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Coliphage Association with Coliform Indicators: A Case Study in Chile
GABRIELA CASTILLO and R. THIERS, University of Chile, Faculty of Physics and Mathematics, Department of Civil Engineering, Casilla 2283, Santiago, Chile; B.J. DUTKA, and A.H. EL-SHAARAWI, De- partment of the Environment, Rivers Research Branch, National Water Research Institute, Canada Centre for Inland Waters, Burlington, On- tario, Canada L7R 4A6
Abstract
This paper summarizes the results obtained from a n International Development Re- search Centre (IDRC), Ottawa, Canada, funded study to evaluate the potential of using coliphage as an indicator of water quality. Raw water sample data indicated that the A-1 test combined with the coliphage test would make an excellent screening program for health hazards in these waters. The superiority of the PresencelAbsence test for detect- ing microbial health hazards in potable water was readily shown, while the HpS paper strip technique was found to be equally efficient for testing potable waters as traditional coliform water quality indicators.
INTRODUCTION In order to attain the goals of the United Nations Programme for the Drinking Water and Sanitation Decade, the developing countries have been trying to supply their populations with water in adequate quanti- ties. In Chile, 90% of the population is supplied with potable drinking waters, mainly through systems operated by the National Public Wa- ter Service. However, 80% of the rural population, which accounts for about one fifth of the total population, lack safe drinking water sup- plies. Also, there are no standards for drinking water quality in rural areas; therefore, this water is not controlled. As a result, local epidemi- ological studies have demonstrated that the lack of good safe potable water is highly correlated to the incidence of enteric diseases in these noncontrolled areas (SENDOS, 1983). A study was undertaken through the support of the International Development Research Cen- tre (IDRC), Ottawa, Canada, to evaluate the use of the coliphage test as an indicator of the water quality of the source water for potable
Toxicity Assessment: An International Journal Vol. 3, 535-550 (1988) 0 1988 John Wiley & Sons, Inc. CCC 0884-8181/88/050535-16$04.00
536/CASTILLO ET AL.
water supplies. The final goal of this study is the development of a classification system for the potable water source waters based on coli- phage counts and sanitary surveys.
Combined with the above study was an attempt to select a set of potable water tests that were sensitive but yet inexpensive and not dependent on costly imported supplies and equipment, and could be carried out in understaffed and underequipped rural laboratories.
The results of these studies are presented.
METHODS
Water Samples
Samples were collected in triplicate from the following potable water sources: rivers and creeks (40), lakes (161, presettled river water (16), and deep wells (6). A total of 150 potable water samples were also examined. The sources of these waters were the Santiago city water pipe network and rural distribution systems. Out of the 150 samples, 134 were collected from chlorinated systems and 16 from nonchlo- rinated well waters. Chlorinated samples were dechlorinated with thiosulphate.
All samples were iced on collection and tested within six hours.
Coliphage Test
The procedure described by Wetsel et al. (1982) and reproduced in section 919C of the American Public Health Association (APHA) Stan- durd Methods (1985), with the addition of 2,3,5-tripheny1 tetrazolium chloride and using Escherichia coli C (ATCC #13706) as host, was used in this study.
Microbiological Tests
Raw water samples were subjected to the following APHA Standard Methods (1985) total coliform and fecal coliform tests: the five-tube most probable number (MPN) procedure using lauryl tryptose broth and brilliant green lactose bile broth with fecal colifomi confirmation in EC broth; the five-tube MPN procedure using A-1 broth; and the membrane filtration fecal coliform procedure using M-FC agar.
Drinking water samples were subjected to the following APHA Standard Methods (1985) total and fecal coliform tests and hetero- trophic plate count test: the five-tube MPN procedure using lauryl tryptose broth and brilliant green lactose bile broth with fecal coliform confirmation in EC broth; the total coliform membrane filtration proce-
COLIPHAGE ASSOCIATION: CHILE/537
dure using solidified m-Endo medium; and the 35°C heterotrophic spread plate count procedure using tryptone glucose extract agar.
All drinking water samples were also tested by the Presence/Ab- sence (P/A) test (Clark, 1969) and all positive tests were subjected to confirmation tests for total coliforms, fecal coliforms fecal streptococci, Clostridium spp., Pseudomonas aeruginosa, Staphylococci aureus, and Aeromonus spp., as detailed by Clark et al. (1982). The drinking water samples were also tested by the H2S technique using chemically inocu- lated paper strips as described by Manja et al. (1982). All positivc samples by the H2S procedure were subjected to similar identificatior procedures as used in the P/A test.
