THE mm GF CHLORENE UPOR UREASE

AND ITS RELATIGN TC} THE BACTERICEML
ACTEGN 0F CMORLNE

Thesis Ear m Degree of Ph. D.
MICHIGAN SVA’FE COLLEGE
M53931 Siiverman
1954

 

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thesis entitled

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The Action of Chlorine Upon Urease And
Its Relation to the Bactericidal Action of Chlorine

 

preeented by

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Milton Silverman ‘ , ’ j

 

he: been accepted town-d: fulfillment
of the requirements for

Ph. D. degree m Bacteriology

“ Ma :- professor

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0-169

 

 

THE ACTION OF CHLORINE UPON UREASE AND
ITS RELATION TO THE BACTERICIDAL ACTION OF CHLORINE

by
Milton Silverman

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

/‘ x
/

DOCTOR OF PHILOSOPHY I

Department of Bacteriology and Public Health
1951+

 

ACKNOWLEDGMENTS

To Dr. W. L. Mallmann who suggested
the problem and who continued to direct
it, and to render aid and encouragement
to the student who pursued it.

And to all others who in their own way
helped him.

3239 34 .1-

VITA

Milton Silverman

candidate for the degree of
Doctor of Philosophy

Final Examination, 3 March 1954, 13:30, Room 325, Giltner Hall
Dissertation: The Action of Chlorine Upon Urease and its
Relation to the Bactericidal Action of Chlorine
Outline of Studies:
Major Subject: Bacteriology

Minor Subjects: Biochemistry, Cytology, Genetics,
Mycology

Biographical Items:
Born: 9 February 1911, New York, N. Y.

Undergraduate Studies: City College of C.C.N.Y., l928é
1933, B. S. 1933

Graduate Studies: Pennsylvania State College, 19u9-1950
M.S. 1950, sponsor Dr. C. D. Cox
Thesis Title: Serodiagnosis of Bru-
cellosis: Attempts to Detect Soluble
Substance of Brucella in Bovine Sera
by the Use of Homologous Antibody

Michigan State College l951-l95h
Ph. D. l95u, sponsor Dr. W. L. Mallmann

Experience: Serologist, N. Y. State Department of Health
19h0-19h2; Bacteriologist, Veteran's Adminis-
tration 19h8-1951

Affiliations: Society of American Bacteriologists
Society of Sigma Xi

II

THE ACTION OF CHLORINE UPON UREASE AND
ITS RELATION TO THE BACTERICIDAL ACTION OF CHLORINE

by
Milton Silverman

AN ABSTRACT

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Bacteriology and Public Health
Year 195A

Approved"Vw1{:tjf¥axlrrvvannrr~\

Milton Silverman

The thesis problem was based upon the distinction be-
tween chlorine that is effective against bacteria, bacteri-
cidal chlorine, and chlorine concentration that is measured
chemically. An attempt was made to evaluate the action of
chlorine upon urease in the hope that such action would
parallel the bactericidal action of chlorine more closely
than a chemical determination of chlorine concentration.

The methods used in the pursuit of this investigation
involved:

1. The maintenance of conditions which provided a
reagent of free chlorine.

2. A method for the evaluation of the bactericidal
activity of free chlorine at different pH values against
Escherichia coli, strain 912_, Non-clumping vegetative
bacterial cells were washed from.agar slants. Small vol-
umes of reagents were used in a repetitive, serological
type test at various contact times. Survivors were deter-
mined by plate counts. Percentage survival logarithms were
plotted against logarithmic time intervals for the ratios
of bacterial nitrogen to the dosage of chlorine used.

3. A parallel method for the evaluation of the inhibi-
tory action of chlorine upon urease. Crystalline urease
was used. Percentage activity remaining was determined by

the action of the inhibited enzyme upon a urea substrate.

The measurement of the ammonia nitrogen evolved was by means

of a Nes
centage

time in:

efficien
at pH h,
their or
ial nitr,
at pH 9,
of 100 p"
2.
ficient L
at pH 4,
oriéinal
nitrogen
pH 9, Ure
original
chlorine J
3. j
for water!
would not A
activity Ci
test DPOCQH
not be 113$

Chlorine .

 

l

Milton Silverman
2

of a Nesslerisation procedure. The logarithms of the per-
centage activity remaining were plotted against logarithmic
time intervals for the ratios of urease nitrogen to chlorine
dosage used.

The major findings were:

1. The action of chlorine upon bacteria became more
efficient as the pH was lowered. In the presence of chlorine
at pH h, bacterial numbers were reduced to .01 percent of
their original value in 40 minutes when the ratio of bacter-
ial nitrogen to chlorine was .1. In the presence of chlorine
at pH 9, bacterial numbers were still present to the extent
of 100 percent with the same nitrogen to chlorine ratio.

2. The action of chlorine upon urease became more ef-
ficient as the pH was raised. In the presence of chlorine
at pH u, urease activity was reduced to .01 percent of the
original activity in 100 minutes when the ratio of urease
nitrogen to chlorine was 3. In the presence of chlorine at
pH 9, urease activity was reduced to .01 percent of its
original activity in seven minutes with the same nitrogen to
chlorine ratio.

3. The effect of chlorine in the concentrations used
for water disinfection upon the viability of a bacterium
would not necessarily be duplicated by its effect upon the
activity of any one single enzyme. On the basis of this
test procedure, the action of chlorine upon urease could
not be used to assay directly the bactericidal activity of

chlorine.

Acknowle

Vita .

I. INTR
II. REVI
III. XPEE
IV. EIPZ‘

7‘" :—
Ve H.332,

A.
B. 1
VI.
VII.
VIII.
IX.
X.

 

 

TABLE OF CONTENTS

Acknowledgments . . . . . . . . . . .

Vita

I.
II.
III.
IV.
V.

VI.
VII.
VIII.
IX.

X.
XI.

INTRODUCTION 0 O O O O O O O O O

REVIEVJ O O O O O O O O O O O O 0

EXPERIMENTAL METHODS - OUTLINE .

EXPERIMENTAL METHODS - EXPLANATIONS

EXPERIMENTAL DATA

A.
B.

EXPERIMENTAL DATA - BACTERICIDAL TEST, Index
EXPERIMENTAL DATA - ENZYME INHIBITION, Index

Explanation of Tables . . .

Explanation of Graphs . . .

EXPERIMENTAL DATA ' ERRORS e e 0

DISCUSSION . . . . . . . . . . .

A.
B.
C.
D.

E.

Measurement of Bacterial Nitrogen

The Urease Preparation . . .

N/Cl Ratics e e e e e e e 0

Relationship of Enzyme Inhibition Test

Bactericidal Test . . . . .

Extrapolation in the Direction of De-

creaSing Time 0 o e o e e e

S UMNLARY O O O O O O O O O O O O 0

LITERATURE CITED . . . . . . . .

to

Page

II

22
23

51
53
77

99
102

102
103
105

112

120

122
12a

I. INTRODUCTION

The experimental work of this thesis concerns itself
with the action of a disinfectant, chlorine, upon a bac-

terium, Escherichia coli and upon an enzyme, urease. It

 

originated from a suggestion of Dr. W. L. Mallmann who re-
ferred this student to a previous doctoral thesis by N. H.
Ozgumus entitled "Evaluation of Chemical and Bacteriological
Methods of Determining Germicidal Activity of Chlorine" (1).
In the summary of this thesis it was stated that in the
presence of certain forms of organic material the values

for free chlorine obtained by chemical tests were in excess
of the germicidal chlorine determined bacteriologically.
Thus the simple chemical tests used to determine free chlor-'
ine did not correctly indicate germicidal activity under
certain conditions. However, the bacteriological test is

of such a complicated nature that it is not practical as a
routine method for determining the germicidal nature of the
chlorine present. It would be necessary to obtain standard
time-survivor curves for a standard strain of micro-organism.
in the presence of different concentrations of chlorine for
each set of determinations. Time-survivor curves in the
presence of test amounts of chlorine would then have to be
interpolated onto the standard curves and the equivalent
germicidal concentration would thereby be determined. The

test is tedious, time consuming, and complicated. It was

suggested that a simpler biochemical method might be sought.
Might not the inhibitory action of chlorine upon an enzyme
duplicate the destructive action of chlorine upon the bac-
terial cell? And might not the degree of inhibition be used

to measure the amount of germicidal chlorine?

II. REVIEW

Bacteriology as a subject of scientific study may be
dated from the middle of the nineteenth century and may be
considered as a direct outcome of the work of Pasteur.
Studies on fermentation were undertaken by him during 1855
to 1860. He was able to demonstrate that the fermentation
of various organic fluids was always associated with the
'presence of living cells and that different types of fer-
mentation were associated with different micro-organisms.
Such investigations of fermentation led to the study of the
behavior of various kinds of natural organic fluids, and of
simple synthetic media, as substrates for micro-organisms.
Different media were found to be suitable for different
micro-organisms to a certain extent. Thus, environmental

factors exercised a selective effect on the type of naturally
occurring bacterial flora. By extension similar ideas were
applied to infection resulting from micro-organisms.

Both desirable and undesirable fermentations were pro-
duced by micro-organisms through the agency of "organised"
ferments. Since such reactions took place only in the pres-
ence of living micro-organisms and were not due to the pres-
ence of certain chemicals as postulated by Liebig, then the
micro-organisms had to be destroyed in order to eliminate any

unwanted reaction. Such destruction by heat could be utilized

for sterilising fermentation media. Lister was struck by
the analogy between changes which occurred in fermenting
organic material and in putrefaction occurring in wounds.
The underlying cause was assumed, analogously, to be living
micro-organisms. The undesirable reactions could be and
were eliminated by the use of chemicals. Carbolic acid
Sprays were utilized successfully as an antiseptic during
surgery by Lister as early as 1867.

The massive figure of Koch dominated the bacteriological
scene for the last two decades of the nineteenth century and
purely morphological studies dominated this field. The ad-
vances in the study of enzymes, as ferments were designated
by Kuhne in 1878, were made in the field of chemistry largely
through investigations upon the so-called "unorganized" fer-
ments which although secreted by living cells, acted in their
absence. However, this distinction of "organized" and "un-
organized" ferments was broken down by Buchner (2) who dis-
covered that cell free extracts of yeast could be made to pro-
duce alcoholic fermentation. Pasteur and Liebig had both
been correct. Living micro-organisms definitely degraded
organic substances, respired, and built up cell substance
by the use of enzymes. But, such enzymes could be isolated
and in the presence of suitable substrates and menstrua could
'be shown to act as a catalyst independently of the cell.
‘Within a short space of time a partial scheme of glycolysis

was formulated by Harden and Young (3) complete with

phosphorylation of sugars and the presence of co-enzymes.
Glycolysis came to be regarded as part of the process by
which a cell made energy available for its needs by means
of intramolecular oxidations not involving oxygen. How
could such oxidations be performed? here these enzyme re-
actions of such a critical nature that their inhibition re-
sulted in the death of the cell?

The concept of enzymes remained a rather obscure one
until.l926 despite the huge amount of work performed upon
them. Also despite the impure nature of the usual enzyme
preparations huge encylopedic volumes were written ascribing
specific preperties and names to such preparations as in von
Euler (h), Oppenheimer (S), Willstatter (6), and Buchanan
and Fulmer (7). Willstatter (8) summarized this obscure
viewpoint when he stated that enzymes did not belong to the
ordinary proteins or carbohydrates nor to any of the known,
large groups of organic compounds with a complex structure.
Yet for the quarter century preceeding 1926 and for about
fifteen years thereafter the work on bacterial enzymes and
the theories of disinfection involving inhibition of such
enzymes was based largely on work with impure preparations
that produced certain Specific reactions.

Wieland (9) provided a theory and a mechanism that was
eagerly seized upon by the investigators in this field. The
essential mechanism of cellular oxidation was regarded as hy-

drogen activation and consequent hydrogen transport. The

removal of hydrogen from a molecule could be preceded by the
addition of a molecule of water or the hydrogen could be re-
moved directly. In either case a suitable hydrogen acceptor
had to be provided. The enzyme that activated the hydrogen
in the substrate to be oxidized and which then acted as a
reversible hydrogen acceptor and donator, thus transporting
the hydrogen, was called a dehydrogenase. When Thunberg (10)
provided a simple technique for the measurement of dehydro-
genase activity, the school of English biochemists under G. F.
Hopkins eagerly adopted it and proceeded to apply it widely

to living tissues and cells. Among these were Stephenson,
Quastel, Whetham, and Wooldridge. The technique was exten-
sively described by Quastel and hhetham (ll). Washed bac-
terial cells suSpended in appropriate buffer solution, inor-
ganic ions, and an indicator dye were placed in a specially
constructed tube, by means of which the tube and fluid could
be evacuated and a specific substrate could be added without
opening the tube to the air. Reactions consisted of reduction
of the indicator dye, methylene blue, which was evident by

the bleaching of the dyed solution. Concentrations were ar-
ranged so that reactions were completed in a short time, 30
minutes or less, to eliminate bacterial multiplication.

Change of color of the dye was taken to indicate dehydrogenase
activity, due to the enzyme itself. It was believed that any
interfering activities due to either living cells or prolifer-

ating cells had been eliminated. Cook and Stephenson (12),

and Quastel and Wooldridge (13) reported that the dehydro-

genases -- washed, resting cell suSpensions -- of Escherichia

 

ggli_differed in their susceptibility to changes in pH, salt
concentration, temperature.and exposure to various chemicals.
Thus, Thunberg tube reactions were obtained when the sus-
pensions were subjected to increasing temperature, pH changes,
and to exposure to nitrite, benzol, toluol, phenol, ether,
chloroform, and propyl alcohol. The most sensitive of the
dehydrogenases were those acting on alanine, glycerol, sugars,
and glutamic acid. Less sensitive were those acting on lac-
tic, succinic, and fumaric acids. Least sensitive were the
formic and acetic dehydrogenases. Potassium cyanide and
hydrogen peroxide gave a reversed picture in which the formic
and acetic acid dehydrogenases were most sensitive. These
investigators pointed out that the effects of such toxic in-
hibitors, as mentioned above, were of interest for two reasons.
They threw light upon the mechanism of action of such inhi-
bitors. EXposure for different periods of time resulted in
step by step repression of the various dehydrogenase activi—
ties. At least two different groups of inhibitors could be
constructed on the basis of this order of suppression. Sec-
ondly, information was obtained concerning the preperties of
enzymes and of bacteria possessing such enzymes.

This work led to the idea that dehydrogenases were criti-
cal enzymes for the vital processes of the bacterial cell.

Partial or complete destruction of some or all of such enzymes

resulted in the death of the cell. Quastel and Wooldridge
(13) had demonstrated that complete inhibition of the sugar
dehydrogenases resulted in greatly reduced numbers of organ-
isms or dead ones. Casman and Rettger (14), Edwards and Rett-
ger (15), and Wedberg and Rettger (16) using different species
of bacteria, including some thermophiles, performed their
experiments in the following manner: the maximum temperature
of growth was determined and concomitantly, the heat resistance
of various enzymes including succinic dehydrogenase. It was
found that the enzymes were inactivated at the same tempera-
ture at which the bacteria ceased to grow. However, the ex-
posure of bacteria for viability occurred in nutrient medium
while the exposure of bacteria for enzyme inactivation was
performed after washing the cells - which process removed
co-enzymes. The period of eXposure was very long - 2h hours~-
long enough for the protein of the enzyme to deteriorate.

