IDE XX
Literature Cover Sheet
IDEXX Library#: 8B
Topic: USEPA Pour Plate vs SimPlate™ for HPC Study
Title: "Comparative Assessment of the Newly Developed SimPlate™
Method with the Existing EPA-approved Pour Plate Method for the
Detection of Heterotrophic Plate Count Bacteria in Ozone-treated
Drinking Water."
Author(s): A. Stillings, D. Herzig, and B. Roll.
Date: October 19-23, 1998
Source: Presented at the International Ozone Association Conference
Vancouver, Canada
Highlights:
• 136 surface and treated water samples were run in parallel on both
SimPlate For HPC and the USEPA Pour Plate method.
• "The SimPlate™ method showed a good correlation with the EPAapproved pour plate method (r2= was 0.96)."
• "Due to the ease of use, SimPlate™ may be an acceptable alternative
to the more time-consuming pour plate method."
• Another SimPlate advantage is that is takes only 48 hours.
Comparative Assessment of the Newly Developed SimPlateTM Method with the
Existing EPA-approved Pour Plate Method for the Detection of Heterotrophic
Plate Count Bacteria in Ozone-treated Drinking Water.
International Ozone Association Conference
Vancouver, Canada
October 19-23, 1998
A. Stillings, D. Herzig, and B. Roll.
Portland Water District, Portland, Maine.
Abstract
Currently, the Surface Water Treatment Rule (USEPA, 1989) of the Safe Drinking Water
Act (SOWA) requires water utilities to maintain a detectable disinfection residual in water
distribution systems, or measure for heterotrophic plate count bacteria (HPC). In addition to
compliance reasons, many water utilities routinely test for HPC in order to monitor water quality
changes as a result of water treatment and distribution. This test is of particular interest to
utilities who use ozone during water treatment, since this process is responsible for generating
assimilable organic carbon (AOC), which in turn, may stimulate growth of heterotrophic plate
count bacteria. Currently, the United States Environmental Protection Agency approves the pour
plate method, a time-consuming and insensitive technique, for the detection of HPC in drinking
water. In order to simplify the existing HPC method, IDEXX Inc. recently developed a new
technique for measuring HPC. This paper will compare this newly developed method with the
existing USEPA pour plate method and the membrane filtration R2A HPC method.
Introduction
Due to the increasing concern over the formation of trihalomethanes (THM) in drinking
water, many water utilities in the United States are abandoning the use of chlorine and are
seeking alternative disinfectants like ozone. Although ozonation reduces the levels of
disinfection by-products like trihalomethanes (THMs), this disinfection process also increases
the levels of biologically active organic carbon (BOC) through oxidation of organic compounds.
BOC is a concern for many water utilities since increases in BOC can stimulate regrowth in the
distribution system. Bacterial regrowth may result in violation of the Surface Water Treatment
2
Rule (USEPA, 1989) and Total Coliform Rule (USEPA, 1989), as well as causing taste and odor
problems, tuberculation of pipes, corrosion problems, and difficulties in maintaining chlorine
residuals throughout the system (Geldreich, 1996).
In February 1994, the Portland Water District switched to ozone as its pnmary
disinfectant after the construction of the Sebago Lake Ozone Treatment Facility. The Sebago
Lake Ozone Facility was the first water treatment facility in the United States to employ "freestanding" ozonation without filtration for primary disinfection purposes. This was due to the
exceptional water quality of the surface water supply (Table I), which allowed the district to
obtain a waiver from filtration in accordance with the filtration requirements of the Surface
Water Treatment Rule (USEPA, 1989). Due to the concern for potential microbial regrowth in
the distribution system, the District also began to incorporate heterotrophic plate counts into their
routine testing as a means of process control.
Currently, SDWA regulations require water utilities to maintain a detectable disinfection
residual or measure heterotrophic plate count bacteria levels (HPC) within the distribution
system (USEP A, 1989). In addition to compliance monitoring, heterotrophic plate counts (HPC)
are also useful for evaluating the efficiency of treatment processes as well as monitoring the
regrowth potential and biofilm development within the distribution systems (Reasoner, 1990).
