A. STUDY OF METHODS OF MEASURING- GERMICIDAL CHLORINE
WITH REFERENCE TO THE OXIDATION--REDUCTION POTENTIAL,
STARCH-IODIDE TITRATION AND ORTHO-TOLIDINE TITRATION

Thesis for degree of Ph.D.
Michigan State College

William B. Ardrey
Department of Bacteriology and Hygiene
1939

ProQuest Number: 10008251

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ACKNOWLEDGEMENT

The writer desires to express grateful acknowledgement
for the valuable assistance of Doctor W. L. Mallmann under
whom this work was carried out.

121959

CONTENTS

ACKNOWLEDGEMENT
INTRODUCTION
HISTORICAL
EXPERIMENTAL AND DISCUSSION
A.

The effect of pH on the oxidation-reduction
potentials of chlorine solutions.

B.

The relationship of the oxidation-reduction
potential to the germicidal activity of chlorine
in the absence of organic matter.

C.

The relationship of the oxidation-reduction
potential to the germicidal activity of chlorine
in the presence of organic matter.

D.

A comparison of the oxidation-reduction potential,
starch-iodide and ortho-tolidine titrations as
measures of germicidal chlorine in the presence
of organic matter.

SUMMARY
CONCLUSIONS
LITERATURE CITED

A Study of Methods of Measuring Germicidal Chlorine
With Reference to the Oxidation-Reduction Potential,
Starch-Iodide Titration and Ortho-Tolidine Titration
Although chlorine has been used for over a century as
a means of disinfection, there are still many factors relating
to its action and methods for its determination which are as
yet not well understood.

At the present time there are two

accepted methods, the starch-iodide and ortho-tolidine tests,
for determining the available chlorine present but there are
very few data showing the relative germicidal value of the
chlorine thus measured.

This is especially true of solutions

in which there is organic matter present.

A few attempts have

been made to correlate the germicidal action of chlorine with
the oxidation-reduction potential but this has largely been
confined to excessive concentrations of chlorine or to a
comparison of the effect of pH on the oxidation-reduction
potential and on the germicidal action of the chlorine.
In general an oxidation-reduction reaction may be con­
sidered as a process where an oxidizing agent gains electrons
and a reducing agent loses electrons.

If an unattackable

electrode, such as platinum, is immersed in such an oxidationreduction system, a potential difference is set up at the
electrode and can be measured by potentiometric methods.

The

more oxidized a substance is, the higher will be the electrode
potential and the more reduced the substance is, the more
negative will be the potential.

Regardless of whether the

nascent oxygen, nascent chlorine or hypochlorous acid theory

- 2 is accepted as the explanation for the germicidal action
of chlorine, it should he possible to measure it hy­
po tentiometric methods.

The present work was therefore

carried out for the purpose of showing the relationship
of the oxidation-reduction potential to varying concentra­
tions of chlorine both with and without the presence of
organic matter, and to present further data relative to
the accuracy of the starch-iodide and ortho-tolidine titra­
tions in the presence of organic matter as means of measuring
the germicidal activity of the chlorine present.

- 3 Historical
For a great many years it was thought that the
germicidal activity of chlorine was entirely due to
oxidation, the chlorine combining with the hydrogen of
water to form nascent oxygen according to the reaction:
Cl2 / H2G

2HC1

0

Nisson (1^) in 1&90 observed an evolution of chlorine
upon the acidification of chloride-of-lime and found that
there was an increased germicidal action which he attributed
to nascent chlorine.

Kronig and Paul ..(.7 ) in 1^97 noted

that solutions of chlorine in water were much more germicidal
than other strong oxidizing agents and concluded from this
that there must be some other bactericidal property present
besides that of oxidation.

Andrewes and Orton (l) in 190*1-

were the first to suggest that hypochlorites owe their germi­
cidal activity to hypochlorous acid (H0C1 ).

They pointed

out that on the acidification of hypochlorite solutions,
hypochlorous acid was liberated and the germicidal activity
greatly increased.

Holwerda (5) and Charlton and Levine (4-)

have presented further data indicating that hypochlorite
solutions owe much of their germicidal value to undissociated
hypochlorous acid.
Rideal and Evans (15 ) in 1921 were the first to present
an oxidation-reduction potential concept of chlorination.
Schiaelkes (17 ) found that the oxidation-reduction potential
of chlorine solutions, having different pH values, ran
parallel to the germicidal activity.

Charlton and Levine (*!•),

-

4

-

in referring to some unpublished work by Hellwig, state that
the oxidation potentials of available chlorine concentrations
of chloramine-T equal to 1,000-, 2,000- and 4,000 parts per
million (p.p.m.) are almost identical at a given pH.

They

therefore conclude, "that even for a specific chlorine
compound the oxidation potential is not a dependable measure
of germicidal efficiency."
The importance of pH changes in relation to the germi­
cidal activity of chlorine has been pointed out by Johns
(6 ), Mallmann (9), Mallmann and Schalm (10) and Charlton
and Levine (3)*

Charlton and Levine (4) showed that a

solution with 100 p.p.m. available chlorine at pH 10.40
gave a killing time of 70 minutes whereas a solution contain­
ing 20 p.p.m. available chlorine at pH S.2 had a killing time
of only 4.3 minutes, thus showing that the influence of pH
is very great.
Since chlorine tends to be taken up very readily by
organic matter many workers, Tilley (19), Beard and Kendall
(2 ) and others have used solutions containing organic
matter when testing the germicidal action of chlorine.
Wright (21) has shown that certain types of organic matter
are more readily attacked by chlorine than are others.
Mallmann (ll) has shown that inert substances such as
charcoal adsorb chlorine whereas other types of organic
matter react with the chlorine and chemically combine with
it.

He also showed that in the presence of suspended

- 5 materials the starch-iodide titration and the ortho-tolidine
titration tend to measure the available chlorine differently.
McOulloch (12) has shown that in the presence of chicken
manure a definite lag precedes the maximum germicidal effect
and attributes it to the formation of a new compound, "probably
a simple chloramine formed by the action of the hypochlorite
upon the ammonia in the chicken manure."
It is generally believed that the chlorine demand must
be satisfied and a small amount of residual chlorine remain
before disinfection will take place.

