AN

INVESTIGATION O F T H E EFFICIENCY OF THE
Q U A T E R N A R Y A M M O N I U M G E R M I C I D E S IN
THE PRESENCE OF HARD WAT ER

By
FRANK

ROBERT

A

PEABODY

THESIS

Submitted to the School of G r a d u a t e Studies of M i c h i g a n
State College of Agriculture and Applied Science
in partial fulfillment of the r e q u i re me nt s
for the degr ee of

DOCTOR

OF

PHILOSOPHY

D e p a r t m e n t of Bacteriology and Public Health

AN

INVESTIGATION
QUATERNARY
THE

OF

THE EFFICIENCY

AMMONIUM GERMICIDES

PRESENCE

OF

HARD

OF TEE
IN

EATER

By/
o1
.

Frank R'i Peabody

AN

ABSTRACT

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

OF

PHILOSOPHY

Department of Bacteriology and
Public Health
1952

AN

INVESTIGATION OF TI-IE EFFICIENCY OF THE
QUATERNARY A: MON IUL1 GERMICIDES IN
THE PRESENCE OF HARD WATER

£

Frank R<s^ Peabody
AN

AESTRACT

The survivor-curve test of Weber and Black was used for
the first phase of the studies.

The interfering acti n of the

college tap water with the germicidal action of a quaternary ammonium
compound (QAC) was shown to be a retarding of the action rather than
a direct neutralization of the germicide.

When the water was soft­

ened by different methods, no difference in the interfering action
was noted except idien using Versene.

The addition of this complex-

ing agent resulted in a marked reduction in the interfering action
of the hard water.

Subsequent testing with Versene in distilled

water proved that the action is more than the softening action
on the water.

A definite increase of germicidal action resulted

from the addition of Versene to the QAC in distilled v/ater whether
the Versene vras added to the organisms or to the germicide.

When repeated dosages of organisms were added to the
same disinfectant solution, a reserve of germicidal action was
demonstrated even in the presence of tap water.

To illustrate

Abstract— F. R. Peabody
Page 2

this more readily, the exhaustion test was designed in which
successive increments of organisms were added to the same dis­
infectant solution and samples taken ten minutes after, the addi­
tion of each increment.

In this way, the number of increments

that could be added before the germicidal action was "exhausted"
could be determined.

By utilizing the strong complexing action of the
Versene, the germicidal action of the QAC which had been inacti­
vated by the hard water could be released again to exert a
germicidal action comparable to that of QAC in distilled water.
This release of germicidal action was also demonstrated without
the use of Versene by allowing an extended period for the germicide
to be released from its combination with the interfering factor
in the hard water.

ACKNOWLEDGMENTS

The
Mallmann,

author

e x p r e s s e s his sincere appreciation to Dr.

Professor

of Bacteriology, for his continuous

a g e m e n t an d gu i d a n c e during
to M r .

Donald Muentener

the c o u r s e

W.

L.

encour­

of this investigation, a n d

for his technical assistance.

TABLE OF CONTENTS

Page
I N T R O D U C T I O N .................................................

1

LITERATURE

4

R E V I E W .........................

EXPERIMENTAL
PRESENTATION

P R O C E D U R E S ...............................
OF

DATA

.............................

17
28

D I S C U S S I O N .....................................................

61

C O N C L U S I O N S ..................................................

77

B I B L I O G R A P H Y ................................................

79

INTRODUCTION

The

rapidly increasing use during the past decade of the

group of g e r m i c i d e s k n o w n as the qu aterna ry a m m o n i u m
pounds has p r e s e n t e d m a n y p r o b l e m s f r o m

com­

the standpoint of p r a c ­

tical applications as well as the laboratory evaluation of their
germicidal efficiency.

Although their first use w a s

in the field

of medicine, as antiseptics for skin and w o u n d treatment, m a n y
of the m o r e

recent applications have b e e n as germicidal agents

in the area of public health; m o r e
dairy e q u i p m e n t both o n the f a r m

specifically, as sanitizers for
a n d in the dairy plants, for

treatment of food -process ing equipment, for use as a c h e m i c a l
rinse treatment in the sanitization of multiple-use containers in
the restaurant, and m a n y

other applications.

of disinfection that m a n y

of the m o s t

It w a s

in this area

controversial p r o b l e m s w e r e

encountered.
Although these difficulties are m a n y

a n d co mpl ex,

one

which is pertinent to this study is the m e t h o d of evaluating the
germicidal action of this gr ou p of c o m p o u n d s .
evident that evaluation of these c o m p o u n d s

It soon b e c a m e

b y the F D A

phenol c o ­

efficient m e t h o d gave misleading results w h e n c o m p a r e d to that of a

2
phenolic type of c o m p o u n d for w h i c h the test w a s
also presented m a n y

technical p r o b l e m s

in duplicating results,

avoiding bacteriostasis, a n d other factors.
velopm ent of m a n y

This led to the d e ­

1'modifications" an d n e w

s o m e germicidal value to these c o m p o u n d s .
sified these m a n y

techniques

fundamental theory involved.
designed to m e a s u r e

designed, a n d

tests for assessing
Wyss

into three g r oups
A t one e x t r e m e

(25) has

clas­

according to the
are e x p e r i m e n t s

the effect of varying individual factors an d

predicting the p e r f o r m a n c e of the c o m p o u n d u n d e r any condition
of application.

A t the other e x t r e m e

is found the test in w h i c h

the c o m p o u n d is taken into the field a n d its effectiveness tested
under the conditions w h e r e

it is to be used.

Intermediate to these

is the phenol coefficient m e t h o d an d its various modifications.
Weber

and B l a c k M e t h o d

The

(23) a n d the Cantor-Shelanski Capacity

Test (3) w h i c h are u s e d in this study are b a s e d to a certain deg re e
on practical considerations of the ultimate use of the c o m p o u n d s
to be tested.
Another p r o b l e m

to receive a great deal of attention w a s

the determination of substances w h i c h w o u l d inactivate these c o m ­
pounds, or at least interfere with their germicidal activity.
ticular p h a s e of this difficulty w a s

A

par­

en cou nte red w h e n the quaternary

ammonium

compounds

were

applied as

sanitizing agents

restaurant,

dairy, a n d ot h e r p l a c e s w h e r e

or p o w d e r s

were

tion.

In these

made

into a u s e

cases w h e n

for evaluation, m a n y

the

c o n c e n t r a t e d solutions

dilution at the t i m e

Weber

of the w a t e r s

and

in the

Black

of applica­

Method was

used

u s e d for the p r e p a r a t i o n of

the sanitizing solutions w o u l d interfere wit h the g e r m i c i d a l action
of the q u a t e r n a r y a m m o n i u m
p rima ri ly with this p r o b l e m
w a t e r s with the g e r m i c i d a l
compounds.
lem,

an

As

a means

" e x h a u s t i o n test"

compounds.

of the interference

Kivela,

and

thesis

is c o n c e r n e d

of certain h a r d

efficiency of the q u a t e r n a r y a m m o n i u m

of studying certain p h a s e s

of this p r o b ­

is u s e d w h i c h is a modification of the

capacity test p r e v i o u s l y m e n t i o n e d
Mallmann,

This

Turney

(11).

a n d a field test m e t h o d of

LITERATURE

Due

to the several fields of application of the q u a t e r n a r y

ammonium

compounds

erature on these

and V a n

this review,

and the m a n y

compounds

eral excellent r e v i e w s
by R a h n

REVIEW

w o r k e r s interested, the lit­

is very

which

cover

extensive.

There

are

sev­

this m a te ri al such as those

Eseltine (17), W y s s

(25), and L a w r e n c e

an attempt ha s b e e n m a d e

(9).

In

to re vi e w only that liter­

ature w h i c h pertains to the interfering action of h a r d w a t e r s u p o n
the germicidal activity of the qua te rnar y a m m o n i u m
The

first m e n t i o n

the diluting w a t e r w a s
compounds

of the possible effect of the h a r d n e s s

made

by H a n n e

suitable for hospital use.

alkyldimethylbenzyl a m m o n i u m

while the

same

He

noted that w h e n

tilled water.

A

using

chloride in a concentration of

coli w h e n

effect w a s

of

(7) in 1937 in a r e v i e w of

1:10,000, a time interval of 16 m i n u t e s w a s
culture of E s c h e ri ch ia

compounds.

n e c e s s a r y to kill a

tap w a t e r w a s

use d as the diluent,

obtained in 8 m i n u t e s w h e n

using dis­

concentration of 1:5,000 w a s n e c e s s a r y to achieve

kill in the 8 - m i n u t e

period w h e n

that this information w a s

using tap water.

not available to w o r k e r s

It w o u l d appear
in this country

5
at that time,

as it w a s

situation w a s m a d e
The

not until 1947 that further m e n t i o n of this

in the literature.

Sanitary En g i n e e r i n g

Service, Washington,

D.

Division of the U.

C., p re se nt ed a statement at the request of

the State Sanitary E n g i n e e r s

at their m e e t i n g in M a y ,

included a section on interfering

substances,

data wh ic h they p res en te d are given in Tab le

compounds

when

1.

that the beneficial effect of detergents

give the opposite
was

of the two tap

compounds

when

In addition, it

a d d e d to the qu at ­

tested in distilled wat er m a y

result in the p r e s e n c e

of tap water.

also called to the fact that the m a n u f a c t u r e r ' s

dilution w a s

The

reduction in the g er mi ci dal action of these

they are u s e d in the p r e s e n c e

ernary a m m o n i u m

u s e d (21).

T h e y indicate that

waters as judged by the tests u s e d for evaluation.
is s h o w n

1947, w h i c h

organic and inorganic,

in the w a t e r s in w h i c h the bactericidal agents w e r e

there is a very m a r k e d

S. Public Health

Attention

recommended

apparently b a s e d u p o n tests p e r f o r m e d with distilled

water, and the m a r g i n

of safety calculated with the pure w a t e r w a s

n o w diminished or lost entirely.

It w a s

concluded that ''pending

the d e v e l o p m e n t of residual tests w h i c h provide an accurate m e a ­
sure of bactericidal efficiency, a n y o n e
such c o m p o u n d s

contemplating the us e of

for disinfection w o u l d be well advised to m a k e

6
Table

1.

Interfering action of tap w a t e r s on g e r m i c i d a l p r o p e r ­
ties of q u a t e r n a r y a m m o n i u m c o m p o u n d s .
(Report of
the U. S. Public H e al th Service, Sanitary E n g i n e e r i n g
Division, p r e p a r e d for C o n f e r e n c e of State Sanitary
E n g i n e e r s meeting, Was hi ngt on , D. C., M a y 7-8, 1947.)

Compound
Designated

P p m R e q u i r e d to P r o d u c e 1 0 0 %
Kill of E. coli in 1 min. W h e n
C o m p o u n d is D i s s o l v e d in

P p m Reccommended
for U s e b y
Manufacturer

Pure
W ate r

Cincinnati
Tap

Norwood
Tap

A

20

100

250

234

B

10

70

220

190

C

5

500

3200 +

78

bacteriological tests of the pr od uct und er consideration in the w a t e r
to be used and u n d e r the conditions in w h i c h it will be u s e d . 1'
This had the effect of practically eliminating the qu ate rnary a m ­
monium

compounds

from m a n y

fields such as the dairy industry,

w h e r e it w o u l d be v e r y impractical to attempt to test the c o m ­
pound in the well wa t e r
was that m a n y

of e a c h individual producer.

health d e p a r t m e n t s

The

result

eliminated t h e m entirely on the

basis of this report.
At about this s a m e

time there w e r e

several other investi­

gators work in g on the p r o b l e m of interfering substances.
Ridenour and A r m b r u s t e r

In 1948

(18) reported their studies on the effects

of various w a t e r s using distilled water, a softened water
soda treatment) with a soap h a r d n e s s

of 80 p p m

hard, untreated water with a soap h a r d n e s s

(lime-

and a m o d e r a t e l y

of 275 p p m .

They

found that to obtain a 99-9 per

cent kill, the following

tions of quaternary a m m o n i u m

c o m p o u n d w e r e n e c e s s a r y in each

of the waters:

34, 75, 95 p p m .

They

co nc e n t r a ­

ran a survivor-curve type

oi test, and a study of the shape of the curves indicates that there
is s o m e

sort of neutralization by the wat er

substances.

This w a s

especially true in the m o d e r a t e l y h a r d water, in which the r e d u c ­
tion in bacteria w a s

v e r y smal l up to a concentration of 50 p p m

8
of the germicide.

Above

cent reduction w a s

quite rapid with an increase in the c o n c e n t r a ­

tion.

