C!:'l.lifornia State Universjty 1 North:ddge
.A stt:!.dy attempting to d(::fine some o:f the factor~~, ·that
{;tl1U!e the d~ath of E. coli in t!1e -~xtern~d- environment
A thesis submitted in partial ss.tisfact~on of the
:e<Bq_uirernents for the degree o.t' J\>laste:r of Sc:i ence j_n
Biology
by
Jeffrey Irwin Daniels
The Thesis of Jeffrey Irwin Daniels is approved:
California State University, Northridge
ii
ACKNO';>lLEDGMENTS
To Dr. C,'harles Spotts I would like to extend my deepest
appreciation for his assistance, guidance, criticsms, and judgment
in the development. of this thesis.
Dr.
Further thanks is extended to
Richard Sv1ade and Dr. David Toppen for serving on my commi tt.ee
and for their valus,l.Jle sugg&stions and assistance.
I am
inrJ.eb~~ed
to l1uth J'1mg and I,inh Nguyen vTho prepared media
:for use in my experiments; to Robert Botts and his staff viho
p:rov-irted me with the departmental equipment necessary to conduct
my study; to the members of the CSUN Chemistry Department for
making materials available for my use; and to the faculty and
staff of the CSUN Biology Department for their interest, encouragement, a.nd support.
A special thanks is extended to my brother,
Clifford, and my father for accompanying me on many late night
excursions to monitor my experiments.
I am grateful for their
curiosity and their inquisitiveness.
Lastly, I vrould like to express my gratitude to my parents,
my brother, and my sister for the confidence they have in me!·
their devotion, and their help in preparing the final manuscript
of this thesfs.
iii
CON'l~ENTS
'I'ABI,E· OF
.ACKNOvli1EDGMENTS • • • • • • • • • • • • • • • •••••••••••••
iii
J... IST OF 'Ii\.BLF.S AN:O FIGURES •••••••••••••••••
v
ABS 'I I{A CT ~
~
"!
vii
e • e .......
~
1
VJETHODS AND MA. TERIALS ••••••••
ORGANISMS •••••••••••• •••••••••••••111•e•••"
8
8
8
1
* ~ ••••
I~-I'l'RODUCTIOl"'I ~
ANALYTICAL
-e
#I . . . . . . . . . 6
•••
~
•
••• - •••
•
..
~
e. "' • • (" 0 • "' • •
•
•
tl
"' • • • • • •
••••eo•c••••••••
~~1RODS .•••.••••••••••••••
GL1JCOSE DETERl{(NATION ••••••••••••••••••
DETERMINATION OF pH •••
H
•••••••••••••••
GROWTH MEASURE~illNTS ••••••••••••••••••••
PHYSICA.'L FACTORS •••••••••••••••••••••••••
RADIATION SOURCE •••
•
• • • • • c • • • c> • • • • •
......... " ...... .
CUJ...TURE PREPARATION ••••••••••••••••••••••
RJE:3ULTS o
••••••••••
(t
••••••••••
DISCtJSSION • ...............
I)
•
~
•••••••••••••
•••••••••••••••••
BIBLIOG·RA.I)HY • ...............................
iv
10
10
10
12
12
12
12
12
14
57
LIST OF TABLES AND
FIGUl~ES
TABLE
1
SUMMl\lW 01'' RESULTS OF NUTRIEN'"r IMBAlANCE EXPERIMENT...
30
2
SUMMARY OF RF..SUL'I'S OF NUTRIENT IMBALANCE E'iCPERIMENT
CONDUCTED AT 3'7°C FOR COl-1PAHISON WITH SIMilAR F..:X:PERIMENT CONDUCTED AT 15°C •••••••••••••••••••••••••••••• ; •
39
Sl..TJVflvJARY OF RE'SUL'J:S OJ.t' NTJTRIENT IMBALANCE :E._'XPERL\1l!."'NT
CONDUCTED AT 15°C li'OR COMPARISON \HTH SIMILl\.R EXPERI~-1ENT CfJNDUCTED AT 37°C. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •
39
4
ST..JMJv.IARY OF RJ<~SULTS FROM pH RECONSTRUC'fiON EXPERIMENT..
43
5
RJWUIBS OF CONTINUED METABOLISM EXPJ!~IMENT............
l+7
1
EXTJ3iNT OF GROWTH OF E. COLI B. • • • • • • • • • • • • • • • • • • • • • • • •
17
2
REil\.TIVE SU1WIVAJ.J OF·E. COLI BAT 37°C, NO GLUCOSE,
AND VARYING-AMOUT~TS OF PHOSPHORUS~ ••••••••••••••••••••
19
RE1ATIVE SURVIVAL OF E. COLI B AT 37°C, 0.2 mgjm1
GLUCOSE, AND VARYING AMOUNTS OF PHOSPHORUS. ·• • • • • • • • • • •
21
RELATIVE SURVIVAL OF E. COLI B AT 37°C, 1.6 mg/m1
GLUCOSE, AND VA...l=\YING AMOU1'ITS OF PHOSPHORUS. • • • • • • • • • • •
23
RELATIVE SURVIVAL OF E. COLI B AT 37°C, NO PHOSPHORUS,
AND VARYING ~IDUNTS OF GLUCOSE ••••••••••••••••••••••••
25
RElATIVE SURVIVAL OF E. COLI B AT 37°C, 1.14 ug/m1
FtiOSPHORUS, AND VARYING AMOUNTS OJ!' GLUCOSE............
27
3
FIGURES
3
4
5
6
7
8
9
Hr]IATIVE SURVIVAL OF E. COLI B AT 37°C, 8. 55 ug/m1
F.HOSFHORUS; AND VARYINGAMOUNTS OF GLUCOSE. • • • • • • • • • • •
29
REIA'l'IVE SURVIVAL OF E. COLI B AT 37°C, 8. 55 ug/m1
PHOSPHORUS, AND VARYING AMOUNTS OF GLUCOSE··-FOR
COMP.AlUSON WTl'H SIMilAR CONDI'riONS AT 15°C. • • • • • • • • • • •
32
RElATIVE SURVIVAL OF E. COLI B AT 37°C, 45.60 ug/m1
PHOSF.tiOHUS, AND VARYING AMOUNTS OJ!' GLUCOSE--FOR
COMPARISON WI'l'H SIMILAR CONDITIONS A'f _15°C ••• ~........
34
v
FIGURES
10
RELA.'I'IVE SURVIVAL Qli' E. COLI B A'I' 15°C, 8.55 ug/ml
PHOSPHORUS, AND VARYING AMOUN'rS OF GLUCOSE............
36
RElATIVE SURVIVAL O:F' E. CO:CI B AT l5°C, 1+5.60 ug/m1
PHOSJ?HORUS, AND VARYING AMOUNTS OF GLUCOSE.............
38
12
DIA.GPu\J.\.1 OF PROCEDURE FOR pH RECONSTRUCTION EXPERH1ENT
42
13
DIAGRA.l.\1
11
OI~
APPARATUS. FOR CON'riNUED METABOLISM
EXPERI~4:E.'NT ••
111.
e . . . . . . . . . e ••••• , ....
~
4
,
tJ
,
"
.......
e ••••••• o • •
SU11VIVAL OF E. COLI B WREN EXBJSED TO LIGHT FROM A
SlJl~LfU~1P • o ~ • o ,. • o • • • • • • • e e • • o • • t:~ ~ e • o
o •
o
----
•
&
6
•
6
"
•
•
•
•
•
•
•
•
• o •
46
50
15
SURVIVAI, OF E. COI,I B WHEN EXPOSED TO NA.TtlRAL SUNLIGHT
16
SURVIVAL OF E. COLI B COMPARED TO SURVIVAL OF E. COLI JD
WHEN BOTH ARE EXWSED TO NATURAL SUNLIGHT. • • • • • • • • • • • • • 56
vi
53
ABSTRACT
TirE SURVIVAL OF NON-GROWING BJPULATIONS OF E. coli
A study attempting to define some of the factors that
cause the death of E. coli in the external environment
by
Jeffrey Irwin Daniels
J4aster of Science in Biology
It has t<een reported in the literature that
E~oli,
a baeter:i.um
normally limited to the gut of warm-blooded animals, dies rather
quickl;y under natural conditions in the external environment.
For
this reason, the occurrence of E. coli in surface waters is thought
to indicate recent fecal contamination of the environment.
It is
also generally accepted that !:_3oli is an extremely hardy organism,
and will live for indefinite periods under certain laboratory conditions.
~'his
study has attempted to define some of the factors that
cause the death of the organism in the external environment.
It has been confirmed in this study that populations of E. coli
will survive indefinitely in laboratory cultures as long as toxic
products are removed or neutralized.
Acids which accumulate as a
result of continued metabolism of substrate have been shown to be
the cause of death frequently associated with densely populated
laboratory cultures.
mates the
natu~ally
When population density more closely approxioccurring conditions of contaminated waters,
vij_
it was shown that starvation, nutritional imbalances, or metabolism
in the absence of growth did not severely affect the viability of
the cultures.
