JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1991, p. 592-594 Vol. 29, No.

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0095-1137/91/030592-03$02.00/0
Copyright 1991, American Society for Microbiology

Comparison of Microbiologic Assay Methods for

Hemodialysis Fluids
MATTHEW J. ARDUINO,* LEE A. BLAND, SONIA M. AGUERO, LORETTA CARSON,
MITCHELL RIDGEWAY, AND MARTIN S. FAVERO
Nosocomial Infections Laboratory Branch, Hospital Infections Program, Center for
Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333
Received 5 October 1990/Accepted 20 December 1990

To help prevent pyrogenic reactions and bacteremia in hemodialysis patients, the Association for the
Advancement of Medical Instrumentation and the Centers for Disease Control recommend microbiologic assay

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of hemodialysis fluids at least monthly. Five commercially available assay systems were evaluated by using the
membrane filtration technique with standard methods agar and trypticase soy agar as the standards for
comparison. Each assay system was challenged with dialysate and reverse-osmosis water from local dialysis
centers, aqueous suspensions of eight laboratory strains of gram-negative bacilli and nontuberculous
mycobacteria, and a mixed microbial flora inoculated into reverse-osmosis water and laboratory-prepared
dialysate. Mean viable counts from triplicate samples were obtained after incubation at 37C for up to 72 h. The
efficiency of recovery varied with the specific type of microbial challenge. The SPC water sampler (Millipore
Corp., Bedford, Mass.) was the most consistent in obtaining the highest viable counts. Other commercial
systems were comparable to each other in overall performance. All assay systems tested provided an acceptable
balance between microbial recovery and required sampling time, equipment, and expertise.

Hemodialysis patients are at risk of developing endotoxin-
mediated pyrogenic reactions (11, 14) and bacteremia caused
by gram-negative bacilli during hemodialysis (8). These
complications are believed to be a result of high bacterial
contamination in dialysis fluids primarily caused by gram-
negative water bacteria (9) or inadequately reprocessed
hemodialyzers (1, 4, 10, 12). The Association for the Ad-
vancement of Medical Instrumentation (AAMI) and the
Centers for Disease Control (CDC) have developed micro-
biologic guidelines for dialysis fluids to protect the hemodi-
alysis patients from these adverse reactions. These guide-
lines recommend that water used to prepare dialysate should
not have viable colony counts that exceed 200 CFU/ml and
that water used to reprocess hemodialyzers should not
contain either bacteria >200 CFU/ml or endotoxin .1 ng/ml
(8). These recommendations suggest that dialysate should
not have total viable colony counts which exceed 2,000
CFU/ml and that dialysate should be cultured at least
monthly (3, 7). AAMI and CDC guidelines list a variety of
different sampling methods for quantitative microbiology
that can be used, such as conventional laboratory proce-
dures (i.e., spread plate and membrane filtration [MF]). In
this study, we evaluated microbial recovery by using several
commercially available samplers and the MF technique.
MATERIALS AND METHODS
Microbial assay systems. Five commercially available mi-
crobial assay systems were evaluated: (i) the SPC sampler
(Millipore Corp., Bedford, Mass.), (ii) the total-count water
tester (TCWT) (Millipore Corp.), (iii) the nutrient pad kit
with standard media (NP) (Nalgene, Rochester, N.Y.), (iv)
NP, with triphenyltetrazolium chloride (NP+) (Nalgene),
and (v) aerobic count plates (ACP), formerly PetriFilm SM
plates (3M Corp., St. Paul, Minn.). Each assay system was
compared with the standard MF technique using trypticase
*
Corresponding author.
592
soy agar (TSA) and standard methods agar (SMA) (2). Each
assay system was subjected to the same microbial challenges
with all samples run in triplicate.
Challenge organisms. Each assay system was challenged
with an aqueous suspension of one of the following bacteria:
Mycobacterium fortuitum, M. chelonae, M. chelonae-like
organisms, Flavobacterium meningosepticum, Pseudomo-
nas paucimobilus, CDC group Ve-1, and two strains of
Methylobacterium mesophilicum. Each strain was first
grown on blood-agar plates and then isolated for purity,
harvested, and resuspended in an aqueous suspension to a
final concentration of approximately 107 CFU/ml. The titer
of this suspension was determined, and a working stock
solution of each test organism was made to contain 100 to
1,000 CFU/ml.
Two of the challenge solutions contained a mixture of
Acinetobacter calcoaceticus subsp. anitratus, Pseudomonas
aeruginosa, P. cepacia, and P. putida. These challenge
solutions were made by first making an aqueous suspension
of each organism containing approximately 107 CFU/ml.
Titers of these suspensions were then determined, and equal
proportions of each suspension were added to 250 ml of
sterile reverse-osmosis water to achieve a final concentra-
tion of 100 to 1,000 CFU of each of the four organisms per
ml. The second was prepared in the same way except that
250 ml of filter-sterilized (0.2-,um-pore-size Nalgene dispos-
able filtration device) acetate dialysate was used.
Hemodialysis fluids. Samples of water, acetate dialysate,
and bicarbonate dialysate were collected from three local
dialysis centers to run as unknowns.
Assay procedure. The SPC and TCWT were tested by
immersing the paddle portion into a 100-ml beaker contain-
ing 90 ml of sample fluid for 30 s. The paddle was then
withdrawn from the beaker, and excess fluid was removed
by gently shaking. The paddle was then replaced into its case
for incubation.
The NP and NP+ were rehydrated with 3.5 ml of sterile
water for injection (Abbott Laboratories, Chicago, Ill.). One
VOL. 29, 1991 MICROBIOLOGIC ASSAY METHODS FOR HEMODIALYSIS FLUIDS 593

