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THE EVALUATION OF THE GERMECiDAL
EFFHCIENCY OF QUATERN‘ARY
AMMOMUM COMPOUNDS

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‘-————_ “flu- --——_-____—_

 

' This is to certifg that the
thesis entitled

The Evaluation of the Gemicidal Efficiency of
. Quaternary Amonium Canpounds

 

presented by

Daniel Paul Rm

has been accepted towards fulfillment '.
of the requirements for

Easter degree in Bacteriology ' 4 ' '

 

14- _ Ma r professor l74."£‘

 

 

The Evaluation of the Germicidal Efficiency

of Quaternary Ammonium Compounds

By
Daniel Paul Roman

w

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

MASTER OF SCIENCE

Department of Bacteriology
1950

THESIS

AC KliO‘v‘IIEDGEMENT
The author wishes to express his sincerest
appreciation to Dr. W. L. Mallmann by whose

able assistance this thesis was made possible .

-1.

Introduction

 

Present methods of evaluating disinfectants leave much to be
desired. The phenol coefficient procedure of the Food and Drug Ad-
ministration yields questionable results when used as a basis of
comparison among chemical agents of different structures and behavoirs.
A phenol coefficient as a comparative evaluation of a quaternary am-
monium compound, sodium hypochlorite, chlorophenate, or a mercurial
is valueless. The use of the phenol coefficient is particularly
inapplicable for testing quaternary ammonium compounds, not only
because of their lack of relationship to phenol but because of the
difficulty of reproducible results. A phenol coefficient is applic-
able only when compared to phenolic derivatives (17).

Several factors contributing to difficulties experienced in
testing surface active agents by the phenol coefficient method have
been recognized. Procedures to compensate for these difficulties
have been advocated, but do not eliminate the basis for error; that
of comparing compounds of different types, and with different modes
of action with phenol.

In order to eliminate bacteriostasis resulting in the carry over
of the disinfectant to the subculture broth, variations based on di-
lution and neutralization have been introduced. Shippen (20) intro-
duced the procedure of carrying over a loopful of the first subculture
into a second tube of broth. Although it was not originally designed
for this purpose, it has found a use in the testing of quaternary am-
monium compounds. Shippen's modification, while diluting out the dis-

infectant has the disadvantage of also diluting out the organisms so

-2...
that the second broth tube may not be inoculated even though the first
tube has a few viable organisms present.

Klarmann and Wright (9) in their "semi—micro" modification of the
phenol coefficient use 200 ml. of broth to dilute their medication
mixtures. They stated that even this high dilution did not eliminate
bacteriostasis.

The use of lecithin as a neutralizing agent in the testing of
these cationic detergents was suggested by Quisno, Gibby and Foter (16).
Pressman and Rhoades (15) neutralized their medication-culture mixtures
with sodium stearate before making subcultures.

Tobie and Orr (22) found that erratic results reported in test-
ing surface active germicides were due partially to the formation
of drops on the transfer loops which were not equal in size. The
resulting reduction in the number of bacteria taken for inoculation
is especially important after the numbers of bacteria have been
greatly reduced. To avoid making the germicides seem better than
they actually are, the authors suggested transferring .02 ml. by
pipette instead of making transfers by loop.

Several writers report that variations in results obtained
when testing quaternary ammonium compounds are due to clumping
(3,8) and to the adherence to the walls of the tubes by the bac-
terial cells (8). This prevents a uniform exposure period and repre-
sentative sampling of the medication mixture.

The hypothesis has been advanced (3,8) that on exposure to cation-
ic germicides a large number of bacteria tend to adhere to each other

forming clumps. Only the outside layers are in direct contact with

~3-
the cations so that complete kill is sometimes impossible in the time
of exposure. However, as reported by Kivela, (7) no clumping of

Salmonella typhosa or Micrococcus pyogenes takes place in any dilutions

 

 

from 1:1,000 to l:lO0,000 using Roccal, Hyamine, and BTC as the cation—
ics.

That the original F. D. A. method is not suitable for measuring
the germicidal action of this class of compounds is claimed by Klarmann
and Wright (8). They noted that suspended bacterial cells enter into
some combination with the quaternary ammonium compounds which causes
them to be attached to carriers, such as glass, making them practically
unavailable for transfer by the F. D. A. loop. Klimek and Umbreit (10)
failed to confirm the results of Klarmann and Wright, finding no evidence
that a significant number of viable organisms remained adsorbed on
glass in a tube to make them unavailable for transfer.

Several methods for the assaying of the effectiveness of sur-
face active agents have been recommended by workers in the field.