Tilley and Chapin (20),

however, showed that tannery effluents which had been
sufficiently ohlorinated to become anthrax free in less than
two hours showed no available chlorine and that more chlorine
could be added without appearing as free or available chlorine.
Nachtigall and Ali (13 ) have shown that some waters can be
disinfected with chlorine in amounts less than the chlorine
demand of the water.

Rudolfs and Ziemba (16 ) found that

in the chlorination of sewage, no matter to what extent of
the chlorine demand, there were certain chloro-products formed
which would bring about bacterial reductions.

On the other

hand, they found that neither ohloro-peptones nor chloro-proteins,
obtained by chlorinating peptone water and nutrient broth, had
any inhibiting effect on the growth of bacteria nor were they
germicidal.

- 6 Experimental and Discussion
There are a number of references in the literature
dealing with the relationship of the oxidation-reduction
potential of chlorine solutions to their germicidal action.
All of these, however, deal with solutions containing such
high concentrations of chlorine that they are impracticable
both from the standpoint of disinfection and accurate
measurement, or they deal with only one concentration of
chlorine at varying pH concentrations.

In the work presented

here an attempt was made to show the relationship of the
oxidation-reduction potential to increasing concentrations
of chlorine (NaOCl) at a constant pH value.

Titration values

not to exceed I .5 0 p.p.m. were used since this is the range
which finds the greatest use in practice.

It was hoped that

the curves derived from this type of determination might act
as the basis for a more accurate method for determining avail­
able or germicidal chlorine.
Increasing concentrations of chlorine (NaOCl) were added
to equal quantities of distilled water and allowed to stand
for 15 minutes to permit the chlorine to come to equilibrium.
At the end of this period voltage determinations were made of
each of the solutions using a bright platinum electrode and
saturated calomel reference electrode.

The pH of the solutions

was adjusted by the addition of .001 N. HOI and .001 N. NaOH.
All pH determinations were made with a glass electrode.

For

convenience the term oxidation-reduction potential in all of

- 7 the studies which follow will be represented by the direct
voltage reading.

Table I and Figure I show these relation­

ships for pH values of 5*00, f.OQ and 9.00.
These data show that at a constant pH, with the addition
of increasing concentrations of chlorine (NaOCl), there is
first a steep rise in the voltage and then a leveling off.

The

voltage continues to rise slowly until about 1 .1 0 p.p.m. of
chlorine have been added when again there is a sharp rise with
each increasing addition of chlorine.

This same relationship

was found to exist for all pH concentrations between 5*00 and
9.00, the limits within which tests were made.

The voltages

at pH 5*00 represent the average of several determinations as
in this acid range the chlorine is so unstable that identical
results could not be obtained from day to day.

Chlorine

solutions with pH concentrations between J.00 and 9*00 are
quite stable and closely-checking voltages were obtained from
day to day.

However, it seems doubtful whether this voltage

determination would be very practical as a means of measuring
available chlorine in water and pool supplies, especially in
the range between 0 .1 p. p.m. and 1 .0 0 p.p.m. of chlorine as
there is too little variation in the voltage readings to give
an accurate evaluation.
In order to demonstrate the relationship between these
oxidation-reductions potentials and the germicidal activity
of chlorine solutions, increasing concentrations of sodium
hypochlorite, not to exceed 1 .5 0 p.p.m., were added to flasks
containing 1,000 mis. of distilled water.

These were then

Table I
The Effect of pH on the Oxidation-Reduction
Potential of Chlorine Solutions

d H S.00
P.P.M. Voltage
ci2

pH 7 .00
p.p .if. Voltage
ci2

oH 9.00
P.P.M. Voltage
012

0.

0 .3S0

0.

0.2 60

0.

O .16 5

0 .1 6

0.6 3 0

0 .1 2

0.440

0 .1 8

0.315

0.34

0.645

0 .2 6

0.450

0.35

0.330

0 .5 2

0 .6 5 5

0.45

0.460

0.53

0.345

0 .7 1

0 .6 6 5

0 .6 7

0.475

0 .7 1

0.355

0.91

0*675

0 .9 2

0.490

0.89

O .365

1 .0 S

0 .6 8 5

1 .1 2

0.535

1 .0 6

0.3 76

1 .2 7

0.7 20

1 .2 7

0.590

1.24

0.465

F/G.I/RE I

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- g allowed to stand for 30 minutes in order that the chlorine
might come to equilibrium.

At the end of this period the

voltage was determined as previously described and the available
chlorine checked by the starch-iodide method.

Five hundred

mis. of each of the solutions were carefully measured and
placed in 1,000 ml. flasks and tests to determine the speed
of disinfection were made on each of the solutions according
to the method of Mallmann and Schalm (10).

This consists in

adding one ml. of a standard suspension of organisms (Escherichia
coli) to the 500

chlorine solution.

At the end of 1 5 ,

30, ^5 , 60, 90, 120 and ISO seconds one ml. samples are removed
and each is placed in nine mis. of peptone water in order to
remove any excess chlorine.

These are then plated, incubated

at 37° C., and counts made of the surviving bacteria.

In the

present study, however, instead of adding a constant number of
organisms to each of the solutions tested a corresponding
increase was made in the number of organisms added with each
increase in chlorine concentration.

Tests were run at pH

concentrations 5*00, 7*00 an<3- 9*00.

The results are presented

in Tables II, III and IV, and in Figs. II, III and IV.
It is quite evident from these data that there is a very
direct relationship between the oxidation-reduction potential
of a chlorine solution and its germicidal action.

This is

especially apparent at pH 7*00 an& pH 9*00 where there is a
leveling off and then a definite rise in the potential tend­
ing towards a step formation.

In each case there is this

same leveling off and then sudden increase in the percent

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of Chlorine at pH 9*00
to the Germicidal Activity

Potentials
of Oxidation-Reduction
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- 9 reduction .of bacteria, especially at the 15 and J>0 second
intervals.

Since, at the end of 30 seconds, all or most of

the bacteria have been killed in the higher chlorine
concentrations, the curves tend to approach a straight line
during the following time intervals thus following the
slowly rising portion of the voltage curve.
There are two probable reasons why this step formation
in the killing action of chlorine and its subsequent relation­
ship to the oxidation-reduction potential have never been
demonstrated before.

