Detection and Enumeration of Salmonella and Pseudomonas Aeruginosa

Author(s): Bernard A. Kenner and Harold P. Clark

Source: Journal (Water Pollution Control Federation), Vol. 46, No. 9, Annual Conference Issue
(Sep., 1974), pp. 2163-2171
Published by: Water Environment Federation
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Detection
and enumeration
of Salmonella
and Pseudomonas
aeruginosa
Bernard

and Harold

A. Kenner

P. Clark

Federal
Pollution
Water
Con
Amendments
of
19721_4
may
The
well
the quantification
and enu
require
meration
of pathogens
such as Salmonella
in all classes of waters.
The re
species
are
described
Shedroff.5
quirements
by
One of the continuing
of the
programs
Environmental
Protection
Agency
(epa)
is a research project
concerned
with
the
of practical
meth
development
laboratory
ods for the isolation,
and
quantification,
enumeration
of pathogens
from polluted
waters.
This paper reports a monitoring
method
for the simultaneous
developed
isolation
and enumeration
of Salmonella
from
species and Pseudomonas
aeruginosa
trol

potable

waters,

reuse

waters,

treatment

and

waters,
effluents,
plant
receiving
sludges.
The method
and de
described
herein,
is
because
Kenner,6
veloped
by
practical
available
media,
readily
bacteriological
are all that are
and equipment
chemicals,
to obtain
the desired
results.
required
These
results are the establishment
of the
or presence
absence
of Salmonella
species
hazardous
bacteria ) and/or
(pathogenic
Pseudomonas
aeruginosa
patho
(potential
are in a
that affect persons who
gens)
debilitated
condition
and are very com
mon
as infectious
in hospitals
be
agents
cause
of
to antibiotic
their
resistance
Potable waters have also been
therapy.7-9
shown

sources
human
waters.11'

to

contain

Ps.

aeruginosa.6'10

The

are
of these potential
pathogens
and
animal
feces
and waste
12

When
the monitoring
method was used,
it was found that 100 percent of municipal
wastewaters
and treatment
plant
sludges

contained
both of these potential
patho
Ps.
has been
found in
gens.
aeruginosa
potable water
supplies of large and small
insufficient
where
residual
municipalities
is evident.
is the
chlorine
Also important
fact that these organisms may be found in
of fecal coliforms,
the absence
whereas
tests
indicator
may
negative
give a false
sense of security.
It is believed
by the
authors that these organisms may be better
indicators
than fecal coliforms
of pollu
tion in potable,
direct reuse, bathing,
and
waters.

recreational

and

Materials

Methods

uses a multiple
The monitoring
method
tube
in
which
dulcitol
(mpn) procedure
13 is
selenite broth
used
for
primary
(dse)
enrichment
and is modified
medium,
by
acid
the use of sodium
selenite
(bbl).
The formula is proteose
peptone
(Bacto),
extract
0.4 percent;
0.15
yeast
(Bacto),
0.4 percent;
0.5
bbl,
dulcitol,
percent;
0.125
and
Na2HP04,
percent;
percent;
in distilled water.
0.125 percent
KH2P04,
are dissolved
in a sterile
The constituents
to
flask, covered with
foil, and heated
88 ?C in a water
bath to obtain a clear
sterile medium
that does not require ad
of
for Salmo
justment
pH.
Productivity
nella species is enhanced
addition
the
by
of an 18-hr, 37 ?C culture
of Salmonella
A (10 percent
in
paratyphi
by volume)
dse broth, killed by heating
single-strength
to 88?C.
Concentration
from
of bacteria
large
volumes

of

water

is necessary

reuse,
ble, direct
treatment
effluents
-Vol.

46, No.

when

pota

and
waters,
receiving
are being monitored.

9, September

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1974

2163

and Clark

Kenner

TABLE

of Several

Characteristics

I.?Retentive

Compared

with

grid, 47 mm, Millipore

984H Ultra Glass
Fiber
Reeve
Angel
Corp.