Physical and Chemical Tests
Turbidity measurements were made on all water samples using the nephelometric procedure and reporting results as nephelometric tur- bity units (NTU; APHA, 1985). Free residual chlorine was assessed in all chlorinated potable water samples using the APHA Standard Methods (1985) amperometric titration method.
Biochemical Identification of Fecal Coliforms
A selected number of samples positive for fecal coliforms were sub- jected to isolation and identification procedures. Usually the last series of positive MPN tubes (EC and A-1 broths) were streaked out onto MacConkey agar and predominant colonies identified. Membrane fil- tration (MF) fecal coliform tests, three or four countable colonies per plate were selected for identification purposes. Isolated, purified colo- nies were subjected to the following tests: oxidase, IMVIC, ornithine and lysine decarboxylase, and growth in Kligler agar.
Statistical Methods
Several nonparametric statistical techniques (Hollander and Wolfe, 1973) were used to analyze the data. The techniques followed were
1. Friedman’s rank sum test and its associated multiple compari-
2. Spearman’s rank correlation. son test; and
RESULTS AND DISCUSSION
Each of the 78 triplicate raw water samples were subjected to fecal coliform and coliphage content tests and to turbidity measurements.
538/CASTILLO ET AL.
Forty samples were positive for coliphage presence in at least one of the triplicate samples and their results are displayed in. Table I. Here it can be seen that within replicate samples there are great variations in all tests, sometimes all three replicates produce the same results, and variations as great as 300% are also seen.
In the 40 samples found to contain coliphage in at least one of the triplicate samples, the density of coliphage ranged from 5 to 1250 plaque-forming units per 100 mL. Fecal coliform densities in the sam- ples positive for coliphage ranged from 2 to 2400/100 mL. In several replicate samples with very high fecal coliform counts and high turbid- ity levels no coliphage were found in one or two of the replicates: e.g., Vizcachitas inlet Maipo River (17-11-86) and Casas Viejas Maipo River (17-11-86) (Table 1). These observations, we believe, are directly re- lated to excess turbidity and adsorption of coliphage to particulates (Ohgaki et al., 1986).
It was observed (Table I) that coliphage levels within triplicates showed better agreement in slightly polluted water (based on fecal coliform densities) than in heavily polluted waters. It was also noted that samples collected in quiet or slow-moving waters (lagoons or creeks) were more likelv to show the presence of coliphage than were samples collected from rapidly flowing rivers. This observation ap- pears to be directly related to turbidity levels. Laboratory experiments performed using autochthonous coliphage inoculated into high turbid- ity waters showed that there was an irregular recovery ilnd significant loss in coliphage viability.
In the 40 samples positive for coliphage, 18 had a fecal coliform mean value of 10/100 mL and a turbidity of <5 NTU. Fecal coliform densities of the remaining 22 positive samples averaged 200/100 mL and their turbidity was >lo0 NTU. The correlation coefficient between fecal coliforms and coliphages was 0.56 for low turbidity and low fecal coliform count samples, and 0.5 in samples with high turbidity and high fecal coliform counts.
Of the 78 raw water samples tested in triplicate, only 1 sample was completely negative for all pollution indicators, the R. Labbe well, which was collected on 06-10-86. The turbidity of this sample was 0.5 NTU. All the other 77 samples were positive for total coliforms, with counts varying from 2 to >16,000.
MPN fecal coliform test results showed that 68 of the samples were positive by the EC test and 67 by the A-1 broth technique. Both had similar fecal coliform count ranges, 2-2400/100 mL. Within the triplicate samples, there appears t o be less variability in the EC broth procedure than with the A-1 broth technique. As for target organisms’ selectivity, 10 samples tested by the EC procedure, which had counts of
COLIPHAGE ASSOCIATION: CHILE/539
10 or less/100 mL, no fecal coliforms were detected in 1 or 2 of the triplicate samples; with the A-1 broth technique 11 samples with 7.8 or less fecal coliforms also showed negative results in 1 or 2 of the repli- cate samples.
