exposed to EPA registered quaternary ammonium
disinfectants.
Phenolic Disinfectants
Table 2 shows the extent of inactivation of the five phenolic disinfectants
challenged with the 3 Use-Dilution Method bacteria. The Letheen Broth
neutralized all tubes containing disinfectant through 1.0 ml that were
subsequently challenged with _S. aureus and S. choleraesuis. Mean numbers of
challenge cells added to tube 5 was 8.4 for S. aureus,and~ 4.6 for S.
choleraesuis. JP. aeruginosa sucessfully grew in all tubes for two of the
disinfectants. For the other three, growth was evident in tubes through 0.8
ml and 0.9 ml. It is felt that those disinfectants would have demonstrated
inactivation through 1.0 ml but the mean inoculum of cells was so low (1.0
per/tube) that one or two tubes may not have been challenged.
In summary, Letheen Broth (Difco) efficiently neutralizes quaternary ammonium
and phenolic hospital disinfectants, and supports the growth of low numbers of
cells.
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132
Table 2. Highest volume (ml) of phenolic disinfectant added to 10 ml Difco
Letheen Broth permitting organism growth.
Disinfectant (use-dilution) S. aureus
S. choleraesuis P. aeruginosa
Lysol (1:100)
Beaucoup (1:128)
0-syl (1:128)
Expose (1:128)
EDC (1:128)
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
.8
.9
.8
1.0
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133
Table 1. Highest volume (ml) of quaternary ammonium disinfectant added to
10 ml Letheen Broth (Difco) permitting organism growth.
Disinfectant (use-dilution) S. aureus S. choleraesuis P. aeruginosa
Tergiquat (1:64)
Quat-128 (1:128)
Virex (1:128)
Organic Quat (1:128)
Tor (1:64)
.3
.2
.2
.2
.7
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
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134
Phenol Resistance Testing: Loop vs. Micropipette
Background;
The method of determination of phenol resistance of organisms used in
use-dilution testing has been criticized, particularly as regards the known
variability of loop sampling, the lack of a suitable number of sample
replicates, and the use of a small sample size. Alternatives, such as the use
of a micropipette and multiple sampling replicates should be considered in
attempting standardization of the phenol resistance test.
Purpose;
To determine variability among replicates of phenol resistance testing by the
recommended loop sampling method as compared to variability using a
micropipette method.
Media and Reagents;
Nutrient Broth (AOAC 4.001a) using Anatone and Beef Extract (Difco) as
specified, Prepare according to directions using glass distilled water.
Adjust pH to 6.8 prior to sterilization.
Letheen Broth (Difco). Prepare as directed using glass distilled water.
Phenol solution, stock 5% (AOAC 4.002f).
Sterile Water for Irrigation, USP (Travenol).
Equipment;
Loops: platinum-rhodium, 0.01 ml, 4 mm id, B & S gauge No. 19 platinum wire
with 3 1/2% rhodium.
Micropipettes: 20 ul (Gilson/Pipetman), Adjustable 50/100 ul (Eppendorf),
with sterile, disposable tips.
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135
Methodology;
1.
Stock culture of £. aureus 6538, £. choleraesuis 10708, and P_.
aeruginosa 15442, are transferred to 10 ml nutrient broth tubes and passed
at 24h intervals with incubation at 37*"
passed 4-6 times before use.
C. Broth cultures for testing are
2. Using 48-54h culture broths, testing is begun with appropriate phenol
dilutions: ^. aureus 1:70, 1:80; £. choleraesuis 1:90, 1:100; P.
aeruginosa 1:80, 1:90. Four replicates are run simultaneously with the
same organism, same broth culture and same sampling method utilizing 30
sec intervals. The resistance testing method as outlined in AOAC 4.009 is
followed.
3. With each organism tested, loop sampling is followed by micropipette
sampling using the same broth culture.
Results:
Procedure A: Loop vs. Micropipette (lOul)
Organism
S. aureus
S. choleraesuis
P. aeruginosa
Sampling
Method
loop
pipette
loop
pipette
loop
pipette
Dilution
1:70
1:80
1:70
1:80
1:90
1:100
1:90
1:100
1:80
1:90
1:80
1:90
Replicates (5,
1 2
-1-00
+++
+++
+++
+++
+++
+00
+00
00+
+00
+++
+++
+00
+++
+00
+++
++0
+++
+00
+++
+00
+00
+++
++0
10, 15
3
+00
+++
+00
+++
++0
+++
+00
+++
000
++0
000
++0
min)
4
+00
+++
+00
+++
+00
+++
+++
+++
000
+00
000
+++
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136
Organism
S.
s.
P.
aureus
choleraesuis
aeruginosa
Sample
Method
loop
pipette
loop
pipette
loop
pipette
Agreement/
Replicates
4/4
3/4
2/4
2/4
0/4
0/4
Results Acceptable/
Replicates
4/4
4/4
4/4
3/4
0/4
2/4
Discussion:
This study was conducted in an attempt to evaluate the reproducibility of
results of the 4mm loop sampling method as currently recommended in
use-dilution testing and to determine if variability could be reduced by using
a micropipetter method of sampling at the same volume. The micropipetter
results actually show slightly more variability than the loop method. Results
with P. aeruginosa show complete disagreement among replicates with both
sampling methods, while results with j». choleraesuis exhibit a replicate
agreement of 50%. _S. aureus demonstrated the best agreement among replicates.