Thus the two parallel tests were not exactly comparable and
the results were not valid. Hahn and Schroeder (17) re-
peated the work of Rettger and his co-workers, but made com-
parative viability and enzyme tests from cells suspended in
buffer. No parallel was found between the two factors of
viability and enzyme inactivation. When more than 99.99
percent of the cells were dead only 50 percent of the succinic
dehydrogenase was inactivated. These workers concluded that
there was no connection between the inactivation of enzymes

and the death of microbial cells. Firstly it was demonstrated

that enzyme action persists in dead cells and secondly, it
was shown that the logarithmic order of death of bacteria,
if such were actually the manner according to which bacteria
died, could occur only if death were caused by the effect
upon a single molecule. It would have been more correct for
Hahn and Schroeder (17) to have said, concerning their first
point, that there was no connection between the death of
microbial cells and those specific enzyme activities which
were studied. The work of Quastel and Wooldridge (13) had
demonstrated that toxic substances killed micro-organisms
but that enzyme activities are only partially suppressed.
Exposure of E. coli to toluene, benzol, ether, chlorform or
cyclohexanol was sufficient to eliminate the power of the
micro—organism to activate sugars, glycerol, or glutamic
acid. The activating mechanisms for succinic, lactic, for-
mic, and alpha glycero-phosphoric, however, remained intact.
Acetone acted similarly to toluene, etc., but in addition
eliminated the activating mechanisms for polyhydric alcohols.
Thus the investigators concluded that dead cells in the
sense that they were incapable of proliferation in known
media still possessed undiminished activating powers. The
action of disinfectants upon cell was a complex one. The
death of cells could not be attributed to the inactivation
of any one specific enzyme but the dogmatic statement that
no enzyme inactivation was involved could not be made. Addi-

tional experimental evidence seemed to bear out this conclusion.

lO

Yudkin (18) demonstrated that silver sulphate was lethal to
suspensions of E. coli in concentrations much lower than those
inhibiting its dehydrogenases. Bucca (19) testing the action

of several drugs upon Neisseria gonorrhoeae found that the

 

death of cells occurred long before any significant enzyme
inhibition took place. One of the drugs, sulphanilamide,
produced practically no enzyme inhibition during an exposure
period of 2h hours yet the cells were dead at the end of the
period.

Hahn and Schroeder‘ (17) in their second conclusion at-
tempted to refute the contention that enzyme inactivation was
the cause of bacterial death on the basis of more theoretical
considerations. These were to the effect the time—survivor
curve of the action of disinfectants upon micro-organisms
could be interpreted only in terms of a monomolecular reaction
which followed the formula, Kt = log a/a-x , where t = time,

a 3 initial population of micro-organisms, and x = the number
of bacteria killed at time t. This relationship was postulated
by Henri (20) for the hemolysis of chicken red blood cells

by dog serum. Madsen and Nyman (21) using the data of Krdnig
and Paul (22) showed that the rate of disinfection followed

the same formula. Chick (23) further confirmed the relation-
ship with additional data. Such a relationship, however, was
based upon the assumption that the cells of a bacterial cul-

ture were of uniform resistance and that death occurred to a

11

certain preportion in each successive time period, due to a
single event. As early as 1889 Geppert (2h) had attributed
the survival of bacteria to individual variation in the popu-
lation under study. This concept was in accord with data
from the pharmacologic field. Heichenbach (25) also pointed
out that organisms varied extensively in regards to the time
needed for their destruction. If this variation had a skewed
distribution then the approximately linear relation between
the logarithm of the survivors and the time might be accounted
for. For the action of heat upon bacteria, time-survivor
curves similar to those with drugs were obtained. Further
cultures of different ages gave time-survivor curves of dif-
ferent shapes, which seemed to indicate that variation in
resistance was occurring. It was concluded that chemical
molecules were so small compared with bacteria and their num-
ber so much greater than the number of bacteria that even in
dilute solutions each bacterium would be surrounded by a
similar number of molecules. They would, therefore, be equally
exposed to the action of the disinfectant. If then, all the
bacteria were of equal resistance, why should any one die be-
fore the other? Buchanan and Enlmer (26) were of the opinion
that the theory of logarithmic death rested on insecure foun-
dations. If the number of cells which died in each equal in-
terval of time were plotted against time, a rate distribution
curve would be obtained. The area under such a curve would

be proportional to the total number of cells. The area to the

12

left of any ordinate would be proportional to the total cells
dead and to the right of any ordinate to the total still sur-
viving. Such a curve presented a distribution of cell resis-
tances - such resistance being measured by the time required
to kill the particular cell. In general, the properties of
living things have been found to be distribu‘ed according to
some form of a probability curve. Such a distribution would
be symmetrical if the constants of the equation of such a
curve were equal. If however, the constants were unequal, the
curve became skewed and if very unequal, so skewed that the
major portion of the curve resembled the logarithmic curve.
Thus, the logarithmic order of death resulted from data based
upon experiments in which rate curves were extremely skewed
and in which the variation of resistance was scattered very
closely about a mean resistance. In support of this conten—
tion data and curves from a paper by Fulmer and Buchanan (27)
showing that time survivor curves of yeast cells in the pres-
ence of methylene blue were not logarithmic, were presented
in (26).

The proponents of the monomolecular theory however, per-
sisted in the belief that their explanation was correct. They
received the support of Arrhenius (28) who regarded the single
event necessary for such action as the ionization of the pro-
tein part of the molecule. Such ionization of but one or two
hydrogen and hydroxyl molecules would open the cell to attack

and result in destruction of the cell. Yule (29) attempted

13

to explain the lag period by the occurrence of a few events
followed by the logarithmic period due to one event. Chick
(30) went so far as to assume that micro-organisms underwent
rhythmical changes and were vulnerable only during certain
phases. Thus a certain percentage was killed in every time
period. But Rahn (31) still insisted that bacterial death
was different from that of multicellular organisms and that
the data of drug action upon multicellular organisms could
not be applied to bacteria. Using arguments based on Yule's
probability calculations, it was shown that a theoretical
logarithmic order could be obtained only if one definite
molecule were affected. Since the order of bacterial death
had been found to be logarithmic, then, Hahn concluded that
one definite molecule had been affected or that one event
had occurred. If curves other than logarithmic ones were
presented, they were to be explained away by such assumptions
as the occurrence of more than one event due to the clumping
of cells. This conclusion has resulted from the commission
of a logical error - that of affirming a consequent, and also
by disregarding other data.

The case for correlation between bacterial cells and en-
zyme inactivation was not proved by such simple experiments
as those performed upon dehydrogenases, but neither was the
possibility of such correlation ruled out by Hahn and Schroe-

der's two arguments.

lb

Perhaps, then, the experimenters who attributed bacterial
death to the inactivation of a single enzyme erred on the side
of simplicity but not on the side of a concept. Disinfectants
did react with the cell and with the proteins of the cell.
This reaction with such complexly arranged organisms would
involve reaction with large numbers of enzymes of which more
than one would be inactivated in the process of killing one
bacterium. Could one say then that the inactivation of one
enzyme would be critical? Apparently not, for it hadtaeen
shown that more than one enzyme was inactivated by any one
toxic substance and the selection of any one enzyme for such
a critical role was dependent upon the available method of
testing rather than upon a complete knowledge of the critical
points of the metabolism of the bacterium. Could it be said,
then, that inactivation of enzymes was not the cause of bac-
terial death? Again apparently not, for no studies had been
performed with enzyme systems of the entire cell but only
with isolated enzymes. Was the assumption of uniform resis-
tance tenable? The evidence that could be presented from dis-
infectant studies was not conclusive enough either to prove
or diaprove the above assumption. In a general way however,
one would not expect bacteria to act differently from other
forms of life and to show a different pattern of resistance.
It would seem that the doctrine of uniform resistance followed
from the monomorphic presumption of the early bacteriologists

that bacteria were perfectly stable, that cell forms remained

15

constant, that cultural characteristics were immutable, and
that cultural reactions were invariable. Variations were
dismissed as the result of poor technique. In the light of
this assumption differentiation and variation of bacteria
would be an impossibility.

Some forms of bacterial variation had been observed to
occur Spontaneously but these were considered exceptions to
the rule of stability. It was difficult to prove in the case
of bacteria that such variation was actually an hereditary
variation and not merely an adaptation to a changed environ-
ment. Luria and Delbrfick (32) were the first to prove defin-
itely by a fluctuation test that variations in phage resistance
arose as a result of a spontaneous mutation rather than as an
adaptation. Newcombe (33) demonstrated that plates cultures
which were allowed to incubate and then spread produced a
greater number of phage resistant mutants than cultures which
were exposed to phage without previous incubation. Since
then numerous investigators have demonstrated that Spontan-
eous mutants accumulate during prolonged cultivation. (Stock-
er, 3h; Novick and Szilard, 35; Atwood, Schneider, and Ryan,
36).

Simplified techniques for the detection of such resistant
mutants have become routine practice. Some were based on the
principle of allowing the growth of mutants but of preventing
the growth of the parents. The penicillin method of Lederberg

and Zinder (37), and Davis (38) depended upon the destruction

16

of dividing parent cells by penicillin in a deficient medium
in which the mutants persisted. Such mutants were then de-
tected on a complete medium. Gradient plates, (Szybalski and
Bryson, 39), easily detected resistant mutants to a variety
of chemotheropeutic agents and antibiotics. Other methods
were more direct and consisted of the isolation of mutants

in the absence of environmental conditions that would favor
their establishment. An example of this would be the replica
plating technique of Lederberg and Lederberg (#0).

The evidence seemed clear that bacterial mutants showing
variable resistances did occur. There was, also, evidence
concerning the frequency of occurrence of such mutants. The
rate of occurrence per bacterium per generation was low,

6

varying from 1 x 10- for penicillin resistance of Micrococcus

 

pyogenes var. aureus to 6 x 10"10 for streptomycin resistance

 

of Hemophilus_pertussis.' (Braun, Al).

 

It would not be stretching the evidence too far to state
that such variation would occur in a bacterial culture during
disinfectant testing but that the mechanics of the experiment
tended to disregard it. Thus the low mutation rate would ac-
count for a low concentration of resistant mutants, and a very
high percentage of sensitive forms with a narrow scatter of
resistances about the mean. Such a distribution when tested
by the ordinary disinfectant testing methods would result in
the appearance of logarithmic time-survivor curves. This would

then lead the investigator to conclude that the culture was of

uniform resistance.

17

Thus, these early attempts to confirm a theory of dis-
infection based upon the inactivation of Specific enzymes of
a cell were not successful. But the counter claims of those
who held that such action could not be responsible for death
also were not proved. The period from 1926 onward, marked
by the first isolation of a pure,crystalline enzyme by Sum-
ner (L2) resulted in a change of attitude toward enzymes.
They were now known to be proteins. They could be purified
of extraneous materials and could be used as relatively pure
reagents to study the mechanism of enzyme action. Enzymes
were found to be organized into systems, the successful action
of which depended not only uponthe protein of the enzyme but
also upon the presence of such entities as coenzymes, high
energy phosphate bonds, inorganic ions, and upon such a vague
concept as the organization of the cell. (Dixon, A3).

One of the more successful workers in the field of en-
zymes attempted a generalization based upon the low concen-
trations of enzymes found in cells. (Green, Ad). This was
to the effect that enzymes were present in cells in such low
concentrations that they had to be called trace substances.
They achieved their effects at such low concentration by acting
reversibly at high turnover rates and by being organized into
systems that tended to maintain a system in one direction.
Consequently, any substance which in low concentration thus
affected cells might be conceived of as affecting an enzyme

or a system of enzymes. This generalization disregarded the

18

distinction that was made by pharmacologists between the
concentration of drug used and the concentration of drug
fixed by the cell. Thus Eichholtz (k5) pointed out that
although the concentration of copper in a solution may be
one part in 108 the concentration within algae in such a
solution is several parts per lOu. DeSpite this oversight,
Green and his coworkers (he) proceeded with a demonstration
that the action of chlorine upon bacterial cells in water
could be explained in terms of the inactivation of enzymes,
because of the low concentrations of chlorine used. These
investigators insisted that critical inactivation occurred
at a single locus -- the sulphydryl group of enzymes possess-
ing such a group. The experimental demonstration was such
that a close correlation was first established between bac-
terial death and the inhibition of glucose oxidation as
measured manometrically. Then an examination of isolated
enzymes of the glycolytic system established that aldolase
was the most sensitive in terms of the amount of chlorine
required to inactivate it. But the time-acbkdwi curve of
the inactivation of aldolase never fell below 70 percent in-
hibition and did not parallel the curve of bacterial death.
Despite this, it was concluded that the action of chlorine
upon aldolase probably disrupts the balanced system of glu-
cose oxidation resulting in its inhibition and consequently
in bacterial death. It seemed that these workers fell into

the same error of simplicity as previous workers in trying to

l9

attribute the complex phenomena of bacterial death to the action
of chlorine upon a single enzyme in the glycolytic system.
There is another point that had not been taken into con—
sideration. This concerned the criteria involved in disinfec—
tant testing in contradistinction to those involved in the
cause of bacterial death. For the purposes of disinfectant
testing and for the uses to which disinfectants are put, the
failure of micro-organisms to reproduce themselves on the
commonly used media is a sufficient and practical criterion
of bacterial death. For the purposes of elucidating the mech-
anism of bacterial death such criteria are insufficient. Thus
Heinmets, Taylor and Lehman (h?) treated E. coli with chlorine
and such treated suSpensions were wholly sterile when incubated
in nutrient broth. However, survival of from 0.3 percent to
20 percent was obtained when such treated cells were incubated
with various metabolites of the tricarboxylic acid cycle
either singly or in combination. The evidence of these ex-
periments was such that many enzymes were inactivated by
chlorine and not just one enzyme. The provision of several
metabolites enabled the damaged organism to resynthesize the
enzymes and to re-establish the various cyclic processes more
readily than the provision of a single metabolite. It is a
question then that the conclusions of Knox, 33 gl., (he) were
tenable. '

Thus once again an attempt to correlate death of bacteria

with enzyme was not successful. But there were several points

20

in the paper of Knox, gt gl., (he) that were significant for
this thesis. One was the demonstration that sulphydryl en-
zymes were extraordinarily sensitive to the action of chlorine.
The choice of an enzyme for the work of this thesis was thus
narrowed down to those containing this group. A purely em-
pirical relationship between bacterial destruction and enzyme
inhibition might be established without implying that such
enzyme inhibition were the cause of bacterial death. Green
and Stumpf (AB) had suggested this but did not proceed to that
end. Papain - a sulphydryl enzyme - was used and the inhibi-
tion of its clotting effect upon milk was used to assay chlor-
ine. But the results were not correlated with bacterial death
due to the action of chlorine.

And so we come to the point of this thesis. It is not
an attempt to locate the cause of death of bacteria. The
action of a disinfectant is not to be thought of as localized
at some locus in the cell. It may affect permeability, mul-
tiplication, and metabolic activities singly or in any possible
combination. The means are not available for determining the
point of Specific activity if such a point does exist. In-
stead the attempt is to be made to establish a purely empiri-
cal relationship between the inactivation of an enzyme -
not even present in the bacterial cell - that is sensitive
to chlorine, and the death of a bacterium also sensitive to
the same chemical. Chlorine will combine with any protein

whether it be part of the bacterial cell or not and the use

21

of a biochemical agent containing extraneous protein as well
as active centres may be thought of an paralleling the exist-
ing structures of the bacterial cell - containing, likewise,
extraneous protein and active centres. In this manner, dis-
crepancies arising from the use of purely chemical tests for

the detection of chlorine might be overcome.

End of Review

I.

J.

III. EXPERIMENTAL METHODS - OUTLINE

Protocols
1. Bactericidal Test
2. Inhibition of Urease Test

Chlorine

Glassware

Water

Zero Chlorine Demand Water
Measurement of Chlorine Concentration
Buffers

The Objects of the Action of Chlorine
l. Bacteria
a. Maintenance of Cultures
b. Preparation of SuSpensions
c. Use of Suspensions
2. Enzyme-Urease
a. Preparation of Crystalline Urease
b. Standardization of Urease
c. USe of Urease

Nitrogen Determinations

1. General Analytical Considerations

2. Total Organic Nitrogen Determination
3. Ammonia Nitrogen Determination

The Bactericidal Test Procedure

1. Plating
a. Surface Plating-Medium Used
b. Manipulation of Dilutions

2. Incubation

. Counting

£0 Plotting
a. Abscissal Units
b. Ordinate Units

The Enzyme Inhibition Test Procedure

1. Enzyme Substrate Reaction

2. Halting of Reaction

3. Ammonia Nitrogen Measurement as Criterion for
Enzyme Inactivation

h. Plotting

22

Page
23

21+
25
2s
27

29
31

3h

41

Ln

23

IV. EXPERIMENTAL METHODS - EXPLANATIONS

A. Protocols

1.

2.

Bactericidal Test

Tubes 1 2 3 Control
Chlorine (buffered) ml. 1 0.5 0.1 0
Water (buffered) ml. 0 0.5 0.9 1.0
Bacterial SuSpension 1.0 1.0 1.0 1.0

(unbuffered) ml.
Variable Contact Time at 30° C.