In all of the previous work, demonstrating

the speed of disinfection of various chlorine compounds, a
constant number of organisms was used for each chlorine concen­
tration so that there was merely an addition effect in germi­
cidal action with each increase in the amount of chlorine.
However, in the work presented above, with each increase in
chlorine concentration a corresponding increase was made in
the number of test organisms added, so that the ratio between
the amount of chlorine present and the number of organisms
was constant at all times.

In this way it was possible to

demonstrate whether one chlorine concentration had greater
germicidal activity than another chlorine concentration and
likewise to demonstrate more clearly the relationship between
germicidal activity and oxidation-reduction potentials at
various chlorine concentrations.

In most of the previous

work in which an attempt was made to show a correlation
between the germicidal activity of chlorine solutions and
their oxidation-reduction potentials the mistake was made

- 10 of using solutions containing too high concentrations of
chlorine, where large variations in potential could not
possibly be expected to occur.
(*f), in referring to

Thus Charlton and Levine

some work by Hellwig in which he used

concentrations of chlorine varying from 1 ,0 0 0 p.p.m. to
^ ,0 0 0 p.p.m., found the oxidation-reduction potentials of

these solutions to be almost identical at a given pH concen­
tration and concluded that the oxidation-reduction potential
is not a dependable measure of its germicidal activity.

They

also concluded from the work of Schmelkes and Homing (13)
“that the oxidation potentials of solutions of different
compounds are not proportional to germicidal action”, in that
the work showed Azochloramid to have a greater germicidal
power than chloramine-T, although the latter had a higher
oxidation-reduction potential.

However, they failed to take

into consideration that this was in the presence of organic
matter whereas Schmelkes and Horning showed very clearly
that in the absence of organic matter chloramine-T had
greater germicidal activity than did Azochloramid.
Neither the starch-iodide titration nor the ortho-tolidine
titration gave any indication of this difference in germicidal
activity between solutions containing different concentrations
of chlorine, as did the oxidation-reduction potential.

It

has been shown by many authors that, in the oase of solutions
containing the same p.p.m. of chlorine but with different pH
concentrations, there was a greatly increased germicidal
activity with those solutions which were more acid.

Here

- 11 again the starch-iodide and ortho-tolidine titrations would
give misleading results as they would tend to give the same
value for each of the solutions*

The oxidation-reduction

potential, on the other hand, would become more positive with
the increasing amounts of acidity and would therefore indicate
the increased germicidal activity.
Mallmann (ll) has shown that in the presence of certain
types of organic matter neither the starch-iodide titration
nor the ortho-tolidine titration tend to measure, with any
degree of accuracy, the amount of germicidal chlorine present.
The following tests were therefore set up to determine further
the limitations of these two tests in the presence of organic
matter and to demonstrate the role of the oxidation-reduction
potential under such conditions.

One-tenth percent agar was

used as a representative type of Insoluble organic matter.
The agar was added to flasks containing 1,000 mis. of distilled
water and was then heated in the steamer for twenty minutes.
After cooling, sufficient chlorine was added to produce the
following residuals as previously determined by the starchiodide titration in distilled water:

0 ., 0 .7 0 , 1 .^0 , 2 .1 0 , 2.30,

3.50, ^.20 and ^.90 p.p.m. respectively.

These were then

allowed to stand at room temperature for 2^ hours so that the
chlorine and agar might come to equilibrium.

At the end of

this period the residual chlorine was determined by the starchiodide titration and voltage determinations were made on each
of the solutions as previously described.

Five hundred mis.

of each solution were then carefully measured out and tests

- 12 to determine the speed of disinfection were made as before.
In these tests a constant number of organisms was used
throughout instead of increasing the number with each increase
in the amount of chlorine added.

These tests were repeated

at pH concentrations of 5.00, 7*00 and 9*00.

The results

are presented in Tables V, VI and VII and in Figs. V, VI and
VII.
The results obtained from these studies are quite interest­
ing even though it is rather improbable that such conditions
would be encountered in the great majority of instances where
chlorine has a practical application as a germicide.

However,

they tend to show clearly some of the difficulties which may
be encountered in successful chlorine treatment and to demon­
strate in a very marked way the relationship between the
oxidation-reduction potential and the germicidal activity of
chlorine solutions.
It will be noted that with successive amounts of chlorine
added there is first an increase in the titratable chlorine
until a maximum is reached.

On

the addition of the next

successive amounts of chlorine, there is a marked falling off
in the amount of titratable chlorine to a minimum value.

In

the case of pH 7,00 this value is 0. or in other words there
is no chlorine present in a form which will show up on
titration.

On the next successive additions of chlorine

the titratable chlorine again increases and continues to do
so indefinitely with each increased addition of chlorine.
Both the voltage curve and the curves derived from the

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- 13 percentage reduction of bacteria in these different chlorine
solutions tend to follow in a general way this same type of
ourve produced by the titratable chlorine.

It will be

noted, however, that the voltage curves in each case give a
true indication of the degree of germicidal action which may
be expected with the various chlorine concentrations whereas
the titration curves give values which would lead to conclu­
sions quite different from those actually observed from the
curves giving percent reduction of bacteria.

For example,

in Table V and Fig. V, on the addition of 2.10 p.p.m. of
chlorine, a titration value of 0.9 9 p.p.m. of chlorine is
obtained whereas on the addition of 3 *5^ p.p.m. of chlorine,
a titration value of 0.6^ p.p.m. is obtained.

This would

indicate that the solution to which 3*50 p.p.m. of chlorine
was added should be much less germicidal than the solution
to which 2.10 p.p.m. of chlorine was added.

On the other

hand, looking at the voltage curve it is seen that on the
addition of 2 .1 0 p.p.m. of chlorine a voltage of 0.6*30 is
obtained and on the addition of 3 *5° p.p.m. of chlorine a
voltage reading of 0.795 is obtained.

This would indicate

that a much better germicidal action should be obtained on
the addition of 3-50 p.p.m. of chlorine than would be obtained
on the addition of 2.10 p.p.m. of chlorine.