(S. aureus, however, exhibits the growth characteristic of clumping which may
have a favorable effect on test results). It thus appears that at the
recommended sample volume of 10 ul, the micropipetter shows no advantage over
the current loop sampling method; additionally, phenol resistance results can
be quite variable depending upon the organism being tested.
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137
Procedure B: Loop vs. 50 ul and 100 ul Micropipette
Organism
S. aureus
S. choleraesuis
P. aeruginosa
Sampling
Method
loop
pipette
(50 ul)
pipette
(100 ul)
loop
pipette
(50 ul)
pipette
(100 ul)
loop
pipette
(50 ul)
pipette
(100 ul)
Dilution
1:70
1:80
1:70
1:80
1:70
1:80
1:90
1:100
1:90
1:100
1:90
1:100
1:70
1:80
1:70
1:80
1:70
1:80
Replicates (5,
1 2
+00
+++
+++
+++
+00
+++
+++
+++
+++
+++
000
+++
++0
++0
000
++0
000
++0
+00
+++
+00
+++
++0
+++
+++
+++
+++
000
000
+++
000
+++
000
+00
+00
+++
10, 15
3
+++
+++
++0
+++
++0
+++
++0
+++
+++
+++
000
+++
000
+00
000
+++
000
+00
min)
4
+0+
+++
++0
+++
++0
+++
+++
+++
++0
+++
000
+++
000
+00
000
++0
000
+++
-------
138
Organism
S. aureus
S . chole rae sui s
P. aeruginosa
Sampling Agreement/ Results Acceptable/
Method Replicates Replicates
loop
pipette (50 ul)
pipette (100 ul)
loop
pipette (50 ul)
pipette (100 ul)
loop
pipette (50 ul)
pipette (100 ul)
2/4
2/4
3/4
3/4
2/4
4/4
2/4
2/4
0/4
4/4
4/4
4/4
4/4
3/4
4/4
2/4
3/4
3/4
Discussion;
This study evaluated the reproducibility of a 50 ul and 100 ul micropipette
method of phenol resistance determination in comparison with the standard
4 mm (10 ul) loop method of testing. The 50 ul pipette showed no advantage
over the loop in regard to agreement among replicates or number of acceptable
replicates with each of the test bacteria. The 100 ul pipette showed slight
improvement over the loop in replicate agreement with S_. aureus and
jS. choleraesuis, but no replicate agreement with P. aeruginosa, although 3 out
of 4 of those results were acceptable toward meeting the required resistance.
P.aer ug ino sa was found to be the most variable of the three test organisms,
regardless of which test method was used.
In conclusion, the loop and micropipette methods are equivalent, with each of
the test bacteria. It is recommended that due to variability of results,
particularly with Pseudomonas, that multiple replicate determinations of
phenol resistance be performed within a single day in order to determine
acceptability of results.
-------
139
Use-Dilution Method Statistics
The primary statistical goal of this research has been to effect a
situation where classical quality assurance techniques can be applied.
Here, an essentially homogeneous batch of disinfectant is tested
repeatedly (i.e., by 60 subtests, the result of each of which may be
"sterile" or "not sterile"), and statistical inference is directed
towards the "long-run proportion of nonsterI Iized carriers."
Decision-making is based on the estimated long-run proportion of
nonsteriIized carriers; this proportion will tend to be low for
efficacious products and higher for less efficacious products. Under
binomial sampling assumptions, the acceptance sampling problem can be
solved in a "forward" manner (i.e., from specifications to decision rule
instead of vice versa), by specifying parameters such as the acceptable
quality level (AQL), producer's risk, and so on. Two major obstacles to
proceeding "forward" are discussed below.
(1) Binomial sampling assumptions require a "comparable" test, both
within and between laboratories. Thus, a comparable (i.e., consistent)
test must be developed.
In statistical terms, binomial sampling assumptions assume
"comparabifity" (i.e., negligible between-laboratory and
within-laboratory variability), by positing that subtest results for any
particular carrier in any given laboratory at any given time are subject
to the same "long-run proportion of nonsteriIized cariers" (i.e., are
subject to the same "carrier failure rate"). Now some reduction in both
elements of total variability (i.e., between laboratory and within
laboratory), should be expected as a byproduct of work on standardizing
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140
and simplifying test conditions. It should be noted that most of the
decisions about how to standardize the test are based on "scientific"
rather than "statistical" grounds in that they are often based on
studies with low power. For example, it was shown that the use of
asparaqine did not have a large effect on the number of organisms seeded
on carriers, although the sample size did not allow for any elucidation
of moderate effects. In addition, using the number of organisms
attached to carriers as a surrogate for the level of biological
challenge, the 1985 Use-Dilution Method collaborative study indicated
that major sources of variability between test results are between
cylinders and between laboratories. Based on this evidence plus
electron microscopy work, it was recommended that the collaborative
study require the culling of suspicious cylinders.
Many statistically equivalent alternatives are available in
attempting to reduce between laboratory variability. AM require the
interested parties to consider the set of laboratories that perform AOAC
Use-Dilution testing within the context of an overall quality assurance
perspective. Some sort of cooperative procedures that help to
"calibrate" test results between laboratories (e.g., running identical
samples through all laboratories from time to time), are recommended.
The cooperation of the interested parties in the design and
administration of an overall quality assurance program should go a long
way towards increasing confidence in the AOAC Use-Dilution Method.