They

kill w a s
course,

that point, the rate of increase

c onc luded that abo v e a certain m i n i m u m ,

about the s a m e ,

although the a m o u n t s

of per

the rate of

req uired were ,

greater in the case of the h a r d water.

of

T h e y continued

this investigation to include an ex a m i n a t i o n of certain factors
which m i g h t be responsible for this interference.
the cations calcium, m a g n e s i u m ,

sodi um,

This included

an d potassium,

anions sulfate, nitrate, chloride, n o r m a l phosphate,

and the

an d carbonate.

T h e y found that the c a l c i u m an d m a g n e s i u m

gave

verse effect on the g e r m i c i d a l action.

s o d i u m and p o t a s s i u m

had little effect.
for all of t h e m

W i t h the anions, the effect w a s

germicidal activity.
range,

about the s a m e

up to a salt concentration of 70 p p m ,

ception of pho sphate and

the p H

The

a definite a d ­

carbonate,

which

Since these two w e r e

9.0 an d

showed

with the e x ­

an increase in

tested at e x t r e m e s

1.0 respectively, this w a s

of

believed to be

the cause of this reaction.
In a c o m p a r a t i v e
ammonium
Sher e

compounds

study of the properties of the quaternary

with the hypochlorites, reported in 1948,

(20) pointed out a m a r k e d

erties of the f o r m e r

reduction of the germicidal p r o p ­

g r o u p v/hen use d in the p r e s e n c e

of h a r d

9
water.

He

tested a series of q u a t e r n a r y a m m o n i u m

in ttvo w a t e r s

of 0 p p m

hardness

a n d 40 0 p p m

germicides

h ardn es s,

and

found that the in c r e a s e in concentration n e c e s s a r y to give kill in
10 m i n u t e s

varied f r o m

the ha r d water.
phosphate) w a s

When

two

to four h u n d r e d

a d d e d in the a m o u n t of ZOO p p m ,

of the h a r d water.

s h o w that an y

Shere

change

Chemical

monium

compound

c o m p o u n d i n c r e a s e d in the case

also noted that in the first part of the

h a d taken place in the p r e s e n c e

tests

same

in both series.

a n d Seeley (15) reported that they h a d

undertaken a study to obtain m o r e

i nfor ma ti on o n the c o n t r o v e r ­

sial subject of the constituents in w a t e r
activity.

They

rep or te d no

ha r d n e s s

(as d e t e r m i n e d b y

differences in p H

compound

as they affect g e r m i c i d a l

close correlation b e t w e e n the w a t e r
soap) an d the g e r m i c i d a l action.

found in the natural w a t e r s tested w e r e

W i t h as m u c h

present, they w e r e

of the h a r d

s h o w e d the concentration of q u a t e r n a r y a m ­

to be the

In 1948, M u e l l e r

monium

the n e c e s s a r y

n o indication other than bacteriological data to

water.

nificant.

of

a water-conditioning agent (trisodium

a m o u n t of q u a t e r n a r y a m m o n i u m

test there w a s

ti m e s in the ca s e

as

1,000 p p m

able to s h o w
was

The

not sig­

of c a l c i u m or m a g n e s i u m

that 2 0 0 p p m

of q u a t e r n a r y a m ­

able to give a p p r o x i m a t e l y

100 p e r cent

10
kill in a period of 8 min ute s.

However,

as m u c h

as

10 p p m

ferric iron c o mp le te ly inactivated the q ua te rn ar y a m m o n i u m
pound.

They

of the g e r m i c i d e ha s

cidal potential for m o s t

following year, M u e l l e r

gent sanitizer,

sufficient r e s e r v e

germi­

jobs.

of the ferric iron ca n be

removed

although h e do e s

later b y M u e l l e r

(14) reported that this effect
by the use of a b al an ce d d e t e r ­

not state w h a t ingredients w o u l d

be included in this formulation.
was m a d e

com­

co ncl ud ed that the results to that date w o u l d indi­

cate that ZOO p p m

The

of

The

report of a detailed study

an d Seeley (16) on the effect of m e t a l ­

lic ions on the g e r m i c i d a l activity of the quatern ar y a m m o n i u m
germicides.

The

list of ions w h i c h they tested against R o c c a l in­

cluded potassium,
ganese, b a r i u m ,

sodium,

cupric

i n u m in the f o r m

calcium, m a g n e s i u m ,

copper, ferrous

of the chloride

lithium, an d ferric sulfates w e r e
T h e test o r g a n i s m

they e m p l o y e d

of about 10 x 10° o r g a n i s m s
(usually 1 an d 5 min u te s)
naphuride.
there w a s

a n d ferric iron, an d a l u m ­

salts.

Potassium,

included,
was

pe r ml.

zinc, nickel, m a n ­

JE.

sodium,

and also s o d i u m nitrate.
coli in a concentration

After varying intervals

the ma t e r i a l w a s

neutralized with s o d i u m

In studying the direct effect of pH,

they found that

no evidence of reduction in bacterial n u m b e r s

between

11
the p H

levels of 2.0 an d

Roccal w a s

10.0.

The

ge rmicid al action of the

d e c r e a s e d in a l o w e r e d pH.

Of the m o n o v a l e n t
with 200 p p m

salts tested, n o n e

of the quatern ar y a m m o n i u m

solution of s o d i u m

chloride gave

a marked

far in excess of any practical situation.
300 to 450 p p m

any

one- and t w o - m i n u t e intervals.
concentration of 8 00 p p m

tity w a s

A

2.0 M

reduction, but this w a s

Of the divalent salts, a

reduction in the germicidal

solution of the Roccal,

minute test.

compound.

concentration of c a l c i u m and m a g n e s i u m

necessary to s h o w
200 p p m

s h o w e d interference

was

action of a

and then only in the 30-second,
It w a s

n e c e s s a r y to go to a salt

to give a reduction of activity in the 5-

In the case of the trivalent ions, only a smal l q u a n ­

n e c e s s a r y to inactivate the germicide.

They

determined

that this effect in cre as ed in a ratio of 1:100:10,000 for the m o n o - ,
di -, and trivalent salts.
ride, 0.02 M

manganese

This m e a n s

that a 2.0 M

chloride and 0.0002 M

wouLd all give about the s a m e

s o d i u m chlo­

a l u m i n u m chloride

interference pattern.

In s u m m a t i o n ,

they state that all of the cations tested interfered with the g e r m i ­
cidal action and the factor of valence had the m o s t
termining the degr ee of this interference.

effect in d e ­

12
A b o u t this t i m e

other w o r k e r s ,

Weber

and Black

(24) and

Foster (5), noted the effect of the h a r d w a t e r ions in interfering
with action of the q u a t e r n a r y
A

ammonium

compounds.

continuation of the studies cited in the statemen t of the

U. S. Public Health Service w a s
and C h a m b e r s

(2), in w h i c h they

six different tap waters.
concentration in p p m

reported by Butterfield, Wattie,
showed

the results of tests on

T h e y found that on these w a t e r s

of g e r m i c i d e

that w a s

per cent kill in a period of 1 m i n u t e

n e c e s s a r y to give

varied f r o m

Using distilled w a t e r buffered to 8.0, 7.0, and
quired w a s

less than 50 in all cases; while

tap water, to w h i c h
quired 500 p p m

but none w a s

6.6, the a m o u n t

a sample

study w a s

re­

of Cincinnati
re­

extended to

of c h e m i c a l constituents in distilled water,

found that could r e p r o d u c e

in natural waters.
natural wate r

This

100

60 to 320 p p m .

a trace of s o d i u m borate had b e e n added,

of the ger mi ci de.

include co mbination s

the

It w a s

consistently the results

also noted that an y modification of the

(boiling, aeration, autoclaving, or filtration) altered

the degree to w h i c h the q u a t e r n a r y a m m o n i u m
fected by the water.

No

accompanying

nor precipitation o c c u r r e d

germicide w a s

c h a n g e in p H

af­

or turbidity

to give visual evidence of alternation.

Thus, they stated that the ch an g e in bactericidal efficiency m u s t

13
be due to factors other than the c h e m i c a l constituents ordinarilydetermined.
The

r e m a i n d e r of this study w a s

carried out o n three

waters--distilled w a t e r buffered with C l a r k and L»ubs buffer to a d ­
just the p H f r o m

7.0 to 9.5; Cincinnati tap water; and N o r w o o d ,

Ohio, tap water.

T hese waters w e r e

to be ap pr oxi mately

155 p p m

of Butterfield e_t a_l. w e r e

reported b y L a w r e n c e

and 445 p p m

hardness.

carried out in a v o l u m e

The

(9)
studies

of 5 00 m l

using

a concentration of E_. coli strain 198 of about 1,000 to 2,000 o r ­
g a n i s m s per ml.

The ir

criterion of satisfactory disinfection w a s

100 per cent kill in 1 minute.
and C o d e

This they b a s e d u p o n the O r d i n a n c e

(22) w h i c h provides for a 2-mi n u t e contact time.

observed that p e r h a p s
point should b e
gin of safety.

this criterion w a s

set at 15 or 30

They

too lenient a n d the e n d

seconds to give a sufficient m a r ­

Bacteriological examinations w e r e m a d e

at the e n d

of 1, 2, a n d 5 mi n u t e s in m o s t of the tests b y pipetting the desired
a m o u n t into a Petri dish a n d pouring the a g a r at the en d of the
exposure period.
In a series of tests on fifteen different quaternary a m m o n i u m
compounds,

it w a s

found that the av er age concentrations in p p m

ne ce s s a r y to give the

100 per

cent kill in 1 m i n u t e on the distilled,

14
Cincinnati,

and N o r w o o d

about 1:4:6.

Thi s

in the 1 - m i n u t e

was

Waters

were

52:211:324,

typical of m o s t

test, but for the 2-

There w a s

one

exception in the

ammonium

chloride w h i c h

required

5-minute

c o m p o u n d plus
the N o r w o o d
give

germicide

a detergent,

water

100 p e r

a lower

In using

this interference w a s

a very m a r k e d

an e x a g g e r a t e d

seemed

to i n c r e a s e

shortest intervals that w e r e

compounds

prepared f r o m
ar e

in g e r m i c i d a l

noted in that
15,000 p p m

to

The

than a v e r a g e

results,

effect.
as to w h e t h e r

a progressive

one.

ca s e in w h i c h

In a

the i n ­

after the first 1 or 2 m i n u t e s — the

tested after the dilutions of the g e r m i ­

the tap waters.

stable in the diluting w a t e r
efficiency h a s

cent

ammonium

intervals.

also investigated the p r o b l e m
an instantaneous or

100 per

effect w a s

series of sixty-six tests, they could find n o

cide w e r e

to give

a quaternary

given only slightly l o w e r

workers

as well.

conc ent ration with the

cent reduction in a n y of the t i m e

but the c o m b i n a t i o n g a v e

terference

results

r e q u i r e d a concentration of o v e r

alone h a d

These

period.

not only

of a n a l k y l d i me th ylben zy l

N o r w o o d w a t e r than with the Cincinnati w a t e r
reduction in the 5 - m i n u t e

a ratio of

of the c o m p o u n d s

and

ca s e

or

taken place.

They

c o n c l u d e d that these

after the first reduction

15
Lawrence

(9) h a s

effect of h a r d w a t e r s
mercial quaternary

r ep or ted findings to c o n f i r m the a d v e r s e

u p o n the g e r m i c i d a l action of s e ve ra l c o m ­

ammonium

bacteria (_E. coli, S a l m o n e l l a
Using the phenol
ents w e r e
another.
where
was

Because

t y p h o s a ) as the test o r g a n i s m s .

about 25 0 to 55 in one

of the u s e

gram-positive

cidal activity w a s
o r g a n i s m in the

predominate,

of M i c r o c o c c u s

of h a r d w a t e r

T h e s e data un e x p e c t e d l y

that h a d

cent w a s

trisodium p h o s p h a t e will e n h a n c e
alkyldimethylbenzyl a m m o n i u m
compares

exception,

group.

the g e r m i
test

reduction

Subsequent

studies

c o n f i r m e d this observation

studies b y this investigator
(0.05 p e r

aureus

of h ardn es s.

shown m a r k e d

with synthetically p r e p a r e d h a r d w a t e r s

of sma ll a m o u n t s

var.

using the g r a m - p o s i t i v e

in the action against the g r a m - n e g a t i v e

Further

22 in

in situations

pyogenes

reveal that, with o n e

waters

an d

a series of tests

containing 350 p p m

not i m p a i r e d w h e n

same

cas e

of these c o m p o u n d s

organisms

strain 209

in the p r e s e n c e

using g r a m - n e g a t i v e

coefficient technique, h e found that the coeffici­

reduced f r o m

run using

compounds

s h o w that the addition

found to be o p t i m u m )

the ger mi c i d a l

chloride h e w a s

with the action in distilled water.

of

activity of the
using

so that it

16
Johns

(8) also o b s e r v e d the effect of h a r d w a t e r s

type of c o m p o u n d .

