APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1989, p. 912-921 Vol. 55, No.

4
0099-2240/89/040912-10$02.00/0

Nonphotosynthetic Pigmented Bacteria in a Potable Water

Treatment and Distribution System
D. J. REASONER,* J. C. BLANNON, E. E. GELDREICH, AND J. BARNICK
Drinking Water Reseairch Division, Risk Reduction Engineering Laboratory, U.S. Environmental Protection Agency,
Cincinnati, Ohio 45268
Received 26 July 1988/Accepted 22 December 1988

The occurrence of pigmented bacteria in potable water, from raw source water through treatment to
distribution water, including dead-end locations, was compared at sample sites in a large municipal water
system. Media used to enumerate heterotrophic bacteria and differentiate pigmented colonies were standard

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method plate count (SPC), m-SPC, and R2A agars, incubated up to 7 days at 35C. The predominant
pigmented bacteria at most sample locations were yellow and orange, with a small incidence of pink organisms
at the flowing distribution site. Seasonal variations were seen, with the yellow and orange organisms shifting
in dominance. SPC agar was the least productive medium for both heterotroph counts and pigmented bacteria
differentiation. At the flowing distribution site, percentages of pigmented bacteria on SPC medium ranged from
2.3 to 9.67 times less than on m-SPC and from 2.3 to 9.86 times less than on R2A. At the same site, seasonal
trends in the percentage of pigmented bacteria were the same for m-SPC and R2A media, and the highest and
lowest percentages occurred in the fall and winter, respectively. At site 6, there appeared to be an inverse
relationship between the yellow and orange pigmented groups, but upon analysis, this did not hold and all
correlations between yellow and orange pigmented bacteria were positive. The study results indicate that
pigmented bacteria could readily be detected by using plate counting media developed for heterotroph
enumeration in potable waters with incubation periods of 7 days. Pigmented bacteria can be used as an
additional marker for monitoring changes in water quality. High numbers of heterotrophs, including
pigmented forms, were found at dead-end locations, usually in the absence of a free chlorine residual and when
the water temperature was greater than 16C. The association of some pigmented bacteria with nosocomial and
other infections raises concern that the organisms may have originated from the potable water supply. High
levels of pigmented bacteria could pose an increased health risk to immunologically compromised individuals.
Therefore, the bacterial quality of the distribution water should be controlled to prevent the development of
high concentrations of heterotrophic plate count bacteria, including the pigmented forms.

