EFFECT

OF

CULTURE

GROWTH OF

M EDIA ON TH E R ESISTA N C E AND

M IC RO CO C CU S PY O G E N E S VAR. AUREUS

By
A N ITA H A R R IET

LEA V ITT

A THESIS
S u b m i t t e d to t h e S c h o o l o f G r a d u a t e S t u d i e s o f M i c h i g a n
S t a t e C o l l e g e of A g r i c u l t u r e a n d A p p l i e d S c i e n c e
in p a r t i a l f u l f i l l m e n t of th e r e q u i r e m e n t s
f o r t h e d e g r e e of

DOCTOR OF

PH ILO SO PH Y

D e p a r t m e n t of B a c t e r i o l o g y a n d P u b l ic H e a l th

19 5 2

i

ACKNOWLEDGMENTS

The author w ishes

to e x p r e s s h e r

sincere

appreciation

to D r . W . L . M a l l m a n n f o r h i s e n c o u r a g e m e n t a n d a d v i c e
th ro u g h o u t this

study.

TABLE

OF

CONTENTS

Page
I.
II.
III.

IV.

V.

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

1

REV IEW

6

QF

L I T E R A T U R E ...........................................

M A TERIA LS

AND

E Q U I P M E N T .............................

16

A.

C ulture

M e d i a .................................................................

16

B.

D i s i n f e c t a n t s .................................................................

19

C.

B acterial

21

C u l t u r e ..........................................................
P R O C E D U R E S .............................

22

A.

M a i n t e n a n c e of C u l t u r e ...........................................

22

B.

"Phenol

T e s t s .............................

22

c u l t u r e s ....................................

22

EX PER IM EN TA L

C oefficient"

1.

U sing b roth

2.

U sing w ashed c u ltu res

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

23

C.

"S em i-m icro"

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

24

D.

G r o w t h C u r v e S t u d i e s ...........................................

25

E.

N e u t r a l i z a t i o n T e s t s ..........................................

26

T ests

R E S U L T S ......................................................................................
A.

27

E f f e c t of C u l t u r e M e d i a on th e R e s i s t a n c e
of h i . p y o g e n e s v a r .

a u r e u s .............................

27

iv
Page
B.

E f f e c t of S u b c u ltu r e

M e d i a o n the

C u ltiv a tio n of D i s i n f e c t a n t - t r e a t e d C e lls
of M . p y o g e n e s v a r .
C.

E ffect

of W a s h e d C u l t u r e s

R esistance
as
D.

of F D A S u b c u l t u r e

V II .

50

E ffect

of A g e

on the

C u l t i v a t i o n of D i s i n f e c t a n t - t r e a t e d

E ffect

of C u l t u r e

M . pyogenes var.

VI.

aureus

C o m p a r e d to B r o t h C u l t u r e s ..............

var.

M edium

aureus

.

.

LITERATU RE

.

57

aureus

.

60

of

to P h e n o l

N e u t r a l i z a t i o n T e s t s ..........................................

SUMMARY

.

M e d ia on B a c t e r i a l

P o p u l a t i o n a n d on th e R e s i s t a n c e

F.

41

on the

o f M_. p y o g e n e s v a r .

C e l l s of M_. p y o g e n e s
E.

a u r e u s .....................

.

.

68

......................................................................................
C I T E D ................................................

74

71

I.

There

INTRO D U CTIO N

are num erous

to b e d i s i n f e c t a n t s , m a n y
order

c o m p o u n d s on the m a r k e t p u r p o r t e d

of w h i c h h a v e q u e s t i o n a b l e v a l u e .

to p r o t e c t th e b u y e r , i t i s n e c e s s a r y t h a t r e g u l a t o r y

agen cies have m eth o d s

of m e a s u r in g

t h e d i s i n f e c t i n g v a l u e of

e a ch and o b tain data th at will r e p r e s e n t t h e ir
The FD A
the

''p h en o l c o e ffic ie n t"

relative

procedure

values.

(3 1 ) h a s b e e n

s ta n d a r d u s e d by r e g u l a to r y a g e n c ie s f o r m a n y y e a r s .

test was

o r i g i n a l l y d e s i g n e d to m e a s u r e

cresylic

type

com pounds.

w h e n a p p l i e d to the
this

In

procedure

relative

U ndoubtedly th is te s t is

e v a l u a t i o n of c o a l t a r

a phenol coefficient is
of w o r t h l e s s

f a c t u r e r in m a in ta in in g

and as

compound,

of p r e v e n t i n g

a n a id to the m a n u ­

t h e u n i f o r m i t y of h i s

The p rese n t FDA p rocedure m ay

reliable

T i l l e y (35) s t a t e d t h a t

of v a lu e o n l y a s a m e a n s

preparations

of

evaluation is

a quaternary am m onium

sodium hypochlorite o r a ch loro-phenol.

values

com pounds; how ever,

fails w oefully w hen a c o m p a ra tiv e

a t t e m p t e d of a m e r c u r i a l ,

the u s e

the

This

product.

give a r e la tiv e

rating

of t h e k i l l i n g d i l u t i o n of a d i s i n f e c t a n t w i t h a s t a n d a r d i z e d t e s t

culture.

In th is way,

compounds

can be c la s s if ie d into g ro u p s

of l o w a n d h i g h k i l l i n g d i l u t i o n s .
W o r k e r s in th e f i e l d of d i s i n f e c t a n t t e s t i n g
aw are

of the d i s c r e p a n c i e s

t h e e f f i c i e n c y of v a r i o u s
there

is no

evaluate

single

that a re

w ell

obtained in testing

c o m p o u n d s in the l a b o r a t o r y .

reliable

disinfectants

in r e s u lts

are

m ethod, it is v e ry

on a c o m p a r a t i v e

Since

d i f f i c u l t to

basis.

I n a d d i t i o n to th e l i m i t e d a p p l i c a t i o n t h a t m a y be m a d e
of a n F D A p h e n o l c o e f f i c i e n t t h e p r o c e d u r e

has been criticized

f o r the f a c t t h a t the u s u a l t e s t o r g a n i s m s f r e q u e n t l y v a r y in
their

resistan ce

to p h e n o l a s

w ell a s

to t h e

compound under

test.
Standards

h a v e b e e n e s t a b l i s h e d by t h e U n i t e d S t a t e s F o o d

a n d D r u g A d m i n i s t r a t i o n (3 1) f o r t h e
organism
ever,

resistan ce

of th e t e s t

to b e u s e d i n r u n n i n g p h e n o l c o e f f i c i e n t t e s t s .

difficulties

sm ooth culture

are

of th e

How­

o f te n e n c o u n t e r e d in t r y i n g to m a i n t a i n a
required

resistance.

C o m p o s i t i o n of t h e

cu ltu re m e d ia has an im p o rta n t influence on this.

N e w l o t s of

peptone and b eef e x tr a c t can c a u se

the a p p e a r a n c e

of r o u g h

strains

(21).

w ith s u b s ta n d a rd r e s is ta n c e

The hyd ro g en ion

c o n c e n t r a t i o n of th e
fluence

culture

m edium

also has

an im portant in­

(26).
S em isynthetic m ed ia have been proposed by K larm ann

a n d W r i g h t (14 ) a n d b y W o l f (3 8 ) t o m a i n t a i n a s t a n d a r d r e ­
sistance

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

and B u rlin g a m e
m edia

pyogenes var. a u re u s.

(2 9) i n t h e i r

c o n c lu d e d t h a t the

studies

resistan ce

to p h e n o l a n d L i q u o r A n t i s e p t i s
f a c to r ily in the

presented

show th at the

Wood

two p u r p o s e d

of M . p y o g e n e s v a r . a u r e u s

was not m aintained as

sem isynthetic m ed ia as

D ata w ere
(19 ) t o

on th e s e

R eddish,

satis­

in FD A b ro th .

by M a llm a n n , L e a v itt and Jo sly n

resistan ce

of M , p y o g e n e s v a r . a u r e u s

to

q uaternary am m onium

co m p o u n d s w as i n c r e a s e d by grow ing the

te s t c u ltu r e s in Difco

D isinfectant T esting m ed iu m

to t h a t r e s u l t i n g f r o m

cultivation in sta n d a rd F D A b roth.

The

w riter

sista n t culture

has

com pared

had difficulty in keeping a s m o o th r e - !

of M . p y o g e n e s v a r . a u r e u s w h e n g r o w i n g i t i n

sta n d a rd FD A broth.

A two p e r c e n t t r y p t i c a s e

by O s tro le n k

c a rry in g his

(20) f o r

p r e s e n t a t i o n of t h i s p a p e r a t t h e

test cultures,

broth was used
and in the

1 9 4 9 m e e t i n g o f t h e S o c i e t y of

A m e ric an B acterio lo g ists, m ention was m ade
sm ooth culture

as

of t h e f a c t t h a t a

could be m a in ta in e d in th is m e d iu m .

4
In o r d e r
pyogenes v ar.
broth

to s e e i f a s m o o t h

aureus

(B altim ore

of t h e

the

could be m a in ta in e d ,

B iological L a b o ra to rie s )

c u l t u r e m e d i a to b e u s e d i n t h i s

T e s t (DT) m e d i u m
to

show the

and

dehydrated form ,

study.

thus

S o y (T S )

s e le c te d a s one
D isinfectant
se le c te d also

e f f e c t of d i f f e r e n t c u l t u r e m e d i a u p o n

T h e f i r s t two m e d i a
ensuring

be u s e d t h r o u g h o u t t h e

course

to r e p r e s e n t a t i v e

c o u l d be o b t a i n e d i n a

th at a u n i f o r m lot of e a c h could
of th e

experim ents.

O n e l o t of

w a s u s e d in the p r e p a r a t i o n of the F D A c u l t u r e m e d i u m .
In o r d e r

upon the killing

to s h o w t h e i n f l u e n c e of t h e
dilutions,

cific n e u tr a liz in g m e d i u m
The

standard

(15) w e r e

used.

The

FD A broth,

"sem i-m icro
"w hole

(24).

DT m e d i u m

and a sp e­

a n d a m o d i f i c a t i o n of
whole

sam ple"

procedure

sam p le"1 m ethod has been c riti­

c i z e d by R a h n a s b e in g too s e n s i t i v e
coefficient technic

subculture m ed ia

w e r e in c lu d e d in the stu d y .