The use-dilution method (12), in which practical working dilutions

are tested under conditions approaching those encountered in the field,
is a relatively simple means of evaluation. The use-dilution was
selected as a test in Part II of this thesis where it is discussed in
greater detail

Johns (S) has developed a glass slide technique in which partial-
ly dried glass slides are immersed in the disinfectant to be tested,
rinsed with tap water to minimize bacteriostatic action and the slide
plated in a nutrient medium. By means of controls, a 99.9 per cent

kill is taken as the end-point.

-L.

Cade (2) uses a combination of the standard phenol coefficient
test combined with a plate count as a means of bringing about the
determination of a more definite end point. The standard F. D. A.
loop does not always pick up viable organisms in the medication mixp
ture, so that a cotton swab is used to remove organisms adhering to
the sides of the tube, the swab is then swished in a tube of melted
agar and plated. Per cent kill is calculated by this method.

Klarmann and Wright (8,9) have developed a semi—micro adaptation
of the phenol coefficient test and a filter paper transfer technique.
In the semi—micro method 0.5 ml. of medication mixture and 0.05 ml.
of the test culture are allowed to react before adding the entire mixp
ture to 20 ml. of nutrient broth. In this way the entire solution is
subcultured resulting in a more representative sample and yet avoid-
ing the necessity of using large volumes of broth. The filter paper
transfer technique introduces pieces of filter paper into the culture
before adding 5 ml. of the various dilutions to the disinfectant. At
the end of five, ten and fifteen.minute intervals, respectively, one
piece of filter paper is transferred to 20 ml. of nutrient broth. These
are retransferred into a second tube, also containing 20 ml. of broth,
to eliminate bacteriostasis.

In vivo methods of evaluating germicides have been devised to
avoid the inconsistencies encountered in the in vitro tests. Nungester
and Kempf (1h) have designed an "infectiondprevention" test using mice
as test animals. In this test the tails of anesthetized mice are
immersed in a culture highly pathogenic to mice. The contaminated tail

is exposed to the disinfectant for two minutes then the tip of the tail

-5-
( 1 cm. ) is cut off and implanted aseptically in the peritoneal cavity.
The efficiency of the disinfectant is determined by survival or death
of the animals.

Green and Birkeland (h) used the developing chick embryo as a
means of determining the effective dilutions of quaternary ammonium
compounds used as wound disinfectants. In this procedure the chorio—
allantoic membrane is inoculated with a 23-25 hour culture of §t§ph.
323222 209, the membrane is then treated with disinfectant for five
consecutive days. On the sixth day, the membrane is stroked with a
moist cotton swab and streaked on nutrient agar for an estimation of
remaining viable organisms.

Although not doing away with variations in the resistance of the
organism, the modification of the phenol coefficient procedure de-
vised by Kenner et al (6) did away with wild plusses and always pro—
duced a definite end point. In this test, using cetyl pyridinium

chloride as the disinfectant, and Salmonella typhimurium as the test

 

organism, subcultures were inoculated into mice instead of into 4
F. D. A. broth. The recovery of organisms from the heart blood of the
infected mice was taken as the end point.

As yet, none of the methods proposed for the evaluation of the
bactericidal efficiency of the quaternary ammonium compounds has been
universally accepted despite the obvious shortcomings of the Food
and Drug Administration method. Some further modifications must be
made in existing tests, or a new test devised, which will compensate
for the peculiarities of this type compound. It is important to re-

cognize that such factors as temperature, variations in number and

.6.
resistance of organisms, and the composition of the culture medium
play an important part in the results obtained in testing disinfect-
ants. The many uses of disinfectants, with their accompanying
different conditions, makes obvious the fact that no one test can
fulfill all the requirements of these conditions. There is, however,
an urgent need for a reliable basis of comparison for even such limit-
ed conditions as can be duplicated practically for a large number
of laboratory tests.

Recognized sources of difficulty in present methods of comparison
which have caused confusion as to the relative merits of quaternary
ammonium compounds have been summarized by Klimek and Umbreit (10).
The difficulties are: variation in composition and components of the
test medium; genetic mutation or transitory dissociation of the test
culture; variations in the number of organisms which may be trans-.
ferred to subcultures when a standard h mm. loop is employed; and
lastly, the habitual comparison of chemically unrelated substances
"with phenol.