The membrane filter fecal coliform (M-FC) test produced a total of 65 raw water samples that that were positive for fecal coliforms. The range of M-FC counts was 1-4000 colony-forming units (CFU) per 100 mL and in several instances great variations were noted between repli- cates, e.g., Casas Viejas Maipo River (01-12-86) had M-FC counts of 100,300, and 1000, and Mapocho River (06-10-86) had replicate counts of 6, 15, and 30. In 11 samples producing M-FC agar counts of 7.8 or less, no fecal coliform counts were detected in 1 or 2 of the replicate samples.
One of the problems frequently encountered during this study was the occurrence of confluent growth on the membranes, which appeared to be related to waters with high turbidity. Colony color variations were frequent, as was size and morphology variations. These varia- tions were not solely related to turbidity.
From the fecal coliform count data (EC, A-1, M-FC), it can be seen that the three methods are similarly sensitive to the fecal coliforms in these raw Chilean waters. From these observations, it would appear that the A-1 broth technique would have a cost advantage over the other two fecal coliform estimating procedures. Also, compared to the EC technique, a maximum of five days or a minimum of three days are required to obtain results. Sample turbidity, and colony overgrowth problems that affect the M-FC test are foreign to the A-1 broth tech- nique.
Isolates collected from the three fecal coliform enumeration proce- dures produced some interesting results. A total of 54 fecal coliform positive A-1 broth samples were tested for E. coli presence and in only 1 sample were no E. coli found. Of the total 218 strains collected from the fecal coliform positive A-1 samples, 87.2% were E. coli with the remainder being, in order of predominance E. aerogenes, Citrobacter spp., E . agglomerans, and some isolates that were not identifiable by the tests used. Forty EC fecal coliform positive samples were also sub- mitted to isolation and biochemical identification procedures for E. coli. In all samples, E. coli was detected; however, out of the 141 strains isolated, only 80.1% were E. coli, and the others found in order of frequency were E. aerogenes, Citrobacter spp., E . agglomerans, Serra- tia spp., and a few nonidentifiable strains.
Fifty membrane-filtered samples containing various types of blue colonies, which were counted as fecal coliforms, were tested for E. coli. In three samples no E. coli were confirmed. Out of the 174 colonies
TAB
LE I
cn 9
rp
0 * m
Col
ipha
ge p
ositi
ve r
aw w
ater
dat
a (7
8 tr
iplic
ate
sam
ples
col
lect
ed a
nd 3
8 tr
iplic
ate
sam
ples
neg
ativ
e fo
r col
ipha
ge)
Tota
l C
olip
hage
d
Dat
e So
urce
Sa
mpl
e N
TU
MPN
/100
mL
EC B
roth
A
-1 B
roth
M
F C
FUa/
lOO
mL
r
E
Feca
l col
iform
s/10
0 m
L T
urbi
dity
co
lifor
ms
02-0
9-86
La
Deh
esa
Lago
on
09-0
9-86
La
Deh
esa
Lago
on
15-0
9-86
La
Deh
esa
Lago
on
15-0
9-86
M
apoc
ho R
.
13-1
0-86
M
apoc
ho R
.
20-1
0-86
A
rray
an R
.
10-1
1-86
E
l Man
zano
R.
17-1
1-86
El
Man
zano
R.
24-1
1-86
E
l Man
zano
R
.
1
2 3 1
2 3 1
2 3 1
2 3 1
2 3 1
2 3 1
2 3 1
2 3 1
2 3
9.5
9.5
9.5
10
10
10
10
10
10
14
14
14
35
35
35
137
137
137 3.
0 3.
0 3.
0 2.
4 2.
4 2.
4 0.
9 0.
9 0.
9
350
220
350 33
33
170 11
11
22
17
14
350
280
1600
28
0 92
0 92
0 22
49
130
540
540
220
170
110
170 4.
5
2.0
<2.0
<2
.0
<2.0
7.
8 <2
.0
4.5
<2.0
4.
5 <2
.0
C2.
0 <2
.0
<2.0
2.