Traditionally, the primary focus of drinking water micro-
biology has been on the total coliform group bacteria as
indicators of potable water quality and disinfection effi-
ciency. The sustained usefulness of the total coliform group
as indicators of the potential presence of human and animal
fecal pollution is based on a substantial body of data derived
from practical application and experience. Another bacte-
riological measurement, the heterotrophic plate count
(HPC), formerly called the standard plate count (SPC), is not
a required test for drinking water quality. However, mea-
surement of the HPC can provide the water plant operator
with valuable information about the effectiveness of treat-
ment processes, including disinfection, and about bacterial
water quality changes during distribution.
After disinfection, water that has received complete treat-
ment will generally have an HPC of <10 to 100 CFU/ml.
After the finished water enters the storage and distribution
system, qualitative and quantitative changes in the bacte-
riological quality may occur and can be monitored by the
HPC procedures. Qualitative changes may be detected sim-
ply by routine observation of HPC culture plates, noting
differences in the variety and appearance of bacterial colo-
nies on the plates.
A characteristic of many heterotrophic bacteria found in
treated drinking water is the ability to form brightly colored,
nonphotosynthetic, nondiffusible pigments. The occurrence
of pigmented bacteria in surface waters, some of which
* Corresponding author.
912
served as sources of potable water or were of potable water
quality, has been documented in the literature (13, 16, 18, 22,
32-34, 40, 42, 44). The pigmented bacteria generally appear
in greatest abundance in clean, low-nutrient (oligotrophic)
waters. Therefore, the observation of pigmented bacteria in
treated distribution waters is consistent with previous obser-
vations on natural oligotrophic surface waters. However, the
occurrence of pigmented bacteria in treated drinking water
generally goes unnoticed because most of these organisms
are "slow-growing pigmented water bacteria" (19) that
require longer than the 48-h incubation period that was used
for so many years for the SPC. Often, these slow-growing
pigmented water bacteria appear to be the predominant
survivors in chlorinated potable water (19, 20). The slow-
growing pigmented bacteria have also been isolated from
drinking fountains, ice machines, sink faucets, distilled
water lines, water baths, humidifying units, and hemodialy-
sis units (7, 10, 11, 19, 20, 25, 28, 35). In a medical
environment, the presence of pigmented bacteria in tap
water used to supply humidifiers or other water-using ther-
apy machines or instruments, or used to clean these ma-
chines, poses a potential health problem that includes gas-
trointestinal upsets, pyrogenic reactions (35), infections, and
possibly subtle responses such as induction of low-level
immunological changes.
The concept of a water distribution system as an ecolog-
ical habitat for bacteria has not been widely acknowledged.
Few or no data are available on in situ conditions conducive
to the bacterial colonization of distribution pipe surfaces, the
VOL. 55, 1989
Sample
Site 3
FIG. 1. Simple schematic showing relative locations of raw
water, Cincinnati water treatment plant, and distribution sites used
during the study.
types of bacteria involved in initial colonization, or the
successional changes in the dominant types of bacterial
colonizers. The chemical and physical factors most impor-
tant to the stability of bacterial populations are presently
unknown.
The purposes of this study were as follows: (i) to develop
data on the occurrence and densities of pigmented bacteria
in treated drinking water, (ii) to compare the efficacy of three
different plate count media for detection and enumeration of
heterotrophic bacteria in water, (iii) to compare the densities
of pigmented bacteria found in the raw source water with
pigmented bacteria densities at various points during treat-
ment and distribution to determine how the treatment and
distribution processes modify the occurrence and densities
of these organisms, and (iv) to evaluate pigmented bacteria
occurrences as an indicator of bacterial quality changes in
treated distribution water.
MATERIALS AND METHODS
The main study of six sample sites was conducted from
November 1978 through January 1980, and samples from fire
hydrants at or near dead-end locations in the distribution
system were collected on an irregular basis until December
1982.
Water samples were collected at 2-week intervals from six
locations as follows (Fig. 1): site 1, raw Ohio River water;
site 2, settled Ohio River water; site 3, flocculated and
settled Ohio River water; site 4, chlorinated influent to rapid
sand filters; site 5, effluent from rapid sand filters; and site 6,
a distribution site approximately 25 miles from the treatment
plant. Sites 7 through 12 were six hydrant sampling points at
or near dead-end areas of the distribution system that were
sampled on an irregular basis. The sample sites shown in
Fig. 1 depict the water treatment configuration at the time of
the study; the configuration has changed since the data
reported here were collected owing to the need to reduce the
trihalomethane formation potential resulting from the use of
chlorine as a disinfectant.
Water samples were collected in sterile 1-liter polypropyl-
ene wide-mouthed bottles containing sodium thiosulfate to
neutralize any chlorine residual present in the water (1). The
PIGMENTED BACTERIA IN POTABLE WATER 913
distribution site sample was collected directly from the water
main with a special sampling ferrule inserted into the pipe.
Prior to sample collection, the ferrule valve was opened and
the water flowed to waste for 2 min to flush the ferrule
thoroughly. Water temperature and free chlorine residual
measurements were made on water collected in a second
sample bottle without a dechlorinating agent. Free chlorine
residual was determined by the N,N-diethyl-p-phenylene-
diamine (DPD) colorimetric method (Hach Co., Loveland,
Colo.), and temperature was measured with a mercury
thermometer. The rapid sand filter influent and effluent, sites
4 and 5, respectively, always contained a total chlorine
residual that was generally greater than 1.0 mg/liter.
At the time this study was initiated, the use of the standard
pour plate procedure for plate counts on drinking water
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samples was being questioned. Therefore, it was decided to
compare bacterial plate counts obtained from other medium-
method combinations with those from the standard pour
plate procedure.
The bacterial density of each sample was determined by
the use of plate count procedures, now referred to as the
HPC. Counts were made by (i) the pour plate procedure
(SPC) with plate count agar (1); (ii) the m-SPC medium
membrane filter procedure of Taylor and Geldreich (45), now
called m-HPC (2); and (iii) R2A medium of Reasoner and
Geldreich (38; Abstr. Annu. Meet. Am. Soc. Microbiol.
1979, N7, p. 180). All plates were incubated at 35C for 168
h (7 days) in a moist atmosphere to reduce dehydration of the
agar.
Differential colony counts were made after 48 h (2 days),
72 h (3 days), and 168 h (7 days) to determine the total colony
count (CFU per milliliter) and the numbers of yellow,
orange, pink, and other pigmented bacteria growing on each
plate. Differentiation of colony pigmentation was best on the
168-h-old plates. Pour plates were counted with the aid of a
Quebec colony counter, and membrane filter plates were
counted with the aid of a binocular dissecting microscope.
No attempt was made to enumerate colonies of different
shades or hues within each color group, although such
differences could often be observed.
Data analysis. Plate count results from the three media
were statistically analyzed with SAS programs (SAS Insti-
tute, Inc., Cary, N.C.). A three-way analysis of variance
(ANOVA) was used to compare medium results while con-
trolling for the effects of both time and site. A two-way
ANOVA with R2A data only was used to compare between
sample sites while controlling for the effect of time. R2A
medium was chosen since it yielded the highest counts of
both total and pigmented heterotrophic bacteria. A Pearson
correlation coefficient was determined to test the strength of
the linear relationship between orange and yellow pigmented
bacteria.
To test the assumptions required for the ANOVA analyses
indicated above, we generated residual plots for each anal-
ysis. Normal probability plots for residuals were also exam-
ined. In addition, formal parametric and nonparametric tests
relating to the normality assumption were conducted. Re-
sults from these analyses (not shown) indicated no gross
discrepancies from the common assumptions for an
ANOVA. Data transformations (log and square root) did not
result in any perceived improvement in the residuals. This
lack of improvement, combined with the positive effects of
both large sample sizes overall and relatively equal factor
level sample sizes, led to an analysis of the untransformed
data.
914 REASONER ET AL.
100000.
1oooo
M
E counts. The results indicated that R2A medium outper-
A
N
C
F that the R2A spread plates gave the best results and that the
U SPC pour plates were the worst. The results were significant
m
L ANOVA to compare sample sites while controlling for time
1 2 3 4 6 6 7 bacteria at sites 1, 2, 3, and 6. The trend indicated by the
SAMPLE SITE
FIG. 2. Summary of 2-, 3-, and 7-day mean HPC at 35C for the
study sample sites. Symbols: C, R2A; 0, m-SPC; E, SPC.
RESULTS
The overall results for the total HPC at the study sites as
determined on the three different media incubated for 2, 3,
and 7 days at 35C are shown in Fig. 2 and Tables 1, 2, and
3. These results indicate that there was a progressive reduc-
tion in the HPC per milliliter from the raw water (site 1)
through the treatment chain (sites 2 to 5), with effluent from
the rapid sand filters (site 5) yielding the lowest counts. The
HPC reduction between site 1 and site 5 was between 1,000
and 10,000-fold on all media. The HPC levels in water
samples taken well out into the distribution system at site 6
and the dead-end site 7 had increased by 100- to 1,000-fold
compared with the levels at site 5, indicating primarily that
bacteria that survived treatment had regrown.
APPL. ENVIRON. MICROBIOL.
Among the three media used to measure the HPC, all
indicated the trends of HPC reduction through treatment and
HPC regrowth in the distribution system. The SPC and
m-SPC counts were similar for all three incubation periods
(2, 3, and 7 days), but the 7-day R2A counts were higher than
the SPC and m-SPC counts at all sites except site 4. The
differences between the R2A counts and the SPC and m-SPC
counts tended to be larger at sites 6 and 7 than at the other
sites. This indicates that the bacteria responsible for the high
HPC counts at sites 6 and 7 were enumerated better on the
low-nutrient R2A medium than on either of the other two
media. The 7-day R2A mean HPC density at site 7 was
nearly as high as the R2A mean HPC density in the raw
water (site 1). However, the qualitative appearance of HPC
plates from site 7 was very different from that of HPC plates
from site 1, and there was also a difference in the overall
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levels of pigmented bacteria.
A three-way ANOVA was run to assess the impact of
time, site, and medium on the HPC and pigmented bacteria
formed m-SPC and SPC media for enumerating both total
HPC and total pigmented bacteria. These analyses indicated
at a. = 0.05 (95% confidence level).
Analysis of the R2A HPC data only by using a two-way
(Table 4) showed significant differences among some of the
sample sites. Overall, after controlling for the effect of time,
sample site 1 total HPC was greater than the total HPC for
each site, from site 2 through site 6. For sample site 7, the
total HPC was greater than that for sites 4, 5, and 6. No other
significant differences between sample sites for total HPC
were found by this analysis.
Figure 3 summarizes the relative differences in the mean
percent occurrence of total pigmented bacteria at each of the
sample sites as determined on each of the three media after
2, 3, and 7 days of incubation at 35C. It is evident that the
SPC pour plate procedure and medium yielded a low per-
centage of pigmented bacteria at all sites regardless of the
length of incubation. After 2, 3, or 7 days of incubation,
m-SPC medium yielded a higher percentage of pigmented
bacteria at all sites, except site 4, than did SPC medium. In
comparison with R2A medium after 3 and 7 days of incuba-
tion, m-SPC medium gave a higher percentage of pigmented
7-day counts on R2A medium showed that the percentage of
pigmented bacteria was stable from sites 1 through 4 and
then progressively increased at sites 5, 6, and 7. The flowing
distribution water at site 6 and the dead-end water at site 7
often showed the highest percentages of pigmented bacteria
after 3 or more days of incubation on either m-SPC or R2A
medium. The rapid sand filter influent (site 4) and effluent
(site 5) both showed the effect of chlorination on the HPC
levels by the very low colony counts (Fig. 2) and the
percentage of pigmented bacteria (Fig. 3). However, it is
clear that a resurgence of bacteria, including pigmented
forms, occurred in the distribution system. Sampling from
other distribution sites between the treatment plant and site
6 might have shown a progressive increase in bacterial
numbers, including pigmented forms, but the need for the
assistance of waterworks personnel to collect flowing water
samples from the mains limited the number of such sites that
could be sampled. Also, collection of dead-end hydrant
samples during the winter was complicated by the need to
pump the hydrants dry to prevent freezing.
VOL. 55, 1989 PIGMENTED BACTERIA IN POTABLE WATER 915