FDA procedure

K la rm a n n and W rig h t's

results

was

of M . p y o g e n e s v a r . a u r e u s

type d i s i n f e c t a n t s .

of M.

T rypticase

standard FDA bro th w ere

com parative

resistan ce

peptone

r e s is ta n t culture

It w a s

as

c o m p a r e d w ith the phenol

d e s ir e d in this

o b t a i n e d by the two m e t h o d s .

s tu d y to c o m p a r e

The

purpose

fe re n t culture
aureus
the

of t h i s

m e d i a upon the

to r e p r e s e n t a t i v e

cells.

s h o w t h e e f f e c t of d i f

resistan ce

of M . p y o g e n e s v a r

type d isin fec tan ts

s u b c u l t u r e m e d i a on . t h e

treated

s tu d y i s to

a n d t h e e f f e c t of

c u ltiv a tio n of the

disinfectant-

II.

Soon a fte r
lished,

REVIEW

the g e r m

efforts w e re m ade

infectants

on b a c te ria .

no a t t e m p t w a s m a d e

t h e o r y of d i s e a s e
tow ard

These

had been

e v a lu a tin g the

the f i r s t to r e a l i z e

and
b asis.

th a t the r e la tiv e
conditions

tested.

In 1903, R i d e a l a n d W a l k e r
testing

crude,

on a q u a n tita tiv e

of d i s i n f e c t a n t s d e p e n d s v e r y l a r g e l y u p o n t h e

un d er w hich they a r e

estab­

a c tio n of d i s ­

e a rly m ethods w ere

at determ in atio n s

K r o n i g a n d P a u l (18) w e r e
value

OF LITER A TU RE

(30) p r o p o s e d a m e t h o d o f

d i s i n f e c t a n t s w h e r e b y t h e g e r m i c i d a l a c t i o n of t h e d i s ­

infectant w as
A

c o m p a r e d w i t h t h a t of p h e n o l .

"phenol

coefficient"

test,

known as the H ygienic L a b ­

o r a t o r y m e th o d , w a s p r o p o s e d by A n d e r s o n and M cC lin tic in
1911

(1).
W o r k e r s i n the U n i t e d S t a t e s D e p a r t m e n t of A g r i c u l t u r e

L aboratories,

a fte r w orking for ten y e a r s

the R id e a l- W a lk e r

with the m e th o d s

and the H ygienic L a b o r a t o r y

tests, proposed

a t e s t w h i c h i n c o r p o r a t e d t h e b e s t f e a t u r e s of b o t h ( 2 7 ).
procedure,

known as the F D A m e th o d

today in te stin g the

(3 1) i s

of

This

com m only used

e ffic ie n c y of d is in f e c ta n ts in the l a b o r a t o r y .

7
In. t h i s t e s t ,

a phenol coefficient fo r a disin fectan t is

by c o m p arin g its
specific

killing

p o w e r w i t h t h a t of p h e n o l a g a i n s t a

organism .
W r i g h t (39 ) p u b l i s h e d

the d i s c o r d a n t r e s u l t s
to

determ ined

slight v a ria tio n s

som e

w o r k to

obtained in testing

show th a t m o s t of

disinfectants

in the t e s t c u ltu r e m e d i a , w hile the

d iffe re n c e s in su b cu ltu re m e d ia w e re not im p o rta n t.
th at this w as
organism .

of the n u m b e r

(9) a l s o

m edium

of o r g a n i s m s

He th o u g h t

u s e d in the t e s t .
s t a n d a r d s f o r the r e s i s t a n c e

pyogenes var, aureus

properties
are

sam e

c a l l e d a t t e n t i o n to t h e i m p o r t a n c e

R e d d i s h (2 6 ) s u g g e s t e d

nutritive

due

c o n n e c t e d w i t h t h e a m o u n t of g r o w t h o f t h e t e s t

G arrod

M icrococcus

were

to p h e n o l .

He

of

s t a t e d t h a t the

a n d the h y d r o g e n io n c o n c e n t r a t i o n of the

i m p o r t a n t f a c t o r s in p r e s e r v i n g the r e s i s t a n c e

of

the o rg a n is m s .
A g r e a t d e a l of w o r k h a s b e e n d o n e o n t h e
various

peptones

on the

c o e ffic ie n t'1 te s ts .
resistan ce

resistance

of o r g a n i s m s

R ed d ish and B u rlin g am e

e f f e c t of

u s e d in

''p h en o l

(28) s t a t e d t h a t t h e

of M . p y o g e n e s v a r . a u r e u s w a s m a i n t a i n e d c o n s i s t e n t l y

when bro th m ade fro m
o th er peptones

w ere

A rm o u r's

used,

peptone w as u sed ,

resistance

w hereas,

of t h e o r g a n i s m

was

when

reduced

i

8
to a s i g n i f i c a n t d e g r e e .
"phenol coefficient"

B rew er

(4) t h o u g h t t h a t v a r i a t i o n s i n

resu lts w ere

phospholipids in som e

due to the p r e s e n c e

l o ts of p e p t o n e .

Q uisno, F o te r

of
and

G i b b y (21) n o t e d t h a t n e w l o t s o f p e p t o n e o r b e e f e x t r a c t s o m e ­
tim es

c a u se d the a p p e a r a n c e

phenol re sista n c e .
trypticase

was

the r e s i s t a n c e

of r o u g h

strains

w ith s u b s ta n d a rd

I t w a s n o t e d b y S t u a r t (34) t h a t w h e n B B L

u s e d i n F D A b r o t h i n s t e a d of A r m o u r p e p t o n e ,
o f th e

organism

varied.

K l a r m a n n a n d W r i g h t ( 1 4 ) a n d W o l f (38) p r o p o s e d
synthetic m e d ia for grow ing M. pyogenes v ar.
to o b t a i n a s t a n d a r d - r e s i s t a n t c u l t u r e .
B urlingam e

R eddish,

in o rd e r

Wood and

(29) p u b l i s h e d d a t a to s h o w t h a t c u l t u r e s

in F D A b ro th a r e m o r e
of t h e t w o

aureus

sem i­

grow n

sa tis fa c to ry than those grow n in e ith e r

sem isynthetic m edia.

O ther fa c to rs b e sid e s m e d ia com p o sitio n have been
s t u d i e d i n a n a t t e m p t to m a i n t a i n a s m o o t h c u l t u r e of u n i f o r m
resistance for

disinfectant testing.

found th a t if M . p y o g e n e s v a r .
s t e a d of a t 3 7 C t h e
tran sfers

Grubb and E d w ard s

a u r e u s w a s i n c u b a t e d a t 40 C i n ­

standard resista n c e

at this te m p e ra tu re ,

(11)

retu rn ed after

one to two

and th a t it w as m a i n t a i n e d a s long

I

as daily t r a n s f e r s

w ere

e v a l u a t i o n of s o m e
ance

i n c u b a t e d a t 40

of t h e f a c t o r s

of M . p y o g en es v a r .

e n c e in the age of the

the a g e

of th e

of t h e

influencing the phenol r e s i s t ­

culture or a o n e -d e g re e

culture.

difference

in

c a u s e d a n i m p o r t a n t d i f f e r e n c e i n th e
It i s i n t e r e s t i n g to n o t e

c u l t u r e u p o n the

ence in the age

S m y t h (33), i n h i s

a u r e u s , noted that a tw o -h o u r d iffe r­

the in c u b a to r t e m p e r a t u r e
resistan ce

C.

resistan ce.

t h e e f f e c t of

A four-hour

differ­

of the t e s t c u l t u r e i s a l l o w e d by th e F D A p r o ­

cedure.'
S o m e a t t e n t i o n h a s b e e n p a i d to t h e v a r i a t i o n i n t h e
a m o u n t of m a t e r i a l r e m o v e d b y t h e 4 - m m

l o o p o w i n g to t h e

different surface

types

( 5).

H ow ever,

num ber

of c e l l s

ten sio n values

of v a r i o u s

of d i s i n f e c t a n t s

R a h n (24 ) b e l i e v e d t h a t t h e f l u c t u a t i o n i n t h e
was

so

s m a l l th a t it could not in flu en ce the

i

results,

as

long a s

stan d ard m ethods

w e r e follow ed.

W ith so m a n y f a c t o r s in flu e n c in g
o r g a n is m , it is
tained.

rem arkable

The author has

the r e s i s t a n c e

that consistent re s u lts

The

e v e r ob­

c o n sisten tly had trouble in m aintaining

a s m o o th r e s i s t a n t c u ltu r e w hen the o r g a n i s m s
in FD A b ro th .

are

of a t e s t

Difco L a b o r a t o r i e s

w ere

cultured

developed a m e d iu m

(D isinfectant T esting m ed iu m ) w hich they claim gives a culture

10
w ith co n stan t r e s is ta n c e .
prepared

a m edium

T he B a ltim o re B iological L a b o ra to ry

( T ry p tic a s e Soy broth) w hich they c la im

w ill m a in ta in a sm o o th cu ltu re

for a co n sid erab le

p e r i o d of

tim e.
T h e a u th o r d e c id e d to c o m p a r e the a b o v e - m e n t i o n e d
t h r e e m e d i a to d e t e r m i n e
a culture

their

re la tiv e v a lu e s in m a in ta in in g

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

It i s

known th a t so m e

bacterio static

disinfectants have

activity that slight t r a c e s

of d i s i n f e c t a n t c a r r i e d

o v e r m a y p r e v e n t g r o w t h of t h e t e s t o r g a n i s m s
them .
m ote

R a h n (24) s t a t e d t h a t t h i s d a n g e r
w hen the F D A

In t h i s p r o c e d u r e ,
from

w ithout killing

seem ed rather

re­

''p h en o l c o e f f ic ie n t'1 tec h n ic w as follow ed.

one

4 m l lo o p fu l (0.01 m l ) i s t r a n s f e r r e d

the m e d i c a t i o n tu b e to

1000 d i l u t i o n of th e

such great

10 m l o f b r o t h ,

disinfectant.

produces bacterio stasis

in

No

r e s u ltin g in a

disinfectant is

1-

known w hich

l/lOOO of th e l o w e s t b a c t e r i c i d a l

con­

centration.
R a h n (25) s t a t e d t h a t s t e r i l i z a t i o n of b a c t e r i a i s b r o u g h t
a b o u t by a c h e m i c a l r e a c t i o n b e tw e e n the d i s i n f e c t a n t and
v i t a l p a r t of th e c e l l .
al steps,

This

som e

r e a c tio n m a y take p la c e in s e v e r ­

the f i r s t of w hich m a y be

reversible.