Filter
Filter,

Corp.
47 mm,

47 mm,
Glass
GF/D
Paper Whatman,!
Reeve
Angel
Corp.
934AH
47 mm,
Fiber Filter,
Glass
Reeve
Angel
Corp.
47 mm,
Glass
GF/A
Paper Whatman,

Percentage
Retention

Angel

Corp.

984H

Ultra

Glass

Fiber

Filter

is flexible

when

readily be bent double with

forceps, and, when
is shaken
bacteria.
tube
and releases
entrapped
E. colt, 0.5 X 1-3 ?t.
t Enteric
bacteria,
? A new

paper

filter GF/F

1,376

100

1,229

25

98

2,698

has better

retentive

99.8
17.4

2,166

2,622

can

Oct.

Papers*

47 mm,

Glass
GF/F
Paper Whatman,t
Reeve
Angel
Corp.

The

Filter

(mf) HAWG 047 HA 0.45 m,white,

Millipore

Fiber

Number Passing
Filter

Total Bacteriaf
Filtered

Filter

Reeve

Glass
Filters

Membrane

1,049

198

81

1,066

680

36

wet,

placed

properties

readily allows filtration

into primary
enrichment

than

the 984H,

of

large volumes

broth,

and has

same

disintegrates

properties

of water,
when

(tested

1973).

is attained
filtration
by
*
fiber
in a membrane
filters
through glass
filter apparatus.
After the desired volume
of water
is filtered through the ultra filter,

Concentration

the flexible filter is folded double with

sterile forceps and inserted into a suitable

dse medium
of
volume
single-strength
in a test tube located
in the
contained
first row of the multiple
tube setup.
The
tube should then be shaken to cause the
filter to disintegrate
(Table I and Figure
To obtain mpn results per one 1 or
1).
per 10 1, 100 ml or 1,000 ml of sample,
are filtered for each tube of
respectively,
dse medium
in the first row of the five
are
Additional
dilutions
tube mpn setup.
made by transferring material
from tubes
in the first row to tubes farther back
in
the setup.
results on a per 1-gal (3.8-1)
Obtaining
basis requires filtration of 380 ml, and on
a per 10-gal (38-1) basis requires filtration
of 3,800 ml for each tube in the first row.
concentration
is not
Where
of bacteria
as in municipal
waste
usually
required,
the
waters,
effluents,
sludges, or primary
10
transfer
of
to
ml
of
each
regular
sample
* Reeve
Reeve
mm,
Mention
of
dorsement

2164

Angel 984H ultra glass fiber filter, 47

Angel
trade

&< Co.,
N.
Inc., Clifton,
J.
not
en
does
constitute

names

or recommendation

by EPA.

tube in the first row of the setup into 10

dse is made,
1 ml
ml of double-strength
dse
in 9 ml of single-strength
of sample
in the second row, and so on. The mpn
14 is
used to
table in "Standard Methods"
of
read directly
the results per volume
sample.

of 40? ? 0.2?C
Incubation
temperature
for 1 and 2 days is critical to obtain opti
mum
of
Salmonella
sp. and
recovery
dse broth
when
Pseudomonas
aeruginosa
is used
After
for primary
enrichment.
at 40? C, surface loop
incubation
primary
or nichrome
fuls (scum)
(7 mm platinum
are
each multi
wire
removed
from
loop)
on each of
and
culture
streaked
ple-tube
two sections of a divided
plate of Xylose
15 in
order
agar (xld)
lysine desoxycholate
to isolate colonial growth.
The numbered
at 37 ?C
plates are inverted and incubated
for a period not to exceed 24 hr.
xld agars (bbl
Commercial
dehydrated
are satisfactory
if they are re
and Difco)
in sterile
in distilled
water
constituted
to 88? or
flasks and heated
foil-covered
is then
92 ?C, respectively.
The
agar
in
to 55? to 60 ?C and distributed
cooled
This
sterile petri dishes.
laboratory pre
in each section of a
fers 10-ml portions
divided
(Figure

sterile

disposable

1).