TABLE 1. Summary of 2-day. 35C mean bacterial plate counts and mean percentages of total pigmented bacteria
on SPC, m-SPC. and R2A media
SPC m-SPC R2A
Sample
site
site,, 7cCUm
CFU/ml 1CF/l%CUm
CFU/mPigmented' Pigmented " CFU/ml Pigmented
1 14 156.5 0.98 15 2,905.3 18.95 12 38,241.7 11.74
2 14 8.8 0.42 15 542.8 31.35 12 6,088.3 12.62
3 14 7.1 0.84 15 580.4 16.40 12 3,742.5 13.28
4 13 0.0 0.0 14 0.44 4.92 10 45.1 1.71
5 13 0.0 0.0 14 0.33 10.45 12 2.0 26.19
6 25 0.04 1.42 25 4.3 32.53 22 358.6 8.08
7 2 0.0 0.0 1 83.0 63.85 3 1,770.0 1.04
8 1 48.0 2.08
9 1 0.0 0.0 2 1,600.0 1.67
10 3 5.1 1.9 3 23,466.7 0.37

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11 1 16.7 75.91
" Percentage of pigmented bacteria.

Because the 7-day incubation period provided both the
highest HPC counts and excellent development of pigmen-
tation that permitted good differentiation of the various color
groups, the following results and discussion focus only on
the 7-day R2A colony counts and pigmented bacterial levels.
Table 4 shows that, in addition to the differences between
sites for the total HPC, there were significant differences
between sample sites for total pigmented bacteria and yellow
and orange pigmented bacteria but not for the pink pig-
mented bacteria. For this analysis, all the dead-end site data
were merged into a single group so that site 7 in Table 4
represents dead-end sites 7 to 12 combined. The significant
results found (x = 0.05) indicated that for the total pig-
mented bacteria, site 7 counts exceeded those of sites 1
through 6 and site 1 counts exceeded those of sites 2 through
6. For yellow bacteria, the site 7 counts exceeded those of
sites 2 through 6 and site 1 counts exceeded those of sites 2
through 6. Finally, for the orange pigmented bacteria, site 7
counts exceeded those of sites 1 through 6 but site 1 counts
exceeded only those of sites 2, 4, and 5.
Figure 4 is a block chart summary of the seasonal percent-
ages of total pigmented bacteria on R2A medium for all
seven sample sites and shows the relative differences in the
mean percentages of pigmented bacteria by site and season.
Although the data for SPC and m-SPC media are not
shown, the choice of medium strongly affected not only the
HPC levels detected but also the numbers and types of
pigmented bacteria detected. The total percentage of pig-
mented bacteria recovered on SPC medium from the raw
Ohio River water (site 1) in any season was about threefold
less than the percentages recovered by m-SPC and R2A (Fig.
4). At site 6, the flowing distribution site, the percentages of
pigmented bacteria on SPC medium ranged from 2.3 to 9.67
times less than on m-SPC and from 2.3 to 9.86 times less than
on R2A (data not shown). However, the seasonal trends
were similar for m-SPC and R2A media, and the highest and
lowest percentages of pigmented bacteria occurred in the fall
and winter, respectively.
Figure 5 shows the seasonal percentages of pigmented
bacteria by color group on R2A medium for sites 1, 5, 6, and
7. At site 1 (raw Ohio River water), the yellow pigmented
bacteria were the predominant color group; all other color
groups accounted for less than 6% of the total percentage of
pigmented bacteria in any season. The results for sites 2 and
3 (data not shown) were very similar to those for site 1, thus
indicating that the settling (site 2) and flocculating (site 3)
processes did not radically change the relative proportions of
pigmented bacteria, although the bacterial counts were re-
duced during these treatment steps. HPC results for sites 4
(chlorinated influent to rapid sand filters) and 5 (chlorinated
effluent from rapid sand filters) were similar; therefore, only
the seasonal results for site 5 are shown (Fig. 5). The data for
site 5 showed that disinfection caused significant changes in
the percentage of pigmented bacteria (Fig. 3) as well as a

TABLE 2. Summary of 3-day. 35C mean bacterial plate counts and mean percentages of total pigmented bacteria
on SPC. m-SPC. and R2A media
SPC m-SPC R2A
Sample
site ! CFU/ml Pigmented" CFU/ml Pigmented " CFU/ml Pigmented

