a membrane filtration technique for the enumeration of.pdf
TRANSCRIPT
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HALLS,
SHEILA AYRES,P. A. (1974).
J .
appl Bad. 37 105-109.
A
Membrane Filtration Technique for the Enumeration
of
Escherichia coli
in Seawater
SHEILA ALLS ND P. A.
AYRES
Ministry of Agriculture, Fisheries and Food, Fisheries Laboratory,
Burnham-on-Grouch,Essex, England
(Received 28 June 1973 and accepted 12 November 1973)
SUMMARY.
Membrane filtration has become an accepted method for enumerating
Escherichia coli
in water, but little published evidence could be found to judge the
specificity of the method t o assess faecal contamination in either fresh or saline waters.
The method is used in our laboratory to monitor the extent a nd degree of sewage pollution
in coastal areas, bu t there is need for information on what proportion of lactose-fcrrnenting
colonies from seawater, developing at 44 on a
4%
enriched Teepol medium, are E. eoli
type
I. A
total of 1352 colonies from seawater was bs te d for production
of
intlole and for
gas from lactoso
at
44 . I n addition, 46% of the colonies were screened by the IMVEC
series of tests. The proportion of colonios tes ted ranged from lO-lOO , depending
on
the
number of colonies on the membrane. Many of tho colonies
(81.9 )
to which
IMVEC
tests were applied were E coli type I ; a further 10.90/, were Irregular type I. The
practical implications of these findings
are
discussed.
MEMBRANE
ILTRATION
has become an accepted method for concentrating bacteria in
liquid samples which, because they contain few cells/unit volume, are not easily
enumerated by other techniques. The method can be adapted to estimate specific
groups of bacteria by the use of seleotive media and choice of incubation temperature.
We have used the technique to enumerate Escherichia coli in seawater samples to
monitor the extent and degree of sewage pollution in coastal and estuarine areas. I n
this work we have assumed that all lactose-fermenting colonies appearing on mem-
brane filters after overnight incubation at
44
re
E .
coli type I . This assumption is
based on the results of t,he examinat>ionof molluscan shellfish by Clegg Sherwood
(1947), who found that 96.9%of red colonies appearing on MacConkey agar incubated
at 44 were eitherE coli type I or Irregular type I. However, little published evidence
exists for judging the specificity of the technique when used to examine fresh or saline
waters. The aim of much
of
the published work on membrane filtration has been to
develop a technique which would enumerate, in 18 h, numbers of
E coli
equal to
or
greater than those obtained by a most probable number (MPN) method employing
incubation for 48
h
a t 37 followed by confirmation a t
44 .
The work described in the present paper was designed to test what proportion of the
lactose-fermenting colonies developing during an 18 h test at
44
on a
4%
enriched
Teepol medium were E . wli type I.
Materials and Methods
Samples
Seawater samples were collected in sterile
8
oz
medical 'flats', using
a
simple weighted
sampler. The majority were taken a t various points in the estuaries of the Rivers
D 5 1
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106
Sheila Halls and
P.
A. Ayres
Roach and Crouch, Essex, where salinities ranged from 26-36%, and the remainder
were taken off the coast a t Whitstable, Kent. Thirty samples were stored overnight
a t 8 prior to examination, but most were examined within 8 h of collection, being
kept in a cool place and out of direct sunlight until testing.
Examination
of
samples
All samples were filtered through 5 cni diam., 0.45
p
pore size, membranes (Oxoid),
using
a
stainless steel filter head (Gallenkamp). Volumes
(100
and 10 ml) of each
sample were filtered through separate membranes and placed on Whatman absorbent
pads previously soaked ui th the cnriclied Tccpol broth. The filters and pads were
incubated in purpose-made plastic trays each holding 8 filters placed in a heavy gauge
polythene bag. A small pad of moistcncd cotton wool was included in each bag to
prevent drying of the membranes, and some lead shot was added to sink it
in
the
water bath. Incubation was carried out in a thermostatically-controlled water bath
fitted with a time switch
so
as to provide resuscitation at
30
for 4 h (Taylor, 1968),
followed by incubatlion for a t least 14 h at 44
0.2 .
Media
Membranes were incubated on
an
enriched Teepol broth (Burman, pers. cornm.) of the
following composition: peptone, 40 g; yeast extract, 6 g; lactose, 30 g Teepol 610
BDH), 40
ml;
0.4%
aqueous phenol red,
50
ml; distilled water,
1000
ml, autoclaved
at
121
for
15
niin (final pH value,
7.4).
The media used for characterizing colonies were tryptone water, brilliant green-bilc
broth, Koser's citrate and MRVP medium (all Oxoid).
Colony characterizatioTL
After incubation, yellow lactose- ermenting colonies on each membrane were counted
and, depending on the numbers present, either
all
or a proportion of them picked off
singly into tryptone water and incubated at
37
for
24
h, when further sub-cultures
were made into brilliant green-bile broth, Koser's citrate and MRVP media. The
tryptone water cultures were incubated for a further
24
h and then tested for indole
production by extraction with xylene and the addition of a few drops
of
Ehrlich's
reagent.