. These are factors which cause wide variations in the results ob-
tained by the phenol coefficient procedure, and.which exist to plague
the worker in other methods devised for testing this type compound.
Variations which occur in the resistance of the test organisms to
the quaternary ammonium type compound'while the resistance to phenol
remains unchanged (1,13), have made the assignment of phenol co-
efficient values meaningless when applied to the cationic class of
disinfectants. A coefficient arrived at today in one laboratory may

not be duplicated in another laboratory tomorrow, and may even differ

-7-
in the same laboratory when tested by the same worker. At present
there is no method of maintaining the disinfectant testing strains

of §almonella typhosa or Micrococcu§_pyogenes var. aureus at a

 

 

standard resistance to the quaternary ammonium compounds. This then
brings up the question of what the one right coefficient is since
so many different values may be obtained by skilled technicians work-
ing under the same conditions.

The search for a basis of comparison which would nullify the
effects of these factors, and produce a method that would yield
reproducible results regardless of the resistance of the test or-

ganisms led to the work done in this thesis.

-8-
Experimental
The selection of a basis of comparison that will yield similar
results regardless of the resistance of the organism is of prime im-
portance in the development of a new method. Not only is the killing
dilution of the quaternary ammonium compounds not comparable to the
killing dilution of phenol in calculating the use-dilution, but the

resistance of the test organisms; namely, Sal. typhosa and fl. pyogenes

 

var. aureus,may vary widely to the quaternary ammonium compounds and
still not vary to phenol. Mallman (11) showed that two strains of

Sal. typhosa obtained from the same culture gave identical resistances

 

to phenol, but gave resistances of l:lS,000 and l:b5,000 to Phemoral,
a quaternary ammonium compound. Mallman, Leavitt, and Joslyn (13)
demonstrated that M. pvovenes var. gpgggg'when grown in F. D. A. broth
and Difco disinfectant testing broth yielded killing dilutions of
l:h,000 and l:20,000 although the resistance to phenol was the same
for both cultures.

Knowing that such marked discrepancies may occur, it is apparent
that unless the approximate killing dilution of quaternary ammonium
compound was known it would be impossible for a bacteriologist to
know whether his results were dependable. The mere fact that his
phenol resistance was standard is of no value in determining that
the resistance to the quaternary ammonium preparation is correct.

In as much as the quaternary ammonium compounds are basically
similar it would seem logical to assume that any shift in the resist-
ance of the test organism to one would be comparable to another, even

though their germicidal activities might be quite different. For this

-9-
reason it was thought that the selection of a pure product such as
Hyamine 1622, a di-isobutyl-phenoxy-ethoxyeethyl—dimethyl-benzyl-
ammonium chloride might be used as a reference compound in the same
manner that phenol has been used in the F. D. A. phenol coefficient.
If, however, the organism need not have a standard resistance to the
Hyamine 1622, then the test would be even more simple than the F. D. A.
phenol coefficient because the standard killing dilution could shift
with the resistance of the test organism.

The selection of a basis of comparison that will give similar
results, regardless of the resistance of the organism, is of prime
importance in the development of a neW'method. To determine whether
resistance of test organisms to all quaternary ammonium compounds
varied.with a constant ratio, several different types of compounds
'were tested against organisms of varied resistance. The compounds
tested were Hyamine 1622, a di-isobutyl-phenoxyeethoxy-ethyl-dimethyl-
benzyl—ammonium.chloride; Bional E. C., an alkyl-dimethyl-ethyl-ammonium
bromide; Tetrosan, an alkyl—dimethyl-dichloro—benzyl-ammonium chloride;
BTC, an alkyl-dimethyl-benzyl—ammonium chloride; and LPG, lauryl
pyridinium ammonium chloride. Hyamine 1622 was chosen as a basis of
comparison since it is a pure compound, readily soluble, and stable
in a stock solution of 1:100. This list includes compounds which have
their highest effective dilutions falling over a wide range; thus
including compounds of varied efficiencies for the test.

Existing tests were chosen for the determination of the in-
fluence of varied resistance on the ability of the compounds to

maintain a constant ratio of effectiveness. The phenol coefficient

—10-
test was chosen for the first set of experiments since it is a
familiar test to all and has the official sanction of the Food and
Drug Administration. In part II the use-dilution method was select-
ed as offering an available method for ascertaining the critical
dilutions with a greater degree of accuracy. This method has been

adopted by the Food and Drug Administration (21).

-11..

Experiment‘z

 

The Determination of a Quaternary_Ammonium Compound Coefficient by
means of the F. D. A. Method.