1 17 13,447.1 2.52 17 18,000.0 27.62 14 28.742.6 16.48

2 17 2,231.7 3.36 17 2,819.7 27.46 13 9,176.9 17.32
3 17 624.9 1.94 17 2.676.8 29.66 14 4,114.3 22.60
4 16 9.6 2.93 16 8.9 6.23 12 11.8 15.50
5 8 1.0 0.69 16 1.5 18.18 14 2.0 19.15
6 26 23.3 3.08 27 79.6 50.60 23 1,262.8 33.52
7 3 380.3 1.06 1 130.0 30.77 5 4,206.0 39.42
8 1 90.0 18.89
9 1 0.30 0.0 4 7,900.0 15.38
10 2 950.2 0.0( 2 37,650.0 3.73
11
"
Percentage of pigmented bacteria.
916

Sample
site

1
2
3
4
5
6
7
8
9
10
site
REASONER ET AL.

nn
19
19
19
18
13
29
3
1
3
CUm
CFU/ml
SPC

15,121.1
3,487.9
1,120.9
20.4
2.4
70.4
2,991.7
0.3
7,078.0
Pigmented"
8.06
14.77
7.58
5.04
6.71
16.51
10.44
0.0
4.34
1

19
19
19
18
18
29
1
CUm
m-SPC

CFU/ml
24,173.7
4,104.4
3,576.2
16.0
3.1
398.1
130.0
Pigmented
26.62
26.62
31.80
5.36
17.84
68.17
15.38
1

16
15
16
14
16
25
6
1
5
3
APPL. ENVIRON. MICROBIOL.

TABLE 3. Summary of 7-day, 35C mean bacterial plate counts and mean percentages of total pigmented bacteria
on SPC, m-SPC, and R2A media

CUm
CFU/ml
59,731.3
12,000.0
18,046.0
R2A

22.3
10.2
3,035.9
46,036.7
250.0
45,820.0
33,566.7
Pigmented
20.04
18.02
19.00
20.92
35.57
62.60
69.96
52.67
28.75
61.42

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11 1 64.0 28.28
" Percentage of pigmented bacteria.

significant reduction in the HPC density (Fig. 2). It is important
to note that the percentage of pigmented organisms can be very
misleading for sites 4 and 5 if considered alone. The reason is
that when HPC counts were low, such as 1 to 10 CFU/ml (or
per 10 ml or some other volume by the m-SPC membrane filter
method), the occurrence of one to a few pigmented colonies
could account for a large percentage of the total HPC. Both
yellow and orange pigmented bacteria appeared to be a signif-
icant proportion of the pigmented flora on R2A medium at sites
4 and 5, but actual densities were low.
The changes that occurred in the pigmented flora between
sites 4 and 5 undoubtedly reflected not only the influence of
the residual disitnfectant but also the contribution of bacteria
from the rapid sand filters. There was no way to differentiate
the relative contributions of each process (filtration and
disinfection) to the changes in the microbial flora. However,
in terms of numbers of bacteria detected by the HPC
procedures used, the disinfection process has been shown to
cause the most significant decreases in HPC levels, and
water from site 5 always carried some free chlorine residual.
Although there were low HPC densities in water from site 5,
including the pigmented bacteria, it appeared that the rela-
tive reductions of HPC and pigmented bacteria were similar.
The flowing distribution water (site 6, Fig. 5) showed the
most interesting changes in pigmented bacterial flora, with
the yellow and orange pigmented organisms again the pre-
dominant flora on R2A medium. The percentage of yellow
pigmented organisms showed peak values in summer and
lowest values in winter, while the orange pigmented bacteria
showed high levels in winter and spring. There appeared to
be an inverse relationship between the yellow and orange
pigmented , density
forms was high the orange pogmented
pgetedfr was high,dthe densit ofofPearson
orang pigmented
bacterlawas low, and vice versa. The correlation
analysis conducted to test this relationship showed that at
site 6 terwas only a slight positive relationship between
the two color groups and that the correlation was not
significant (Pearson r = 0.06896, P = 0.7545). The Pearson
correlation analyses for all sites and for the combined
dead-end sites (sites 7 to 12) indicated that the yellow and
orange pigmented groups were positively correlated and that
the values were significant (all sites, Pearson r = 0.65953, P
= 0.0001; dead-end sites, Pearson r = 0.66995, P = 0.0088).
The pink pigmented bacteria were present, usually in very
low numbers, throughout the year and rarely accounted for

TABLE 4. ANOVA table for bacterial count (HPC) results over time between sample sites, R2A medium only
Dependent variable Source df Sum of squares Mean square F value
Total HPC Model 6 41,160,546,253.7 6,860,091,042.1 6.66"1
Error 66 68,036,265,131.6 1,030,852,502.0
Total 72 1.091968113853E + 11
Total pigmented HPC Model 6 1,890,955,511.6 315,159,251.8 9.30"
Error 66 2,237,436,371.9 33,900,551.1
Total 72 4,128,391,883.5
Yellow Model 6 1,472,956,566.2 245,492,761.0 8.56"
Error 66 1,893,163,384.0 28,684,293.7
Total 72 3,366,119,950.2
Orange Model 6 23,106,083.5 3,851,013.9 6.46"
Error 66 39,320,292.2 595,762.0
Total 72 62,426,375.7
Pink Model 6 802,455.7 133,742.6 1.35
Error 66 6,547,322.9 99,201.9
Total 72 7,349,778.5
"Significant at a = 0.05.
VOL. 55, 1989 PIGMENTED BACTERIA IN POTABLE WATER 917