Exposu:re of both growing and
non~growing
eu.lttiTes of
E. coli to either artificial or natural sunlight did, however, kill
the populations quickly.
Contrary to our expectations, and to the
generally accepted notions, bubbling of air seemed to impart some
protection against the adverse effects of solar radiation.
These results '\vere interpreted to mean that nutritional or
metabolic processes were not the prima1•y factors involved in the
death of
Ji~.
coli populations in surface waters, but that sunlight
probably plays a major role in the elimination of this organism
from the external emrj_ronment.
viii
INTRODUC'riON
Among Americans today there exists a broad environmental
concern, particularly for preserving the
11
pri~tine
11
quality of
Berry and Horton (1974) proposed that the American
water resources.
public's interest in protecting the environment and maintaining
high standards of water quality \ras promoted by four principle factors:
(1) Many people have died from pathogenic microorganisms
carried by vlater.
Hence, care must be taken in preventing water
from being contaminated.
(2) 'l'he productivity of aquatic environ-
ments and the terrestrial ecosystems associated with them has
decreased as pollution from industry, agriculture, and metropolitan refuse and sewage has increa,sed.
Furthermore, the public
health hazar·d is thereby intensified sjnce effluent can contain
nutrients, such as fertilizer run-off (Clapham, 1973) which promotes
the productivity of pathogenic agents, or effluent can contain the
disease producing microorganisms themselves which were introduced
as feed-lot run-off or sewage out-flow.
(3) The greater affluence
and increased leisure time possessed by Americans has created a
demand for the
11
outdoors" and has produced a public outcry for
unpolluted and undisturbed natural waters.
Ironically, as this
demand for the "outdoors" j_ncreases so does the public health hazard
associated '\'11th the inability of rural recreational sites to properly
assimilate the increasing amounts of human excrements.
(4) Man's
affinity for v.•ater as a result of the human need to satisfy physiological requirements or find aesthetic gratification was being
l
2
~leopardizc~d.
All of these factors emphasized to the public the
importance of preserving the pristine nature of the aquatic environmen·t and its integrally related ecosystem.
Burnet a.nd White ( 1974) use historical records and scientific
evidence to describe how the microorganisms responsible for such
fatal diseases in man as polio, hepatitis, typhoid, and cholera,
can be introduced into ·water ways.
These and. other disease producing
microorganisms proliferate in the gastro-intestinal tract of warmblooded organisms.
Therefore the fecal material of the infected
host -vrill contain these disease producing agents.
Any stream, lake,
or pond where warm-blooded organisms congregate for drinking, bathing, or recreation, or which is susceptible to contamination from
sewage discharge or feed lot run-off can transfer disease producing
microorganisms from host to host.
Efforts to eliminate disease producing agents from water have
been employed since the time of Koch and Pasteur.
The detection
of enteric pathogens in water was ma,de easier and safer with the
description of the bacterium Escherichia coli by the pediatrician
Theodore Escherich in 1885 (Clark and Kabler,
determined. tha.t
!E_s_ch~ic~_ia
1964)~
Escherich
coli was always present in the fecal
discharge of warm-blooded animals because it was a natural inhabita.nt of the intestine.
Since E. coli shared a common niche with
enteric disease producing pathogens, Escherich pointed out that the
presence of E. coli in water was associated with the fecal discharge
of warm-blooded organisms and indicated the likelihood that enteric
3
pat.hogenn also contaminated the watex-.
characteristJes which made it easy to
guishable.
~coli
also possessed
and readily distin-
cu~ture
Fll!·thermore E. coli was found to be innocuous as an
infectious agent under ordinary circumstances.
All of these prop-
erti.es mee,nt that >vater could be analyzed for fecal contamination
and the possible presence of enteric pathogens simply by sampling
for the presence of E.
coli~
Escherichia coli is an aerobic and facultatively anaerobic,
gram negative,
rod~shaped
bacterium which produces gas from lactose
in forty eight ho"i.U'S or less >-Then incubated at 35°C (Clark and
Kabler, 196l.-1).
Categorized as a fecal coliform bacterium because it
possesses the aboV'e characteristics,
~
can be easily distin-
guished from other fecal coliforms by utilizing a variety of physiological and biochemical tests and by growth on selective (EiviD) media
(Dii'co I-1anual, 1974).
Geldreich (1970) reviewed evidence which showed that fish do
not contribute significantly to the fecal coliform contamination
of an aquatic environment.
Clark and Kabler (1964.) inferred from
this same eYidence, reviewed by Geldreich, that fecal coliform
bacteria which represent a public health hazard are primarily· from
warm-blooded animal sources.
According to McFeters and Stuart
(1972) E. coli and fecal coliform bacteria in general are "the
single most important indicators of public health hazard from
infectious agents" in surf'ace waters.
The information presented
by these authors emphasizes the vTarm blooded animal source of
infectious microorganisms and the important role E. coli plays
indicating the presence of fecally dishcarged pathogens.
Fecal contamination can pollute aquatic environments not only
with pathogenic bacteria such as members of the genus
and Vi ~rio
chol~_:r~,
Salmonel~~'
but also vli th viruses, and intestinal parasites.
E. co]_j_ has been established as an indicator of the presence of
these disease producing microorganisn1s only because they are
discharged in the feces of warm-blooded organisms.
According to
information proYided by Geldreich (1970) and 1'1cFeters and Stuart
(1972) the coliforms can be among the first to die of all the
microorganisms present in the aquatic environment.
has been proposed in the literature that
of
.::_~~~;t.
~tlso
I~. ~li
Therefore it
is an indicator
stream pollution. Hm'fever, McFeters and Stuart (1972)
point out recent studies which show that the chemical and
physics,l natu.re of the aqueous environment influence the survival of
E. coli.
E.
col~
Obviously factors vThich improve the survivability of
reduce the reliability of E. coli as an indicator of recent
fecal contamination.
In order to improve the reliability of E. coli as an indicator
of fecal pollution and enteric pathogen contamination it is important
to determine the factors present in water which change the survival
of E. coli.
Experiments present in the literature which deal with
this topic can be separated into two categories:
(1) Those investi-
gat:tng the chemical constituents of the aquatic environment, and
(2) those assessing the role of physical factors in the aquatic
environment.
5
Hanes and Fragala (1967) sho111ed that decreased survival of
E. coli is directly proportional to increasing concentrations of
seawate~
in the grm1th medium.
These authors also discovered that
33% to 67% seawater concentrations can prevent growth of E. coli
completely.
E. coli survival is also known to be affected by nutrient
limitation.
Clapham. (1973) explains that nutrient levels are
customarily lmv in aquatic environments.
When concentrations of
these limiting nutrients are increased the survival of aquatic
organisms is signific:antly affected.
that,
11
Clapham (1973) concludes
as a general rule, ho-vrever, it seems reasonably certain that
the element that is most consistently suboptimal in aquatic ecosystems, and thus is the most likely candidate for a limiting factor,
is phosphorus, followed by ni tr·ogen, sulfur, and calcium.
Of the
four, phosphorus and nitrogen are used in the greatest quantity ••• 11
Data from the experiments of Slezak, and Sikyta (1967) suggest
that phosphorus limitation leads to the reduction of growth rate
but does not necessarily affect the survival of E. coli.
In their
experiments the survival of E. coli was not determined over an
extended period of time for the condition where only phosphorus was
completely absent from the medium.
Therefore, the possibility
remains that long term phosphorus starvation could affect the su:rvival of E. coli.
Gerba and McLeod (1976) examined the effect of sediments on
the survival of E. coli in marine waters.
They observed that
E. coli "always persisted much longer when sediment was present."
6
Gerba and McLeod (1976) state that this increased persistence could
be due to the fact that organisms can more readily utilize the
nutrients present on sediments to which the organisms attach
selves.
availablE~
them~
111entioned also is the possibility that the nutrients
on the sediments afford E. coli a competitive advantage
over other organisms due to the proximity of the nutrients thereby
One other explanation offered by
increasing E. eoli survival.
these au.thors is that the sediment interferes
elements which allows
~.::_coli
''~'i th
antagonistic
to survive longer.
Looking at the survival of
E._~
in membrane·-filter chambers
pla.ced in two different creeks McFeters and s·tuart (1972) observed
that the survival of E. coli was different in the two creeks.
These
investigators then conducted laboratory experiments to elucidate
the reasons for the differential survival of E. coli.
The labora-
tory studies showed that the optim'l1ffi pH for E. coli survival was
between 5.5 and 7.5.
A decrease in viable cells was· demonstrated
to be inversely proporttonal to temperatures between 5°C and 15°C,
with temperatures above.l5°C being substantially less influentia.l.
The ionic composition of water was also found to influence survival
of E. coli.
vfuen the creek conditions were reproduced in the labcra-
tory using actual creek waters the resulting differential survivals
seen in situ did not occur.
McFeters and Stuart concluded that the
flow rate across the membrane-filter chamber was presumably responsible for the difference in survival of E. coli in the two creeks.
