T0 DEVISE Es WEEK?!) OF EXPRESSENG

THE BACTENCIDAL EFFEClENCY DE A
DISIRFECTMT ESTHER TM??? THE STEREBEBD
HM. PfiEE‘éDL EUEFFEEEM E’EETHQE

THEStS FOR THE DEGREE 0F M.S.
MICWGAN STATE COLLEGE

fiNDREW M. fiYMA
1940

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TO DEVISE A METHOD OF EXPRESSING
THE BACTERICIDAL EFFICIENCY OF A
DISINFECTANT OTHER THAN THE STANDARD
F.D.A.‘PHENOL COEFFICIENT METHOD.

by .g. ’ .JN
9

Andrew M; E198“

A THESIS
Submitted to the Graduate School of Michigan State
College of Agriculture and Applied Science
in partial fulfilment of the .
requirements for the degree of

MASTER OF SCIENCE.

Department of Bacteriology
Year 1940.

THESIS

Attempts at disinfection are nearly as old as
the human race. We read of the effects of Boreas in
the plague stricken quarters of Babylon; and Kemer-
wrote about wood smoke and sulfur fumes. As time
went on many attempts were made to compare the
effectiveness of various substances in preventing
putrefaction. It was not, however, until the epoch
making discovery of Koch and his method of pure
culture of the disease germ during the early part of
the last century that the science of disinfection
came into existence. The classical experiments of
Koch with impregnated silk threads are well known.
The discoveries of other factors affecting germicidal
comparisons soon followed. In 1897 Kroning and Paul (1)
published their classical paper describing a new
method for the quantitative study of disinfection and
demonstrated that the death of bacteria under the
influence of a germicidal agent is a gradual process
and that it follows an orderly sequence. They also
recognized the influence of temperature upon a
disinfectant.

The introduction of phenol as a basis of comparison
was first seriously prOposed by Rideal and Walker (2).

They made parallel tests with various concentrations

,6 5 (3’1
s- ' 's-

1 ”54
a: J -. ._ I

Q
_ 1‘,
.4.

of phenol and the disinfectant thus establishing a
"carbolic acid coefficient" this being the ratio of
the concentration of the two disinfectants which will
kill all the test organisms in the same length of
time.

The variable factors as time, temperature and
culture media, nature and condition of test organisms
are controlled as closely as possible. These factors
are arbitrarily fixed. In 1908 Chick and Martin (3)
added another element. Instead of noting the time
required for disinfection with varying concentrations
of the disinfectant the concentration necessary to
disinfect in a given time is determined. They used
30 minute eXposures in most of their experiments.

Anderson and McClintic (4) modified and improved
the technique devised by Rideal and Walker. The phenol
coefficient determined according to their method is
known as the Hygienic Laboratory Method. A still later
method has been prepared by Shippen which combines the
best features of the Rideal Walker and the Hygienic
Laboratory Methods. Reddish later published Shippen's
Method under the name of "R - W'Modified Method" (5).
The procedure of Shippen has been little changed and

it has come to be used for testing the great majority

of the germicides now received at the Food and Drug
Administration. The three methods, namely, R-W
Method, Hygienic Laboratory Method and the Food and
Drug Administration show only slight differences.
The F.D.A. Method is considerably superior to the
R-W method in producing consistent results as the
medium employed is better adapted to bacterial growth
and one is not restricted to the use of only one
test organism as in the case of the R-W and Hygienic
Laboratory methods. The R-W broth is not well
adapted for Optimum growth of the test cultures
producing negative subcultures indicating that the
organism has been killed when in fact the organism
is incapable of growing in this culture medium.

The "phenol coefficient" as described by the
Food and Drug Administration in its final anaylsia
is an expression of the ratio, showing the germicidal
power of dilutions of phenol under standard conditions
with comparative dilutions of the disinfectant tested
under the same standard. It depends upon the fixation
of one variable and that incompletely, namely, the
reaction velocity at a given temperature and during
a particular interval of time. This limits the value

of the phenol coefficient to only one temperature

and one period of eXposure.

Compounds chemically related to phenol react
favorably under the prescribed conditions and may
show a favorable coefficient as demonstrated by Ruehle
and Brewer (6) when applied to coal tar disinfectants.
Other compounds, on the other hand, equally valuable
as disinfectants may react unfavorably under the
prescribed conditions and show a low phenol coefficient.
The only variable allowed by the F.D.A. phenol coefficient
method would.be the concentration of the disinfectant.
Thus, if compounds are selected for test which vary in
concentration and reaction time the phenol coefficient
will not show true comparative values. The phenol
coefficient ratio would increase as the period of
exposure is increased, conversely, the shortening of
the period of exposure would lessen the coefficient
ratio. Chick (3) instead of noting the time required
for disinfection with varying concentrations of the
disinfectant determines the concentration necessary
to disinfect in a given time. She selects 30 minutes
arbitrairly. In discussing the time element it is
pointed out that comparing the action of phenol and
mercuric chloride upon Salmonella paratyphi at 20°C.

the concentrations necessary to kill in 2.5 minutes

have a ratio of 13.6 while that necessary to kill in

30 minutes have a ratio of 550. The selection of a
constant time factor is therefore necessary if a constant
phenol coefficient is to be obtained. Such a coefficient
would indicate the relative value of the disinfectant

in question only at the concentration at which the

test was made. New coefficients would be obtained at
other concentration.

Chick establishes three arbitrary conditions of
experiment for comparison of disinfectants. Temperature
must be standard; the initial number of bacteria must
be the same in each comparison; and the time required
for disinfection must be fixed. Thus, it is possible
to determine relative values as phenol coefficients
under restricted conditions of comparison. However,
the arbitrary fixed conditions in comparisons would
deprive the results of their practical value.