U s i n g both g r a m - n e g a t i v e

on this

a nd g r a m - p o s i t i v e

organisms, he noted that in the case of the latter, there w a s

no

appreciable reduction in the g e r m i c i d a l efficiency f r o m that o b ­
served in distilled water.

When

organism, his results w e r e
One

of the m o r e

comparable

to those previously cited.

recent reports on the p r o b l e m of h a r d

waters is that of B o t w r i g h t (1).
c o m p a r i n g the use

using the jE. coli as a test

This is p ri mari ly a field study

of an alkaline cleaner a n d hypochlorite

system

against the u s e of a detergent-sanitizer u s e d on a g r o u p of dairy
farms.

However,

the observations o n the effect of the h a r d w a t e r s

encountered is pertinent to this discussion.
waters varied f r o m
350 p p m

14 to 180 p p m

at one

station, and f r o m

of the
2 4 to

( d e t e r mi ne d b y the soap h a r d n e s s test) at the other.

both cases,

the h a r d n e s s

of the w a t e r

on f a r m s

sanitizer #5 e x c e e d e d that of the f a r m s
He

The hardness

stated that the h a r d n e s s

In

using the cleaner-

using the chlorine

syst em .

did not a p p e a r to affect adv er sely the

quality of the m i l k as m e a s u r e d b y the pasteurized m i l k counts.
As

a m a t t e r of interest, the p r o d u c e r s

tended to p r o d u c e

a better milk.

having the h a r d w a t e r

E X P E R I M E N T A L

There
first w a s

were

typ es

a survivor -curve

given by W e b e r
much

two

of the i n f o r m a t i o n

obtained b y the u s e

of tests u s e d in these

type

and Black

PROCEDURES

This

test w a s

the int erf er en ce

of this technique.

of g e r m i c i d e s

The

an

test u s e d

was

a modification of the
Shelanski

(3) a n d

sanitizers u s e d

tests using a n

and

100 x

number

as

15 m i n u t e s .

lOO /l

tyPe

test, w h i c h is

10

was

Turney

Cantor

and

(11).

Escherichia

the test o r g a n i s m .
organisms

per

ml

coli, C i n c i n ­

The

conc entrat ion

in the reaction

selected on the basis
dish w a t e r

of lab or at ory
and

w h i c h h a d b e e n d i p p e d in it a n d
By

making

that the ratio of bact er ia p e r m l
glass w a s

and

artificially c o n t a m i n a t e d

rinse count of g l a s s e s
drain for

Kivela,

Black method,

is u s e d

of the i n o c u l u m is
This

to evaluate

the field test for the evaluation of r e s t a u r a n t

In the W e b e r

mixture.

was

in the food industry.

''capacity'1 test p r e s e n t e d b y

by Mallmann,

nati strain #198,

waters

designed

used

"exhaustion"

selected b e c a u s e

of h a r d

It w a s

the practical p e r f o r m a n c e
second

The

of test following the p r o c e d u r e s

(23).

on

studies.

plate

counts

of dish w a t e r

in both heavily a n d

a swabal l o w e d to

of both, it w a s
to bacteria p e r

lightly c o n t a m i n a t e d

water.

found

18
They consid er ed that high counts
be f r o m

several h u n d r e d

the 100 x 10

6

organisms

s w a b - r i n s e plates w o u l d

t h o u s a n d s to on e or t w o millions,
per m l

contamination in dishwashing.
an agar

on the

slant b y w a s h i n g

would

The

and

represent a heavy

organisms

were

and

load of

taken f r o m

s u s p e n d i n g in the w a t e r to be tested

and then diluted s u c h that the final concentration will h a v e
sired n u m b e r

of o r g a n i s m s .

nique w a s intr od uc ed h e r e

recommended.

This w a s

A

by

dilution bottle for 2 m i n u t e s

slight variation f r o m

their t e c h ­

in place of the twenty-five

done

times

as

only in the one dilution prior to

the standard technique for m a k i n g
in a n y type

the d e ­

shaking the culture in the st and ard

adding the s u s p e n s i o n to the m e d i c a t i o n pot.

As

so

A t all other t i m e s

dilutions w a s

followed.

of a s p e e d reaction test, it is n e c e s s a r y to

have an effective neutralizer to stop the action of the g e r m i c i d e
at the exact end of the t i m e interval being tested.
been m a n y

s ug g e s t e d for this pur pose ,

T h e r e have

but the W e b e r

and Black

m e t h o d indicates that asolectin in T w e e n

80 is satisfactory.

Since a study of neutralizers w a s

beyond

the ran g e of this study,

this m e t h o d

adopted for all of the tests p e r ­

formed.

of neutralization w a s

19
The

exposure

and 300 s e c o n d s

times

in o r d e r

they

selected w e r e

to give data c o m p a r a b l e

encountered in sanitizing dishes.
the test is
bath at 25

10 m l ,

and

15,

The

30,

to the

total v o l u m e

all of the solutions

60,

120,
situations

of m a t e r i a l in

a r e held

in a w a t e r

C.

The

testing p r o c e d u r e

bacterial s u s p e n s i o n

is as follows:

containing

double

a 5 ml

the a m o u n t

desired is p l a c e d in a m e d i c a t i o n pot, being
the o r g a n i s m s

ar e

splashed

m ed ic at ion pot in the w a t e r
g e r m i c i d e being

tested,

tion in thew a t e r

under

solution is

then

on the

amount

of o r g a n i s m s

careful that n o n e

sides of the tube.

bath is p l a c e d

adjusted to twice
consideration.

of the

about

Five m l

In a s e c o n d

10 m l

the des i re d

of the

concentra­

of the

germicide

rapidly pipetted into the s u s p e n s i o n of o r g a n i s m s

and the tube quickly

s w i r l e d to aid in m i xin g.

pension is then r e m o v e d ,

a n d at 15

One

seconds f r o m

ml

of the s u s ­

the t i m e

of m i x ­

ing, it is quickly d i s c h a r g e d into the dilution blank containing
of the asolectin
120, a n d

300

dilutions are

sample

solution.

seconds

surviving

taken f r o m

This

from

then m a d e

counting of the

of

is r e p e a t e d

the initial t i m e

from

at the en d of 30,
of mixi ng.

e a c h of the blanks

organisms,

A

60,

Appropriate

to insure

count is also m a d e

the u n u s e d portion of the

9 ml

accurate
of a

su sp e n s i o n w h i c h h a d

20
not bee n in contact with the g e r m i c i d a l agent a n d this is u s e d as
the initial count, or n u m b e r

of o r g a n i s m s

at " 0

the plates w e r e p r e p a r e d with tryptone glucose
(Difco) with no a d d e d milk, but containing
(asolectin in T w e e n

80) p e r

100 m l

seconds."
extract ag a r

100 m g

of lecithin

of m e d i u m .

Additional controls ar e d e s c r i b e d b y W e b e r
wo r k by M a l l m a n n ,

Alegnani,

indicated that these g a v e

an d C o p e

The

organisms

results m a y

The

(13) using this technique

at the e n d of 2 4 hours' incubation

be repo rt ed as actual densities of

surviving or as p e r

of exposure.

an d Black, but

little additional information.

Plate counts w e r e m a d e
at 37 C.

All

"endpoint"

cent reduction after e a c h interval
is

100 p e r cent kill of the test

organisms.
The

"exhaustion"

to the " ca pa ci ty "

test e m p l o y e d in these

test of C a n t o r

a n d Shelanski.

their technique (3), they defined the t e r m

studies is similar
In describing

"ca pa ci ty"

tribute of g e r m i c i d e s w h i c h d e t e r m i n e s the m a x i m u m
incrementally a d d e d m i c r o o r g a n i s m s ,
ous m a t t e r in solution or suspension,
tity of the g e r m i c i d e . "

plus

" a n at­

number

of

rigidly defined e x tra ne ­

sterilizable b y

Their test w a s

as

a given q u a n ­

des igned to simulate m o r e

closely the actual conditions u n d e r w h i c h a g e r m i c i d e

is used.

21
As

two e x t r e m e s

of this they

treatment an d the u s e
the contamination,

cite the u s e of antiseptics in w o u n d

of sanitizers in dishwashing.

both bacteria a n d e x t r a n e o u s

added incrementally.

W i t h this in m i n d ,

In both c a s e s

matter,

they outlined a p r o c e d u r e

which w o u l d a t t e m p t to evaluate a situation a na lago us
laboratory.

Inasmuch

from

is the standard S_„ t y p h o s a g r o w n

the test, the culture
in F D A

In those

under

organic m a t t e r

was

adverse

cases

in w h i c h

plus

it w a s

conditions, the a m o u n t

i n c r e a s e d by m i x i n g

recommended

phenol coefficient broth.

This furnishes a s ta nd ard i n o c u l u m of o r g a n i s m s

the g e r m i c i d e

to this in the

as they are not interested in eliminating the

presence of org ani c m a t t e r

extraneous matter.

is being

controlled

desired

to test

of soil or

an equal quantity of

*

sterilized w h o l e m i l k

with the broth

This resulted in a broth
per m l

test w a s

8

s u s p e n s i o n containing 2 x 1 0

an d a p p r o x i m a t e l y
The

culture of the test o r g a n i s m .

6 pe r

performed

cent w h o l e m i l k

bacteria

solids.

b y adding i n c r e m e n t s

of 2.5 m l

g
of the broth

suspension

of the g e r m i c i d e
Ten minutes

and

(a total of 5 x 10

sampling

organisms)

at the e n d of 15 a n d

to 500 m.1

30 seconds.

after the addition of e a c h i n c r e m e n t another w a s

adde d a n d the

15-

and

30-second

samplings

continued for a total of ten i n c r e m e n t s .

repeated.

For

purposes

This w a s
of their

discussion, they also a d d e d
10

8

.

organisms)

.

at o n e t i m e

cide under these

two

types

the s a m p l e s in o r d e r

the total of the ten i n c r e m e n t s
'

to c o m p a r e

the ability of the g e r m i ­

of situations.

to d e t e r m i n e

(50 x

Plates w e r e

the p e r

made

of

cent reduction in e a c h

case.
A lt ho ug h the
some
ent.

''exhaustion'1 test parallels

extent, the f u n d a m e n t a l in for mation
In this case,

it w a s

d e sir ed

could be a d d e d to a g e r m i c i d a l
activity h a d b e e n depleted,
which the c o m p o u n d
ence of s o m e
was

such that 1 m l

to

sought is slightly differ­

to d e t e r m i n e

solution before
''exhausted.''

how many

organisms

all of the g e r m i c i d a l

Since the

sp e e d with

could react is not as i m p o r t a n t as the p r e s ­

germicidal

10 min utes .

or

this technique

The

action, the ti m e interval u s e d in this ca s e

organisms

were

contains the n u m b e r

p r e p a r e d in a su spensi on

of cells

suitable for addition

g

at one time — usually as close to 2 0 0 x
added to 20 0 m l
of 100 x
Weber

1 0 6 , w h i c h is c o m p a r a b l e

as possible.

solution,

When

this gives an i n o c u l u m

to the concentration u s e d in the

and B l a c k tests.
The

the r e m o v a l
of 1 m l

of the g e r m i c i d a l

10

volume

of 2 0 0 m l

of 1 m l

of the

of the g e r m i c i d e

was

solution for s a m p l i n g

of the bacterial s u s p e n s i o n for e a c h

selected so that

and the addition

stage of the test w o u l d

23
not appreciably alter the concentration of the g e r m i c i d e in the
reaction flask.

In a series

of fifteen i n c r e m e n t s ,

in concentration of the g e r m i c i d e
cent.

The

successive

actual concentrations
stage

cide w e r e m a d e

in the vari ou s

added w o u l d not c h a n g e

made

pensating errors.

in distilled w a t e r

no

was

by

in the Z5 C
1 ml

which

1 minute

elap se d b e t w e e n

after sampling,

1 ml

com­

a d d e d in this

of the bacterial

well by

w a t e r bath for

is r e m o v e d

not c o n s i d e r e d important,

added

adding

swirling,

a n d al­

10 min ut es.