Utilities that experience difficulty in maintaining chlorine residuals within their systems may
choose to run HPCs more frequently to determine the extent of microbial regrowth within the
system.
Heterotrophic bacteria are organisms that require organic compounds for growth and
reproduction and are found in water, soil and vegetation. These organisms may be introduced
into the water distribution system in a number of ways: some survive the disinfection process,
some enter through open reservoirs or other storage units, and some are introduced when repair
work is done or new mains are added to the system (Geldreich, 1996). Elevated levels of
heterotrophs are a general indication of microbial regrowth within the distribution system and a
decline in overall water quality. Most heterotrophs are not a health risk, but they may cause taste
and odor problems.
Some heterotrophs, however, are opportunistic pathogens, such as
Klebsiella, Legionella, Pseudomonas and Staphylococcus, and increases in heterotrophic
bacterial levels may indicate conditions that are favorable to the growth of these pathogenic
3
organisms. Elevated HPC levels may suppress the detection of coliform bacteria present in the
distribution system, resulting in an increase in waterborne disease in the absence of detectable
coliforms (LeChevallier, 1984).
A number of factors may affect bacterial regrowth in a system: the initial quality of the
finished water entering the distribution system, nutrient supply, water temperature, disinfection
residual (chlorine or chloramine), residence time, and water usage (Geldreich, 1996 and
Reasoner, 1990). Use of ozone as a disinfectant adds nutrients to the system by breaking down
long-chain carbons into smaller organic molecules, which can stimulate bacterial growth. Longterm use of chloramines to maintain a disinfection residual within the system can also increase
bacterial concentrations in the distribution system (Geldreich, 1996). Long retention times in the
distribution system may also produce elevated counts of HPC.
Methods for the detection of heterotrophic bacteria were being standardized as early as
1905, when the first edition of Standard Methods was published (Reasoner, 1990). Procedures at
that time incorporated plate count techniques using nutrient gelatin as a medium. Over time, the
procedures were amended, with changes in the media, temperature of incubation, and time of
incubation.
By 1985, several methods for evaluating heterotrophs were available.
These
included the traditional pour plate method using plate count agar, as well as membrane filter and
spread plate techniques, utilizing several types of media (including low nutrient such as R2A)
and several incubation temperatures and times of incubation. Currently, the USEPA approved
method for compliance monitoring is the pour plate method. This method utilizes plate count
agar, and incubation of plates at 35°C for 48 hours. Although this method is used by many water
utilities, there are drawbacks to the pour plate method (Geldreich, 1996, Reasoner, 1990, and
Reasoner and Geldreich, 1984). The pour plate technique involves tedious and time-consuming
media preparation and plate count agar is a less sensitive medium than other media like R 2A. In
addition, the US EPA approved method uses a relatively high incubation temperature (35°C) and
short incubation period (48 hours) when compared to other HPC methods. Depending of the
application, each HPC method has advantages and disadvantages, which are summarized in
Table 2.
4
Objectives
The objectives of this project were to:
•
compare the USEPA approved pour plate method with the newly developed SimPlaterM
(IDEXX Inc., Westbrook, Maine) method.
•
evaluate the sensitivity of R2A membrane filtration, pour plate and SimPiaterM methods
using drinking water samples with varying disinfectant concentrations.
Materials and Methods
Sample collection and preparation. Samples were collected from several water sources-
raw waters (lakes, streams, water treatment plant influent) and finished process waters (water
•
treatment plant effluent, distribution sites, water storage tanks). Each sample was collected in a
sterile 250mL bottle containing approximately 0.2mL of a I 0% sodium thiosulfate solution (for
neutralization of chlorine).
Sampling occurred over several weeks during the spring/early
summer of 1997, from April 28th through June 4th. Sample water temperatures ranged from 4°C
to 19°C. A total of 136 samples were analyzed (44 raw waters and 92 finished/process waters).
Samples were held at 4°C during transport and refrigerated upon receipt by the laboratory. All
samples were analyzed within 24 hours of collection. When necessary, serial dilutions were
made. A 10 1 dilution was created by aseptically adding I OmL of sample to 90mL of sterile
phosphate buffered water and mixing well. Higher dilutions were created by further diluting the
10 1 solution.
