Evaluation of Factors Affecting Survival of

Escherichia coli in Sea Water : V. Studies with

Heat- and Filter-sterilized Sea Water
A. F. Carlucci, P. V. Scarpino and David Pramer
Appl. Microbiol. 1961, 9(5):400.

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These include:

Evaluation of Factors Affecting Survival of

Escherichia coli in Sea Water
V. Studies with Heat- and Filter-sterilized Sea Wateri
A. F. CARLUCCI,2 P. V. SCARPINO, AND DAVID PRAMER
Department of Agricultural Microbiology, Rutgers, The State University, New Brunswick, New Jersey

Received for publication December 9, 1960

and, thereby, to cause an increase in concentration of

available nutrients in sea water. It was concluded
that the bactericidal action of sea water is not explicable in terms of the destruction or inactivation by heat
of bacteriophages or antibiotics. Although added
organic matter influenced the survival of E. coli, the
test organism was not an effective competitor in sea
water and the nutrient levels required to offset the
bactericidal action were excessive.
Artificial sea water was demonstrated to exert a
bactericidal action comparable to that of natural sea
water. Low levels of cysteine which favor survival of
E. coli in natural sea water had a similar effect in
artificial sea water. Nevertheless, it is not at this time
possible to conclude that the factors responsible for
the bactericidal action of artificial sea water are identical with those responsible in natural sea water.

It has been observed repeatedly that bacteria survive to a greater extent in heat-sterilized sea water
than in untreated sea water (Nicati and Rietsch, 1885;
De Giaxa, 1889; Kiribayashi and Aida, 1934; ZoBell,
1936; Krassilnikov, 1938; Ketchum, Carey, and Briggs,
1949; Vaccaro et al., 1950; Nusbaum and Garver,
1955; Richou, Neant, and Richou, 1955). This thermolabile bactericidal action of sea water varied with
season of the year and was greatest during summer
months (Vaccaro et al., 1950). Furthermore, the
survival time of bacteria in sea water has been extended by filtration, chlorination, and treatment with
organic matter as well as by heating (Beard and
Meadowcroft, 1935; Krassilnikov, 1938; Vaccaro et
al., 1950; Williams, 1950; ZoBell, 1936). However, our
present knowledge provides little insight into the
nature of the factors responsible for the phenomenon
observed. Although numerous explanations for the
beneficial effect of complete or partial sterilization on
the survival of bacteria in sea water have been suggested (Carlucci and Pramer, 1959), they have not
been tested experimentally and the identity of the
factors responsible for the more rapid death of bacteria
in natural than in sterilized sea water remains to be
established.

I Paper of the Journal Series, New Jersey Agricultural

Experiment Station, Rutgers, the State University, Department of Agricultural Microbiology, New Brunswick, N. J.
This investigation was supported in part by research grant
E1437 from the National Institute of Allergy and Infectious
Diseases, National Institutes of Health, U. S. Public Health
Service.
2 Present address: Research Department, United Fruit
Company, La Lima, Honduras, C. A.

400

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ABSTRACT
CARLUCCI, A. F. (Rutgers, the State University,
New Brunswick, N. J.), P. V. SCARPINO, AND DAVID
PRAMER. Evaluation of factors affecting survival of
Escherichia coli in sea water. V. Studies with heatand filter-sterilized sea water. Appl. Microbiol. 9:400404. 1961.-The bactericidal action of sea water was
measured as the difference in survival of cells of Escherichia coli in untreated and autoclaved portions of water
samples. The beneficial effect of sterilization by heat
on the survival of E. coli in sea water varied with
season and was most marked during summer months,
however, the magnitude of the effect differed greatly
from sample to sample. The more obvious and commonly suggested explanations for the bactericidal
action of sea water were tested experimentally. The
pH and salinity of sea water were changed by autoclaving, but the direction of the former was detrimental rather than beneficial and the significance of
the latter was not clarified. The survival of cells of E.
coli in filtered portions of some water samples was
greater than that in untreated portions and equal to
that in autoclaved portions, indicating that predators
and competitors removed by filtration had contributed
significantly to the rapid death of the bacterium in the
untreated water. However, in the majority of samples
tested, survival of E. coli in autoclaved water was considerably greater than survival in filtered water.
The possibility that the beneficial effect of autoclaving over and above that of filtration resulted from
inactivation or destruction by heat of bacteriophages
and thermolabile toxic substances such as antibiotics
was considered. Moreover, the suggestion was tested
that the increased survival of E. coli in autoclaved sea
water was due to the ability of heat to disrupt and
degrade microbial cells and thermolabile compounds