The percentage

reduction curve shows that after an exposure of 15 seconds
with the addition of 2.1 0 p.p.m. of chlorine there was 66.70
percent reduction of bacteria whereas on the addition of 3.50
p.p.m. of chlorine there was 100 percent reduction of bacteria.

-

-

From this it is quite evident that the oxidation-reduction
potential represents a truer picture of the degree of
germicidal activity which may he expected than does the
starch-iodide titration method.

Table VI and VII and Figs.

VI and VII show that this same relationship holds good at
pH concentrations of 7 .00 and 9.00.
In order to demonstrate whether these solutions were
more or less germicidal than chlorine solutions in the absence
of organic matter (agar), tests were run as before comparing
the germicidal action of chlorine in the presence of 0 .1 percent
agar with solutions made up in distilled water.

Solutions of

chlorine in the presence of agar were used in concentrations
which represented both those before and after the minimal
titration value had been reached.

These determinations were

run at pH concentrations of 5*00 > 7*00 and 9*00.

The results

are shown in Tables VIII, IX and X and in Figs. VIII, IX and
X,
Table VIII and Fig. VIII show this relationship at pH
5.00.

It may be seen that 2.20 p.p.m. of chlorine in the

presence of agar before the minimal titration value is
reached has less germicidal action than has 0 .5 3 p.p.m. of
chlorine in distilled water and that 0.7*3 p.p.m. of chlorine
in the presence of agar after the minimal titration value
has been reached has greater germicidal action than has 0 .9 6
p.p.m. of chlorine in distilled water.

Likewise at pH 7*°0»

Table IX Fig. IX, 0.2>1 p.p.m. of chlorine in the presence of
agar before the minimal titration value has been reached has

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- 15 less germicidal value than 0.4-9 p#p.m. of chlorine in
distilled water and 0 «72> p.p.m. of chlorine in the presence
of agar after the minimal titration has been reached shows
much more germicidal action than does 0 .g>2> p.p.m. in distilled
water.

This same relationship also holds good at pH 9.00,

Table X Fig. X, where 0.92 p.p.m. of chlorine in the presence
of agar after the minimal titration value has been reached had
much more germicidal action than had 0 .9 9 p.p.m. of chlorine
in distilled water.

As shown in the previous experiment, the

oxidation-reduction potential gives the true evaluation of
the amount of germicidal action which may be expected to take
place whereas the starch-iodide titration method gives very
misleading values.
This same relationship, where in one case the chlorine
and agar solution is less germicidal than a solution of
chlorine alone and in the other case more germicidal than
chlorine alone, has been noted previously by Mallmann (S).
He

found that the addition of 0.3 p.p.m. of chlorine (NaOCl)

to

a solution containing 3*0 p.p.m. of agar or of a commercial

hydrophilic colloid would cause a great increase in the amount
of

germicidal activity.

He also found that if the concentration

of

agar was greatly increased in proportionto the amount of

chlorine, the solution tended to show a greatly decreased
germicidal action.

This same picture is shown in the present

study except that much larger quantities of both agar and
chlorine were used.

-

16

-

Table XI and Fig. XI show the effect of 0.01 percent
peptone on the germicidal action of chlorine at pH J.00 and
the relationship of the oxidation-reduction potential and
starch-iodide titration for residual chlorine to the germi­
cidal action.

Sufficient chlorine was added to flasks of

sterile peptone solution to give final concentrations of
O.7O, 1.4-0, 2.10 etc. up to 11.90 p.p.m. of chlorine.

These

were then allowed to stand for 24- hours at room temperature
to allow the chlorine and peptone to come to complete
equilibrium.

At the end of this period the oxidation-reduction

potential, residual chlorine by the starch-iodide titration
method and germicidal action were determined as previously
described.
Here again there was shown to exist a very definite
relationship between the oxidation-reduction potential and
the germicidal activity of a chlorine solution.

The peptone

tended to exert what appears to be a poising effect on the
voltage, tending to hold it down in a region which is nongermicidal.

This poising effect was continued until 9.10

p.p.m. of chlorine had been added; the voltage increasing
in a slowly rising line from 0.24-0 to 0*355*

On the next

two additions of chlorine up to 10.50 p.p.m. the voltage
rose sharply from 0 .3 5 5 to 0.4-95 and then leveled off again.
The percentage reduction of bacteria tended to follow this
voltage curve very closely.

Up through the addition of 9*10

p.p.m. of chlorine there was no reduction in the number of
bacteria.

On the next two additions up to 10.50 p.p.m. of

Table XI
The Relationship of Oxidation—Reduction Potentials
to the Germicidal Activity of Chlorine at pH 7.00
in the Presence of 0.01 Percent Peptone*
P.P.M. Titrated
Voltage c i 2
p.p.m.
added
Clg

Time of Exposure in Seconds

15

30

45 ! 60 ' 90

120

|1S0

Percentage Reciuction of Bacteria

0.240

0.

0.

0.

0.

0.

0.

0.

0.

0.

0.250

0.70

0.

0.

0.

0.

0.

0.

0.

0.

0.250

1.40

0.1s

0.

0.

0.

0.

0.

0.

0.

0.255

2.10

0.35

0.

0.

0.

0.

0.

0.

0.

O.265

2 .SO

0.53

0.

0.

0.

0.

0.

0.

0.

0.272

3.50

0.71

0.

0.

0.

0.

0.

0.

0.

O.27S

4.20

1.10

0.

0.

0.

0.

0.

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0 . 2S5

4.90

1.55

0.

0.

0.

0.

0.

0.

0.

Q.290

5.60

1.95

0.

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0.,

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0.300

6.3O

2.30

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0.

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0.

0.

0.

0.

0.310

7.00

2.65

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0.325

7.70

3.0s

0.

0.

0.

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0.

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0.

0.335

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0.

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0.355

9.10

4.07

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0.415

9.30

4.43

0 .5

7.S 12.1

I S . 2 24.6

29.2

37.3

0.495 10.50

4.79

s.o 1 3 .s 1 7 .s

22.7 29.2

44.2

66.1

0.500 11.20

5.12

10.5 I S . 2 22.1

26.7 32.7

61.9

96.5

0.505 11.90

5.49

12.1 16.5 24.0

29.2 4i . i

64.4

92.3

30,000 organisms per ml.