At the

an d placed in an asolectin-

80 dilution blank containing 9 m l

though ti m e

c o n s i d e r e d as

of the bacterial cells alone.

reaction flask, m i x i n g

end of this interval,
Tween

in concentration of g e r ­

d e si red to follow the interaction of the g e r m i ­

test is p e r f o r m e d

lowing it to r e m a i n

The

to an a p p r e c i ­

ext ra ne ou s m a t t e r

cide and tap w a t e r in the p r e s e n c e

suspension to the

test.

so the a m o u n t s

of the wa t e r

of the w a t e r m i g h t be
was

7 per

Dilutions of the g e r m i ­

types of w a t e r u n d e r

the h a r d n e s s

There

case b e c a u s e it w a s

The

2.

In addition, the d e c r e a s e

micide and h a r d n e s s

approximately

resulting in the flask at e a c h

ar e given in T a b l e

culture s u s p e n s i o n w a s

able degree.

would be

the reduction

of the neutralizer.

Al­

the individual stages of the test

the following i n c r e m e n t w a s
or at intervals

usually

of 11 m i n u t e s

24
Table 2.

A c tual concentrations of g e r m i c i d e p r e s e n t in e a c h
s u c c e s s i v e stage of the e x h a u s t i o n test w h e n the initial
concentration w a s 2 0 0 p p m ,

Increment

Concentration
in p p m

1

200.

2

199.

3

198.005

4

197.060

5

196.075

6

195.095

7

194.115

8

194.145

9

192.180

10

191.219

11

190.263

12

189.312

13

188.366

14

187.425

15

186.489

25
throughout the testing period.
were m a d e

a n d plated o n

the asolectin in T w e e n
100 m g

per

100 m l

ial suspension w a s

Suitable dilutions of the s a m p l e s

tryptone glucose extract a g a r to w h i c h

80 h a d b e e n a d d e d in the concentration

of m e d i u m .

The

concentration of the b a c t e r ­

d e t e r m i n e d b y taking a s a m p l e

about m i d w a y

in the testing p e r i o d a n d plating as for the other s a m p l e s .
This
Kivela, and

type of test is-similar to the one u s e d b y M a l l m a n n ,
T u r n e y in a s s e s s i n g the value of sanitizers in b e v e r ­

age establishments
an 11exhaustion"

(11).

A l t h o u g h not specifically d e s c r i b e d as

test, their m e t h o d w a s

glass as it w e n t through the w a s h i n g
determine

the n u m b e r

of glasses

concentration a n d v o l u m e
tivity b e g a n to diminish.

to s a m p l e

e v e r y tenth

a n d sanitizing p r o c e s s

that could b e

of g e r m i c i d e before
In this way,

sanitized in a given
the g e r m i c i d a l a c ­

as high as eight h u n d r e d

glasses could be e x a m i n e d consecutively to d e t e r m i n e
not the sanitizer w o u l d b e c o m e

The

water hardness

determinations w e r e

Company

will f o r m

made

using the

Technical Bulletin No.

(26).

the principle that the salts of ethylene
(referred to as V e r s e n e )

w h e t h e r or

exhausted.

Versenate m e t h o d as outlined in the
the B e r s w o r t h C h e m i c a l

to

The

2, of

test is b a s e d upo n

d i a m i n e tetra acetic acid

v e r y stable chelates with the

26
calcium, a n d m a g n e s i u m .

Other

c i u m and m a g n e s i u m

decomposed

are

to this there is a n

azo

a colored c o m p l e x

compound

less stable
to f o r m

complexes,

the m o r e
The

magnesium

dye,

dye

to a solution

stable
by

wi t h m a g n e s i u m .

a deep

formed

containing

the

is w i n e

red.

red-colored

color to the blue is n o t e d w h e n
chelate all of the m a g n e s i u m .

down

color

by

the

In addition

which
the
the

at a p H

forms

other
Versene

of 10, but the

Versene

chelate,

is a d d e d

a sharp

change

sufficient V e r s e n e

Although

the c a l c i u m

does

ions.

since

In m a k i n g

sma l l a m o u n t

only

The

Versene

dye

calcium

method

soap h a r d n e s s
the a m o u n t

the

Versene
standard

of m a g n e s i u m

reaction with the
contains

the

will chelate first with the
Versene

chloride

not f o r m

in c a s e

the w a t e r

or

solution being

the

calcium

solution for titration,

is a d d e d to f o r m the

a

color
titrated

and no m a g n e s i u m .

is m u c h

simpler

titration f o r m e r l y

of h a r d n e s s

titration m e t h o d

in the
has

and m o r e

used.

samples
been

For

accurate

employed.

t h a n the

the d e t e r m i n a t i o n of

u s e d in these

in

to

this chelate with the dye, it is i n c l u d e d in the titration with
magnesium,

cal­

chelate.

When

the r e is

T,

Like

magnesium
blue

with

Versene.

Eriochromeschwartz

Versene

itself h a s

chelates

the

this also is b r o k e n

stable

complex

less

studies,

the

27
The

determinations

were m a d e

using a B e c k m a n

values w e r e
of the p H

of the p H

in the r a n g e

pH

of the v a r i o u s

meter.

6.9 to 7.5, n o

in the results e x c e p t in those

test w a t e r s

Since m o s t of these
specific m e n t i o n is m a d e
c a s e s w h i c h deviate f r o m

this range.
E a c h table giving results of a s u r v i v o r - c u r v e test r e p r e ­
sents a typical set of data selected f r o m

many

consistent results a n d is representative of m a n y

trials to insure
replicates.

The

data o n the e xhaustion tests correlate well with others in the
series a n d r e p r e s e n t f e w trials, in addition to those
Other equipment and m e t h o d s
standard for bacteriological w o r k .

used w e r e

reported.

those a c c e p t e d as

PRESENTATION OF DATA

According

to the literature p r e v i o u s l y cited s h o w i n g the

interference of the activity of the q u a t e r n a r y a m m o n i u m
(QAC)

in h a r d w a t e r s ,

eliminated f r o m

use

it w o u l d a p p e a r that this g r o u p

in this class of w a t e r s

i n c r e a s e d to the point w h e r e

sults c o m p a r a b l e

to those obtained with distilled water.
shown,

associates

Specific c a s e s

on milking m a c h i n e

sanitation (12), a n d the
There

on its p r o d u c e r s '

re­

Practical

are the

a r e a s with v e r y

studies of M a l l m a n n

satis­
and

sanitizers (10), in restaurant utensil

studies of B o t w r i g h t p reviously cited (1).

is also the e x p e r i e n c e

b e e n using a Q A C

they will give

h o w e v e r , that this type of c o m ­

p o u n d h a s b e e n u s e d routinely in h a r d w a t e r
factory results.

sho u l d be

or that the c o n c e n ­

tration s h ould b e

experience in the field h a s

compounds

of o n e

of the local dairies that h a s

in a h a r d w a t e r a r e a for the pas t five y e a r s
farms

p h a s e of this w o r k

was

wi t h v e r y

satisfactory results.

T h e first

a n a t t e m p t to investigate the r e a s o n s for

these apparently conflicting situations.
As
determine,
QAC

an a p p r o a c h to this p r o b l e m ,

it s e e m e d desirable to

if possible, w h e t h e r o r not the reaction b e t w e e n the

a n d the interfering factor in the w a t e r w a s

a direct c h e m i c a l

29
neutralization.

It w a s p os t u l a t e d that if this w e r e

following situation w o u l d exist:

When

QAC

the g e r m i c i d a l

is m a d e

up in h a r d w a t e r ,

c o m p a r a b l e to that obtained w i t h a
same

c o m p o u n d in distilled w a t e r

w a t e r used.

centration of 350 to 400 p p m
parable to a 200 p p m

depending

of Q A C
of the

in distilled w a t e r gives results
ppm

concentration u s e d in the

of 3 85 p p m .
by

150 p p m ,

there is m o r e

c o m p l e t e kill in 60 seconds.
of Q A C

of the

lost, then a c o n ­
results c o m ­

distilled water.

50 p p m

college

was

s h o u l d give

Table

that a r e

B y increasing the

activity m i g h t b e

u p o n the particular

which

solution tested in

data s h o w that u s i n g

of

r e a c t i o n b e t w e e n the factor

QA.C

is not the case m a y be o b s e r v e d - i n
These

solution of 200 p p m

c o n c e n t r a t i o n of 50 p p m

If there existed a dir e c t

in the w a t e r a n d the 150 p p m

a

the case, the

T h a t this

3.
c o n centration of Q A C

c o m p a r a b l e to the 200

t a p w a t e r havi n g a h a r d n e s s

c o n c e n t r a t i o n in the distilled w a t e r

than e n o u g h

g e r m i c i d a l potential to give

Starting f r o m

in the tap water, a n i n c r e a s e

is not sufficient to give this d e g r e e

of

the point of 200 p p m

in concentration of 300 p p m
kill in the s a m e

t i m e in­

terval.
A s further evidence

of the

in distilled water as c o m p a r e d

r e l a t i o n s h i p b e t w e e n the activity

to tap

water,

the data in Table

4

30
Table 3.

Exposure
T i m e in
Se c o n d s

T h e effect of increasing the concentration of q u a t e r n a r y
a m m o n i u m c o m p o u n d s in the p r e s e n c e of both distilled
w a t e r a n d tap water.

Distilled W a t e r
50 p p m *

100 p p m

(organisms per m l

200 p p m

r e m a ining)

0

165,000,000

165,000,000

165,000,000

15

55,000,000

26,300

40

30

39,000,000

2,480

270

60

4,700,000

350

0

120

36,000

10

0

300

1,150

0

0

* Concentration of q u a t e r n a r y a m m o n i u m

compound.

31
Table 3 (continued)

Tap
100 p p m

Water

(385 p p m

200 p p m

(organisms per m l

hardness)
300 p p m

500 p p m

remaining)

34,000,000

34,000,000

34,000,000

34,000,000

64,000,000

17,500,000

8,000,000

3,400,000

12,700,000

10,000,000

700,000

620,000

10,000,000

710,000

80,000

38,000

3,500,000

98,000

38,000

0

950,000

180

0

0

32
Table

4.

Exposure
T i m e in
Seconds

A c o m p a r i s o n of the g e r m i c i d a l effect of Q A C
distilled a n d tap w a t e r as the test waters.

using

Distilled
W ate r
(0 p p m )
100 p p m *

100 p p m

(organisms per m l

remaining)

0

107,000,000

76,000,000

15

1,960

170,000,000

30

620

100,000,000

60

70

130,000,000

120

10

56,000,000

300

0

145,000

* Concentration of Q A C .

33
Table 4 (continued)

Tap

200 p p m

Water

(330 p p m )

300 p p m

(organisms

400

per m l

ppm

remaining)

76,000,000

76,000,000

76,000,000

33,000,000

5,800,000

680,000

15,000,000

110,000

245,000

7 0 0 ,000

2,000

1,000

320

60

10

0

0

0

34
are presented.

The

time to 330 p p m ,

hardness

of the w a t e r h a d d e c r e a s e d at this

bat the results a r e

c o m p a r a b l e to those at the

slightly h i g h e r level.
These

data all tend to c o n f i r m

that there is a certain d e g r e e
the Q A C

when

the reports

in the literature

of interference with the action of

u s e d in certain h a r d wate r s .

In o r d e r to find h o w

high a concentration w o u l d b e n e c e s s a r y to achieve the s t a n d a r d
of c o m p l e t e kill in 60

seconds,

nique, additional ru n s w e r e
this point w a s

reached.

made

These

indicate that in o r d e r to u s e
of this area,

using the W e b e r

and B lack tech­

at increasing concentrations until

data are given in Tab l e

5.

They

a c o m p o u n d of this type in the w a t e r s

it w o u l d be n e c e s s a r y to increase the concentration

of g e r m i c i d e to a p p r o x i m a t e l y

800 p p m

or about four t i m e s

the

concentrations usually e m p l o y e d .
Additional

study of these tables w o u l d indicate that in place

of a direct "neutralization11 of the Q A C
in the h a r d water,
action.

Thus,

the g e r m i c i d e

b y the interfering substances

there is rather a retarding of the g e r m i c i d a l

in a situation w h e r e

there is a n e x t e n d e d p e r i o d for

to exert its effect { m o r e

than

1 o r 2 minutes)

it

m i g h t b e possible that the e n d result w o u l d b e entirely satisfactory
as far as

a practical application of a Q A C

is concerned.

35
Table

5.

T o d e t e r m i n e the c o n c e n t r a t i o n n e c e s s a r y to obtain
p e r cent kill in 60 s e c o n d s , u s i n g tap w a t e r as the
diluent.