1961]

SURVIVAL OF E. COLI IN SEA WATER

The present report describes the influence of season,

pH, salinity, filtration, and organic matter on the
bactericidal action of sea water. Moreover, artificial
sea water was observed to exert a bactericidal action
comparable to that of natural sea water.

water is increased rather than decreased the bactericidal

3

Millipore Filter Corporation, Bedford, Mass.

action cannot be attributed to the influence of autoclaving on pH alone.

The effects of sterilization by filtration and autoclaving on the salinity of sea water were examined
and although there was tendency for filtration to cause
a decrease and autoclaving to cause an increase in
salinity, the magnitude of the changes never exceeded
1.0%. The nature of the changes was not determined
and therefore their significance is not known.
Filtration. Water samples collected during July and
August of 1957 were divided into three portions. One
was sterilized by filtration, the second was sterilized
by autoclaving, and the third served as an untreated
control. The survival of cells of E. coli in these waters
was determined for each of six different collections.
The results listed in Table 1 show that in each of the
six samples tested E. coli died more rapidly in untreated
than in autoclaved water but the magnitude of the
effect varied greatly. Likewise, the influence of filtration
was not constant. In four of the six experiments survival
of the test organism was significantly greater in filtered
than in untreated sea water. If it is assumed that the
favorable effect of filtration resulted from removal of
predatory and competitive organisms, then the numbers
and activities of these organisms varied from sample
to sample. Waksman and Carey (1935) considered
predation as a factor contributing significantly to the
death of bacteria in sea water. The decreased survival
of E. coli in the filtered portion of water sample no. 5
can be explained by the possible but improbable assumption that a beneficial substance or organism was
removed during filtration.
In two of the samples tested (no. 1 and 6) the bactericidal action of the water could have been fully accounted for by predatory and competitive organisms
that were eliminated by filtration; survival of E. coli
was the same in filtered and autoclaved water. In two
other samples (no. 3 and 5) filtration had no influence
on survival of the test organism and it appeared unlikely that predation and competition contributed
significantly to the rapid death of E. coli in the untreated water. In four of the six samples tested (no. 2,
3, 4, and 5), predation and competition were not an
adequate explanation for the rapid death of E. coli in
untreated water, since survival in autoclaved water was
considerably greater than survival in filtered water.
TABLE 1. Survival of Escherichia coli in untreated, filtered, and
autoclaved portions of six sea water samples collected during
July and August, 1957
Survival after 48 hr in sea water sample no.
Treatment

None

2.2

8.3
Filtered
Autoclaved 8.4

Sot2
0.7
3.8
30.9

4.6
4.8
64.6

22.8
30.1
53.6

4.4
0.6
69.6

2.7
39.6
38.5

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MATERIALS AND METHODS

The bactericidal action of sea water was measured
as the difference in survival of cells of Escherichia coli
in untreated and autoclaved portions of the same water
sample. Detailed descriptions of the methods employed
for preparation and use of inocula, treatment and
storage of water samples, and of the enumeration and
calculation of survival of cells of E. coli in sea water
were presented previously (Carlucci and Pramer,
1960a).
Sea water samples were heat sterilized by autoclaving at 121 C for 15 min. Sterilization by filtration
was performed using glass filter holders and type HA
Millipore membranes3 having a pore size of 0.45 ,u.
RESULTS AND DISCUSSION
Seasonal variation. The bactericidal action of sea
water was determined at irregular intervals for 2 years
by measuring the survival of cells of E. coli in untreated
and autoclaved portions of each water sample. It was
first demonstrable in April, increased in magnitude to
a maximum in July, then decreased, and was absent
from water collected in November. The greatest
bactericidal action was exerted by water sampled
during July 1957 when the ratio of per cent survival
after 48 hr in autoclaved to that in untreated water
exceeded 24. These results were consistent with those
of Vaccaro et al. (1950) who reported that the bactericidal action of sea water was most pronounced during
the summer months and absent in the winter. Water
collected during the winter months exerted no bactericidal action and it was not unusual for cells of E.
coli to die more rapidly in autoclaved than in untreated
portions of these samples. This has been observed
(Williams, 1950) but not commented on by previous
investigators.
pH and salinity. The influence of autoclaving on pH
and salinity and the possible contribution of these
factors to the bactericidal action of sea water were
considered experimentally. Electrometric determinations demonstrated that the reaction of sea water
increased from 0.5 to 1.0 pH unit during heat sterilization and that equilibrium was not re-established for 24
to 48 hr. It has been noted previously that the survival
of E. coli in sea water varied inversely with hydrogenion concentration (Carlucci and Pramer, 1960b) and
on the basis of pH alone sterilization by heat would be
expected to have an adverse rather than beneficial
effect. Since the survival of E. coli in heat-sterilized

