Disinfectants during Clinical Use - A Suspension Method To Determine Reuse Life of Chemical
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1988, p. 158-164 Vol. 54, No. 1
0099-2240/88/01158-07$02.00/0
Copyright © 1988, American Society for Microbiology
A Suspension Method To Determine Reuse Life of Chemical
Disinfectants during Clinical Use
RICHARD A. ROBISON,'* HOWARD L. BODILY,2 DAENA F. ROBINSON,' AND RELLA P. CHRISTENSEN'
Microbiology Section, Clinical Research Associates, Proovo, Utah 84604,1 and Department of Microbiology, Brigham
Young University, Provo, Utah 846022
Received 15 June 1987/Accepted 28 September 1987
In-use testing of disinfectants is necessary to ensure efficacy over time. The current official procedure for
testing disinfectants, the Association of Official Analytical Chemists (AOAC) use-dilution method, cannot be
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adapted to repeated sampling techniques of use-life testing. It is therefore necessary to use an alternative
method when evaluating the activity of a disinfectant under actual use. The Clinical Research Associates (CRA)
suspension method was developed to fill this need. It consists of adding 0.5 ml of a standard culture to 5.0 ml
of test disinfectant and sampling the mixture after 10 min for surviving bacteria. When this test was compared
with the AOAC use-dilution method under a simulated use situation, the two methods were generally
equivalent in identifying disinfectant inactivation. In addition, the CRA method was less time consuming,
easier to perform, and less variable than the AOAC method. Use of the CRA method in a clinical study
demonstrated the need for reuse claims to be based on clinical use studies rather than on laboratory testing
only.
Reuse of chemical disinfectants for periods ranging from cannot be used for this purpose because the volume of
days to weeks has been a routine practice throughout the disinfectant required is too large to allow repeated testing
health care industry of the world. Many disinfectants, how- over time without requiring an immense quantity of disin-
ever, were never intended for prolonged, repeated use, and fectant to be dispensed initially. Such a large volume would
they lose their ability to inactivate microorganisms after a give misleading results since the ratio of disinfectant to
short time (5). Because clinicians have no way to assess the instruments would be unrealistic. The AOAC method is also
potency of a disinfectant, they unwittingly use solutions qualitative, very time consuming, and difficult to standard-
which may have little or no antimicrobial activity. This ize. Therefore, a quantitative suspension test (hereafter
practice has important implications to the well being of large referred to as the Clinical Research Associates [CRA] sus-
numbers of people who are treated daily in hospitals and pension method) was developed to circumvent these prob-
outpatient medical and dental facilities, as well as the clinical lems. In the development of such a method, an effort was
personnel performing the treatment. made to adhere to the same culture-to-disinfectant ratio and
In actual clinical use, chemical disinfectants are subjected exposure time used in the United States standards. The
to many conditions that challenge their antimicrobial stabil- culture-to-disinfectant ratio used is identical with that of the
ity. Some of these include dilution, age, and contamination AOAC phenol coefficient test, and the exposure time (10
with chemicals and organic matter (6, 9, 15). While many of min) is that specified in the AOAC use-dilution method.
the clinical challenges can be mimicked in laboratory testing, Other suspension-type tests such as the standard methods of
the true nature of many other potential inactivators can only France, Germany, The Netherlands, and the United King-
be surmised. Dental office environments offer a particular dom are methodologically more difficult. Some use washed
problem owing to the broad range of possible contaminants bacterial suspensions, which have been shown to be less
contained in the large number of dental materials used resistant than untreated broth cultures (13), or multiple
routinely (i.e., resin polymers, glass filters, metal alloys, exposure times which make interpretation of the results
viscosity modifiers, etc.). more complex (4, 13). The CRA method was designed to be
These facts reinforce the need for reliable disinfectant as simple as possible to perform and still give quantitative
efficacy testing. Many evaluation methods exist (4). Most of results.
these, however, are designed to test only the initial activity The purposes of this study were to compare results
of an unused product. Although new Environmental Protec- obtained with the CRA suspension method with those of the
tion Agency criteria now specify that documentation must be AOAC use-dilution method to establish a point of reference
provided by vendors if reuse claims are made, an accepted, between the two procedures and to use the CRA suspension
standardized protocol has not been specified. Test protocols method in a clinical comparison study to determine its
used currently may not reflect clinical reality since stressing feasibility as a method for reuse testing.
agents are limited to microorganisms and proteins (8, 11).