Sulfite-Buffer 2.0 2.0 2.0 2.0
(pH 7.0) mle

Plating by a Surface Plating Method
Incubation for 2h hours at 37°C
Counting of Plates

Inhibition of Urease Test

Tubes 1 2 3 Control
Chlorine (buffered) ml. .5 .25 .10 0
water (buffered) m1. 0 .25 .hO .5
Ureaae (unbuffered) ml. .5 .5 .5 .5

Variable Contact Time at 30°C

Urea-Sulfite Buffer 1 1 1 1
(pH 7e0) ml.

Incubation Time at 30°C - 15 minutes
N-HCl - 1 1 l l
Nesslerization of Portion of Reaction Mixture

Photelometric Measurement of Nessler Reaction

21+

The protocols are presented here to provide an intro-
duction to the nature of the tests used. Following is a
more detailed description of the methods used and some of

the reasons for using them.

B. Chlorine

Chlorine was prepared as a concentrated solution of
chlorine gas by reacting potassium.permanganate with concen-
trated hydrochloric acid in a suitable generator under a
hood. The gas so generated was passed through two flasks
of distilled water and then collected in a third flask of
double distilled water. This latter was transferred to a
dark brown, glass stoppered bottle and stored in a refrigera-
tor at about 5°C. Its uses were to prepare test solutions of
chlorine in zero demand water and to prepare the zero demand

water 0

C. _§lassware

All the pyrex glassware that was to be used in any test
involving chlorine was used only for this purpose and was
washed in a prescribed manner. That used to store zero de-
mand water was treated several times with chlorinated water
and then it was used for no other purpose. The four ounce
bottles for the preparation of test chlorine solutions were
treated similarly. Test tubes and pipettes were washed with-
out detergents in tap and distilled water, autoclaved if

necessary, and soaked in chlorinated water before using. The

25

test tubes were dried in an inverted position in a 37° incu-
bator. The pipettes were dried in a vertical position. In
addition any pipette used to deliver a chlorine solution was
rinsed several times with the chlorine solution before an
actual transfer was made.

Wherever possible glass stOppered bottles were used for
all chlorine solutions. If not possible, paper closures se-
cured with rubber bands were used. No cork or rubber stoppers

were ever used on a bottle containing a solution of chlorine.

D. Water

The water used in this investigation had to be prepared
with the following points in mind: the water had to possess
a zero chlorine demand for any test procedure that involved
the use of chlorine. The water had to be freeeof any heavy
(metal ions that have been proved to be inhibitory for urease.
(Sumner and Somers, A9). For the urease experiments double
distilled water would be needed. It was also used where small
amounts of zero demand water were required in the bacterial
experiments. Where large amounts of zero demand water were

required, singly distilled water was used.

E. Zero Chlorine Demand Water

A method for preparing zero chlorine demand water is
described with a minimum of detail in APHA Manual (50a).
It was prepared _ in the following manner for both double
distilled and singly distilled water: large amounts were

 

 

 

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26

started by chlorinating two and one-half liters of water in
three liter Erlenmeyer flasks to give a chlorine residual of
3-5 ppm. The flasks were closed with paper covers and stored
in a dark cabinet for several days. For the preparation of
buffer solutions, or zero demand water in small,known volumes,
this treated water was transferred to smaller bottles, re-
chlorinated to give a residual of about 2 ppm, and stored again
in a dark cabinet. Prior to the use of water as zero demand
water, any excess chlorine was removed by expoSing the small
bottle to sunlight for about three hours or to germicidal
ultra-violet light for about eight hours. ‘Water prepared in
this manner was found to possess neither residual chlorine
nor chlorine demand.

This rather involved procedure had.been followed to re-
move from.the water any extraneous substances with which.
chlorine might react. By this method the germicidal activity
of chlorine could be directed wholly against the experimental
objects that were used.

Chlorine can react with.many types of substances such as
reducing agents; non-nitrogenous, organic compounds; thio-
cyanates; proteins and peptides; amino acids; and ammonia.
'The presence of such substances would reduce the amount of
chlorine that would be available for action against micro-
organisms. In this investigation, the distilled water used
was presumed to contain ammonia. The amount of chlorine that

reacted with this ammonia and that would be removed from the

27

water as a result of the reaction is called the chlorine de-
mand of the water. This action in an excess of chlorine re-
sults in the eventual chlorination of the ammonia to a vola-
tile nitrogen trichloride and in a residual of chlorine called
free chlorine. The destruction of this residual by sunlight
provides a water free of any ammonia or chlorine. Chlorine
may now be added so that its concentration is proportional

to the volume of concentrated chlorine used and so that it

is present in the form known as free chlorine. This latter
is conceived of as existing in water as an equilibrium.mix-
ture of hypochlorous acid and hypochlorite ion. This was

the form of chlorine sought after by means of the rather in-
volved procedure described above. It is analogous to the

use of an analytical, reagent grade chemical. It provides

a known factor in the investigation. Free chlorine is ex-
pected to act to the full extent of its concentration against
the objects presented to it. Chlorine that exists in comp
bination with ammonia or organic nitrogen is called combined
chlorine. This is a far less suitable reagent and was not

used. (Phelps, 51).

F. Measurement of Chlorine Concentrations

The methods by which chlorine concentration is usually
measured is some form.of an oxidation-reduction reaction.
In this investigation concentrations of chlorine were measured
in distilled water. Under these conditions any of the chemi-

cal methods would be satisfactory. The one chosen was the

28

amperometric method which gives values of chlorine concen-
tration accurate to tenths of a part per million (ppm.) and
which enabled one to distinguish clearly between free and
combined chlorine. The amperometric titrator and the method
of its use is completely described by Wallace and Tiernan (52).

Test concentrations of chlorine were made by adding drops
of concentrated chlorine to 100 ml. of buffered, zero demand
water. Twenty-five ml. of this test solution was diluted to
200 ml. with zero demand water. This diluted chlorine solu-
tion was titrated with a prepared phenylarseneoxide solution
in the amperometric titrator. At the end point, the volume
in milliliters of the arsenic solution used, was equivalent
to the ppm. of free chlorine. This was multiplied by eight
to compensate for the dilution of the test solution. The
dilute solution could be titrated further for the presence
of combined chlorine. One dr0p of five percent potassium
iodide was added to the solution whose free chlorine content
had Just been determined. Phenylarsenoxide was again used
to titrate to another and point. If combined chlorine were
found to be present in an amount greater than .01 ppm. the
test solution was discarded and a new one prepared.

A satisfactory test solution, containing free chlorine
in a known concentration was used in either a bactericidal or
the enzyme inhibition test within 10 minutes of its preparation
and within five minutes of the measurement of its concentration.

It was used only once.

29

G. Buffers

All buffers used in the experimental work were phOSphate
buffers. In the range of their most effective buffer action
the salts, potassium acid phosphate and dipotassium phosphate
were used. Each of these was prepared as an M/lS solution,
using double distilled, zero demand water. Only 100 ml. of
such stock solutions was made up at any one time to elim-
inate long storage periods and possible mold growth. Usually
such an amount lasted for ten days. During that time it was
stored at 5°C. For pH values outside the most effective range
of the phosphate buffer salts, phosphoric acid was used with
potassium acid phosphate for the lower pH range and potassium
hydroxide was used with dipotassium phosphate for the higher
pH range .

The buffer solutions that were to receive chlorine and
that were used as buffered water in the protocols were made

up as follows:

pH H3P0u(M) KHéPOu(M/lS) KZHP04(M/15) KOH(M) (zerflngggand
double dist.)

t.0 0.1 ml 4,m1 ---- ---- 96 ml

7.0 -- 1.9 " 2.1 ml ---- 96 "

7.5 -- 1.0 " 3.0 " ---- 96 "

9.0 -- --- u.o " 0.1 ml 96 "

The above made buffers were chlorinated to a residual two ppm.
to secure zero demand and to disinfect the buffer solutions.

Such solutions were stored in the dark for several days. They

30

were exposed to sunlight or ultraviolet light before use.
Measurements for pH were made before chlorination and Just
before use. Differences of pH amounting to one-tenth of

a unit were found usually in the direction of a lower pH.
The zero demand buffers were used to prepare test concen-
trations of chlorine as described under F.

The buffer solutions that were used to neutralise chlorine-
bacteria mixtures or to neutralise chlorine-urease mixtures
were of a higher buffer index and were of pH 7.0. In view
of the dual purpose of such'buffers it was deemed simpler to
continue the use of zero demand, double distilled water in
their preparation.

The sulfite buffer, pH 7.0, for the bactericidal test

was made as follows:

Potassium.Acid Phosphate (M/lS) 10 ml.
Dipotassium Phosphate (M/lS lO "
Sodium Sulfite (0.5 gm/lOO ml) 10 "
Water 20 "

Fifty ml. were prepared in glass stoppered bottles.
Pipettes used for transferring were sterile. Sodium sul-
fite solution had previously been autoclaved. Only enough
for one day's testing was prepared and was discarded at the
end of the day's work.

The urea-sulfite buffer pH 7.0 for the enzyme inhibition
test served the additional function of providing a substrate
for the enzyme that had not been inactivated by the action of

chlorine. It was made up as follows:

 

31

Potassium Acid Phosphate M/lS 10 ml.
Dipotassium Phosphate M 15 10 "
Sodium Sulfite (0.5 gm 100 ml) 10 "
Urea (20 gms/lOO ml) 10 "
Water 10 "

Fifty ml. were prepared in glass stOppered bottles.
Pipettes used for transferring were sterile. Sodium sulfite
solution had previously been autoclaved. The urea solution
had been kept under refrigeration for one month before use.
Only enough for one day's testing was prepared and was dis-

carded at the end of the day's work.

H. The ogjects of the Action of Chlorine

l. Bacteria. One species of bacterium, E. coli, strain
ngQ, originally obtained from the Environmental Health Cen-
ter of the U. S. Public Health Service was used. The stock
culture was subcultured every three weeks on nutrient agar
slants, Difco (533). The inoculated slants were incubated
for twenty-four hours at 37°C. The culture was then sealed
by impregnating the cotton plug of the culture tube with
paraffin. The sealed culture was stored in the dark at room
temperature for three weeks and then subcultured.

A working culture was prepared in nutrient broth, Difco
(53b). This was started from one of the slants mentioned
above. After twenty-four hours of incubation at 37°C trans-
fers were made by sterile pipette for the purpose of sub-
culturing into nutrient broth and of preparing a culture for
use in the bactericidal test. Subculturing was performed

every twenty-four hours. Cultures for bactericidal testing

32

were prepared the day before such tests were to be run. One
tenth ml. of the nutrient broth culture was pipetted onto

the surface of a sterile, nutrient agar slant in a 16 x 150 mm
pyrex tube. The inoculum was distributed over the entire
slant by tilting the tube. The inoculated tube was incubated
in an horizontal position for 2h hours at 37°C.

Previously a McFarland Nephelometer, (Kolmer, Sh), had
been prepared. This was a series of tubes containing differ-
ent concentrations of barium sulfate. The graded turbidities
of the successive tubes are taken to indicate different num-
bers of bacterial cells per ml. Also, some Pasteur capillary
pipettes had been plugged, individually wrapped, and steril-
ized. Finally, 10 ml. of zero demand water, containing no
chlorine, had been dispensed in tubes the same size as the
nephelometer.

With a capillary pipette, to which a rubber bulb had
been attached, some of the water was removed from one of the
tubes containing 10 ml. of zero demand water. This water
was delivered to a slant containing a 24-hour growth of the
test organism. This growth was quickly washed off the slant
- within 30 seconds. With the tube held horizontally and the
slant uppermost, the capillary pipette was inserted into the
small volume of thick suspension and manipulated to take up
and expel the suspension rapidly for a two-minute period.
This procedure was used in order to break up clumps of cells

and prepare a suspension of single sells. The entire sus-

pension was then drawn up into the pipette.

33

The suspension was added drop by drop to the same tube
from which the small amount of water had.been withdrawn.
Comparison with the proper nephelometer tube was made after
the addition of each drOp. The process continued until a
satisfactory match had been obtained.

This standard suspension of micro-organism was usually
between 100 x 106 and 300 x log/ml. It was the suspension
upon which bacterial nitrogen measurements were made directly.
It was also the suspension from which suitable dilutions were
made for the bactericidal test. Consequently the bacterial
nitrogen content present in the bactericidal test was derived
by multiplying the bacterial nitrogen determination figure
by certain dilution factors. The usual dilution factor was
1000.

2. Enzyme, Urease. The enzyme used in these experi-

 

ments was urease. As noted in the review, sulphydryl enzymes
are especially sensitive to chlorine. Urease possesses
sulphydryl groups and gives tests for such groups, (Sumner
and Poland, 55). Crystalline urease was prepared from Arlco
jack bean meal by the method of Sumner as described by
Sumner and Somers (56). The purification did not proceed
further than the first crystallisation due to the sensiti-
vity limits of the method used for the detection of organic
nitrogen. As soon as the solution of these crystals had

been obtained, dilutions of it were made and these were

tested in the following manner:

34

Urease Dilution 0.5 ml.

Buffer pH 7.0 0.5 "

Urea-Sulfite Buffer 1.0 "
Incubation 30°C 15 minutes

N'HCl l .0 "

Nesslerisation of a portion of the acid reaction
mixture '
Photelometric reading of the Nessler Reaction
That dilution which produced a photelometer reading of
between u5-50 with a 0.3 - O.h ml. portion of the acid re-
action mixture was chosen as satisfactory for the tests to
follow. This was found to be a.l/&25 dilution. The entire
lot of crystalline urease solution was then diluted in 125 m1.
batches using zero demand water free of chlorine. The di-
luted urease was dispensed in six ml. amounts in four inch
test tubes. Each tube was corked and the entire lot was
quickly frozen and kept frozen at about ~5°C. For use, only
the required volume of urease was thawed. It was used once
and discarded.
The urease nitrogen figure was derived from determina-
tion upon various volumes of the diluted preparation. This

diluted preparation was also used in the enzyme inhibition

tests.

I. Nitrogen Determination

The usual organic nitrogen determinations by the Kjehldahl
technique detect 0.5 mg. or more of organic nitrogen. The
method desired for this work was the one which would detect
at least .05 mg. and possibly .02 mg. The method chosen was
that described in Manual TM8-227 (57). This was a colorimetric

35

method that was described as capable of detecting .05 mg.
to 0.2 mg. of nitrogen. In its application it was variously
modified.

The ammonia formed by the digestion with sulfuric acid
and catalysts of a sample containing 0.05 - 0.2 mg. of nitro-
gen was estimated from the color obtained by adding Nessler's
reagent to the diluted digest. It was recommended that potas-
sium.persulfate be used during the final stages of the digestion.
to hasten the oxidation of organic matter. However, the sam-
ples of persulfate that were available contained more nitrogen.
than the sample being analysed. It was, therefore, omitted.

A uniform procedure of digestion with sulfuric acid and copper
sulfate was used for all samples. Fifteen minutes from the
time of the appearance of the first dense,white fumes was
found to give a water clear digest. Silica chips, glass beads
or any other form of anti-bump material was eliminated as it
proved to be but another source of extraneous nitrogen. Care-
ful heating over a small flame prevented any accidents due to
bumping. ,Large pyrex tubes, 25 x 200 mm., were used.

Nessler's reagent was prepared as described in the APHA
Manual (50c). The water clear digest was allowed to cool.
Twenty-five ml. of recently boiled and cooled distilled water
was added to the digest. Five ml. of Nessler's Reagent was
added as the tube was undergoing a swirling motion. Twenty
ml. of water made the final volume 50 ma. For organic nitro-

gen detenminations five ml. of Nessler's reagent was found

36

necessary in order to completely neutralise the acid of the
digest.

The mixture was allowed to stand for ten minutes and then
examined in a Cenco Photelometer equipped with a number 65
Wratten gelatin film filter. A blank prepared by digesting
and Nesslerizing the reagents was run along with the sample.

The procedure described in Manual TM8-227 (57) did not
prescribe any definite filter but recommended any suitable
one in the range from 450 to 500 millimicrons. Three such
filters were tested using Nessler's reagent and standard
ammonium chloride. Data for the filters was obtained from
"wratten Light Filters," a data book of the Eastman Kodak
Company, and are presented in Table I along with the phote-
lometric determinations. These latter are graphed in
Figure 1. Number A9 filter proved to be too sensitive and
the plotted points non-linear. Number h? filter proved to
be too insensitive and the plotted points, slightly non-
linear. Number 65 filter proved to have a sensitivity inter-
mediate in value and the plotted points resulted in a straight
line over the concentration of nitrogen measured. The data
of Table l for the number 65 filter were replotted in Figures
2 and 3. Assuming a zero value of nitrogen for the value of
the blank, a table was created and inserted in each figure
giving nitrogen values equivalent to photelometric readings.
These standard values were used to obtain all values of organic
and urea nitrogen in subsequent tests.