(2) The interpretation of "pass" and "fail" on the test must be
clari f fed.
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141
Even assuming that the variability in test results may be reduced
to an acceptable level, the setting of test specifications must begin
from consideration of the meaning of a disinfectant "passing" or
"failing" a particular test. In this regard, perhaps a "5/5/5" test,
which requires a disinfectant to kill 5 fogs of 5 different organisms in
5 minutes may have a more direct interpretation than the present test.
If the parties involved are willing to specify what constitutes
"acceptable" performance on a 5/5/5 test then this methodology should be
given serious consideration. Finally, it may be noted that while
agreement on the meaning of "pass" and "fail" is desirous when
attempting to set specifications, such agreement is not necessary in
order to proceed. For example, the recently completed collaborative
study evaluated AOAC Use-Dilution testing on six randomly chosen
products. The results of this study will be used as a starting point in
the development of specifications. That is, the discussion may be based
on how typical disinfectants actually perform when subjected to the
Use-Dilutfon Method rather than how such products "should" perform. It
is interesting to note that not all of the six randomly chosen
disinfectants consistently passed the Use-Dilution tests in the
collaborative study even though all six products are registered and
presumably passed the test in some laboratory at some time.
Finally, it may be noted that it is reasonable to expect that
progress should simultaneously occur on all of the above issues. What
is required is:
(a) continued research supplemented by more inter laboratory
studies;
-------
142
(b) some administrative arrangement for between laboratory quality
assurance; and
(c) agreement on what "pass/fail" actually means for a test, either
under the present methodology or an alternative one.
-------
143
Appendix
The statistical methods used in the analysis of this project were
straightforward. The binomial distribution was used for an analysis of
the power of the present Use-Dilution test (assuming for the moment
consistent subtests). Analysis of variance (ANOVA) methodology was used
for the analysis of pilot studies and inter laboratory studies. In a
previous inter laboratory study, the standardization of bacterial numbers
on penicylinders was analyzed using a nested random effects ANOVA model
applied to the logarithm (base 10) of the number of organisms observed,
where the goal was to apportion overall variability Into components of
variance that were due to laboratories, days, cylinders and replicates
(components of variance and variance proportions were calculated). The
recently completed collaborative study of bactericidal activity of
randomly selected germicides will be analyzed using a two-factor random
effects ANOVA model (with one observation per cell), applied to the
arcs in-transformed proportion of nonsterfIized carriers for each
product-laboratory combination. An adjustment will be made for
proportions of zero, and parallel analyses will be performed using
"robust methods" (e.g., truncating extreme responses), in order to
assess the effects of outliers on the final result.
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144
PRELIMINARY REPORT
A.O.A.C. USE-DILUTION METHOD
COLLABORATIVE STUDY 1985
Division of Infectious Diseases
547 Clinical Sciences Building 229H
University of North Carolina
School of Medicine
Chapel HI I I, NC 27514
919-966-2536
Eugene C. Cole, Dr.P.H.
Research Associate
Co-Associate Referee
A.O.A.C. Use-Dilution Method
Gregory P. Samsa, M.S.
Blostatlstlclan
Edith M. Alfano, M.S.P.H
Research Assistant
William A. Rutala, Ph.D, M.P.H.
Research Associate Professor
-------
145
QUATERNARY AMMONIUM DISINFECTANTS
1. n-A!kyl (60* CM, 30* C16, 5* C^)
dimethyl benzyl ammonium chlorides 6.25*
n-Afkyt (68* C}2> 32* C^) dimethyl
ethyI benzyl ammonium chlorides
Tetrasodium ethylenediamine tetraacetate
Sodium sesquicarbonate
Inert Ingredients
2. Sodium carbonate
n-Alky! (60% CH, 30* C16, 5% C]2, 5% C^Q)
dimethyl benzyl ammonium chlorides
n-Alkyl (50% C]2, 30% GU, 17* Ct6, 5% C]Q
dimethyl ethylbenzyl ammonium chlorides
Sod i um metas i t i cate
Inert Ingredients
3. OctyI decyf dimethyl ammonium chloride
Oioctyl dimethyl ammonium chloride
Didecyl dimethyl ammonium chloride
Alkyl (50* CM, 40* C12, 10* C^)
benzyl dimethyl ammonium chloride
Tetra sodium ethylenediamine tetraacetate
Inert Ingredients
6.25*
3.60*
3.00*
80.90*
3.0*
1.6*
1.6*
1.0*
92.8*
3.750*
1.875*
1.875*
5.000*
3.420*
84.080*
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146
PHENOLIC DISINFECTANTS
4. Isopropanol 15.00?
Potassium orthobenzyl para-chlorophenate 5.38?
Potassium para-tertiary-amyIphenate 5.19?
Sodium dodecyI benzene suffonate 3.36?
Potassium orthophenyIphenate 2.94?
Tripotass turn salt of ethylenediamine
tetraacettc acid 0.74?
Inert Ingredients 67.39?
5. Sodium xylene sulfonate 10.8?
Triethanolamine dodecyfbenzene sulfonate 6.3?
o-PhenyI phenol 5.7?
Trisodium ethylene dfamine tetraacetate 3.0?
p-tert-amy(phenol 1.8?
Inert Ingredients 72.4?