100 -
TWO DAY COUNT AT 35'C
80 -

0o - 5 / i 3
/ 29 5/S1.07 / 932 2.97/

40 - 35.83 4.08 / 29.51 29.62

3
/9. 1
3g
/
///2./
/
/221 1.24/ 28/
20 2 & 5 /&
23.27 18.15 20.01 14.03

23.89 17.18 22.63 19.57

2 3 4 5 e 7 WIN SPR SUm FALL
SEASON

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M
E 100 FIG. 4. Summary of seasonal percent occurrence of total pig-
A
N THREE DAY COUNT AT 35 C mented bacteria by sample site (7-day [168-h] incubation. R2A
medium, 35C).
80
p
G the lowest percentages of pigmented bacteria did not neces-
M 60 sarily occur under these same conditions.
E
N At sample site 7, located near sample site 6, the yellow
T pigmented bacteria peaked in the summer and the orange
E 40
D pigmented bacteria predominated in the spring (Fig. 5);
B again, the positive Pearson correlation coefficient indicated
A 2 0O that as the yellow pigmented group increased, so did the
C
T
E
orange pigmented group. The pink pigmented bacteria
R peaked in the fall at a level (18.9%) that was higher than that
A 2 3 4 6 6 7 of either the yellow or orange pigmented group. This peak in
pink pigmented bacteria represented the highest mean per-
10c SEVEN DAY COUNT AT 35 C centage of pink bacteria found at any of the sample sites
throughout the study. Overall, the large variability in the
HPC levels and the percentage of pigmented bacteria at the
soI- dead-end sample sites suggest that there was also great
variability in the water conditions that contributed to. or
BC
inhibited, the development of the heterotrophic bacterial
populations.
40 DISCUSSION
The results of this study indicate that pigmented bacteria
20 can readily be detected by using plate count media and
membrane filter media such as those developed for HPC
determinations on potable water samples. The fact that all
2 3 4 6 6 7 three media used in this study could recover pigmented
SAMPLE SITE bacteria, although not equally well, when an extended
FIG. 3. Summary of 2-, 3-, and 7-day mean percentage of total incubation period was provided illustrates that these organ-
pigmented bacteria at 35C for the study sample sites. Symbols: O. isms can be used as an additional marker for monitoring
R'A; 0. m-SPC; *. SPC. changes in the bacterial quality of the distribution water.
Since pigmented bacteria may compose a high percentage of
the HPC in treated distribution water, monitoring specific
more than a low percentage of the total pigmented bacteria at pigmented bacterial groups throughout the year could permit
site 6. additional characterization of the system. Sharp excursions
The R2A HPC and total pigmented bacteria results from in the percentage of total pigmented bacteria or of the yellow
fire hydrant samples at or near dead-end locations (including or orange pigmented subgroups could signal a change in
site 7) are shown in Table 5. These data indicate that the water quality, perhaps owing to a posttreatment contamina-
HPC levels varied significantly, both at the same sample site tion event or to a change in disinfectant residual. However,
as well as from one sample site to the next. At sites 7, 9, and characterization of the system by the pigmented population
10, for example, the HPC varied by 10,000-fold, 100-fold, would need to be done over a prolonged period to provide a
and 10-fold, respectively. Also, the percentage of total good data base from which to judge the significance of the
pigmented bacteria varied from a low of 7.4% to a high of changes in the pigmented HPC flora. R2A medium and
61.2% at site 7, from 9.2 to 98.9% at site 9, and from 6.6 to m-SPC medium were found to be better media for enumer-
91.5% at site 10. The lowest HPC levels observed generally ating pigmented bacteria and total heterotroph levels than
occurred when the water temperature was less than 16.5C was SPC medium. Also, studies done since these data were
and there was a free chlorine residual in the water. However, collected have shown that extended incubation (5 to 7 days)
918 REASONER ET AL. APPL. ENVIRON. MICROBIOL.

SITE 1 SITE 5
SEASONX
FALL 4 /D /

SLJM / Y X / Dg <

SPR / fJf D 4~-

WINON 4.0 3 0. 0.17.8 WI N

ORANGE OTHER PINK YELLOW ORANGE OTHER PINK YELLOW

COLOR COLOR

SITE 6

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SFEA.SON X D X X 7
SITE 7
FAL 1 1j=3js< SEASON
FALL /f
5
2
/
/
.
S
8.9
19 //
fi

I=X
W~~ ~. 1 J2/ O. -3

WIN ~ / =//- A / I=D

ORANGE OTHER PINK YELLOW ORANGE OTHER PINK YELLOW
COLOR COLOR
FIG. 5. Seasonal mean percent occurrence of pigmented bacteria by color groups at sites 1, 5, 6. and 7 on R2A medium spread plates
(7-day [168-h] incubation, 35C).

of R2A medium or m-SPC medium at 20 or 28C resulted in
higher HPC recoveries than did incubation at 35C (38).
The fact that all media used in this study exhibited high
and low recovery extremes at different times during the
study shows that shifts occurred in the physiological types of
bacteria in the distribution system. While no single medium
is suitable for recovery of every type of bacteria at any given
time, some media are much better than others.
The pigmented bacteria found in the distribution samples
(site 6) and in dead-end samples (site 7 and others) were
similar in appearance to some pigmented forms that were
seen in the raw water and in the treatment chain samples.
However, there were many yellow and orange pigmented
bacteria, and there was no simple way to screen the colonies
to determine whether they were the same organism or
several different organisms. The role of pigment-producing
bacteria in the ecology of potable water and water distribu-
tion systems is unknown.
Although there were changes in the levels of pigmented
bacteria that appeared to be related to seasonal effects, for