(continue reading on page 41)

37

 

 

 

 

 

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FIGI
CHOICE OFA SUITABLE FILTER

+WRATTEN Q!

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x wanna 55,5ML. NESSLER +
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, LOG PHOTELOMETER READING

39

FIG 2 _
STANDARD CURVE roe BACTERIAL NITROGEN AID LREBE NITROGEN
«AMMONIUM CHLORIDE OJ IO. N I I.

NESSLER SOLUTION 5m. .
TOTAL VOLUME 50 It. EWIVAIENTS

PHOTELOIETER was WRATTEN 65 FILTER um “N.

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FIG 3

STANDARD ClRVE FOR ‘UREA NITROGEN
AMMONIUM CHLORIDE ONION/MI.
NESSLER SOLUTION IML.

TOTAL VOLUME SOIL. EQUIVALENTS

.m AMMONIA NITROGEN

 

 

 

 

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30 91-92 .91
88-90 .02
85-87 .03
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77-79 .96
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67-68 .19
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55-56 .15
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50 .18
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46-47 .20
44-45 .21 -
43 .22
11-12 .23
40 .21.
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38 .26
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LCB PHOTELOMETER READING

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The two determinations for organic nitrogen were those
for bacterial nitrogen and urease nitrogen. Five ml. of
Nessler's reagent was used. The determination of urea ni-
trogen as a result of the action of urease upon urea was

performed using one ml. of Nessler's reagent.

J. The Bactericidal Test Procedure

Upon completion of the preparatory work described in sec-
tions A through I above, the bactericidal and enzyme inhibition
tests were set up according to the Protocols Al and A2. The
bactericidal test involved the action of variable chlorine
concentrations acting upon a constant number of bacterial
cells during six different time periods - l, 2.5, 5, 10, 20,
and 30 minutes. At the end of each time period the action
of chlorine was stopped by the addition of the sulfite buffer,
pH 7.0. This buffer also changed any adverse pH to a favor-
able one. As rapidly as possible the contents of each reaction
tube were suitably diluted and plated out - five .02 m1. drops
of each dilution onto the surface of a dry Eosin Methylene
Blue Agar Plate. The medium used was that described in Difco
(53c). The inoculated plates were allowed to stand at room
temperature for about one hour until the surface inoculated
drops dried down. The plates were then inverted and incubated
at 37°C for 2h hours. At the end of this period the plates
were counted. The numbers of cells were recorded. The numbers
of cells present in the medication tubes surviving the action

of chlorine were calculated. Log time - log survivor curves

1.2

were plotted for different ratios of bacterial nitrogen to
chlorine dosage used. This ratio of N/Cl bore the units,
micrograms of ammonia nitrogen per micrograms of chlorine.
Suitable dilutions of the medication tubes were based
upon the method of counting and the contact time interval
of the action of chlorine. The method of plating by drops
upon the surface of a plate was described sketchily by
Pomales-Lebron and Fernandez (58) and more fully and criti-
cally by Reed and Reed (59). Two hundredths of a ml. was
the volume dropped upon the plate surface and five such
drOps were used from each dilution tube. The dilution sought
after was one which with a drop volume of .02 ml. would pro-
duce five to 75 colonies. In order to be sure to obtain
numbers within this range, three dilutions were made from
each medication tube. Tubes that had beenuexposed to low
concentrations chlorine for short periods of time were di-
luted as were the controls. Dilutions became progressively
lower as the concentration of chlorine increased and as the
.time of contact increased. Reed and Reed (59) pointed out
that the drOp plate method could satisfactorily replace pour
plates used for counting micro-organisms from milk and water
if sufficient attention was paid to details of the test and
if a sufficient number of micro-organisms was present. Fur-
ther advantages were that it was somewhat more accurate than
conventional pour plates, that it gave higher counts, that
differential media could be used, and that it was less

#3

laborious. Snyder (60) in a relative study of plate counting
methods concluded that it was possible to recommend more ex-
tensive use of surface plate counting methods which1were con-
ducive to a greater replication of plates. The drOp plate
method was considered a satisfactory procedure for this re-
search.prob1em as it gave a consistent body of data. It

was felt that a greater accuracy was achieved by counting

one plate with five drops than would have been achieved by
counting two pour plates. And this feeling was no doubt
accentuated by the fact that less work was involved.

The incubation of plates for 2h hours at 37°C was con-
sidered standard practice. The drop plate procedure,however,
produced countable colonies in 12 hours. If contamination
could be ruled out these counts were as valid as those at
24 hours. However, the colonial appearance of the early
colonies was not distinctive enough for this purpose. Only
the results of the count at 2A hours were presented in the
data tables. Drops that presented between 5 and 75 separate
colonies were counted. The average of the five drop-counts
was entered into the data sheet. These counts were then
corrected for various dilutions to give the actual number
of organisms present during the bactericidal test.

Once the data were obtained, how could they be presented
significantly? .The usual method of plotting the results
of experiments, involving the killing of micro-organisms,would

be to use arithmetic time units as abscissae and logarithmic

so»: ..

 

percentage survival units as ordinates. This method of pre-
senting data from disinfectant testing originated with
Madsen and Nyman (21) who used the data of Kronig and Paul
(22). Chick (23) confirmed this exponential relationship
and postulated it as a universal law of disinfection. For
micro-organisms at least, death was supposed to occur to
a definite percentage of the survivors in each succeeding
time period. The multitude of investigations that followed
only partly confirmed this relationship. Hewever, the ten-
dency of the investigators was to assume that the exponential
relationship was the correct one and that the discrepancies
observed were due to the conditions of performing the ex-
periment, the nature of the bacterial suspension, or the
peculiar nature of the bacterial cell at a certain period.
The typesof curves found by these investigations fell into
three classes:

Exponential time-survivor curves;

Sigmoid time survivor curves;

Exponential time-survivor curves withLan initial lag phase.

These classes of curves were obtained with individual
data or with data representing the means of large numbers of
determinations. Thus, it was not Just the experimental data
or the conditions of the experiment that were at fault. The
basic assumptions made by those who postulated an exponential
relationship of bacterial death were that the organisms of a

culture were essentially uniform in resistance; that their

15

death was due to one event; and that the times of survival
were not normally distributed. Could the data of the non-
exponential relationships be explained away by such simple
assertions that the organisms were clumped together or by

such complex assertions that the nature of such reactions

changes under the experimental conditions or that the re-

sistance of the organism presents different aspects during
the killing process?

Data from other sources than bacteriology indicated that
variation is a rather fundamental attribute of living matter.
Therefore the postulation that a culture of bacterial cells
was of uniform resistance would run counter to the expected
variation of living organisms. In line with this viewpoint,
the resistances exhibited by a culture of micro-organisms
would be a variable. The length of time that any one organism
could survive in a bactericide would be prOportional to its
resistance. But the obJection was raised that if such sur-
vival times were plotted as frequency distributions then
extremely skewed curves of resistance are obtained. Thus
it was argued by the proponents of the exponential relation-
ship that there was no normal distribution of resistances but
rather, there was only one uniform resistance. Hewever, this
argument supposed that when any attribute were measured then
this attribute itself had to be normally distributed. That
this was not necessarily so had been pointed out by Gaddum (61).
In.many cases it was not the attribute itself but the logarithm

A6

of the attribute that was distributed normally. Withell

(62) proceeded to show that by adopting this point of view
all of the previous conflicting data could be harmonized

and the dynamics of bacterial death was brought into con-
formity with that of other forms of life. The plot of log
time-survivor curves from all types of data produced but

one type of symmetrical S-shaped curve from which, normally
distributed frequency diagrams of survival times could be
plotted. Thus, resistances of a culture were not essentially
uniform but variable. The degree to which these varied would
be expressed by the slope of the S-shaped curve. When the
slope of this curve was steep then the variation in the re-
sistance of the culture would be small. The bulk of the
resistances would be clustered about the mean. This type

of distribution in a culture had provided the exponential
curves of the experimenters. If, however, the slope of

this curve was flat, then the variation in resistance would
be great. A much larger percentage of organisms would possess
resistances far from the mean. Such cultures would give sig-
moid curves and curves with a lag phase proceeding the logarith-
mic phase.

Fbr the plotting of curves derived from the data of the
bactericidal test a logarithmic time scale was used to con-
form.with the above point of view. A logarithmic survivor
scale was used as a matter of convenience. Its use enabled

the plotting of four decade cycles of survival from 100

In

percent to .01 percent on one piece of paper. The signifi-
cance attached to the log time scale could be given to the

log survivor scale.

L. The Enzyme Inhibition Test

 

The protocol A.2. indicates the analogy between the
bactericidal and enzyme inhibition tests as performed in this
work. I

At the outset, though, a difference in the manner that
enzyme inhibition test was used in this investigation from
the usual enzyme inhibitor tests might be stressed. Enzyme
chemists provide optimal conditions for the activity of the
enzyme and note inhibition under such conditions. (Heller-
man, 63). The enzyme inhibition test used here subjected
the enzyme to adverse conditions and then restored optimal
conditions for the measurement of the activity remaining.
Thus, it was not analogous to the tests chemists have per-
formed but it was analogous to the tests that bacteriolo-
gists have performed. It was the latter point of view which
was held to be significant for this thesis.

Concerning the test itself, similar precautions were
taken in so far as zero demand, double distilled.water was
involved. Precautions concerning sterility were relaxed
during the final phases of the test which lasted about ah
hour. There was less flexibility in this test in so far as
organic nitrogen was concerned, since once the enzyme concen-

tration had been standardized it could not be altered. Thus,

us

although one was working with a simpler system, the data
obtained were not so widely distributed as that of the
bactericidal test.

Instead of 2h hour incubation of the bactericidal test,
15 minute incubation with a urea substrate took place. To
insure that measurements of activity remaining occurred only
during this 15 minute interval, normal hydrochloric acid
was added to stop the enzyme-substrate reaction at the end
of this period.

The degree to which the activity of the enzyme had been
impaired by its exposure to chlorine was measured indirectly.
After a stated contact period, optimal conditions were re-
stored and the enzyme was allowed to act upon its substrate.
The amount of ammonia nitrogen liberated from the substrate
was taken to be prOportional to the percent activity remain-
ing in the enzyme preparation. The method for obtaining am-
monia nitrogen values was based upon the standard curve for
one m1. of Nessler's reagent (Figure 3) from which an.un-
corrected value for ammonia nitrogen was obtained. This was
corrected for the volume of reaction mixture and for the por-
tion of the sample used in the determination. Finally the
value was converted to mg. of ammonia nitrogen per ml. of

urease 3

Uncorrected mg.NH3-N x volume Of test (ml.) x 1
portion analysed (mIFJ urease (1111.)

0.0.1

 

1+9

Comparison with the control value provided the figure
for the percent activity remaining. Again, the logarithms
of these values were used to provide the plotting of four
decade cycles from 100 percent to .01 percent activity re-
maining. In accord with the discussion under the bacteri-
cidal test the logarithms of the time intervals were plotted.
These curves were plotted for different values of the ratio
of organic nitrogen in the urease preparation to the dosage

of chlorine used. This was expressed as an N/Cl ratio.

End of Experimental Methods

50

V. EXPERIMENTAL DATA - EXPLANATIONS

A. Explanation of the Tables Containing the Data of the
Bactericidal and Enzyme Inhibition Tests as Well as Calcula-
tions Made From Such Data

The tables which follow are in three parts - the left
hand side contains experimental data, the right hand side
contains derived data, the lower part of the tables of the
bactericidal test contains additional experimental data.

The left hand side of tables for the bactericidal test
uses chlorine concentration as the main horizontal heading
and contact time as the main vertical heading. For each con-
tact time there is a subdivision of dilutions to which each
medication tube was subject. The figures within the body of
this portion are averages of actual counts made. The lines
of data in the lower part give the control count, and the
amount of bacterial nitrogen. The main horizontal headings
of the right hand portion are the N/Cl ratios of the corres-
ponding medication tubes of the experimental data. The ver-
tical headings summarize the experimental data for each con-
tact time by providing the count in each medication tube during
the actual contact time; the percent survival that such a count
represents when compared to thecontrol count; and the logarithm
of the percent survival. The general plan of the enzyme in-

hibition test table is similar to that of the bactericidal test

51

but the details that are inserted are different. The main
difference to be noted is.the subdivision of the contact
time. Here in place of dilutions there are the volume of
acid digest mixture used for Nesslerization (V), the phote-
lometer reading (R), the uncorrected nitrogen equivalent of
the reading (Un), and the corrected nitrogen equivalent of
the reading (Corr). The right hand portion of the table
summarizes the observed data under N/Cl headings. Percent
Activity Remaining is obtained by dividing each corrected
nitrogen value by the control value. Log10%AR is the logarithm
of this number. I

The sole organism used in the bactericidal test was E;
22;}, strain g;3_,

The sole enzyme used in the enzyme inhibition test was

once crystallised urease.

B. Explanation of the Graphs that Were gpnstructed From The
Tables of the Bactericidal and Enzyme Inhibition Tests

Fbr both tests the abscissae are logarithms of the con-
tact time interval expressed in minutes. Fbr the bactericidal
test the ordinates are logarithms of the percent survival.
Fbr the enzyme inhibition test the ordinates are logarithms
of the percent activity remaining. Each line represents the
complete vertical summary data under one N/Cl ratio. This
ratio is printed near each curve.

Negative numbers have been derived by subtracting the

positive mm‘z‘ssa- ‘ from the negative W151i of the loga-
P1th1fle

52

Extrapolations were made from the curves that actually
fitted the points. Such extrapolations are represented by
dashed lines. Two such dashed lines are appended to each
solid curve wherever such extrapolation falls in with the
observed data. One is to the line of .01 percent survival
(99.99 percent kill). The other is to the point of 100 per-
cent survival (0 percent kill). These extrapolations will

'be treated further in the discussions.