401

402

A. F. CARLUCCI, P. V. SCARPINO, AND D. PRAMER

duced benefit derived by cells of E. coli from peptone

added to water which was otherwise untreated was due
in part to competition, since turbidity measurements
and plate counts showed that the indigenous microbial
population of the sea water increased with an increase
in the amount of peptone added.
The survival of cells of E. coli in sea water that was
not sterilized but contained 10 ppm of peptone did not
differ significantly from that in unsupplemented autoclaved sea water; the addition of 10 ppm of peptone to
sea water that was otherwise untreated eliminated the
bactericidal action of the water. However, the effect
lasted only 48 hr, after which cells of E. coli were observed to die off more rapidly in the peptone supplemented unsterilized water than in the unsupplemented
autoclaved water. It appeared that survival of the
test organism in natural water would have been comparable to that in autoclaved water for the duration of
the experiment if the former had been supplemented
with more than 10 ppm but less than 100 ppm of peptone. Although available organic matter in the form of
peptone favored survival of E. coli in sea water the
magnitude of the effect was not great. The number of
cells that persisted in sterilized sea water supplemented
with 10 ppm of peptone was approximately 10 times
that in sterilized but unsupplemented water 4 days
after treatment, whereas the latter was more than
1,000 times the number of cells that survived in water
which was not sterilized or supplemented. If the
favorable effect of autoclaving on the survival of cells
of E. coli in sea water is to be explained on the basis of
an increase in concentration of available nutrients,
it is necessary that the heat treatment release more
than 10 but less than 100 ppm of organic matter, and
this is unlikely, since the required range exceeds the
organic matter content of sea water (ZoBell, 1946).
To better evaluate the importance of competition
and predation, the survival of E. coli was measured in

DAYS
FIG. 1. Influence of concentration of peptone on the survival
of Escherichia coli in untreated and autoclaved sea water.

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The beneficial effects of autoclaving over and above

those resulting from filtration may have been due to
any one or a combination of possibilities that include:
(i) destruction of bacteriophages; (ii) inactivation of
thermolabile toxic substances such as antibiotics; (iii)
increase in available nutrients.
The influences of bacteriophages and antibiotics
on the survival of E. coli in sea water were tested and
discussed previously (Carlucci and Pramer, 1960c, d).
The effectiveness of bacteriophages depends on growth
and multiplication of host cells. Since sea water does
not support development of cells of E. coli, coliphages
have no significant influence on survival of the test
bacterium and it is doubtful that they contribute to
the bactericidal action of sea water. The biological
effects of antibiotics in sea water vary with the nature
and concentration of the antibiotic. It was observed
that, although some antibiotics adversely influenced
the survival of cells of E. coli in sea water, the result
was of academic interest only since relatively high
concentrations were required and there is no evidence
that antibiotics are produced under natural conditions by marine microorganisms.
Nutrients. Since heat kills and disrupts microbial
cells and degrades thermolabile compounds the third
possibility listed above appeared reasonable, namely,
that the concentration of available nutrients in sea
water was increased by autoclaving. Previous studies
(Vaccaro et al., 1950; Orlob, 1956; Carlucci and
Pramer, 1960b) have shown that the addition of organic
matter to sea water which was otherwise untreated
increased the survival of E. coli. The effect varied with
the composition and concentration of the substance
tested. Relatively high levels were required and it was
demonstrated that the ability of E. coli to utilize
organic matter added to sea water was limited by pH
and salinity, as well as by competition from the indigenous micropopulation (Carlucci and Pramer,
1960b). Since sterilization by heat would simultaneously eliminate competitors and increase the amount of
available organic matter in sea water, lower nutrient
levels may be required for a beneficial effect in autoclaved than in untreated sea water. Therefore, the
influence of concentration of peptone on the survival
of cells of E. coli in untreated and autoclaved portions
of the same water sample was determined at daily
intervals for 4 days. The results are illustrated in Fig. 1.
The bactericidal action of the water was apparent
from the more rapid death of cells of E. coli in the untreated than in the autoclaved unsupplemented portion
of the water sample. Peptone at levels of 1 and 10 ppm
favored survival of the test organism in both untreated
and autoclaved sea water but the effect was greater
in the autoclaved portions. A peptone concentration
of 100 ppm supported growth of E. coli in both untreated and autoclaved water and the inerease in cell
numbers was greater in the sterilized water. The re-

