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Appl Environ Microbiol. 2000 January; 66(1): 453-454.
PMCID: PMC91848
Copyright © 2000, American Society for Microbiology
Multiregional Evaluation of the SimPlate Heterotrophic Plate Count
Method Compared to the Standard Plate Count Agar Pour Plate Method in
Water
R. Wayne Jackson,1* Karen Osborne,1 Gary Barnes,1 Carol Jolliff,2
Dianna Zamani,3 Bruce Roll,4 Amy Stillings,4 David Herzog,4 Shelly
Cannon,5 and Scott Loveland5
Cobb County-Marietta Water Authority, Acworth, Georgia 301011;
Clinical Laboratories of San Bernardino 2, Research and Development,
Lompoc, California 934362; Indiana Department of Health, Indianapolis,
Indiana 462023; Upper Guadalupe River Authority, Kerrville, Texas
780285; and Portland Water District, Standish, Maine 040844
*Corresponding author. Mailing address: Cobb County Marietta Water,
3728 Mars Hill Rd., Acworth, GA 30101. Phone: (770) 974-4286. Fax:
(770) 975-7011.
Received June 4, 1999; Accepted October 22, 1999.
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Abstract
A new SimPlate heterotrophic plate count (HPC) method (IDEXX
Laboratories, Westbrook, Maine) was compared with the pour plate method
at 35°C for 48 h. Six laboratories tested a total of 632 water samples.
The SimPlate HPC method was found to be equivalent to the pour plate
method by regression analysis (r = 0.95; y = 0.99X + 0.06).

Water utilities are required to maintain a detectable disinfection
residual in water distribution systems or measure for heterotrophic
plate count (HPC) bacteria (6). The standard HPC pour plate method
is an approved U.S. Environmental Protection Agency USEPA method
(5) for reporting HPC in lieu of testing for residual disinfectant
concentration or for testing when residual disinfectant levels are less
than 0.2 mg/liter in finished waters (4). This method, as well as
other HPC methods, such as membrane filtration or spread plating, may
be also used to collect data for internal purposes (nonreporting). All
the methods (1) for testing of heterotrophic bacteria require
time-consuming preparation of media and can be difficult to read.
Recently, the SimPlate total plate count method for determining the
most probable number (MPN) of microorganisms in food was developed by
IDEXX Laboratories, Westbrook, Maine (3), and approved by the
Association of Official Analytical Chemists (AOAC) International
Research Institute (2). The formulation was modified to allow for
the detection of heterotrophic bacteria in water. The test known as
SimPlate for HPC medium contains substrates that are hydrolyzed by
microbial enzymes to release 4-methylumbelliferone, which fluoresces
blue under a long-wavelength (365-nm) 6-W UV light after incubation for
48 h at 35°C. The bacteria are detected as fluorescent wells on the
SimPlate. The bacterial density of a water sample is determined by
determining the number of positive wells and by using the MPN table
provided for SimPlate. This format will allow a MPN/milliliter value up
to 738 without any dilution. The objective of this study was to compare
the performance of SimPlate and the HPC pour plate method (1) for
the enumeration of heterotrophic bacteria. Six laboratories in
different regions of the United States participated in this study.
Between May and June 1997, naturally occurring heterotrophic bacterium
samples were collected in sterile vessels at each site. The samples
consisted of chlorinated drinking waters (neutralized with sodium
thiosulfate) (1), well waters, untreated natural (raw) waters
(lakes and streams), and secondary chlorinated sewage effluents
(neutralized with sodium thiosulfate). Since chlorinated drinking
waters have relatively few or no heterotrophic bacteria, the sites
prepared composites of raw waters and/or secondary effluent with
neutralized chlorinated drinking water. The composite samples were
prepared in ratios of 1:1, 1:2, and 2:1 to allow as broad a range of
bacterial counts as possible to be represented in the study. Each site
was requested to test 100 samples for the evaluation. Approximately 40%
of the samples were to be from finished waters (defined in this study
as chlorinated drinking water and well water), 30% were to be from raw
waters, and 30% were to be from composites of finished and raw waters.
No dilution was required for finished waters and was labeled as 100;
dilutions of 10-1 and 10-2 and/or 10-3 were required for raw waters
and composites. All samples were tested in duplicate.
SimPlate for HPC is available as a unit-dose (1 test) or as a
multi-test (10 tests) medium (used for this study) from the
manufacturer. This kit consists of 10 foil-wrapped, sterile polystyrene
vessels with dehydrated medium and four plastic sleeves holding 25
sterile SimPlates each. The foil wrap enclosing the vessel was removed,
and the vessel was opened aseptically to hydrate the medium with 100 ml
of sterile diluent (sterile deionized water or sterile buffered water).
SimPlates to be used were removed from the sterile plastic sleeve, and
the remaining SimPlates were stored in the sleeve sealed with tape. A
blank was performed with each run using sterile diluent to verify
SimPlate sterility.
One milliliter of the water sample was placed on the center-landing pad
of the SimPlate by using a sterile pipette. Nine milliliters of
hydrated medium was then placed on top of the sample, by using a
sterile pipette, to achieve a total volume of 10 ± 0.2 ml. The mixture
of the sample and the medium was distributed in all wells by gently
swirling and tilting the plate. Air bubbles were observed in some of
the wells, although IDEXX Laboratories indicated that this would not
have any effect on the test (3). This was not evaluated during the
study. Approximately half of this mixture was removed by pouring off
all of the excess through the indented spout on the base of the
SimPlate. The SimPlates were inverted to prevent condensation on the
covers and incubated at 35 ± 0.5°C for 48 ± 2 h. The number of wells in
each SimPlate exhibiting a blue fluorescence when exposed to a
long-wavelength (365 nm), 6-W UV light was recorded as positive
results. The MPN/milliliter was obtained by using the IDEXX MPN table
provided, and where applicable, this value was multiplied by the
dilution factor to obtain the corrected MPN/milliliter.
The pour plate count agar was prepared as described in Standard Methods
(1). One milliliter of the sample was placed on the center of a