Finished process water samples were analyzed for total chlorine residual according to the
DPD Colorimetric Method (4500-Cl G.) in Standard Methods 19th edition.
SimPiate™.
SimPlaterM is a substrate-based medium in which the substrates are
hydrolyzed by microbial enzymes, causing a release of 4-methylumbelliferone, which fluoresces
(after 48 hours of incubation at 35°C) when exposed to long wavelength ultraviolet light
5
(365nm).
The SimP!aterM test kit contains I 0 foil-wrapped, sterile, polystyrene vessels
containing medium, and 4 plastic sleeves containing 25 sterile SimPlates each. The foil was
removed, and the vessel was opened aseptically to hydrate the medium with 1OOmL of sterile
distilled water, providing enough re-constituted media for 10 tests. Aliquots (0.1mL and l.OmL)
of each sample were placed on the center of the sterile SimPlate dish. Appropriate volumes of
SimP/ate for HPC reagent were then placed on top of the sample to achieve a final volume of 10
± 0.2mL. The lid was replaced on the SimPlate and the mixture of the sample and the medium
was distributed in all wells by gently swirling the plate. The plate was gently tapped on the
benchtop and then allowed to sit for a few minutes to eliminate any air bubbles trapped in the test
wells. Excess liquid was removed through the pour spout located on the base of the plate. The
SimPlates were inverted to prevent condensation on the covers and incubated at 35± 0.5°C for 48
± 3 hours. All samples were run in duplicate. At the completion of the incubation time, those
wells exhibiting a blue fluorescence when exposed to a long wavelength (365nm) ultraviolet
light (UVL-56 BLAK-RA Y®, UVP, Upland, CA) were documented as positive. The MPN/mL
was determined by using the IDEXX MPN table provided. Diluted samples were multiplied by
the appropriate dilution factor to obtain the final MPN/mL.
Standard Method Pour Plate. Plate count agar was prepared as described in Standard
Methods 19th edition (9215 A). Solid plate count agar was re-liquefied in boiling water and
tempered to 44-46°C. Aliquots (O.lmL and l.OmL) of each sample were pi petted into the center
of a sterile petri dish (IOOx15mm). Approximately 12mL of the tempered liquid agar was added
to the petri dish and mixed by swirling the plate.
The plates were allowed to solidify
(approximately 10" minutes), then inverted and incubated at 35± 0.5 ° C for 48 ± 3 hours. All
samples were run in duplicate. Colonies formed in or on the plate count media within 48 ± 3
hours were counted and the results reported as CFU/mL. Diluted samples were multiplied by the
appropriate dilution factor to obtain the final CFU/mL.
R2A agar- Membrane Filtration. R2A media was prepared according to manufacturer's
instructions. Tempered agar (44-46°C) was poured into 9x50mm petri dishes and allowed to
solidify. Samples were analyzed utilizing the membrane filter technique described in Standard
Methods 191h edition (9215 D). All samples were analyzed in duplicate. The R2A plates were
incubated at room temperature (23± 2°C) for seven days. All colonies were counted and results
6
were reported as CFU/mL. Diluted samples were multiplied by the appropriate dilution factor to
obtain the final CFU/mL.
Results & Discussion
Comparison of pour plate technique to SimPiateTM, Water utilities are required to
maintain a detectable disinfection residual within the distribution system or test for heterotrophic
bacteria (USEPA, 1989). Currently, the USEPA-approved pour plate method must be utilized to
for compliance monitoring. In an effort to offer a simpler and faster method, IDEXX inc.
developed the SimPlateTM method. In order to compare the pour plate and SirnPlateTM methods,
water samples from surface waters (n=44) and the distribution system (n=92) were submitted to
a paired comparison. As seen in Figure I, the SirnPlateTM method showed a good correlation with
the EPA-approved pour plate method (~=0.96). These results would indicate that the newly
developed Sirnplate"' is comparable to the USEPA-approved pour plate method for the waters
tested in this study.