Intervals of
Exposure
in S e c o n d s

Tap Water
(300 p p m ) ;
QAC
(200 p p m )

Tap Water
(330 p p m ) ;
QAC
(400 p p m )

(organis m s

per

ml

100

Tap Water
(310 p p m ) ;
QAC
(800 p p m )

surviving)

0

93,000,00 0

76,000,000

40,00 0 , 0 0 0

15

10,300,000

6 80,000

10

30

4,500,000

245,000

0

60

3,100,000

1,000

0

120

820,000

10

0

300

0

0

0

36
The

ne x t phase, of the w o r k

associated with w a t e r
might h a v e

hardness

to d e t e r m i n e

series w a s

to d e t e r m i n e

be due to the p r e s e n c e

of s i m p l e

this p u r p o s e

and Black

the

Weber

solution containing 250 p p m

T he

peptone

results

to study v a r i o u s factors
the i m p o r t a n c e

in c o n n e c t i o n wi t h the over-all p r o b l e m .

step in this

Z50 p p m

was

are

(16) in r e g a r d

salts a n d o r g a n i c
technique w a s

The

in a c c o r d a n c e

to m o n o v a l e n t

T h e first

the interference that m i g h t

of s o d i u m

in the other.

each

chloride

data ar e

matter.

For

r u n with the test
in o n e

series

given in T a b l e

with the . work of M u e l l e r

and
6.

a nd Seeley

ions h a v i n g v e r y little inhibiting ef­

fect on the Q A C .
The

n e x t point of attack w a s

tionship b e t w e e n

the h a r d n e s s

terference with the g e r m i c i d a l

of the w a t e r
action.

of this type, the tap w a t e r w a s
amounts

of the c o m p l e x i n g

of ethylene d i a m i n e
had a h a r d n e s s
there a r e n o

220,

outstanding

To

and

acid.

The

140 p p m ,

differences,

softer water.

of in­

obtain a series

of w a t e r s

This

the tetra s o d i u m

salt

resulting w a t e r s
respectively.

it c a n b e

that there is a consistent tre n d t o w a r d
efficiency in the

a n d the d e g r e e

softened b y the addition of v a r y i n g

age n t V e r s e n e ,

tetra acetic

of 270,

a d e t e r m i n a t i o n of the rel a ­

tested

Although

s e e n in T a b l e

7

an increased germicidal

is in a c c o r d with p r e v i o u s

37
Table

6.

Exposure
T i m e in
Seconds

T h e effect of the addition of s o d i u m chloride a n d
p e p t o n e in v a r i o u s co n c e n t r a t i o n s of Q A C .

Distilled
W ater
(0 p p m )
100 p p m *

NaCl

100 p p m

(organisms
0
15
30
60
120
300

21,000,000
16,800
3,600
2,250
670
550

Exposure
T i m e in
Seconds

20 0 p p m

per

25,000,000
38,000
28,000
2,250
30
0

Distilled
Wate r
(0 p p m )
100 p p m *

91,000,000
11,500
1,700
3,950
750
20

ml

Peptone

100 p p m

98,000,000
24,000
51,000
5,100
2,950
20

* C o n c e n t r a t i o n of Q A C .

25,000,000
10
40
0
0
0

(250 p p m )

200 p p m

ml

400 p p m

remaining)

25,000,000
40
100
0
0
0

(organisms per
0
15
30
60
120
300

(250 p p m )

400 p p m

remaining)

98,000,000
360
390
0
0
0

98,000,000
530
10
0
0
0

38
Table 7.

Exposure
T i m e in
Seconds

Effects of v a r y i n g a m o u n t s of V e r s e n e a d d e d to tap
w a t e r u s i n g a c o n e en.tr ati o n of 100 p p m Q A C .

Versene

Distilled
Wate r

140 p p m

Softened T a p
220 p p m

(organisms per

ml

Water
270 p p m

s urviving)

0

95,000,000

93,000,000

86,000,000

90,000,000

15

1,980

21,500,000

64,000,000

56,000,000

30

840

1,400,000

25,000,000

52,000,000

60

30

160,000

1,700,000

1 8 ,500,000

120

90

310,000

36,000

1,200,000

300

0

0

10

1,320

39
work, but other data in this

study did no t s e e m

effect with the

of c h a n g e

To d e t e r m i n e
was

same

amount

of tap a n d distilled (165 p p m ) ,
and a V e r s e n e - s o f t e n e d w a t e r
indicate that there is n o
"softened"

of softening, a series

tap w a t e r

(315 p p m ) ,

a

1:1 ratio

a zeolite - softened w a t e r (180 p p m ) ,
(160 p p m ) .

The

data in T a b l e

w a t e r s , but in the c a s e of the V e r s e n e - t r e a t e d
in the g e r m i c i d a l

was

of w a t e r s

r epeated o n this g r o u p

any differences
enough to b e

significant.

the question as to w h e t h e r

However,

of 34 5 p p m )

the p r e v i o u s

there w a s

which was

action.

using 400 p p m

(including the tap w a t e r

case of the V e r s e n e

8

ap preciable difference in the first three

there is a definite i n c r e a s e

of the Q A C

as m u c h

in the a m o u n t of h a r d n e s s .

the effect of other m e t h o d s

tested using distilled water,

to s h o w

some

sample,

When

this

of the Q A C ,
were

series

not great

did raise

factor p r e s e n t in the

e n h a n c i n g the g e r m i c i d a l activity

independently of the effect u p o n the h a r d n e s s

of the

water.
To

investigate this possibility, t w o

up using only distilled w a t e r
T h e concentrations
T h e data in Table

series of tests w e r e

a n d adding the V e r s e n e

of g e r m i c i d e

used w ere

50,

set

to one group.

100, a n d 200 p p m .

9 illustrate v e r y clearly the increase in the

g ermicidal action of this Q A C

in the p r e s e n c e

of the V e r s e n e .

It

40
Table

8.

Exposure
T i m e in
Seconds

T h e effect of softening m e t h o d s o n the interference with
g e r m i c i d a l activity of a 200 p p m concentration of Quater­
nary A m m o n i u m Compound.

Di s tilled*
(6 p p m )

(organisms per

Tap*
(315 p p m )

ml

surviving)

0

500,000,000

530,000,000

15

40

> 100,000

30

20

> 100,000

60

0

> 100,000

120

0

> 100,000

300

0

290

* p H value of solutions:
Distilled
7.5
Tap
7.2
Zeolite softened
7.2
T a p + distilled
7.2
Tap + Versene
6.4

41
Table 8 (continued)

Zeolite
Softened*
(180 p p m )

Tap +
Distilled*
(165 p p m )

(organisms per
470,000,000

ml

Tap +
Versene*
(160 p p m )

surviving)

650,000,000

480,000,000

> 100,000

>

100,000

40,000

> 100,000

>

100,000

50,000

> 100,000

>

100,000

1,900

5,700

530

500

0

0

< 1 0 0

42
Table

9.

Exposure
T i m e in
Seconds

Effect of V e r s e n e o n G e r m i c i d a l Activity of a Quaternary
A m m o n i u m C o m p o u n d in distilled water.

Distilled W a t e r - p H
“
'
100 p p m

50 p p m *

(organisms

per

ml

6.9
'

~
200 p p m

remaining)

0

165,000,000

165,000,000

165,000,000

15

55,000,000

26,300

40

30

39,000,000

2,4 80

270

4,700,000

350

0

120

36,000

10

0

300

1, 1 50

0

0

60 •

* Concentration of Q A C .

43
Table 9 (continued)

Distilled W a t e r
50 p p m

and Versene, p H

6.9 - 7.0

100 p p m

(organisms per
95,000,000

ml

200 p p m

remaining)

95,000,000

95,000,000

190,000

9,000

30

13,000

30

0

4,000

0

0

0

0

0

0

0

0

44
may

also b e n o t e d that at a concentration of 200 p p m

water, there

is sufficient g e r m i c i d a l p o w e r

of the V e r s e n e
Table

is not noticeable,

the s a m e

in distilled

that the a d d e d effect
thing that w a s

n o t e d in

8.
Since only the e n d result w a s

there w a s
the Q A C

studied in e a c h of these cases,

n o indication as to w h e t h e r the V e r s e n e w a s
or w h e t h e r

w a y as to m a k e

it w a s

it m o r e

altering the bacterial cell in s u c h a

susceptible.

This w a s

distilled a n d tap w a t e r b y adding the V e r s e n e
one series

a n d to the

suspension of o r g a n i s m s

each of these test waters.

These

for e a c h water,

are given in Table

indication f r o m

this one

and culture g a v e
e n o u g h to d r a w

trials w e r e
This
Versene

studied in both

to the g e r m i c i d e

in

in the other for

data, together with the controls
10.

A l t h o u g h there is s o m e

trial that the c o m b i n a t i o n of the V e r s e n e

a greater

reduction, the differences are not great

a n y definite conclusions.

of a digression f r o m

acting u p o n

the m a i n p r o b l e m

Since this w a s

somewhat

of the thesis, no additional

made.
i n c r e a s e d activity of the Q A C

is in a c c o r d a n c e

Technical Bulletin N o .

w h e n c o m b i n e d with

with the data cited in the m a n u f a c t u r e r s

2 (26).

45
Table

10.

Effect of varying the initial contact point of Versene.

Distilled W a t e r (5 p p m ) ; Q A C (50 p p m )
E x p o s u r e _________ _______________________________________________
T i m e in
,,
,
_
.
Versene. +
Versene +
Seconds
Control
QAC
Culture

(organisms

per

ml

remaining)

0

210,000,000

210,000,000

180,000,000

15

10,000,000

13,600

400

30

6,000,000

40

20

60

1,900,000

10

20

120

148,000

0

0

300

280

0

0

46
Table 10 (continued)
Tap

Water

(300 p p m ) ;

QAC

Versene
QAC

C o ntrol

(organisms

per

(200 p p m )

+

ml

Versene +
Culture

remaining)

170,000,000

170,00 0 , 0 0 0

240,000,000

11,600,000

1,850,000

220 , 0 0 0

900,000

11,000

6,400

19,000

360

1,850

320

20

10

0

0

0

Briefly,
that
QAC

there
in

seem

the

to

water.

is

tlie
an

work

The

of

propor t i o n a l l y
method

by

which has been

the
the

although
degree

water

of

is

have

the p h y s i c a l

not

hardness

of

has

the
little

effect h a s b e e n
However, from

after it h a s

so that it c a n n o t give the s a m e
is still p r e s e n t in the

g e r m i c i d a l potential.

of three c o n s e c u t i v e

survivor-curve

cidal solution.

This

c o u l d also b e

mine

organisms

could b e

tion b e f o r e the action b e c a m e

removed

does

this

the

is a possibility that the

of this u n u s e d potential w a s

as the total v o l u m e

of

softened

Thi s

shown

action

state of the Q A C

There

in distilled w a t e r ,

the s a m e

many

has

indication of the reaction w h i c h m i g h t o c c u r

"tied u p "

that takes p l a c e

amount

germicidal

of the V e r s e n e .

b e e n a d d e d to the h a r d w a t e r .

how

the

water,

to

which

in this interference, n o r

the a m o u n t

presented

i n d e p e n d e n t of the softening p r o c e s s .

these data the r e is n o

and m a y

been

with

hard

effect e x c e p t in the c a s e
s h o w n to b e

has

i n t e rferen c e

presence

vary

that

made

An

centration of the g e r m i c i d e

reaction
solution

a t t e m p t to a s s e s s

b y running

tests using the

same

a series
germi­

c o n s i d e r e d as a test to d e t e r ­
a d d e d to the g e r m i c i d a l s o l u ­

depleted— or

exhausted.

in the m e d i c a t i o n po t is only

for s a m p l i n g

QAC

in e a c h

10 ml,

series is 5 ml,

in e a c h s u c c e s s i v e

Inasmuch
a n d the

the c o n ­

series w o u l d b e

48
50 p e r cent of that in the p r e c e d i n g
ture s u s p e n s i o n

added

control for e a c h
Table

each

time.

stage of the test.

solution w a s

volume

of s u s p e n s i o n

reduced

germicide

a n d bacterial

These

data a r e

of c u l ­

separate

presented

in the

Several trials w e r e

made

results p r e s e n t e d
activity of the
As

solutions

the Q A C

This

r a i s e d the

in three

purpose

15-

and

in

and Black

the s a m e
100 p e r

the

same

technique.
and

the

that the g e r m i c i d a l

cent kill, there
curve

is

is e x a m i n e d ,

survivors

indicate that the g e r m i c i d a l

stages

of b o t h

in e a c h stage of the

if the entire

wi t h e a c h

the
the

concentrations

data s h o w

30-second

i n c r e a s e wit h
capacity of

i n c r e a s i n g i n c r e m e n t of bacteria,

to s h o w

question of h o w

repeated before

and

a d j u s t e d to give

Weber

These

However,

would

is d e c r e a s i n g

but not e n o u g h

12.

is a l m o s t

s e e n that the

stage.