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- 17 chlorine, where the voltage rose sharply into what appears
to he the germicidal range, the reduction of bacteria
became quite marked, reaching 26.1 percent on the addition
of 10.50 p.p.m. of chlorine.

On the next two additions of

chlorine the germicidal activity only increased slightly,
thus tending to follow the portion of the voltage curve as
it leveled off.

Observation of the curve representing

p.p.m. of titratable chlorine shows that there was no
correlation between it and the germicidal activity.

After

the addition of 0 .7 0 p.p.m. of chlorine the titration curve
rises steadily to 4.20 p.p.m. of chlorine without showing
any germicidal action in the time limits employed.

It is

quite evident from this that the starch-iodide titration
method does not measure germicidal chlorine alone but also
measures that chlorine which has been absorbed or is chemi­
cally combined and is not free to act in a germicidal
capacity.
Since such high titration values were obtained with the
peptone solutions up through the addition of 9*10 p.p.m.
of chlorine, it was thought that possibly there had been a
combination between the chlorine and the peptone to form
chloro-peptones, which might act similarly to chloramines.

If this were the case they would tend to show little or no
germicidal action in the three minute time.period employed,
whereas if they were allowed to remain in contact with the
organisms for a longer period of time they might show
germicidal action similar to that shown by chloramines.

-

12

-

Rudolfs and Ziemba (16) showed that peptone solutions
chlorinated to different percentages of their one-hour
chlorine demand have no germicidal properties.

They did

show, however, that samples of sewage which had been
chlorinated to different percentages of their one-hour
chlorine demand tended to form certain ”chloro-productsM
which would bring about bacterial reductions.

The work

presented here, however, was conducted with peptone solutions
which had been chlorinated above their 24-hour chlorine
demand and which showed as high as 2 .5 5 p.p.m. of chlorine
as measured by the starch-iodide titration.
Chlorine was added to solutions containing 0.01 percent
peptone to give final concentrations of 0.24, 1.62, 2 .5 2 ,
3 .3 6 and 4.20 p.p.m. of chlorine.

These were allowed to

stand for 24 hours in order for the peptone and chlorine to
come to equilibrium.

One ml. of a suspension of Esch. coli

was then added to each of the flasks and well mixed.

At the

end of 30 minutes, 1 , 2 , 3 , 4 and 5 hours, samples were with­
drawn and plated out as before.

Table XII shows the results

of these tests.
On the first three additions of chlorine up through 2.52
p.p.m. of chlorine the voltage remained constant at 0 .260.
On the next two additions it rose to 0.225 and 0*310 respectively.
It is interesting to note that as long as the voltage remained
constant at 0 .2 6 0 for the three different amounts of chlorine
added, the number of organisms remained relatively constant
for each of the time periods at which tests were made.

The

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- 19 reductions in bacteria in these three concentrations
of chlorine may probably best be explained by the plasmopp-tysis due to the action of the distilled water over this
long period of time and not to any germicidal action due
to the presence of "chloro-peptones11.

The control, made

in distilled water, showed a much greater germicidal effect
than did these first three solutions containing peptone.

The

peptone probably in part tended to reduce the difference in
osmotic pressure between the organisms and the solution and
at the same time offered a substrate suitable for the growth
of organisms, such as Esoh. ooli, so that the killing in
these instances was less than that shown by the control.

In

the two solutions containing the highest concentrations of
chlorine where there was an increase in the voltage reading,
there was also a marked increase in the amount of germicidal
activity.

Since the three previous solutions showed titration

values as high as 1.42 p.p.m. of chlorine by the starch-iodide
method but still showed no germicidal value, it seems probable
that this was due to free chlorine which remained in excess
of that required to completely chlorinate the peptone or to
the formation of small amounts of chloramine.

Since the

starch-iodide titration gave a value of 1.42 p.p.m. of
chlorine and the ortho-toiidine titration gave a value of
0 .5 0 p.p.m. of chlorine, these tests present a very striking
example of the complete inadequacy of these two methods as
measurements of the germicidal action of chlorine in the

- 20 presence of organic matter.
Table XIII and Fig. XII give a comparison of the
oxidation-reduction potential, starch-iodide titration and
ortho-tolidine

titration as means of measuring the germi­

cidal activity

of chlorine in the presence of 0 .1 percent

agar at pH

The chlorine was added to the agar

solution and allowed to stand for 12 hours before the
determinations

were made.

It may be seen from Fig. XII that

the values for the ortho-tolidine titration and the starchiodide titration were almost identical up to the addition of
2 .1 0 p.p.m. of chlorine at which concentration all the
titratable chlorine disappeared.

From this point on the

values obtained by these two methods of titration varied
widely, the ortho-tolidine titration always giving a much
lower value than that obtained by the starch-iodide titration.
Since, as was previously shown, the starch iodide-titration,
in this particular range, gave values much too low for the
actual germicidal value, it is apparent that the orthotolidine titration in this particular instance was of much
less value since it gave much lower results than the starchiodide titration.
Table XIV and Fig. XIII give a comparison of these same
relationships in the presence of 0.01 percent peptone at
pH 7.00.

The tests were made as with agar in the above

experiment.

Here again it is shown that there was no corre­

lation between the three methods for measuring the germicidal
activity of a chlorine solution.

The voltage which, as

Table XIII
The Oxidation-Reduction Potential,Starch-iodide and
Ortho-Tolidine Titrations as Measures of Available Chlorine
in the Presence of 0.1 Percent Agar

1
P.P.M. Cl2
added

Voltage

!

Titrated p.p.m. Cl2

Starch-iodide

Ortho-tolidine

0.

0 .2 6 0

0.

0.

0 .7 0

0.490

0.53

0.55

1 . 4o

O.3SO

0.35

0.35

2 .1 0

0.295

0.

0.

2 .SO

0.410

0 .2 5

0.10

3.50

0.6 90

O .63

0 .3 5

4.20

O.72O

1.06

0.60

T/OM
T E E OX/DAT/OE-EtEDi/CT/OM
E C TEMT/AL , STA EC E - / D D / D E A M D
O/R TEO - TOL /D/ME T/T/RA T/DMS A S M E A S E / R E S O E A V A / L A S L E
CELOE/ME /// T E E E/RES E M C

O/RTHO-TOL/D/ME T/TEAT/DM
STA/RCM-/OD/DE T/T/RA T/OA/
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0.J5

0,25

777.