0 <2
.0
<2.0
<2
.0
<2.0
<2
.0
23
33
23
17
>160
0 39
92
0 24
16
00
17
33
49
22
10
17
6.8
7.8
il
4.5
23
13
6.8
13
13
4.5
13
4.5
17
13
13
13
23
2.0
-
-
8 10
4 2 2 3 0 5 2 <
5 <
5 <
5 20
0 15
0 17
0 16
24
10 9 6 11
20 6 12
20
n
P r
0 0 0 5 0 5 5 0 5 0 0 0 5 0 10 5
0 0 5 55
55
50
15
30
65
01-12-86
09-12-86
12-0 1-87
10-11-86
12-01-87
10-11-86
17-11-86
24-11-86
09-12-86
10-1 1-86
17-1 1-86
El M
anza
no
R.
El M
anza
no
R.
El M
anzo
R.
El C
anel
o R.
El C
anel
o R.
Lag
una
Neg
ra D
.
Lag
una
Neg
ra
Dm
Lag
una
Neg
ra
Dam
L
agun
a N
egra
Dm
Cas
as
Vie
jas
Maipo
R.
Ca
w
Vie
jas
Mai
po R
.
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
1.7
1.7
2.1
1.3
1.4
1.3
0.7
0.6
0.7
3.0
3.0
3.0
1.2
1.0
1.1
5.0
5.0
5.0
5.0
5.0
5.0
3.7
3.7
3.7
9.5
9.9
9.8
>800
>800
>800
275
275
275
110
130
110 49
23
79
540
540
350
540
130
240
350
540
1600
23
23
23
49
170
170 49
49
49
170
130
170
1400
1300
1300
790
2400
1700
13
13
33
<2.0
4.5
6.8
<2.0
2.0
7.8
<2.0
<2.0
2.0
4.5
4.5
23.0
7.8
2.0
2.0
2.0
4.5
2.0
4.5
6.8
13 2 4 7.8
1300
1300
1300
220
340
260
13
13 2.0
2.0
<2.0
<2.0
4.5
4.5
4.5
<2.0
<2.0
<2.0
23
23
23
<2.0
4.0
<2.0
4 11 2.0
4.5
4.5
7.8
2.0
<2.0
2.0
1700
2400
1100
790
260
490
20
10 6 0 0 0 0 1 0 0 0 0 1 0 9 2 0 14 2 6 4 2 12 6 0 0 0
900
1800
1200
1200
1000
500
5 85 5 0 0 5 0 5 10 0 10 0 0 5 0 10 0 20 5 0 5 5 0 0 0 10 0
100
100
300
100 0
100
TAB
LE I (Continued)
01
Tot
al
A e 2
Col
ipha
ge
Feca
l col
iform
s/10
0 m
L T
urbi
dity
co
lifor
ms
Dat
e So
urce
Sa
mpl
e N
TU
MF"
/lO
O m
L EC
Bro
th
A-1
Bro
th
MF
CFU
a/lO
O m
L $
24-1
1-86
01-1
2-86
09-1
2-86
15-1
2-86
12-0
1-87
10-1
1-86
17-1
1-86
24-1
1-86
01-1
2-86
09-1
2-86
Cas
as
Vie
jas
Mai
po R
. C
asas
V
ieja
s M
aipo
R.
Cas
as
Vie
jas
Mai
po R
. C
asas
V
ieja
s M
aipo
R.
Cas
as
Vie
jas
Mai
po R
. V
izca
chas
In
lets
M
aipo
R.
Viz
cach
as
Inle
ts
Mai
po R
. V
izca
chas
In
lets
M
aipo
R.
Viz
cach
as
Inle
ts
Mai
po R
. V
iz c a
c has
In
lets
M
aipo
R.
1
2 3 1
2 3 1
2 3 1 2 3 1
2 3 1
2 3 1
2 3 1 2 3 1
2 3 1
2 3
420
400
440
128
128
132
690
700
680
660
650
640
310
300
320
>SO
0 >8
00
>SO
0 28
0 28
0 28
0 42
0 41
0 40
0 13
6 13
6 13
2 71
0 71
0 70
0
790
1300
79
0 11
00
2300
17
00
3500
22
00
1400
17
00
3500
24
00
2400
54
00
1100
24
00
790
330
1700
17
00
1100
46
0 14
00
1100
17
00
1700
2400
17
00
700
2200
490
300
490
460
490
700
490
490
700
230
490
790
330
490
330
330
330
330
1700
17
00
1700
33
0 49
0 79
0 79
0 11
00
1400
23
0 17
00
700
790
490
790
330
230
790
230
230
230
700
330
700
790
230
330
1100
13
00
1100
24
00
1300
12
00
790
790
1100
13
00
1700
79
0 23
0 13
00
1700
1000
>
1000
60
0 10
0 30
0 10
00
200
200
300
300
400
CGb
300
200
300
1400
13
00
1900
C
G
CG
40
00
700
180
300
900
200
900
500
1000
10
00
40
d b
0
5 M
25
0
@ * r
15
0 5 20
10 0 0 5 25
10
15
100 0
150 25 5 0 15
15 0 20
15
10
20
30
15
15-12-86
12-01-87
20-01-87
10- 11-86
17-1 1-86
24-1 1-86
01-12-86
09-12-86
15-12-86
12-01-86
Viz
cach
aa
Inle
t. M
aipo
R.