This theory was not tested by these investigators because the
different flow rates could not be obtained in the laboratory due
to physical limitations of their equipment.
Light
the sun.
!=._~
if:{
an integral part of every environment exposed to
Hollaender (1943) determined that the survival of an
population could be reduced by focusing an artifictal light
possessing long ultraviolet, and short visible light, on the vessel
containing the bacterial population.
Harrison (1967) comprehensively
reviewed other studies demonstrating the reduced su.rvival of E. coli
due to the effects of light.. Similar results were observed by
other j_nvestigators ( C'nichester and MaXivell, 1969; 1-Tebb and ll.talina,
1967; Kubitschek, 1967; Futter and Richardson, 1967; Muller and
Schicht, 1965) examining how E. coli survival changed when exposed
to wavelengths of light from artificial sources.
This thesis examines parameters that can affect the long
term survival of E. coli under ecological conditions.
The
results fi•om the experiments discussed in this thesis verify the
relationships reported by other experimenters and illustrate the
importance of each factor in determining the survival of E. coli
under natural conditions in fresh water resources.
Parameters
measured include phosphorus and carbon limitation, pH and temperature changes, continued metabolism in -the absence of growth, and
exposure to artificial and natural sunlight.
MATERIALS AND METHODS
ORGANISMS:
E. coli B, obtained from a stock culture maintained by the
Biology Department at California State University, Northridge,
was one of two strains employed in this study.
~,!-_coli
:IQ, iso-
lat8d f'rom the gut of thj_s investigator, "Vras the other strain
that 'Vras used.
'l'he purpose of utilizing the freshly isolated
E. coli JD vias to prove that the effect of sunlight on bacterial
survi va.:'. was not restricted to the laboratory strain.
The .identity of the cultures were frequently confirmed by
standard procedures of coliform analysis including assay for gas
production from lactose, green sheen on EMB (Difco Manual,
1971~),
and characteristic reactions with the IW!iC tests (Clark and
Kabler,
196~.).
MEDIA:
Davis Minimal:
Davis Minimal medium without glucose (Difco)
contains the minimum array of nutrients which can support E. coli
growth.
The ingredients per liter are:
7 grams dipotassium phos-
phate, 2 gl'nms rnonopotassium, 0. 5 grams sodium citrate USP, 0.1
gram
magm~s:tum
sulfate, 1.0 gram ammonium sulfate.
medium contains no organic compounds.
Davis Minimal
The chemical composition of
this medium made it suitable for use as the cultivation medium
for supplying a population of E. coli cells for experimental
purposes.
8
9
Trls;
MgSOl~
Tris medium consists of 0.1% (NH4)2S04 and O.OJ.%
and is buffered with 0.1 ,!:i Tris ((Hydroxymethyl) Amino
Methane) adJusted to a pH between 7. 2 and 7. 5 "t>Ti th concentrated
HCL
The basic purpose of this medium is to provide a phosphorus-
free minimal base as a substitute for Davis Minimal medium.
Tris
medium is used in tv10 circumstances.· (1) To permit E. coli cells
transferred fJ:'()m Davis Minimal medium to grow to a sufficient
concentration for experimental purposes while using up the excess
nutrients introduced into the Tris medium wi-th the bacterial
i.noculum; and (2) to provide
phosphorus~free
medium that possesses
a buffering capability for maintaining optimal pH conditions
(McFeters and Stuart, 1972) for determining the effect of measured
amounts of phosphorus and/or glucose on E. coli survival and growth.
No~-~E_owtl::
MgS04 only.
lbis medium contains 0.02$ (Nfi4)2S04 and 0.005%
Non-grovTth medium allmrs E. coli to remain viable for
an indefinite period of time without supporting growth.
Therefore
it is suitable as a suspending medium for examining the effect of.
sunlight on non-growing cultures of E. coli, a
situation~~
cells experience in nature when nutrients are absent.
TSY/2_:
TS (tryptic soy) broth (Difco) was diluted 1:5 with
distilled water and supplemented with 0.1% yeast extract.
For
plates themedium was supplemented with 1.5% Difco-Bacto Agar.
This medium was utilized for determining the viability of E. coli
cells in conjunction w:t th the pour pla,te technique.
EMB:
Levine EMB (Difco).
'l1his is a selective and differential
10
medium for E. coli.
Since E. coJ2:_ can easily be distinguished from
other organisn1s by a characteristic green sheen when grown on this
medium, it '\'las employed to confirm the presence of E. coli.
All media were dissolved in distilled water and sterilized
by autoclaving.
When indicated, media were supplemented with glucose separately sterilized by autoclaving (4 to 8 n~/ml unless otherwise
specified) or \vith phosphorus (added as KH2P04) and sterilized
with the medium.
ANALYTICAL ME1RODS:
All glassware utilized for experimental purposes or media
preparation, or for an analytical assay, was always washed in
2.5
,!:!
HCl and rinsed thoroughly with distilled water.
This pro-
cedure removed any residues of phosphorus accumulating on the
glasmvare (ASTM, 1975).
Glucose Determination:
described in
~1anometric
1'he Ferricyanide Reduction Method
Biochemical Techniques (Umbrei t, Burris,
and Stauffer, 1972) was employed.
Results were read at 690 nm
on a Beckman Model 24 Spectrophotometer using quartz cuvettes.
Determination of pH:
Approximate pH values of cultures were
determined with pH paper, or in some experiments, by including a
pH indicator in the growth medium.
Bromcresol green indicator
was employed at a concentration of 0.0268 mg/ml to indicate pH in
the range of
4 to 5; phenol red was used at a concentration of
0.018 mg/ml to indicate pH in the range of 7 to 8.
The incorpora-
tion of these indicators in the experimental culture medium did not
11
affect the grO\>lth or the survival of' the cultures.
Exact pH determinations were made with a Beckman model 76 pH
meter • . In the range of a particular indicator, the color was
standardized to agree with the pH value determined by the pH meter.
Determination of Viability:
Viability was determined as the
ability of E. coli cells to form colonies on agar plates.
The
pour plate method, or occasionally the spread plate method was
used.
In both cases, serial dilutions were made in sterile distilled
water and measured samples of each dilution transferred to the
agar growth medium.
The pour plate technique involves placing
one milliliter of culture, removed from a serial dilution bottle,
on the bottom of a sterile plastic petri dish.
Melted TSY/5 agar
is then poured on top of the culture at a temperature of approximately 40°C.
The suspension of bacteria is then gently swirled so
that the bacteria will be uniformly distributed in the medium.
After allowing the medium to solidify the petri dish is turned
upside down and incubated at 37°C for
24
to
48
hours during which
time each organism will have developed into a visible colony.
Duplicate plating was generally perfonaed unless otherwise noted.
Viable organisms were counted on EMB plates were indicated, using
the spread plate technique.
The spread plate technique involves
depositing 0.1 milliliter of culture removed from a serial dilution
bottle in the center of the EMB agar.
An "L" shaped, sterilized,
glass rod is then used to spread the bacteria uniformly over the
surface of the ID4B gel.
Colonies which develop from E. coli cells
12
during 24 to 48 hours of incubation at 37oc are characterized by a
green sheen.
Growth Measurements:
The cell population density was measured
turbidimetrically for the purpose of ascertaining the phases of
cell population growth.
arm culture flasks.
Cells were grmm in 125 ml or 250 ml side
The side arm was a 16 mxn test tube capable of
being used as a spectrophotometric cuvette in the Bausch and Lomb
Spec 20 Spectrophotometer.
Turbidimetric measurements were made at
600 nm.
Piff.SICAL FACTORS:
Radiation Source:
Natural sunlight or a Sylvania Model RllO
275 watt (110 to 125 volt) sunlamp were used to examine E. coli
survival v1hen exposed to- artificial or natural solar energy.
Air Souxce:
The laborat-ory air supply was used as the source
of oxygen for experiments examining the effect of artificial sunlight on the survival of E. coli with bubbling oxygen present.
A
common aquariUJn air pump provided the oxygen for bubbling air in
E. coli culttiTes on the roof of the CSUN Science Building.
In
addition, air from either source was humidified to prevent evaporation of the culture, and passed through sterile cotton fibers
before reaching the cultures.
CULTURE PREPARATION:
The standard procedure for preparing cell cultures for experimental utilization was to grow overnight cultures in Davis Minimal
medium supplemented with
~·
to 8 mg/ml glucose.
This ten to fourteen
hour period permitted the cells to reach late log phase/early
13
stationary phase growth.
on a rotary shaker.
Cells were grown at 37°C in culture flasks
After the initial growth period, cells were
transferred to Tris medium.
Cells harvested at this time resume
growth with little delay when transferred to another growth medium,
thus lengthy lag periods did not have to be anticipated and a uniform response time could be expected.
Volume of culture mediu."ll
never exceeded 2o% of the culture flask volume in any circmnstances
in order to ensure adequate aeration.
Turbidimetric measurement
of cell density determined approximate time for transfer from Tris
medimn to the experimental flasks containing measured amounts of
nutrients.