TABLE 1. Recovery of laboratory challenge organisms by seven assay procedures tested

Mean bacterial count (CFU/ml) by assaya
Organism
SMA TSA SPC TCWT NP NP+ ACP
Mycobacterium fortuitumb 425 307 567 558 71 143 527
Mycobacterium chelonae 111 148 83 76 73 76 121
Mycobacterium chelonae-like organism 106 81 113 107 107 120 NTC
Methylobacterium mesophilicum 1 22 0 12 9 15 26 NT
M. mesophilicum 2 36 35 39 39 43 45 NT
Pseudomonas paucimobilus 85 49 106 98 104 105 NT
GroupVe-1 145 149 212 189 112 145 NT
Flavobacterium meningosepticum 26 22 21 20 23 25 26
Reverse-osmosis water miXd 126 335 348 119 220 210 159
Dialysate miXd 182 335 150 77 199 255 205
" Assays: SMA, standard methods agar; TSA, trypticase soy agar; SPC, SPC sampler (Millipore Corp.); TCWT, Total count water tester (Millipore Corp.); NP,
nutrient pad (Nalgene Corp.); NP+, nutrient pad plus triphenyltetrazolium chloride (Nalgene Corp.); ACP, aerobic count plate (3M Corp.).

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b Nontuberculous mycobacteria incubated for up to 7 days.
c
NT, Not tested.
d The reverse-osmosis and dialysate mixes contain the following organisms: A. calcoaceticus, P. aeruginosa, P. cepacia, and P. paucimobilus.

milliliter of sample fluid was filtered through a 0.45-,um-pore-
size sterile cellulose nitrate filter (Micro Filtration Systems,
Dublin, Calif.) and aseptically placed on the rehydrated pads
for incubation.
The ACP were tested by placing 1 ml of sample fluid
directly between the two pieces of medium-coated polypro-
pylene and spreading the sample to a fixed diameter with a
special tool supplied with the plates.
MF of samples was done by previously described methods
(2). The 0.45-p.m-pore-size sterile cellulose nitrate filters
were placed on either SMA or TSA plates.
All assay systems were incubated at 37C for 72 h and up
to 7 days for test solutions containing mycobacteria. AAMI
and CDC both recommend incubation of cultures at 37C for
48 h. Colonies were counted by using a dissecting micro-
scope (15x objective).
Statistical analysis. Statistical evaluation of the different
assay methods was done by using Waller-Duncan grouping
based on the Waller-Duncan K-ratio t test and the Student t
test for paired samples.
RESULTS
The performances of five different commercial bacterial
recovery systems were compared against the MF technique.
Each system was challenged with a variety of organisms
commonly found in hemodialysis fluids and with water and
dialysate from three dialysis centers. All challenge solutions
were assayed in triplicate by each bacterial recovery
method. There was no significant difference in the overall
recovery of specific challenge organisms in all methods
tested. Recovery rates varied from system to system de-
pending on the microorganism used in the challenge solution
(Table 1). SPC had the highest recovery in 40% of the
challenges, followed by TSA (MF), which had the highest
recovery in 20% of the challenges. ACP and NP+ both had
the highest recoveries in 10% of the challenges.
When dialysate from three local dialysis centers (one using
bicarbonate dialysate) was used as unknowns, center-to-
center variation was noted. Microbial recovery from acetate
dialysate in centers A and B was best achieved using the MF
method employing SMA, although no significant difference
was seen between the standard procedure MF and the NP
commercial system in samples from center A. The ACP
plates were superior for bacterial recovery from bicarbonate
dialysate samples from center C (P < 0.03). When all 27
dialysate samples were analyzed, the SPC and ACP systems
were the best overall recovery systems for bicarbonate
dialysate and SMA was best for acetate dialysate (Table 2).
Overall bacterial recovery from 21 water samples from
centers A, B, and C was best achieved by the SPC system.
There was no significant difference between the overall
bacterial recovery rates of SMA, TCWT, and NP+. When
analyzed separately by center, the SPC and TCWT systems
had the highest recovery rates, followed by either SMA or
the NP+ system (Table 3).
DISCUSSION
Hemodialysis patients can be exposed to 400 to 600 liters
of dialysis fluids per week during dialysis (6). The quality of
the water and the dialysate is important because of the
nature of the contact between the dialysate and the patient's