[VOL. 9

1961]

SURVIVAL OF E. COLI IN SEA WATER

TABLE 2. Influence of organic matter on the survival of Escherichia

coli in untreated, filtered, and autoclaved sea water
Survival after 48 hr after
water treatment:

Supplement

None
Peptone, 200 ppm
None
Glucose, 200 ppm +

None

Filtered

4.6

4.8

Autoclaved

64.6

272.7* 2,873.6* 2,631.2*

2.2
8.4
8.3
712. 1* 3,305.9*
17.6

(NH4)2HPO4-N, 4 ppm
None
Sewage (volatile solids), 200
ppm

0.7
5.0

3.8
18.6

30.9
20.2

be offered for the comparable survival of E. coli in

sewage-supplemented, autoclaved, and filter-sterilized
water. On the other hand, growth of E. coli in autoclaved sea water supplemented with glucose was much
greater than that in filtered water that received the
same supplement, suggesting that development of the
test organism in sea water containing glucose was
limited not only by predators and competitors, but also
by other factors not removed by filtration but destroyed
by the autoclaving process.
From these and other studies it is apparent that
organic matter can influence the survival of E. coli in
sea water. However, the concentration required to
obtain an effect of sufficient magnitude to overcome
the bactericidal action is in the range of 10 to 100 ppm
and exceeds the total organic matter content of pelagic
water that is free of pollution. The report of Johannesson (1957) is unique in that the amino acid cysteine
was demonstrated to be particularly effective, and to
favor survival of E. coli in sea water at organic matter
levels that are conceivable under natural conditions.
The beneficial effect of cysteine on survival of cells of
E. coli in sea water was confirmed (Carlucci and Pramer,
1960b) and studies of the mode of action of cysteine
are in progress.
On the basis of the present investigations it is concluded that the bactericidal action of sea water as
measured by the difference in survival of cells of E.
coli in untreated and autoclaved portions of water is
not explicable in terms of the destruction or inactivation by heat of bacteriophages or antibiotics. Although
autoclaving kills and disrupts microbial cells and degrades thermolabile compounds, the resulting increase
in nutrient level is not adequate to explain the favorable
effect of heat sterilization. Further evidence in support
of this conclusion was obtained from studies with
artificial sea water.
TABLE 4. Survival of Escherichia coli in untreated and autoclaved
portions of four artificial sea waters

* E. coli multiplied.
TABLE 3. Cosnposition of four artificial sea waters
Constituent

NaCl

MgCl2-6H20
Na2SO4
CaCl2-2H20
KCI

NaHCO3
KBr

SrCl2*6H20
H3BO3

Na2SiO3,9H20
NaF

NH4NO3
FePO4-4H20
MgSO4-7H20
NaBr

ZoBell

(1946)

2.4
1.1
0.4
0.1
0.07
0.02
0.01
0.004
0.003
0.0005
0.0003
0.0002
0.0001

Tomlinson
and MacLeod
(1957)

2.4
1.1
0.4
0.07
0.02
0.01
0.004
0.003

Sverdrup
et

al.