A fte r a definite

«

11
p e r i o d of c o n t a c t w i t h t h e d i s i n f e c t a n t ,

all c e lls

contain a

c e r t a i n a m o u n t of t h e d i s i n f e c t a n t , b u t a f a t a l r e a c t i o n m a y
not have b een p ro d u ced .
cause
the

T r a n s f e r into f r e s h b ro th m a y not

th e d i s i n f e c t a n t to d iffu s e q u ic k ly o ut of the

c e ll m a y die in th e n ew m e d i u m

at the tim e

of t r a n s f e r . -

ism

w h ich w ould be

For

this

the u se

of a n a n t i ­

w o u l d s a v e t h e l i f e of t h e

c o n s id e r e d dead by the

re a so n a n eutralizing m edium ,

b ro th and DT m ed iu m , w as u s e d fo r

and

although it w as not dead

In s u c h a c a s e ,

dote o r n e u tr a liz in g m e d i u m

cell,

organ­

standard technic.

as w ell as S pecial FD A

s u b c u lt u r i n g the t e s t o r ­

ganism .
Jam es
tralizer:

(13) l i s t e d t h e

(1) i t d e s t r o y s

specifications for an ideal neu -

a l l t h e g e r m i c i d a l e f f e c t s to b e

e x e r te d by d is in fe c ta n t r e m a in in g in
from

unkilled b a c te ria l

ing in flu e n c e s ,
e f f e c t a n d (4)

cells

solution,

(2) i t r e m o v e s

all the d is in fe c ta n t and r e s t r a i n ­

(3) i t i n t r o d u c e s no g e r m i c i d a l o r b a c t e r i o s t a t i c
it

resuspends

unkilled

b acteria

in

fluid su b ­

strate .
Gegenbauer

(1 0 ) f o u n d t h a t s t a p h y l o c o c c i t h a t h a d b e e n

tre a te d w ith m e r c u r i c

chloride

w a s h in g , p r o v i d e d t h a t the

could be re v iv e d by r e p e a te d

c o n ta c t w ith the d isin fe c ta n t had

f

12
been less
save the
the

than one h o u r.
cells,

as

the c e ll.
duction,

w ashing

could not

the m e r c u r y io n s had r e a c te d with s o m e

cell co n stitu e n ts.

chem ical

A fter that tim e,

sulfide

r e v e r s e d this

re a c tio n by fo rm in g in so lu b le m e r c u r i c

sulfide w ithin

The
was

H ow ever, hydrogen

reversion,

w h i c h r e s t o r e d t h e p o w e r of r e p r o ­

still p o ssib le

after

sixteen, but not th ir ty - th r e e ,

hours,

if the d i s i n f e c t a n t c o n ta in e d one p e r c e n t m e r c u r i c

ride.

F ild es

(7) a l s o u s e d h y d r o g e n s u l f i d e f o r t h e

i n a c t i v a t i o n of t h e m e r c u r y i o n .
containing

There

are m any

com p o u n d s w hich can n e u tr a liz e

a c t i v i t y of m e r c u r i a l s .
Salle,

of

M e ta p h e n in this

chem ical

sulfur-

the b a c te r i o s ta t ic

Sodium th io g ly co llate w as u s e d by

C atlin and W estley

glycollate b ro th w as

chlo­

(32) a n d b y H e i n e m a n n ( 1 2 ).

selected as

study b e c a u s e

T hio­

the n e u tr a liz in g m e d iu m fo r
of i ts

availability in a po w d ered

form .
W ith p h e n o l i t h a d n o t b e e n t h o u g h t n e c e s s a r y to u s e
neutralizing
conducted

a g e n t in the

subculture m ed iu m .

studies in w hich they u se d f e r r ic

c h e m ic a l in a c tiv a to r fo r phenol.
d i l u t i o n of p h e n o l w a s
to t h e

chloride

(8)

as a

T h e y found th a t the killing

low ered when fe rric

subculture m ed iu m ,

F l e t t et al.

a

chloride w as added

T i l l e y (36) w a s u n a b l e to d u p l i c a t e

13
these

resu lts.

He found no d iffe re n c e w ith f e r r i c

a d d e d to t h e F D A b r o t h a s
In this
ride

chloride

c o m p a r e d with FD A b ro th alone.

s t u d y , i t w a s d e c i d e d to c h e c k t h e u s e of f e r r i c

as a n in a c tiv a to r for the p henolic

compounds,

chlo­

D o w i c i d e A.

a n d D o w i c i d e C.
M any

substances have been

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

am m onium

suggested for

com pounds.

the i n a c t i v a ­

D o m a g k (6) w a s t h e

f i r s t t o c a l l a t t e n t i o n to t h e f a c t t h a t a n i o n i c d e t e r g e n t s
inactivate
H arrison

the

surface-active

and M iller

d etergents

cationic

compounds.

(3) s h o w e d t h a t c e r t a i n

B aker,

s y n th e tic anionic

a ls o in a c t iv a t e d the g e r m i c i d a l p r o p e r t i e s

com pounds.

will

K l e i n a n d K a r d o n (1 6) s h o w e d t h a t

of c a t i o n i c

1' r e v e r s a l 1' of

q uaternary

a m m o n i u m a c t i v i t y c o u ld n o t be p r o d u c e d o n c e the

organism s

h a d b e e n e x p o s e d to g e r m i c i d a l c o n c e n t r a t i o n s of

the co m p o u n d s.

T h e y fo u n d t h a t t h e a d d it io n of a n a n io n ic

d e t e r g e n t to the t r e a t e d b a c t e r i a l
a c t i o n of t h e
organism

s u s p e n s i o n s i n t e r r u p t e d the

cationic d isin fectan ts,

thereby p erm itting

surviving

t o gr ow ..

W e b e r a n d B l a c k ( 37 ) t e s t e d l e c i t h i n , n a p h u r i d e
D u p o n a l W. A.,
sodium oleate,

T e rg ito l W. A .

7, T r i t o n x 2 0 0 ,

so d iu m thiosulfate,

am ong other

sodium ,

T r ito n x300,
com pounds.

They

1

14
found th at le c ith in and n a p h u rid e
the in h ib ito rs
a m ixed

tested that

s o d i u m w e r e t h e o n l y o n e s of

s h o w e d no b a c t e r i o s t a t i c

effect a g a in s t

culture.

F avorable

resu lts

o n t h e u s e of l e c i t h i n a l o n e o r i n

com bination w ith other

s u b s t a n c e s f o r t h e i n a c t i v a t i o n of c a t i o n i c

disinfectants

r e p o r t e d a lso by Q uisno, G ibby and F o te r

have been

(2 3) , A r m b r u s t e r
and F o te r
as

the

and R id e n o u r

(2) a n d o t h e r s .

Q uisno,

Gibby

(2 3 ) f o r m u l a t e d L e t h e e n b r o t h i n w h i c h l e c i t h i n a c t s

chief n e u t r a l i z e r

and d isp e rsin g

while

agent for

T w e e n 80 a c t s

the le c ith in .

The

as a solubilizing

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

l e c i t h i n a n d T w e e n 80 u s e d d i d n o t i n h i b i t t h e g r o w t h of a n y
organism

th a t w ould g ro w

in F D A b r o th .

Therefore

b ro th w as p ick ed as the in activ atin g m e d iu m
testing

q u a te rn ary am m onium
There

has

sam pling technic
w orkers

’’L e t h e e n "

to b e u s e d f o r

c o m p o u n d s in this

study.

b e e n a g r e a t d e a l of d i s c u s s i o n a b o u t t h e
of t h e

"phenol coefficient"

procedure.

(15) h a v e f e l t t h a t i n t h i s m e t h o d of " r a n d o m

a representative

sam ple

not rem o v ed for

subculturing.

w h ic h the w h o le m i x t u r e
(2 4) f e e l t h a t t h i s

"w hole

Some
sam p lin g "

of t h e d i s i n f e c t a n t - c u l t u r e m i x t u r e

is

M ethods

c u l t u r e d (15 ,

sam ple"

is

h a v e b e e n p r o p o s e d in
22).

Some w orkers

m e t h o d i s too

sensitive

a

c

15
test as
the

c o m p a r e d to t h e

relative

coefficient"
technic

value

standard FDA procedure.

of t h i s t e c h n i c

procedure,

as

To d e te r m in e

c o m p a r e d to t h e

a s lig h t m o d i f i c a t i o n in the

''p h en o l

"sem i-m icro "

of K l a r m a n n a n d W rig h t w a s u s e d along w ith the F D A

procedure.

«

III.

M A T E R IA L S AND E Q U IP M E N T

A.

C ulture

M edia

The follow ing m e d ia w e r e u s e d fo r daily t r a n s f e r s
M . pyogenes var.
organism s
1.

aureus.

grow n in these

Subsequent tests

w ere

ru n upon the

th ree m edia.

FDA broth
A r m o u r peptonum

siccu m ,

Lot No.

100976

1 0 .0 g m

Bacto b e e f e x tra c t

3.0 g m

Sodium

5.0 g m

chloride

D istilled w ater
(pH w a s a d j u s t e d to 6 . 8 to 7.Q)
2.

of

Difco D is in f e c ta n t T e s t m e d i u m
dium ), L ot No. 380974
P ro teo se

peptone

1000 m l

(DT m e ­

10.0 g m

Bacto beef e x tra c t

3.0 g m

Sodium

2.0 g m

chloride

L actose
A scorbic

5.0 g m
acid

D istilled w ater
(final pH w a s

0.025 g m
1000 m l
7 .0 ± )

17
3.

T r y p t i c a s e Soy b r o t h (TS b r o t h ) , L o t No.
2 75 1 ( B a l t i m o r e B i o l o g i c a l L a b o r a t o r i e s )
T rypticase

17 .0 g m

Phytone

3.0 g m

Sodium chlo rid e

5.0 g m

D ip o tassiu m phosphate

2 .5 g m

D extrose

2.5 g m

D istilled w a te r
(final p H w a s

10 00 m l
7.2-7.4)

The follow ing m e d i a w e r e u s e d a s
'phenol c o e f f i c i e n t '' and
1.

"sem i-m icro "

subculture m e d ia for

tests.

Special F D A b ro th
D ifco p ep to n e

10.0 g m

B acto beef e x tra c t

3.0 g m

Sodium

5.0 g m

chloride .