53

.11. BACTERICIDAL TEST DATA

 

 

 

 

pg gggggg 3352, GRAPHS (Fig.) ngg
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Interpretations of Graphs Page 73

5h

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55

saxw: ooo. n west os x m. a mnos a ss\ws oso.
poo» Heosoanopoon was so usomesm unsoSd nonesseaoo an

oso. mso. oo oo ss.s
mso. mso. mmo. no. mm mm ss m

 

 

 

 

 

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.oos o o .>ssm s oosx . oosx o .o snos
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o.~ o.mx o.wa mmoswos om Nnos
.oos o o .>ssm a oosn o o snos
o.m o o mnos a oosoo o o oos z m
so.s mm.n mo.n mmosmos om mnos
.mo m. ms. .>ssm a oosx o s snos
m.m so. moo. .mnos a posoo ms m oos g m.m
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56

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mac. mo. mm cm H5 mm as non as nowoppan Hessopoem
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o.o o.mx o.mx macsmos os mnos
m.m o o .psso a o o snos
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ms. o.wx o.mx mmosmos m Nnos
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mo.wm o m o m «Nana o owes o wnos
m.s o o mnos a scoop o oos = os
mo.s o.m. o.wA mmoswos ‘ss o mnos
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m nos
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.m asses

59

FIG 5
BACTERICIDAL TEST PH 7.0

 

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41

 

 

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om.s om.s mo.n mmosmos om o : mnos
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mo.s so.s o.m. .maosoos mm so ss o-os
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s.m s.s o mnos a oesoo s .oos a os
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m.m o.s ooo. mnos a onooo o oos z m
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.m os.s mm.n mmosmos om mm ms Mnos
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62

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o.m m.s s. mnos a ooooo oosx oos g os
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oo.s oo.s oo.s msoswos om om om Nnos
I.oo .oo .oo .sasm m oos. oos. oos. snos
o.m o.m o.m mnos a ocsoo -oos g m.m
o.m oo.s oo.s mmosmos mm om sm os mnos
.oos .oo .os .pssm m oos. oos. oos. oos. nos .
m.m d.N o.s mIoa K assoc dooa .nflfi a
moospssso amass
uczoo posucoo
os.o mmo. oso. soxz
mo.\ooo. mm.\ooo. m.\ooo. soxz nn mo. mm.o m.o soo oossosso
....,m._ _.m_ .. s A. . moose o m .m . s moose

 

 

ooom m.s me some saososmmeoom
r o msmoa

O"

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ssst soo. u ws\ms mos a m. s mnos a oso.
poop Hmososnopomn wssaso anemone unsosd oopoazoaeo An

sso. mso. so. oo. ss :s ss :
mNHo. mNo. pm we HE N...
. a .
HS\zImmzws znmmzw: somwmwflwmmnm smmeNSMMM mcospwesasepoa so nomosusz Hessopoem
mos K,N u “pesos Hoapnooo poop Heososaopooo sou some oneness sesaouoem

 

 

osus o.m. oowA mmosmos ss ms Nnos
.oo o o .pssm a oos. oos. o o snos
m s o o mnos s pcooo o o oos = on
as. a”- ... ammo is: o
A o nos
s.s soo. o mnos a scooo o o soos = om
Nous o3” o.wx mmosmos om ss s Nnos
.mm m.m o spam a oos. oos. m o snos
s s mo o mnos a oesoo mm s oos = os
moms osns oo. mmoswos ss m mnos
s.mm m.ms m.m .pssm s oos. mm o snos
mo mnos s ocooo m oos = m
nonmo oonmm .o mmosmos os o o mnos
o.s . .s peso a . oos. mm m snos
o mo mnos a ocsoo om oos = m.o
.m os.s oo.s mmoswos m os
”oos Moo ”ms .sssm m om om ms o muos
o m m s mm mnos a sesoo oosa oos. oos. mm +oos .oss s
.oosoosso amass
. __., I. . I . I. . canoe penance
mo.. ooo. moo. so\z
m~.\smo. s.s\soo. m.m\soo. so\z nn mm.o s.s m.o soo ocssosso
“ ‘. . .. I‘m m ...... s..hay.. (noose s. I m ..‘m. s, . , noose

 

III‘II
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ssst ooo. n ms\ws os x m. a mnos a saxwa sso.
poop HooHoHAouoon mamsao usomoa assess oouoasodeo An
sso. so. ss ss :
sso. mmo. em . mo sa m
we no access
”W HS\zImmwa zlmmzms somofiwmwwmfim HM oSSHoM maospmcaafionon as nowOAsz Heahopoem
mOH.R m u Aussoo Hosesoov use» Hoowosaopoen sou poms maoossa seasopoem
.m so.s. mo. mmosmos mm om es m Nnos
.oos .s: .o .pssm m oos. oos. oos. om snos
o.m s.s om. mnos a Wazoo ooze oesa ooze oos a on
.m os.s oo.s mm was on as : mnos
.oos .oo .ms . .>ssm s oosx oos. sm snos
o.m o.s sm. mnos a oasoo ooze oasa oase -oos = om
.m mo.s oo.s mxoswos .mm Mm om o mnwm
.oos .so .om .>ssm m oosA oos. oosx oos“ snos
o.m o.o o. mnos a oosoo ooze ooze oos a os
.m oo.s mo.s mmosmos m m mnos
.oos .mo .os .>ssm m om os Ism nos
o.m o.s s.m mnos a oosoo oos. oosx mnos = m
.m. mots. mo.s msosts : os
”oos .oo .so .>nso a om sm om muos
m .sm o.m mnos a ossoo oos. oos. oosx snos = m.m
.m .m oo.s mmosmos m s s m mnos
”oos .oos .mo .pssm m on om om om mnos
m .m m.m mnos a osooo oos. oos. oosx oos. snos .css s
cosossso amass
assoc peeacoo
oo. oso. ooo. so} .
ss.\ooo. mm.\ooo. s.s\ooo. so\z nn ss.o mm.o s.s sen osssosno
.m . . m ..w ‘ s ‘, noose : . .m m s noose

 

 

OH mqmda

66

HO 7
BACTERICIDAL TEST PH 9.0

 

 

 

 

 

 

 

oos
.oos
‘I
I
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LOO TIME (we)

 

67

peace on some cos n 0929

ds\ws sso. u ws\ws os x m. a mnos a sexes mmo.
poop Hoosossopeon memsso gnomes assess oouddsoaao an

 

 

 

omo. o. so ss_:
mmo. o. os oo oo ss m
we no a a :
HEKEImmzwz zImmzmz somefiwwwmmnm HMmoMsmom ocospossEAopon as nomoapsz sesaoponm
000.00 . MOH N 0. u.nud5oo Homecoov.poop.amososaopooo 90% com: opossum sessouowm
mo. oo.mx mmosmos s Nnos
m.: o .pssm m oo. s o o nos
mmo. o mnos a oesoo oasa mm o o oos = om
oo. oo.n mmosmos s Nnos
.o os. .25 a m o o snos
mo. soo. mnos w oasoo om s o .oos a om
mm.s om. msoswos m s Nnos
.mm o.s .pssm a oo om o o snos
om. so. mnos a ocsoo oasa osss os o oos a os
os.s os. onwx ososwos .o mnos
.om .m o .pssm a on o o snos
on. no. o mnos a ossoo oaze on o oos = m
oo.s. oo. o.mx mmosmos : mnos
.ms .w o .pssm a m: s o snos
mo. mo. o mnos s oosoo oesa om o os a m.o
oo.s o~.s o-wx mmoswos m N s o mmos
“mo .os o :55 m mm om os, o snos
m s. o mnos s sesoo ooze ooze ooze o oos .sss s
, meosossso amass
assoc pesusoo
ss. «mo. sso. so\z
s.\sso. m.\sso. s\sso. so\z nn s.o m.o o.s Iago osssosso
H. “m... N. .,..~.s .opr s m m s moose

 

Emma AdQHonmBodm
Ha mqmdfi

68

 

 

HE\ws 000. u wa\ws moa N m. K MIOH N HS\wS 0H0.
use» Hoososaouooo wasnso anomosm pcsoad oopsasoaeo an
oso. oso. ss ss 3
sso. mmo. ws s3 m
oso. mmo. mm o in seam
sS\ZI mzmz zImmwa sQMMMWwwmmnm cmwewnsmwm acospsaaasouom As Gomospaz asasopowm
000.mo mos H mo. u Aucsoo Honpcoov poop Hausoaaopomn you does oneness seahopodm
os. som.n mmosmos ms Nnos
.o m. .sssm a mo o o o snos
oo. moo. . mnos a oosoo oasa oo m o oos g om
oo.s om. mmoswos o mnos
.sm .m .>ssm m on o o snos
om. mo. mnos a oesoo ooze om o oos = om
ms.s os. msoswos os o Nnos
.Nm o.m .pssm s oo om m o snos
om. mo. mnos a oosoo oase oesa om o oos a os
oo.s mm.s oo.mx mmosmos ss s Vnos
.ms .sm o .>asm m os om o +nos
os. om. o mnos m.onooo oase oasa o oos = m
mo.s mo.s o.sn macawos os s snos
oo . m: s. . Saw a mo o: o nnos
mo. . oo. soo. mnos a ocsoo ossa oasa s oos = m.o
mo.s oo.s om.n mmosmos s os o o nos
.oo .mo m. .>ssm m mo oo oo o snos
oo. oo. moo. mnos a ocsoo oesa ooze oase m oos . .oss s
sosossso amass
andou poepcoo
oo. omo. oso. so\z
s.\ooo. mm.\ooo. m.\ooo. soxz nn s. mm. m. .sao cosmosco
.m I .N n._ .H . _ noose . :7 m I. N . A sense

 

 

 

 

 

 

 

 

Doom mm UOHHthdoonb amme AdQHOHmmaodm
NH mqmdfi

69

' m ' TEST

PH WCONTRDLLED

 

it use +
/’V .,

 

 

 

 

 

HE\ws 00. u wE\ws mos K m. N Nos K Ha\wfi oso.
poop Hoososaouosn was so anemone pesosd oopuasoaoo An
oso. mo. os ss m
sso. mo. mmo. mm so so ss N

Go a mu:
HE\2Immzws ZImmzwz aonMwmwwmflm thomsaoM acospdcfiaaouon As nowoapsz seasouosm

000.0ms moa N m.s a finance Hoaugoov poop Hooaosaopown 90% noon uaonasn adssoposm

 

 

e e I e I HWO . l

oo.ss mm.s m.s mmmsm w mwss mo M m wnwm
mo. so. mo. mnos a oqsoo oass mo om oos = on

. o nos

om.n ms.sn owosmos mm m o mnos

.s: m. so. .>ssm a ossa o: s snos
m.m oo. mo. mnos a pcooo oase om oos = om

s nos

oo.s so.s so. «mosses ms oo o m mnos

.oo .ss .m .pasm m oasa osss mm mm nos
o.o mm. mm. mnos a ossoo ooze os oos = os

so.s mm.s om.s mmoswos s o mnos

.oo .mm .om .>s=m m os ms ms Mnos
o.s m.m m.s mnos s ocsoo ooze oesa ooze +nos = m

o.m oo.s mo.s mmosmos os o snos

.oos .oo .sm .ssam s ms oo om snos
m.s o.o o.m mnos a ooooo oaze ooze oasa snos = m.o

o.m oo.s mm.s muosmos s o os s nnos

.oos .so .mm .pasm m ms ms mo mm snos
m.s m.o m.m mnos x ossoo case oese oese ooze snos .oss s

eosossso mossy
pcsoo peauqoo
o. os. mo. so\z

4.\00. m.\00. a\wo. H0\z II 4.0 m.o 0.H Ema cashedno
m m s moose : m m s moose

 

 

o on ma oossosooooqo some saososmmsoom
ms msm<s

71

 

 

 

 

 

HE\ws 0H. u m. N I0H K w8\ms 0H K H5\wa Nmo.
- poop HooHoHsopown mamaso anemone masoad popstoHeo An
Nm0. moo. wmdsowwm GOHMMommsm

s5\2nmmzms znmmzms coooaosooons so ossso> cososcsssoooo so cowosssz sassoooom
OOOAOOOAM MOH K OM. u Auflfloo HOEDGOOV umou HNUHOHRmnown’ .HOIH Umwfi MQODESG Addhopowm

mm.s ms. ms.sn maosoos wmso os mums

.so .m so. .>ssm m om m nos
.om .s mo. mnos a ucsoo osse om oos = on

‘ os om m mnos

oo.s ss.s mm.n mm mos oszs o: s mnos

.so .ms m. .>som a ooze os snos
.om .: s. mnos a oosoo osss oos z om

0H N is :IOH

.mos mm.s om.s mm mos om mm os mnos

.mo .mm .os .>som m ooze oo om nos
.mm .os .m mnos a ossoo ooze oese snos = os

oo.s oo.s om.s mmosmos mm om mnos

.mo .so .os .pasm m ooze ooze mm Nnos
.mm .om m.m mnos s oosoo oese ooze snos = m

so.s mo.s os.s mmoswos m snos

.mo .so .mm .>ssm s mm om o mnos
.om .om m.s mnos a oosoo ooze osss ms Nnos : m.m

oo.m oo.m mm.s mmosmos m m : snos

.oos .oos .mm .>ssm m mm on on os mnos
.om .om .os mnos s ocsoo ooze Nnos .css s

moosossso case
MMfioo uodpdoo
o.s mm. os. so\z
s.\os. m.\os. s\os soxz -n s. m. s ass .cssosso
.m _.N H moose : m N H weeps
ooom mo oossosoeooso some sansosmmsodm

:s msm<a

 

LOSS SURVIVORS

72

FIG 9
BACTERICIDAL TEST
PH “CONTROLLED

 

 

“ta ‘
K
‘ ‘. .
'n‘!
e

‘0

 

 

 

 

 

 

2.0

73

Interpretation of the Graphs Derived from Bactericidal Test Data

 

1. Individual Curves from any one Figure.

At least one curve in each.figure could be plotted
through the six points of the contact periods. This deter-
mined the general shape of the curve given by the data. Data
for any N/Cl ratio which provided fewer points were plotted
to conform to this general shape. Extrapolations were also
drawn to conform to this shape to the points of .01 percent
and 100 percent survival.

For any one N/Cl ratio, an increase in contact time re-
sulted in a decrease of the number of survivors.

2. Groups of Curves from any one Figure.

In any one figure, an increase of the N/Cl ratio re-
sulted in a shift of the group of curves to the right and
upwards. Since an increase of the N/Cl ratio could mean
either an increase of the micrograms of bacterial nitrogen
or a decrease of the micrograms of chlorine, then it could
be concluded:

That in the presence of an increased number of bacterial
cells as represented by an increase in bacterial nitrogen
or in the presence of a decreased concentration of chlorine,
the time to obtain a specified percentage survival of bac-
teria was increased (rightward movement) and the percentage
survival of bacteria at a specified contact time was greater
(upward movement).

On any one figure the extrapolated values for .Ol per-

cent survival could be obtained. These values were in accord

7h

with the above conclusion. The times for .01 percent sur-
vival increased with an increase in the N/Cl ratio.

On any one figure the extrapolated values for 100 per-
cent survival could be obtained. These values correspond
to the values obtained for .01 percent survival in the sense
that a short 100 percent survival time was followed by a
short .01 percent survival time and a long 100 percent sur-
vival time by a long .01 percent survival time.

3. Groups of Curves from all the Figures for Controlled pH
Values.

From all these figures together a comparison of corres-
ponding N/Cl ratios provided comparative data for the bac-
tericidal activity of chlorine at different pH values.

It was noted that as the pH increased in value any one
N/Cl ratio provided a curve that was shifted to the right
and upwards. Thus with increasing pH value but in the pres-
ence of relatively comparable amounts of bacterial nitrogen
and chlorine, the percent survival of bacteria was greater
and the time to obtain a specified percent survival was in-
creased.

At high pH values lower amounts of bacterial nitrogen
or increased chlorine concentrations were required to secure
percentage survivals and time intervals comparable to those
at low pH values..

A. Curves obtaines with uncontrolled conditions of pH.

Figure 8 represented the data obtained with bacterial

numbers that were comparable to those that were used to

7S

obtain Figures u to 7. Prom Figure 8 it could be concluded:

a. That discrepancies in the grading of the curves

may arise due to failure to control pH. Thus N/Cl .09

is further to the right and higher than N/Cl .11.

b. That the curves presented values of N/Cl ratios

that fitted best onto the curves with values of N/Cl

ratios obtained at pH u.0 and pH 7.0.

Figure 9 represented data obtained with bacterial num-
bers that were much greater than those used to obtain Figures
h to 7.

If these curves wer: compared to Figures H to 7 it could
be seen that they couldjbe fitted even roughly to any of the
groups of curves for controlled pH values. Thus the use of
N/Cl ratios is restricted to the comparison of groups of
data that have been derived from tests performed with com-
parable amounts of bacterial nitrogen.

The use of increased amounts of bacterial nitrogen and
corresponding increases of chlorine concentration which.main-
tained a numerically similar ratio resulted in a shift of
N/Cl curves downward and to the left. Thus, the somewhat
paradoxical result was obtained that with large bacterial
numbers and with higher chlorine concentrations, smaller per-
centage survivals and shorter time intervals were obtained.

It must be stated, however, that this conclusion is not

too reliable since it was based on results obtained with un-

controlled pH data.

76

A summary table of the points of .01 percent survival
is presented in Table 3; and of the points of 100 percent

survival in Table 39.