(1942)

2.7
0.5
0.2
0.07
0.02

0.7
0.008

MacLeod
and Onofrey
(1956)

2.4
1.1
0.4
0.2
0.07
0.02
0.01
0.004
0.003

Survival after 24 hr
Artificial sea water

Untreated

Autoclaved

0.01
<0.01
< 0.01
<0. 01

2.3
0.7
5.1
0.1

ZoBell (1946) .................<........

Tomlinson and MacLeod (1957) .......
MacLeod and Onofrey (1956) .........
.........
Sverdrup et al. (1942) .......

TABLE 5. Influence of cysteine on the survival of Escherichia coli

in artificial sea water
Treatment

Survival after 24 hr

Autoclaved ..............................
Untreated .........................<......
Untreated + cysteine, 0.035 ppm ..
Untreated + cysteine, 0.35 ppm
Untreated + cysteine, 3.5 ppm ...........

13.6
0.01
<0.01
0.3
20.3

..........

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untreated, filter-sterilized, and autoclaved portions

of sea water samples with and without addition of 200
ppm of glucose, peptone, and sewage volatile solids.
The results summarized in Table 2 show that suirvival
of the test bacterium varied with the composition of
the organic material tested and the treatment to which
the water sample was subjected. In all but one case,
survival of E. coli was greater in supplemented than in
unsupplemented water. The exception was autoclaved
water that received sewage. E. coli multiplied in sea
water supplemented with 200 ppm peptone. The addition of glucose and sewage to sea water that was otherwise untreated increased survival but did not result in
multiplication of the test organism. Removal by filtration of predators and competitors caused an increase
in growth of the test organism in water treated with
peptone and permitted multiplication in water that
received glucose. The survival of E. coli in water supplemented with sewage was increased by filtration.
Development of the test organism was similar in peptone-supplemented, autoclaved, and filter-sterilized
water, which might suggest that competitors and predators were of primary importance in limiting utilization of peptone by E. coli. The same explanation may

403

404

A. F. CARLUCCI, P. V. SCARPINO, AND D. PRAMER

LITERATURE CITED
BEARD, P. J., AND N. F. MEADOWCROFT, 1935. Survival and
rate of death of intestinal bacteria in sea water. Am. J.
Public Health 25:1023-1026.