'l'he amount of culture transferred never exceeded 0.2
ml on any occasion.
Growth in the Tris medium allowed complete
assimilation of any residual phosphorus present in the inoculum.
Plate counts determined the maximum population density of E. coli B
in the Tris medium culture under these conditions to be between
1.0 to 2.0 x 109 cells/ml.
RESULTS
~osphorus
and carbon are two essential nutrients contributing
to the productivity of aquatic environments.
Growth limiting
concentrations of these nutrients were determined for E. coli B
by growing cultures at 37°C on a rotary shaker in Tris medium
(unless
otherwise indicated) supplemented vJ:lth different amounts
of either glucose (at excessive phosphorus concentrations) or
phosphor11s (at excessive glucose concentrations).
The viable count
assay was used to measure the extent of growth in each case.
Figure 1 shows the concentrations of glucose and phosphorus
that are:
(1) severly growth limiting; (2) non-limiting, a term
used in this thesis to describe the concentration that allows the
maximum population size to be reached without any free
nut~ient
remaining, and (3) excessive, such that free nutrient remained
after maximum population size was reached.
Glucose is found to
be limiting at concentrations below 1.6 mg/ml, and phosphorus is
limiting at concentrations lower than 8.55 ug/ml, with growth of
E. coli B proportional to lesser concentrations of these nutrients.
This information was experimentally utilized to ascertain
the influence of phosphorus and glucose nutrient limitation and
deprivation on the long term survival of E. coli B populations.
Survival of E. coli B after growth in Tris medium containing a
severly limiting (0.2 mg/ml glucose;
1.14 ugfml phosphorus),
non-limiting (1.6 mg/ml glucose; 8.55 ug/ml phosphorus), or
15
excessive quantity (8.5 mgjml glucose; 45.6 ug/ml phosphorus) of
either glucose or phosphorus or both was measured using the viable
count assay.
shakers.
All growth flasks were incubated at 37°C on rotary
Portions of this experiment were repeated at 15°C to
investigate temperature effects.
The results of those experiments which examined the effects
of nutrient limitation and deprivation on the survival of E.
~
for an extended period of time are presented in Figures
2 through ll, and SUmmarized in Tables 1 through 3.
The data
indicate that when populations of E. coli B are depriv·ed of glucose
or phosphorus there will be an initial decrease in viable cells
averaging 1 to 3 decades of killing follmved by population stabilization which is characterized by no further evidence of cell
death for periods as long as 20 to 25 days which was when the
experiment was terminated.
A notable exception to this generali-
zation is evident when glucose is present in an excessive amount.
Under such conditions cell death is continuous and rapid until
no survivors are detectable {Figures 6 through 11).
These
results were determined to be unaffected by temperature since
0
0
the same reslts occur at both 15 C and 37 C.
It was also observed
that in those experiments determining the effect of temperature on
survival .under nutrient imbalance conditions E. coli populations
reached their maximum size less quickly at 15°C than at 37°C.
Furthermore Figures 8 through 11 indicate that die-off is slower
at 15°C than at 37°C when the culture contains excess glucose.
16
FIGURE 1:
Extent of Growth of E. coli B populations exposed
to different amounts of phosphorus (at excess glucose concentrations) and glucose (at excess phosphorus concentr&,tions). Initial E. coli B. population -vras 0.50 to 1.00 x 107 cells71nL Davis minimal medium 'ViaS used for glucose concentrations of
0. 05, 0. 20, 0. 30 mg/ml. Tris medium supplemented
with 45.60 ug/ml of phosphorus w·as used for glucose concentrations of 0.00, 0.20, 1.60, and 8.50
mg/ml. Tris medium containing either 8.0 or 8.50
mg/ml glucose was used to determine the extent of
growth at phosphorus concentrations of 0.00, 1.11{.,
2.85; 5.70, 8.55, and 45.60 ug/ml.
PHOSPHORUS:
GLUCOSE:
17
PROS PHORUS
2
(ug/ ml)
4
2.0
1.5
,_
&.
I
0
r-1
X
0.5
.._...;,_.......,.,. .,r-·. . . . . . .'". .--.'¥"'<13\11•......- ..-..a--~,---.-.~(f--;-1h-q
0.5
1.0
1.5
GLUCOSE (mg/ml)
8.5
9.0
wo-o
18
.FIGURE 2:
Relative survival of E. coli B at 37°C.
Tris medium was supplemented with vary~
·ing amounts of phosphorus and no glucose.
0.00 ug/ml phosphorus:
Cll-e-e
1.11+ ug/ml phosphorus:
o-o-o
8.55 ug/ml phosphorus:
i:.-l;g-f¥.
45.60 ug/ml phosphorus:
A-A-b.
19
f
~~
-2
..::..-10
H
~
>
~
!§
00
~
- 3
10~
E---1
<I'
.::t
iil
P'<
-
10
10
-
4
-6
,-
5
10
15
DAYS
20
25
30
'
20
FIGURE: 3:
Relative survival of E. coli B at 37°C.
Tris medium was supplemented with varying amounts of phosphorus and 0.20 mg/ml
glucose.
e-G
0. 00 ug/ml phosphorus:
@-
1.14. ug/ml phosphorus:
o- 0
8.55 ug/ml phosphorus:
A- A-A
45.60 ug/ml phosphorus:
A -A-A
-
0
21
·-----
•
-2
10°
10- 1 .
,--...
1!,$!.
..,_,
~
10-
2
~
~
t:f.}
~ 10- 3
~
b
lli
10- 4
. 10
-6
5
10
15
20
DAYS
25
30
22
FIGURE 4:
Relative survival of E. coli B at 37°C.
Tris medium was supplemented with varying amounts of phosphorus and 1.60 mg/ml
glucose.
0.00 ug/ml phosphorus:
e-o-~
1.14 ug/ml phosphorus:
o- o - o
8.55 ug/ml phosphorus:
£-A-A
45.60 ug/ml phosphorus:
A-A-A
. 23
0~0
0
10
10-1
10-4'
5
10
15
20
DAYS
25
30
24
FIGURE 5:
Relative survival of E. coli B at 37°C.
Tris medium was supplemented with varying amounts of glucose and no phosphorus.
0.00 mg/ml glucose:
<9-0-G
0.20 mg/ml glucose:
o-o-o.
1.60 mg/ml glucose:
A-A-A
8. 50 IDgjml glucose:
A-A-A
25
5
t
10
15
20
DAYS
25
30
26
FIGURE 6:
Relative survival of E. coli B at 37°C.
Tris medium was supplemented with varying amounts of glucose and 1.14 ug/ml
phosphorus.
0.00 mg/ml glucose:
e-e-e
0.20 mg/ml glucose:
o- o- 0
1.60 mg/ml glucose: A -A-A
8.50 mg/ml glucose: L!.-A-11
27
----o
10-7
5
10
15
20
DAYS
25
30
c
28
FIGURE 7:
Relative Survival of E. coli B at 37°C.
Tris medium was supplemented with varying amounts of glucose and 8.55 ug/ml
phosphorus.
0.00 mg/ml glucose:. ~--8-8
0.20 mg/ml glucose: 0 - 0 - o
1.60 mg/ml gluco~e: A -A-A
8.50 mg/ml glucose: A-A-A
20
/
...-...
"\,..Q..
"--'
H
~
I>
lo- 2 ·:
,_
~
ro
~
H
E-1
1o- 3
<li
1-i
IZ-1
p:;
1o- 4
-~
5
~
10
15
20
DAYS
25
30
30
TABLE 1:
Summary of experimental parameters used in examini.ng
the long term survival of E. coli B when exposed to
nutritional imbalances, at 37°C.
GLUCOSE
(mg/ml)
0.00
0.20
1.60
8.50
PHOSPHORUS
(ug/ml)
MAX. roP.
o.oo
(cfu/ml)
9.6 X 106
1.14
5.8
X
8.55
5.4
X
45.60
5.3
X
0.00
7.2
X
1.14
1.7
X
8.55
2.8
X
45.60
1.4
X
o.oo
9.0
X
1.14
2.5
8.55
6
10
6
10
6
10
6
10
108
8
10
8
10
FINAL :OOP.
(cfu/ml)
REL. SURV.
(%)
FINAL pH
3.2
7.5
X
105
105
4.3
7.5
2.4
X
105
4.lt
7.5
3.0
X
105
5.7
7.5
6.6
X
9.0
7.5
1.8
X
1.0
7.5
2.5
X
0.9
7.5
1.9
X
105
6
10
6
10
103
0.001
7.5
103
0.05
7.5
5.0
7.5
0.2
7.0
3.1
X
2.5
5.1
X
X
6
10
108
1.3
X
2.0
X
109
4.2
X
107
106
45.60
1.9
X
109
7.1
X
6
10
0~4
7.0
o.oo
9.5
X
3.6
X
103
0.04
7.3
1.14.
2.4
X
6
10
108
8.55
2.0
X
45.60
2.1
X
<1/ml
4.5
109
<1/ml
4.5
109
<1/ml
4.5
31
FIGURE
8:
Relative surv:i.val of E. coli B at 37°C.