TABLE 2. Recovery of microorganisms from the dialysate of

three dialysis centers by six assay methods TABLE 3. Recovery of microorganisms from the reverse-osmosis
water of three dialysis centers by six assay methods
Mean bacterial count (CFU/ml) by assay"
Center Mean bacterial count (CFU/ml) by assaya
SMA SPC TCWT NP NP+ ACP Center
SMA SPC TCWT NP NP+ ACP
A (n = 3) 56 34 23 48 42 25
B (n = 3) 42 6 1 17 10 0 A (n = 9) 168 178 98 110 130 68
Cb (n = 3) 206 691 130 136 57 1,526 B (n = 9) 207 334 229 173 254 86
Meanc 101 244 51 67 36 517 C (n = 3) 2,303 3,630 2,267 1,107 1,020 703
" Assays are described in Table 1, footnote a.
Meanb 490 738 464 280 310 166
b Bicarbonate dialysate used. "Assays are described in Table 1, footnote a.
' Means based on loglo CFU per milliliter from each center. b Mean based on A+B+C (n = 21).
594 ARDUINO ET AL.
blood system. High concentrations of bacteria can pose risks
of bacteremia or endotoxemia to hemodialysis patients be-
cause of possible passage of bacterial endotoxin across the
membrane or because of transmembrane stimulation of
macrophages and subsequent cytokine production by endo-
toxin or bacteria. Early studies have shown that the attack
rates for pyrogenic reactions can be directly proportional to
the level of bacterial contamination in the dialysis fluid (7, 9).
As a result of these studies, AAMI and CDC made the
following recommendations regarding the bacterial content
of hemodialysis fluids: (i) water used to make dialysate
should contain <200 CFU/ml; (ii) dialysate should not con-
tain >2,000 CFU/ml; and (iii) water used to prepare germi-
cide for the reprocessing of dialyzers should contain <200
CFU of bacteria per ml or <1 ng of endotoxin per ml (3, 12).
Testing of water and dialysate should be done at least
monthly (3, 13).
A variety of bacteriologic samplers designed primarily for
water testing are commercially available. We evaluated five
of these sampling systems for their possible use in the
sampling of hemodialysis fluids compared with the recom-
mended MF technique using TSA and SMA. The bacterio-
logic samplers are readily available to the dialysis facility
and easy to use by dialysis personnel with little laboratory
training or equipment. All tests however, require an incuba-
tion of culture at 37C as per AAMI guidelines (3), and a
dissecting microscope to count viable bacterial colonies is
strongly recommended. In our evaluation, we found perfor-
mance of each system varied depending on the microbial
flora of the solution being tested. This would also explain the
center-to-center variation noted in the bacterial recoveries
from water and dialysate samples from these dialysis cen-
ters.
The SPC sampler consistently had higher microbial recov-
ery rates than the four other assay systems included in this
study. In overall performance, when compared to MF with
SMA or TSA, all of the commercial systems that were tested
produced an acceptable range of microbial recovery. Differ-
ences in the ability of each method in recovering organisms
may be partly due to the matrix (i.e., water, acetate, or
bicarbonate dialysate) in which the organisms had adapted.
For example, recovery of organisms isolated from bicarbon-
ate dialysate will depend on the salt content of the medium
because of the presence of haloduric or halophilic organisms
(5). In this case, ACP, which are inoculated by adding a 1-ml
sample to the system, had the best recovery. This inocula-
tion ensures that the salt content in the recovery medium
remains close to that of the original matrix.
However, differences in the numbers of organisms recov-
ered from system to system were generally close when
logarithmic levels were compared. Thus, any of the assay
systems could be easily used by dialysis system personnel
for microbiologic monitoring of dialysis fluids. All of these
commercial assay systems are relatively easy to use and do
not require extensive knowledge or expertise in the field of
J. CLIN. MICROBIOL.
microbiology. Two of the tests (SPC and TCWT) involved
simply dipping a paddle containing dehydrated media into
the test solution for 30 s, while the other four tests involved
rehydration of media with 1 ml of test solution. The costs per
test range from approximately $0.60 for ACP to approxi-
mately $3.00 for SPC and TCWT. The only additional
equipment that would be needed by the dialysis center would
be a 37C incubator and a dissecting microscope. We con-
clude that all of these commercial assay systems provide an
acceptable balance between microbial recovery and required
sampling time, equipment, and expertise.
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