Viz
cach
as
Inle
t Maipo
R.
Viz
cach
aa
Inle
t M
aipo
R.
Viz
cach
itas
Inle
t M
aipo
R.
V i z
c a c h
itas
Inle
t M
aipo
R.
Viz
cach
itas
Inle
t M
aipo
R.
Viz
cach
itas
Inle
t M
aipo
R.
Viz
cach
itas
Inle
t M
aipo
R.
Viz
cach
itas
Inle
t M
aipo
R.
Viz
cach
itas
Inle
t M
aipo
R.
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
640
16,000
660
>16,000
660
3500
290
2200
300
2400
320
1100
420
>16,000
430
5600
400
2800
>800
1700
>800
1700
>800
1100
300
1100
300
5400
300
1700
420
700
430
700
420
1700
128
1700
124
1300
128
1100
720
>1600
700
5400
690
1700
650
>16,000
640
>16,000
650
>16,000
310
9200
310
1700
330
3500
490
330
490
330
230
490
790
490
330
1700
1300
790
330
170
170
460
330
490
790
330
490
2400
1300
330
490
490
790
2400
790
1300
490
CG
490
1000
490
CG
1100
400
1300
600
1100
200
790
500
490
400
1300
200
1300
1100
1420
400
700
600
790
1000
190
1000
1100
2000
490
CG
790
1000
330
1000
790
1000
490
400
700
400
1100
1000
1100
CG
790
300
330
CG
790
CG
330
CG
490
400
490
400
490
200
5 20 0 5 20
10
10 0 25
1250
400
100 5 0 0 45
15
Q
10 5 0
8 5
+ 15
15
tll
15
0" d 5 2
15
0
10
?
0 Q
5 0 10
E m
&
CG
conf
luen
t gro
wth
. w
a CFU: co
lony
form
ing
units
. I@
544lCASTILLO ET AL.
examined, 68.3% were E . coli, and the other organisms found in order of frequency were E . agglomerans, Citrobacter spp., E . aerogenes, Aero- monas spp., Proteus spp., and many strains not identifiable by the techniques used.
The development of atypical colonies on M-FC agar was common. Grey, pink, and light-blue colonies were mixed among the typical blue colonies, these colonies were predominantly Pseudomonus spp. and other nonfermenting bacteria. Some sky-blue colonies were also con- firmed as E . coli.
Based on these fecal coliform raw water isolate data, the A-1 broth technique is the most selective procedure for E . coli, with the M-FC procedure being the least selective. These observations along with den- sity study data provide very strong support for the use of the A-1 broth technique in all Chilean fecal coliform studies. It is the least expen- sive, it provides results within 24 h, it is easy to read, and it is the most selective for the target organism, E. coli.
A total of 150 drinking water samples, not replicated, were col- lected from the Santiago city drinking water distribution system and rural distribution system as well as from individual rural community drinking waters. Of these 150 potable water samples, 134 were from chlorinated systems and 16 were from nonchlorinated wells.
Table I1 presents a summary of the drinking water samples col- lected from “chlorinated” sources, as well as the tests used. Twenty-six potable water samples were found to contain contaminating bacteria by one or more of the tests used. The P/A test was the most sensitive, producing 21 positive tests and the total conform (TC) m-Endo LES test was the next most sensitive procedure with 19 positives. In Table I1 it can also be seen that the P/A test and the TC m-Endo LES agar test were the sole positive tests on each of three occasions, while the TC MPN and 35°C HZS strip tests were the only positive indicator tests on one occasion each.