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plastic

dish

Pathogen
i
Sterile
polypropylene
container

Detection

Filter funnel
for 47-mm 984 H
filter pad

n
for
10-liter
test

vacuum
flask

1000ml pad
1xDSE broth

into 20ml
inserted
for each of 5-tubes
row
1st
^_
Jn
2ml to
8ml 1xDSE in
2nd row

is
After filtration filter-pad
folded double with forceps
4

1ml from
2nd row to
9ml 1xDSE
^3rd row etc.
MPN incubated at 40C for
^Completed
medium
streaked
for
1- and 2- days-Secondary
from surface MPN tubes with
isolated colonies
22 gauge
7 mm Nichrome
loop
6

XLD Agar plate

cap & incubate
6-20 hrs
mm

Loosen

invert plates

incubate

37C

hrs

20-24

Loosen

caps

Pick Black centered

flj
to KIA
colonies
l^
Red or
1000 A
slants
<?no-change
'Pink colonies
.slant
rarely
Salmonella
sp.
'H2S
Streak and Stab butt
Black
Yellow
Incubate Slants at 35
Acid
Pick flat erose edge
37C 18-20 hr.
wButt
grayish alkaline colonies
to Tech Agar streak & stab
KJ W
KJ
Blue Green
, typical slant
Ps. aeruginosa
Salmonella
sp.
y/
Slide Serology
aerology
on XLD Plate
^Purify
Salmonella
"O" poly A-l
U rease>
''for isolated pure strains
f^Urease
T est
or Salmonella
"H" poly
Negative
a-z\

HI

Chloroform
extract
blue

FIGURE

Positive
incubated
contain
clear,
typical
centered
Salmonella

xld

40C

I1000A
King A
Tech Agar

1.?Procedure

cultures
plate
black
pink-edged,
and
colonies,
flat,
mucoid,
grayish alkaline, pink erose-edged
Ps. aeruginosa.
The Salmonella
colonies
are picked
or
to Kligler
iron agar (kia)
iron
slants
for
agar
sugar
Triple
typical

for isolation

of pathogens.

and identity tests.

appearance,
purification,
to King
Ps. aeruginosa
colonies are picked
A agar slants (Tech agar bbl)
for obtain
confirmation
pyocyanin
ing the bluegreen
at40?C
(Figure 1).
Salmonella
sp. slant cultures
Typically,
over
and
incubated
(streaked
stabbed),
-Vol.

46, No.

9, September

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1974

2165

and Clark

Kenner

TABLE

of Ultra-filter
984H
for Salmonella

IL?Advantage

Use

Waters

Type

5.

4.5

runoff

Stormwater

Serotypes Found
(no./100 ml)

Salmonella
(no./100 ml)

of Sample

in Monitoring

Suspected

species
Salmonella
(no./gal)

210

bareilly1

Serotypes Found
(no./gal)
S.

kottbus10

S.

sludge effluent
wastewater

<3.0

Municipal

Municipal

wastewater

<3.0

Activated

sludge

Activated

7.3

<3.0

runoff

Stormwater

3.6

6.2

Arizona3

1,500

110

bareilly11
S. java*
S. muenchen2
S. group G4
Arizona4
S. anatum2
S. newport4
S. san diego1
S. worthington2
S. anatum3
S. derby1
S. newport3

River
Mississippi
403.1
mile

Municipal

43

water,

28

none

<3.0

effluent

S.

> 11,000

ohio10

S.

blockley1
S. newport3
S. ohio19
S. derby2
S. meleagridis*

S.