A method is needed that will permit field testing of MATERIALS AND METHODS
commercial disinfectants to establish reliable reuse recom-
mendations and substantiate reuse claims of manufacturers Media and chemicals. Tryptic soy broth (TSB; Difco
(16). The only current standard test method, the Association Laboratories, Detroit, Mich.) was used for the CRA suspen-
of Official Analytical Chemists (AOAC) use-dilution method, sion method cultures. Tryptic soy agar (TSA) was made by
the addition of 1.5% Bacto-Agar (Difco) to the TSB de-
scribed above. Both the TSB and TSA used in the CRA
*
Corresponding author. suspension method contained the neutralizers specified by
158VOL. 54, 1988 REUSE LIFE OF CHIEMICAL DISINFECTANTS 159
the AOAC for letheen broth (2). These were 0.07% lecithin the manufacturers in preparing these solutions. All glutaral-
(Sigma Chemical Co., St. Louis, Mo.) and 0.5% Tween 80 dehyde-containing disinfectants were at a concentration of
(Fisher Scientific Co., Pittsburgh, Pa.). Nutrient broth used 2% before dilution. The acid glutaraldehyde was diluted 1:4,
in the AOAC use-dilution method was prepared as specified the alkaline glutaraldehyde was not diluted, and the gluta-
by the AOAC (2). It consisted of 0.5% beef extract (Difco), raldehyde-phenol was diluted 1:16. Only the alkaline gluta-
0.5% NaCl (Mallinckrodt, Inc., St. Louis, Mo.), 1% peptone raldehyde and glutaraldehyde-phenol solutions required ac-
(Difco), and the same concentration of neutralizers as de- tivation. The phenolic and quat preparations were diluted
scribed above. 1:32 and 1:64, respectively. The label on the quat suggested
Preparation of frozen stock cultures. Pseudomonas aeru- that distilled water be used as a diluent in areas of known
ginosa ATCC 15442, Salmonella cholerae-suis ATCC 10708, water hardness. Therefore, to comply with label instructions
and Staphylococcus aureus ATCC 6538, three standard and to provide a point of comparison with the other disin-
strains used in disinfectant testing, were obtained directly fectants, which were diluted with tap water, the same quat
from American Type Culture Collection (Rockville, Md.) for was included twice, once with tap water and once with
use in this study. Since S. choleraesuis and P. aeruginosa deionized water as the diluents.
produce both smooth and rough colonies, mixtures of these A 3-liter batch of each solution was prepared according to
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colony forms were used to prepare TSB cultures which were the label instructions of each manufacturer. Table 1 shows
grown at 37°C for 24 h without shaking. These cultures were the suggested reuse life, use-dilution, and final active ingre-
mixed with equal volumes of sterile 20% glycerol (Sigma) dient concentration of each solution. All disinfectants were
and dispensed in 1.5-ml aliquots to sterile Nunc tubes stored in covered plastic containers (Surgikos model 2010) at
(Vangard International, Neptune, N.J.). All tubes were room temperature (approximately 22°C) for the duration of
stored in liquid nitrogen until used. the study.
