A Membrane Filtration Technique for the Enumeration of.pdf

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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

BARRITT,M.

M.

(1936). The intensification of the V-P reaction by the addition

of

a-naphthol.

CLEW,

L.

F.

L. SHERWOOD,

.

P.

(1947).

The bacteriological examination

of

molluscan shellfish.

HOLDEN,

.

S.

(1970). Water Treatment and Examination.

London: Churchill.

MUSHIN, R . ASHBURNER,. M. (1964). Studies on coli-aerogenes and other Gram-ncgative

intest inal bacteria in various animals and birds.

J pp l . Bact .

27

392.

R E P O R T

71. (1969).

The Bacteriological E xamina tion of Water Suppl ies ,

4th ctl. London: H.M.S.O.

TAYLOR,E. W. (1968). Progress with membrwe filtration. 42nd

Rep . Me t ro p . Wa t . Bd .

for

WOOD,P. C. (1964). The effect of water temperature on the sanitary quality

of Oslrea edulis

and

Crmsostrea angulata

held in polluted waters.

Comm ission Internationale pour Exploration

Scienti que

de

Mer Mkditerranke, Symposium Pollulion Marines par les Microorganismes

et lea Produits Petroliere, Monaco.

J .

Pa th .

Bact. 42, 441.

J . H y g . ,

Camb.

45, 604.

1965 66.

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