TABLE 5. Summary of R2A heterotroph counts, total pigmented bacteria counts, and percentage of pigmented bacteria from
dead-end fire hydrant sample site
Temp residuaZl
~Freechlor-ine HPC Total pigmented Pimne
Date Site (OC)Temp (CFU/ml) T cteUrmi
Date ('C)
~~~~~~(mg/liter) (F/l
bacteria
8-31-81 7 ND' ND 76,000 31,000 40.8
4-19-82 7 10.5 0.0 390 29 7.4
8-6-82 7 24.6 0.0 170,000 104,000 61.2
10-18-82 7 20.2 0.0 1,900 560 29.5
11-17-82 7 14.5 0.25 19 3.4 17.9
11-17-82 8 14.5 0.0 250 130 52.0
8-31-81 9 ND ND 60,000 26,000 43.8
4-27-82 9 11.5 1.2 1,90( 1,800 94.7
8-6-82 9 23.7 0.0 150,000 98,000 65.3
10-18-82 9 20.0 0.0 6,200 1,700 27.4
10-25-82 9 ND 0.0 9,100 840 9.2
4-27-82 10 11.5 0.6 4,700 4,300 91.5
4-28-82 1t) 11.5 0.0 86,000 5,700 6.6
11-10-82 11 15.5 0.0 10,000 8,000 80.0
11-17-82 11 13.0 0.0 1,400 380 27.1
11-10-82 11 16.5 0.7 64 21 32.8
11-17-82 11 14.0 0.8 83 48 57.8
11-10-82 12 15.3 0.4 28(0 61 21.8
" ND, Not done.
VOL. 55, 1989 PIGMENTED BACTERIA IN POTABLE WATER 919

example, the orange and yellow bacteria at site 6 (Fig. 5), the TABLE 6. Reported occurrences of pigmented bacteria
overall data analysis did not bear this out. The strongest in potable water
seasonal relationship was for the summer total HPC and Bacterial count % Refer-
summer total pigmented counts, which were greater than the Water source (CFU/ml) Pigmented ence
total HPC and total pigmented counts for fall, winter, and
spring. Follow-up pairwise tests (summer versus winter, Distilled water 50,000-2,000,000 NG" 25
etc.) indicated that there was probably only a weakly signif- Tap water 4,000 NG 19, 20
icant difference in total HPC owing to seasons. To establish Wells, springs (Cl.) <10->1,000 NG 6
Bottled water 140-570,000 0-100 14
the existence of true seasonal effects, it is necessary to study Distilled water 30,000 NG 19
the total and pigmented HPC populations over a period of Distribution water 1,000 80-90 37
several years. In such a study, it would be necessary to Distribution water (Cl) 200 62 30, 31
provide additional intense monitoring of temperature and Distribution, well water 500 35 30, 31
disinfectant residual to try to distinguish between the indi- Reservoir water (Cl,) 5-150,000 55-90 41
vidual and combined effects of these factors on the bacterial Well water 30-690 10-14 43
flora of the finished distribution water. ' NG, Not given.
Pigmented bacteria frequently have been identified as

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species of Flavobacterium. However, one of the primary
characteristics used for identification is pigment production,
and the genus Flavobacterium turns out to be a mixed group
taxonomically. Many of the pigmented bacteria isolated
from the treated distribution water have been found to be
aerobic, oxidase-positive, nonfermentative, nonglucose ox-
idizers (41). Also, they generally did not demonstrate typical
reactions (or any at all) in most of the biochemical identifi-
cation media used for characterization. Thus, these bacteria
are very difficult to identify.
The fact that pigmented bacteria were present in the raw
water suggests that the pigmented forms found in the treated
distribution water originated as organisms that survived the
treatment process(es), including disinfection. Regrowth of
heterotrophic bacteria occurred in the distribution system as
evidenced by the increased HPC levels at the treated distri-
bution water sample site (site 6) compared with levels at
treatment site 5, the chlorinated effluent from the rapid sand
filters. Some may also have been introduced by posttreat-
ment contamination events. The pigmented bacteria, in
general, appear to adapt well to the distribution system
environment, as evidenced by their sustained presence at
site 6 and in the dead-end areas that were sampled during the
study. At least two populations, the yellow and orange
pigmented groups, were always present at site 6. Each of
these groups exhibited population maxima and minima at
different times, which indicates that suitable conditions
existed for their maintenance and regrowth.
Little or nothing is known about the actual conditions in
the distribution system that favor the maintenance and
growth of these pigmented bacteria and other heterotrophs
that are found in treated distribution water. The absence of
a free chlorine residual and elevated water temperatures
during the warmer periods of the year certainly appear to
contribute to increased numbers. The types and concentra-
tions of nutrients, inorganic and organic, that stimulate
growth of these organisms are unknown.
In nonpotable waters, pigmented bacterial levels can be
interpreted to be an indicator of the cleanliness of the water
since the percentage of pigmented forms present is inversely
proportional to the pollution level of the source water.
Guthrie et al. (17) hypothesized that when chromogenic
bacteria compose 25% or more of the aerobic, heterotrophic
bacterial populations in an aquatic environment, it is indic-
ative of a nonpolluted body of water, in that the bacterial
flora is a naturally occurring population. Treated drinking
water would certainly be considered unpolluted by most
standards.
Occurrences of pigmented bacteria in potable water have
been documented in other studies. Table 6 shows reported
incidences of pigmented bacteria in potable waters, includ-
ing distilled and bottled water. In some cases, the percentage
of pigmented organisms was not given and could not be
calculated from the data. However, the range of the percent-
age of occurrence of pigmented bacteria is broad and,
depending on the water source, reached as high as 100%.
The percentage of pigmented bacteria is highly variable, and
in bottled water there was often a dominant population of
one pigmented type. The dominant color group reported was
usually the yellow to yellow-orange group; this is true for
both natural and potable waters.
During studies of point-of-use granular activated carbon
cartridge water treatment units, pigmented bacterial levels in
the product water from the test units varied with sample time
(a.m. versus p.m.) and from test unit to test unit (36). The
mean percentage of pigmented bacteria present in product
water from each of the test units was variable, ranging from
a low of about 4% to a high of about 42%. Levels of
pigmented bacteria in the dechlorinated tap water used as
the influent to the test units averaged about 60% of the HPC.
In general, the presence of pigmented bacteria in the
distribution water does not appear to demonstrate that a
problem exists, except the likelihood that the disinfectant
residual is too low to inhibit the growth of the organisms.
The treatment processes, particularly disinfection with chlo-
rine, may be a selective factor for pigmented bacteria. There
is evidence to indicate that pigmented bacteria may be more
chlorine tolerant than nonpigmented forms. Herman and
Himmelsbach (20) and Herman (19) reported the survival of