Preparation of CRA suspension method cultures. A thawed The disinfectants were challenged by the addition of 10 ml
stock culture (1 ml) was added to 9 ml of TSB. This primary of whole human blood at the end of each working day (5 days
culture was incubated without shaking at 37°C for 3 h to per week) for 4 weeks. Twice a week, in the morning, each
generate log-phase cells. The secondary or test culture was disinfectant was sampled. This involved the removal of 1,800
prepared with a 10% inoculum from the primary culture and ml for AOAC testing (3 organisms x 60 replicates x 10 ml
incubated for 20 to 24 h at 37°C without shaking. per tube) and 45 ml for the CRA method (3 organisms x 3
Selection of disinfectants. The choice of which solutions to replicates x 5 ml per tube). AOAC use-dilution and CRA
use in the methods comparison was based on what is suspension tests were performed on each sample. The dis-
currently being used in dentistry (5; Clinical Research As- infectant from the AOAC tubes was returned to the batch
sociates, Clin. Res. Assoc. Newsletter, 9:1, 1985). Environ- after testing (5); even so, approximately 175 ml of each
mental Protection Agency-registered disinfectants with var- disinfectant was lost each sampling day owing to the two
ious active ingredients were purchased for use in this study assay procedures. This loss was taken into consideration
(see Table 1). Six solutions (acid glutaraldehyde, alkaline when the percentage of blood in each disinfectant was
glutaraldehyde, glutaraldehyde-phenol, phenolic, and two computed for each sampling day.
solutions containing the same quaternary ammonium com- AOAC use-dilution method. The standard test employing
pound [quat]) were mixed and/or diluted by the directions of organisms dried onto stainless-steel penicylinders was used
the manufacturer. Even though quats have lost the approval (2). Each disinfectant sample was tested against 60 carriers
of the American Dental Association owing to their relatively prepared from each of the three standard organisms listed
low level of activity, at least 30% of practitioners in the above (180 total). The carriers were coated with suspensions
United States continue to use quats because they are rela- prepared by daily transfers as specified by the AOAC. All
tively nontoxic and inexpensive (5; Clinical Research Asso- transfers were performed in a 20°C water bath. A secondary
ciates, Clin. Res. Assoc. Newsletter, 9:1, 1985). Therefore, subculture was performed on each carrier (as recommended
some low-level disinfectants were included in this study. by the AOAC when disinfectant residual effects are sus-
Disinfectant preparation, loading, and sampling for meth- pected). Both subcultures were incubated at 37°C for 48 h.
ods comparison. Care was taken to follow the directions of Growth in either tube was scored as a positive for that
TABLE 1. Reuse life,a use dilution, and active ingredient concentrations of the disinfecting solutions used in the methods comparison
Disinfectant Sugested reuse
life (days) Suggested
use dilution Diluent Use concn of active ingredient(s)
Acid glutaraldehyde 21 1:4 Tap water 0.5% Acid glutaraldehyde
Alkaline glutaraldehyde 28 Undiluted 2.0% Alkaline glutaraldehyde
Glutaraldehyde-phenol 30 1:16 Tap water 0.44% Phenol
0.15% Sodium tetraborate
0.13% Glutaraldehyde
0.08% Sodium phenate
Phenolic 1 1:32 Tap water 0.28% o-Phenylphenol
0.03% o-Benzyl-p-chlorophenol
Quat-deionized water 1:64 Deionized water 0.2% Isopropyl alcohol
0.1% Cetyldimethylethylammonium bromide
0.1% Benzalkonium chloride
Quat-tap water 1:64 Tap water 0.2% Isopropyl alcohol
0.1% Cetyldimethylethylammonium bromide
0.1% Benzalkonium chloride
a Reuse life is defined as the length of time a disinfectant may be reused clinically, as opposed to shelf life, which is the stability of an unused solution.160 ROBISON ET AL. APPL. ENVIRON. MICROBIOL.
carrier. Broth from all tubes showing growth was streaked lines on a graph denotes a different test organism. A lower
on TSA to confirm the presence of the test organism and the log reduction (CRA method) translates into a higher viable
absence of contamination. count and thus a lower level of disinfectant activity. Simi-
CRA suspension method. A 0.5-ml sample of test culture larly, a lower number of negative tubes (AOAC method)
was added to 5.0 ml of disinfectant in a 20°C water bath. The corresponds to an increase in the number of positive tubes
suspension was mixed thoroughly and returned to the bath. and also represents a lower activity. A downward trend in
After a 10-min exposure, a 1-ml sample was removed and any line represents a loss of antimicrobial activity for a
diluted in TSB (with neutralizers). Samples of 1 ml from each particular disinfectant-method-organism combination.