The

ml

wit h this technical a r r a n g e m e n t

the e nd p o i n t of

v e r y little difference.
it will b e

1 ml.

standard

in T a b l e

judged by

to

i n c r e a s e d to 20 0

suspension w e r e

ratio as that s o u g h t

be

5 ml

of the difficulties i n v o l v e d in this technique,

of g e r m i c i d a l

This

to the

This necessitated a

volume

each

due

1 1.
Because

test.

one,

long

the g e r m i c i d e

of studying this question,

up

5-minute

c a n this d o s a g e

becomes
the

at the

exhausted.

period.

of o r g a n i s m s
For

" e x h a u s t i o n ' 1 test w a s

the
evolved.

49
Table

11.

T h e effect of a d d i n g three s u c c e s s i v e
b a c t e r i a to a solution of Q A C .

Decreasing
Co nce n t r a t i o n
of Q A C
Dilution

E x p o s ure
T i m e in
Seconds

(1st i n c r e m . )

200 p p m

15
30
60
120
300

(3rd in c r e m . )

15
30
60
120
300

of

C o n t r o l for
Each Q A C
Concentration
(org./ml)

9 1,000,000

(200 p p m )
9 1 , 0 0 0 ,0 0 0

120
40
10
0
0

30
0
10
10
0

91,000,000

(100 p p m )
9 1 ,0 0 0 , 0 0 0

Innumerable
Innumerable
260
10
0

23,600
520
270
0
0

9 1 ,0 0 0 , 0 0 0

(50 p p m )
91,000,000

15
30
60
120
300

(2nd i n c r e m . )

100 p p m

Surviving
O rganisms
After A d d e d
Inc r e m e n t s

increments

Innume
Innume
Innume
Innume
Innume

rable
rable
rable
rable
rable

Inn u m e r abl e
I n n u m e rable
I n n u m e rable
2,900
300

50
Tab l e

12.

Germicidal

efficiency after r e p e a t e d d o s a g e s

of o r g a n ­

isms.

Exposure
T i m e in
Seconds

Distilled W a t e r ;
QAC

(100 p p m )

Trial

1

( o r g a n i s m s p e r ml)
1st I n c r e m e n t
0
15
30
60
120
300

83,000,000
14,300
1,110
0
0
0

110,000,000
171,000
14,600
2,000
110

2nd Inc r e m e n t

83,000,000

110,000,000

16,100
120
0
0
0

Innumerable
3,600,000
25,000
140
0

15
30
60
120
300

3rd I n c r e m e n t

15
30
60
120
300

83,000,000

6,200
170
0
0
0

51
Table 12 (continued)

Tap
Trial 2

Water

(315 p p m ) ;

QAC

(200 p p m )

Trial 3

Trial 4

( o r g a n i s m s p e r ml)

84,000,000
185,000
21,000
2,400
580
0

790,000,000
45,000
9,200
60
0
0

800,000,000
360,000
430
40
0
0

84,000,000

790,000,000

800,000,000

14,000,000
210,000
3,500
_

6,000,000
20,000
600
4,000
0

7,800,000
570
60
240
10

84,000,000

790,000,000

800,000,000

42,000,000
2,250,000
113,000
1,800
2,750

26,000,000
2,460,000
170,000
10
0

33,000,000
2,100,000
41,000
180
0

-

52
Since the
but rather the

interval of
culture.

of kill w a s

the

series

survivor-curve
of t i m e

10 m i n u t e s

The

no t the i m p o r t a n t factor here,

ability of the g e r m i c i d e

length of time,
place of the

speed

type of test w a s

intervals, tests w e r e

specific techn i q u e s

of this

section.

on distilled w a t e r ,

tap w a t e r ,

and two

these t w o w i t h the

resulting levels

hardness.

Each

of the t w o

is r e p o r t e d in T a b l e
able

sample

was

the s e venth i n c r e m e n t

as

tion point of

of the Q A C

added.

same

intermediate

degree

a total of m o r e

test h a v e

115,

220,

run

and

of

315 p p m

a n d the a v e r a g e

of g e r m i c i d a l

the g e r m i ­

activity in

In other w o r d s ,

the e x h a u s ­

u s e d is b e y o n d the limit of this

than

500 x

10

8

organisms

differential information,

to g o t h r o u g h m o r e

was

mixtures

In all fo u r cases,

in the first.

In o r d e r to obtain a n y

be n e c e s s a r y

In

o n l y at a m

T h e first series

of 5,

13.

the

series in w h i c h

made

"exhaustion"

plat e d in duplicate,

to give

100 p p m

not used.

after the addition of e a c h i n c r e m e n t of the

b e e n d e s c r i b e d in a p r e v i o u s

cide w a s

to kill in a r e a s o n a b l e

increments

than w a s

was

it w o u l d
done

in

this case.
A

series

of these

ferent w a t e r s

using

determine

endpoint of these v a r i o u s

the

75,

e x h a u s t i o n tests w a s
100,

150,

and

200 p p m

performed
of the Q A C

on dif­
to

concentrations of the

4

53
Table 13.

Increment

Results of the exhaustion test applied to four differ­
ent waters of varying hardness, using 200 p p m QAC.

300 m l
Distilled
(5 p p m )

200 m l D i s ­
tilled +
100 m l T a p
(115 p p m )

100 m l D i s ­
tilled +
200 m l T a p
(220 p p m )

300 m l T a p
(315 p p m ) 4

( o r g a n i s m s p e r m l r e m a i n i n g 10 m i n u t e s
after e a c h s u c c e s s i v e dosing)*
1 st

25

10

0

30

2nd

150

10

10

320

3rd

5,000

0

5

20

4th

0

10

50

10

5th

10

0

2,300

10

6th

5

0

35

20

7 th

0

10

-

460

❖ Average

of t w o plates.

54
germicide.
A

These

data a r e p r e s e n t e d in T a b l e s

study of these tables

germicide

increases,

b e r of i n c r e m e n t s
period.

This

also s h o w s

there

that, as

is a c o r r e s p o n d i n g

in the c a s e

killed in a

dition of the Q A C
Since p r e v i o u s

these tests

tests h a d

which m i g h t have

these

still g a v e n o

shown

and

some

removed

the activity to b e

several i n c r e m e n t s .

Then

another

As

controls,

three other

These

consisted of one
containing the

some

series

of the V e r s e n e

might
run.

w o u l d be

to 0 p p m

hard­

a c c o m p a n i e d this one.

a m o u n t of V e r s e n e

third series, w h i c h w a s

which

the

agent

series w a s

series using distilled water,
same

action.

exhaustion test a n d continue

a d d e d in a quantity sufficient to soften the w a t e r
ness.

from

increased b y

effect in releasing a n y Q A C

to start a n

in tap

indication as to the c o n ­

since this is a strong c o m p l e x i n g

the plan w a s

It

conditions.

be tied up but still unaltered chemically,

a d d e d to the

10-minute

efficiency of the Q A C

w h i c h obviously h a d b e e n

addition of V e r s e n e ,

tap w a t e r

in the n u m ­

of the highest concentrations

series, the g e r m i c i d a l

However,

it for

increase

is true of b o t h the distilled a n d the tap water.

w a t e r is rapidly e x h a u s t e d u n d e r

In this c a s e

17.

the c o n c e n t r a t i o n of the

of culture w h i c h c a n b e

that e v e n

tested in the

reveals

14 t h r o u g h

a s e c o n d with

that w a s

to b e

started in tap w a t e r alone,

55
Table 14.

Tbe exhaustion test applied to two waters using a
concentration of Q A C of 75 ppm.*

Increment
Numbe r

Distilled W a t e r

T ap W ate r
(310 p p m )

(organisms per m l remaining
after e a c h successive dosing)
1

20

Innumerable

2

0

I n n u m e rable

3

0

I n n u m e rable

4

Innumerable

I n n u m e rable

5

16,000

I n n u m e rable

6

240

Innumerable

7

26,500

I n n u m e rable

8

40,000

I n n u m e rable

9

I n n u m e rable

I n n u m e rable

10

Innumerable

Innumerable

11

Innumerable

I n n u m e rable

12

Innu m e r abl e

I n n u m e rable

* Concentration of o r g a n i s m s p e r m l
beginning of e a c h i n c r e m e n t

a d d e d at the

- 51,000,000.

A

56
Table 15.

The exhaustion test applied to two waters using a
concentration of Q A C of 100 ppm. *

Increment
Number

Distilled W a t e r

T ap W ate r
(310 p p m )

(organisms per m l remaining
after e a c h s u c c e s s i v e dosing)
1

0

Innumerable

2

500

I n n u m e rable

3

40

I n n u m e rable

4

0

I n n u m e rable

5

30

I n n u m e rable

6

20

Innumerable

7

14,000

I n n u m e rable

8

5,800

Innumerable

9

0

Innumerable

10

30

I n n u m e rable

11

500

I n n u m e rable

12

35,000

I n n u m e rable

* Concentration
b e ginning of e a c h

of o r g a n i s m s

increment

per m l

- 48,000,000.

a d d e d at the

57
Table 16.

The exhaustion test applied, to two waters using a
concentration of Q A C of 150 ppm.*

Inc r e m e n t
Number

Distilled W a t e r

T ap W ate r
(310 p p m )

(organisms per m l remaining
after e a c h s u c c e s s i v e dosing)
1

0

60,000

2

0

9,800

3'

10

300, 0 0 0

4

10

I n n u m e rable

5

0

I n n u m e rable

6

0

I n n u m e rable

7

0

I n n u m e rable

8

0

I n n u m e rable

9

10

I n n u m e rable

10

10

I n n u m e rable

11

0

I n n u m e rable

12

30

I n n u m e rable

13

0

Innumerable

14

0

I n n u m e rable

15

0

I n n u m e rable

* C o n c e n t r a t i o n of o r g a n i s m s p e r m l
beginning of e a c h i n c r e m e n t - 76,000,000.

a d d e d at the

58
Table 17.

The exhaustion test applied to two waters using a
concentration of Q A C of 200 ppm.*

Increment
Numbe r

Distilled W a t e r

Tap Wate r
(310 p p m )

(organisms per m l remaining
after ea c h successive dosing)
1

0

3 70

2

0

300

3

0

660

4

0

8,400

5

10

60,000

6

10

I n n u m e rable

7

4,100

I n n u m e rable

8

20

I n n u m e rable

9

0

I n n u m e rable

10

0

Innumerable

11

0

I n numerable

12

0

I n n u m e r ab 1e

13

0

I n n u m e rable

14

0

I n n u m e rable

15

0

I n n u m e rable

* Concentration of o r g a n i s m s p e r m l
beginning of each i n c r e m e n t - 58,000,000.

added at the

59
and a. fourth series using only tap water.

The results are given

in Table 18.
In the three
expected f r o m

"control”

the p r e v i o u s

out to s e v e n i n c r e m e n t s ,
to r e a c h

an

endpoint.

data.

as

The

runs,

the

The

results

are

tap w a t e r w a s

this h a d b e e n

parently,
water

a d d e d at that t i m e

been
the

which

is still c a p a b l e

Not

only w e r e

data is

to the reaction
a l m o s t all of

d estroyed, bu t also those w h i c h

accumulating f r o m

QAC

sufficient

interesting thing abo u t these

flask p r i o r to the fifth inoculation.

m u s t have

only c a r r i e d

s h o w n to b e

the reaction following the addition of the V e r s e n e

the o r g a n i s m s

as w o u l d be

the first f o u r i n c r e m e n t s .

is inactivated b y

of exerting

some

germicidal

Ap­

factor in the h a r d

action if it c a n b e

released.
It is interesting to note
in w h i c h

the h a r d w a t e r

increments,
this m i x t u r e

the
had

also that in the c a s e

sample

count w a s

had been

of the flask

discontinued after

r e d u c e d to 60 o r g a n i s m s p e r

s t ood for a p p r o x i m a t e l y 90

minutes.

ml
Thi s

seven
after
would

i
s u g gest the possibility that o v e r
that the g e r m i c i d a l

a n e x t e n d e d p e r i o d s u c h as this

activity of the Q A C

is also

released.