D istilled w a te r
( p H a d j u s t e d t o 6 . 8 to 7 .0 )
2.

Difco D is in fe c ta n t T e s t m e d iu m
F orm ula

3.

1000 m l

the

sam e

as given in A

L etheen broth
P art A
T w e e n 80 ( A t la s P o w d e r

Co.)

5.0 g m

I

18
A zolectin (A m erican L ecithin

Co.)

0.7 g m

Hot distilled w a te r

400 m l

Part B
Beef extract

5..0 g m

Sodium ch lo rid e

5 .0 g m

A r m o u r peptonum
100976

s ic c u m , L o t No.
10.0 g m

D istilled w a te r

600 m l

M ix p a r t s A a n d B and b o il fo r te n
m inute s .
(p H a d j u s t e d t o 6.8 t o 7 . 0 )
4.

Difco B r e w e r ' s

thioglycoliate m e d iu m

B eef, infusion f ro m
Sodium

50 0

chloride

5 .0 g m

D ip o tassiu m phosphate

2.0 g m

P ro teo se

1.0 g m

peptone

D extrose

5 .0 g m

Sodium thioglycoliate

0 .5 g m

B acto-agar

0 .5 g m

B a c t o - m e t h y l e n e blue

0.002 g m

D istilled w ater
5.

gm

S p ecial F D A b r o th plus

1000 m l
0.03%

ferric

chloride

19
T h e follow ing m e d i a w e r e u s e d in S e c tio n E u n d e r R e ­
s u lts , f o r the

s tu d ie s on g r o w th c u r v e s

pyogenes var. aureus

using

No.

and resistan ce

of M.

to p h e n o l .

1.

Difco D is in f e c ta n t T e s t m e d i u m ,

L o t No.- 3 65 85 3

Z.

Difco

L o t No. 380974

3.

D isinfectant T e s t m edium , m ade

D isinfectant T e st m ed iu m ,

Difco P e p t o n e ,

Lot No.

in the l a b o r a t o r y

406935

4.

FDA

broth m ade

with Difco p e p to n e , L o t No. 406935

5.

FDA

broth m ade

w ith D ifco p e p to n e , L o t N o . 40593 4

6.

FDA

bro th m ad e

with A r m o u r p e p to n u m

siccum ,

Lot

100976
7.

T rypticase

Soy b r o th , L o t N o. 3639 ( B a ltim o r e

B iological L a b o ra to rie s)
T he follow ing S o r e n s e n 's b u ffe r, pH

7.0, w a s u s e d f o r

the w a s h e d c u ltu r e s
6.0 m l N a H P O
(11.876 g m N a H P O
• H O in one
Z
4
Z
4
Z

1 of

solution)
4.0 m l KH P O
(9.078 g m
Z
4
Tryptone

glucose

KH P O
in one
Z
4

1 of solution)

e x tra c t a g a r w as u se d for

a ll plating

purpose s .

I

B.

The
1.

d i s i n f e c t a n t s u s e d in the
D ow icide A F la k e ,

sodium -o-phenylphenate
f a c tu r e d by the
2.
adding

D ow icide

d i r e c t e d by the
product.
active

Dow C h e m ic a l

D ow icide

study w ere:

which is

as the

c o m p o s e d of 97 p e r c e n t

active in g re d ien t.

31 w a s

It is m a n u ­

Com pany..

C, w h i c h w a s m a d e

s o d iu m hydroxide..

soluble,

D isinfectants

In o r d e r

from

to m a k e

c o n v e r t e d into

the

D ow icide
the

compound w ater

sodium

salt as

Dow C h e m i c a l C o m p a n y , m a n u f a c t u r e r s

It c o n ta i n s

31 b y

of th is

85 p e r c e n t c h l o r o - o - p h e n y l p h e n o l a s

the

ingredient.
3.

m ercuri-2

M etaphen,

an anhydride

of 4 n i t r o - 3 , 5 - b i a c e t o x y ft
c r e s o l , w h ic h is m a n u f a c t u r e d by A bbot P h a r m a c e u ­

tical Com pany.
4.

B T C , w h i c h i s c o m p o s e d of

benzyl am m onium
alkyl ra d ic a l

chloride

ranging f r o m

O n y x Oil an d C h e m i c a l
5.
tained fro m

as

10 p e r c e n t a l k y l - d i m e t h y l -

the a c tiv e i n g r e d i e n t w ith th e

C8 to C18.

It is m a n u f a c t u r e d by

Company.

P h e n o l (B a k e r and A d a m s o n Q uality),
the A llied

C h e m ic a l and Dye

p e r c e n t solution w as m ad e

and

which w a s

C orporation.

A

ob­
5-

s t o r e d i n a d a r k b o t t l e a t 4 C.

21
C.

The te st o rg an ism
as

B acterial

C ulture

was M. pyogenes var.

a u r e u s N o . 209,

a p p r o v e d by the F o o d an d D r u g A d m i n i s t r a t i o n .

I

IV.

EX PER IM EN TA L PROCEDURES

A.

The
agar

M aintenance

stock b a c te ria l

slants.
The

of C u l t u r e

culture w as

Stock su b c u ltu re s w e re

kept on plain n u trie n t

m ade

e v e ry four w eeks.

c u lt u r e s u s e d in the e x p e r i m e n t s w e r e

only a f te r a m in i m u m

of f o u r d a i l y s u b t r a n s f e r s
the

of u s e , the t e s t o r g a n i s m

w as once again taken fro m

stock cu ltu re.

test organism
aureus.

m edium

w as a 22-26 hour

The ex p erim en ts

of, a t l e a s t ,
before

C ultures

three

the te s t w a s

w ere

w ere
culture

slant.

in the p r o p e r

bro th after tra n s fe r fro m

old a g a r

stock a g a r

em ployed

A fter four w eeks
the m o n th -

i n c u b a t e d a t 3 7 C.

The

of M . . p y o g e n e s v a r .

conducted using pooled c u ltu res

t u b e s of the a p p r o p r i a t e b r o t h c u l t u r e s .

Just

s t a r t e d t h e p H of t h e c u l t u r e s g r o w n i n D T

a n d TS b r o t h w e r e

a d j u s t e d to 6 . 8 - 7 , 2 , u s i n g

a Beckm an

potentiom eter.

B.

1,

" P h e n o l C oefficient"

U sing b ro th c u l t u r e s .

by R uehle and B r e w e r

(31).

T ests

The technic u se d is

This tech n ic is known as

described
the F D A

23
m ethod.

In o r d e r

to h a v e

com parable

results,

dilutions

th e d i s i n f e c t a n t to be t e s t e d w e r e m a d e up in s t e r i l e
E rle n m e y e r flasks

and 5 -m l am o u n ts

p i p e t t e d i n t o e a c h of t h r e e

sterile

One test w as

s u b c u ltu re m e d iu m ; in the

250-m l

of e a c h d i lu t io n w e r e t h e n

seeding pots.

seeding pots w e r e p r e p a r e d fo r th re e
at one tim e .

of

In t h i s m a n n e r ,

1' p h e n o l c o e f f i c i e n t ' ' t e s t s

r u n u s in g S p e c ia l F D A b r o th a s the
second test,

DT m e d iu m w as

used for

s u b c u ltu rin g a n d in the t h i r d t e s t a n e u tr a li z in g m e d i u m w a s
ployed.

The

s a m e pooled cu ltu re

w as u se d in all th re e

em ­

tests.

S e e d i n g p o t s w e r e h e l d i n a w a t e r b a t h a t 20 C f o r t h e tes t. .
A fte r the te s t s

w ere

organism

plated.

w ere

com pleted,

suitable

T h e s e p la te s w e r e in c u b a te d a t 37 C

f o r 48 h o u r s

an d then the

m ade

' ' p h e n o l c o e f f i c i e n t 1' t e s t s

on the

colonies w e re

and 6 d a y s ' in c u b a tio n a t 3 7 C.
as the final r e s u l t .

var. aureus,

The

counted.
after

six -d ay

The killing dilution w as

dilution w hich c a u s e d

2.

d i l u t i o n s of t h e t e s t

"k ill"

f o r 30 m i n u t e s
the o r g a n i s m s

old, w e r e

to one h o u r
w ere

2 4 , 48 ,

72 h o u r s '

read in g w as taken
reported as

that

in te n but not in five m i n u t e s . .

U sing w ash ed c u l t u r e s .
22-26 h o u rs

R eadings w ere

B r o t h c u l t u r e s of M . p y o g e n e s

c e n t r i f u g e d a t 1 80 0 R P M

d e p e n d in g u p o n the m e d i u m in w h ic h

grow ing.

T hirty m inutes

w ere

s u f f i c i e n t to

24
th ro w dow n the o r g a n i s m s

g ro w in g in D T m e d i u m

w h ile i t t o o k one h o u r to t h r o w
FDA broth.
ism s

The

s u s p e n d e d in

again w e re
ism s

down the

supernatant was

centrifuged,

the

pH 7.0.

three

coefficient"
m anner

as

tubes w ere

used as

tests

conducted and r e s u lts

w ere

w ith the b r o t h

cultures
and the o r g a n ­

sam ples

the t e s t o r g a n i s m .

of, a t

The

r e a d in the

"phenol
sam e

cultures.

C. " S e m i - m i c r o "

A

Pooled

g ro w n in

and the o r g a n ­

These

supernatant discarded,

s u s p e n d e d in b u ffer fo r the te s t.

least,

organism s

then d isc a rd e d ,

10 m l o f b u f f e r ,

and TS b r o th ,

T ests

s lig h t v a r i a t i o n in the te c h n ic

of the

"sem i-m icro "

m o d i f i c a t i o n b y K l a r m a n n a n d W r i g h t (15 ) w a s u s e d .
O n e m l of e a c h d i s i n f e c t a n t d i l u t i o n w a s p l a c e d i n e a c h
of t h r e e

large

test tubes.

Three

dilutions

of the d i s i n f e c t a n t to be t e s t e d w e r e u s e d .
vals

0.1 m l of a c u l t u r e

to e a c h t u b e a n d t h e t u b e
15 m i n u t e s '
broth,

o r the

seven

At 15-second in te r ­

o f M.. p y o g e n e s v a r . a u r e u s w a s a d d e d
was

sw irled.

e x p o s u r e to th e d i s i n f e c t a n t ,

DT m e d iu m

of p h e n o l a n d

A t t h e e n d of 5,

10 a n d

20 m l o f S p e c i a l F D A

ap p ro p riate neutralizing m ed iu m was

25
added.