dilution tube were assayed with duplicate pour plates in TSA Figures 1 and 2 demonstrate the antimicrobial activity of
(with neutralizers) poured at 45°C. The dilutions were ex- the acid glutaraldehyde (1:4) and alkaline glutaraldehyde
tended as far as necessary to include the expected counts. (undiluted), respectively. Neither solution showed signifi-
The CRA procedure was repeated three times for each cant inactivation over time when subjected to the organic
standard organism, making a total of nine determinations on loading. Both the CRA and AOAC methods predicted good
each disinfectant sample. A standard plate count employing disinfectant activity against all three organisms up through
serial 1:10 dilutions in TSB (with neutralizers) was per- 24 days and showed that the two solutions were not affected
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formed on each test culture to establish a base-line concen- substantially by the addition of blood (6.3%).
tration of viable organisms. All plates were incubated at 37°C The performance of the glutaraldehyde-phenol (1:16) is
for 48 h. shown in Fig. 3. This preparation was affected noticeably by
Log reductions were calculated by the following formula: organic stress. Both the CRA and AOAC methods indicated
log reduction = log (number of organisms per milliliter a sharp decline in effectiveness after the blood reached a
before exposure) - log (number of organisms per milliliter concentration of about 3.2%. However, some organism-
after exposure). method differences were apparent. The AOAC method
Disinfectant preparation, sampling, and testing for clinical showed the day 17 solution to be effective against S. chole-
comparison. Three commercially available glutaraldehyde- raesuis, whereas the CRA method indicated that this same
based disinfectants (acid glutaraldehyde, diluted 1:4; alka- solution was almost completely ineffective against this organ-
line glutaraldehyde, undiluted; and glutaraldehyde-phenol, ism. Conversely, the CRA method indicated the day 17
diluted 1:16) were evaluated during actual clinical use. A solution to have some activity against S. aureus; however,
latin-square study design was used. Three busy dental offices the AOAC method showed little activity.
known to use liquid disinfectants instead of heat sterilization
were sought as field-testing sites. Selection was based on the
number of patients treated per day. The study was con- CRA
ducted in three 5-week periods. Each disinfectant was ro- >8 FB
tated randomly through the three offices, such that each
agent was used in every office. 7
A 2.5-liter batch of each solution was prepared according 6
to the instructions of the manufacturer. A 250-ml sample was c
removed from each batch and placed in a sterile dark glass .9 5
bottle which was kept in the laboratory at room temperature -g4
to serve as an unused control. The remaining 2,250 ml was a:
dispensed to a clinical site for use. Each office used identical 0 3
-j
plastic containers with removable trays (Surgikos model 2
2010).
Twice a week, for 5 weeks, 23 ml was removed from each
solution (and its unused control) for testing. The activity of n
each solution was assayed by the CRA suspension method,
using each of two separately prepared cultures of S. chole- AOAC
raesuis. With the means at our disposal, it was not possible
to test each sample against all three standard organisms. 600
Therefore, to provide replication, we decided to use one
organism in duplicate. At the end of the study, the effective- (n
0
50F
ness of each solution at each time point was determined from Dg 40
I-
the average of six log reduction values (two determinations
at each of three sites). *' 30
Z 20
RESULTS
10
Methods comparison. Figures 1 through 7 compare the
CRA and AOAC methods in their ability to detect disinfec- 0 I I I
tant inactivation over time. The activity of each disinfecting 0 3 7 10 14 17 21 24
solution is represented by two plots. The upper graph (0.9) (1.5) (2.5) (3.2) (4.3) (5.1) (6.3)
demonstrates results obtained by the CRA suspension Sample Day (%Blood)
method, and the lower graph shows results of the AOAC FIG. 1. Comparison of the CRA and AOAC methods in moni-
use-dilution method. The log reduction values (CRA toring the inactivation of a 0.5% acid glutaraldehyde solution during
method) and the number of negative tubes/60 (AOAC simulated clinical use. Three standard organisms were used. Each
method) were both plotted against time (and percentage of CRA value is the mean of three separate determinations. Symbols:
blood) to generate comparable curves. Each of the three 0, S. aureus; 0, S. choleraesuis; A, P. aeruginosa.VOL. 54, 1988 REUSE LIFE OF CHEMICAL DISINFECTANTS 161
CRA When the data were further collapsed across disinfectants, a
single line for each method was produced (Fig. 7). This
overall comparison shows that disinfectant activity is pre-
7 dicted equivalently by both the CRA and AOAC methods.