Comparison of pour plate/SimP!ateTM to R 2A. Numerous studies have indicated that
the pour plate method is not a sensitive method for measuring HPC (Reasoner, 1990, Reasoner
and Geldreich, 1984). Other methods such as membrane filtration using R2A agar have been
shown to be 100 to 1000 times more sensitive when compared to the pour plate method
(Reasoner and Geldreich, 1984). R2A is a low-nutrient medium that was developed in the late
1970's for heterotrophic plate counts, as an alternative to standard plate count agar or rn-HPC
medium. While it is a low-nutrient medium, it has a greater variety of nutrients than the other
commonly used media (Reasoner, 1990). Due to this lower nutrient level, bacteria take longer to
fully develop on R2A media. In order to obtain optimum bacterial concentrations on R 2A media,
it is necessary to incubate for a longer period of time. For this study, samples were analyzed
using the membrane filtration technique, placed on R2A agar, and incubated at 21-25°C for 7
days. As seen in Figure 2, results obtained by the membrane filtration technique using R2A were
significantly higher than results obtained by either the standard plate count pour plate technique
or the SimP lateTM method. Of the total number of samples tested by PCA, SimPlateTM, and R2A
methods, 90% of the samples analyzed yielded R2A results that were a I 00-fold higher than the
other two methods. Other studies comparing heterotrophic bacteria methods have shown similar
7
results.(Reasoner, 1990, Reasoner and Geldreich, 1984). As seen in Figure 2, HPC results
(geometric mean) for the R2 A method were lower at sites having higher disinfection residuals,
while PCA and SimP late TM concentration remained relatively consistent regardless of the
disinfection residual.
Summary and Conclusions
Results from this study indicate that the SimPlaterM product gives heterotrophic bacterial
results comparable to those of the EPA-approved pour plate technique. Due to the ease of use,
SimPlateTM may be an acceptable alternative to the more time-consuming pour plate procedure.
Utilities that use the PCA pour plate technique for compliance monitoring of their distribution
system may underestimate actual HPC concentrations. As presented in this study, R 2A results
indicated bacterial concentrations in the distribution system are much higher than the
concentrations obtained by the pour plate and SimPlaterM methods.
Currently, the USEPA-approved pour plate method utilizes plate count agar and
incubation at 35°C for 48 hours. Many utilities, including the Portland Water District, have
distribution water temperatures that are <20°C, which may select for HPC bacteria that are
capable of growth at these lower temperatures. Indigenous heterotrophic bacteria in such systems
may be less tolerant to the higher incubation temperatures employed by the pour plate method.
Incubating samples at 35°C selects for bacteria that may represent only a small percentage of the
overall bacterial population (Reasoner and Geldreich, 1984).
Utilities who rarely find themselves out of compliance (with no detectable disinfection
residual) may choose to continue to employ the pour plate technique for analysis of heterotrophs.
If the SimPlaterM product is approved by the USEPA, then utilities may decide to switch to this
simpler and faster method. With no media prep involved, this method is easy to use and gives
results comparable to those of the pour plate method.
Due to its higher sensitivity, the membrane filtration technique utilizing the 7-day RzA
method may be useful to utilities for process control measures.
R2A results may aid in
determining potential areas for regrowth in the distribution system as well as determine if
chlorine dosage in the system is sufficient to keep biofilm levels down. Portland Water District's
ozone treatment facility utilizes R2A results for process control purposes (e.g. off-line contact
8
chambers are subjected to R2A analysis to determine tbe levels of heterotrophs present). In
addition, samples from the distribution system are routinely submitted for R2A analysis in order
to monitor changes in the distribution system water quality resulting from the presence of BOC,
different retention times, and varying disinfection residuals.