Tris medium was supplemented with varyj_ng amounts of glucose and 8.55 ugfml
phosphorus. An identical culture was
simultaneously incubated at 15°C (Pigure
10) so that those results could be compared \'lith these.
0.00 mg/ml glucose:
(1}-~-e
0.20 mg/ml glucose:
0
-c-o
1.60 mg/ml glucose:
A -A-A
8.50 mgfm1 glucose:
A -.A-A
32
0
10
10
-4
A
JJ
5
·10
20
15
DAYS
25
30
33
. FIGURE 9:
Relative survival of E. coli B at 37°C.
Tris medium was supplemented with varyj_ng amounts of glucose and 45.60 ug/ml
phosphorus. An identical culture was
simultaneously incubated at 15°C (Figure
11) so that those results could be compared with these.
0.00 mg/ml glucose:
e. --:..e - e
0.20 mg/ml glucose:
0 .:__ o-o
1.60 mg/friJ. glucose:
4- A-A.
8.50 mg/ml glucose: 6.-A-~
o~~e-:-=- •
•----
~
o-o~ -o
.
-o .
•
0
10 -
-1
10 -·
,......
A
~
..........
~
-?...
~
10
H
>
~
,..:>
Cll
~
H
E-1
10- 3
;:S
rx:l
~
1o- 4
10- 7
A
0
5
10
J
15
20
DAYS
25
30
35
FIGURE
10~
Relative survival of E. coli B at 15°C.
Tris medium was supplemented with varying amounts of glucose and 8.55 ugjml
phosphorus. An identical culture was
simultaneously incubated at 37°C (Figure 8) for comparison purposes.
0.00 mg/ml glucose:
G-e-e
0.20 mg/ml glucose:
o-o-o
1.60 mg/m1 glucose:
A --A-lA
A- A-A
8.50 rng/ml glucose:
36
lol
0
10
I
15
20
DAYS
I
25
I
30
3?
FIGURE 11:
0
Relative survival of E. coli B at 15 C.
Tris medium was supplemented with varying amounts of glucose and 1t.5. 60 ug/ml
phosphorus. An identical culture was
simultaneously incubated at 37°C (Figure 9) for comparison purposes.
0.00 mg/ml glucose:
0.20 mg/ml glucose:
1.60 mg/ml glucose:
e -t) -G
o-o-o
A. -JJ. - A
8.50 mgfml glucose: A-f:l-~
10-!
,......_
"'...........
<f?..
..:I
~
:>
lo- 2
t;!
~
p
r.lJ.
~ lo-3
~
;$
~
10M4
5
l
10
15
20
DAYS
25
30
39
7
¢
TABU~
2:
Summary of experimental parameters used in examining
the long term surv·ival of E. coli B when exposed to
nutritional imbalances, at;r7°C. (Compare to 15°C)
GLUCOSE
PHOSPHORUS
MAX. POP.
FINAL FOP.
REL. SURV.
(mg/ml)
(ug/ml)
(cfu/ml)
(%)
o.oo
8.55
(cfu/ml)
6
2.6 X 10
108
FINAL pH
6.9
7.4
7.2
X
6
10
4.0
7.4
4.0
X
107
5.2
7.0
0.20
1.8
X
1.60
7.7
X
8.50
8.3
X
108
8
10
2.5
X
6
10
2.9
X
105
11.6
7.4
0.20
1.4.
X
108
1.1
X
107
7.9
7 .~.
1.60
8.6
X
3.5
X
6
10
0.4
7.0
8.50
9.0
X
108
8
10
o.oo
},~5.60
~1/ml
4.5
.( 1/ml
4.5
TABLE 3:
STh~ary of experimental parameters used in examining
the long term survival of E. coli B when exposed to
nutritional imbalan~es, at 15°C. (Compare to 37°C)
GLUCOSE
PHOSPHORUS
MAX. POP.
FINAL POP.
(ug/ml)
(cfu/ml)
6
3.1 X 10
_(cfu/ml)
(mg/ml)
o.oo
8.55
2.9
X
8
10
8.50
0.20
3.0 X
6
10
8
10
1.60
1.2
109
8.50
9.0 X 10
o.oo
45.60
3.1
X
X
8
FINAL pH
6
1.6 X 10
51.6
7.4
3.0 X 107
10.3
7.4
84.6
7.0
0.6
4.5
1.1 X 109
6
6.8 X 10
1.60
REL. SURV.
(~)
1.7
X
6.0
1.3
6
54.8
7.4
X
10
7
10
20.0
7.4
X
109
108.0
7.0
<1/ml
4.5
.
40
These experiments provide evidence that the absence of phosphorus
or glucose does not adversely affect the survival of E. coli B.
Routine pH measurements performed using pH paper revealed
that a marked decrease in E. coli B population Stlrvival is always
associated with a precipitous drop in pH.
The drastic pH change
from an optimal value of 7.2 to 7.5 (McFeters and Stuart, 1972)
to
4.5 manifests itself only in those cultures containing excess
amounts of glucose ( •rables 1 through 3).
A pH reconstruction experiment was conceived to examine the
effect of pH on E. coli· B survival.
grown in two flasks:
Cultures of E. coli B were
Jt'1ask A contained Tris medium supplemented
with a non-limiting concentration of phosphorus (8.55 ug/ml), a
non-limiting amount of glucose (1.60 mgfml), and bromcresol green
indicator (0.0268 mg/ml); Flask B contained a non-limiting concentration of phosphorus, an excessive amount of glucose (8.50 mg/ml),
and phenol red indicator (0.018 mgfml).
When the pH began to drop
(flask B) or cells began to die (flask A).the cultures were divided
into subcultures and additions were made to produce deliberate
pH changes.
Survival was then measured in all flasks.
When the initial cell death was noted in flask A the contents
were divided into four equal subcultures and labelled numbers: 1,
2,
3, and 4. When the pH drop occt'lrred in flask B as observed by
a noticeable change in color of the indicator, the contents of
that flask were divided into four equal subcultures and labelled
numbers:
5, 6, 7, and 8. Additions were made to these eight
(
41
as
~lasks
~ollows:
Flasks number 1 and 5 received no additions.
Flask number 2 received citric acid to reduce the pH to
4.5.
Flask number 3 received hydrochloric acid, '\oThich reduced the pH
to 4.0o
Flask number
4 received sodium citrate which was added
in an amount which made the concentration
that in flask number 2.
Flask nunber
o~
citrate
e~ual
to
4 provided the control for
determining if citrate was responsible for any toxic effects.
Flask number 6 received sodium hydroxide to restore the pH to
7.5.
Flask number 7 also had its pH restored to 7.5 by an addi-
tion of NaOH.
Flask number 7 differed from flask nmnber 6 because
the NaOH was not added until 5 days after the pH change was noted
in flask B instead of immediately after as was the case for flask
number 6.
This delay in adding NaOH permitted detection of
whether or not cell death could be arrested once initiated at
the low pH of 4.5.
Flask number 8 received sodium chloride so
that possible inhibiting or beneficial effects from the salt
produced in flasks 3, 6, and 7 could be detected.
The pH changes
in each flask were recognized by the changes in color of the pH
indicators, and the final pH was determined using a pH meter.
The artificial pH changes induced in the subcultures were maintained throughout the course of the experiment which lasted 30
days.
Table
Figure 12 is a diagram
o~
the procedure for this experiment.
4 summarizes the results which were obtained.
The data collected in this experiment reveal that ~ions and
not the salts are responsible for the reduction of E. coli cell
FIGURE 12:
Diagram of' procedure for conducting pH reconstruction
experiment. When initial cell death occurred in Flask
A the contents were divided into four equal subcultu~es:
#1, #2, #3, #4. When a pH change was observed in Flask
B the contents were divided into four equal subcultures:
#5, #6, lh, #8. Flasks #1, and #5 receiv·ed no additions.
Flask #2 received an amount of 10 M citric acid capable
of' changing the pH to 4.5. Flask i3 received an amount
of 12 M hydrochloric acid capable of' reducing the pH to
4.0. Flask #4 received an amount of 2 M sodium citrate
which made the concentration of sodium citrate in Flask
#l~ equal to the concentration of citric acid in Flask
#2. Flask #6 received an amount of 10 M sodium hydroxide capable of returning the pH to a value near 7.4,
and subsequent additions of sodium hydroxide were made
to maintain the 7.4 pH level. Flask #7 received an
amount of 10 M sodium hydroxide capable of' returning
the pH to a value near 7.4 but this addition was not
made until 5 days after the pH change was observed
in Flask B. Flask #8 received an amount of 5 M sodium
chloride which made the concentration of' sodimn chloride in Flask #8 equal to the concentration of sodiu~
hydroxide ih Flask #6. This addition of sodium chlorlae to Flask #8 was repeated whenever sodium hydroxide
was added to Flask.#6 •
.-
i ii
iii
iii
iii
iii
iii
iii
iii
;;;;;
A
iiiii
i ii
;;;;;
iiiii
!!!!