6. Sodium orthophenyIphenate 5.5?
Sodium 0-benzyf-p-chlorophenate 4.4?
Sodium p-tertiary amyIphenate 1.1?
Inert Ingredients 89.0?
-------
147
Collaborative Study Report
The protocol (attached) for the Collaborative Study of the AOAC Use-Dilution
Method to Assess Inter laboratory Variabilllty of Results and to Set
Specifications for Pass/Fail was mailed to more than 50 laboratories in the
continental United States on August 12, 1985. Eighteen laboratories submitted
study data. These laboratories represent disinfectant manufacturers,
independent testing facilities and federal and state governments.
The final protocol was a second draft that had been approved by the General
Referee, the Statistical Consultant for Committee A and the Steering Committee
of the AOAC Use-Dilution Task Force.
For this blind collaborative study, disinfectants currently available in the
marketplace were used. Three randomly selected quaternary ammonium and three
phenolic disinfectants (all with hospital and distilled water claims only),
were sent to participating laboratories that routinely performed the
Use-Dilution Method. They were asked to test the products as they normally
would using only those modifications of the Method approved by the Task Force.
The purpose of the study was to assess the degree of variability in
disinfectant efficacy test results among the participating laboratories. It
is expected that the collaborative study will also aid in reviewing current
pass/fall criteria and will serve as a basis of comparison when the revised
method has been finalized and submitted for its own collaborative study.
-------
Co-General Referee:
Statistical Consultant:
Committee A
AOAC Use-Dilution:
Task Force Steering
Committee
148
Mr. Aram Betoian (TS-768-C)
Benefits and Use Division
Office of Pesticide Programs
U.S. Environmental Protection Agency
401 M Street, S.W.
Washington, D.G. 20460
Dr. Bertram D. Litt (TS-769-C)
Hazard Evaluation Division
Office of Pesticide Programs
U.S. Environmental Protection Agency
401 M Street, S.W.
Washington, D.C. 20460
Mr. Aram Beloian
U.S.E.P.A.
Co-General Referee, Disinfectants
Mr. Gayle K. Mulberry
Co-Associate Referee, Use-Dilution Method
HI I I Top Research, Inc.
P.O. Box 42501
Cincinnati, OH 45242
Dr. Eugene C. Cole
Co-Associate Referee, Use-Dilutfon Method
University of North Carolina
Division of Infectious Diseases
547 Clinical Sciences Bldg. 229H
Chapel Hi II, NC 27514
Dr. Fred Boyd
Huntington Laboratories
968 E. Tipton Street
Huntington, IN 46750
Dr. R.S. Dahiya
State of N.C. Department of Agriculture
Food and Drug Divison
Raleigh, NC 27611
Dr. George Walter
S.C. Johnson and Sons,
1525 Howe Street
Racine, Wl 53403
Inc.
Ms. Betsy Woodward
Florida Department of Agriculture
and Consumer Services
Laboratory Complex
3125 Conner Blvd.
Tal lahassee, FL 32301
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149
Ruggedness Testing
AOAC Use-Dilution Collaborative Study
Background
Following the final technical and statistical reviews of the proposed
collaborative study, ruggedness testing was performed as recommended by the
AOAC. In ruggedness testing, the initiating laboratory performs testing and
follows the approved collaborative study protocol after the introduction of
minor reasonable variations in the method. These departures are of the
magnitude that a scientist might well expect to find among laboratories.
Normally, two values of seven variables are selected for scrutiny. If the
procedure Is "rugged" and thus immune to modest and inevitable departures from
the usual routine, then the results obtained should not be altered by these
minor departures. Presumably most minor departures should show negligible
effects. If, however, a sensitive condition fs discovered, then considerable
effort that would have been expended In a disappointing collaborative study is
spared.
Method
In this disinfectant evaluation study, eight combinations of seven factors
were used to test the ruggedness of the Use-Dilution study protocol. The
combinations were those found In the Statistical Manual of the AOAC and are
reproduced in Table 1. The seven factors tested, with their variables, are
contained in Table 2. Two disinfectants (one quat and one phenolic) from the
six chosen for the collaborative study were randomly selected for the
ruggedness testing. The organism used was the resistant Pseudomonas
aeruginosa ATCC 15442, one of the Use-Dilution Method specified strains. Each
disinfectant was tested on a single day, representing 8 trials of 20
pen icy Iinders each using the same broth culture.
Results and Discussion
Use-dilution ruggedness testing with the phenolic disinfectant (use-dilution
1:100) yielded no positive tubes (0/159). Testing with the quaternary
ammonium disinfectant (use-dilution 1:256) yielded three positive tubes
(3/100), each occurring randomly in a separate set of replicates. There was
no association with a particular variable.
Results showed that the collaborative study protocol to assess Inter laboratory
variability and set specifications for pass/fail was:
(1) extremely rugged and/or (2) the products tested were very effective. On
the basis of these results It was determined that the test method was
acceptable and the collaborative study was Initiated.
-------
150
Table 1. Eight combinations of seven factors used to test the ruggedness of
an analytical method.
Combination or Detn No.
Factor VaIue
A
B
C
D
E
F
G
or
or
or
or
or
or
or
a
b
c
d
e
f
g
A
B
C
D
E
F
G
A
B
c
D
e
f
9
A
b
C
d
E
f
9
A
b
c
d
e
F
G
a
B
C
d
e
F
9
a
B
c
d
E
f
G
a
b
C
D
e
f
G
a
b
c
D
E
F
g
Observed result
-------
151
Table 2. Ruggedness testing variables for the Use-Dilution Method
Collaborative Study.