Flas'obacteriu,n sp. after 10 min of exposure to 10 mg of
chlorine per liter. Graf and Bauer (15) found that red
pigmented Cotynebacteriuim rubrium from tap water had
thermotolerance to 80C and chlorine resistance of up to 0.3
mg of free chlorine per liter. Du Moulin (7) reported that the
Flavobacterilmn strain implicated in airway colonization
could survive exposure to 1.0 mg of chlorine per liter for 24
h. Ridgway and Olson (39) found that bacteria of the
Flavobacteriumn-Moraxella group isolated from a chlorinated
distribution system showed smaller zones of inhibition when
exposed to chlorine in a disk assay procedure than did
bacterial isolates of the same groups obtained from a non-
chlorinated distribution system. However, in a membrane
filter assay of chlorine resistance, the isolates from the
chlorinated distribution system did not survive exposure for
2 min at a free chlorine concentration greater than 0.1
mg/liter.
The most important aspect of the occurrence of pigmented
bacteria in distribution water is related to the potential of the
organisms to cause human health problems. Pigmented
920 REASONER ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 7. Pigmented bacteria isolated from water supplies emphasizes the need to control bacterial regrowth in the
Source Organism" Reference
distribution system. In many water distribution systems,
control of bacterial growth can be managed by maintaining
Distilled water Flavobacterium capsulatum 26 the disinfectant residual above 0.2 mg/liter and by pursuing a
Tap water Corynebacterium species 15 vigorous, regularly scheduled program of hydrant flushing to
Distribution water Flavobacterium species 46 reduce stagnation in the dead-end sections of the system.
Mycobacterium species Other strategies to minimize bacterial growth, or regrowth,
Open reservoir Flavobacterium species 7 ultimately may involve the use of alternative disinfectants
Distribution water, Flavobacterium species 31
such as ozone, chlorine dioxide, and chloramines or some
Cl2 and non-Cl2 Serratia species
Tap water, water Micrococcus species 5 combination of these disinfectants. Also, since little is
reservoirs Flavobacteriuim breve known about the nutrient conditions that favor the growth of
Flavobacterium detvorans pigmented bacteria, information about the levels of assimi-
Chromobacterium species lable organic carbon in potable source water and treated
Flavobacterium-Cytophaga species drinking water is needed. Control strategies may eventually
Distribution water Mycobacterium gordonae 8 need to include ways of reducing the assimilable organic
carbon levels in the treated water before it enters the

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" Genus and species names indicated are those given in the referenced
citations and are not necessarily the currently accepted designations. distribution system.

bacteria that have been isolated from waters of potable
quality are shown in Table 7. Of the organisms shown in this
table, several have been associated with human disease.
Table 8 lists examples of pigmented bacteria isolated from
disease cases and certainly does not represent an exhaustive
list. However, it illustrates the point that some of the
pigmented bacteria are opportunistic pathogens. This may
be particularly important where there are populations of
compromised individuals, such as in nursing homes, hospi-
tals, and other institutional settings. It is possible that many
nosocomial infections in hospitals result from bacteria that
originated from the hospital water supply and plumbing
devices or from water-using machines such as vaporizers
and humidifiers (26). Despite the potential health association
concerns expressed here and by other workers, direct evi-
dence of the link of pigmented organisms in water with
human infections and disease is sparse. The exception to this
is the pigmented mycobacteria, which do not grow out
during the short incubation period used for enumeration of
HPC levels in drinking water. These organisms have been
shown to pose a problem for immunodeficient patients and
have been linked directly to the water supply (8, 9).
Recognition of the pigmented bacteria as a significant
proportion of the HPC population that can develop in treated
distribution water and recognition that some pigmented
bacteria have been implicated in human health problems
TABLE 8. Pigmented bacteria identified as pathogens
Organism" Disease(s)
Flavobacterium meningo- Meningitis
septicuum
Flavobacteriuim meningo- Meningitis
septicum
Flavobacterium ineningo- Meningitis
septic um
Flavobacterium species Septicemia
Flavobacterium odora- Surgical infection
tum
Serratia species Pneumonia
Serratia marcesc ens Urinary tract infection
Mycobacterium gordonae Nontuberculous disease
Chromobacterium viola- Septicemia, chromobac-
ceum teriosis
"Genus and species names given are those indicated in the referenced
citations and are not necessarily the currently accepted designations.
ACKNOWLEDGMENTS
We thank Susan Campbell and Jeff Finkeldey for their expert
technical assistance in data entry and analysis, Deanna Wild for her
assistance in statistical analyses, and Virgil Lakes for his help in the
preparation of some of the figures. We gratefully acknowledge the
cooperation and assistance of the Cincinnati Water Works, without
which this study could not have been done. We particularly thank
Charles Cobb, James Gilday, and Mike Piepers, who provided
assistance in collection of the flowing main samples through instal-
lation and operation of the ferrule valve.
LITERATURE CITED
1. American Public Health Association. 1975. Standard methods for
the examination of water and wastewater, 14th ed. American
Public Health Association, Washington, D.C.
2. American Public Health Association. 1985. Standard methods for
the analysis of water and wastewater, 16th ed. American Public
Health Association, Washington, D.C.
3. Berry, W. B., A. G. Morrow, D. C. Harrison, H. D. Hochstein,
and S. K. Himmelsbach. 1963. Flavobacterium septicemia fol-
lowing intracardiac operations: clinical observation and identi-
fication of the sources of infection. J. Thorac. Cardiovasc. Surg.
45:476-481.
4. Cabrera, H. A., and G. H. Davis. 1961. Epidemic meningitis of
the newborn caused by flavobacteria. I. Epidemiology and
bacteriology. Am. J. Dis. Child. 101:289-295.
5. Dott, W. 1983. Qualitative and quantitative examination of
bacteria found in aquatic habitat. VI. Communication: bacterial
regrowth in drinking water. Zentralbl. Bakteriol. Parasitenkd.
Infektionskr. Hyg. Abt. 1 Orig. Reihe B 178:263-279.
6. Druce, R. G., and S. B. Thomas. 1970. An ecological study of
the psychrotrophic bacteria of soil, water, grass and hay. J.
Appl. Bacteriol. 33:420-435.
Refer- 7. du Moulin, G. C. 1979. Airway colonization by Flavobacterium
ence in an intensive care unit. J. Clin. Microbiol. 10:155-160.
23 8. du Moulin, G. C., I. H. Sherman, D. C. Hoaglin, and K. D.