CL A D D E D

VOATAGE

T/T/RAE^ED

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0.55

Table XIV
The Oxidation-Reduction Potential, Starch-iodide and
Qrtho-Tolidine Titrations as Measures of Available Chlorine
in the Presence of 0 .0 1 Percent Peptone

P.P.M. Cl2
added

Titrated p.p.m. Cl2
Voltage

Starch-iodide

Ortho-tolidine

0.

0.2 60

0.

0.

0 .7 0

O .265

0.

0.

1.40

0.2 70

0 .2 0

0.

2 .1 0

O .270

0.40

0 .0 5

2 .SO

O.27O

0 .6l

0 .1 5

3.50

O.27O

O.76

0 .2 0

4.20

0.275

0.92

0 .3 0

4.90

0 .2 6 0

1.17

0 .5 0

VOLTAGE

O R T H O - T O L / 0 / N E T / T R A T /O /V
S T A R C / V - / O 0 / 0 S T / T R A T /O A '
VO L T A G£T

-

21 -

previously shown, gave a true indication of the amount of
germicidal activity to be expected in a peptone solution,
remained almost constant, changing only from 0.2 6 0 to 0 .22>0
thus indicating that there was no germicidal activity.

In

contrast, the p.p.m. of chlorine as measured by the starchiodide titration increased steadily after the addition of
the first 0 .7 0 p.p.m. of chlorine to give a final titration
value of I .1 7 p.p.m. of chlorine on the addition of ^ .9 0
p.p.m. of chlorine.

Although the ortho-tolidine titration

gave a much lower value than did the starch-iodide titration
it still did not represent anywhere near the true germicidal
activity of the chlorine solutions tested.

The chlorine

demand was 1 .^ 0 p.p.m. of chlorine instead of 0 .7 0 p.p.m. as
measured by the starch-iodide method but from this point on
the p.p.m. of chlorine rose steadily up to 0 .5 0 p.p.m. on the
addition of ^.90 p.p.m. of chlorine.

Both the starch-iodide

and ortho-tolidine, therefore, gave values which would
indicate a high degree of germicidal activity whereas actually
there was no germicidal activity at least for the time periods
used.
In the great majority of instances where chlorine finds
application as a disinfectant, such as in water and sewage
treatment the chlorine is added directly to solutions already
containing organisms so that destruction of organisms starts
at once before the chlorine has had opportunity to come to
complete equilibrium with any organic matter present.

Since

-

22

-

the previous work was conducted with solutions in which
this equilibrium had been allowed to take place, tests
were next made to demonstrate the effect of chlorine on
adding it to solutions already containing organisms and
to demonstrate the value of the starch-iodide titration,
ortho-tolidine titration and oxidation-reduction potential
as methods of measuring this germicidal action.

These

determinations were made by adding one ml. of a suspension
of Esch. coli to flasks holding 500 “Is- ot distilled water
containing organic matter.

After these had been thoroughly

mixed, varying amounts of chlorine were added and one ml.
samples were withdrawn at the end of 1 5 , J>0,

9 0 , 120

and 1£>0 seconds and placed in nine mis. of two percent peptone
water to remove any excess chlorine.
out as before.

These were then plated

The determinations of p.p.m. titratable

chlorine by both the starch-iodide and ortho-tolidine titra­
tions and the oxidation-reduction potential were made at the
end of the three-minute contact period between the chlorine
and organic matter.
Table XV and Fig. XIV show these relationships in the
presence of 0.1 percent agar at pH 7 .00.

The results obtained

here are quite different from those obtained where the chlorine
and organic matter had first been allowed to come to equili­
brium.

Here there was no rise and then fall in the titratable

chlorine, as in the previous experiments, with the addition
of increasing amounts of chlorine but a constant and steady

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- 23 rise in the titratable chlorine.

The starch-iodide and

ortho-tolidine titrations agree very closely in all cases
instead of diverging in the higher concentrations.

Likewise

with the voltage curve there is no rise and fall as was the
case when the chlorine and agar were first allowed to come
to equilibrium.

Instead the voltage rose immediately into

the germicidal range and then tended to level off, producing
a curve much like the one formed in the complete absence
of organic matter except that the voltage readings were
higher due to the larger amounts of chlorine added.

Here

again the curve derived from the percentage reduction of
bacteria tended to follow in a general way the oxidationreduction curve and not the titration curve, in that it rose
steeply at first and then leveled off whereas an inspection
of the titration curve would lead one to suspect that there
would be twice as much germicidal action on the addition of
1 .6 0 p.p.m. of chlorine as was shown with 0.30 p.p.m. of
chlorine.
Table XVI and Fig. XV show these same relationships
using 0.01 percent peptone in the place of agar.

Here again

the picture was quite different from that where the chlorine
and peptone were first allowed to come to equilibrium.
Although the starch-iodide and ortho-tolidine titration
curves present much the same relationship as they did after
the chlorine and peptone had been allowed to come to equili­
brium, the voltage curve and the curve derived from the

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2k

-

percentage reduction of "bacteria present an entirely different
picture.

After the first two additions of chlorine up to

2 .2*1- p.p.m, the starch-iodide and ortho-tolidine c^pves ran
parallel to each other, rising steadily with each addition
of chlorine.

The ortho-tolidine titration, however, as before

gave a much lower value than that obtained with the starchiodide titration.

Here as in the previous cases cited the

oxidation-reduction potential seemed to give a more accurate
estimation of the relative amounts of germicidal activity
which may be expected than did either the starch-iodide or
ortho-tolidine titrations.

The voltage reading remained

almost constant up through the addition of 1.1 2 p.p.m. of
chlorine, rising only from 0.260 to O.27O.

Upon successive

additions of chlorine the voltage rose rather sharply and
steadily up to 0.^ 5 0 on the addition of 5*60 p.p.m. of
chlorine.

The curve derived from the percentage reduction

of bacteria at 15 seconds followed this voltage curve very
closely.