Tubes

w e r e i n c u b a t e d a t 37 C a n d r e a d i n g s m a d e

24 , 4 8 ,

72 h o u r s

and 6 days.

The te s ts
p a r e d in the

at

w ere

sam e

c o n d u c t e d a t 20 C.

w ay a s th o se f o r the

C ultures

w ere

pre­

"phenol coefficient"

te s t s .

D.

G row th C urve

Studies

A s u s p e n s i o n of M i p y o g e n e s v a r . a u r e u s
taking

a s m a l l a m o u n t of g r o w t h f r o m

s la n t and putting it in
b r e a k up all the
suspension w as
of w h i c h a r e
propriate
These

saline.

clum ps.

six h o u r s '

m ade

seven m edia,

hour

the n a m e s

and E quipm ent.

Ap­

counts.

A t t h e e n d of

a n d 2 4 h o u r s ' i n c u b a t i o n a t 37 C, d i l u t i o n s w e r e
A fter

24 h o u r s ' i n c u b a t io n , one

lo o p fu l of c u l t u r e w a s t r a n s f e r r e d f r o m
into n e w b r o t h tu b e s

24 h o u r s .

l o o p fu l of t h i s

a n d p l a t e d of e a c h n e w c u l t u r e .

c o n s i d e r e d to be z e r o

again p lated.

dilutions

s h a k e n w e l l to

standard 4 -m m

g i v e n i n (C) u n d e r M a t e r i a l s

by

stock n u trie n t a g a r

T he tube w as

p l a c e d i n e a c h of t h e

dilutions w e re

w ere

One

the

was m ade

of t h e s e n e w
A fter

of the

sam e

standard 4 -m m

e a c h of t h e

k i n d of m e d i u m . .

cultures w ere

plated at zero ,

24 h o u r s ' i n c u b a t i o n , o n e

seven tubes
Proper
six and

s ta n d a r d loopful w as

I

i

26
transferred from
of th e

the

seven m edia.

24 h o u r s .

second tra n sfe r
Platings

A ll p lates

cultures

into a n e w s e t

again w e re m a d e

at zero,

six and

w e r e i n c u b a t e d a t 37 C a n d c o u n t e d a t t h e

e n d o f 48 h o u r s .

E.

N eutralization

M. pyogenes v a r. a u re u s,
a s the t e s t o r g a n i s m .
broth,

DT m e d iu m

The

an d the

T ests

g r o w n in DT m e d i u m ,

was used

su b c u ltu re m e d ia w e re Special FDA
s p e c if ic n e u t r a l i z i n g m e d i u m f o r the

d isin fe c ta n t being u sed .
O ne m l of e a c h d i s i n f e c t a n t d ilu tio n w a s p l a c e d i n a
large

t e s t tube.

p o u re d into

T w e n t y m l of t h e

each tube.

subculture

The m ix tu re

m e d iu m then was

of d i s i n f e c t a n t d i lu t i o n s

s u b c u l t u r e m e d i a w a s i n o c u l a t e d w i t h 0.1 m l o f t h e c u l t u r e .
am inations for grow th w ere m ad e
days'

after

24, 4 8 ,

72 h o u r s '

and
Ex­

and 6

i n c u b a t i o n a t 3 7 C.

I

V.

A.

E f f e c t of C u l t u r e M e d ia on the R e s i s t a n c e
M . pyogenes v a r ; a u re u s

FDA
w ere

RESULTS

"phenol c o e ffic ie n t'1 te s ts

ru n using a s

the t e s t o r g a n i s m ,

and

"sem i-m icro "

a n d (C) T S b r o t h .

subculture m ed iu m

used,

FDA b ro th was u se d as
while DT m e d i u m
the

D ow icide

the

C (sodium

disinfectants, w ere
The

results

E x a m i n a t i o n of t h e
compounds

shows

compound.

Special

su b c u ltu re m e d iu m in one test,
in o r d e r

to s e e if

com parative

resu lts.

D ow icide A ( s o d iu m -o - p h e n y l phenate),

c h l o r o - o - p h e n y l p h e n a t e , M e t a p h e n (4 n i t r o -

3, 5 - b i a c e t o x y - m e r c u r i - 2
b en zy l-am m o n iu m

r u n on e a c h

w ould a ffe c t the

compounds,

(B) D T

d iff e rin g o n ly in the

w as u s e d fo r the o th e r,

subculture m e d iu m
Four

Two te s ts ,

w ere

tests

M. pyogenes v a r. au reu s

w h i c h h a d b e e n g r o w n i n (A) s t a n d a r d F D A b r o t h ,
m edium ,

of

c re so l) and B T C

chloride) w hich w e re
u s e d in the

chosen as

com parative

obtained a re

(alkyl-dim ethyl-

study.

show n in T ables

"phenol coefficient"

representative

I-V III in c lu s iv e .

t e s t v a lu e s f o r the four

that lo w er phenol coefficient v a lu e s

ta in e d w h e n the t e s t o r g a n i s m

was

w ere

g r o w n i n DT m e d i u m ,

ob­

showing

28
a m ore

resistan t organism

FD A broth o r

than w h en it w as grow n in

standard

TS b r o t h .

A lth o u g h m u c h h a s b e e n s a id about the i m p o r t a n c e
th e a m o u n t of g r o w t h of th e t e s t c u l t u r e u p o n i t s
(3 9 ), t h e n u m b e r o f o r g a n i s m s

to be th e in flu e n c in g f a c t o r h e r e .

w ere m ade

of e a c h

case
was

a t the t i m e

B a c t e r i a l counts

of t e s t i n g a n d i n e v e r y

a h i g h e r b a c t e r i a l c o u n t w as o b ta in e d w h e n the
g r o w n i n TS b r o t h t h a n in e i t h e r F D A b r o t h o r

T a b l e IX l i s t s
cases

the

som e

culture

of t h e b a c t e r i a l

E x a m i n a t i o n of T a b l e s

show s th at the c u ltu re
th a n the one
phenolics.

DT m edium .
In m o s t

count t h a n the one
I-IV in c lu s iv e

g r o w n in F D A b r o th w a s m o r e

g r o w n i n TS b r o t h w h e n the c o m p o u n d s

resistant
tested w ere

,

W hen M etaphen and BTC w e re
would a p p e a r
or m ore

organism

counts o b tain e d .

g row n in FD A h ad a lo w er

g row n in DT m e d iu m .

draw

resistance

p r e s e n t in the t e s t c u lt u r e would

not appear

culture

of

that the

so than the

culture

the c o m p o u n d s

g r o w n i n TS b r o t h w a s

one g r o w n in F D A b r o th .

It i s

tested, it
as

resistan t

d i f f ic u lt to

c o n c l u s i o n s w h e n no n e u t r a l i z i n g m e d i a a r e u s e d w ith

t h e s e two c o m p o u n d s .

I

29
B oth M etap h en and B T C have high b a c te r io s ta tic
ties,

c a u s i n g i n h i b i t i o n of t h e t e s t o r g a n i s m

tions.

In h i g h d i l u t i o n s ,

"skips"

m e a n t t h e o b t a i n i n g of a n e g a t i v e
is indicated,

and

"w ild p lu s e s ,"

the d a ta w h e r e n e g a tiv e
The b a c te rio s ta tic
"skips"

and

two

g ro w n in DT m e d iu m
tested,

compounds

technic

c o u p led with

not as easily in te rp re te d

C.

H o w e v e r , the c u l t u r e
resistance

to a l l t h e
cultures

TS b r o t h w h e n the F D A

did no t affect the

was used as

o b tain e d by the

com parative
was

the s u b c u l t u r e m e d i u m

used.

"sem i-m icro "

technic w e r e not

c o n s is te n t a s the o n es o b tain ed by the F D A p r o c e d u r e .

killing d ilu tio n s fo r

ob­

was used.

than when Special F D A b ro th w as

as

often o c c u r .

although a lo w er phenol coefficient value

obtained w hen DT m e d i u m

R esults

are

showed a g re a te r

subculture m ed iu m

obtained,

result

r e s u l t s in

p h e n o lic s and n o n p h e n o lic s , than the

"phenol coefficient"

results

a positive

a c c o u n t f o r the f a c t th at r e s u l t s

grow n in sta n d a rd F D A b ro th or

The

re su lt w here

a re indicated,

as w ith D ow icide A and D ow icide

compounds

in the data, by w h ic h is

p r o p e r t y of M e t a p h e n a n d B T C ,

"w ild p lu s e s "

tain e d with th e s e

in v e ry high dilu­

w hich a re positive

results

proper­

D ow icide A and Dow icide

The

C checked fairly

30
w ell w ith the ones
organism s
ca n n o t be

o b ta in e d by the F D A p r o c e d u r e .

g ro w n in any one
s a i d to b e

M uch higher
and B T C by the
FDA procedure.

consistently m o re
killing

culture m e d ia te ste d

resistant.

dilutions w e re

obtained fo r M etaphen

11 s e m i - m i c r o ' ' t e c h n i c t h a n w i t h t h e

substance,

p roperties,

and w ith o u t the u s e

of t h e t e s t w a s

com pleted.

the w hole

c u lt u r e d in the

"sem i-m icro "

sam ple is

b acteriostatic

ratio

of t h e

activity exists

''p h e n o l c o e f f ic ie n t''
disinfectant-culture

d i s i n f e c t a n t to the
to a g r e a t e r

tech n ic in w hich one
m ixture

a n d U m b r e i t (17) f o u n d t h a t
w ith the

"sem i-m icro "

of

inhibiting actio n w ould p e r s i s t a fte r

the e x p o s u re p e rio d

allow s a g r e a te r

standard

T h is w ould be e x p e c te d a s both M e ta p h e n and

BTC have high b a c te rio s ta tic
a neutralizing

of t h e t h r e e

How ever,

is

technic

and

technic

d ilu e n t so th a t

e x te n t t h a n in the
4-m m

rem o v ed for

"skips"

The fact that

l o o p f u l of

subculturing.

"w ild p lu s e s "

K lim ek
occurred

a s w ell a s w ith the FD A p r o ­

cedure.
W ith M e ta p h e n and B T C
D ow icide

as w ell a s w ith D ow icide A and

C, the k illin g d i lu tio n s w e r e

was used as

the

subculturing m e d iu m

low er when DT m e d iu m
than w hen Special FD A

31
broth was

used.