Stottmeier. 1985. My(obiccteriium aviurm complex, an emerging
29 pathogen in Massachusetts. J. Clin. Microbiol. 22:9-12.
9. du Moulin, G. C., and K. D. Stottmeier. 1986. Waterborne
4 mycobacteria: an increasing threat to health. ASM News 52:
525-529.
3 10. Favero, M. S., N. J. Peterson, K. M. Boyer, L. A. Carson, and
21 W. W. Bond. 1974. Microbial contamination of renal dialysis
systems and associated health risks. Trans. Am. Soc. Artif.
12 Intern. Organs 20-A:175-183.
28 11. Favero, M. S., N. J. Peterson, L. A. Carson, W. W. Bond, and
8 S. H. Hindman. 1975. Gram-negative water bacteria in hemodi-
24 alysis systems. Health Lab. Sci. 12:321-334.
12. Feeley, T. W., G. C. du Moulin, J. Hedley-Whyte, L. S. Bushnell,
J. P. Gilbert, and D. S. Feingold. 1975. Aerosol polymyxin and
pneumonia in seriously ill patients. N. Engl. J. Med. 293:
471-475.
VOL. 55, 1989
13. Fred, E. B., F. C. Wilson, and A. Davenport. 1924. The
distribution and significance of bacteria in Lake Mendota.
Ecology 5:322-339.
14. Geldreich, E. E., H. D. Nash, D. J. Reasoner, and R. H. Taylor.
1975. The necessity of controlling bacterial populations in
potable waters-bottled water and emergency water supplies. J.
Am. Water Works Assoc. 67:117-124.
15. Graf, W., and L. Bauer. 1973. Red bacterial growth (Coryne-
bacterium rubrum, n. spec.) in tap-water systems. Zentralbl.
Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe B
157:291-303.
16. Graham, V. E., and R. J. Young. 1934. A bacteriological study
of Flathead Lake, Montana. Ecology 15:101-109.
17. Guthrie, R. K., D. S. Cherry, and R. N. Ferebee. 1974. A
comparison of thermal loading effects on bacterial populations
in polluted and non-polluted aquatic systems. Water Res. 8:
143-148.
18. Henrici, A. T. 1939. The distribution of bacteria in lakes, p.
39-64. In Problems of lake biology. Publication no. 10. Ameri-
can Association for the Advancement of Science, Washington,
D.C.
19. Herman, L. G. 1976. Sources of the slow-growing pigmented
water bacteria. Health Lab. Sci. 13:5-10.
20. Herman, L. G., and C. K. Himmelsbach. 1965. Detection and
control of hospital sources of flavobacteria. Hospitals 39:72-76.
21. Holmes, B., J. J. S. Snell, and S. P. La Page. 1979. Flavobacte-
riuim odoratium: a species resistant to a wide range of antimi-
crobial agents. J. Clin. Pathol. 32:73-77.
22. Jordan, E. 0. 1903. The kinds of bacteria found in river water.
J. Hyg. 3:1-27.
23. King, E. 0. 1959. Studies on a group of previously unclassified
bacteria associated with meningitis in infants. Am. J. Clin.
Pathol. 31:241-245.
24. Koburger, J. A., and S. 0. May. 1982. Isolation of Chroinobac-
terium spp. from foods, soil, and water. Appl. Environ. Micro-
biol. 44:1463-1465.
25. Leifson, E. 1962. The bacterial flora of distilled and stored
water. I. General observations, techniques and ecology. Int.
Bull. Bacteriol. Nomencl. Taxon. 12:133-153.
26. Leifson, E. 1962. The bacterial flora of distilled and stored
water. III. New species of the genera Corynebacteriium, Flai'o-
bacterium, Spirillum, and Pseiudomonas. Int. Bull. Bacteriol.
Nomencl. Taxon. 12:161-170.
27. Matsen, J. M. 1975. Hospital infections with pigmented water
bacteria. Health Lab. Sci. 12:305-310.
28. Okuda, T., N. Endo, Y. Osada, and H. Zen-Yoji. 1984. Outbreak
of nosocomial urinary tract infections caused by Serratia mar-
cescens. J. Clin. Microbiol. 20:691-695.
29. Olsen, H. 1969. FlIi'obacterium meningosepticium isolated from
outside hospital surroundings and during routine examination of
patient specimens. Acta Pathol. Microbiol. Scand. 75:313-322.
30. Olson, B. H. 1982. Assessment and implications of bacterial
regrowth in water distribution systems. Project report EPA
R-805680010. Order no. PB 82-249368. National Technical In-
formation Service, Springfield, Va.
31. Olson, B. H., and L. Hanami. 1980. Seasonal variation of
PIGMENTED BACTERIA IN POTABLE WATER 921
bacterial populations in water distribution systems, p. 137-151.
Proceedings of the 8th Annual American Water Works Associ-
ation Water Quality Technology Conference, December 7 to 10,
Miami Beach, Fla. American Water Works Association, Den-
ver.
32. Potter, L. F. 1964. Planktonic and benthic bacteria of lakes and
ponds, p. 148-166. In H. Heukelekian and N. C. Dondero (ed.),
Principles and applications in aquatic microbiology. John Wiley
& Sons, Inc., New York.
33. Potter, L. F., and G. E. Baker. 1956. The microbiology of
Flathead Lake, Montana. I. Preliminary survey of the microbial
populations. Ecology 37:351-355.
34. Potter, L. F., and G. E. Baker. 1961. The microbiology of
Flathead Lake, Montana. 11. Vertical distribution of the micro-
bial populations and chemical analyses of their environments.
Ecology 42:338-348.
35. Quarles, J. M., R. C. Belding, T. C. Beaman, and P. Gerhardt.
1974. Hemodialysis culture of Serratia marcescens in a goat-
Downloaded from http://aem.asm.org/ on February 22, 2017 by guest
artificial kidney-fermentor system. Infect. Immun. 9:550-558.
36. Reasoner, D. J., J. C. Blannon, and E. E. Geldreich. 1987.
Microbiological characteristics of third-faucet point-of-use de-
vices. J. Am. Water Works Assoc. 79:60-66.
37. Reasoner, D. J., and E. E. Geldreich. 1979. Significance of
pigmented bacteria in water supplies, p. 187-196. Proceedings
of the American Water Works Association Water Quality Tech-
nology Conference, December 8 to 12, Philadelphia, Pa. Amer-
ican Water Works Association, Denver.
38. Reasoner, D. J., and E. E. Geldreich. 1985. A new medium for
the enumeration and subculture of bacteria from potable water.
Appl. Environ. Microbiol. 49:1-7.
39. Ridgway, H. F., and B. H. Olson. 1982. Chlorine resistance
patterns of bacteria from two drinking water distribution sys-
tems. Appl. Environ. Microbiol. 44:972-987.
40. Snow, L. M., and E. B. Fred. 1926. Some characteristics of the
bacteria of Lake Mendota. Trans. Wis. Acad. Sci. Arts Lett.
22:143-154.
41. Staley, J. T. 1985. Enumeration and identification of heterotro-
phic bacteria from drinking water. EPA 1600/2-85/061. MERL-
Ci-2428. U.S. Environmental Protection Agency, Cincinnati,
Ohio.
42. Stark, W. H., and E. McCoy. 1938. Distribution of bacteria in
certain lakes of Northern Wisconsin. Zentralbl. Bakteriol. Par-
asitenkd. Infektionskr. Hyg. Abt. 2 98:201-209.
43. Stetzenbach, L., L. M. Kelley, and N. A. Sinclair. 1986. Isola-
tion, identification and growth of well-water bacteria. Ground-
water 24:6-10.
44. Taylor, C. B. 1942. Bacteriology of freshwater. 111. The types of
bacteria present in lakes and streams and their relationship to
the bacterial flora of soil. J. Hyg. 42:284-296.
45. Taylor, R. H., and E. E. Geldreich. 1979. A new membrane filter
procedure for bacterial counts in potable water and swimming
pool samples. J. Am. Water Works Assoc. 71:402-405.
46. Water Research Center (United Kingdom). 1976. Deterioration
of bacteriological quality of water during distribution. Notes on
Water Research, no. 6, October. Water Research Center, Med-
menham, United Kingdom.