’0

77

 

VII. ENZYME INHIBITION TEST DATA

 

 

 

 

 

 

pH TABEES Page GRAPHS (Fig.) Page

h-0 15 78 10 81
16 ' .79
17 80

7.0 18 82 11 85
19 83
20 8LI

7.5 21 86 12 89
22 87
23 88

9.0 24 90 13 93
25 91
26 92

Uncontrolled 27 94 1h 96
28 95

 

 

 

Interpretations of Graphs Page 97

78

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poo

commas Ho .HS sea unoHo>Hsvo somospss concessoo u aaoo

Q
use

nouHAonmoz mg»

on» so paonpssuo GomospHn oopoossoosb u up
so waspwoa sopoEOHoponm a m

onpomHsonmoz sou new: pmowso oHos Ho oSdHo> I >

 

 

 

 

ms mamas

oonHnan th>Hpos psooosmIMMEMPHsemoHI at amen wchHdEmm th>Hwo4 useoaom I #MHIR
. -. Mum o. wo. so. ssoo
. e o oo. o. moo. on
s2.s om. mm.- .ss. m.s .ss .ss .so .so a
.om .m «. ems m a. o. o.s o.m > = o:
I- . . . .HRO
- n om.s mmm. mmm. soo
oo.s me. o same mos .ms .mo .mo m
.os o.m .s smo a o. :. o.s > = om
o.m m.s ms ms. saoo
o moan sm.m: o.M mo. mo. o no
o.s oo.s oo. - 6s . ,. o .oo .o .m
.om .os .sm as and m .2. is +1 o.s > .. os
s.s o. ms. ssoo
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I . . . . . .m .o m
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s.m o. o ssoo
2 es. mo. o no
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.os .om o ems s o. s. s. > = m.m
.. o.m 0.N 0. N. ssoo
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oo.s om.s mo. ssmawos .oo .mm .mo .mo m
eNQ eON 0N1 *g R :0 .30 .3. .Je > enhHE H
amass
. _. posunoo
ms o so sonz
sm.\m mo.\m s.s\m soxz nn in. mm. s.s soo cosmosso
. m I. m. . o m s Images

 

 

79

 

 

 

 

 

 

OH mqmda

 

0e 0e me mHOe ennHOU
2. om. so. mo. moo. so
mm.s o.s o.o we won .oo .os .om .mo x
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om.s am. mm. on
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.om .om .m as m o. o. m. > g om
N.m o.s o. N. naoo
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os.s om.s mo. m4.mos .mo .oo .mo .mo m
.mo .om .s as m s. s. o. s. > = os
o.m o.s s. taco .
s seem ms.om oo. s mo. so
mo.s o.s ss.s as . .o .oo m
.so .ss .ms me a o. .s. s. o a m
o.m s.s m. ssoo
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oo. s os. s om. s ms. was .sm . mo . mo m
.oo .sm .os me a o. o. o. > a m.m
o.m o.m 0.N 0. ssoo
om. om. ss. so. so
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.oos .so .om m4 a o. o. s. s. > .sss s
amass
poopcoo
mm ss m.m so\z
ms.\m mo.\m o.\m so\z oo ms.o m».o m.o _soo osssosso
. \.mn m s H.+n...oooa. :. I _m . m s moose
o om o.:_mn some zosssmstH msswzm

80

 

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.oos

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mo.s
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s.o\m mm.\m m\m so\z o s.o m~.o m.o
ooom o.s mo some zosasmsmzs mssuzm

sH

mqmdfi

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manna

 

 

81

 

 

 

 

 

 

 

82

 

 

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2 _ om. oo. moo. moo. no
oo.s oo.w. oo.wn m4 was .0: .ms .oo .oo m
.om o o m4 m d. o. .m .m > = o:
s.s mo. o naoo
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«m.s ms.u oo.wA m<.mos .ms .oo .so m
.mm s. o os.s +1 .m .m > z ow
s.m o.s no. mo. unoo
2 sm. oo. moo. moo. no
NOOH o.o mafio-I m4 moq cm: 000 omw .mo m
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s.m ms. no. anoo
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mo.s oo. o.o m<.wos .sm .oo .oo m
.os .: .s m4 a d. .s .s > .. m
m.m ms. mo. unoo
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mo.s ms. o.o ms was .sm .oo .mo m
0mm .0 ON a R :0 0H OH > 8 mom
o.m s.N m.o m.o hhoo
9. om. ms. ss. mo. no
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amass
poduaoo
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m.\m ms.\m m.s\m so\z o m. ms. m.s _aoo onsnosno
m N s nonoa : m N s nonsa

 

ooom o.s mo ammo zosasmsmzs mssuzm

 

- os msmsa

83

 

 

 

 

 

o.m o.s m. No. agoo
e m. ms. mo. moo. co
oo.s oo.s ms.- m4 mes .s: .oo .oo .oo m
.mo .os s. m< m d. o. :. o.m > = o:
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ms. so. moo. no
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.ms o.m s. m4 m o. o. o.m s z om
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om. os. so. moo. so
oo.s mm.s .o msgmos .ss .om .ms .oo m
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o.m o.s ms. “goo
a as. ms. mo. so
so.s os.s oo. m4 mas .o: .mo .oo m
.mo .oo .: m4 m o. :. o.s > = m
o.m m.m om. gnoo
‘ os. ms. do. no
oo.s oo.s oo. mosoos .oo .om .mo m
.so .ms .o m4 m o. :. o.s > = m.m
o.m o.m m.m o. “goo
a om. om. ss. do. no
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.oos .os .om m¢ R o. s. o. o. > .sss s
@0238
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ms.\m om.\m ms.\m suxz o ms. on. ms. ago wasposno
m m s moose : m m s moose
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os msmsa

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0 sm a mdewoq Hm.mi Hm.m# ss.mm wo.ms gm
0N moH o o o o O
.oos .os .sm m4 m z. o. s. o. > = os
m.m o.m o.s .88
mo ms H mdemoq smnmi os.m: NH.H© aw
..N CH O O O O
.00H .00 .om E R +7 .3. +7 > = m
m.o o.m o.m anoo
. sm. om. ss. no
.N so.s so.s m<bwos .mo .so .mm m
.oos .mo .so m4 R o. d. o. > = m.m
m.m m.o o.m s.m gnoo
mm s mm s mdawoq om.o: Hm.mi os.m: ms.om gm
ON 0 O O O O 0
.oos .oo .oo m< m o. o. o. o. s .asa s
amass
poaucoo
oslw mm :s so\z
so.\m os.\m mm.\m soxz o so. ms. mm. .aao oasnosno
m m .s o m m s

moons

 

o.s mo some zosasmsmzs mzwuzm
om moose

 

“0959

85

 

 

 

 

 

 

 

 

 

LOOTIKM

86

 

 

 

 

 

o.n1. no. o so. nooo
e om. moo. o moo. oo
coo oo.m. oo.m. m4 mos .so .mo .so .oo m
o.s m .o 5 R :. o.s o.m o.m o = o:
so. so. so. onoo
a moo. moo. moo. no
om. mm.- oo.w. mo mos .oo .oo .oo m
m.m m. o 5 s :. o.m o.m > m om
o.m m. so. o aooo
. 0. mo. moo. o .oo
oo.s mm.- oo.m. m<.woo .oo .oo .oo .so m
.os m. o mo m o. :. o.m o.m > = os
os. ms. so. omoo
.. oo. moo. moo. so
om.s o.o mm.- m< woo .mo .mo .mo m
.om .s m. mo.& 3. o.s o.m > = m
m.s NH. mo. naoo
.. os. mo. so. no
oo.s oo. o.o m¢ mos .so .oo .so m
.om is .s mom :. o.s o.m > .. m.m
o.m s.m us. no. oooo
g. 2 ON. MM. HO. MOO. GD.
oo.s oo. o.o ms moo .so .oo .mo .oo m
oON. 0.: 0H m4 R :0 :0 mo OOH > OQHS H
moawe
audpcoo
ms mwm o.m soxz
om.\m o.\m o.s\m soxz o om.o m.o o.s goo oosoosno
m N a moans : m N H monoe
ooom m.s mo some zosesmsmzs msswzm

AN mqmdfi

 

2"

87 '

 

 

o.m n. . no. mo. anoo
A w m.s: mo. o moo.mo moo.mo .nm
oo.s o.o o.m on co . .o . .
.os .s o oo.s :. o. n. o.s o = on
o. mo. mo. unoo
.. no. moo. moo. no
om.s om. o.o on mos .oo .mo .oo m
.om .m .s os.n :. o. o.s o = om
o.m o.s :m. «s. nnoo
. m. so. mo. mo. no
«m.s oo. oo. on was .s: .:s .oo .oo o
.mm .o .: m4 m n. n. m. o.s > = os
m.s o. os. anoo
. . os. no. mo. no
os.s om.s os. no mo .oo .mo .mo m
.om .om .o oo.s :. n. o.s o s m
H.N o.s m. nnoo
.. ms. so. mo. no
mo.s oo.s oo.s m4 mos .oo .ms .oo o
.os .om .os mn_m :. :. o.s o = m.m
, o.m o.m m.s m. Ahoo
e , om. as. os. mo. no
oo.m oo.s oo.s m4 moo .so .oo .so .oo m
.oos .om .os m4 m n. n. n. n. > .nsa s
mass
ooavcoo
mm os m so\z
m.\m m.\m os\m soxz o m.o m.o o.s _aoo onsnosno
m m s o m. m

 

NN mqmda

I u‘l u. , .llll‘llll’l'lfll

 

moans

88

 

 

 

 

 

o.m m.s mo. ‘wm. voo
2 ms. oo. moo. no
mo.s ms. mm. on mas .o: .os .oo .oo n .
.m: o.m s.s on m n. n. n. o. > = on
m.s o. mo. nnoo
s on s m: n¢.wos os.oo oo.:o moo.mo nm
H- OH O O 0 O 0
.sm .om o.m oo.s :. n. n. o = om
o.m o.s s. m. nnoo
:s m mmemoo os.o: ss.mo so.os mo. o no
OH m CH OOH O O O Ow m
.mm .:m .os ma.m :. n. o. n. > = os
o.s o.s ss. anoo
2 ms. ss. mo. no
oo.s os.s os.s n4 woo .om .mo .so n
.8 .mm .sm 5. m s. n. s. s .. m
o.m o.s m.s nnoo
2 ms. ms. os. no
oo.s os.s ss.s on was .mm .mo .so m
.os .mo .sm on m n. n. n. > = m.m
o. o.m o.m o.s nnoo
e as. os. os. ms. no
.N mo.s os.s m4 mos .oo .oo .om .mo m
.oos .mm amo on m n. n. o. 3. > .nna s
mofisa
. nouuaoo
oo Wm ms so\o
mo.\m N.\m d.\m Ho\z 0 mo. N. 3. Eng cashedzo
m‘ m _s ..lnnoona : m m s moons
ooom m.s on some zosasmstH oosnzm

MN mqmda

 

 

59

 

 

”luull‘ll‘ J

2! fl-

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9O

 

 

 

 

 

 

 

 

s.m so. so. so. nnoo

2 oo. moo. moo. moo. no

oo.wA ooswn oo.w5 on mos .om .mo .mo .oo o
o o o on m n. o.m o.m o.m > = on

so. so. so. snoo

. moo. moo. moo. no

mm.a oo.mn oo.wA on was .mo .mo .oo . m
m. o o on m o.m o.m o.m > = om

s.m mo. o so. nnoo

. os. moo. o moo. no

no. mm.: oo.w. mn.moo .om .oo .so .mo m
m.s o. o on m n. o.s o.m o.m o = os

N. mo. so. Ahoo

.. so. moo. moo. no

so. no. oo.wA on moo .mo .mm .mm m
n.s s.s o on m n. o.s o.m > = m

m. oo. so. nnoo

. no. so. moo. no

oo.s nm. mm.- nn.won .mo .so .no n
.os m.m m. on m :. o.s o.m > = m.m

o.m 3.4 m. no. snoo

2 ms. oo. no. moo. no

ms.s om.s no. on mos .oo .os .mo .oo o
.mm .os s.s on m n. n. m. o.s > .nsn s

amass
.. . oodpcob
.os o n so\o
mm.\m o.\m o.s\o soxz o mm. o.o o.s .ann onsnosno
m ,m s manna : m m s manna
ooom o.o on some zosesmsozs mzsozo

om moons

91

 

 

 

o.mx .so. o so. nnoo
. os. moo. o moo. no
mm.- oonmn oo.mA m<.mos .o: .oo .so .oo m
m. o o on m n. o.m o.m o.m > n on
mo. so. o nnoo
.. moo. moo. o no
mmn. mm.a oo.mm m4 moo .mo .mo .so m
o. m. o on m o.s o.m o.m > = om
o.m no. mo. so. nnoo
.. 8.. w - moo. moo. moo. no
on. mm.- oo.m« on was .so .oo .oo .mo m
.m o. o on m n. :. o.s o.m > = os
. :s. os. so. anon
no. mo. moo. no
sis os. mm... 563 .so .so .mo m
.ms o. m. on m n. o.s o.s > = m
o. oo. ms. .88
‘ oo. moo. mo. no
oo.s on. ow. mnewos .os .oo .oo o
.om .m -. on m n. :. o.s > = m.m
Mom. 00H 00 mmo. aHoHoo
\ O O NH. oo. 000 AND
os.s oo.s on. mnomos .om. .so .os .mo m
.00 00“ OM E R :0 .30 .30 .30 > OHHHE H
amass
. nooonoo
mm o m.: so\z
sm.\m mm.\m s.s\m so\o o sm. mm. s.s soo onsnosno
m .m M_.s..~.._w.noonew. n m m s moons
. [I E
ooom o.o on some zososmsozs mosuzm

 

 

 

mm moons

 

 

92

 

 

o.m so. . so. so. .38

.. os. moo. moo. moo. no

No... No... o.mA on mos .3 .oo .oo .mo n
m. m. .o on m :. o.m o.m o.m o = on

mo. mo. so. nnoo

.. moo. moo. moo. no

mm.- mm.- mm.u on was .mo .mo .oo m
o. o. . m. 5m o.s o.s o.~ o .. om

o.m oo. o oo. nnoo

om. moo. o so. no

on. oo.mA- om. mnewos .sn .mo .so .mo m
.m o .m 5;“ n. n. :. o.s o .. os

s. o. oo. nnoo

.. mo. .mo. moo. no

oo.s ss.s on. on mos .oo .so .oo o
. .mm m.s .m on m n. n. n. o .. m

o.s o. :. Ahoo

. ss. 00. mo. no

ms. s on. s ss. s on. was .mo .os . mm m
.mm .om .ms 5m 3. n. n. o n m.m

o.m s.m o.s o.s nnoo

.. om. ns. ms. 3.. no

mos os.s ms 5 won .3 .om .mo .ms n
.os .00 .mm md.m #. d. d. o. > .oos s

noose
podonoo
oo mm ms so?

oo.\m m.\m :.\m 8% o oo. m. n. nan onsnosno
manna moans

 

 

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93

 

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m. 4 oo. s oo. on. .88
.m os.nm os.so ms.so oo.os nm
\0 0H 0H 5 O1H 0 o o o

QWOWN mflomfl OOH m4 R ON mo OH OH > = mom

m.m m. m.s ms. .28

2 ss. os. ms. ss. no

os.s oo.s nm.s on mos .mo .mo .om .oo m
.om .oo .mm mn.m m. n. o. o. > = m.m

m.o mo.s os.s on. nnoo

2 ms. ss. os. oo. _no

sm.s oo.s .s on mos .om .mo .3 .ms m
.sm .mm .os on m m. n. .s .s o n m.m

o.m m. m.s oo. nnoo

.. ms. os. om. os. no

os.s oo.s om.s on woo .om .oo .om .sm m
.om .oo .om on m m. m. .s .s o n s

o.: o.m o.s m.s nnoo

e om. m. o. om. no

os.s oo.s oo.s m4 mos .mo .sm .sm .mo m
.om .04 .Om. m4 R m. #. o. .H > = H

m.s mo.s oo.s on.s nnoo

oo.s mo.s om.s mnemos smumn mnom osuon osuom nm
.oo .mn .om on m m. n. o. .s o .nsn s

nonso
. _. nonono
om om os so\o row
m.\os m.\os s\os so\z o m. m. s non onsnosno
m , w. s moons n m m s manna
ooom on nossonnnonno some zososmsoos ooswzm

sN mqm4a

9S

 

 

 

 

 

 

 

s.m om. ms. oo. nnoo
9m oo.s mmo.m mo. o so. o nm
0 OF. om. m4 OuH o O o w to OH
.JOOMH om 0N m4 R N. m. 0H OH > 2 OH
mo+~ 00 oo. ANN. ““00
.. ms. o. oo. .so. no
oo.s ss.s os. on was .mm .oo ..os .oo m
.om .ms .m on m m. m. o. o.s o n os
o.m oo. ms. ms. nnoo
2 ms. ss. so. mo. no
oo.s no.s on. on man .mo .mo .os .oo o
.os .ss .m 55s m. .s .s .s o n os
o.m oo. on. os. nnoo
. ss. mo. mo. mo. _no
oo.s o.s ms. on mos .mo .oo .so .oo o
.om .os .o mnnm m. m. .s .s o = m
m.o oo. oo. mo. nnoo
. 9 ns. oo. oo. oo. no
«m.s mn.s o.s on mos .mm .ms .os .ss m
.sm .os .os os.s m. n. o. .s o g m
o.m 3w. mi. :N. onoo
\ . ms. ns. so. no. no
:FOH JOOH 0N0 awoq 000 .mm omN. .mm m
.mm .ss .o os.s N. .s .s .s o .nsn m
, noose
podpnmw
om om os so\m.
m.\os m.\os s\os soxo o m. m. s non onsnosno
m _,m. s moons : m m s moons
ooom on nossononoono ammo oosssmsoZH mosnzm

mm mamas

PIC M
MNTION OF mus:
PR WCONTROLLED

96

 

mL

 

 

LDC‘ACTIVEI’YMM

 

 

 

on 0
L00 TIRE mun

 

97

Interpretation of Grgphs derived from Enzyme Inhibition Test Data

The remarks under the interpretation of graphs derived
from bactericidal test data are applicable to the tests for
enzyme inhibition insofar as points 1 and 2 of that discussion
are concerned.