Twenty-seven of the 134 “chlorinated water supply samples were found to have no free residual chlorine and 17 of these samples were part of the 26 samples showing bacterial contamination. The remain- der of these 134 “chlorinated” samples had free residual chlorine levels varying from 0.1 to 0.6 mg/L. There appeared to be no relationship between free residual chlorine levels and heterotrophic plate counts. Samples 1 and 2 and 48 and 79 are illustrative of this point. Turbidity at the levels encountered in these samples did not appear to play a role in the quality of the potable water examined.
All of the nonchlorinated well water samples (16) were positive for indicator bacteria (Table 111). Here again, the P/A test was the most productive (16 positive) followed closely by the TC m-Endo LES agar
TAB
LE I1
C
hlor
inat
ed d
rink
ing
wat
er S
ampl
e 13
4: S
umm
ary
of p
ollu
ted
sam
ples
(26)
Free
H
eter
otro
phic
pl
ate
TC
TC
FC
coun
ts
no.
NTU
mgl
L
(day
) 20
°C
35°C
m-E
ndo
MPN
M
PN
Col
ipha
ge
perm
L
HSS
, test
re
sidu
al
Sam
ple
Tur
bidi
ty
chlo
rine
P/A
(d
ay)
1
1.7
2 1.
7 8
1.9
18
1.5
27
0.5
44
3.2
45
0.9
46
2.6
47
1.5
48
3.2
52
1.6
53
1.4
54
3.2
55
1.5
56
1.3
57
1.1
58
1.7
60
2.4
68
0.4
79
0.7
95
3.2
111
1.8
120
0.8
140
0.6
142
0.4
145
0.8
Tot
al (
+)
posi
tive
Tot
al (-
) ne
gativ
e
+ +
+ +
+ + + +
-
-
-
- +
+ +
+ +
+ + +
+ +
+ +
+ +
+ +
+ +
+ +
-
-
-
- +
-
-
+ + +
-
-
- -
+ +
+ +
+ -
19
18
7 8
1700
33
50
6100
13
0 2 25
800
1200
70
0 1
750
920
1300
77
0 13
0 30
30
140 10
<1
30 1
1
1000
14
0 3
546lCASTILLO ET AL.
TABLE III Well water samples, all polluted (16)
Hekro-
H2S test
SamDle Turbiditv P/A (day) TC TC- FC
trophic plate
counta no. NTU (day) 20°C 35°C m-Endo MPN MPN Coliphage per mL
4 5 6 7 9 71 72 73 74 75 77 131 133 139 147 150
+ + + + + + + + + +
+ + + + + + + + + + +
-
-
-
5800 1500
33,000 54,000
680 700 1400 2000
16,000 4000 2100 110 330 1160 350 580 - Total + 16 13 12 15 13 10 0
Total - 0 3 4 1 3 6 16
technique (15). The H2S strip test was as sensitive as the TC MPN procedure and more sensitive than the fecal coliform (FC) MPN proce- dure. No coliphage were found in these waters. Heterotrophic plate counts, an excellent indicator of water quality, ranged from 110 to 5400 per mL.
In Table IV all the potable water samples positive for coliphage are displayed as well as those samples that gave a positive reaction in only one test. The table presents a representative view of the type of data obtained in this study, the number of positive tests occurring in tests with delayed incubation, and the type of organisms isolated from positive PIA and H2S strip tests.
Bacterial identification procedures were carried out on all positive PIA samples (37) and in all 61 (34-20°C and 27-35°C) positive H2S strip tests. Out of the 37 P/A positive samples, coliforms were con- firmed in 35: E. coli, E. aerogenes, and Citrobacter freundii. The other bacteria identified in order of frequency were fecal streptococci, Clos- tridium spp., P . seruginosa, and Aeromnas spp.
In positive H2S strip tests the most commonly isolated organisms
COLIPHAGE ASSOCIATION CHILE/547
were Clostridium spp., Citrobacter freundii, Proteus spp., and H2S pro- ducing E. coli. Typical coliform organisms were also recovered such a8 E. aerogenes, E. agglomerans, E . coli, and Klebsiella spp. Occasionally Aeromow, Serratia, Pseudomow, and Alkaligenes species were found. No differences were noted between bacteria isolated from the 20 and 35°C H2S strip test.