Up through the addition of 1.12 p.p.m. of chlorine

there was no reduction in the number of bacteria.

From this

point on the curve rose steadily to 62.90 percent reduction
on the addition of 5.6 0 p.p.m. of chlorine.

Judging from the

starch-iodide and ortho-tolidine titrations which gave values
of 5.3$ and ^ .3 0 p.p.m. of chlorine respectively on the
addition of 5 .6 0 p.p.m. of chlorine, it would be expected that
100 percent reduction would be obtained in 15 seconds.

Tests were next made to determine the relative value of

- 25 the starch-iodide titration, ortho-tolidine titration, and
oxidation-reduction potential as means of determining the
germicidal activity of chlorine (NaOOl) in the presence of
sewage.

The sewage was well mixed by shaking to insure

even distribution of organisms and organic matter and 500
mis. measured out into flasks.

Sufficient chlorine was then

added to each of the flasks to give final concentrations of
O.S*!-, 1.6S, 2 .5 2 ,

and *l-.20 p.p.m. of chlorine.

At the

end of 3 > 5 > 1 0 , 15 and 20 minutes one ml. samples were
withdrawn into 2 percent peptone solution and plated out as
before.

Oxidation-reduction potentials, starch-iodide and

ortho-tolidine determinations were made at the three and 20
minute periods.

Table XVII and Fig. XVI are representative

of the results obtained in this work.
The data show that here again there was a very definite
correlation between the oxidation reduction potential and
the germicidal activity of the chlorine added.

Rudolfs and

Ziemba (16) have shown that high reductions in the number of
bacteria in sewage may be obtained by chlorinating to different
percentages of the ten-minute chlorine demand.

The data pre­

sented here give this same picture and also show the probable
explanation for it.

On the first two additions of cnlorine

up to 1.63 p.p.m. the voltage curve rose sharply whereas on
the next three additions of chlorine up to *4-.20 p.p.m. it
rose less rapidly but nevertheless tended to rise steadily.
Fig. XVI shows that the curves derived from the percentage

of
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reduction of bacteria tended to follow this voltage curve
very closely, as in every case the greatest relative amount
of reduction took place on the first two additions of
chlorine up to 1 .6$ p.p.m. and then increased gradually
up through the addition of 4,20 p.p.m. of chlorine.

As

before, there appears to be no correlation between the starchiodide titration and the ortho-tolidine titration nor between
these two tests and the amount of germicidal activity produced.
The starch-iodide titration gave a three-minute chlorine
demand of 1.47 p.p.m. of chlorine and the ortho-tolidine
titration a demand of 2.52 p.p.m.

From this it would be

inferred that little or no germicidal activity would take
place till after the addition of 1.47 or 2 .5 2 p.p.m. of
chlorine whereas it is seen that the greatest percent reduc­
tion took place before the addition of 1 .6$ p.p.m. of chlorine.
This is probably due, as pointed out by Rudolfs and Ziemba
(l6 ) to the formation of certain "chloro-products” which
are very germicidal and yet are not demonstrable by either
the starch-iodide titration or the ortho-tolidine titration.

- 27 Summary
With increasing concentrations of chlorine below I .50
p.p.m. in the absence of organic matter the oxidation-reduction
potential indicates the relative amount of germicidal activity
which may be expected for each concentration.

With increasing

concentrations both the oxidation-reduction potential and
germicidal activity tended toward a step formation, rising
sharply at first, leveling off and then rising sharply again.
Neither the starch-iodide nor the ortho-tolidine titrations
demonstrated this relationship between different concentrations
but gave steadily increasing values with each successive
amount of chlorine added.

The same type of relationships

existed at pH values of 5*00 > 7 »00 and 9*00.
When increasing amounts of chlorine were added to
solutions containing 0 .1 percent agar and allowed to stand
for 24 hours to come to equilibrium the titratable chlorine
first increased, then decreased sometimes to 0 . p.p.m., and
then increased again.

The oxidation-reduction potential and

the percentage reduction of bacteria tended to follow this
same type of curve.

However, the starch-iodide and ortho-

tolidine titrations gave too high values before the disappear­
ance of chlorine and too low values afterwards.

The oxidation-

reduction potential in each case gave a true evaluation of the
amount of germicidal activity which may be expected.

The

starch-iodide and ortho-tolidine titrations agreed with each
other up to the point where the chlorine disappeared but from

- 2g this point on the ortho-tolidine titration gave a lower
value than did the starch-iodide titration.
Solutions containing 0.1 percent agar and chlorinated
beyond the point where the chlorine disappeared were found
to be much more germicidal than solutions containing no agar;
whereas solutions which had just been chlorinated up to this
point were much less germicidal than solutions containing no
agar.
When increasing amounts of chlorine were allowed to
come to equilibrium for 2)\ hours in the presence of 0.01
percent peptone there was found to be a poising effect on the
oxidation-reduction potential, tending to hold it down in a
non-germicidal range.

When sufficient chlorine had been

added to overcome this effect, the voltage rose sharply and
was accompanied by a sharp increase in the percentage reduction
of organisms.

There was found to be no correlation between

the starch-iodide and ortho-tolidine titrations and the
amount of germicidal activity.

The ortho-tolidine titration

gave a much lower value than the starch-iodide titration but
was still much too high for the corresponding germicidal
activity.

Apparently some inert "chloro-productsn , without

germicidal activity but still able to show up on titration,
were formed, since no reduction in bacteria was observed even
with time periods up to five hours as long as the voltage
remained constant.

On adding increasing amounts of chlorine to solutions of

- 29 agar and peptone containing organisms without allowing the
chlorine and organic matter to first come to equilibrium
the oxidation-reduction potential gave a better relative
idea of the amount of germicidal activity than did either
the starch-iodide or ortho-tolidine titrations.

In the

case of agar there was not a rise and fall in titratable
chlorine as was the case where chlorine and agar were first
allowed to come to equilibrium.