3. Groups of Curves from all the Figures for Controlled
pH Values.

From all of these figures together a comparison of cor-
reaponding N/Cl ratios provided comparative values for the
enzyme inhibitory action of chlorine at different pH values.

It was noted that as the pH decreased in value any one
N/Cl ratio provided a curve that was shifted to the right
and upwards. Thus with decreasing pH value but in the pres-
ence of relatively comparable amounts of urease nitrogen and
chlorine, the percent activity remaining of urease was great-
er and the time to obtain a specified percent activity re-
maining was increased. This was the reverse of the result
obtained with the bactericidal test.

At low pH values lower amounts of urease nitrogen or
increased chlorine concentrations were required to secure
percentages activity remaining and time intervals comparable
to those at high pH values.

u. Curves obtained under Uncontrolled conditions of pH.

The curves of Figure 14 present values of N/Cl ratios
that fitted best on to the curves with values of N/Cl ratios

obtained at pH 9.0.

98

A summary table of the points of .01 percent activity
remaining is presented in Table 32 and of the points of 100
percent survival in Table 34.

A discussion of the behavior of urease in the presence

of chlorine is presented under section D of the discussion.

99

VIII. EXPERIMENTAL DATA - ERRORS

Discussion of the Errors Involved in the Data as Represented
by the Deviations of Points Shown on the Graphs

Deviations on the graphs reflect back upon the experi-
mental data and the methods used for obtaining such data.
The sources of error may be divided into two categories for
the purpose of this discussion. One was the counting method
which gave the numbers on the data sheet. The other was all
other experimental procedures. It will be assumed that
these procedures provided a constant source of error which
affected the entire body of data in a uniform manner. The
Nesslerisation procedure which corresponded to the counting
procedure was not considered to be a source of error of any
magnitude. In comparison with a biological method, any
chemical quantitative procedure is to be considered a mar-
vel of precision. Instead the wide variations of the enzyme
inhibition test data were considered to be due tp an inherent
inconstancy of the action of chlorine in alkaline media.
This role of the chloramines will be discussed more fully
towards the end of the discussion. I

In general the bacterial count data provided points
that could be used to fit curves of a certain form. There

was one discrepancy in Figure 7 which can be attributed to

an error of recording more readily than to an error of method.

The other two serious discrepancies occur in Figure 5.

100

For the curve representing the N/Cl value of .07, the
point at the 20-minute time interval is about 1 percent lower
than the line itself. The data from which the point was de-
rived showed a count of 12 colonies from a 1/10 dilution of
the medication tube. This represented six percent survival.
If, however, only three more organisms had been counted, the
percentage survival becomes 7.5 and the point falls in the
curve. This difference of 3 in 12 is a 25 percent error
which is about within the accuracy of any plate counting pro-
cedure.

For the curve representing the N/Cl value of .011, the
point at the 20-minute interval is about .6 percent higher -
than the line itself. The data from which the point was de-
rived showed a count of 15 colonies from an undiluted medi-
cation tube. This represented .8 percent survival. In order
to fit a point to the line, the figure for the percentage
survival would have to be .2. The colony count for this
figure would be h, which is below the limit of accuracy of
the counting method. The error of 11 in 15 is a 70 percent
error. This was possible because of the low numbers of or-
ganisms and the small size of inoculum used for counting. It
was one of the limitations of the method used. It led to the
most careful attention to those medication tubes that con-
tained low numbers of organisms and which.would be expected

to yield low counts.

3,01

It should also be recognized that the method of presenting
data as logarithms actually provided four uneven intervals

of percentage survival as follows:

-2.0 to -l.0, .Ol% - .l% or a .09% interval;
-l.0 to 0.0, .10% - 1% or a .9% interval;
0.0 to 1.0, 1% - 10% or a 9% interval;
1.0 to 2.0, 10% - 100% or a 90% interval.

In the regions of high numbers of organisms or high sur-
vival values, any error in the plating and counting procedure
made a slight difference in the percentage figure. In the
region of low numbers of organisms or low survival values,
errors in the plating and counting procedures tended to be
higher and differences in the percentage figures larger.
However the use of logarithms tended to make such differences

less apparent.

End of Experimental Data

102

IX. DISCUSSION

A. Measurement of Bacterial Nitrogen

Although it was stated that a uniform procedure was
used in the determination of bacterial nitrogen and that
therefore the results were relatively significant, other
evidence is provided below concerning the correctness of
the results of this procedure.

The value for the weight of a bacterial cell will be
calculated from the data of this thesis and compared with
a calculation made from data presented by Clark (6A) in a
table "0n the Dimensions of Cells." For E. coli these di-

mensions are:

a) linear dimensions 3 x 1.2 micra;
b) surface area 11.6 square micra;
c) volume 0.26 cubic micra.

Using this last figure the following calculation was made on
the assumption that the density of bacterial cells is approxi-
mately equal to that of water:

0.26 u3 1 6 3 _ -7
6311 x 7% x 10 g x 16% - 2.6 x 10 ug/cell
' ....(2)

From... data in this thesis the average figure of .007 ug.
NH3-N/ml for 2 x 105 cells/ml was chosen. Further it was as-

sumed that the percent of nitrogen in bacterial cells was

approximately 16. Thus:

103

 

.007 usNH -N 1 ug(cell) m1 - -7
51 x I5 ug NH3-N x 2 x 105 cells - 2.:87C:$1

0000(3)

B. The Urease Preparation

The following calculations are provided to indicate
that the urease preparation conformed to data reported in
the literature for other urease preparations.

One pound or approximately 450 grams of Arlco jack bean
meal was used as the starting material. From this an un-
weighed amount of urease crystals was obtained. _This was
dissolved in 12 ml. of sterile water and this was further
diluted 1/125 to give the urease test preparation. The or-
ganic nitrogen determination of this diluted preparation was
found to be S micrograms/ml. Sumner (65) reported that the
percent nitrogen in urease that had been crystallised once
was 15.4 percent. Assuming an approximate factor of 6 then
the amount of urease in the diluted preparation was 30 ug/ml.

The total amount of urease obtained was

30 ug/ml x 125 x 12 m1 = usooo ug = as mg ....(4)

The percent of urease in the original jack bean meal was

s I
figfiflgfig x'iggsfié x 100 .01 percent ....(5)

This was somewhat low in comparison with the figures given by

Sumner who quotes figures ranging from .07 - .13 percent.

104

For the determination of the activity of this preparation a
urea-phosphate buffer of pH 7 was used. The concentration of
urea in this solution was .66M or 660uM/ml. The hydrolysis

of urea was performed at 30°C giving the following results.

 

 

 

 

TABLE 29
Time mgNHB-N m§.Urea uM.Urea mUrea Urea
(Min.) ml.urease ml.urease ngaggase %I%%I%f—' fiydrolysed
1 .20 .%3 7.1 7.1 1.08
205 .38 e 3 1309 5.5 2.1
5 .88 1.88 31.3 6.2 .8
10 1.65 3.50 58.3 5.8 .9
20 3.80 8.00 133.3 ,6.6 20.
30 5.70 12.00 200. 6.6 30.
Av.= 6.3

 

From this table it could be seen that the rate of hy-
drolysis of urea by urease was linear at a rate of 6.3 micro-
moles of urea per milliliter per minute. Such a relationship
for a reaction indicated that the rate of reaction was con-
stant and was not influenced by the concentration of reactants.
This result had been previously reported by Sumner and Hand
(66) for crystalline urease.

The same authors had also expressed urease activity in
units. A unit of urease activity was that amount of urease
which would form 1 mg. of ammonia nitrogen from a urea-
phosphate buffer at pH 7.0 at 20°C in five minutes. Since
the above hydrolysis was run at 30°C it will be assumed that

half the amount of ammonia nitrogen would have been formed

105

at 20°C. This amounted to .hu.mg. NHB-N/ml. Since the

amount of urease was 30 micrograms/ml or .03 milligrams/m1,

 

 

then:
.03 mg. Urease/ml = .066 m8- Urease ....(6)
mm mg. NH3-N fms- NHB-N

Thus the unitage was .066 per milligram or 66 per gram.
But this was the diluted preparation. Therefore the original

crystalline precipitate possessed:
66 x 125 x 12 = 99,000 units per gram ....(7)

Sumner (65) reported upon a once crystallized urease prepara-
tion as having 115,000 units per gram. The result of this
extraction and crystallisation was considered to be in good
agreement with Sumner's values when it was recalled that

the percentage figure for urease in jack bean meal for this

preparation was lower than the amount usually present.

0. N/Cl Ratios

The use of this ratio was suggested in the paper of Knox,
g£_gl, (M6). These investigators, however, reversed the
fraction and used it as a chlorine:nitrogen ratio. It was
claimed that although the bactericidal concentration of
chlorine varied with the number of bacteria, the ratio of
chlorine to bacteria was constant. This dependence of bac-
tericidal action upon the amount of chlorine for a given a-

mount of bacteria was experimentally useful since comparable

106

results could be obtained under widely different conditions.
It also provided a value for the bactericidal amount of
chlorine which could be applied to bacteria and to enzyme
proteins.

The same reasoning may be applied to the use of the
reversed ratio, nitrogen:chlorine, with the note that the
ratio now indicates the amount of organic nitrogen present
per unit of free chlorine concentration. It was expected
that curves for similar nitrogenzchlorine ratios would co-
incide closely and that there would be an orderly stepwise
arrangement of such curves. For the controlled pH experi-
ments, this was generally true. There was only one serious
discrepancy occurring in the group of bactericidal tests at
pH h (Table 2, N/Cl .02). However, this curve was not plotted
as another curve with the same N/Cl ratio was available. When
it was realized that these ratios represent microgram quan-
tities of nitrogen per microgram quantities of chlorine and
that the method of counting organisms involved about a 25
percent error then it was apparent that the ratio was not a
precise one but rather a representation of a tendency. Simi-
lar comments might be applied to the enzyme inhibition curves.
It was noted that the curves for the uncontrolled pH experi-
-ments could be presented in an orderly progression only if
numbers of bacteria fell between certain levels. Thus in
Figure 8 the data were based on bacterial numbers that did

not exceed 100,000, and in Figure 9 the data were based on

-1..-

 

107

numbers that varied from 7.5 x 105 - 3 x log/ml. If, however,
the two groups are combined then serious discrepancies were
noted and there was no orderly progression of curves in terms
of N/Cl ratios. It would seem then that the significance
of these ratios is dependent upon the manner of obtaining
them - pH had to be controlled, and the bacterial nitrogen
could not vary too greatly. The statement in Knox, 33.75;.
(#6) appeared to have been based upon very dense suspensions
of g, ggli_that were used for manometric determinations.
Such suspensions‘possessed high nitrogen values. Two sets
of data were presented and from them the concentrations of
chlorine could be calculated as 10 ppm. and no ppm. Such high
concentrations of chlorine were well beyond those used in the
disinfection of water. This leads one to cast additional,
doubt upon the applicability of these data toward this specific
problem.

Returning to the work of this thesis and remembering
the possible limitations of such ratios, what has been found?
It should be noted that the N/Cl ratios for the enzyme in-
hibition test were much larger than those for the bactericidal
test. It was mentioned in the Experimental Methods that this
followed from the nature of the reagents used. A standard
amount of enzyme had to be present in all test procedures in
order for any comparisons to be made. Once set, this could
not be varied. The criterion for the bactericidal test was

more flexible -- that of viability -- and some variation in

108

the numbers of organisms was permissible and was even in-
evitdble.

Yet despite this difference in the magnitude of the
N/Cl ratios the data of both tests appeared to be comparable.
The curves obtained in both tests were of similar shape, and
they were arranged in a stepwise progression of N/Cl values.
The ranges of the N/Cl ratios in both tests as well as the
excess of that of the enzyme inhibition test over that of
the bactericidal test follow:

109

 

 

 

 

 

TABLE 30
pH u Bactericidal Range of N/Cl .01 - .2
Enzyme Inhibition Range of N/Cl 3 - 5O
Ratio of E/B 300 - 250
pH 7 Bactericidal Range of N/Cl .007 - .11
Enzyme Inhibition Range of N/Cl 3 - 70
Ratio of E/B #00 - 700
pH 7.5 Eactericidal Range of n/01 .005 - .16
Enzyme Inhibition Range of N/Cl 2.8 - 60
Ratio of E/B 600 - loo
pH 9.0 Eactericidal Range of N/Cl .003 - .08
Enzyme Inhibition Range of N/Cl 3 - 60
Ratio of E/B 1000 - 800

 

W

What is the meaning of this excessive ratio of the enzyme

inhibition test to the bactericidal test?

Consider the data of the two tests at pH 7.0:

 

 

 

 

TABLE 31
N701 M°:;CU195 Molecules chlorine 38:31:31 831%
hypochlorite bacterial cell Activity Rem.
.011 .6 ug/ml 6 x 1015 3 x 1010 15 min.
Molecules chlorine
molecules urease
3 1.5 us/ml 1.5 x 1016 5 x 102 20 min.

 

110

The excess of the ratio of enzyme N/Cl over that of
bacterial N/Cl arises from relative figures of organic
nitrogen for the enzyme preparation and for the bacterial
suspension. Although bacteria are giants compared to the
molecules of urease there are more molecules of the latter
than there are cells of the former. In the case of the
enzyme it is known that the chlorine is acting upon a par-
ticle of known weight which for urease is #83,000. (Sum-
ner, Gralen, Eriksson, and Quensel, 67). In the case of
the ail-l bacteria it can only be assumed that all the
chlorine molecules are distributed equally among the cells.
~If the figure for the distribution of chlorine molecules
per molecule of urease were taken as the approximate dis-
tribution of chlorine per large protein molecule and if
the same distribution were assumed for the bacterial cell
then the 3 x 1010 molecules of chlorine would be distri-
buted among 108 protein molecules of the bacterium. As-
suming the volume of.a protein molecule to be about 5 x

10-723, then the total volume of 108 protein molecules

 

 

would be:
-0 -
5 x 10 7A3 x 108 protein molecules x 1 u3 8 5 x102uu3
protein molecule 1012A3

....(8)
And since the volume of the E, coli cell was 2.6 x 10'1113 the
percent fraction of the bacterial cell ocCUpied by the af-

fected protein molecules is

.00g02005 = ca.0000002% ....(9)

111

This would further assume that chlorine acted by dif-
fusing through the cell as a neutral hypochlorous acid
molecule and that it acted upon many loci within the cell
some sensitive and some not. The large N/Cl ratio for
urease might then be taken to indicate that many molecular
centers of one specific enzymatic activity are present.

The nature of the experimental test for its detection was
such that only a few needed to be inactivated to reduce
the activity of the enzyme and to lower its activity towards
a specific substrate. The small N/Cl ratio for bacteria
might be taken to indicate that many molecular centers of
one Specific enzyme were affected or that many molecular
centers of diverse enzymes were affected. It has already
been indicated in the review that the latter seems more
probable. Chlorine is not a Specific reagent and it would
tend to oxidize any protein with which it came in contact.
The nature of the test for the detection of its activity,
i.e. viability of living organisms, was such that complete
disruption of enzyme systems would be necessary to detect
its effect. The small percentage of such centers affected
in relation to the entire cell is reflected in the small
N/Cl ratio.

Some confinmation of the amount of chlorine required to
kill a bacterial cell comes from the work of Heinmets, Taylor
and Lehman (87) whose measurements indicated that over 10

hypochloride molecules are required to kill one organism,,

112

E, coli, strain B/r. Since the calculation in Table 31 had
provided a figure of 3 x 1010 hypochloride molecules per
cell then there would be 106 such molecules available for
the destruction of the cell. Such correspondence of con-
clusions should be considered good in view of the different
techniques involved and the different strains of organism
used.