Only 5 drinking water samples contained coliphage, and these samples were also found to have fecal coliforms and E. coli (from iso- lates). Fecal coliform densities ranged from 13-350 MPNI100 mL for the coliphage-positive samples, and the correlation between fecal coli- forms and coliphage varied from 1 :2 to 1:35. In no instance were coliphage recovered from coliform-free potable waters as has been noted recently in Singapore (Sim and Dutka, 1987) and Egypt (El- Abagy -~~ et al , 1987).
The association of coliform tests, coliphage, and turbidity in raw water are summarized in Table V, which gives the Spearman’s rank correlation matrix. All correlations, except that between coliphage and total coliform tests, are positive and significant ( p < .Ol), which indi- cates that the general pattern of the water quality variability can be characterized using any of these tests.
Friedman’s rank sum test was applied to test the equality of the estimates of the fecal coliform densities that were determined by the EC, A-1, and M-FC tests. The calculated value of the test is 41.98, which is significant at the 1% level. The multiple comparison test associated with the rank test is 41.98, which is significant at the 1% level. The multiple comparison test associated with the rank test shows that the EC test is equivalent to the A-1 test and both are superior to the M-FC test.
The A-1 test has a cost and time advantage over both of the other compared fecal coliform tests, and should be, based on these data, considered as the preferred bacteriological test for examination of raw water supplies in Chile. The A-1 test combined with the coliphage test would make an excellent screening program for health hazards in Chilean raw water supplies.
The superiority of the sensitivity of the P/A test for monitoring potable water samples can easily be seen in this study. This test is relatively inexpensive, simple to perform, and we recommend it with- out reservation for all routine potable water quality analyses. We be- lieve this procedure can enhance water quality testing procedures, especially when cost is a factor. This procedure, along with the H2S paper strip technique, are both very amenable for use in remote areas or in field studies.
The H2S paper strip technique has been shown to be equally sensi-
TABLE IV
Dri
nkin
g w
ater
sam
ples
sho
win
g a
posi
tive
resu
lt in
onl
y on
e te
st a
nd th
ose
with
pos
itive
col
ipha
ge te
sts:
Exa
mpl
es o
f pat
tern
s H
fi t
est
TC
Cal
i- m
-End
o H
etem
- Fr
ee
phag
e A
gar
TC
FC
hp
hic
T
ur-
resi
dual
Sa
mpl
e (d
ays)
TC'
FCb
FCc
Cl.p
d P.
ae S'
Ae
d 2
0°C
35
°C
20°C
35
°C
PF
U/1
00
CF
U/1
00
MPN
/100
M
PN
/100
pl
ate
bidi
ty
chlo
rine
num
ber
Sour
ce
mL
mL
mL
m
L w
unt/m
L N
TU
mg/
L
P/A
test
.+
or
- (d
ays)
B
acte
ria i
sola
ted
t or
-
27
Com
mun
al
-(5)
A
A
A
A
A
A
A
-(
5)
-(5)
-
dish
i-
butio
n sy
stem
dist
ri-
butio
n sy
stem
dish
i-
butio
n sy
stem
dist
ri-
butio
n sy
stem
dist
ri-
butio
n sy
stem
dist
ri-
butio
n sy
stem
44
Mun
icip
al
+(4
) P
A
A
A
A
A
A
45
) -(
5)
-
48
Mun
icip
al
-(5)
A
A
A
A
A
A
A
-(
5)
-(5)
-
58
Mun
icip
al
-(5)
A
A
A
A
A
A
A
-(
5)
-(5)
-
60
Mun
icip
al
+(3
) P
P P
P P
A
A
-(5)
-(
5)
-
111
Mun
icip
al
-(5
) A
A
A
A
A
A
A
-(5)
t(
5)
-
133
Wel
l +
(3)
P A
A
A
A
A
A
-(
5)
-(5)
-
P. ae
rugi
nosa
C
lost
ridi
um
<1
<
1 2
<2
2 0.
5 0.40
<1
<
1 <2
<2
<1
1.
8 0.
30
<2
<2
1 3.
2 0.
60
<1
1
<1
1
<2
<2
30
1.7
0.00
<1
<1
<2
.<2
14
0 2.
4 0.
00
<2
1
3.2
0.60
<1
1 <2
<1
<1
<2
12
33
0 0.