-
1.6 mg/ml GLUCOSE
+
!!!!
8. 55 Uf6./ml PHOSPHORUS
i
+
:
0. 0268 mg/ml Bromcresol Grn. ~
8.5 mgfml GLUCOSE
+
8.55 ugjml PHOSPHORUS
+
0.018 mgfml Phenol Red
iiiii
~~~~~~~~~
-HCi
HCl
NaCi
NaOH
*NaOH*
NaCl
TABLE 4:
FLASK
NO.
1
2
Summary of results of pH reconstruction experiment~ *NaOH*: NaOH added after 5 days elapsed.
pHMP: Population maximum pH measurement. pHa.~: After addition pH. PHasA: Aft~r. Subsequent
add1.t1.on pH.
GLUCOSE MAX. POP.
ADDITIONS pHaA SUBSEQUENT pH~s~ FINAL POP.
REL.
FINAL
(mg/ml)
(cf'u/ml)
ADDITIONS
__, •
(cfu/ml)
SURV.
pH
(days after
(%)
1st add 1 n.)
6
1.2 X 109 7.4
1.60
5.0 X 10
0.4
7.2
7.4
-----------------3.4 X 103 0.0003 4.8
HCi
1.60
1.2 X 109 7.4
4.5
PHMP
-----
-----------------·----------------------------------
1.2 X 109
9
1.2 X 10
7.4
HC1
4.0
7.4
NaCi
7.4
4.5
----·
4.5
------------------
4.5
NaOH
7.4
1 thru 5
7.4
-<.1/ml
7
1.1 X 10
2.5
-3
1.60
4
1.60
5
8.50
6
8.50
1.0 X 109
1.0 X 109
7
8.50
1.0
X
109
4.5
*Na.OH*
7.4
1 thru 3
7.4
8
8.50
1.0
X
109
4.5
NaCl
4.5
1 thru 5
4.5
.( 1/ml
2.1 X 106
X
6
10
.( 1/ml
3.1
0.18
7.4
----
4.5
1.0
8.3
0.2
8.1
----
4.5
+="
l;J
44
population density.
Furthermore, only w·hen excess glucose is
present can the cells manufacture enough
~ions
to overcome the
Tris buffer and change the pH to a suboptimal value.
E. coli
cell die-off initiated at low pH can be stopped if the ~ions
are neutralized.
Death occurs in flasks 2, 3, 5, and 8 in which HCi, HCl,
or metabolism is responsible for reducing the pH to
4.5. Death
did not occur in flasks 1 and 4 which contained non-limiting
amounts of' glucose and no addition or the addition of NaCi.
Die·-off was e.lso not recorded in f'lasks 6 and 7 which received
NaOH to restore the pH to
7.4 but also contained glucose in an
excess amount.
An experiment was developed which investigated the possibility that glucose was directly responsible for the decrease in
survivorship noted when glucose was present in an excess ruaour1t.
It
~~s
considered possible that glucose metabolism that continued
in the absence of phosphorus would cause the essential metabolic
machinery for survival and reproduction of E. coli to wear out.
The experiment involved suspending E. coli B in Non-growth medium
containing approximately 0.5 mg/ml glucose within a dialysis bag.
The bag was then suspended in a chamber filled with the same
medium and this chamber was continuously flushed with fresh medium
flmving at a rate of 50 to 80 milliliters per minute from a 25
liter reservoir.
The continuous flow also removed any ~ions
accumulating during the continued metabolism of glucose.
A
sketch of the apparatus is shown in Figure 13.
Table 5 presents the evidence that establishes the fact
that cell population decline was absent even though the glucose
assay revealed that E. coli B was continuously exposed to an
excess·quantity of glucose.
1be reservoir was shown to contain
a glucose concentration of 0.47 mg/ml while the effluent still
possesseO. 0.41 mg/ml of glucose.
The results of this experiment
indicate that continued metabolism of glucose in the absence of
growth does not lead to the death of E. coli B.
In batch cultures it is obvious that pH changes may represent a major factor involved in the decline of population density.
These pH changes are characteristic of high density, closed
culture systems where waste products can accumulate.
Since sunlight is an integral part of any natural aqueous
environment an investigation was conducted to determine the effect
of solar emissions and those parameters intimately associated
with them on the survival of E. coli populations.
The general procedure for this investigation was to suspend
a low density population of E. coli (approximately 1 x 107 cells/
ml) in 500 ml capacity specimen jars containing 200 ml of Nongrowth
m~di·am.
The specimen jar containing the eulttiTe was then
placed in a water bath capable of maintaining the temperature of
the culture at 15°C.
The culture was then exposed to either
light emitted from an artificial source or light emitted from
the sun.
FIGURE 13:
Diagram of apparatus which examined the effect of the continued metabolism
of glucose in the absence of gro~~b on the survival of E. coli B. A- Effluent;
B- :ft~. coli Sampling Port; C- Dialysis Bag containing E. coli; D- Influent;
E- Purr~; F- Chamber; G- 25 Liter Reservoir.
-B
D
G
~
0'\
47
TABLE 5:
Survi.val of E.. coli B and glucose utilization
during continued metabolism in the absence of
growth.
TIME (days)
POPULATION DENSITY (cfu/ml)
GLUCOSE (mg/ml)
RESERVOIR EFFLU~~T
0
13
0.47
0 .. 41
48
Three separate experimental protocols were employed to
identify the exact circumstances when solar energy might influence the survival of E. coli.
The particulars of those three
protocols are discussed below.
The first experiment was designed to verify the existence
of solar emissions as a factor affecting the survival of E. coli.
Fonr cultures were used:
!
populations.
Numbers 1 and 4 contained only E. coli
Numbers 2 and 3 contained E. coli B populations
ana were also sparged \vith oxygen.
Numbers 3 and 4 were wrapped
in tin foil to prevent the penetration of any light.
All four
cultures \·rere then placed in the water bath capable of maintaining
a 15°C temperature.
A Sylvania sunlamp was positioned 40 em
above these four cultures and turned on for
Fib~re
12 consecutive hours.
14 displays the results obtained from this experiment.
The evidence contained in the results verify that solar
errdssions produced by a sunlamp can adversely effect the survival of E. coli B.
E. coli B cell die-off occurred only in
cultures number 1 and 2 which were the only ones exposed to
the artificial sunlight.
Comparing the die-off of E. coli B
in cultures 1 and 2 (Figure 14) indicates that bubbling of
oxygen in the culture may provide E. coli cells some protection
against the harmful effects of artificial sunlight.
A second protocol was developed to determine if natural
sunlight would produce the same results obtained when the
sunlamp was used.
An identical experiment to the one just
FIGURE ll~:
Survival of E. coli B populations at 15°C when
exposed to light from a sunlamp positioned 40 em
above the culture. rnitial concentration of E.
coli B was 9. 2 x 106 cells/ml.
.
Exposure to light emitted from sunlamp:
Exposure to light emitted from sunlamp
and introduction of bubbling oxygen into
the culture from laboratory air source:
Light from sunlamp prevented from shining
on culture due to tin foil covering but
bvbbling oxygen was introduced into this
culture from laboratory air source:
No exposure to light from sunlamp, and
no introduction of bubbling oxygen:
A ~A-IJ.
,A -JA -A
o-o-o
8 -Ct-8
50
7
10 Lv'l.
~ ~-
,.O-.
0
®
-~~·~0
e=-o
'-CD
---
Q
=--~-----------
o0
105
6-------2
4
6
8
HOURS
A
I
10
12
51
described was conducted on the roof of the Science Building at
California State University, Northridge.
Ideal weather
condi~
tions prevailed although smog was noticeably present in the sky.
The experiment began at
l-1-
pm and lasted for 45 consecutive hours.
The cultures were monitored at periodic intervals using the viable
counting procedure and EMB plates to verify the results.
Figure
15 shows the results obtained from this experiment.
The results shm·l that in the absence of sunlight E. coli B
is capable of continued survival.
When E. coli B populations
are exposed to solar emissions a dramatic population decrease
results.
The bubbling of oxygen in a culture containing an
E. coli B population once again seems to offer protection against
the severest effects of solar radiation.
responsible for E.
~l~
Thus solar energy is
cell die-off.
The third experimental protocol was employed to confirm
that the harmful effect of sunlight could be generalized to
wild-type E. coli, and to determine if
40
mg/ml of glucose would
provide protection against the harmful effects of solar radiation.
Six
culttL~es
were used in this experiment.
contained only E. coli B.
coli JD.
Numbers 1 and 5
Numbers 2 and 6 contained only E.
Number 3 contained E. coli B and the Non-growth medium
was supplemented with
40
mg/ml glucose.
Number
4
contained E.
coli J~ and the Non-growth medium was supplemented with
glucose.
40
mgfml
Cultures number 5 and 6 were wrapped in tin foil to
prevent the penetration of any light.
All six cultures were
52
FIGURE 15:
Survival of E. coli B at l5°C when exposed to
natural sunlight. Initial population of E. coli B
was 1.0 x 107. cells/ml.