This result also lends support to a theme of the review.
Bacterial death is not the simple result of the inactivation

of one enzyme.

D. The Relationship of the Enzyme Inhibition Test to the
Bactericidal Test

 

The following table summarizes the results of both
tests in terms of the average time required to reach the

point of .01 percent survival or activity remaining.

113

 

 

 

TABLE 32
EXTRAPOLATIONS TO THE .01 PERCENT SURVIVAL POINT
W
_ Time of .01 Percent Survival (min)
N/Cl Cl/N' pH 4 pH 7 pH 7.5 pH 9.0 uncontrolled
pH
.005 200
.006 170 -- 2 5 30
.007 140
_____ ,- _._._._._._._._..._._._._._._._._._..._..._._._._.
.01 100 3 15 30 100 5 - 60
.02 50
.0 30 u E5 50 >200
.0 25
.1 10 R0 >100 >100 >200 60 ~>100
.2 1? >100
.8 i
1.6 100 -)100
=-==-%’p-==-=-==-=-=B---=-=-----.
Time of .01 Percent Enzyme Activity
Remaining (min.)
.33 100 20 10 7-10 100
.16 ca500 no to 20
1° '1 Vegggd 1E8; 100 20 beyond 500
n
15 .06 begggd 100 ho
25 .04 " " beyond 00
500 l
50 .02 ” " " 100 beyond 500

 

 

The data of the bactericidal test seemed to fit well
with the prevailing concept of the bahavior of chlorine as a

water disinfectant. (Fair, Morris, Chang, Weil, and Burden, 68).

114

Chlorine in water has been considered to act primarily as
an oxidizing agent. When chlorine is dissolved in water it

undergoes hydrolysis as follows:

012 + H20 __.— H001 . H’ + 01' ....(10)

At the concentrations employed in water disinfection this
hydrolysis is complete and rapid, in a very few seconds at
ordinary temperatures. The hypochlorous acid undergoes a

further reaction with water as follows:

HOCl 3’: H‘ + 001' ....(11)

This process is reversible giving the equilibrium expression:

(H+)(001') = K
(H001)

 

0000(12)

At 25°C this ionization constant has a value of 3.7 x 10’8.
The significant part of this equation is considered the ratio
of (0C1')(HOC1). The equation can be rearranged to give:

(001')(H001) = K/(Hi) ....(13)

Thus the ratio is dependent inversely upon the hydrogen ion
concentration and directly upon the pH. Since it has been
found that chlorine in acid solution is more bactericidal
than in alkaline solutions a relationship can be established
between the decrease of hydrogen ion concentration and the
increase of the concentration of undissociated hypochlorous

a01do

115

   

 

TABLE .33
pH Percent Free Chlorine Required
‘ HOCl to give an Equivalent (HOCl)
h 100 1.0
7 75 1.3
7.5 50 2.
9 3 31+.

 

Also since the measurement of free chlorine by a chemical
test is the sum of both the undissociated hypochlorous acid
and the hypochlorite ion concentration, such measurement

does not express bactericidal efficiency which is supposed

to depend upon the concentration of hypochlorous acid alone.
Thus,with a pH greater than u more free chlorine is necessary
to give a concentration of undissociated acid equivalent to
the concentration of undissociated acid at pH 8. Up to pH 8
this amount is no more than four times that required at pH h.
However beyond pH 8 the amount becomes very great.~ The dis-
infectant action of solutions of chlorine may be further
modified by the reaction of hypochlorous acid with ammonia

to give chloramines. However since precautions were taken

to eliminate this type of reaction in this investigation this
subject will not be discussed here. It will suffice to men-
tion that the chemical tests will measure a chlorine residual
due to chloramines with the same efficiency that they measure

chlorine residual due to hypochlorous acid and hypochlorite.

116

Yet the disinfectant efficiency of chloramines is much less
than that of hypochlorous acid.

The data in the paper of Fair, gg.‘gl. (68) demonstrate
the marked decrease in the disinfection efficiency of chlorine
with increase of pH, and the marked increase of contact time
required to reduce the numbers of micro-organisms as the
chlorine concentration was lowered. In the first instance
at pH 7, .03 ppm of free chlorine resulted 1 percent sur-
vival in.u minutes; at pH 8.5, .03 ppm required 30 minutes
for the same survival figure and at 9.8 about 150 minutes
were required. Second, to secure a 1 percent survival at
4 minutes, about .15 ppm of chlorine had to be used at pH
8.5, and 1 ppm had to be used at pH 9.0. Similar results
have been noted in this investigation except that N/Cl ratios
were used. The ratio provided a missing factor from the a-
bove data -- the effect of organic material upon the efficiency
of the germicidal activity of chlorine. At an N/Cl value of
.02 the time for .01 percent survival increased from h min-
utes at pH 0, to 45 minutes at pH 7.0, to 50 minutes at
pH 7.5, and to more than 200 minutes at pH 9.0. To secure
an equivalent survival time to that of pH 4 the N/Cl had to
be reduced to .007 for 7.0 and .005 for pH 7.5 which meant
either that the chlorine concentration had to be increased
n times or that the amount of organic nitr0gen had to be re-
duced h times. This agreed with the efficiency figures for
HOC1 cited at the beginning of this section, Table 32. Thus

the data of the bacteriological test are confirmed by reliable
reports in the literature.

117

The results of the enzyme inhibition tests were, however,
contrary to the results of the bactericidal test. The times
to reach points of .01 percent Activity Remaining are much
greater and the results at the ends of the pH scale were
actually the inverse of the bactericidal test. Thus there
was the result that chlorine at pH 9 is more inhibitory than
at pH k. This result, of course, ruled out any possibility
of the use of this enzyme as_a biochemical reagent for the
assay of bactericidal chlorine. It was the result of at-
tempting to set up an empirical relationship. There was no
generalization at the start of the work that could have pre-
dicted the occurrence of such a finding. There was also
little or no indication for an explanation of the failure

to find a direct relationship. However, one might be at-
tempted.

Urease possesses an isoelectric point of pH 5.1.
(Sumner and Hand, 69). Thus, at pH H the reactive groups
should show a different distribution of charges than at
the other experimental pH values and it might be possible
that under such conditions the molecule of urease becomes
more resistant to the action of hypochlorous acid. Similar
arguments could not be applied to bacteria. The proteins
of the cell are more complex and their isoelectric points
differ. FUrther, the inactivation of a few enzymes under
adverse conditions, might entirely disrupt a delicately

balanced, multi-enzyme system. Sumner and Myrbéck (70)

: 1-; L

.44? .

 

118

presented uncited data of the action of an acid pH upon urease.

At pH k in water the unitage of urease activity was reduced

to 0.8 of its value after 15 minutes exposure of the enzyme

but within five minutes after neutralization the unitage rose

to 0.9 of its original value. Comparative values at other

pH values were lacking. Chlorine at pH 4 did not act destruc-

tively upon urease in the short time periods allowed. It

should also be remembered that the urea-phosphate buffer that

was added to the chlorine-enzyme mixture contained sulfite

which not only reduced the hypochlorous acid to chloride ion

but also provided a poised potential of +200 mv. According

to Sizer and Tytell (71) urease showed a maximum activity

when the potential of the medium was poised at +150 mv. Thus

a chance possible combination of a change in the isoelectric

point and a quick re-establishment of optimal conditions

might account for the persistence of urease activity at pH 4.
For the behavior of urease in the presence of chlorine

at pH 9 another explanation might be offered for the increased

sensitivity of the enzyme to chlorine. At alkaline pH values

some ammonia would be liberated from the protein. This

would combine with the free chlorine present to form mono-

chloramine:

NH +H001 4.- NH

3 201 4 H20 ....(11)

This would be formed very rapidly in the neighborhood of
pH 9. Although monochloramine is a very inefficient disinfectant

119

for bacteria it may or it may not affect the enzyme at this
stage. However, when the pH is lowered some of the mono-

chloramine would change to dichloramine

‘- —-¢- +
2NH2C1 + H «—~— NHu + NHCl2

At pH 7, 35 percent of the chloramine would be present as

....(15)

this form. The dichloramine in the presence of the sulfite
of the buffer Iii would be reduced but slowly to chloride
ion and ammonia. Thus the actual contact period for the
action of chlorine as chloramine would not be known but

it may well extend for some irregular time interval into

the fifteen.minute incubation period. A combination of the
action of chloramines during both the contact period and the
incubation period would result in a prolonged period of
activity of chlorine against the sensitive loci of urease.
The result would be an apparent increase in the sensitivity
of the enzyme at pH 9.0. Now with low N/Cl ratios the con-
‘centration of dichloramine per urease nitrogen would be
relatively greater than with high N/Cl ratios. Thus in the
former case a greater number of the active centers would

be affected and the time to reach .01 percent activity re-
maining would be short. In the latter case the relatively
greater concentration of urease in proportion to chlorine
would provide many more active centers. A lesser percentage
of these would be inactivated and the time to reach .01

percent activity remaining would be extended. This also has

120

been observed and has been summarised in tables at the be-
ginning of this section. (Table 32). The irregular action
of the chloramines during the contact and incubation periods
would also account for the deviations of the points of the
N/Cl curves presented for the enzyme inhibition test. The
observation that the deviations were greater at alkaline pH
values might also be accounted for by the loss of ammonia ”
from the reaction mixtures at the temperature (30°C) of the A
test procedure. In order to explain why the action upon

bacteria is so different one would be forced to fall back

 

upon the argument that more than one enzyme would be in— "w=
volved and that the slow disinfectant action of chloramines

would be balanced by the ability of bacteria to replace any

of the inactivated centers more rapidly than they would be

destroyed.

E. Exprgpolation of Curves in tEe Direction of Decreasinngime

 

This was done merely as a matter of interest and it really
did bear upon any of the significant points of the thesis.
The action of chlorine is so rapid that measurements could
not usually be made at such short intervals. By following
the general shape of the curves they may be extrapolated
back without unnecessarily forcing the data from which they
have been derived. When this was done the intercept at the
100 percent survival line would indicate the time to which
the entire inoculum maintained itself completely. The graded
series of times thus obtained paralleled the points of .01

"IY‘

 

-1

121

percent survival for curves of similar N/Cl value. Short
100 percent survival times corrSSponded with short .01
percent survival times. Long 100 percent survival times

corresponded with long .01 percent survival times.

TABLE 34
EXTRAPOLATIONS TO THE 100 PERCENT SURVIVAL POINT

L E

Maintena 6 Ti e of 100 Percent Survival
n/01 01/11 H" m

 

 

pH (4 pH 7 pH 7.5 pH 9.0
.005 200 1 sec. 20 sec.
.006 170
.007 140 1 sec.
.01 100 .012 sec. 25 " l " 1 min.
.02 50 .06 " 15 " 1 "
.0 30 1 min.
.0 25 1 "
.07 l " l min.
.1 10 2.5 " 20 " 10 " 30 "
.2 10 "

Maintenance Time of 100 Percent Enzyme Activity

3 .33 .03 sec. .001 see. .0001 sec. <.0001 sea
.16 .06 " .06 " .006 " 2.006 "
10 .1 2 " .06 " <.06 "
15 .06 15 " 6 " .6 " .06 "
25 .ou 1 min. 1 min. 10 " .6 "
50 .02 20 u 20 " 1 min. .6 "

 

End of Discussion

122

X. SUMMARY

A comparison was made between the action of chlorine
upon an enzyme urease and upon a bacterium, E, ggli, strain
glgé, . . --—-

The experimental method was set up to conform with the
procedures of a bactericidal test in whichtaacteria were
treated with a bactericide over different contact periods.
and in which the survival of bacteria was determined by a
plate counting method. A parallel enzyme inhibition test
was develOped and used. The activity of the enzyme was es-
timated by.a Nesslerisation procedure.

The experimental procedure used differed from the pre-
viously reported.bactericidal tests in two respects. Small
volumes were used for each combination of chlorine concen-
tration and contact time. Meticulous attention to such de-
tails as cleanliness of glassware and pipettes and the use
of reagents that possessed no chlorine demand provided a
body of experimental data that was consistent and was pre-
sumed to be reliable. .

The action of chlorine upon urease at different pH
values provided results that differed from the action of
chlorine upon the complex bacterial enzyme system.

In the presence of chlorine at pH 8, urease activity

was reduced to .01 percent of the original activity in 100

123

minutes when the ratio of urease nitrogen to chlorine was
three. In the presence of chlorine at pH 9, urease activity
was reduced to .01 percent of its original activity in 7
minutes with the same N/Cl ratio. Intermediate values were
obtained for the intermediate pH values used. Corresponding
results were obtained at different N/Cl ratios. Urease Showed p-«
its highest rate of inactivation at pH 9.

In the presence of chlorine at pH h bacterial numbers
were reduced to .01 percent of their original value in i0

minutes when the ratio of bacterial nitrogen to chlorine was

 

.1. In the presence of chlorine at pH 9, bacterial numbers
were still present to the extent of 100 percent with the
same N/Cl ratio.

The results of the bactericidal test are Similar to
those reported in the literature.

The results of the enzyme inhibition test were inversely
related to the results of the bactericidal test. These re-
sults could not be confirmed directly but there was no reason
to expect close correspondence between the activity and re-
actions of a single enzyme and the multiple enzyme activity
of a complex, living cell.

0n the basis of these studies the urease inhibition test

could not be recommended for the assay of bactericidal chlorine.

End of Summary

l.

9.

10.

ll.

12.

124

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Kolmer, J. A., Spaulding, E. H., Robinson, H. W. 1951.
Approved Laboratory Technique. Fifth Ed. Appleton-
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Sumner, J. B. and Poland, L. O 1933. Sulfhydryl Com-
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Med. 30:553-555.

Sumner, J. B. and Somers, F. G. 1949. Laboratory Experi-
ments in Biological Chemistry. Second Ed. Academic
Press. N. Y. 566 Pp. 89-91.

Manual TM8-227. 195T. Methods for Medical Laboratory
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ing Office. Washington. See Pp. 212-216.

Pomales-Lebron, A. and.Fernandez, C. 1952. A Method
for Estimating the Number of Bacteria in Liquids and
Tissues. J. Bact. 64:837-839.

Reed, R. W. and Reed, G. B. 1948. The Drop Plate Method
of Counting Viable Bacteria. Can. J. Research 26, Sec.
E:317-326.

Snyder, T. L. 1947. The Relative Errors of Bacterio-
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Gaddum, J. H. 1937. Discussion on the Chemical and
Physical Basis of Pharmacologic Action. Proc. Roy.
Soc. London. Bl2l:598-60l.

Withell, E. R. 1942. The Significance of the Variation
in the Shape of Time Survivor Curves. J. Hyg., Cam-
bridge. 42:124-183.

Hellerman, L. 1939. Reversible Processes in the Control
of Activity of Certain Enzymes with a Preliminary Note

on the Oxidation of Urease by Prophyrindine. Cold Spring
Harbor Symposia on Quantitative Biology 7:165-173.

64.

65.

66.

67.

68.

69.

70-

71-

129

Clark, A. J. 1933. The Mode of Action of Drugs on
Cells. 298 Pp. Williams and Wilkins. Baltimore.
See p. 10.

Sumner, J. B. 1930. ufiber die Reinigung der Urease durch
Kristallisation und uber die Elementare Zusammensetzung
der Kristalle. Ber. 63:582-586.

Sumner, J. B. and Hand, D. B. 1928. Crystalline Urease
II. J. Biol. Chem. 76:149-162.

Sumner, J. B., Gralen, N., Eriksson-Quensel, I. B. 1938.
The Molecular Weight of Urease. J. Biol. Chem. 125:

37-44 .

Fair, G. M., Morris J. C., Chang, S. L., Weil, I., and
Burden, R. P. 1948. The Behavior of Chlorine as a
Wager Disinfectant. J. Am. Water Works Ass'n. 40:1051-
10 1.

Sumner, J. B. and Hand, D. B. 1929. The Isoelectric
Point of Urease. J. A. C. S. 51:1255-1260.

Sumner, J. B. and Myrback, K. 1951. The Enzymes. 2
Vol. Academic Press, New York. See. Vol. 1, part 2,
p. 884.

Sizer, I. W. and Tytell, H. H. 1941. The Activity of

Crystalline Urease as a Function of the Oxidation Re-
duction Potential. J. Biol. Chem. 138:631-642.

End of Literature Cited
End of Thesis

 

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