M ore

about the

e ff e c t of the

subculture m ed ia

w ill be p r e s e n t e d in th e next s e c tio n .
To
it can be

sum m arize

the m a t e r i a l p r e s e n te d in this

s t a t e d t h a t w i t h th e F D A p r o c e d u r e ,

ant culture

of M . p y o g e n e s v a r .

aureus

organism s

w ere

they w e re

g r o w n in e i t h e r F D A b r o t h o r

No d e fin ite
the t e s t

culture

a m ore

can be

when the

draw n as

"sem i-m icro "

resist­

w a s o b ta in e d w h e n the

grow n in D isinfectant T e s t m e d iu m

conclusions

section,

T rypticase
to t h e

than when
Soy b ro th .

resistan ce

technic is u sed .

of

32
TABLE
EFFECT

OF

CULTURE

I

M EDIA ON TH E RESISTA N CE

M. PYOGENES

OF

VAR. AUREUS

T e st com pound--D ow icide A
T est organism --M .

pyogenes var.

A.

FD A broth

B.

DT m edium

C.

TS b ro th

Subculture m e d iu m - - D T

Phenol

A

12700

B

> 1 -3 00< 1-400

C

1-900

g ro w n in:

m edium

C oefficient T e s t

K illing D ilution

aureus

S em i-m icro

Phenol
.
C oefficient

K illing D ilution

10

> 1 -200<1-300

Test
Phenol
C oefficient
5

5

1-400

10

15

1-400

7

T A B L E II
EFFECT

OF

CULTURE

M EDIA ON TH E R E SISTA N C E O F

M. PYOGENES

VAR. AUREUS

T e s t c o m p o u n d - - D ow icide A
Test organism --M .

pyogenes

A.

FDA bro th

B.

DT m e d iu m

C.

TS b r o th

var. aureus

g ro w n inr

Subculture m e d iu m --S p e c ia l FD A b ro th

P h en o l C oefficient T e s t

Semi -m ic ro

Test

K illing D ilution

Phenol
C oefficient

A

>1 - 1 0 0 0 < 1 - 1 2 0 0

12

> 1 -800<1-900

10

B

1-400

5

> 1 - 4 0 0 < 1 - 500

6

C

>1 - 1 2 0 0 < 1 - 1 40 0

16

> 1 - 6 0 0 < 1 - 700

8

K illing D ilution

Phenol
C oefficient

f

l

34
TABLE
EFFECT

OF

CULTURE

M E D IA ON T H E R E S IS T A N C E O F

M. P Y O G E N E S

T est com pound--D ow icide
Test organism --M .
FD A broth

B.

DT m e d iu m

C„

TS b r o th

Subculture m e d iu m - - D T

C

D ilution

aureus

g r o w n in:

m edium

P h en o l C oefficient T e st

K illing

VAR. AUREUS

pyogenes v ar.

A.

III

Semi -m icro

Test

Phenol
C oefficient

K illing D ilution

Phenol
C oefficient

A

1-4000

57

> 1 -2 0 0 0 < 1 -4000

42

B

1-2000

33

1-2000

36

C

1-4000

61

> 1 -2 0 00< 1-4000

75

35
TABLE
EFFECT

OF

CULTURE

M ED IA ON TH E

M. PYOGENES

T est com pound--D ow icide
Test organism --M .

IV

VAR. AUREUS

C

pyogenes v a r. aureus

A.

FDA bro th

B.

DT m e d iu m

C.

TS b r o t h

R E SISTA N C E O F

g r o w n in:

Subculture m e d iu m --S p e c ia l FD A b ro th

P h en o l C oefficient T e s t

K illing

D ilution

Phenol
C oefficient

S em i-m icro

K illing

D ilution

Test
Phenol
C oefficient

A

> 1 - 4 0 0 0 < 1 - 6000

71

> 1 -2 0 0 0<1-4000

50

B

> l-2 0 0 0 < l-4 0 0 0

46

> l-2 0 0 0 < l-4 0 0 0

60

C

1-6000

85

>1-4000<1-6000

71

36
TABLE
EFFECT

OF

CULTURE

V

M EDIA O N TH E

M. PYOGENES

R E SISTA N C E

OF

VAR. AUREUS

T est com pound--M etaphen
T est organism --M .

pyogenes var.

A.

FD A broth

B.

DT m ed iu m

C.

TS b r o th

S ubculture m e d iu m - - D T

D ilution

g r o w n in :

m edium

P h e n o l C oefficient T e s t

Killing

aureus

Phenol
C oefficient

Semi -m icro

Test

Killing D ilution

Phenol
C oefficient

A

> 1-30,000< 1-40,000

46 6

<1-50,000

<769

B

> 1 - 2 0 , 0 0 0 < l - 3 0 , 000

384

> 1 - 5 0 , 0 0 0 < l - 6 0 , 000

13 75

C

> 1 -3 0 ,0 0 0 < 1 -40,000

46 6

> 1 - 6 0 , 0 0 0 < l - 7 0 , 00 0

T625

37
TABLE
EFFECT

OF

CULTURE

VI

M E D I A ON T H E R E S I S T A N C E

M. PY O G EN ES

OF

VAR. AUREUS

T e st com pound--M etaphen
T est organism --M .

pyogenes var.

A.

FDA

B.

DT m ed iu m

C.

TS b ro th

aureus

g r o w n in:

broth

Subculture m e d iu m - - S p e c ia l FD A b ro th

Phenol

K illing

C oefficient T e s t

D ilution

Phenol
C oefficient

S em i-m icro

K illing D ilution

Test
Phenol
C oefficient

A

> 1 - 5 0 , 0 0 0 C 1 - 6 0 , 00 0

687

>

1 - 100,000

>1300

B

> 1 - 3 0 , 0 0 0 < l - 4 0 , 00 0

466

>

1- 100,000

>1300

C

1-50,000

768

>

1- 100,000

>1300

TABLE
EFFECT

OF

CULTURE

VII

M E D IA ON

M. PY O G E N E S

THE RESISTA N CE OF

VAR. AUREUS

T est com pound--B TC
T est organism --M .

pyogenes var.

A.

F D A .broth

B.

DT m edium

C.

TS b ro th

.Subculture m e d i u m - - D T

aureus

m edium

P h e n o l C oefficient T e s t

Killing

D ilution

g r o w n in :

Semi -m icro

Phenol
K illing
C oefficient

D ilution

Test
Phenol
C oefficient

A

>1 - 1 5 , 0 0 0 < 1 - 2 0 , 0 0 0

Z50

> 1 - 2 0 , 0 0 0 < l - 3 0 , 000

384

B

1-8,000

100

> 1 - 2 0 , 0 0 0 < l - 3 0 , 000

415

C

1-15,000

214

> 1 - 2 0 , 0 0 0 < l - 3 0 , 000

6 25

39
TABLE
EFFECT

OF

CULTURE

VIII

M E D IA ON T H E

RESISTA N CE O F

M, PY O G EN ES VAR, AUREUS

T est com pound--B TC
T est o rg an ism - - M. pyogenes v ar. aureus
A.

FDA bro th

B.

DT m edium

C..

TS b r o th

g r o w n in:

S ubculture m e d iu m - - S p e c ia l FD A b ro th

P h e n o l C oefficient T e s t

K illing

D ilution

Phenol
C oefficient

S em i-m icro

K illing

Dilution

Test
Phenol
C oefficient

A

> 1 -2 0 ,000<1-25,000

321

1-50,000

833

B

1-10,000

125

> 1 - 4 0 , 0 0 0 C 1 - 5 0 ,000

750

C

1-20,000

26 6

> 1 - 3 0 , 0 0 0 C 1 - 4 0 , 000

636

f l

40
TABLE
BA C TER IA L

COUNTS, E X P R E S S E D

CULTURES OF

TEST

IN M IL L IO N S,

ON

TEST

M. PY O G E N E S VAR. A U R E U S , C U LTU R ED

IN (A) S T A N D A R D F D A
ANT

IX

BROTH,

(B) D IF C O

M E D I U M A N D (C) T R Y P T I C A S E

D ISIN FE C T ­
SOY B R O T H

A

B

C

130

140

775

140

480

1070

140

300

760

82

380

650

98

570

9 80

200

400

810

480

210

90 0

56

530

740

I

41
B.

E f f e c t of S u b c u ltu r e M e d ia on the C u ltiv a tio n
o f D i s i n f e c t a n t - t r e a t e d C e l l s of
M . pyogenes var. aureus

In the
organism s
various

a fte r they had been

disinfectants,

experim ents
organism
dium

s t u d y of s u b c u l t u r e m e d i a u s e d f o r g r o w i n g

s u b j e c t e d to th e a c t i o n of th e

o n ly the d a ta a r e

in w hich DT m e d iu m

p reparatory

resu lts

w ere

for

The

was used for

w ere

b ro th plus

of c o m p o u n d u s e d in th e
percent ferric

n eutralizing m e d iu m
w hich a r e

for

coefficient w as
Special FD A

that was

S pecial

specific for

Standard FDA

(8) w a s u s e d a s

D ow icide A and D ow icide

the

C, b o t h of

T h i o g l y c o l l a t e b r o t h (32) w a s

a n d L e t h e e n b r o t h (2 3) w a s u s e d f o r B T C .

E x a m i n a t i o n of T a b l e s
dium w as u se d as

DT m e ­

the o r g a n is m s

DT m e d iu m ,

study.

chloride

phenolic d e riv a tiv e s .

u sed fo r M etaphen,

The

the

the m o s t r e s i s t a n t .

subculture m ed ia used w ere

0.03

the

grow ing

the d i s i n f e c t a n t t e s t s .

FD A b ro th and a neutralizing m ed iu m
e a c h type

presented fro m

selected p rim a rily because

grow n in this m e d iu m

the

the

X a n d XI s h o w s t h a t w h e n D T m e ­

subculturing m ed iu m , a low er phenol

obtained w ith the FD A p r o c e d u r e

b r o th o r the F D A b r o th p lu s f e r r i c

than w hen
chloride

was

I

42
us ed -.