Factor
Standard
Variable
Dry time (min)
Carrier
Test temp.
Broth •
Exposure
Water
(For dl lutfon)
Pellicle
(Removal)
A = 40
B = S&L
C = 20
D = nutrient
E = 10
F = commercial
G = suction
a = 35
b = Fisher
c = room
d - synthetic
e = 9.5
f -. lab
g = decant
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152
AOAC Use-Dilution Collaborative Study
Preliminary Results and Discussion
Each participating laboratory tested each of the six unknown disinfectants
against each of the Use-Dilution Method test bacteria. Sixty pen icy IInders
(replicates) were processed for each disinfectant/organism combination (a
total of 1,080 for each laboratory). At present, 18 laboratories have
submitted test results with Salmonella choleraesujs, Staphylococcus aureus and
Pseudomonas aeruginosa.
Table 1 presents an overview of how well the disinfectants have performed.
Most laboratories have passed the test disinfectants when challenged with
_S. choleraesuis. Fewer products pass when challenged with S_. aureus, and
Tewer stM I when tested with £. aeruginosa.
S. aureus
Table 2 shows each laboratory's performance against the six products. Some
variability Is apparent, particularly with a number of significant failures
(> 4/60). Of interest is the number of marginal (2,3/60) failures. This Is
shown In Table 3, and may reflect the need for change in pass/fall criteria.
The number of clear failures (> 4/60), however, Is of concern. Table 4
Indicates the number of passing Use-Dilution tests, based on different
pass/falI criteria.
S. choleraesuts
Table 5 shows that, with a few exceptions, the six disinfectants are effective
against the organism, the same number of marginal failures as
S. aureus but fewer significant failures. Table 7 shows the number of passing
Use-Dilutfon tests, based on different pass/fall criteria.
P. aeruginosa
Table 8 demonstrates significant variability of results from lab to lab and
product to product. An examination of the data reveals that two products (#4,
5) consistently inactivate P_. aeruginosa and one product (#6) consistently
falls by significant margins. The other three products (#1,2,3) do not
perform consistently well against £. aeruginosa. It is noteworthy that these
three products are quaternary ammonium compounds, and
P_. aeruginosa has demonstrated intrinsic resistance to their action.
Interlaboratory variation in results is evident as the data is viewed
horizontally. It can be seen that some laboratories failed all of the
products (e.g., #3, 17, 18), while others passed most or all of them
(#5, 7, 13). Of particular concern Is the situation where disinfectant #6
significantly failed in 12 labs yet passed In 4. Table 9 shows high numbers
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153
of marginal and significant failures, while Table 10 shows how the overall
passing rate wouid change with different pass/fall criteria.
Overall variability may be assessed In regard to four areas:
(1) methodology, (2) laboratories, (3) disinfectants and (4) pass/fail
criteria.
MethodoIogy
At least 15 presumptive deficiencies or inadequacies have been identified in
the Use-Dilution Method, All have the potential to influence final test
results independently or in conjunction with other Inadequacies. These
deficiencies Involve: non-quantitative results, Pseudomonas pellicle
removal, choice of bacterial culture broth, penlcylinder drying conditions,
loss of phenol resistance by test strains, testing for effective
neutralization and the absence of bacteriostasis in subculture, use of
different penicyUnder brands, use of secondary subcultures, lack of a
glassware cleaning protocol, lack of specifications for distilled water,
non-unfform storage of test bacteria, bacterial attachment to penfcyIinders,
use of asparagine, propogation of freeze-dried cultures, and an overall lack
of appropriate quality control procedures.
Laboratories
20 C room. Many labs
, freezing in liquid
,), rather than the
Method. Water quality
In a preliminary review of the returned collaborative study questionnaires, it
is evident that quality control practices differ from lab to lab, in addition
to differences in the actual performance of the test. Most laboratories use a
20 C water bath to perform the test, whlle some prefer a
use their own method of preserving stock cultures (e.g.
nitrogen, freezing on glassbeads at -70 degrees C, etc.
refrigerated slant start procedure as specified in the
differs from lab to lab. Some use glass distilled water, while others use
non-glass distilled, or only deionlzed water. Many laboratories have but one
person to perform use-dflution testing, while other laboratories use two or
three people with the degree of expertise possibly varying. Some
laboratories test 10 penicyIinders at a time, while others test 20 or 30.
Most labs do not calibrate thermometers with an NBS traceable thermometer.
Laboratories also vary the number of phenol resistance test replicates for
each organism. In summary, departure from the "standard" method is
commonplace. There is a need for standardization within laboratories,
especially in regard to quality assurance practices and procedures.
Disinfectants
It would appear from the test data that some disinfectant products are
ineffective against the test bacteria when using the manufacturers'
recommended use-dilution.
-------
154
Pass/Fat I Statistics
The Use-Dilution Method currently states "while killing in 10 of 10 replicates
specified provides a reasonably reliable index in most cases, killing fn 59
out of 60 replicates is necessary for confidence level of 95%." The rationale
for these figures Is not understood. Pass/fail criteria must be redefined,
beginning with a study of how disinfectants in the marketplace perform when
tested by a standard method. Thus, the present Use-Dilution Method requires
standardization prior to a collaborative study of EPA registered disinfectant
products. When the standardization and the study have been completed, then a
rational approach to the setting of specifications, with resultant pass/fail
criteria, can be undertaken. Since these disinfectants do not consistently
meet their product label claims, the current pass/fail criteria must be
modified or the products need to be reformulated to meet existing criteria.