Any method for expressing the comparative value of
various disinfectants that takes sterilization as its
end point is apt to be fallacious due to the fact that
the rate of kill varies with various types of disinfectants
and further the so called resistant minority behave in
a radical manner with various types of disinfectants.

Three constants, one for each variable condition
met with, are necessary completely to describe a

disinfectant. An expression of bactericidal efficiency

should show factors as velocity of reaction or rate

of disinfection, dilution factor, temperature and
toxicity coefficients. A method of standardization
should comprise the determination of these factors

for each disinfectant. All these determinations should
be made in the presence of organic matter so as to
simulate as near as possible conditions as they are

met with in disinfection. Calculations also should

be made in the absence of organic matter as this infor-
mation is desirable in certain cases of disinfection.

The death of bacteria under the influence of a
germicidal substance is a gradual process the rapidity
of which decreases with advancing time.

Disinfection is comparable to a chemical reaction
in which the disinfectant represents one of the reacting
substances and the protOplasm of the bacterial cells
the other. It can be accelerated by increasing one
of the reacting substances and 13's gradual process
similar to a unimolecular chemical reaction. In any
chemical reaction the amount of change in the reacting
substance in unit time is directly prOportional to
some power of the concentrations of these substances.
In a unimolecular reaction it is the first power that

is involved and it may be stated that the velocity of

the reaction or the amount of change in unit time is
directly pr0portional to the concentration of the
reacting substances.

The reaction velocity of the disinfectant can be
calculated by enumerating the bacteria surviving at
successive intervals of time. The disinfectant in all
cases is present in excess so that its concentration
is not appreciably reduced. The concentration of
disinfectant plus the suspension of organisms equal
the number of surviving organisms or number of organisms
killed in any given period of time plus excess disin-
fectant. Again as in the law of mass action the rapidity
with which the reaction proceeds is dependant upon the
concentration of the substance at any given moment,
temperature and other environmental conditions remaining
constant.

An equation representing the reaction velocity may
be written as follows:

k= 35.1%.];
k = constant depending upon the nature of the substance.
t = time.
B = the original amount of substance undergoing change

(bacterial suspension).

b = the amount (bacteria in suspension) remaining after

time (t). \

It will be found if 10g b is plotted against time in 5

this equation the resulting graph will be a straight

line. we may assume and experimentally it has been
proved that at any time the reaction velocity is pro-

portional to the number of surviving organisms per

unit of volume. Other factors such as temperature,

culture media are controlled.

This constant, then, will serve as an expression
of the reaction velocity or rate of disinfection for
any germicide under consideration. The ideal method
would be to take two or three points on the disinfection
curve and ascertain the concentration of disinfectant
necessary to bring about a given decrease in the number
of viable bacteria. Then, the larger the value of k
the more rapid the disinfection and the more efficient
the disinfectant. Knowing this value we can then
determine the reaction velocity of a disinfectant under
conditions desired.

The second factor to be considered is the effect
of varying the concentration. The reaction velocity at
any concentration can be calculated.

Chick, 1908, found that the relationship between
the concentration of a disinfectant is not a simple but

an exponential one. The exponent of the concentration

being a factor varying for each disinfectant. Watson, (7)
1908, working on Chick's figures found that the relation
could be expressed by the formula out as a constant.
Where 0 is the concentration; n is a constant varying
for each disinfectant and indicating the order of the
reaction. In a monomolecular reaction n is considered
as unity; t represents time necessary for disinfection.
Formula: Cnt equals K then represents the relation
when one molecule of one certain substance reacts with
n molecules of a second, the latter being present in
excess.
Returning to the original equation k 2 %-log €-
and integrating log %'= Kent. Ken is the k of
previous expression. It varies with the concentration
and may be determined for two concentrations of like
disinfectant. Let these values be k and k1 and c
and 01. Then,

k1 c1
E— ? log 6--

Having C and n it is then possible to calculate K.
For calculation it may be expressed as n log C-+ log
t equals K. The value for exponent n will first have
to be determined. This is done as follows: Taking

the concentration into account we may say that

KCnt = log %-. K is the true velocity constant of

the disinfectant being independent of the concentration
thus differing from k formerly expressed which is

a constant at only a given concentration. k1 is
determined from concentration 01 and k2 from concen-

tration Cg in any given experiment. Then n a log

- 1 B
kZ-flogfi
1 B
k1=flogfi

- k c
n - 10 lo
, Ski-9 86*:-
Having established the value for n we calculated the

relationship of one molecule of one substance reacting
with n molecules of another substance by formula:
out = K or n log C'f log t = a constant.

In order to arrive at true evaluation of the
result of disinfection it will be necessary to consider
the effect of temperature upon the rate of disinfection
as the third factor. A knowledge of the temperature
coefficient as well as the coefficient of dilution is
of great importance in evaluating the ability of a
disinfectant to destroy bacteria.

There are two commonly used methods of calculating
the temperature coefficient. One is simply to determine

the periods of exposure necessary to sterilize two

portions of a culture when exposed to equal concen-
tration but held at different temperatures. Under
these conditions the time required for sterilization
is inversely proportional to the velocity of disin-
fection. For instance if the temperature is varied,
other factors remaining constant, and 10 minutes is
required to sterilize a culture at 30°C. and sixty-
two minutes 20°C. the temperature coefficient
(signified by O) can be determined as follows:

9 = gg-or 6.2 for 19°C.

for 1° would be 6.2O°1.

To determine the temperature coefficient for
each degree of temperature divide the logarithmof
the temperature coefficient by the number of degrees
the coefficient represents and the resultant will be
the logarithm of the temperature coefficient for
each degree; e.g., log of 6.2 I 0.7924.

1 9:;%§2-= .07924 mantissa = 1.2
0 = 1.2 for each degree.