No d iffe re n c e

either

D ow icide A o r D ow icide

FDA broth

w as o b s e r v e d in the

plus f e r r i c

chloride.

and FD A broth.

r id e h ad no e ffe c t a s
compounds

as

This

i n a c t iv a t e d by the
less

than

was

obtained fro m

with F D A

would in d ic a te

chlo­

th at the f e r r i c

chlo­

highly b a c te r io s ta tic ,

thioglycollate

broth.

t h e w o r k of

plus f e r r ic

the phenolic

s u g g e s te d by F l e t t et a l.

w hich is

broth and

su b stan tiates

a chem ical in activ ato r for

previously

M etaphen,

(8 ).

was

T he killing

effectively
dilution w as

1-500 w h ic h is the d ilu tio n at w h ich the m e r c u r i a l

The
to i t s

T his

d i l u t i o n of

C with S p ecial F D A

T i l l e y (3 6 ) w h o f o u n d n o d i f f e r e n c e
ride

killing

the m a n u f a c t u r e r .

a n t i b a c t e r i a l a c tio n of m e r c u r y

c o m b in atio n with the

thus d e p riv in g the
m etab o lism

(7 ).

i s t h o u g h t to b e d u e

-SH g ro u p s in the b a c t e r i a l cell,

cell of-S H g ro u p s w hich a r e

e s se n tia l fo r its

T h e a n ti b a c t e r i a l actio n of m e r c u r y h a s b e e n

sp ecifically n e u tra liz e d by

-SH c o m p o u n d s .

Thus,

it would be

e x p e c te d th a t a m u c h lo w e r killing dilu tio n would be ob tain ed
when thioglycollate
or

DT m e d i u m

a low er

b ro th w as u s e d than w hen S p e c ia l FD A b ro th

was used.

killing d ilution w a s

O b s e r v a t i o n of T a b l e X I I s h o w s t h a t
obtained when DT m e d iu m

was used

43
for
is

subculturing

than w hen S p ecial F D A b ro th w as u sed ,

w hich

c o n s is te n t w ith data p re v io u s ly p re s e n te d .
The

broth w as
this

d a ta on B T C

are

d e m o n s t r a t e d to be a m o r e

com pound than w as

phenol c o efficien t value
broth,

p r e s e n t e d in T a b le XIII.

w hile one

DT m ed iu m
o f 85 w a s

effective in a c tiv a to r for

o r S pecial FD A b ro th .

N egatively

of n e u t r a l i z i n g
cationic

germ icides

organism s
broth as

w ere

also

The

as

the

chief n e u tr a l-

c h a rg e d fatty a c id ions have b e e n found

the g e r m i c i d a l p r o p e r t i e s

A low er

a n d o n e of 125 w i t h

resulted.

L e c ith in in the L e t h e e n b r o th a c ts
izer.

A

o b ta in e d with the L e th e e n

of 100 w i t h D T m e d i u m

Special F D A b ro th

L etheen

of p o s i t i v e l y

dilution w as

o b tain ed w hen the t e s t

s u b c u ltu r e d in DT m e d i u m

occurred

t e n d to s u b s t a n t i a t e

than in S p ecial FD A

w ith the o th e r d isin fe c ta n ts

o b t a i n e d w ith the
those

FD A b r o th plus f e r r i c

"sem i-m icro "

tested.
technic

o b tain e d w ith F D A p r o c e d u r e

chloride

vating D ow icide A and D ow icide
Special F D A bro th .

charged

(23).

killing

resu lts

capable

i s no m o r e

in that

effective in in a c t i ­

C than w as DT m e d iu m

or

44
A lthough a lo w e r

killing dilu tio n w as o b tain ed fo r D o w i­

cide A w h e n DT m e d i u m
Special FD A

was used for

broth w as used,

a n t to p h e n o l , w h i c h c a u s e d

the

subculturing than w hen

culture used was m o re

a higher

"phenol

resist­

coefficient"

value

to r e s u l t .
Low er
resulted

w hen DT m e d i u m

w ith D ow icide
R esults
late

killing d ilu tio n s

and

"phenol coefficient"

w a s u s e d in the

C, M e t a p h e n a n d B T C

"sem i-m icro "

c o r r e la t e d with those

the phenol c o e ffic ie n t te c h n ic .
phenol coefficient v alu es

the n e u tr a liz in g m e d i a w e re
S um m arizing

thioglycol­
obtained by

killing dilutions

and

M etaphen and B T C w hen

used.

the m a t e r i a l in th is

th at F D A b r o th plus f e r r ic

chloride

FD A b ro th and in m o s t c a s e s
a subculture m ed iu m

Low er

resu lted for

tests

a s the t e s t c o m p o u n d s .

o b ta in e d w ith the n e u tr a liz in g m e d ia ,

b ro th and L e th ee n broth,

values

less

sectio n , it can be

w a s no m o r e

stated

effective than

effective than DT m e d iu m

in in a c tiv a tin g

Dow icide A and D ow icide

as
C.

T h io g ly c o lla te b r o t h w a s e ffe c tiv e in both m e t h o d s u s e d in
neutralizing

the M e ta p h e n .

A low er

killing d ilution fo r B T C

obtained when L eth een b ro th was used for
FD A bro th or

DT m e d iu m

was used.

subculturing

was

than when

45
DT m e d iu m
culture m ed iu m
f i c i e n t 11 t e s t s

was

shown

to be m o r e

effective

than w as S p ecial FD A b ro th in

on all fo u r

as

a sub­

"phenol coef­

compounds.

f

l

46
TABLE
EFFECT

X

OF

SUBCULTURE

OF

D ISIN FEC TA N T-TR E A TED
M.

M E D IA ON T H E

PYOGENES

C U LTIV A TIO N

C ELLS OF

VAR. AUREUS

T est com pound--D ow icide A
Test o rg an ism --M .

pyogenes v ar.

T est M edium

aureus

g ro w n in D isin fe c ta n t

(DTM )

Subculture m e d ia
A.

S pecial FD A b ro th

B.

DT m e d iu m

C.

FDA

Phenol

b ro th plus

C oefficient T e s t

K illing D ilution

A
B
C

1-400
> 1 -3 0 0 < 1 -400
1-400

0.03%

Phenol
C oefficient
5.7
5.0
5.7

F eC l3

S em i-m icro
K illing D ilution

> l-4 00< l-500
1-400
> 1 -4 0 0 < 1 -500

Test
Phenol
C oefficient
6.4
10.0
9.0

TABLE
EFFECT

OF SUBCULTURE
OF

M E D IA ON T H E

D ISIN FE C T A N T -T R E A T E D
M. PYOGENES

Test

XI

com pound--D ow icide

T est organism --M .

C U LTIV A TIO N

CELLS

OF

VAR. AUREUS

C

pyogenes var.

aureus

grow n in D isin fectan t

T e s t M edium
S ubculture

m edia

A.

Special FD A b ro th

B.

DT m ed iu m

C.

F D A b r o th plus

0.03%

P h e n o l C oefficient T e s t

K illing

D ilution

Phenol
C oefficient

F eC l^

S em i-m icro

K illing

D ilution

Test
Phenol
C oefficient

A

> 1 - 2000<1-4000

46

> l-2 0 0 0 < l-4 0 0 0

60

B

1-2000

33

1-2000

40

C

> l-2 0 0 0 < l-4 0 0 0

42

> 1 -2 0 00< 1-4000

50

f

l

48
TABLE
EFFECT

OF SUBCULTURE
OF

XII

M ED IA ON TH E

D ISIN FEC TA N T-TR E A TED
M.- P Y O G E N E S

C U LTIV A TIO N

CELLS OF

VAR. AUREUS

T e st com pound--M etaphen
T est organism --M .

pyogenes var.

aureus

g ro w n in D isin fe c ta n t

T e s t M edium
S ubculture m e d ia
A.

S pecial FD A b ro th

B.

DT m e d iu m

C.

T hioglycollate

Phenol

K illing

broth

C oefficient T e s t

D ilution

Phenol
C oefficient

Semi -m icro

K illing

D ilution

Test
Phenol
C oefficient

A

> 1 -3 0 ,000<1-40,000

466

>1-100,000

B

> 1 - 2 0 , 0 0 0 C 1 - 3 0 ,000

384

> 1 - 5 0 , 0 0 0 < l - 6 0 , 000

1375

C

<1-500

<7.0

<1-500

<9.0

>1428

f

l

49
TABLE
EFFECT

OF

X II I

S U B C U L T U R E M E D I A ON

OF

THE

D ISIN FEC TA N T-TR E A TED
M. PY O G EN ES

C U LTIV A TIO N

CELLS OF

VAR. A U R EUS

T est com pound--B TC
Test organism --M .

pyogenes var.

aureus

grow n in D isin fectan t

T e st M edium
S ubculture m e d ia
A.

S pecial FD A b ro th

B.

DT m edium

C.

L eth een b roth

Phenol
Killing

C oefficient T e st

D ilution

Phenol
C oefficient

Semi -m icro
Killing

D ilution

Test
Phenol
C oefficient

A

1-10,000

125

> 1 - 4 0 , 0 0 0 C 1 - 5 0 , 00 0

750

B

1-8,000

100

> 1 - 2 0 , 0 0 0 < l - 3 0 , 000

454

>1 - 5 0 0 0 < 1 - 1 0 , 0 0 0

187

C

>1-4000<1-8000

85

50
C.

E f f e c t of W a s h e d C u l t u r e s on the R e s i s t a n c e
of M . p y o g e n e s v a r . a u r e u s a s
C o m p a r e d to B r o t h C u l t u r e s

As

early as

1 89 7 , i t w a s

te r played an im p o rta n t ro le
a c tiv ity of d is in f e c ta n ts
proteins
ganic

are

present,

substances.

(1'8).

In m o s t c a s e s

in terfere

of d i s i n f e c t i o n ,
and i n o r ­

w ith s te r iliz a tio n .

of d i s i n f e c t a n t s

sam e foreign

are

w ith the g e r m i c i d a l

T h e r e f o r e , i t i s i m p o r t a n t to k n o w t o w h a t

D ifferent types

why th e re

in i n t e r f e r i n g

and f r e q u e n tly o th e r o r g a n ic

extent fo re ig n m a te r ia ls

ently by the

reco g n ized that organic m a t­

substance..

will be affected d i f f e r ­
T h i s i s o n e of t h e

so m a n y k in d s of d i s i n f e c t a n t s

reasons

on the m a r k e t t o ­

day.
M ost m ethods
involve the u se
FDA

of t e s t i n g th e

of a b r o t h c u lt u r e

"phenol coefficient"

culture

e f f i c i e n c y of d i s i n f e c t a n t s
of t h e t e s t o r g a n i s m .

procedure

In the

0.5 m l of the b a c t e r i a l

i s p l a c e d i n 5.0 m l of d i s i n f e c t a n t

solution.