-------
155
Table 1. Percentage of laboratories (n«18) passing (0, 1/60) test
disinfectants by the Use-Dilution Method
Disinfectants
Ouats Phenol Ics
S. choleraesuis
S. aureus
P. aeruginosa
a=60 pen icy 1 Inders test*
72
50
22
1
61
61
28
234
83 94 83
78 67 72
44 44 67
5 6
83
67
22
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156
Table 2. Use-Dilution Method Collaborative Study
Lab
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Pass (0, 1/60)
Fai 1 (> 1/60)
Number
+/60 for
S. aureus
Disinfectant
1
1
0
9
1
0
0
1
3
2
1
3
26
3
1
2
6
10
0
50?
50?
2
0
1
12
0
1
1
3
1
1
1
4
16
1
1
2
4
4
0
61?
39?
3
2
0
0
0
0
20
1
3
1
1
1
1
1
0
0
0
7
0
78?
22?
4
2
0
2
0
0
4
1
0
2
0
5
0
0
0
0
0
4
0
67?
33?
5
1
0
1
1
0
'2
1
2
4
0
6
1
0
1
0
1
8
0
72?
28?
6
3
0
1
0
0
1
1
1
5
0
7
4
1
1
0
4
8
0
67?
33?
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157
Table 3. Staphylococcus aureus pass /faiI*
Disinfectant
Pass (0,1/60)
Marginal (2,3/60)
Fall O4/60)
1
9
5
4
2
11
2
5
I
3
14
2
2
• 4
12
3
3
5
13
2
3
6
12
1
5
*Eighteen testing laboratories for each disinfectant.
Table 4. Staphylococcus aureus pass/fail
Pass/Fa I I
% Pass*
0-1
0-2
0-3
65.7 (71/108)
74.0 (80/108)
79.6 (86/108)
*No. of use-dilution tests (60 carriers) passing out of 108,
-------
Table 5. Use-Dilution Method Collaborative
Study
Number +/60 for S
Lab
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Pass (0, 1/60)
Fail O1/60)
158
. choleraesuis |
Dfsinfectant •
1
0
0
8
0
1
0
1
0
1
0
1
0
1
1
2
5
2
2
72*
28*
2
0
0
2
4
0*
1
0
1
4
1
0
4
1
0
2
3
2
0
61*
39*
3
0
0
1
1
0
1
0
1
2
0
0
1
0
0
0
0
6
2
83*
17*
4
0
0
1
0
0
1
0
1
1
0
1
0
1
0
0
1
2
1
94*
6*
5
0
0
0
0
0*
0
0
0
3
0
1
1
0
1
0
0
2
2
83*
17*
5 1
0 1
0 1
1 1
0 1
0 1
0 I
0 I
1 I
3 1
0 1
0 1
0 I
0 I
1 1
0 I
1 1
4 1
3 1
83* 1
n* 1
*No. positive out of 59 rather than 60.
-------
159
Disinfectant
Pass (0,1/60)
Marginal (2,3/60)
Fat 1
1*
13
3
2
i
2
11
4
3
3
15
2
1
4
17
1
0
5
15
3
0
6
15
2
1
Table 7. Salmonella choleraesuis
Pass/Fat I
% Pass*
0-1
0-2
0-3
79.6 (86/108)
89.8 (97/108)
92.6 (100/108)
*No. of use-dilution tests (60 carriers) passing out of 108.
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160
Table 8. Use-Pilutton Method Collaborative Study
Number +/60 for P. aerugfnosa
Lab
,
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Pass (0, 1/60)
Fall O1/60)
Disinfectants
1
t
15
1
14
7
0
4
1
7
6
10
5
8
1
3
8
10
60
10
22*
78*
2
9
0
8
1
0
7
1
J>
5
1
2
5
2
3
8
8
59
15
28*
72*
3
6
0
11
2
0
3
1
4
0
0
0
1
1
2
10
3
59
3
44*
56*
4
1
0
2
0
0
3
1
3
2
0
2
1
1
3
2
2
56
11
44*
56*
5
1
0
4
0
0
0
0
0
5
1
4
0
0
2
0
0
26
4
67*
33*
6
52
16
13
22
2
1
1
40
18
7
2
58
1
1
5
6
59
5
22*
78*
!
1
-------
Table 9. Pseudomonas aeruginosa pass/fall*
*Eighteen testing laboratories for each disinfectant.
161
Disinfectant
12345i
Pass (0,1/60)
Margina !
Fall O4/60)
4
1
13
5
4
9
8
5
5
8
8
2
12
1
5
4
2
12
Table 10. Pseudomonasaeruginosa
Pass/Fa I
% Pass*
0-1
0-2
0-3
38.0 (41/108)
50.0 (54/108)
58.3 (63/108)
*No. of use-dilution tests (60 carriers) passing out of 108.