It has been found in most of the chemical reactions
that the velocity increases from two to threefold for
each rise of 10°C. Among bacterial reactions, however,
the increase may be greater. The agreement of the

disinfecting reactions with the temperature law of

chemical reactions has been demonstrated in many
instances and may be assured in all cases. Comparisons
are usually made of two temperatures at 100 apart
as a basis for calculation. Having determined the
temperature coefficient the value for the velocity
constant at any temperature may be calculated from
the following:
KTO = K200 x 9(r - 20)

The three constants, velocity constant, the
concentration exponent and the temperature coefficient
define far more efficiently a disinfectant than the

standard F.D.A. Method.
Experimentation.

Test organism used is a standard strain of
Eberthella typhosa furnished by the Bacteriology
Department of Michigan State College. This was
cultured in F.D.A. broth for T successive 24 hour
periods at 37°C. and the seventh day culture was used
in the following determination.

A 0.5% and a 1% concentration of phenol were
prepared from a 5% phenol solution standardized as

per F.D.A. requirements.

Before adding the disinfectant the number of
bacteria in the initial broth culture was first determined
by suitable dilution and plating. This was found to
be 2,400,000 per standard loopful. The amount of
culture used throughout the experiment was a standard
F.D.A. loOpful. The culture and phenol dilutions are
each placed inéwater bath one at 25°C. and one at 35°C.
until they have reached the temperature of the bath.
Fivetenthsof one cc. of culture is then added to 5 cc.
of each dilution as in the F.D.A. phenol coefficient
method and transfers made at specified time intervals
to nutrient broth. Suitable dilutions as l - 1,000
and l - 10,000 are made and then plated on nutrient
agar. Determination of the reaction velocity or
velocity constants was first determined on~l% solution
of phenol at 25°C., a 0.5% solution of phenol at 25°C.
and a 0.5% solution of phenol at 55°C. with the following
results.

1.24

1% phenol at 25°C. k
0.5% phenol at 25°C. k - .02

0.5% phenol at 55°C. k .098

1% 06H50H 25°C. 2,400,000 control.
Time Count Value k.
0.5 min. 440,000 1.47
1.0 min. 88,000 1.43
2.5 min. 6,400 1.05
5.0 min. 20 1.02
10.0 min. --- ----

Average Value k = 1.24 = reaction velocity

p — 1 B
from iormula k - {-10g 5'

0.5% 06H50H 25°C. 2,400,000 control

Time Count Value k
0.5 min. 2,340,000 .022
1.0 min. 2,300,000 .019
2.5 min. 2,140,000 .020
5.0 min. 1,880,000 .021

10.0 min. 1,660,000 .016
15.0 min. 1,180,000 .020
20.0 min. 955,000 .020
30.0 min. 479,000 .022
60.0 min. 60,300 .026
120.0 min. 8,000 .020
240.0 min. 210 .020
480 min. 10 .015

Average Value k = .021 = reaction velocity

0.5% C6H50H 5500. 2,400,000 control

Time Count Value k
0.5 min. 2,200,000 .076
1.0 min. 1,920,000 .097
2.5 min. 938,000 .123
5.0 min. 720,000 .105

10.0 min. 300,000 .090
15.0 min. 240,000 .066
20.0 min. 156,000 .096
30.0 min. 120 .131

Average Value k = .098 = reaction velocity

Culture - E. typhosa
Media - Nutrient broth and nutrient agar.
Incubation on agar plate 24 hours.

Experiments previously cited have shown that k
varies with the kind and concentration of the disin-
fectant, with the temperature, and with the bacterial
species; but otherwise it is a constant as shown in
this experiment. It is a definite measure of the
value of the disinfectant and indicates the rate of
disinfection. The larger the value of k the more

rapid the disinfection. Constant k is also independent

of the initial number of bacteria present and need
not involve complete disinfection, as the end point
of complete disinfection is indefinite.

From the above experimentation it is shown that
the rate of disinfection or constant k of a 1% phenol
at 25°C. = 1.24 of 0.5% phenol at 2500. is .02 and of
0.5% phenol at 55°C. is .09 and that rate of disinfection'
of a 1% solution of phenol is 62 times that of 0.5%
solution of phenol. Also that an increase of 10°C.
in temperature will increase the rate of disinfection
about five times in the comparison of the two 0.5%
solutions of phenol, one at 25°C. and one at 35°C.

k is only constant at a given concentration and varies
at different concentrations and temperatures as shown
by experiment.

In the disinfection of paratyphoid bacilli at
0.5% phenol at 20°C. Chick found the value of k to be
.027 and at 50°C. k equals 0.7.

After the determination of the reaction velocity
or rate of disinfection for the two concentrations of
phenol the next step considered is the concentration
coefficient.

From the tables shown it was found that with a

concentration of 1% phenol or 10 parts per thousand,
10 minutes was required for the end point of disin-
fection and at 0.5% or, 5 parts per thousand, 480
minutes was required. The relationship between the
concentration is exponential and varies for each
disinfectant. This relationship can be expressed as
previously shown by the formula

out = K

K=nlogC+1ogt
KCntzlog-E-
The value for the exponent n must first be determined.
This can be found from the formula

n = 10g §§»é- g?-
Finding the value for kg and k1

'k2= %log%
B = 2,400,000, original number of
bacteria per loopful.
b 2 may be taken as one.
t

= 10 minutes, time of disinfection.

1/10 10g 2,400,000 = .6380.