Exposure to natural sunlight:
f:l-A-A
Exposure to natural sunlight and
the introduction of bubbling oxygen
into the cultm·e:
A -A -A
Exposure to sunlight prevented by
tin foil wrapping but bubbling
oxygen was introduced into the
culture:
Exposure to sunlight prevented by
tin foil wrapping and no bubbling
oxygen was present:
o-o-o
53
~
50
HOURS
then placed in the 15°C water bath and exposed to sunlight on
the roof of the CSUN Science Building.
The experiment lasted
39 consecutive hours including periods of darkness.
Ideal weather
conditions once again prevailed although smog was present in the
sky.
The l'JMB spread plate technique was used to verify the
results obtained using the viable counting method.
The results
of this experiment are presented in Figure 16.
Once again E. coli B die-off is recorded after exposure
to sunlight.
In addition E. coli JD also is killed by exposure
to sunlight.
40
mg/ml glucose concentration serves no protective
functton according to these results.
The fact that both E. coli
populations seem to stabilize at night and die-off when exposed
to sunlight indicates that sunlight probably plays a major role
:in the elimination of E. coli from natural surface "Vmters.
5. )r::
FIGURE 16:
Comparison of the su..rvival of E. coli B with
E. coli J"D ivhen cultures of both are- simultaneously-exposed to natural sunlight at 15°C.
Initial cell population of E. coli B was l.2 x
10'7 cells/ml. Ini ti'l cell population of .!!·
coli JD was 1. 5 x 10 cells/ml.
E. co~.~ ~exposed to sunlight:
~ -~-~
E. coli JD exposed to sunlight:
A-£ -A
E. coli B in culture supplemented
with 40 mg/ml glucose exposed to
sunlight:
~---<)---~
E. coli JD in culture supplemented
with -40 mg/ml glucose exposed to
sunlight:
·-~-
E. coli B culture containing no
glucose prevented from exposure
to sunlight by tin foil wrapping:
0 - 0 --0
E. coli JD culture containing no
glucose prevented from exposure
to sunlight by tin foil wrapping:
e---~---~
+
101!.
lol -
10
~
~~
~:717§3
20
NIGHT
~
I
30
~~
HOURS
~ 40
~~ NIGHT
DISCUSSION
Physical and chemical factors associated with aquatic environments have been shown to influence the survival of E. coli
popu~
lations.· The previous series of experiments were designed to.
determine if nutritional imbalances could lead to the death of
1!~.
coli, and to identify other factors which might be responsible
for the elimination of' E. coli from fresh \'rater habitats.
T.he information presented in Figure 1 supports the statements
of Claphe.m CJ-973), and Berry and Horton (1974) that ·phosphorus
is usually a limiting factor.
The results reported here also show
that E. coli growth is Umited by concentrations of phosphorus
and glucose higher than those typically fom1d
atid Eddy, 1972).
~ sit~
(Metcalf
Therefore, E. coli grmvth is expected to be limited
:tn unpolluted natural waters.
Gerba and McLeod (1976) implied that die-off of E. coli can
be a function of a limited nutrient supply.
Various concentrations
of glucose and phosphorus were tested for their effect on the long
term survival of E. coli.
Analysis of these results showed that
at least under laboratory conditions the absence of phosphorus and/
or glucose or: the presence of these nutrients in limiting amounts
does not lead to the death of E. coli at 37°C or 15°C.
If the
results reported in this laboratory exercise (Figures 2 through 11)
can be extrapolated to nature then this evidence is environmentally
important because it indicates that
57
~
does not die from nutrient
58
starvation even over long time periods.
Consequently E. coli would
remain viable even if these nutrients are prevented from polluting
a fresh water environment.
The experiments revealed that high glucose ·concentrations, far
above those determined to be growth limiting, are somehow linked to
the death of E. coli populations.
Evidence presented in Tables 2
and 3 indicates that the killing of E. coli occurs regardless of
temperature if the population is allowed to continue to metabolize
glucose at concentrations exceeding 1.6 mg/ml.
~:uggest
These results
that the rate of E. coli die-off is temperature sensitive.
Since temperature does influence metabolic activity, toxic metabolites manufactured from the continued assimilation of excess glucose
by means of metabolic processes susceptible to temperature changes
could account for the temperature related differences in die-off.
This conclusion implies that excess glucose is being converted to
harmful products.
It is important to note that there is no evidence which can
satj_sfactorily explain the fluctuations in stability which appear
prior to the final stabilization of E. coli populations shown in
Figures 2 through 11.
It is a possibility that the variation reflects
the fragile nature of cells placed under extreme starvation conditions
and therefore some variable killing might result from the plating
procedure during determination of viability.
It is also interesting
to note that this anomaly appears only in those experiments conducted
at a temperat1rre of 37°C.
Whatever the actual causes are, relative
59
to the final level of survival the fluctuations are insignificant.
Thus, the conclusions drawn from these results are not affected
b;~r
this variability.
In addition, in isolated instances a culture unexplainably
behaved abnormally, but since this did not represent a consistent
behavior the results ·were not considered meaningful ;.rhen this
happened.
l'"or example, in :F'igure 3 the culture containing
~ 5. 6
· ug/ml phosphorus and 0.2 mg/ml glucose behaved abnormally; however,
in Figllre 9 a culture posSE-)Ssing identical concentrations of
phos~
phorus and glucose behaved predictably.
~1e
results presented in Tables l through 3 which summarize
the pertinent parts of the experiments dealing with the affect of
nutrients on E. coli survival reveal that there was a correlation
between cell death and a precipitous pH drop to
4.5.
This pH is
characteristic of the normal accumulation of acids which typically
result from E. coli fermentation of glucose. ,A pH reconstruction
experiment demonstrated that Wions accumulating due to the production of acids can be responsible for E. coli cell die-off.
Both
HCi and HCl produced this effect indicating that n+ions, not the
anions are responsible.
The normal death which occurred j.n excess
glucose was prevented by oH- indicating again that the anions produced from fermentation (Ci, etc.) are not responsible for the
death of E. coli.
Information provided in Tables 2 and 3 and in
Figures 8 through 11 which show E. coli die~off at 15°C and.37°C
argue that only when the bacteria are present in large numbers, or
60
allowed to metabolize for long periods of time will the change in
pH to q.• 5 occur and subsequent cell death result.
These results
were interpreted to mean that the pH change occurs when enough
acids have been manufactured to overcome the Tris buffer.
The rate
of acid production appears to be a function of popule.tion size and
temperature.
There are a number of reasons which su.ggest that the relationships just discussed are of little environmental importance.
these are:
Among
(1) concentrations of glucose or an easily assimilated
carbon· gou_rce that could account for a dramatic pH change are
rarily found in aquatic environments; (2) concentrations of
~:..
coli
tha,t are responsible for reducing pH to 4.5 in batch culture
experiments are environmentally unrealistic because the diluting
capabilitJ' of the aquatic em;ironment is substantial (Chanlett,
1973); and (3) compounds, colloids, and other substances such as
ions, individually and together, represent a significant buffering
agent in natural waters which will prevent the reduction in pH
to the value occurring under batch culture conditions.
These
reasons indicate that it is highly unlikely that the factors
responsible for lowering the pH to a suboptimal level for E. coli
survival U!'lder laboratory conditions play a significant role in
eliminating E. coli in situ.
A second possibility was also considered to explain E. coli
cell death at high glucose concentrations.
It was believed that
E. coli cell death could result from exposure to a continuous
61
supply of glucose and be unrelated to the accumulation of toxic
metabolites.
This situation was thought to exist if the metabolic
machinery necessary.for the routine maintenance and survival of E.
coli cells was not replaced or supplemented when necessary because
critical elements such as phosphorus v1ere absent.
A
metabolic
suicide vTould occur if the metabolic machinery processing the
11
continuous supply of excess glucose
>V"ore out," and was not replaced
due to the lack of essential.replacement elements.
The long term
survive,l of an E. coli population contained in dialysis tubing
continuously bathed by a steady flovr of non-growth medium supplemented vTith excess glucose demonstrated that metabolic suicide was
not responsible for any reduction in the size of an E. coli population.
Table 5 contains the data on which these conclusions are
be,scd.
Laboratory studies have demonstrated the lethal effects of
near visible light on the survival of microorganisms including
~i
(Chichester and Maxwell, 1969; Flutter and Richardson,
1967; Harrison, 1967; Kubitschek, 1967; Webb and Malina, 1967; MUller
and Schicht, 1965; and Hollaender,
19~J).
These investigations
emphasize that near visible light from solar energy could play an
important role in eliminating E. coli from fresh water environments.
Experiments conducted in this study have verified and extended these
observations.
Evidence presented here demonstrated that E. coli
cells suspended in a non-growth medium were killed when exposed to
visible light produced by a sunlamp.
The common conception that
62
bubbling water aids in purification seems to be contradicted because
the E. coli population was observed to be protected from elimination by.the light when bubbling was employed (Figltre
14).