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162
THE UNIVERSITY OF NORTH CAROLINA
AT
CHAPEL HILL
School of Medicine
Department of Medicine
Division of Infectious Diseases
919-966-2536
The University of Nonh Carolina al Chapel Hill
547 Clinical Sciences Building 229 H
Chapel Hill, N.C. 27514
TO : Participants in Collaborative Studies of the AOAC
Use-Dilution Task Group
FROM: Eugene C. Cole, Dr. P.M.
Co-Associate Referee
AOAC Use-Dilution Method
DATE: August 12, 1985
RE : Collaborative Study of the AOAC Use-Dilution Method
to Assess Inter laboratory Variability of Results and
to Set Specifications for Pass/Fall.
Method Rev?sion
In regard to revision of the AOAC Use-Dilution Method, many
modifications are currently under consideration and/or Investigation.
of variablIity fn
ly employing the
I laboratories
same disinfectants.
in disinfectant
aid In setting the
I I serve as a basis
zed and submitted
Before a finalized revision is considered, the extent
use-dilution testing among those laboratories current
method must be known. This can be accomplished by al
performing the Use-Dilution Method on samples of the
This will assess degree of agreement or disagreement
efficacy testing among performing laboratories, will
specifications to establish suitable pass/fall and wl
of comparison when the revised method has been final!
for its own collaborative study.
Participation
If your laboratory routinely performs the Use-Dilution Method you are
encouraged to participate. !n addition to providing valuable data
needed for method revision, you will be able to assess your accuracy
relative to the performance of other analytical laboratories.
Hopefully the study design has minimized the time commitment and
laboratory cost for those participating without sacrificing the Intent
of the study In providing valuable data.
-------
163
Procedure
You will receive shortly, under separate cover, six allquots of
concentrated hospital disinfectants (3 phenol Ics and 3 quats with
distilled water claims only), as supplied by the manufacturer. The
products will not be Identified by brand name. You are asked to process
each disinfectant (at Its stated use-dilution concentration In distilled
water) by performing the use-dilution method as norma I I y done in your
laboratory.
Each disinfectant is to be tested with
three recommended test strains. There
tested for each organism.
60 penicylfnders for each of the
will thus be 360 penicy I inders
Test all 6 disinfectants against one organism within a 2 week period,
and proceed In the same manner for the other test bacteria. Please
perform testing in the following order: S_^ choleraesuis, S. aureus, and
FV.^ aeruglnosa.
Modi f lea t? on s
The AOAC Use-Dilution Task Group has tentatively approved the following
modifications for Inclusion in the revised Method (third draft
10/25/84). Please adhere to these In the performance of the
collaborative study.
1. Carrier screening. Discard carriers that are visibly
unacceptable (gouged, dented, etc.) Test those remaining with
S. aureus ATCC 6538 and 500ppm alky/dimethyl/ammonium
chloride, alky/chain distribution of C14, 50?; C12, 40?; C16,
(Onyx orLonza) and discard those giving positive results.
2. Pseudomonas pellicle. Remove pellicle by vacuum suction with
sterile Pasteur pipet, followed by decanting Into sterile tube or
bottle.
3. Carrier inoculation. Remove asparagine from tube of sterile
carriers and aseptically transfer carriers to another sterile
tube to which the correct amount (12,24,or 36ml) of thoroughly
mixed broth culture Is added. Inoculate carriers In multiples
of 12.
4. Carrier drying. Dry inoculated carriers In multiples of 12
at 37C for 40 mln.
Most laboratories perform sequential testing in batches of 20, 30, etc.
Thus when reporting results for each 60 carriers please report the
number of positives per batch tested, e.g., 0/20, 1/20, 0/20 or 1/30,
2/30, etc., for each organism. Also Include the number of broth
transfers that each organism used for testing has undergone, as well as
the phenol resistance data.
-------
164
Etvclosures
Enclosed please find:
1. Statistical basis for this study.
2« Report -forms for xour results (one for each organism).
3. Collaborative Studx Questionnaire.
4, Post-paid envelope.
Please return your results and questionnaire by October 4th.
Thank xou for your time and effort in participating.
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165
Statistical Issues of the AOAC Use-Dilution Collaborative Study to
Assess Interlaboratory Variation and Set Pass/Fail Specifications
Dr. Trout's paper has been discussed at several AOAC Use-Dilution Task
Force meetings and recommends that our group apply acceptance sampling
methodology to the AOAC Use-Dilution test. His underlying goal is to
determine efficient sample sizes and roles for accepting or rejecting
disinfectants based on test results. For example, a typical industrial
application of acceptance sampling methodology pertains to a
manufacturer's decision whether or not to accept a shipment of
materials, such as parts, from a supplier. The manufacturer's decision
will be based on the results of a sample of parts from the shipment.
Economic considerations often suggest that, say, the manufacturer deems
shipments with 1% or fewer defective parts to be clearly acceptable,
shipments with 5% or more defectives to be clearly unacceptable, and
shipments with between 1 and 5% defectives somewhere in between. Since
statistical sampling procedures are by nature an imperfect decision
making tool, a shipper's risk (the likelihood that a clearly acceptable
shipment will be rejected on the basis of sample results) and a
manufacturer's risk (the likelihood that a clearly unacceptable shipment
will in fact be accepted) must be specified, again based on economic
considerations. In the present application, the AOAC test corresponds
to the sample of parts and Dr. Trout describes how the actual producer's
risk (i.e., "shippers risk") and consumer's risk (i.e., "manufacturer's
risk") may bs derived from the mathematical properties of the present
decision rule (i.e., "pass" if 59 or 60 out of 60 tubes evidence no
growth).