60
Table 18.

Inc r e m e n t
Numbe r

The exhaustion test as applied to several waters
using 100 p p m concentration of Q A C . *
Distilled
Water
(0 p p m )

Tap +
Versene^
(0 p p m )

Tap
W ate r
(3 10 p p m )

Tap
Water
(310 p p m )

(organisms per m l remaining
after e a c h s u c c e s s i v e dosing)
1

0

0

I n n u m e rable

I n n u m e rable

2

0

0

I n n u m e rable

I n n u m e rable

3

20

0

I n n u m e rable

I n n u m e rable

4

0

0

I n n u m e rable

I n n u m e rable

(V e rse n e
added^)
5

0

0

170

I n n u m e rable

6

0

10

0

I n n u m e rable

7

50

0

0

I n n u m e rable

8

310

0

0

9

0

0

0

10

10

10

20

11

0

0

60

12

10

0

20

13

10

0

70

14

2,100

0

20

15

25,000

0

0

C o n c e n t r a t i o n of o r g a n i s m s p e r m l a d d e d at the
beginning of e a c h i n c r e m e n t - 18,000,000.
2
500 m g V e r s e n e a d d e d in e a c h case.
3

90 m i n u t e

sampling

- 60

o r g a n i s m s p e r milliliter.

3

DISCUSSION

A
stances

study of the literature o n the
in h a r d water,

especially in those

a n d B l a c k technique w a s
ammonium

compounds

subject of interfering s u b ­

employed,

studies w h e r e

the W e b e r

indicates that the q u a t e r n a r y

a r e not suitable for u s e

in a h a r d water.

U s i n g this m e t h o d of testing, it h a s b e e n

s h o w n that the w a t e r

the L a n s i n g

D u r i n g the p e r i o d of test­

a r e a falls in this category.

ing, the h a r d n e s s
varied f r o m

300

of the M i c h i g a n State College w a t e r
to 3 85 p p m .

this w a t e r
(Table

unle s s

5).

supply has

A c c o r d i n g to the W e b e r

test, the q u a t e r n a r y a m m o n i u m

compounds

the concentration w e r e

in

and Black

could not b e

u s e d in

i n c r e a s e d to 800 p p m

It h a s b e e n p r e v i o u s l y stated that this is not in a c c o r d ­

ance with the actual e x p e r i e n c e

in. the.field with this g r o u p

of c o m ­

pounds .
The

results

technique w h i c h
would be
to the

of l a b o r a t o r y tests b y

and Black

indicate that the q u a t e r n a r y a m m o n i u m

inc o m p a t i b l e

in the college w a t e r

results of field tests

cent a r e a a r e

the W e b e r

showing

supply are

that dai r y f a r m s

compounds
in opposition

in the adj a ­

successfully using q u a t e r n a r y a m m o n i u m

compounds.

H o w e v e r , these t w o findings ar e not incompatible, b e c a u s e the d e g r e e of

62
kill attained and the initial inoculum m u s t be considered.

Th e

death curves obtained b y applying a germicide to a bacterial culture
are always characterized b y a so-called resistant minority of o r ­
ganisms

that extends the time of total kill m a r k e d l y b e y o n d the

n o r m a l curve.

In the case of death curves obtained with quater­

nary a m m o n i u m

compound,

the resistant minority appears to s u r ­

vive for a longer period than with m o s t germicidal agents, although
this group is relatively small in n u m b e r - - s o m e t i m e s less than 10
or g a n i s m s

out of an original inoculum of 100,000,000 or m o r e .

To avoid this seemi n g l y long period for total kill, m a n y w o r k e r s
have suggested using an endpoint of 99, 99.9, or even 99.9999 per
cent reduction as the endpoint.
results m o r e

This w ould bring the laboratory

in line with the field experience in this case.

A

closer examination of the data presented s h o w s that in the case of
the tests m a d e

using 200 p p m

of the germicide, there w a s

almost

always at least a 50 per cent reduction at the end of 15 seconds.
B y the end of the 1-minute interval, the kill had reached a point
of approximately 99 per cent.

Looking n o w at the practical side

of the situation, there are two factors to be kept in mind.

The

first is that in m a n y instances, even w h e r e only a contact time of
1 or 2 minutes is allowed, a reduction of 99 pe r cent is a very

acceptable

result.

The

s e c o n d is the g e n e r a l

a sanitizer w o u l d b e used.
cede

the u s e

to r e m o v e

Thorough

cleaning

of a sanitizing solution.

the o r g a n i c

matter,

This

debris,

soil m i g h t b e p r e s e n t o n the

surface

not done,

amounts

there will b e

lar g e

will either rea c t with the g e r m i c i d e
o r act as

a barrier betw e e n

which

that w h e n
there

the

stroyed.

dealing h a v e

if there

is s o m e

the

thus b e e n

action.

the p r o p e r

if the rate of kill f r o m

vivor c u r v e

is applied to a theoretical i n o c u l u m of

the following m i g h t occur:

de­

a re n o w
a per

e v e n less w o u l d give a v e r y

For

ml,

conditions,

to a v e r y l o w n u m b e r ,

e n d result.

of

Th i s m e a n s

of bacteria with w h i c h w e

reduced

example,

germi­

soil that is present, m o s t

a v e r y l o w concentration of bacteria to be

reduction of 90 o r p e r h a p s

per

If this is

a n d the bacterial cell

r e m o v e d by mechanical

Since the n u m b e r s

of

to c h e m i c a l l y inactivate it,

sanitizer is applied u n d e r

should be

type

a result of this t h o r o u g h cleaning

is n e c e s s a r y to r e m o v e

the bacteria will b e

sanitized.

the g e r m i c i d e

As

or w h a t e v e r

of m a t e r i a l p r e s e n t that

so that it c a n n o t get to the bacteria e v e n
cidal potential r e m a i n i n g .

should a l w a y s p r e ­

is n e c e s s a r y in o r d e r

grease,
to be

situation in w h i c h

cent

satisfactory

an actual s u r ­
1,700 o r g a n i s m s

64
T i m e in
Seconds

Actu a l
“ Death.
Curve"

Theoretical " C u r v e "
if Initial Inoculation
w e r e 1,700 o r g . / m l

0

170,000,000

1,700

15

11,600,000

116

30

900,000

9

60

19,000

120

320

300

0

Thus, the

100 p e r cent endpoint w o u l d h a v e b e e n a c h i e v e d in 60

seconds

using the s m a l l initial inoculum,

number

was

attained in 30

seconds.

a n d a n insignificant

Another w a y

this situation is that the a m o u n t of g e r m i c i d e
e m p l o y e d is far in e x c e s s
w o u l d therefore h a v e
used.

that is usually

of that w h i c h is actually needed,

a reserve

It is a p p a r e n t f r o m

distilled water,

of looking at

and

of g e r m i c i d a l action w h i c h is not

the data p r e s e n t e d that in the case of

the use-dilution of 200 p p m

quaternary a m m o n i u m

c o m p o u n d is far in e x c e s s of that n e e d e d e v e n w h e n the initial
dosage

is about

100 x

10

this r e s e r v e is reduced,
curve

.

In the p r e s e n c e

of the h a r d water,

but it is a p p a r e n t f r o m the survivor-

tests in w h i c h three s u c c e s s i v e i n c r e m e n t s w e r e

a d d e d that

the g e r m i c i d e h a s

a l m o s t the s a m e

teria has b e e n destroyed.

activity after e a c h load of b a c ­

If the s a m e

p e r cent reduction w e r e

applied in a situation in w h i c h the initial i n o c u l u m w a s
less--as w o u l d b e the ca s e with p r o p e r
the e n d result w o u l d b e

much

cleaning m e t h o d s - - t h e n

s u c h that there w o u l d b e n o public health

hazard.
A n o t h e r observation m a d e

from

these tables is that the

interfering action of the h a r d w a t e r has m o r e
of kill than o n the total kill.

effect u p o n the rate

This w o u l d indicate a slightly dif­

ferent type of r e s e r v e w h i c h is not i m m e d i a t e l y available but m a y
be released m o r e

slowly to exert its g e rmicidal action o v e r a

longer p e riod of time.

This being the case, there is the p o s s i ­

bility that in m a n y practical situations the actual contact time w o u l d
be m u c h

greater than the ti m e allowed for the sanitizer to be in

contact with the objects being treated.
is no attempt m a d e
contact t i m e

bers

to stop the g ermicidal action at the e n d of the

although there are

That the g e r m i c i d e

Tha t is, in the field there

still has

situations w h e r e

this w o u l d occur.

a potential capacity to kill large n u m ­

of cells is d e m o n s t r a t e d b y the results given in Table

the case of the tap w a t e r

sample.

at the e n d of the seventh increment,

When

the test w a s

the o r g a n i s m s

18 in

discontinued

surviving w e r e

66
such that a plate p r e p a r e d f r o m
overgrown.

However,

to r e d u c e the n u m b e r

a dilution of 10

after 90 minutes,

10

-1

the g e r m i c i d e

small n u m b e r

icidal rinse.

Assuming

from

instances w h e r e

glasses m a y

be

six

of bacteria w h e n

the v o l u m e

a rel­
situation.

cited as an e x a m p l e .

cleaning h a s b e e n a c c o m p l i s h e d ,

ca r r y a v e r y

glasses

able

10^ that

a sanitizer is a v e r y acceptable

sanitization of b e v e r a g e

5 gallons,

was

dilution plate).

atively s l o w action f r o m

After the p r o p e r

completely

(as r e p r e s e n t e d b y

In actual practice, there ar e m a n y

The

was

of cells f r o m the total of 126 x

h a d b e e n a d d e d to 60 o r g a n i s m s p e r m l
colonies o n the

-2

the glasses

they r e a c h the g e r m ­

of the rinse

tank to be

about

the m a j o r i t y of the bacteria r e m a i n i n g o n the first

to b e

introduced into a fresh

the glass b y dilution.

off w o u l d then b e

These

solution w o u l d b e

organisms

attacked b y the germi c i d e .

g e r m i c i d a l action w o u l d be

taking place

o n the surface of the glasses.

As

removed

that h a d b e e n w a s h e d
That is, m u c h

of the

in the solution as well as

long as the bacterial count of

the rinse w a t e r a p p r o a c h e d zero, the dilution effect w o u l d continue
to r e m o v e

organisms fr o m

the surface of the glasses a n d the e n d

p r o d u c t — the bacterial count o n the g l a s s — w o u l d be
T h e function of the g e r m i c i d e

satisfactory.

in this case w o u l d b e to k e e p the

bacterial count of the

rinse

solution v e r y low.

this, only a sufficient s p e e d of kill w o u l d b e
all of the o r g a n i s m s
before the n e x t w a s
rinse w a t e r
or s o m e
just as

does

removed from
p l a c e d in the

not b e c o m e

one

To

n e c e s s a r y to d e s t r o y

glass, o r g r o u p

rinse water.

overloaded due

As

of glasses,

long as

other factor, a relatively s l o w - a c t i n g g e r m i c i d e
effective as o n e with a v e r y

the

to insufficient w a s h i n g

rapid kill.

would be

If this situation

of o v e r l oading did exist, the fast-acting c o m p o u n d
not b e

accomplish

would probably

effective either d u e to the inactivating action of the

soil

present.

In e a c h of these cases

discussed, there is a portion of the

c o m p o u n d w h i c h has b e e n m a d e
of Sal ton in Australia (19) has
adsorbed m o r e

were

Micrococcus pyogenes

s h o w n that the bacterial cell

Pseudomas

var.

5.2 to 8.2 a n d g e r m i c i d e

aureus.

flourescens

concentrations

of q u a t e r n a r y a m m o n i u m

for all four p H

p e n d e n t u p o n the p H

levels f r o m

of 100, 200,

compound,

he w a s

This

400, a n d
able to s h o w

salt a d s o r b e d w a s

levels, but the g e r m i c i d a l
of the solution.

542, a n d

U s i n g four p H

that the a m o u n t of q u a t e r n a r y a m m o n i u m
same

The w o r k

of the g e r m i c i d e than w a s n e c e s s a r y to kill it.