3.0

wastewater

21

cholerasuis
var. kunzendorf2

S.

cholerasuis
var. kunzendorf5

5.

or alka
night at 37 ?C, give an unchanged
is
line red-appearing
the
butt
black
slant;
ened by H2S, is acid-yellow,
and has gas
bubbles,
except for rare species.
Typical
are purified
slant cultures
appearing
by
them to xld agar plates
for
transferring
the development
of isolated colonies.
The
flat or umbonated-appearing
colonies with
and clear pink edges
large black centers
then are picked to kia slants ( streaked and
at 37?C,
incubated
stabbed),
tested before
the identification

and

urease

procedure
tubes are re

1). Urease-negative
tests
for presumptive
serological
and serotype
identification.
Tech agar slant cultures for Ps.
Typical
at 40?C
that are incubated
aeruginoca
a
turn
color
from
overnight
bluegreen
(Figure
tained

pyocyanin,
this species.

a pigment
produced
A reddish-blue
color

only by
is caused

of pyorubin.
presence
by the additional
is extractable
in chloro
The blue pigment
form and is light blue in color after a few
hours at room temperature.
No
further
tests are necessary.
rectly from the mpn
2166

The
table.

count

is read di

Justification

for

newport*

Procedures

Choice
of primary
enrichment
medium
and secondary
isolation agar. Most of the
in contem
enrichment
media
described
were
literature
for the
porary
designed
isolation of pathogens
from clinical
speci
mens
from ill persons or from samples of
foods, and they work quite well
suspected
for those types of samples.
When
they
are used, however,
for the isolation
of
from polluted
pathogens
types of environmental

waters

and other
such as
samples,
En
soils, they do not prove
adequate.
richment media that were tested and found
in regard to detection
and selec
wanting
tetrathionate
tivity were
and without
brilliant
selenite

(tt), with
at
41.5 ?C;
green
at 37?C;
selenite
broth

broth
cystine
at 37?C; selenite brilliant
green,
with and without
sulfa, at 37? and 41.5?C;
and Gram-negative
broth
(gn) at 40? and
F broth

41.5?C.

None of the media

named worked well
at 37 ?C for the isolation of Salmonella
sp.,
and isolation
from wastewaters
only oc

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Pathogen
TABLE

No.

Liquid Samples

wastewater

Municipal
Stockyard
Rivers

of Colony Picks
for Salmonella

III.?Percentage

wastewater

Ohio
Activated
sludge
effluent

110
18

84
14

26

76
78

386
103
83
41
17

306
78
55
37
13

80

37
17

16
10

21

20
16

14

80
15
78

66
13
65

9
189

3
155

6
2
2

(1.6 km) below

plant
package
cisterns
R-0
Dupont

outfall

2
2

Home

Feed

Reject
Product-negative
Raw
sludge
primary

4
sludge

digester

sludge

digester

sludge

activated

(28 days)
Activated

secondary
Total

biological
20

sludge

6
84

1,223

curred by chance and was purely qualita

tive. Of the above-named
used in
media
selenite
brilliant
tests,
green
preliminary
at 41.5 ?C gave
the
sulfa broth
(sbgs)
best isolation of Salmonella
sp. from waste
waters
the addition of
(with and without
in known numbers).
S. typhimurium
Of
sbgs
in
thirteen wastewater
tested
samples
or 46
at 41.5?C,
six contained
Salmonella
were
dse
at
With
broth
percent
positive.
40 ?C, 28 of 28, or 100 percent
of waste
water
results.
gave positive
samples,
on sbgs me
Studies were not continued
it was noted that some lots of
dium when
sbgs seemed
to be
available
commercially
for Salmonella
selective
while
others
sp.
were
was
not.
The medium
then pre
to the original
formula16
pared according
with
six different
lots of brilliant
green
selec
(certified),
only one of which was
tive.
The use of brilliant
green agar as
a selective medium
is subject to the same
to Read and Reyes.17
variability,
according