W
\7
ll

- 1/480 10g 2,400,000 = .0155

W
l—‘
I

Then to find the value for n from equation

a. k C‘
n - 100 9 100
° 1 ° 1
n = log Lg%%%-+ log lg

log 48 9 log 2

= 1.681 = .
n‘730‘1'56

The exponent n may be regarded as a concentration

coefficient varying with each disinfectant. The value
of exponent n is very important because it gives
information that is not shown by k, the simple reaction
velocity exponent. A high value of exponent n is
actively germicidal above a given concentration,
however, a low degree of dilution will greatly diminish
or abolish its germicidal activity entirely. A low
n coefficient while being actively germicidal in
solutions above a given concentration exercises an
inhibitory effect even in high dilutions. This
constant n expresses mathematically, as Chick pointed
out experimentally, that mercuric chloride may have
a phenol coefficient of 13 at one concentration and
550 at another concentration. If the value of n can
be determined it will not be necessary to adopt a
standard time of testing.

The value for constant n or coefficient of dilution
in the above eXperiment was found to be approximately 6.

This would show that doubling the concentration of

phenol or 2 times the concentration exponent n (6) or

(2°) will diminish the time taken for disinfection
64 times while halving the concentration will increase
the time for disinfection 64 times.
We can now calculate the value for K at concentration

of 10 parts phenol per thousand. This value would be

K = n log C + log t
K = 5.6 log 10 -+ log 10
K = 5.6 X l + 1 = 6.5

For 5 parts phenol per thousand.
5.6 X log 5 + log 480

K

K 5.6 X .699 + 2.681 = 6.59
Value for K then equals 6.6 which is the true velocimz
constant of phenol and is independent of the concentration.
The value of k previously found is only constant at
constant concentration. The coefficient of dilution
n is found to be 5.6. It may also be called the
concentration exponent. These two constants, K and n,
will show the fundamental characteristics of a dis-
infectant at any given temperature.

The third factor to be considered is the temperature
coefficient.

The same concentration of phenol (0.5%)(at 25°C.

and 35°C.) shows a killing time of 480 minutes at

25°C. and 60 minutes at 35°C.

0 (Temperature coefficient) = égg = 8 for 10°C.

The temperature coefficient for 1° may be
determined as follows. To determine the temperature
coefficient for each degree of temperature divide
the logarithms of the temperatura coefficient by the
number of degrees the coefficient represents and the
resultant will be the logarithm of the temperature
coefficient for each degree.

Then, log 8 = .9030 e 10 = .0903

mantissa .0903 = 1.23
0 for 1°C. = 1.25

It has been found in the case of most reactions
that the velocity increases from two to threefold for
each rise of 10°C. Among bacterial reaction this may
be greater and according to Chick may vary from two to
fourfold in the case of metallic salts and seven to
eight fold for phenolic compounds. This makes necessary
a temperature coefficient to express this characteristic
of a disinfectant. In the above experiment the
temperature coefficient was found to be 8 for each

10°C. This then gives us the following values for phenol.

K = a true velocity coefficient of 6.6.
n - a dilution coefficient of 5.6
O = a temperature coefficient for 10°C. of

8. or 1.23 per degree.

This is in accord with the finding of Phelps (8).
According to Phelps the value of n for phenol was

found to be 6 and the value of 0 between 7 and 8.

The values, n coefficient of dilution and 0 temperature
coefficient, should not appear to vary widely for
different organisms. The value K or true velocity
coefficient will vary with each organism.

The essential characteristics of phenol as a
disinfectant are defined by these three constants;
the velocity constant, the concentration exponent
and the temperature coefficient of which the concen-
tration exponent and temperature coefficient are
independent of experimental conditions. The velocity
constant is dependent upon the type of test organism
employed and standardization of a disinfectant should
be referred to a particular organism. For practical
purposes it will suffice to work these values for

one organism such as Eberthella typhosa.

 

Now, having established these values for phenol
we can determine the reaction velocity of any given
disinfectant under any conditions as desired in like
manner. We can then make direct comparison with phenol

and set up a ratio or coefficient comparing the true

velocity coefficient of the disinfectant With that of

phenol. Also a comparison of the values for the
dilution and temperature coefficients can be made.

Such a determination was made in the case of bichloride
of mercury as follows:

A 1 to 1,000 and a 1 to 10,000 HgClg solution is

first prepared.

The value k or velocity constant is determined for

a l - 1,000 dilution at 25°C.

a l - 10,000 dilution at 25°C.

a 1 - 100,000 dilution at 25°C.

a 1 - 100,000 dilution at 55°C. with the following results.

1 - 1,000 HgClg 25°C. 5,280,000 control.
Time Count Value k.
0.5 min. 2,640 6.4
1.0 min. ————— ---
1.5 min. ----- _--

Average value for k - 6.4

1 - 10,000 HgClz 25°C. 5,280,000 control

Time Count Value k.
5.0 min. 2,800 0.65
7.5 min, 500 : 0.67.

10.0 min. --- ----

Average value for k = 0.66

1 - 100,000 23013 25°C. 5,280,000 control

Time Count . Value k.
5 min 2,004,000 .084
10 min. 547,000 .09
15 min. 95,000 .11
30 min. 2,800 .11
50 min. 10 .11
60 min. ----- ---

Average value for k = .10

1 - 100,000 HgClg 35°C. 5,280,000 control
Time Count Value k

10 min. 1,280 .26

15 min. 580 .26

20 min. 50 .25

25 min. -—- ---

Average value for k a .26.

Mercuric chloride, due to its coagulating action,
on protein gives a high inhibitory value in preventing
growth in subcultures. High phenol coefficients
often result which may show a false value. This
difficulty is largely overcome in plating out the
subculture as done in the proposed method.

The following values for k were obtained.

1 - 1,000 HgClg k = 6.4 at 25°C.
1 — 10,000 Hg012 k = .67 at 25°C.
1 - 100,000 23012 k = .10 at 25°C.
1 -2100,000 HgClg k = .26 at 55°C.

This shows that the rate of disinfection of a
l - 1,000 solution of Hg012 is 10 times that of a
10,000 solution and a l - 1,000 solution is 64 times
that of a 1 - 100,000 solution.

This factor in no way can be expressed by the

F.D.A. phenol coefficient method.