The test organisms used were 22-26 hour broth cultures of a
standard disinfectant testing strain of Salmonella typhosa, (Hopkins)
and Micrococcus pyogenes var. gggggg (209) incubated at 37° C. To
obtain organisms of a greatly varied resistance to quaternary ammon-
ium compounds M. pyogenes var. gpgggs was grown in Difco disinfectant
testing broth (13), and in F. D. A. broth. The presence of lactose
in Difco broth enables M. pyogenes var. gpggpg to withstand substantial-
ly higher concentrations of quaternary ammonium disinfectants. First

tests with Sal. typhosa were carried out with an organism transferred

 

for five consecutive days in F. D. A. broth from an agar slant stock
culture of the organism. Daily transfers for several weeks signifi-
cantly altered the resistance of the organism, and it was this less
resistant form that was used in a second series of tests. A resis-
tant and a susceptible form of each organism was used in this experi-
ment, although both forms originally came from the same source.

Tests were carried out at 20° C. following the directions set
forth in the F. D. A. Circular 198 (19-), except that Hyamine 1622
was used to check the organisms for standard resistance. In all
cases the test organisms showed the following resistance to phenol

at 200 C.

-12...

E, pvocenes var. aureus
Dilution 10 15
41:60

5
/

1:70 / - -
;

 

1:80 / #
Sal. typhosa

Dilution S 10 15

1:90 — - _

1:100 / - —

1:110 / ¢ 2

Five m1. of appropriate dilutions was transferred to medication
pots (l in. by 3 in.) and placed in a 200 C. water bath until the
temperature of the bath was reached. Five-tenths ml. of the test
culture, previously shaken for fifteen minutes, was added to each of
the dilutions at a suitable time interval. At five, ten, and fifteen
minute intervals from the time of seeding the medication pots, trans-
fers were made to tubes of broth. The broth was of the same composi-
tion as that used to grow the organisms.

From the results of the tests quaternary ammonium compound co-
efficients were calculated for the various types of compounds. The
The quaternary ammonium compound coefficients are calculated by di—
viding the highest dilution of disinfectant capable of killing in
ten minutes but not in five minutes, by the greatest dilution of
Hyamine 1622 showing the same results. The quaternary ammonium com-
pound coefficients are expressed to the nearest whole number to avoid
fictitious accuracy. Hereafter, these will be referred to as QAC co-

efficients.

-13-

Examination of the data in Tables I and II does not show any
basis for the establishment of a QAC coefficient. 'Variations in the
resistance of the organism to the quaternary'ammonium compounds used
in this series of tests gave as widely divergent results in the cal-
culation of a QAC coefficient as for a phenol coefficient. In the
case of Bional E. C. a constant ratio was maintained when compared to
Hyamine 1622 regardless of the resistance of the organism. ‘With BTC,
Tetrosan, and LPG the coefficients obtained varied by as much as 33
to 50 per cent. Phenol coefficients calculated from the killing di—
lutions of the disinfectants against organisms grown in F. D. A. broth
varied to a slightly greater extent than did the QAC coefficients,
ranging from 33 to 56 per cent.

A quaternary ammonium compound coefficient determined by the
F. D. A. procedure is as accurate a method for evaluating this type
compound as a phenol coefficient. It varies over almost as wide a
range, but there is a more solid basis in comparing like compounds
'with each other.

Differences in the QAC coefficients obtained may be due to
factors inherent in testing by the F. D. A. phenol coefficient proé
cedure. The effect of surface tension on the size of the drops on
the transfer loops, clumping, adherence to the side of the vessel,
and bacteriostasis still exist to make the use of the phenol co-
efficient impractical. Wide ranges of dilutions are necessary to
avoid wild plusses which make it impossible to obtain a definite end
point. All these factors exist to make it impossible to ascertain the

exact killing dilutions for these compounds, thus making this method

-11,-
inappropriate for determining either a phenol or a QAC coefficient

of any value.

_15-

 

 

 

 

 

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-17-

Fingering}: E

 

The determination of a Hyamine Coefficient by means of the
UseéDilution'Method

 

A second set of tests were run using the use-dilution (12)
method as a check on the results obtained by the phenol coefficient
method.

The test organisms used were the same as those in the tests
using the F. D. A. phenol coefficient method.

In the use-dilution method 22-26 hour broth cultures were shaken
for fifteen.minutes to break up bacterial clumps. Sterile glass rods
:3; inch in diameter and 1 inch in length having a flattened "nail" head
for handling were dipped into the broth culture and then laid on sterile
filter paper in petri dishes for drying. Care was taken to avoid roll-
ing while drying. A drying period of thirty'minutes was allowed at
room temperature.

Desired dilutions of the disinfectant were made and 10 ml.
amounts placed in each of the medication pots (1 inch by 3 inches).

A 10 m1. amount of sterile physiological saline was placed in another
set of medication pots. The pots were then placed in a 20° C. water
bath and brought to the temperature of the bath.