28
4
4

2,100

2
13

3-43
4.5-12
0.26-1.1
4.3
0.91
13-700
23
79-170

33
82

6
34

average

1.5

1.8-620

83
87
83

14

>300

43-240

70
50

1.5
0.2

0.1-1,100
0.35-140

43
59

347

3.0-1,500

79
76
66
90
76

25

1,570

Range of
Salmonella
counts/100 ml

Percentage
Positive

79
100

Package
plant effluent
sludge
Package
plant
outfall
Chlorinated
primary

Anaerobic

No.
Negative
65
0

Primary
Anaerobic

No.
Positive

Total Picks
from xld

250
36

Trickling filter effluent

Creek 1mile

Positive

315
36

runoffs

Combination

species

15
1

Mississippi
Stormwater

from DSE-XLD

Detection

ll->

11,000

78

reasons
of tt,
for rejection
The main
and
for
brilliant
and without
with
green,
selenite broth's using brilliant
agar
green
are not
and xld agar as secondary media
sp., but
only fewer isolations of Salmonella
of these combina
also the poor selectivity
tions when
they are used for monitoring
waters.
These combinations'
poor
polluted
at
in the
41.5
?C is apparent
selectivity
results of Dutka
and Bell,18 where
the tt
26 percent
broth-xLD combination
yielded
confirmation
of colonial picks, and selenite
broth-BGA and selenite broth-XLD gave 55
and 56 percent
confirmations,
respectively.
The authors had similar results.
The gn
xld
was
water
combination
for
poorest
less
samples at 40? and 41.5?C, yielding
than 10 percent
isolations
from waste
waters.

on iso
Effect of incubation
temperature
a
In
lation of Salmonella
sp.
study of
was
26 wastewater
that
conducted
samples
with
the dse multiple
tube setups at three
-Vol.

46, No.

9, September

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1974

2167

and

Kenner
TABLE

Clark

IV.?Serotypes
Polluted

1 typhimurium*
2 derby
3 cubana
4 ehester
5 newport
6 kottbus
7 blockley
8 infantis
9 enteritidis
10 anatum
11 Heidelberg
12 manhattan
13 paratyphi B
14 illinois
15 thornpson
16 livingstone
17 montevideo
18 muenchen
19 oranienberg
20 saw ?tego
21 barielly
22 tshiongwe
23 orion
24 senftenberg
25 schwarzengrund
26 lexington
27 cholerasuis
28 fcittsa
29 cholerasuis var.
kunzendorf

Salmonella

Found

in

Waters

No. of
Strains

Serotype

Other serotypes
30 albany
31 benfica
32 braenderup
33 brancaster
34 bredeney
35 california
36 dry Pool
37 friedenau
38 gw
39 grumpensis
40 ?fli/a
41 Hartford
42 Havana
43 indiana
44 java
45 javiana
46 litchfield
47 lomita
48 meleagridis
49 mission
50 newington
51 newlands
52 norwich
53 oftt'o
54 preston
55 reading
56 rubislaw
57 sainf ?)awi
58 schleissheim
59 simsbury
60 taksony
61 tennessee
62 typhi-suis var.
voldagsen
63 uzaramo
64 ??>?7
65 worthington

of

375
287
223
203
188
158
157
141
128
127
110
97
91
77
63
52
47
45
44
44
42
41
41
39
37
33
30
29

Rank in
Water
Isolations
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
19
20
21
21
22
23
24
25
26

29
:

Sub total
Arizona
Incomplete serology
Total
* Rank

20
10
13
1
8
2
14
4
25
10
2
2
16
10
15
13
17
26
18
14
2
8
14
19
2
26
15
21
12
9
16
12
2
28
3
10

31
40
38

Results
37
29
40
35
40
36
38
34
28
33
37
37
32
28
36
30
39
35
39
27
40

3,417
151
232
3,800

in human occurrence Table I, Martin and Ewing.19

f Separation of S. typhimurium and var. Copenhagen not
done after initial identifications.
in humans,
1965-1971, Center for
t Serotypes
occurring
Disease Control, Salmonella Surveillance, Annual Summary
1971, Table IX, U. S. DHEW/PHS
DHEW Publ. No. (HSM)
73-8184 (Oct. 1972).