Hollaender (194-3) predicted that light energy from the sun
would produce a similar germicidal action as that observed for
artificial sunlight in the laboratory.
This predicition was tested
and shown to be accurate in the experiments presented in this thesis
fer both the laboratory strain
~~'
E.~o1i
B, and a strain
fresh1y isolated from this investigator, E. coli JD.
Both strains
\<Tere el:tminated from non-grm-1th cultures exposed to direct sunlight
(Figu.:re 16).
This exper1ment continued for tvm days which permitted
detern1ination of E. coli population size during periods of darkness.
Although the data are sparse, the fact that E. coli populations
seem to stabilize in the dark (Figm·es 15 and 16) supports the
contention that solar energy is an effective germicidal agent in
nattiTe.
Re~examination
of the affect of bubbling on E. coli B
populations exposed to natural sunlight showed that bubbling was
also effective in offering some protection against the harmful
effects of sunlight (Figure 15).
It is evident E. coli can be killed when exposed to sunlight.
Educated speculation suggests that photochemical oxidative solar
radiation can be instrumental in the elimination of E. coli.
For example, free radicals formed by photochemical oxidation could
adversely affect the survival of E. coli.
Ozone, a highly effective
disinfectant used in water treatment for years (Metcalf and Eddy,
63
1972) can form from the combination of molecular oxygen and atomic
oxygen.
Atomic oxygen is formed by the splitting of molecular
oxygen in water as a result of the impingement of solar energy on
the molecule.
It is also possible that singlet oxygen, a very
reactive free radical, is responsible for E. coli die-off which
is observed when cultures are ex.posed to the sunlight.
The
protective effect of bubbling might be explained by the fact that
bubbling increases the probability that free radicals
'\'rould eombine
with oxygen, the subsequent creation of ozone would reduce the concentration of free radicals and the ozone concentration might be
reduced by the evaporative loss of the ozone in the bubbles.
These
ideas regarding the mechanism of E. coli cell death could be tested
by performing an experiment in which a photodynamically unreactive
gas, such as argon or nitrogen, was bubbled in a non-growing culture
of E. coli.
If free radicals associated with the interaction of
oxygen \'lith solar emissions were responsible for E. coli cell death
and no other effect of sunlight reduced E. coli population size,
then the E. coli population of such a culture would remain viable.
Obviously argon or nitrogen would not be detrimental to the culture
of E. coli because the bacterium is facultatively anaerobic.
However, there is evidence in the literature that near visible
light emitted from the sun is capable of disrupting the physiological
integrity of an E. coli cell by acting directly upon cellular
components.
Observations by Kubitschek (1967) and Webb and MaUna
(1967) indicate that near visible light (300 to 400 nm) can func-
Data provided by Harrison (1967)
t1.on as a mutagenizing agent.
shows that E. coli is killed by long exposure to white
light~
and
prior to being killed demonstrates signs of under going mutation.
The acttw,l photosensitive cellular component of E. coli is
not precisely identified by any investigator.
Nevertheless, all
of the investigators seem to agree that wavelengths of near-visible
light do not effectively damage bacterial nucleic acid, since
nucleic acids are not highly absorbent at near visible light
\·m.velengths (Hollaender, 1943).
Webb and Malina (1967) suspected
that a photodynamically active molecule closely associated with
the DJifA, such as riboflavin, is able to absorb the light and ultimately can cause cell death.
This is consistent w·i th information
prov·j_ded by Hollaender (1943) concerning the fact that riboflavin,
'\'ihich is present in bacterial cells, is know·n to become toxic
after irradiation from near visible light.
There are a variety of factors present in water which might
protect bacteria from harmful solar radiation.
Geologic processes
have distributed sediments throughout every aquatic environment on
earth.
In addition to sediment fresh water often contains dissolved
organic compounds (Crosby, 1976).
Macroscopic and microscopic
organisms both alive and dead appear in water under natural conditions
due to their ubiquity as members of the aquatic biota (Crosby, 1976;
Berry· and Horton, 1974; Clapham, 1972).
Furthermore it is not
unusual f'or fresh water that is close to shore to also contain
natural and synthetic organic compounds introduced as pollution
by man.
Among these are pesticides, detergents, agricultural manure,
and even female sex hormones excreted in the urine of women on the
pill (Crosby) 1976; Berry and Horton, 1974).
Metcalf and Eddy (1972)
point out that it is dtfficult to use ultraviolet radiation as a
sterilizing agent for aqueous systems because in addition to the
microorganisms absorbing the UV radiation, the organic molecules
and suspended matter will also, thus protecting the microorganisms
from elimination.
Crosby (1976) explains that organic molecules,
such as pesticides, undergo environmental transformation from the
absorption of solar radiation in the near visible \'lavelength range
of 300 to L!·OO nm.
Since free radicals have a proclivity for surface
absorption it can even be argued that the inert sediments described
by Gerba and McLeod (19'76) provide an absorption surface which can
remove free radicals from the water thereby shielding E. coli
populations from elimination.
This information implies that a
non-bacterial organic or inorganic suspension represents a protective
factor against bacterial death from sunlight.
In order to test one of these possibilities, the protective
effect of organic material, wild-type E. coli JD, and laboratory
strain E.
co~i
~
were suspended in a non-growth medium supplemented
with a copious quantity of glucose.
to sunlight.
Tile cultures were then exposed
The results from this experiment failed to provide
evidence that an excessive quantity of glucose could protect
E. coli cells from the deleterious affects of solar radiation
(Figure 16).
It is entirely possible that glucose vras not the
best choice as a typical organic molecule.
It is recommended
66
that further studies be conducted to determine if substances which
naturally extst are far more effective so that a precise statement
can be made regarding the protection of
!.:_~
by organic matter.
The lag period which appears before E. coli cells begin to
die after exposure to sunlight (Figures 15 and 16) was also
observed by Chichester and Maxwell (1969) and Hollaender (191.13)
"lhen the yeast Rhodotorula f?lutinis, and E. coli, respectively, were
exposed to short wavelength visible light.
These investigators
account for the lag period as a result of the fact that multiple
sites of damF;,ge must occur in the cell before the cell is destroyed.
Hollaender
(1943) concluded that " ••• up to a certain limit (the
affect of the killing agent) does not permanently destroy the ability
of the cell to divide and further develop."
It is obvious from the
results of experiments presented here assessing the affect of sunlight on the survival of E. coli (F'igures 15 and 16) that the
photodynamic reaction mechanisms killing the bacteria are complex.
There is a good possibility that the lethality of the photodynamic
process could be due to several mechanisms acting simultaneously
to destroy the E. coli cells' capability to survive.
These
mechanisms by which the photodynamic process kills the bacterial
cells are probably acting at multiple sites to eventually annihilate
the metabolic systems responsible for cell maintenance and proliferation and/or disrupt the physical integrity of the cell.
Further studies are required to define the wavelength of solar
energy most important in killing E. coli.
One possible approacl1
is to determine the wavelength using an action spectrum.
This
technique would permit an investigator to associate a measurement
of cell death with a specific wavelength of light emitted from the
sun.
The use of filters to restrict the 'vavelength of light to
narrow bands would be a simple way of performing an action spectrum.
'Yhis type of experiment would provide insight as to the nature of
the cellular substance possibly affected by solar radiation.
Evi-
dence presented in the literature implies that several cellular
components of E.
Among these are:
~oli
are more likely to be involved than others.
(1) riboflavin \vhich Holla,ender (1943) and
l'Jebb and 1:1a1ina (1967) state to be a photodynamically active
molecule; and (2) sulfhydryl compounds, such as cysteine, which
~1ichester
and Maxwell (1969) discuss as a possible cellular
component capable of interaction with free radicals.
Other experiments should be designed to determine the nomenclature of the free radicals which can appear in the medium during
exposure to Sut1light.
An experiment needs to be conducted to deter-
m:tne the maximum depth to which solar energy is an effective killing
agent in an aqueous environment.
This information should then be
extended to determine the response of actual fecal pathogenic
bacteria and perhaps viruses to solar energy •.
Finally field studies should be carried out to assess the
exact nature of the response to sunlight of E. coli under in situ
conditions.
The pre.ctical outcome of this recommended battery of
experiments is the elucidation of the effectiveness of E. coli as
an indicator of recent fecal pollution and pathogenic contamination
of fresh water environments in the United States.
68
Water bodies are capable of undergoing a process Chanlett
(1973) terms "self-purification" or the elimination of bacterial
pollutants by the dynamic properties inherent in fresh waters.
Therefore the role of sunlight as a disinfecting agent may be
included \vi th a varJety of other factors such as toxicity to bacterial contaminants by salt concentrations ( Gerba and MciJeod, 1976;
Hanes and F'ragala, 1967), predation, competition from native microflora, or the presence of heavy metals (Gerba and McLeod, 1976).
Thus the exJx:riments presented in this study showed that nutrient
limitation does not affect the su-rvival of
~coli,
and confirmed
what He;tcalf and Eddy (1972) state emphatJcally, that "sunlight is
also a good disinfectant."
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