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At present, two major statistical difficulties are apparent in applying
these theories to the AOAC test methodology. First, there exists no
counterpart to the "economic considerations" of the industrial example.
That is, a priori considerations do not clearly dictate that, for
example, "acceptable disinfectants" should sterilize 90% of tubes
tested, or 97% of tubes tested, etc. Second, classical acceptance
sampling methodology assumes that sample test results are always correct
and thus may be perfectly replicated. For example, it seems relatively
simple to test if an automobile door fits according to specifications as
one simply measures the door. So long as different divisions use
calibrated measuring devices, a certain door should suggest the same
pass/fail decision regardless of what division performs the sampling or
when the sampling is performed. It is not clear to what extent AOAC
test results may be replicated on identical disinfectants, either from
laboratory to laboratory or even from day to day within a given
laboratory. The results of Dr. Walter's collaborative study are
encouraging with respect to consistency, although Dr. Cole's research
suggests that disinfectants are often subject to differing microbial
loads. Of particular concern are the results of Dr. Cole's examination
of the surfaces of typical penicylinders by scanning electron microscopy
suggesting that penicylinder quality may potentially be a major source
of variability for test results.
The proposed collaborative test addresses both of the above statistical
issues. Rather than attempting to make an a priori decision as to an
acceptable "passing vote" for penicylinders, a random sample of
disinfectant products from major producers will be used to generate
baseline test data for typical disinfectants. These test data may then
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be used as a standard when discussing acceptable disinfectant
performance under the present AOAC test. Although the primary purpose
of this collaborative study is to aid in determining appropriate
pass/fail standards, data will also be generated as to the consistency
of results between laboratories. Also, a questionnaire about laboratory
techniques and specifications may aid in uncovering sources of between
laboratory variability, if such variability is in fact observed.
A number of steps will be taken to help to insure the validity of the
findings from the proposed collaborative study. First, disinfectants
will be randomly selected (using a random numbers table) from a list of
products available from major producers. Thus, the sample is expected
to be representative of typical disinfectants. Second, the
collaborative will be "blinded" in that collaborating laboratories will
not be told the identity of the disinfectants tested, only the
appropriate use-dilution. The name of the disinfectants will not be
disclosed.
Finally, Dr. Cole will subject the sample disinfectants to "ruggedness
testing" before sending them to the participating laboratories. That
is,, the effects of slightly changing laboratory conditions will be
considered. After passing the "ruggedness tests", it will be expected
that reasonable between laboratory consistency will be observed.
Gregory P. Samsa, M.S.
Biostatistician
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Disin.
Collaborative Study
AOAC USE-DILUTION METHOD
Salmonella choleraesuis ATCC 10708
#Broth Transfers
Quat Disinfectants
Positives/Batch Date
Phenol Resistance
Dilution 5 10 15
1.
2.
3.
Phenolic Disinfectants
4.
5.
-j-.* _/__. _/
6.
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#Broth Transfers
Collaborative Study
AOAC USE-DILUTION METHOD
Staphylococcus aureus ATCC 6538
Quat Disinfectants
Pos i tives/Batch Date
Phenol Resistance
Dilution 5 10 15
1.
2.
3.
4.
5.
6.
Phenolic Disinfectants
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Disin.
Collaborative Study
AOAC USE-DILUTION METHOD
Pseudomonas aeruglnosa ATCC 15442
Quat Disinfectants
#Broth Transfers
Positives/Batch
Phenol Resistance
Dilution 5 10 15
1.
2.
3.
Phenolic Disinfectants
4.
5.
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Collaborative Study Questionnaire 171
AOAC USE-DILUTION METHOD
MgdJ.a;
1.
Type of Broth. S. aurcus;
S. choleraesu i s;
P. aerucii nosa:
H Nutrient Broth -
Anatone lot tt and date of purchase:
Di-fco Bee-f Extract lottt and date o-f purchase;
I-f other than above brands, were used, indicate:
3. If Synthetic Broth, was it Lab prepared or commercial?
4. ]f commercial Synthetic Broth -
Brand, lottt and date of purchase:
Water
1. Was the same type of water used for media and disinfectant
di lut ion?
2. Is water distilled, deionized/disti11ed, or deionized?
3. Is the water lab prepared or commercial water?
4. If lab prepared, glass distilled?
- conductivity in micromhos:
- resistance in megohms:
5. If commercial, brand and lottt:
Carr iers
1. S&L, Fisher, Colgate planchet, other:
2. Approximate date of purchase of carriers used:
3,. Describe carrier treatment between decontamination and NaOK soak
(e.g., exposure to cleaner, surfactant, sonication, and distilled
water r inse , etc .) :
Describe the finish of the carriers used:
dull .
j>o1 i shed |
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Equipment
1. Are all thermometers calibrated periodically against NBS certified
ones (those traceable to National Bureau of Standards thermometer)?
2. Pipettes used (plastic disposable, glass reusable, etc.):
3. How often is the ph meter standardized?
4. Analytical balance used to weigh reagents
Manufacturer:
Model No:
Sensitivity:
When caIi brated:
5. Describe procedure for cleaning reusable glassware:
Personnel:
1. How many technicians performed this testing?
Laboratory:
1. Approximately how many Use-Dilution tests are performed in your
laboratory per year?
Other Comments:
4-
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