2168

it was
found that
different
temperatures,
of the samples contained
Sal
100 percent
at 40?C.
monella
sp. and Ps. aeruginosa
At 41.5?C, however,
only 50 percent or 13
of the samples yielded
Salmonella
sp., and
at 37 ?C only 8 percent or 2 of the samples
sp.
yielded Salmonella
Effect of enhancement
of dse broth with
a killed culture of S. paratyphi
A.
In a
84
of
activated
of
sludge
samples
study
effluents,
trickling filter effluents, package
dse
and stream waters,
effluents,
plant
a killed
broth enhanced
culture
with
of
A in dse broth
S. paratyphi
(10 percent
in 64 sam
isolations
by volume)
yielded
isolated Salmonella
sp.,
ples or 74 percent
48 samples or 57 percent
compared with
isolations when
the dse broth was
used
iso
An improved
without
enhancement.
lation of 17 percent was
achieved
with
enhanced dse broth.
Ultra-filter.
of ultra
The
advantages
filter use in testing water
samples are illus
trated in Table II.
and

Discussion

use
to those who must
importance
to
tests
Salmonella
obtain
bacteriological
Of

sp.

and

Ps.

aeruginosa

counts

from

waters

is the amount of work that must be done

to secure accurate
III pre
results.
Table
sents the percentage
of colony picks made
to
with
that proved
the described method
If there are black
be Salmonella
sp.
on the xld plates, more
centered
colonies
than 75 percent of the picks will prove to
be Salmonella
leads
sp.; thus, the method
to less unproductive
work.
When
other
were
methods
the authors have at
used,
times had to pick 50 black-centered
col
to obtain
onies
sp.
only 5 Salmonella
strains.
This
work
type of unproductive
in the
has given the search for pathogens
an undeserved
environment
bad reputation,
and it has caused some to give up.
In Table
II it may readily be seen that
cases the fault with many
in many
tests
has been the testing of an insufficient vol
ume of sample.
think that
people
Many
it involves too much work,
and that only
fluorescent
expensive
techniques
antibody
to
will work.
The problem
is, however,

Journal WPCF

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Pathogen
TABLE

of Various

V.?Percentage

Type

Before
chlorination
After
chlorination,
5 min
residual,
filter

Package
plant
1 mile
Creek
Ohio

plant
River

Wabash

Septic

effluents
effluents
(1.6 km)

below

Cincinnati

28

40

29

11

26

15

11

15

public

after

heavy

rain

suburban
cisterns
tank sludges

20

1*

31

11

18

13

6
4

3t
0

3?
4

114

69

183

Totals

Municipal
f Positive
? Negative

Number
Negative

0
above

collective
runoff

species

package

River

Stormwater
Farm wells

Number
Positive

for Salmonella

mg/1

landing
River

Mississippi
Streams

Home

1.4-2.0
contact

Positive

Samples

28

effluents
primary
Municipal
(chlorinated)
effluents
Activated
(clarified)
sludge
effluents
Activated
sludge

Trickling

of Water

Number of
Samples

of Sample

wastewaters

Municipal

Types

Detection

intake.
technique.
by per-gallon
by per-100 ml technique.

concentrate
in a 10-gal (38-1)
the bacteria
or
a
of
(380-1)
sample
100-gal
sample
or
reuse
to
water
obtain
results,
potable
and still not require even more expensive
or centrifugation
filtration
It
equipment.
to test only extremely
also seems unrealistic
small samples of the water being examined,
because
they may not be representative.
a list of Salmonella
IV contains
Table
serotypes isolated from polluted waters and
ranked according
to the frequency
of sero
It will be noted
that all
type isolations.
of the serotypes
iso
except S. typhi were
lated from environmental
samples by the
and
that
method,
monitoring
only 6 of the
65 serotypes
were
not reported
reported
as occurring
in humans
in the U. S. over
the period from 1965 to 1971.
Table V summarizes
the percentage
of
various
water
of
types
positive
samples
for Salmonella
sp. Of interest is the fact
that 100 percent
of the municipal
waste
waters
tested contain Salmonella
sp., that
56 percent of chlorinated
primary effluents
tested contain the pathogens,
and that 100

of chlorinated
effluents
percent
secondary
are negative
are
for pathogens.
There
more
studies scheduled
for testing of sec
to obtain
and tertiary
effluents
ondary
et
minimal
chlorine
residuals.
Calabro
that more
than 50 attempts
al.20 reported
at isolating Salmonella
sp. from septic tank
samples

using

sbgs-bgsa

combinations

were

unsuccessful.