6 Comparison of method variability. Table 2 shows the
C
.Q 5 coefficients of variation for each disinfectant-method combi-
nation. The CRA coefficients were consistently smaller than
-S4 those of the AOAC method. The AOAC coefficients for the
a: acid and alkaline glutaraldehyde solutions could not be
o0 3 estimated reliably because of the small numbers of positive
-j
2 tubes obtained with these solutions. This did not prevent the
assessment of the overall coefficient of variation for the
I I
AOAC method, which was almost six times greater than that
of the CRA method.
Clinical comparison. Figure 8 shows how three glutaralde-
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AOAC hyde-based disinfectants performed under actual clinical
use, as monitored by the CRA suspension method. The acid
(1:4) and alkaline (undiluted) glutaraldchyde solutions re-
mained effective for nearly the entire sampling period. The
0
0
glutaraldehyde-phenol (1:16) preparation, however, steadily
A lost activity after the eighth day of use. The unused control
solutions from all disinfectants showed full effectiveness
throughout the 36-day sampling period.
DISCUSSION
The purpose of this study was not to suggest a replace-
o ment for the AOAC use-dilution method. Rather, it was to
0 3 7 10 14 17 21 24 validate a procedure that could be used in cases that are
(0.9) (1.5) (2.5) (3.2) (4.3) (5.1) (6.3)
Sample Day (%Blood) CRA
FIG. 2. Comparison of the CRA and AOAC methods in moni-
toring the inactivation of a 2% alkaline glutaraldehyde solution
during simulated clinical use. Three standard organisms were used.
Each CRA value is the mean of three separate determinations.
Symbols: 0, S. aureus; 0, S. choleraesuis; A, P. aeruginosa.
Figure 4 shows the performance of the phenolic prepara-
tion. This solution lost activity against S. aureus only. The
AOAC method showed an earlier inactivation which was
more erratic than that depicted by the CRA method; how-
ever, both identified the ineffectiveness of the solution
against S. aureus after day 17 (4.3% blood). Adequate
activity against the other two test organisms was detected
equally by both methods.
Figure 5 indicates the activity of the quat diluted in AOAC
deionized water. This solution showed inactivation over
time with respect to S. aureus and P. aeruginosa. Neither
method indicated any substantial loss of activity against S.
choleraesuis. The CRA method depicted a more pronounced
inactivation over time than the AOAC method, especially
against S. aureus.
The activity of the quat diluted with tap water is shown in
Fig. 6. This preparation exhibited a pattern of effectiveness
much different from that of the quat diluted with deionized
water. The early lack of activity against P. aeruginosa was
similarly detected by both the CRA and AOAC methods;
however, a steady decline in effectiveness against the other
two organisms is more clearly represented by the CRA data. 3 7 10 14 17 21 24
A condensation of methods comparison data allows an (0.9) (1.5) (2.5) (3.2) (4.3) (5.1) (6.3)
easier interpretation of the results. When the data from each Sample Day (%Blood)
method were averaged across the three organisms used, FIG. 3. Comparison of the CRA and AOAC methods in moni-
eight values (one for each sample day) for each disinfectant- toring the inactivation of a glutaraldehyde-phenol solution during
method combination were obtained. A correlation analysis simulated clinical use. Three standard organisms were used. Each
of this data produced a Pearson product moment of 0.91, CRA value is the mean of three separate determinations. Symbols:
indicating a strong agreement between the two methods. 0, S. aureus; O, S. choleraesuis; A, P. aeruginosa.162 ROBISON ET AL. APPL. ENVIRON. MICROBIOL.
CRA the organism-related differences observed between the two
methods.