In the presence of agar

the starch-iodide and ortho-tolidine titrations were the
same but in the presence of peptone the ortho-tolidine
titration again gave a lower value than did the starch-iodide
titration.
In the presence of sewage the oxidation-reduction
potential again tended to measure the relative germicidal
activity of different chlorine concentrations whereas there
was no correlation between the starch-iodide and orthotolidine titrations and the amount of germicidal activity.
Likewise there was no correlation between the starch-iodide
titration and ortho-tolidine titration in the measurement
of the chlorine demand, the starch-iodide titration in every
case giving a much lower demand than the ortho-tolidine
titration.
From these results it is obvious that the present
methods of measuring available or germicidal chlorine are
of little value, especially in the presence of organic
matter.

The oxidation-reduction potential gives a true

indication of the relative germicidal activity of different

- 30 concentrations of chlorine whether in the presence or
absence of organic matter.

Conclusions
The oxidation-reduction potential gives an accurate
indication of the relative amounts of germicidal action
which may be expected between different concentrations
of chlorine (NaOCl) both in the presence and absence of
organic matter.
Neither the starch-iodide nor the ortho-tolidine titrations
give an accurate indication of the relative amounts of
germicidal action between different concentrations of
chlorine (NaOCl) either in the presence or absence of
organic matter.
There is little or no correlation between the values
obtained by the starch-iodide and ortho-tolidine titra­
tions in the presence of organic matter.
The chlorine demand of solutions containing organiG matter
is much higher as measured by the ortho-tolidine than by
the starch-iodide titration.
With solutions containing identical amounts of chlorine
(NaOCl) at different pH concentrations, the oxidationreduction potentials indicate the increased germicidal
activity with increasing acidity whereas the starch-iodide
and ortho-tolidine titrations give the same values at
different pH concentrations.
With the addition of increasing amounts of chlorine (NaOCl)
to solutions containing agar, a point is reached where the
agar exerts an increased germicidal effect over that of

- 32 solutions containing the same amount of titratable chlorine
but in the absence of agar.

- 33 Literature Cited
1.

Andrewes, F. W., and Orton, K. J. P. - HA Study of
Disinfectant Action of Hypochlorous Acid with Remarks
on Its Practical Application” - Centralhl. of Bakt.
IAbt• Originale, 3 5 :645-6 5 1 , 611-615. 1904.

^2.

Beard, P. J. and Kendall, N. J. - ’’Sterilizing Velocities
of Chlorine and Chloramine under Varying Conditions of
Organic Load and pH” - Jour. A m .

Water Works Assn.,

2 7 :676-677. 1935.
3.

Charlton, D. B. and Levine, Max - "Some Observations on
the Germicidal Efficiency of Chloramine-T and Calcium
Hypochlorite1.' - Jour, of Bacty., 3 0 :l6l-171. 1935*

4.

Charlton, D. B. and Levine, Max - "Germicidal Properties
of Chlorine Compounds" - Engr. Exp. Sta. Bui. 132. 1937*

5.

Holwerda, K. - "On the Control and the Degree of Reliability
of the Chlorination Process of Drinking Water in Connection
with the Chloramine Procedure and the Chlorination of
Ammoniacal Water" - Mededeelingen van den Dienst der
Volksgenzondheid in Ned-Indie, 1 7 :251-297» 1922>.

^ 6.

Johns, C. K. - "Germicidal Power of Sodium Hypochlorite:
Effect of Alkali" - Ind. and Engr. Chem., 2 6 :767-766. 193*1-.

7.

Kronig, B. and Paul, T. - "Die Chemischen Grundlagen der
Lehre von der Giftwerkung und Disinfection” - Ztschr. f.
Hyg. w. Infeklwnskrankheiten, 25:1-112. 1697•

6.

Mallmann, W. L. - (Unpublished work)

9.

Mallmann, W. L. - "Hydrogen Ion Concentration in Disin­
fection by Chlorination" - Jour. A m .
2 4 :1054-6 1 . 1932.

Water Works Assn.,

-

10.

Mallmann,

34

-

W. L. and Schalm, 0 . - "The Influence of

the Hydroxyl Ion on the Germicidal Action of Chlorine
in DiluteSolution"

- Mich. State College, Engr. Exp.

Sta. Bui. 44. 19 3 2 .
11.

Mallmann,

W.L. - "The Germicidal Activity of Available

Chlorine as Measured by the Ortho-Tolidine and Iodometric
Tests for Chlorine" - MiGh. State College, Engr. Exp.
Sta. Bui. 59. 1934.
12.

McCulloch, E. C. - "Disinfection and Sterilization" Chap. XV. 1936.

^1 3 .

Nachtigall, G. and Ali, M. - "Chlorine Fixing Velocity
and Bactericidal Chlorine Quantity" - Jour. Am. Water
Works Assn., 26:430. 193^*

14.

Nissen, Franz - "Ueberdie desinficirende Eigenshaft
des Chlorkalks" - Zeitschr. J* Hyz., 6 :62-7 7. 1690.

15.

Rideal, E. K. and Evans, V. R. - "The Effect of Alka­
linity on the Use of Hypochlorites" - Jour. Soc. Chem.
Ind., 4o:64-66r.

^1 6 .

1921.

Rudolfs, Willem and Ziemba, J. V. - "The Efficiency of
Chlorine in Sewage Disinfection as Affected by Certain
Environmental Factors" - Jour, of Bacty., 27**419-442.
193^.

17.

Schmelkes, F. C. - "The Oxidation Potential Concept of
Chlorination" - Jour. Am. Water Works Assn., 25**695~7°3«
1933-

16.

Schmelkes, F. C. and Horning, E. S. - "Bactericidal

- 35 ~
Action of Azoohloramid (N-N Dichlorazodicar bonamidine)" Jour, of Bacty., 2 9 :323-331. 1935.
19•

Tilley, F. W. - "Investigations of the Germicidal Value
of Some of the Chlorine Disinfectants" - Jour. Agr. Res.,

20 :65-110. 1920.
20.

Tilley, F. W. and Chapin, R. M. - "Germicidal Efficiency
of Chlorine and the N-Chloro Derivatives of Ammonia,
Methylamine and Glycine Against Anthrax Spores I'- Jour,
of Bacty., 19:295* 1930*

21.

Wright, N. C. - "The Action of Hypochlorites on AminoAcids and Proteins',' - Biochem. Jour., 20:524-532. 1926.