VI
summarizes
of
Table
the isolation
Ps. aeruginosa
from potable water
supply,
that is, wells,
cisterns, and small municipal
water
It should be noted
that
supply.
in most
fecal coliforms were not detected
of these samples.
Fecal streptococci
counts
were
than
counts
fecal
coliform
higher
where both tests were used.
Ps. aeruginosa
were present
in all but three of the tests,
and Salmonella
from
isolated
sp. were
two different
cistern samples.
It is of importance
to the user of patho
In
gen tests that the test be quantitative.
initial studies on the dse-xld
combination,
it was important to know if the enrichment
broth would
support the growth of a wide
-Vol.

46, No.

9, September

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1974

2169

Kenner

and

Clark

TABLE

Type

Well
Well
Well
Well

of Pseudomonas

VI.?Isolation

Ps. aeruginosa
Isolation

of Sample

8/16/71
8/25/71
3/27/72
3/27/72

Suburban

8/23/72
10/ 4/72

+
+

22

Population

Fecal
Coliforms

0.25

<2

180
15
<2
<2
3

supplies
served

54,700

served

<1
<1
<1

+
+
+
+

0.26

+
0

<1

<1

<1

14,000

<

served

<1

+
10,000

11/27/72
sp. also

<1

present

in samples.

range of Salmonella
serotypes.
Laboratory
cultures of S. paratyphi A, S. typhimurium,
S. bredeney,
S. oranienberg,
S. pullorum,
S.
S. give, and S. worthington
were
anatum,
in three enrichment
tested
broths.
The
time required to isolate each of the above
cultures from an estimated
10 to 20 orga
in
was 48 to 72
ml
water
buffer
nisms/100
hr for S. paratyphi A in tt broth, 24 hr for
dse broth, and 36 to 48 hr for sbgs broth.
The rest of the cultures were
isolated in es
timated numbers
in 14 to 24 hr in tt and
dse broths.
In sbgs broth, S. typhimurium?,
S. bredeney,
S. anatum,
S. give,
and S.
worthington
required 36 to 48 hr incuba
and S. oranienberg
tion, and S. pullorum
required 48 to 72 hr incubation.
It is impossible
to know if 100 percent
of Salmonella
water
sp. in a polluted
are
In
tests
isolated.
where
lab
sample

2170

ml

<1

5/ 8/72
10/24/72

Salmonella

Indicators/100

<1

+
+

3/17/71
6/21/71
7/19/71
6/19/72
10/ 9/72
5/ 8/72

Supply

cisterns

Municipal

Population

Water

8/ 4/72
10/ 9/72*
11/ 6/72*
11/ 6/72
11/26/72

Population

Total
Coliforms

(chlorinated)

Well
Well

from Potable

aeruginosa

in low
oratory cultures have been added
to wastewater
numbers
and
treatment
effluent samples, all of the numbers added
were detected,
as well
as the Salmonella
The
sp. that were
occurring.
naturally
of the water
the quality
higher
(for ex
or tertiary
treatment
ample,
secondary
or
even
the
better
effluent,
potable waters),
the possibility
pf isolation of all the Salmo
as well
as Ps.
nella
serotypes
present,
a potential
aeruginosa,
pathogen.
Summary
A practical
is pre
laboratory method
sented for the simultaneous
and
isolation
enumeration
of Salmonella
sp. and Pseudo
monas aeruginosa
from all classes of waters,
a
including
potable water
supplies, with
minimum
of interfering
false positive
iso
lations. The method
allows for the testing

JournalWPCF

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Pathogen
of large volumes
of high quality waters,
the absence of indicator bacteria
wherein
fecal coliforms,
is, total coliforms,
(that
and fecal streptococci),
may give a false
sense
low
of the
of security
because
tested.
of water usually
volumes
Justifica
tion for each step of the procedural method
is

presented.