Growth and the product'ion of gas in brilliant green-bile broth, after
18
h at 44
(Eijkman test) , were recorded. Koser's citrate medium was incubated a t 37 for 3-5
days. Cultures in MRVP medium were incubated a t 30 for 5 days and the contents of
each tube divided into two; one half was
tested
with 0.04%mcthyl red solution and
thc othcr by Barritt's
(1936)
method for the Voges-Proskauer reaction. These
5
tests
formed the IMVEC series (Mushin Ashburner,
1964).
The IMVEC reactions of the coli-aerogenes solates are given in Table 1 (taken from
Mushin Ashburner,
1964).
Since it was not possible to distinguish between
E coli
type
I1
and Irregular type
I11
using this scheme, colonies giving the reactions of these
groups were placed together as E .
coli
type 11.
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Enumeration of
E. coli
in
seawater
107
Results
Numbers and percentages of E . coli type I amongst colonies tested, separated on the
ba.sis
of our tests,
are
shown in Table 2 . The percentage recovery of E .
coli
type
I
obtained by applying the full IMVEC series of tests to
622
colonies from 30 samples
was Sl.9yo.
TABLE
Characterization of colonies
from
seawater sam ples by I M
VE C
tests
IMVEC* reactions Number
Colonies testod
E
oli type
I1
and Irregular 111
I , coli type I - -
+
-
erobacter aerogenes I
E. coli
Irregular
I
- - -
I1
- - -
I
VII
- - -
C -
D -
J - - -
Unidentified
622
510
4
68
18
7
1
2
2
4
4
1
-
100
8 1 . 9
0 . 3
0 . 6
10 9
2 . 8
1 . 1
0 . 2
0 . 3
0 . 3
0 . 6
0 . 6
* I, Indole; M, Methyl rod; V, Voges Proskauer; E, Eijkman; C, Citratc.
A further 730 colonies from
58
samples were tested only for production of indole and
of gas from lactose at
44
(i.e. I E tests). Of these,
78.4%
gave positive results with
both tests and were regarded as
E oli
type
I
(Report
71 , 1969). If
either or both
tests were negative,
a
culture
was
not characterized further.
TABLE
Isolation of E. coli type
I
fr o m seawater samples
Tests applied
IMVEC*
IE
No. of samples examined 3 58
No.
of colonies testod 622 730
yo of
E.
coli
type
I 8 1 . 9 78.4
No.
of
E
coli typo I 510 573
* Abbreviations as in Table 1
Table
1
shows the number and percentage of colonies other than
E
coli type
I
from the results
of
IMVEC tests. The largest single group
was
E .
coli
Irregular type
I
occasional isolates of Irregular types
11,VT,
VII,
C,
and
J
were found,
as
well as
Aerobacter aerogenes type
I
and E . coli type
11.
Four colonies out of the
622
tested
could not be placed in any group on the basis of the tests used.
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108
Sheila Halls and
P.
A. Ayres
Discussion
The percentage recovery of colonies identified as E .
coli
type I using the I W E C
and I E tests was very similar.
It
is not clear why the more rigorous IMVEC tests
should identify more colonies as type
I
than the I E tests. However, the close agree-
ment between the
2
methods suggests tha t identification on the basis of I E tests only
is a valid one, with the limitation tha t the remaining organisms can be described only
as 'other than
E
coli type
1 .
Of the colonies subjected to the full IMVEC test ,
16.2%
were classified as irregular
coli-aerogenes, but they could not be distinguished from
E .
coli type
I
by their appear-
ance on membrane filters incubated at 44 . The public health significance of these
organisms in estuarine water is uncertain, but they are known to occur in the intestinal
tracts of man and animals (Holden, 1970 . It would seem, therefore, that there may
be some merit in assuming that all lactose fermenters growing under these conditions
are of faecal origin.
Aerobacter aerogenes
constituted only 0.6y0of the lactose fer-
menters, and the errors of including non-faecal organisms would, therefore, appear to
be low. Although there are no British bacteriological standards for shellfish-growing
uaters, and the relationship between the level of pollution in shellfish and in the
surrounding water is complex (Wood,
1964),
water sampling can be valuable in
identifying the source, extent and degree of sewage pollution in estuarine waters.
Since a survey of this type depends on comparisons of the E . coli content
of
samples
taken a t the same time from different stations, or a t different times from the same
station, absolute numbers are not required. Provided tha t each sample is heated
similarly, it does not seem unreasonable to assume that the proportions of colonies
which are not
E .
coli
type I are of the same order. However, when comparisons are
made with other techniques (MPN, roll tube or plate count),
or
where an absolute
estimate of the number of E. coli in seawater is required, the fact that
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Enumeration
of
E
coli in seawater
109
References
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CLEW,
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F.
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.
P.
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HOLDEN,
.
S.
(1970). Water Treatment and Examination.
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MUSHIN, R . ASHBURNER,. M. (1964). Studies on coli-aerogenes and other Gram-ncgative
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