Since the end-point in these tests was to be the highest dilu-
tion to kill in ten minutes but not in five, a departure was made
from the method set up by Mallman and Hanes (12). Only two glass
rods were immersed in the disinfectant to be tested, eliminating the
1 min. and 30 min. exposure periods. At the end of the five and ten

minute exposure periods a rod was removed and immersed in the physio-

-18-
logical saline for one minute. It was then placed in a tube contain-
ing 10 m1. of nutrient broth. The tube was shaken vigorously to re-
move all organisms from the rod and 1 ml. amounts plated.

Controls were run by dropping rods covered with the dried or-
ganisms into the saline tubes for one minute and then into tubes
containing 10 ml. of nutrient broth. Suitable dilutions of the
broth were plated.

Since the data obtained from the plate counts is not pertinent
to this paper the results are not included. Due to errors in random
sampling some negative plates were obtained where the tubes showed
growth at the end of fortybeight hours. All plates and tubes con-
taining the rods were incubated at 37° C. for fortyaeight hours.

The organisms were checked for standard resistance by the phenol
coefficient method.

The use-dilution was designed originally to evaluate disinfectants
under conditions approximating those encountered in astual use, and
was meant to divide disinfectants into two categories: satisfactory
and unsatisfactory. However, in these tests a comparison was set
up between BTC, Bional, Tetrosan, and LPG; again using Hyamine 1622
as a basis of comparison. No standards have been set up for the

resistance of M. pyogenes var. aureus and Sal. typhosa to phenol when

 

tested by the use-dilution method. In this group of experiments only
a QAC coefficient was calculated.

Examination of the QAC coefficients obtained (Tables III and IV)
by the use-dilution.method show a close correlation regardless of the

resistance of the organism. Only with LPC did the relative ratios

-19-
of the disinfectant powers of the compounds fail to remain constant.
The coefficients obtained with Lauryl Pyridinium Chloride against
E, pyogenes var. aggggg differed by twentyafive per cent, against

Sal. typhosa only twenty per cent. With the other compounds tested

 

an exact ratio was maintained when tested against these organisms.
Although the resistance to Hyamine 1622 differed from 1:5,000 to
l:10,000 in the case of each organism, the resistance of the organisms
to BTC, Bional E. 0., and Tetrosan changed to the same degree so that
a constant ratio was maintained.

The data indicate that a reliable evaluation of quaternary
ammonium.compounds was possible by a QAC coefficient obtained by
the use-dilution method, using Hyamine 1622 as a basis of compariSOn.
By this method the need for standardizing the test organism has been
eliminated. Since the resistance of the organism to the reference
compound varies to the same degree to the compound being tested, a
coefficient representing their relative merits may be obtained that
will remain constant even when tested against organisms of widely

varied resistance.

 

 

 

 

 

 

 

 

 

 

 

 

 

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l. The Food and Drug Administration phenol coefficient procedure,
and any modifications of it yet prOposed, are not suitable for evalu-
ating the germicidal efficiency of quaternary ammonium compounds.

2. A quaternary ammonium compound coefficient obtained by the
use—dilution method offers a reliable means of obtaining a quantita-
tive and qualitative evaluation of the germicidal efficiency of

quaternary ammonium compounds.

-22-

Discussion

 

'The development of a suitable method for the evaluation of
quaternary ammonium compounds is of considerable interest. That
the present F. D. A. technique is found lacking as a basis of com-
parison is obvious from a study of the literature. In this thesis
Hyamine 1622 was used as a basis for comparison of other compounds
of the same type and of the same mode of action. In the first group
of experiments the use of Hyamine 1622 was the only departure from
the standard phenol coefficient procedure. The results of the first
experiment show no basis for the establishment of a QAC coefficient
in preference to the F. D. A. procedure, except as a comparison of
similar compounds.

In the group of experiments using the use-dilution as a test
method many of the sources of error which produced variable results
have been eliminated or reduced. 'Variations in the resistance of the
organism have become of lesser importance since the organism changes
its resistance to the standard in approximately the same ratio as
it does to the disinfectant to be tested. By rinsing the rods in
physiological saline the need for excessive dilution or for the
addition of chemical neutralizing substances is eliminated. A
representative sample is assured by removing all viable organisms
with the glass rod.

Since the conditions of the test more closely resemble those en-
countered in practice it is felt that a QAC coefficient obtained by
the use-dilution method offers a more practical method for the evalua-

tion of cationic germicides.

1.

2.

3.

h.

7.
8.

9.

10.

11.

Bibliography

 

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