Calculation of Concentration exponent.

k‘ 02
= 10
n 8 Ef" all
Let 1 part per 1,000 (1 - 1,000) 3 Ca and
t = 1.5 minutes and 0.1 part per 1,000 (1 - 10,000) =

Cl and t - 10 minutes.

1 B
k2 ='f 10g 5'

tfl
ll

5,280,000
1

b
t I 1.5 minutes
k2 = 1/1.5 10g 5,280,000 = 4.476
k1 = 1/10 10g 5,280,000 = 0.6722
n = 100 k e 0

log W. 1- 6T.
10g 6.66 9 log 10 = 0.823

I].

n
of

The action of the simple compoundamercury depends
upon the concentration of mercuric ions in solution.
The ionization of the simple salts of mercury in
solution is far from complete and mercuric chloride
behaves like a non-electrolyte.

f the Hg ions only are considered, the value for

n will be greater than 1, found above, considering

only the molecular concentration.

%-1og.%

l/l.5 log 5,280,000

W
N
II

P?
to
II

4.476

W
M
II

l/lo 10g 5,280,000

N
[.1
II

.6722

3’7
H
N

concentration Hg ions (1 - 1,000) = 63

O
N
I!

concentration Hg ions (1 - 10,000) a 42.5

0
l-'
ll

v-

k
n=10g :10

5‘. g .-
“=103m22 10842035
n.- log'6.66 4 log 1.48 = :%%§.= 4.75

At 0.1 parts per 1,000 HgClz t 10 minutes and
.01 part per 1,000 HgClz t = 65 minutes.
Hg ion concentration 1 - 1,000 HgCl2 a 42.5

Hg ion concentration 1 - 100,000 Hg012 = 23

22 =.6722
k1 8 e1028
z 108 gfaz * 108 325‘

Average value for n = 3.8.
If the molecular concentration of H3012 is considered
the value for n is equal to about 1.

This would indicate that doubling the concentration
of HgClg would halve the time taken for disinfection.
For phenol it has been shown that doubling the
concentration would decrease the time taken for disin-
fection 64 times. However, if we halve the concentration
of HgClg the time for disinfection would be increased
only twice while doing the same for phenol the time
would be increased 64 times.

This very important factor is in no way expressed

or shown in the F.D.A. phenol coefficent method.

for l - 1,000 HgClZ solution

n log C + 10g t
508 10g 65 + 103 le5 = 7.01

NNNN
u

for l - 10,000 Hg012 solution
. 308 10g 4205 + 108 10 = 7.18
for 1 - 100,000 HgClz

NW P9

= 3.8 log 23 -+ log 65 = 6.98

Average value K-- 7.06.
The average value of K, or true velocity constant
findependent of the concentration)for HgClz, then is
found to be 7.06. In comparing this value with K 6.6
as found for phenol it is found that the speed of
action, or rate of disinfection HgClz is 1.7 times as
great as that for phenol.

Again this important factor is in no way shown

by the F.D.A. phenol coefficient method.

Temperature coefficient for

1 - 100,000 23012 at 25°C. killing time 60 minutes.
1 - 100,000 H5012 at 35°C, killing time 2 15 minutes.
9 = 2%.: 2.4 for 1000. I
log 2.4 = .580 e 10 = .0580
Mantissa .0380 = 1.09

O for 100. . 1.09

The influence of temperature upon the time
required for mercury salts to kill was found for 1 -
10,000 at 50°C. to be g;5-minutes while at 20°C. it
was found to be 11.5 minutes. Madsen and Hyman (9)
found that the velocity at which anthrax spores were
destroyed by Hg012 was about 2.5 fold for a rise of
10°C.

A This is in accord with the findings above for

a 1 - 100,000 HgClz Solution, or O for 1°C. 8 1.09.

The temperature coefficient for phenol was found to

be 1.23 for each degree of temperature. This indicates
that phenol would have a higher temperature coefficient
than HgClZ. Phenol is much more active at high
temperatures than at low temperatures. This is another
important factor not shown by the F.D.A. phenol
coefficient method.

The following values were found for HgCl

2
K = 7.06
N = 3.8 as Hg ions and 1 as molecular
concentrate.
O - 1.09

A corresponding determination was made on Liquor Cresolis
Compositus U.S.P. using a
1% solution or 1 - 100

0.5% solution or 1 - 200

0.33% solution or 1 - 300.

The cresols, ortho, meta and para, are only
moderately soluble in water but in the presence of
soaps and alkalies they readily form emulsions.
U.S.P. Liquor Cresolis Compositus forms a clear
solution in distilled water, however, an emulsion
is formed with the bacterial suspension in nutrient

broth.

1‘- 100 Liquor Cresolis 25°C. 6,240,000 control

Time Count Value k.
0.5 min. 400 8.2
1.0 min. --- _--

Average value for k = 8.2

1 - 200 Liquor Cresolis 25°C. 6,240,000 control

Time Count Value k.
1.0 min. 336,000 1.3
2.5 min. 8,000 1.15
5.0 min. I ..... ----

Average value for k = .88

l - 500 Liquor Cresolis 25°C. 6,240,000 control

Time . Count Value k
0.5 min. 2,800,000 .69
1.0 min. 1,290,000 .69
2.5 min. 140,000 .65
5.0 min. 2,400 .68
7.5 min. 10 ---

10.0 min. ---- ---

Average value for k = .68

l - 300 Liquor Cresolis 35°C. 6,240,000 control

Time Count Value k
0.5 min. 121,600 2.4
1.0 min. 8,800 2.8
2.5 min. 10 2.4
5.0 min. ---- ---

II‘
to
e
(D

Average value for k

The rate of disinfection or reaction velocity of
1 - 200 solution Liquor Cresolis is 1.3 times as fast
as l - 300 solution and a 10° rise in temperature from
25°C. to 35°C. would speed up the rate of disinfection

4 times in a 1 - 300 solution.