7. Moody,
M. R.,
in a Center

et

al, "Pseudomonas
for Cancer
Research.

tribution

of

Human

and

Intraspecies
Environmental

1. Federal
PL

Appl Microbiol, 24, 219 (1972).

9. "New Hospital Controls Urged
to

Water

Pollution
86 Stat.

Control
816,

33

Amendments,
U.
S. Code

Sec. 1151 et seq. (1972).

2. FWPCA, Section 504 as amended (1972).
3. FWPCA, Section 307 (a) (1972).
4. FWPCA, Section 311 (1972).
5. Quality
and

Assurance
Monitoring,

III.

Analysis
for
Salmonella
U.
Tool."

10. Reitler,
monas

of Waste

S.
Research

species
EPA,
Center,

Treatment
Sludges
a Surveillance
as
National
Cincinnati,

Environ

Clin.

Lab.

Stem
Forum

2, 1 (May-June 1970).

and
R.,
aeruginosa

"Pseudo

R.,
Seligmann,
in Drinking

Water."

145
Jour. Appl.
Bacteriol,
20,
(1957).
11. Ringen,
L. M.,
and Drake,
C. H.,
"A Study
of the Incidence
of Pseudomonas
aeruginosa
from
Various
Natural
Sources."
Jour.

64, 841

Bacteriol,
12. Drake,
for

C.
the

(1952).

"Evaluation

H.,

of Culture

and

Isolation

Pseudomonas

Media

Enumeration

of

Health

aeruginosa."

Lab.

Sei., 3, 10 (1966).
13. Raj, H.,
"Enrichment
Medium
of
Salmonella
from
Fish
"Standard
Water

for

Methods

the

for

Examination
of
13th Ed.,
Amer.
York, N. Y. (1971).
of
I.
Shigellae.

and Wastewater."
Health
Assn., New

Pub.
15. Taylor,
W.
"Isolation
I.,
New
Xylose
Lysine
Agars;
lation
of Enteric
Pathogens."

Reg. Med. Technol,

16. Osborne,
fied
the

W.

Isolation

of

Read,
in

R.

B.,

Jr.,

Iso
Bull.

35, 161 (1965).

J. L., "A Modi
Medium
for

Salmonella
and

for

Tech.

Stokes,
Brilliant-Green

ucts." Appl. Microbiol,

17.

Media

and

W.,
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Selection

Homogenate."

14, 12 (1966).

Appl. Microbiol,
14.

Office
of Research
Division,
1st
U.
S. EPA,
"Proc.
on
Seminar
Standardization

Microbiology
of Methods."
EPA-R4-73-022
(Mar. 1973).
B. A., et al, "Simultaneous
6. Kenner,
Quantita
tion of Salmonella
and Pseudomonas
species
I. Polluted
II. Persist
Waters.
aeruginosa.
ence
in Sludge
of Pathogens
Treated
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mental

Rise."

(Eli Lilly),

92-500,

Jour.

of Pseudo
"Epidemiology
in a Burns Hospital.
Sur
aeruginosa
veillance
by a Combined
Typing
System."

Acknowledgments

References

from

Types
Sources."

et al,

P.,

Pseudomonas

assistance
of
Credits.
The
technical
in performing
the neces
Pauline C. Haley
for identifying many
of the
sary serology
is gratefully
Salmonella
serotypes
reported
acknowledged.
is super
Bernard A. Kenner
Authors.
and
Harold
research
microbiologist,
visory
is biological
P. Clark
technician, Waste
and Analysis Activity
of the
Identification
Treatment
Advanced Waste
Research Lab
Research
Environmental
oratory, Natonal
S. Environmental
Protection
U.
Center,
Ohio.
Agency, Cincinnati,

aeruginosa
I. Dis

125, 95 (1972).

Inf. Diseases,
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Appl.

(1968).

B.
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of
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J., and Bell,
J. B.,
Salmonellae
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Polluted
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45, 316 (1973).

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46, No.

9, September

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All use subject to JSTOR Terms and Conditions

1974

2171