Since these microorganism differences were method de-
pendent it was necessary to average the data across orga-
nisms to compare the two methods more accurately. When
c
0
this was done, the CRA and AOAC methods were highly
correlated (Pearson product moment = 0.91). A further
compaction of the data across disinfectants produced the
lines in Fig. 7. This graph shows the CRA values to be
.0 slightly lower than those of the AOAC method, suggesting
that the AOAC method is slightly more restrictive. It also
shows that the two lines follow each other quite closely,
indicating that disinfectant activity is equivalently predicted
C,)I by both methods. Figure 7 was constructed by equating a
0) CRA log reduction greater than or equal to 8 with 60 negative
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AOAC AOAC tubes. These numbers represent the maximum
achievable values of each method.
The concept of log reduction used here as a quantitative
measure of disinfectant efficacy has been used by other
0
investigators (14, 17). The minimum acceptable log reduc-
tions for several standard methods were summarized by
CD
Reybrouck (12). A value between 5 and 6 is representative of
1_ I most suspension tests. A log reduction of 6 was the sug-
Q) gested minimum in a clinical study by Christensen et al. (3).
z d1 However, a log reduction of 8 was established as the
minimum acceptable level for the CRA suspension method
to come as close as possible to the pass-fail criterion of the
AOAC use-dilution method (Fig. 7). Therefore, any disin-
3 7 10 14 17 21 24
(0.9) (1.5) (2.5) (3.2) (4.3) (5.1) (6.3)
Sample Day (%Blood) CRA
FIG. 4. Comparison of the CRA and AOAC methods in moni-
toring the inactivation of a phenolic solution during simulated
clinical use. Three standard organisms were used. Each CRA value
is the mean of three separate determinations. Symbols: 0, S.
aureus; O, S. choleraesuis; A, P. aeruginosa. C
0 5
-g4
0)3
inappropriate for the AOAC method, such as testing a ,m
0 3
-j
disinfectant over time as it undergoes clinical use. This 2
validation was accomplished by comparing the log reduction
curve from the CRA suspension method with the plot of
negative tubes from the AOAC use-dilution method.
The ideal disinfectant for comparing these two methods is
one which exhibits a linear deterioration over time. The AOAC
solution that most nearly fit this pattern was the glutaralde-
hyde-phenol (Fig. 3). This disinfectant had a high initial
activity, lost activity through the course of the test period,
and exhibited low activity at the end of the study. The
shapes of the CRA and AOAC curves for this solution are
very similar, indicating a general agreement between the two
methods.
A closer inspection of these curves, however, reveals an
inversion of the S. aureus and S. choleraesuis lines. This is
presumed to be due to a fundamental difference between the
methods. The AOAC method requires drying of organisms;
the CRA method does not. Our experience with environmen-
tal surface disinfection procedures, in which dried test 0 3 7 10 14 17 21 24
organisms are used, has confirmed the well-established fact (0.9) (1.5) (2.5) (3.2) (4.3) (5.1) (6.3)
that most gram-positive organisms survive desiccation better Sample Day (%Blood)
than gram-negative organisms. The number of viable S. FIG. 5. Comparison of the CRA and AOAC methods in moni-
aureus organisms is almost 1,000 times greater than that of toring the inactivation of quat diluted in deionized water. Three
S. choleraesuis when these two organisms are dried under standard organisms were used. Each CRA value is the mean of three
identical conditions (unpublished data, Clinical Research separate determinations. Symbols: 0, S. aureus; O, S. cholerae-
Associates). This disparity could be responsible for most of suis; A, P. aeruginosa.VOL. 54, 1988 REUSE LIFE OF CHEMICAL DISINFECTANTS 163
CRA TABLE 2. Coefficients of variation of CRA and AOAC methods
Coefficient of variation
Disinfectant
CRA AOAC
Acid glutaraldehyde 3.04 __a
Alkaline glutaraldehyde 3.04
Glutaraldehyde-phenol 10.07 33.19
Phenolic 3.73 37.35
Quat-deionized water 5.59 40.40
Quat-tap water 8.35 21.25
Overall 5.64 33.05
a-, The number of positive tubes/60 was so small that the coefficients
could not be estimated reliably.
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AOAC
findings confirmed this fact, with the overall coefficient of
variation of the AOAC method being almost six times
greater than the corresponding CRA value. One contributing
factor to the variability associated with the AOAC method is
the fluctuating organism load on the penicylinders. The
number of organisms attached to carriers is highly dependent
on the bacterial species used. Differences up to 100-fold have
been reported (E. C. Cole, W. A. Rutala, and J. L. Carson,
Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, Q41, p. 264).