8 N
ot
&lo
r-
inat
ed
145
Rur
al
diStTi-
butio
n sy
etem
distri-
butio
n sy
stem
distri-
butio
n sy
stem
dist
ri-
butio
n sy
stem
&ti-
bu
tion
system
distri-
butio
n system
45
Mun
icip
al
47
Mun
icip
al
52
Mun
icip
al
53
Mun
icip
al
64
Mun
icip
al
-(5)
A
A
A
A
A
A A
3
5) -6)
-
-
<1
2 <2
<2
3 0.8
0.20
+(1
) P
P P
A
A
P A
+(2)
+(1) C
itmba
cter
C
itmba
cter
65
>80
360
350
800
0.9
0.0
Clo
stri
dium
C
lost
ridi
wn
E. e
oli
E. c
oli
Ent
emba
cter
E
ntem
baet
er
Pmte
us
Pmte
us
Clo
stri
dium
C
loat
ridi
um
Ale
alig
enes
Clo
stri
dium
E
. col
i E
ntem
bact
er
Ent
emba
cter
C
ibob
acte
r
Clo
stri
diw
n E
ntem
bact
er
Ent
emba
cter
Pm
teua
C
losi
ridi
um
Clo
stri
dium
C
lost
ridi
um
Ent
emba
eter
+(1)
P P
P A
A
A
A
+(2) +(1)
E. c
oli
E. c
oli
10
280
240
170
700
1.5
0.0
+(1
) P
P P
P A
A
A +(
2) +(1)
E.d
i C
itmba
cter
10
280
540
350
750
1.6
0.0
+(1)
P P
P P
A
A
A
+(2)
+(1) E
. col
i E
.col
i 15
>80
240
240
920
1.4
0.0
+(1)
P P
P P
A
A
A
+(2)
+(1)
E. c
oli
E.c
oli
110
280
240
130
1300
3.2
0.0
a T
otal
col
iform
. Fe
cal c
olifo
rm.
Feca
l str
epto
cocc
i. * C. perfnngens.
P. aeruginosa.
‘S. a
ure
u.
B Ae
romo
nas spp.
55o/CASTILLO ET AL.
TABLE V The correlation matrix for the different water quality tests
(raw water)
Fecal coliforms Turbidity Total EC A-1 M-FC
Total coliforms .382 EC .396 .444 A-1 .632 .313 .677 M-FC .766 .336 .588 .826 Coliphage .376 .029 .350 .308 ,387
tive in potable water testing as the traditional water quality bacterial indicator systems. Furthermore, it is probably the best and simplest method to test remote water supplies, as well as for use in city and town laboratories. These two procedures combined with the coliphage test we believe would provide an excellent assessment of the safety of potable waters from bacterial and virus contamination.
References
American Public Health Association. 1985. Standard methods for the examination of water and wastewater (16th edition). Washington, DC.
Clark, J.A. 1969. The detection of various bacteria indicative of water pollution by a presence-absence (P-A) procedure. Can. J. Microbiol. 15771-780.
Clark, J.A., C.A. Burger, and L.E. Sabatinas. 1982. Characterization of indicator bacte- ria in municipal raw water, drinking water and new main water samples. Can. J. Microbiol. 281002-1013.
El-Abagy, M.M., B.J. Dutka, and M. Kamel. 1987. Incidence of coliphage in potable water supplies. Appl. Environ. Microbiol. 54:1632-1633.
Hollander, M., and D.A. Wolfe. 1973. Nonparameteric Statistical Methods. John Wiley & Sons, New York.
Manja, K.S., M.S. Maurya, and K.M. Rao. 1982. A simple field test for the detection of fecal pollution in drinking water. Bull. WHO 60(5):797-801.
Ohgaki, S., A. Ketratanakul, S. Suddevgrai, U. Prasertson, and 0. Suthrenkul. 1986. Adsorption of coliphages to particulates. Water Sci. Technol. 18:267-275.
SENDOS, Chilean National Public Works Service. 1983. Drinking water: A factor of health. Foro. Mund. Salud. 4(2):198-199.
Sim, T.S., and B.J. Dutka. 1987. Coliphage counts: Are they necessary to maintain drinking water safety? MIRCEN J. 3:223-226.
Wetsel, R.S., P.E. @Neil, and J.F. Kitchens. 1982. Evaluation of colphage detection as a rapid indicator of water quality. Appl. Environ. Microbiol. 43:430--443.