1 - 100 = 10 parts per M. Time = 1
l - 200 I 5 parts per M. Time = 5
1 - 300 = 3.33 parts per M. Time = 10.

kz'flog-fi-

B
b

B

6,240,000
1

k2 = 1/1 10g 6,240,000

k1

n

n

n

1 x 6.795 = 6.795
1/5 10g 6,240,000
0.2 x 6.795 = 1.5590

10g Ef- . log gi-

10g ff§§§6 . 10g 1% = log 5 9 log 2 = §§%§-- 2.5
2.5

The dilution coefficient of phenol was found to

be about 6. as compared to 3 for Liquor Cresolis

Compositus. This would indicate that Liquor Cresolis

Compositus would be more effective in lower dilution

than phenol.

K = n log 0'? 10g t for 1 to 100 solution I

2.3 log 10 + 10g 1 = 2.3

K

n log 0 + log t for l - 200 solution =

2.3 log 5-+ log 5 I 2.3

K = n log C +-log t for l to 300 solution =
'2.3 log 3.3 + log 10 = 2.3

Average value for K = 2.3.

The true velocity constant K is about 2.4 compared
with phenol as 7. The speed of action of Liquor Cresolis
Compositus is not as rapid as that of phenol.

Calculation for O.

0 = 10/5 = 2. for 10°C.

102 2 = .501 e 10 = .0501

Mantissa .0380 = 1.09

O = 1.09

Liquor Cresolis Compositus shows a relative high
temperature coefficient as do all compounds related to
phenol.

O for 1° of temperature for phenol = 1.23

K for Liquor Cresolis Compositus 3 2.4

n = 3.3

O I 1.09

These values would indicate that the speed of
action of Liquor Cresolis Compositus is not as great
as phenol. The coefficient of dilution, however, shows
it to be more germicidal in far greater dilutions.

The germicidal properties are not as greatly increased

by 100 rise in temperature as in the case of phenol.

Determination on Acriflavine. Acriflavine has
been reported to be more actively germicidal in the
presence of blood serum than in peptone water and
thus it would appear that it is active in the presence
of organic matter. All the coal tar dyes on the other
hand are much less effective in the presence of blood
serum. However, there appears to be much controversy
on the matter and the increased action of acriflavine
in blood serum has been shown to be due to an increase
of alkalinity on loss of 002.

Churchill (10) recognizing that the action was
more inhibitory than germicidal coined the term
bacteriostasis. Gildersleeve suggested the adjective
term bacteriostatic.

A l - 500 and l - 1,000 solution prepared using

the same procedure with the following results.

l - 500 Acriflavine

Time

1.0
2.5
5.0
7.5
10.0

min.
min.
min.
min.

min.

1 - 1,000

Time

01

10
15
30
45
60
75

min.
min.
min.
min.
min.
min.

min.

25°C.
Count
58,400
6,400
720
460

10,560,000 control
Value k.
2.44
1.28
.83
.58

Average value for k = 1.28

Acriflavine 25°C.
Count
7,840
2,640
560
480
100

90

10,560,000 control
Value k.
.62
.36
.28
.14
.11
.09

Average value for k = .27

Acriflavine l - 1,000 55°C. 10,560,000 control.

Time - 9 Count Value k.
5 min. 'l5,600 ‘ .58
10 min. 11,200 , ~ .29
15 min. . 4,860, .22
30 min. . 200 .16
60 min. 160 , .07
90 min. 70 .05
120 min. --- ---.

Average value for k I .23

The rate of reaction between the disinfectant and
microbial p0pulation in the case of Acriflavine more
closely approximates a bimolecular reaction.

The average values found for k indicate, however,
that acriflavine in a 1 - 500 solution is 4 times as
rapid as in a 1 - 1,000 solution. A ten degree rise
in temperature, however, will decrease the speed of

action.

Calculation of coeffigent of dilution
k 92
nlog Eng-.901
1 - 500 or 2 parts 1,000 = 02 and t = 10.
l - 1,000 or 1 part to 1,000 = C1 and t = 75.

kg 3

B
- b

d‘
ll

1 B
:6- 10% '5'
10,560,000

 

.0133 x 7.027 =.0954

n

n
log

n:

log Ea.9 gfi
10g m: 027 9 log g-

705‘ f 108 2 = 208
2.8

7.027

8 01 X T“. 07027

This would indicate that the bacteriostatic power

of acriflavine with a dilution coefficient of 3 would

be increased 8 times on doubling the concentration and

would be

NNNN
u u a ll

K
The rate

rapid as

decreased 8 times on halving the concentration.

n log C +-1og t(l

2.8 log 24-log 10

n log C +-log t(l
2.8 log 1 + log 75
1.87

of disinfection for

phenol or HgCla.

500)
1.84
1,000)

acriflavine is not as

Calculation for 0.

_ 75 at 25°C.
9 ' I20 at 5500.

o = Egv'3 for 10°C.

 

o .6 for 10°C.

102 .6 = ’1.778 4 10 = -.1778

Mantissa =1.50

O for 1°C. = -1.5

The temperature coefficient of acriflavine was
found to be less than 1 and shows that acriflavine
is an exception to the general rule. The acridine
dyes are more actively bacteriostatic at low temperature
than at high temperatures. This is in accord with the
findings of Cameron (11) who observed that dyes are

more actively bacteriostatic at low temperatures than

at high.

Values found for acriflavine.

' 1.8

Pt.
I

n=2e8
0

-1.5
In comparing these values as found with phenol it
is shown that the velocity or rate of disinfection

for acriflavine is not as great as that of phenol.