CARLUCCI, A. F., AND D. PRAMER. 1959. Factors affecting the

survival of bacteria in sea water. Appl. Microbiol. 7:388392.
CARLUCCI, A. F., AND D. PRAMER. 1960a. An evaluation of
factors affecting the survival of Escherichia coli in sea
water. I. Experimental procedures. Appl. Microbiol. 8:243247.
CARLUCCI, A. F., AND D. PRAMER. 1960b. An evaluation of
factors affecting the survival of Escherichia coli in sea
water. II. Salinity, pH, and nutrients. Appl. Microbiol.
8:247-250.
CARLUCCI, A. F., AND D. PRAMER. 1960c. An evaluation of
factors affecting the survival of Escherichia coli in sea
water. III. Antibiotics. Appl. Microbiol. 8:251-254.
CARLUCCI, A. F., AND D. PRAMER. 1960d. An evaulation of factors affecting the survival of Escherichia coli in sea water.
IV. Bacteriophages. Appl. Microbiol. 8:254-256.
DE GIAXA, V. 1889. Ueber das Verhalten einiger pathogener
Mikroorganismen im Meerwasser. Z. Hyg. Infektionskrankh. 6:162-224.
JOHANNESSON, J. K. 1957. Nature of the bactericidal agent in
sea water. Nature 180:285-286.
KETCHUM, B. H., C. L. CAREY, AND M. BRIGGS 1949. Preliminary studies on the viability and dispersal of coliform
bacteria in the sea. In Limnological aspects of water supply
and waste disposal, p. 64-73, AAAS, Washington, D. C.
KIRIBAYASHI, S., AND T. AIDA. 1934. A study of the fate of
cholera vibrio in the sea water of Keelung Port, Formosa.
U. S. Public Health Eng. Abstr. 14:61.
KRASSILNIKOV, N. A. 1938. The bactericidal action of sea water.
Mikrobiologiya, 7:329-334 (English summary).
MACLEOD, R. A., AND E. ONOFREY, 1956. Nutrition and metabolism of marine bacteria. II. Observations on the relation
of sea water to the growth of marine bacteria. J. Bacteriol.
71:661-667.
NICATI, W., AND W. RIETSCH. 1885. Experiences sur la vitalit6
du bacille virgule cholerigene. Rev. hyg. et police sanit.
7:353-378.
NUSBAUM, I., AND R. M. GARVER, 1955. Survival of coliform
organisms in Pacific Coastal waters. Sewage and Ind.
Wastes 27:1383-1390.
ORLOB, G. T. 1956. Viability of sewage bacteria in sea water.
Sewage and Ind. Wastes 28:1147-1167.
RICHOU, R., M. NEANT, AND H. RICHOU. 1955. Sur le pouvoir
bactericide de l'eau de mer a l'6gard dii staphylocoque.
Rev. immunol. 19:64-68.
SVERDRUP, H. V., M. W. JOHNSON, AND R. H. FLEMING. 1942.
The oceans. Prentice-Hall, Inc., New York. p. 1087.
TOMLINSON, N., AND R. A. MACLEOD. 1957. Nutrition and
metabolism of marine bacteria. IV. The participation of
Na+, K+, and Mg+ salts in the oxidation of exogenous
substrates by a marine bacterium. Can. J. Microbiol.
3:627-638.
VACCARO, R. F., M. P. BRIGGS, C. L. CAREY, AND B. H.
KETCHUM. 1950. Viability of Escherichia coli in sea water.
Am. J. Public Health 40:1257-1266.
WAKSMAN, S. A., AND C. CAREY. 1935. Decomposition of organic matter in sea water by bacteria. II. Influence of
addition of organic substances upon bacterial activities.
J. Bacteriol. 29:545-561.
WILLIAMS, F. W. 1950. Survival of Escherichia coli in sea water.
MSc. Thesis, Univ. of Washington, Pullman.
ZoBELL, C. E. 1936. Bactericidal action of sea water. Proc. Soc.
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Co., Waltham, Mass. p. 240.

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Artificial sea water. Several formulations of artificial

sea water have been used for various purposes by
numerous investigators (ZoBell, 1946). To determine
if artificial sea water exerted bactericidal action, the
survival of E. coli in autoclaved and untreated portions of four different formulations was determined.
The composition of the waters employed is presented
in Table 3. The simplest formulation contained 7 salts,
whereas the most complex contained 13. They differed
primarily in the composition and concentration of
trace constituents.
The results listed in Table 4 show that survival of
the test organism was consistently greater in autoclaved than in untreated water but the magnitude of
the effect varied. Artificial sea water prepared according to MacLeod and Onofrey (1956) had the greatest
bactericidal action and the formulation of Sverdrup,
Johnson, and Fleming (1942) had the least effect. The
water chosen for further use was that of MacLeod
and Onofrey (1956). It was more convenient to prepare
than that of ZoBell (1946), more complete in major
elements than that of Tomlinson and MacLeod (1957),
and demonstrated a marked and consistent bactericidal
action. Artificial sea water is a more simple system for
study than natural sea water. It is of known composition, and lacks bacteriophages, predators, competitors, antibiotics, and organic nutrients that are or
may be present in natural sea water. However, the
bactericidal action of artificial sea water may be caused
by factors which differ from those responsible for the
effect in natural sea water and, in such a case, identification of the former will not clarify the latter.
Evidence that the bactericidal action of artificial
and natural sea water have a common basis was obtained from studies with cysteine. Since this compound is known to offset the bactericidal action of
natural sea water (Johannesson, 1957; Carlucci and
Pramer, 1960b), its effect on the survival of E. coli in
artificial sea water was determined. The results listed
in Table 5 show that survival of the test organism was
greater in autoclaved than in the untreated artificial
sea water. Cysteine at a concentration of 0.035 ppm
was without effect, but survival was increased by 0.35
ppm and in water that received 3.5 ppm of cysteine
but was otherwise untreated, survival of the test
organism exceeded that in autoclaved water. Further
study is required to identify the factors responsible
for the bactericidal action of artificial sea water and
to establish that the same or different factors are
responsible for the more rapid death of E. coli in untreated than in autoclaved natural sea water.

[VOL. 9