Large differences in the bacterial load from carrier to carrier
were also seen by Ascenzi et al. (1). In addition, they
showed that most of the viable organisms were washed from
0 3 7 10 14 17 21 24 the carrier into the disinfecting solution. This compounds the
(0.9) (1.5) (2.5) (3.2) (4.3) (5.1) (6.3) reproducibility problem, since the majority of the test or-
Sample Day (%Blood) ganisms remain in the solution and are never subcultured.
FIG. 6. Comparison of the CRA and AOAC methods in moni-
A comparison of disinfectants with respect to their inac-
toring the inactivation of quat diluted in tap water. Three standard tivation over time was not the primary intent of this study.
organisms were used. Each CRA value is the mean of three separate However, some interesting differences between solutions
determinations. Symbols: 0, S. aureus; 0, S. choleraesuis; A, P. were apparent. The glutaraldehydes are known for their
aeruginosa. ability to continue to disinfect in the presence of high levels
of organic contamination (7, 10). Our study confirmed this
point. Neither the acid (1:4) nor alkaline (undiluted) glu-
fectant which could not equal or exceed this value during use taraldehyde solution showed any significant inactivation
was considered to have questionable clinical efficacy. owing to organic load during the study. Even at the minimal
Although the CRA and AOAC methods have similar concentration of 0.5% active ingredient, the acid glutaralde-
predictive abilities, the AOAC use-dilution method has the hyde solution remained effective under the stress of 6.3%
major disadvantage of being highly variable (1, 8). Our human blood and exposure to 1,260 organism-coated peni-
cylinders. The other four solutions experienced some degree
of inactivation. The phenolic solution lost activity against S.
a 8 60
_ ,-- s
7 ^- ^ ^ - A ^ A *
*6 45 0
v w \-
7
.Q 5
t5 6
_vo~V~V
V---VV
'4 30 , c
133 ,_z .g 4
2
1
15
_? 3
2
\vv\
0
I I I I I I
1
0 3 7 10 14 17 21 24
(0.9) (1.5) (2.5) (32) (4.3) (5.1) (6.3) 12 15 19 22 26 29 33 36
0 5 8
Sample Day (%Blood) Use - Day
FIG. 7. Overall comparison of the CRA (log reduction) and FIG. 8. Log reductions for three glutaraldehyde-based disinfec-
AOAC (negative tubes) methods averaged across three standard tants during actual clinical use. Each value is the mean of six
organisms and the six disinfecting solutions used in the methods determinations; two from each of three clinical sites, evaluated by
comparison. Symbols: *, CRA log reduction; *, number of AOAC separate cultures of S. choleraesuis. Symbols: alkaline glutar-
0,
tubes showing negative growth. aldehyde; *, acid glutaraldehyde; V, glutaraldehyde-phenol.164 ROBISON ET AL. APPL. ENVIRON. MICROBIOL.
aureus only, and this did not occur until the blood concen- data. We thank Kelly Lundeen and Debbie Cox for their technical
tration reached approximately 4.3%. The inactivation of the assistance.
glutaraldehyde-phenol (1:16) solution was more extensive. A This study was supported by Clinical Research Associates.
decline in effectiveness against all three test organisms was LITERATURE CITED
observed after the blood concentration reached approxi- 1. Ascenzi, J. M., R. J. Ezzell, and T. M. Wendt. 1986. Evaluation
mately 4%. The relative ease with which some quats are of carriers used in the test methods of the Association of Official
inactivated is well known (5). The quat-deionized water Analytical Chemists. Appl. Environ. Microbiol. 51:91-94.
solution showed a decrease in activity against S. aureus after 2. Association of Official Analytical Chemists. 1984. Disinfectants,
only 0.9% added blood. This solution, however, was much p. 65-77. In W. Horwitz (ed.), Official methods of analysis, 14th
more effective than the quat-tap water solution, which had a ed. Association of Official Analytical Chemists, Washington,
very low activity against P. aeruginosa before any blood was D.C.