The coefficient of dilution on the other hand destroy

the effici ncy of acriflavine as rapidly as decreasing
the phenol concentration. The effect of a 100 rise
of temperature decreases its bacteriostatic action of
acriflavine while for phenol the germicidal action

is greatly increased.

Determinations on a 1 - 2,000 and 1 - 20,000
solution of Chloramine T U.S.P.

Chloramine T does not combine with organic matter
as rapidly as do other forms of chlorine and it
exerts its germicidal action much more slowly and
while it is not as active a disinfectant as other
compounds of chlorine it is more stable when applied
to wounds. It maintains its strength for considerable
periods of time.

The depressive influence of organic matter upon
the germicidal activity of chloramine T is very much

less than in the case of other types of chlorine compounds

according to Johns (l2).

Chloramine T 25°“

1 - 2,000 i 0.
Time Count

0.5 min. 152,000
1.0 min. 2,400
2:5 min. .....

Average value for k

l - 20,000 Chloramine s 25°C.
Time Count
5. min. 256,000
10. min 8,000
15 min. 240
20 min. 20
25 min. ---

Average value for k

l - 20,000 Chloramine T

Time Count
5. min. 30
10. min. ---

Average value for k

55°C 15,040,000

13,040,000 control
Value k
3.8
3.6

3.7

13,040,000 control
Value k.
.34
.32
.30
.28

3.7

control
Value k

1.13

= 1.13

The rate of disinfection is 12 times as great for

a l - 2,000 solution of Chloramine

a 10° .
rise in temperature increases

T as 1 — 20,000 and

the rate of

disinfection 3.3 times.

n I log Ei-e 93

C1
1 B
k2 “flog-B-
B = 13,040,000
b = 1
t : 2.5 min.
k2 = 2%3-x 7.115 = 2.846
k1 = g%- x 7.115 = .2846
- k C2
n-10gE29-C—i-
- 20846} 05
n-logmelogm=l
n = l.

The coefficient of dilution is the same for
chloramine T as for HgClg and indicates that dilutions
have far less effect Upon the germicidal power of

chloramine T than upon phenol.

Calculation for O.

25 min. at 25°C.
5 min. at 35°C.

log 5 = .698 e 10 = .0698 = 1°C.

 

5 for 10°C.

This is in accord with the findings of Charlton
and Levine (13). "For a given concentration of available
chlorine as chloramine T a rise of 10°C. resulted in

a decrease in the killing time of about 82%".

As the germicidal activity of chloramine T is
due to its available chlorine content the value for
K as the true velocity coefficient should thus be
considered. A l - 2,000 solution of chloramine T
containing 15% available chlorine = 150 parts per
2,000,000 or 75 parts per million.

Let C2 = 75 parts per million.

Cl = 7.5 part per million

K = n 10g 0 + log t

K 1 X 1.875 +.397 = 2.27

1 X .875 4- 1.397 = 2.27

Velocity Coefficient K = 2.27

Temperature coefficient 0 = 1.17

n - dilution coefficient I 1.
This would indicate that while the rate of disinfection
of chloramine T is not as rapid as phenol. It has
a decided advantage over phenol in having a low
coefficient of dilution. Halving the concentration
of Chloramine T would only reduce its germicidal

activity by one half while that of phenol would be

reduced 64 times.

Summary of Results.

Summary of Results.

An attempt has been made to show the three
most essential characteristics of a disinfectant;
namely, the velocity constant, the concentration
exponent or coefficient of dilution and temperature
coefficient of four various types of disinfectants.
Experimental determinations were made on mercuric
chloride as a salt of a heavy metal; Liquor Cresolis
Compositus U.S.P. as a phenolic compound; acriflavine
as a medicinal dye; and chloramine T as a chlorine
compound. Similar determinations were also made
for phenol. A

The value for K or true velocity constant was
found to be about 6.6. The coefficient of dilution
was found to be approximately 6 and the temperature
coefficient for each 10°C. rise of temperature was
found to be 8.

The following results were obtained.

Compound Velocity Dilution Temperature
coefficient coefficient control

K n 0 10° 0 1°
Phenol 6.6 6. 8. 1.23
Hg012 7.06 1. 2.4 1.09
Liquor Cresolis 2.3 2.3 2. 1.09
Acriflavine 1.8 2.8 0.6 1.5
Chloramine T 2.27 1. 5. 1.17

The value of K for any disinfectant may be determined
and divided by the value of K as simularily determined
for phenol.) This gives a coefficient independent of
all variables. The value of K for phenol was found to
be 6.6. The value of K for Hg012 was found to be 7.06.
This yields a coefficient as compared to phenol Z%%%.=
1.07 and indicates that the rate of disinfection or
velocity coefficient of HgClz is 1.07 times that of
phenol.

Approximate comparative velocities were likewise
found to be for

Liquor Cresolis Compositus éfg-3 .34

Acriflavine 1'8 a .28
(3.6

Chloramine T 553-131 = .54

This would indicate that the speed of action of these

compounds is not as rapid as that of phenol or Hg012.

This one factor alone will show more effectively
the value of Hg012 as disinfectant than the standard
F,D.A, phenol coefficient method which only tells us
the highest dilution of Hg012 that will kill the
micro-organism under test after ten minutes but not
in five, as compared toklike solution of phenol. The
phenol coefficient in no way will show the rapidity of
action of a disinfectant. Disinfectants vary in their
speed of action as is shown by the value found for K
or the velocity Constant of HgClB as compared to phenol.

Neither does the F.D.A. phenol coefficient methci
in any way show the change in efficiency with change
of concentrations. The method as proposed showing that
coefficient of dilution is very valuable and in
conjunction with the velocity constant as shown
describes far more fully the value of a disinfectant
then the F.D.A. phenol coefficient method.