His test o r g a n i s m s

800 p p m

germicidally inactive.

action w a s

the
de­

indicates that the

68

process of adsorption on the surface of the cell is somewhat inde­
pendent of the germicidal action taking place.
Based

u p o n this w o r k

a n d the fact that the adsorption is

dependent u p o n the isoelectric point of the cell w h i c h c a n be
altered b y

a change

in pH,

an atte m p t w a s

material in the following m a n n e r :
B lack m e t h o d ,

justed to 3.8.
there w a s

made

One

data p r e s e n t e d in Table

ad­

19 are evidence that
levels in two s e p ­

A l t h o u g h these data are inconclusive insofar as

this particular p r o b l e m

is concerned, they are p r e s e n t e d her e

s h o w that the neutralizer is apparently doing a g o o d job.
there w e r e

of these

of 7.2, a n d the other w a s

little difference b e t w e e n the two p H

arate trials.

and

run in parallel,

simultaneously into each.

kept at the usual p H
The

to release this

Using a modified W e b e r

t w o neutralizing solutions w e r e

and inoculations w e r e
solutions w a s

made

some

cide that w a s

bacteriostasis talcing place b e c a u s e

to

If

of the g e r m i ­

a d s o r b e d o n the cell surface, then the inocula in the

acid blank should h a v e

s h o w n increased growth because

lease of the quater n a r y a m m o n i u m
a n d their reaction with asolectin.

molecules f r o m

of the r e ­

the surface

69
Table

19.

Exposure
T i m e in
Seconds

A

c o m p a r i s o n of acid a n d neutral inhibitor solutions

Distilled W a t e r (0 p p m ) ;
Q A C (100 p p m )

Tap

W a t e r {300 p p m ) ;
Q A C (200 p p m )
r

pH

7.2

pH

3. 8

pH

(organisms per

ml

7.2

pH

3.8

surviving)

0

98,000,000

120,000,000

66,000,000

66,000,000

30

120

550

1,250,000

830,000

60

20

0

120,000

98,000

120

0

0

2,500

1,700

300

0

0

0

0

Second

Trial

0

240,000,000

240,000,000

250,000,000

250,000,000

30

21,000

19,000

140,000

120,000

60

300

230

11,000

7,200

120

30

0

100

70

300

0

0

0

10

70
Dennis
is

used

two

showed

concurrently

highly

strain

(4)

what

with

an

mouse-virulent

C-203,

and

isms.

Using

an

to s h o w

that the

in

Pasteurella
exposure

germicide

in w h i c h

seem

these

test a s

compound
more

than

actual

confirm

the

minutes,

employed
pyogenes,

test
he

organ- .

was

of N o r w o o d

able

water

gave

action of the h a r d w a t e r

did not

indicate

and

Weber

that the

survivor-

w o u l d not b e

a

satis­

e v a l u a t i o n of a q u a t e r n a r y a m m o n i u m
especially w h e n

the c o n t a c t p e r i o d

is

2 minutes.
correlation b e t w e e n

application

seems

the fact that t h e r e

cidal action of this g r o u p
water.

He

test

diluted in distilled w a t e r - - a n o t h e r

Black

in the

in h a r d w a t e r ,

A l t h o u g h the
the

2

as

vivo

result.

outlined b y

1 or

in

Streptococcus

in the p r e s e n c e

factors p r e s e n t e d

f a c t o r y test to u s e

of

an

method.

boviseptica

period

interfering

to affect the e n d

All
curve

the

when

vitro

organisms,

a greater protection than w h e n
case

happens

this l a b o r a t o r y m e t h o d

questionable,
is

some

the

data p r e s e n t e d

interf e r e n c e

of c o m p o u n d s

by

and

do

w i t h the g e r m i ­

the p r e s e n c e

of h a r d

It h a s also b e e n s h o w n that b y the u s e of V e r s e n e , the g e r m ­

icidal efficiency of t h e s e c o m p o u n d s c a n b e i n c r e a s e d in h a r d w a t e r
s o that the s p e e d of kill is c o m p a r a b l e to that of the s a m e c o n c e n t r a t i o n

71
of q u a t e r n a r y a m m o n i u m

c o m p o u n d in distilled water.

«

formulations of this type of combination,
to the bulletin of the m a n u f a c t u r e r

is also a n increa s e

quaternary a m m o n i u m
the Y e r s e n e

is added.

p o u n d is m o r e

This

made

(26).
(Table

in the g e r m i c i d a l

compounds

several

reference m a y b e

Additional studies o n this c o m p o u n d
that there

For

9) h a v e

shown

activity of the

diluted in distilled w a t e r w h e n

indicates that the action of this c o m ­

than a s i m p l e

r e m o v a l of the h a r d w a t e r ions.

satisfactory explanation of this is k n o w n

No

at the p r e s e n t time.

It

is r e p o r t e d (6, 26) that there is a zone at w h i c h the concentration
of q u a t e r n a r y a m m o n i u m
other to f o r m

c o m p o u n d and Vers e n e

react with e a c h

a cloudiness or precipitate a c c o m p a n i e d b y a s h a r p

d r o p in g e r m i c i d a l

activity.

This

c a n be

ing the concentration of the V e r s e n e

recovered by increas­

b e y o n d the zone.

C o n s i d e r i n g again the reaction of the Y e r s e n e
water

solutions, there are

as a result of this action.
reaction of this c o m p o u n d

Versene + metal

This

several things w h i c h m i g h t be p r o p o s e d
A

s i m p l e equation for the chelating

ca n b e

given in this way:

ions --- •> M e t a l - V e r s e n e

is given m o r e

in the h a r d

in detail in F i g u r e I.

complex + sodium

Two

points f r o m

ions

the

NaO-C-CH

C H -C-ONa
N - C H - C H - N'

/

II

O

2

+

2 Na

2OI

o

O

I

C H — C-ONa

NaO-C-CH.
\

N-CH,— C H

/

2

CH

CH

O-C:

C-O

Figure

~ a ++
C

\ CH,— C-ONa

NaO-C-CH,
•

+

I.

Tlie chelating

r e a c t i o n of V e r s e n e .

73

Technical

Bulletin N o .

nonspecific

in their

2 are

complexing

practically a n y m e t a l l i c
Versene

will b r e a k

t h o u g h th e y m a y

be

the

seems

this

complexes

shown

by

release

was

simple

that the r e

added

is s u c h that the
metal

salts

even

is m o r e

to the action

activity of the

can

of a l a r g e

sible to d e t e r m i n e

to f o r m

only b e

a Versene

quater­

it is ev i ­

alteration of the g e r m i c i d a l

of the

it

complex.

s p e c u l a t e d upon,

compound

amount

taking place.

Versene

of the

information given
whether

increments,

interfering factor a n d the

is little, if any,

the addition

after f o u r

to this p r o p e r t y of d e s t r o y i n g

nature

of the

compound

S t u d y i n g the

d u e to s o m e

and

it is likely that the m a i n

quaternary a m m o n i u m
shown

complex

water

of a m o r e

ammonium

of the

diate

simpler

the V e r s e n e

dent that there

been

Also,

been

A l t h o u g h the c o m b i n a t i o n
nary

are

action in that they will inactivate

likely that the action is d u e

complexes

Versenes

is a c o m p l e x i n g out of the factor that is
\
substance.
T h u s , in the c a s e of the e x h a u s t i o n

interfering

test in w h i c h

The

insoluble.

than that of softening,
in h a r d

ion.

down

A l t h o u g h it h a s

Versene

of interest h e r e .

action o n the bacterial

has

resulting in the i m m e ­

germicidal
in T a b l e

the e n h a n c e d

Thi s

action

agent.

10, it is not p o s ­

action of the V e r s e n e

cell o r

an

alteration of the

is

74
germicide
ences

itself to m a k e

it m o r e

germicidal.

The

only differ­

indicate that there is slightly better g e r m i c i d a l action w h e n

the V e r s e n e

is a d d e d to the bacterial culture first.

This w o u l d

be indicative of a n action w h i c h altered the bacterial cell in s o m e
w a y to m a k e

it m o r e

vulnerable.

However,

able to d r a w a n y definite conclusions f r o m
especially since

some

From

the V e r s e n e

thing is the

results; h o w e v e r ,

a continua­

the standpoint of

of action of this p h e n o m e n o n .

use of the exhaustion test for the evaluation of q u a ­

ternary a m m o n i u m
more

is a d d e d to the g e r m ­

studies m i g h t b e of interest f r o m

d e t e r mining the m o d e

The

(6) w o u l d indicate

a practical standpoint, the m a i n

i ncreased g e r m i c i d a l activity w h i c h
tion of these

these limited data,

of the w o r k b y Geot c h i u s

that the action is better w h e n
icide first.

it w o u l d b e inadvis­

compounds

in the p r e s e n c e of h a r d w a t e r gives

information w h i c h correlates with the practical situation

than does the su r v i v o r - c u r v e type of test of W e b e r

a n d Black.

It is possible to s h o w that there is a r e s e r v e of g ermicidal p o ­
tential p r e s e n t ev e n in the cas e of h a r d w a t e r that is not evident
from

the results of the W e b e r

an d B l a c k m e t h o d .

This is d e m ­

onstrated b y the fact that the s u b s e q u e n t i n c r e m e n t s

of culture will

be killed until an endpoint is r e a c h e d with the quaternary a m m o n i u m

75
c o m p o u n d in the " n o r m a l "

state in the h a r d water.

T h e test w a s

easily adapted to the technique with the addition of V e r s e n e w h i c h
has b e e n discussed previously.
T h e time interval w a s

selected with the idea that 10 m i n ­

utes w o u l d give sufficient ti m e for the action of the g e r m i c i d e to
be completed.
"total"

In this w a y an evaluation could be m a d e

action taking place.

That this w a s

of the

a false a s s u m p t i o n can

be seen b y the results with the tap w a t e r as s h o w n in Table
W h e n the solution w a s
there w a s

a marked

10-minute period.

18.

allowed to stand for about 90 minutes,

increase in the germicidal action over the
T h e s e time intervals could be adjusted to give

information for a n y time period desired f r o m the

15- a n d 3 0 - second

intervals u s e d b y Cant o r and Shelanski up to any desired interval.
T h e repeated increm e n t s

could be u s e d in the s a m e w a y with any

interval.
B y the interpretation of the results of the exhaustion test,
it is possible to obtain an evaluation of a c o m p o u n d in the presence
of h a r d wat e r w h i c h is m u c h

closer to the actual conditions than

the survivor-curve type of test.

Although extensive tests wou l d

be necessary, it m i g h t be possible to establish a certain criterion
as an endpoint b y a c o m p a r i s o n with the results-of actual field

76
studies.

Thu s ,

compounds

a laboratory evaluation of the q u a t e r n a r y a m m o n i u m

in the p r e s e n c e

of h a r d w a t e r

could b e m a d e .

CONCLUSIONS

1.

The

supply w a s
monium

interference of the h a r d w a t e r of the college w a t e r

shown

compounds

2.

to affect the rate of kill of the q u a t e r n a r y a m ­
to a g r e a t e r extent than the total kill.

D a t a a r e p r e s e n t e d to s h o w that the g e r m i c i d e h a d

b e e n inactivated, but not c o m p l e t e l y destroyed.

3.

The

addition of V e r s e n e

after several stages of the e x ­

haustion test rel e a s e d the q u a t e r n a r y a m m o n i u m

compound

that

h a d b e e n inactivated so that it e x e r t e d a g e r m i c i d a l action c o m ­
p a rable to that o b s e r v e d in distilled water.

4.
monium

U s i n g a concentration of 200 p p m

compound,

the addition of 870 x

in fifteen i n c r e m e n t s
this c o m p o u n d
in tap water,

10

of q u a t e r n a r y a m ­
organisms per m l

did not exhaust the g e r m i c i d a l capacity of

in the p r e s e n c e of distilled water.
the endpoint of g e r m i c i d a l action w a s

the addition of 340 x

10

S

o r g a n i s m s p e r ml.

When

diluted

noted after

78
5.
taining a m o r e

T h e exhaustion test is p r e s e n t e d as a m e t h o d of o b ­
realistic evaluation of the g e r m i c i d a l properties

of the q u a t e r n a r y a m m o n i u m
wate r .

compounds

in the p r e s e n c e of h a r d

BIBLIOGRAPHY

1.

Botwright, W . E.
1952.
Sanitization of dairy f a r m uten­
sils. A c o m p a r i s o n of a cleaner-sanitizer containing
H y a m i n e 1622 with an alkaline cleaner and h y p o ­
chlorite sanitizer.
J. M i l k and F o o d Tech.
15 , 29-33.

2.

Butterfield, C. T., E. Wattie, and C. W . C h a m b e r s .
1950.
Bactericidal efficiency of quaternary a m m o n i u m c o m ­
pounds,
Pub. Health Rpt.
6j3, 1039-56.

3.

Cantor,

A., and H. A. Shelanski.
germicidal activity.
Soa p
133.

1951.
A capacity test for
an d Sanit. C h e m .
2J7, (2)

4.

Dennis,

E. E.
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