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The second purpose of this study was to use the CRA 4. Cremieux, A., and J. Fleurette. 1983. Methods of testing disin-
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solutions are the current state of the art in liquid disinfec- evaluation of germicidal solutions used for the sterilization or
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clinical comparison. The results of this study are shown in ADM Lab. J. 12:1-8.
Fig. 8. This represents data from the clinically used solutions 6. Gelinas, P., and J. Goulet. 1983. Neutralization of the activity of
only. Results from the unused (control) portions showed no eight disinfectants by organic matter. J. Appl. Bacteriol. 54:243-
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is important since suggested reuse lives in general are not 7. Gorman, S. P., E. M. Scott, and A. D. Russell. 1980. A review.
based on clinical use studies; rather, they are derived from Antimicrobial activity, uses and mechanism of action of glutar-
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lated use only. It can be seen that the reuse life claims in capacity use-dilution test for disinfectants. Methods Bull. Min-
Table 1 have no correlation with the performance of a istry Health 24:152-160.
disinfectant. The acid glutaraldehyde solution has a sug- 9. Kortenbout, W. P. 1982. Some factors influencing the effective
gested reuse life of 21 days, but its activity remained high use of disinfectants and cleaning agents. Curationis 2:29-33.
throughout the 36-day sampling period. Conversely, the 10. Miner, N. A., J. W. McDowell, G. W. Willcockson, N. I. Bruck-
glutaraldehyde-phenol is labeled with a 30-day reuse life, but ner, R. L. Stark, and E. J. Whitmore. 1977. Antimicrobial and
under heavy use, it lost activity much sooner, probably other properties of a new stabilized alkaline glutaraldehyde
owing to the high dilution (1:16) and thus a lower initial disinfectant/sterilizer. Am. J. Hosp. Pharm. 34:376-382.
11. Miner, N. A., E. Whitmore, and M. L. McBee. 1975. A quanti-
glutaraldehyde concentration. The alkaline glutaraldehyde tative organic "soil" neutralization test for disinfectants. Dev.
had a reuse life claim more appropriate for its performance. Ind. Microbiol. 16:23-30.
This solution did not experience a loss of activity until 1 12. Reybrouck, G. 1975. A theoretical approach of disinfectant
week after its labeled reuse life claim. These inconsistencies testing. Zentralbl. Bakteriol. Hyg. Abt. 1 Orig. B 160:342-367.
reinforce the need for a reliable method of clinical in-use 13. Reybrouck, G. 1980. A comparison of the quantitative suspen-
testing which can establish proper reuse life claims. The sion tests for the assessment of disinfectants. Zentralbl. Bakte-
CRA suspension method fills this need. riol. Hyg. Abt. 1 Orig. B 170:449-456.
A modification of the CRA suspension method is currently 14. Reybrouck, G., J. Borneff, H. Van de Voorde, and H. P. Werner.
being tested in a field evaluation of disinfectants in which 1979. A collaborative study on a new quantitative suspension
test, the in vitro test, for evaluation of the bactericidal activity
pour plates are replaced by membrane filtration. The number of chemical disinfectants. Zentrabl. Bakteriol. Hyg. Abt. 1 Orig.
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bacterium bovis and poliovirus type 1. Results of field testing 15. Russell, A. D. 1974. Factors influencing the activity of antimi-
with these additional organisms should provide an even crobial agents: an appraisal. Microbios 10:151-174.
more detailed picture of how disinfectants perform over time 16. Sykes, G. 1962. The philosophy of the evaluation of disinfec-
in clinical environments. tants and antiseptics. J. Appl. Bacteriol. 25:1-11.
17. Van Klingeren, B., A. B. Leussink, and L. J. Van Wingaarden.
ACKNOWLEDGMENTS 1977. A collaborative study on the repeatability and the repro-
ducibility of the Dutch standard-suspension-test for the evalua-
We thank Melvin Carter of the Center for Statistical Research, tion of disinfectants. Zentralbl. Bakteriol. Hyg. Abt. 1 Orig. B
Brigham Young University, for his help in the interpretation of the 164:521-548.You can also read