9
Table I. Sebago Lake Water Quality
Constituent
Microbiological
Fecal Coliform
Unit
Sebago Lake
CFU/100 ml
<1
su
Physical
Color
Conductivity (micromhos/cm)
Total Residue
Turbidity
mg/1
NTU
5
49
31
0.25
Metals
Arsenic (Note 2)
Cadmium
Calcium
Chromium
Copper
Iron
Lead
Magnesium
Manganese
Mercury (Note 2)
Nickel
Potassium
Silver
Sodium
Zinc
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
<0.005
<0.0003
2.6
<0.002
<0.010
<0.025
<0.005
0.56
<0.002
<0.0005
<0.005
0.45
<0.0005
3.2
<0.005
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
mg/1
4.0
3.5
<0.10
6.2
12.1
<0.10
9.5
<0.2
<0.001
6.57
2.2
3.9
0.013
Inorganic Non-metals
Alkalinity (as CaC03)
Carbon dioxide
Ammonia as N
Chloride
Dissolved Oxygen
Fluoride
Hardness (as CaC03)
Nitrate as N
Nitrite as N
pH
Silica
Sulfate
Total P
su
mg/1
mg/1
mg/1
10
Table 2. Comparison of Methods
Method
Pour plate
Spread plate
Membrane filtration
SimPlaten•
Advantages
Easy technique to learn
Disadvantages
May be unsuitable for heat-sensitive or
nutrient-stressed organisms
Results in 48 hours
Time-consuming media prep
Growth/morphology of colonies inhibited
by submersion in media
Low sample volumes
Plate count agar is a less sensitive media
than other media used to enumerate
heterotrophs
Tempered media may begin to re-solidify
before it can be poured
Fairly easy technique to learn
Time-consuming media prep
Potential contamination of pre-poured plates
Results in 48 hours
Necessary to have the plate warm enough so
Bacteria grow on surface of
sample goes into agar completely but not so
media; allows for ease in
observing differences in
dehydrated that bacterial recovery is
morphology
impaired
Low sample volumes
Loss of bacterial colonies due to spreading
technique (bacterial cells remain on glass
rod)
Easy technique to learn
Smaller surface area, less clearly defined
colonies
Larger volumes
Limited to 200cfu per plate
R 2A media is lower nutrientR2A 7-day technique- long time to wait for
higher sensitivity than plate count results
agar
Pre-made, need only re-constitute Sensitivity similar to PCA
Results in 48 hours
Some difficulty in determining slight
fluorescence
Low sample volumes
Cost/sample
II
Figure 1. Heterotrophic Plate Count Com paris on
SimPiate vs. Pour Plate Method
10000
y = 0.9546x
R 2 = 0.9602
..J
-..."
....E
1000
:I
ci
•
100
D..
:I:
-a:.."'
10
~
••
:I
0
D..
• •
1
•
10
100
1000
10000
SimPiate, HPC, cfu/1ml
Figure 2. Comparison of HPC Concentrations In Sam pies
Having Different Disinfection Residuals
1 0000
..J
."'
1000
·;:
1 00
c
- ."
-.
....E
:::J
LL
E
0
c.i E
0
D..
:I:
C)
10
0.00-0.20
(n=15)
0.21-0.50
(n = 16)
0.51-0.75
(n=14)
0.76-1.00
(n=27)
Chlorine residual range, ppm
12
>1.00 (n=7)
References
American Public Health Association-American Water Works Association-Water Pollution
Control Federation: Standard Methods for the Examination of Water and Wastewater,
19th ed. Washington D.C., APHA. 1995
Geldreich, Edwin E. 1996. Microbial Quality of Water Supply in Distribution Systems. Lewis
Publishers, Boca-Raton, Florida.
LeChevallier, Mark W., and Gordon A. McPeters. 1984. Interactions between Heterotrophic
Plate Count Bacteria and Coliform Organisms. Appl. Environ. Microbial., 49:1338-1341.
LeChevallier, Mark W., William Schulz, and Ramon G. Lee. 1991.
Drinking Water. Appl. Environ. Microbial., 57:857- 862.
Bacterial Nutrients in
Reasoner, Donald J. 1990. Monitoring Heterotrophic Bacteria in Potable Water.
Water Microbiology. G.A. McFeters, Ed., Springer-Verlag, New York.
Drinking
Reasoner, D.J., and E.E. Geldreich. 1984. A New Method for the Enumeration and Subculture
of Bacteria from Potable Water. Appl. Environ. Microbial., 49:1-7.
U.S. Environmental Protection Agency. 1989. Drinking Water: National Primary Drinking
Water Regulations; Filtration, Disinfection, Turbidity, Giardia !ambia, Viruses,
Legione/la, and Heterotrophic Bacteria. Federal Register, 54:27486-27568, Juiy 29,
1989.
Water Research Centre. 1976. Deterioration of bacteriological quality of water during
distribution. Notes on Water Research No.6, October.
13