Values for the concentration coefficient or

dilution exponent n were found to be for

Phenol, an a c6
Acriflavine, On 3 0°
Liquor Cresolis, on = C2.6
Chloramine T, ch = Cl

H8912. 0n = Cl

The coefficient of dilution for HgClz was found to

be 1 while that for phenol was found to be 6. A

1 - 1,000 solution of HgClz for instance is twice as
efficient as l - 2,000 solution (will do the work in
half the time) while a 1% solution of phenol is 64
times as efficient as a 0.5% solution. The same holds
true for'Chloramine T. The converse is also true;

a 1 - 2,000 solution HgClg is only half as efficient
as a 1 - 1,000 solution and a 0.5% solution is 64 times
less efficient than a 1% of phenol. Likewise halving
the concentration of acriflavine would decrease its
efficiency four times while doubling would increase

it four fold.

This shows exactly what to expect on diluting
concentrations of a disinfectant and does away with
the necessity of first having to determine the concen-
tration necessary to come within the killing range of
a 1 - 90 or 1 - 100 solution of phenol. It shows
definitely that a disinfectant with a high value for
n losses efficiency rapidly with dilution.

Another factor, which is highly important and
is in no way shown by the F.D.A. phenol coefficient
method, is the relation of temperature to disinfectants.

increasing the temperature 1° may increase the rapidity

'7

of disinfection from 1.07 to 1.23 fold, as shown by
the comparative values found for a rise of 1° of

temperature.

Phenol 0 = 1.23
Liquor Cresolis O = 1.09
Hg012 0 - 1.09
Chloramine T o = 1.17
Acriflavine O = minus 1.5

The temperature coefficient of phenol was found to be
1.23 and for Hg012 1.09. An increase in temperature
increases the bactericidal action of phenol to a .
greater extent then it does for HgC12. (The temperature
coefficients of HgClg (o = 1.09) and phenol (o = 1.25)
would indicate that a 1° rise in temperature would
increase the bactericidal action of phenol 1.13 times
over that of HgClz. The ratio or phenol coefficient
only shows the value of the disinfectant at 57°C. and
would give no indication of the effect of temperature
upon the disinfectant. The temperature coeffident

of acriflavine (minus 1.5) shows that a rise in
temperature would greatly reduce its efficiency as

a disinfectant and, as has been stated, is an exception
to the rule.

If a comparison with phenol is desired comparative

ratios of these factors can be shown as, for instance,
in the case of H3012. The comparative ratio of speed
of action or velocity coefficient K will equal 1%%%.

or 1.07. The coefficient of dilution or concentration
exponent of phenol was found to be 05 while that for
H3012, Ci. The temperature coefficient was found to
be 0 = §f§§~or 1.13. This would then indicate to us
that the speed of action of Hg012 is 1.07 times that
of phenol and that the rate of dilution of Hg012 is to
the first power as compared to the sixth power for
phenol or that HgClz would be effective in far greater
dilutions than phenol. The temperature coefficient
shows that a 1° rise in temperature would increase

the bactericidal power of phenol 1.13 times that of
HgClz. This will tell us far more effectively what

we wish to know about a disinfectant than a mere
statement of its F.D.A. phenol coefficient which
depends solely upon fixation of the reaction velocity

of a given dilution at a given temperature and

during a given period of time.

Conclusion.

For practical purposes the employment of such
mathematical formulae as proposed seems to fit fairly
well. The employment of such formulae does not in
the least necessitate the acceptance of a strictly
chemical, physical or biological explanation of forces
that control the rate of disinfection as for instance
the monomolecular law. However, a strictly methematical
interpretation of numerical data based upon a limited
number of determinations appears to be without
justification due to experimental errors which may
arise. It shows, however, far more effectively what
we desire to know about a disinfectant, namely,
speed of action, rate of dilution, and effect of
temperature than does the present F.D.A. Method.

At a latfer date it is proposed to make a further

and more detailed study of the subject in hand.

3.

4.

5.

6.

7.

References.

Kroning B and Paul T.L.

(ztschr- f. Hyg. und Infect. 25; 1, 1897).
Rideal S. and Walker J.T.

The Standardization of Disinfectants.

Jour. Roy. San. Inst.~ 24: 424, 1903.

Chick, Hariette and Martin C.J.

The Principle Involved in the Standardization
of Disinfectants.

Jour. Hyg. 8: 654, 1908.

Anderson & McClintic. '

A Method for the Bacteri010gical Standardization
of Disinfectants.

Jour. Infect. Dis. 8:1, 1911.

Reddish, G.T.

Examination of Disinfectants.

Jour. of Public Health. 17:320: 329, 1927.
Buehle, G.L.A. and Brewer.

U.S. Food and Drug Administration Method of
Testing Antiseptics and Disinfectants.
U.S.D.A. circular 198, 1931.

Watson, H.E.

A Note on the Variation of the Rate of Disinfection

with Changes in Concentration of the Disinfectant.

T Ans. Trun- Q- R112 1 GHQ

8.

10.

11.

12.

13.

Phelps, E.B.

The Application of Certain Laws of Physical
Chemistry in the Standardization of Disinfectants.
Jour. Infect. Dis. 8: 27, 1911.

Madren, T. and Nyman, N

Zur Theorie der Disinfection.

Zea. hr. f. Hyg. 57: 388, 1907.

McCulloch, E.C.

Disinfection and Sanitation.

Chap.XIII , page 325, last edition.

Cameron, E.F.

The Bacteriostatic Effect of Gentian Violet on
ThermOphilic Spore forming Bacteria.

Jour. Bacteriology. 19: 52, 1935.

Johns, C.K.

The Evaluation of the Germicidal Potency of
Chlorine Compounds.

Scientific Agr. 15: 218, 1934.

Charlton, D.B. and Levine, Max.

Some Observations on the Germicidal Efficiency
of Chloramine T.

Jour. Bacteriology. 30: 163, 1935.

 

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