A co n sid er­

able

a m o u n t of o r g a n i c m a t t e r i s i n t r o d u c e d ,

ppm

of p e p t o n e a n d m e a t e x t r a c t .

fere

w i t h t h e a c t i o n of d i s i n f e c t a n t s t h a t o t h e r w i s e w o u l d b e

This is

approxim ately

15 00

s u f f i c i e n t to i n t e r ­

c a p a b le of k illin g b a c t e r i a in v e r y low c o n c e n t r a t io n s

(25).

f

l

51
In o r d e r to
w ould be

elim inate

tests

w ere

pyogenes var. aureus
p hosphate

buffer,

"sem i-m icro "

subculturing

show n in the

preceding

m ethod

the

cultures

the w a sh e d cells

w ere

cultures

w ere

in w hich DT

presented.

This m e ­

s e c t i o n to be m o r e

are

resu lts

effective

obtained using w ash ed

recorded

E x a m i n a t i o n of t h e d a t a o n t h e

shows th at a h ig h er

the b ro th

are

and the

than w as Special F D A b roth.

com paring

and b r o th

inclusive.

of M .

re s u s p e n d e d in S o re n s e n 's

O n l y t h e d a t a of t h e t e s t s

a subculture m e d iu m

cultures

cells

c o n d u c te d u sin g the F D A p r o c e d u r e

technic.

D ata for

conducted using w ash ed

that w ere

was u sed for

dium w as

c e ll s of the

pH 7.0, f o r th e t e s t .

T ests w ere

as

e f f e c t of f o r e i g n m a t e r i a l t h a t

in tr o d u c e d by th e b r o t h along w ith the

test organism ,

m edium

the

in T a b le s

XIV-XVII

"phenol coefficient"

killing dilution w as o b tain ed w hen

u s e d a s the te s t o r g a n i s m
used.

This was

true

than when

f o r all of the

com pounds tested.
R esults

o b tain ed by the

show c o n sisten t d iffe re n c e s
cells and those
with the r e s u l ts

u sin g the

"sem i-m icro "

betw een te s ts

technic

ru n using the w ash ed

reg u lar broth cu ltu res.

obtained in p rec e d in g

do n o t

This

co rrelates

sections.

f

l

52
W ashed

cells

of M . p y o g e n e s v a r . a u r e u s t h a t h a d b e e n

grow n in DT m edium , w e re
cases

than the w a s h e d

a nd TS b r o th .

s h o w n to b e m o r e

r e s i s t a n t in m o s t

ce lls th at had b een grow n in F D A b ro th

53
T A B L E XIV
EFFECT

OF

OF

WASHED

C U L T U R E S ON T H E R E S IS T A N C E

M. PY O G E N E S
PARED

VAR. A U R E U S AS C O M ­

TO BROTH

CULTURES

T e s t com pound--D ow icide A
T est organism --M .

pyogenes var. aureus

A.

FDA broth

B.

DT m edium

C.

TS m e d i u m

Subculture m e d iu m - - D T
PHENOL

B roth

A
B
C

m edium
C O EFFIC IEN T

C ulture

Killing D ilution

W ashed C ulture

K illing D ilution

Phenol
C oefficient

10
5
15

1-1000
1-900
> 1 - 1 4 0 0 < 1 - 1600

14
13
21

SEM I-M IC R O

K illing

A
B
C

> 1 -2 0 0 < 1 -300
1-400
1-400

TEST
W ashed

C ulture

D ilution

TEST

Phenol
C oefficient

1-7000
> 1 -3 0 0 < 1 -400
1-900

B roth

g ro w n in:

Phenol
C oefficient
5
10
7

C ulture

K illing D ilution

> l-300< l-600
> l-500< l-600
1-600

Phenol
C oefficient
10
8
11

I

54
TABLE
EFFECT

OF

OF

WASHED

CULTURES

TO B R O T H

T e st com pound--D ow icide
Test organism --M .
A.

FD A broth

B.

DT m edium

C.

TS m e d iu m

PHENOL

B roth

CULTURES

C

pyogenes v ar.

Subculture m e d iu m - - D T

A
B
C

ON TH E R E S IS T A N C E

M . P Y O G E N E S V A R . A U R E U S AS C O M ­
PARED

K illing

XV

aureus

m edium
C O EFFIC IEN T

C ulture

D ilution

57
33
61

K illing D ilution
> 1-8000< 1-10,000
1-6000
1-8000

S E M I- M ICRO
B roth

K illing

A
B
C

> 1 - 2 0 0 0 < 1 - 4000
1-2000
> 1 -2000< 1-4000

Phenol
C oefficient
100
85
100

TEST
W ashed C ulture

C ulture

D ilution

TEST

W ashed C ulture

Phenol
C oefficient

1-4000
1-2000
1-4000

g r o w n in:

Phenol
C oefficient

K illing D ilution

Phenol
Coefficient

42
36
75

1-4000
> 1 - 1000<1-2000
> l-2000< l-4000

53
25
60

I

TABLE
EFFECT

OF

OF

XIV

W A SH E D C U L T U R E S ON T H E

M. PYOGENES
PARED

R E SISTA N C E

VAR. A U R E U S AS C O M ­

TO B R O T H C U L T U R E S

T est com pound--M etaphen
Test organism --M .

pyogenes var.

A.

FD A broth

B.

DT m edium

C.

TS b ro th

Subculture m e d iu m - - D T

aureus

m edium

PHENOL

CO EFFICIEN T

TEST

B ro th C ulture

K illing D ilu tio n

A
B
C

W ashed

Phenol
C oefficient
466
384
466

> l-3 0 ,000< l-40,000
> 1 -20,000< l-30,000
>1 - 3 0 , 0 0 0 < 1 - 4 0 , 0 0 0

SEM I-M IC R O
B roth

A
B
C

< 1-50,000
> 1 - 5 0 , 0 0 0 < l - 6 0 , 00 0
> 1 - 6 0 , 0 0 0 < l - 7 0 , 000

Phenol
C oefficient
625
46 1
53 3

1-50,000
1-30,000
1-40,000
TEST
W ashed

Phenol
K illing
C oefficient
<769
13 75
1625

C ulture

Killing D ilution

C ulture

K illing D ilution

g r o w n in:

C ulture

D ilution

> 1 - 5 0 , 0 0 0 < l - 6 0 , 000
140T
> 1 - 5 0 , 0 0 0 < l - 6 0 , 000

Phenol
C oefficient
1100
2000
1375

TABLE
EFFECT

OF

OF

WASHED

XVII

C U LTU RES ON THE

RESISTA N CE

M. P Y O G E N E S VAR. A U R EU S AS C O M ­
PARED

TO BROTH CULTURES

T est com pound--B TC
T e s t o r g a n is m - - M. pyogenes v a r.
A.

FD A broth

B.

DT m ed iu m

C.

TS b ro th

Subculture m e d iu m --D T

m edium

PHENOL

B roth

Killing

A
B
C

CO EFFICIEN T

TEST

C ulture

D ilution

W ashed

Phenol
C oefficient

> 1 - 1 5 , 0 0 0 < l - 2 0 , 00 0
1-8000
1-15,000

250
100
214
SEM I-M IC R O

K illing D ilution

> 1 -2 0 ,0 0 0 < 1 -30,000
> 1 - 2 0 , 0 0 0 < l - 3 0 , 000
> 1 - 2 0 , 0 0 0 C 1 - 3 0 , 000

C ulture

K illing D ilution

Phenol
Coeffi cien t
266
133
28 6

1-20,000
1-10,000
1-20,000
TEST
W ashed C ulture

B ro th C ulture

A
B
C

a u r e u s g r o w n in:

Phenol
C oefficient
384
415
625

Killing D ilution

> 1 - 2 0 , 0 0 0 < l - 3 0 , 000
> 1 - 2 0 , 0 0 0 < l - 3 0 , 000
> 1 - 2 0 , 0 0 0 < l - 3 0 , 000

Phenol
C oefficient
384
31 2
625

57
D„

E f f e c t of A g e of F D A S u b c u l t u r e M e d i u m o n t h e
C u l t i v a t i o n o f D i s i n f e c t a n t - t r e a t e d C e l l s of
M. pyogenes v a r. a u re u s

D u r i n g the c o u r s e of t h i s
tim es

wide

discrepancies

study, it w as n o ted th at s o m e ­

o c c u r r e d in the

phenol coefficient

killing d ilu tio n w hen S p e c ia l F D A b r o th w as u s e d a s the
culturing m e d iu m .
DT m e d i u m

No

was used.

when duplicate tubes
FDA

as the

the a g e s
tors.

w ere

sam e

lots

w ere noticed when

phenom enon was firs t observed

r u n o n D o w i c i d e A u s i n g tw o l o t s of
As far

subculturing m ed ia w ere

in th e p r e p a r a ti o n
the

T his

subculture m ed iu m .

of t h e

The

such d iscrep an cies

sub-

of p e p t o n e

as

could be d e t e r m i n e d

the d istin g u ish in g f a c ­

and beef e x tra ct had been used

of the m e d ia .

T h e p H of t h e t w o l o t s w a s

sam e.
E x a m i n a t i o n of T a b l e

XVIII s h o w s t h a t the

t i o n of D o w i c i d e A r a n g e d f r o m
w h en the o r g a n i s m

1 - 4 0 0 to m o r e

w a s g ro w n in DT m e d iu m .

FD A broth w ere u se d .

than
Three

dilu­

1-2400
lots

of

A k i l l i n g d i l u t i o n of 1 - 4 0 0 w a s o b t a i n e d

w h e n a l o t of F D A b r o t h t h a t w a s
a killing dilution

killing

18 d a y s o l d w a s u s e d w h i l e

1-800 w as o b tain ed on the

d i f f e r e n t l o t of s u b c u l t u r e m e d i u m ,

sam e

test when a

f o u r d a y s old, w as u s e d .

A

I

58
d i l u t i o n of m o r e
broth,

one

than

day old,

1-2400 w as

o b t a i n e d w h e n a l o t of F D A

was used.

"P h en o l coefficient"

tests

also

w ere

the te s t o r g a n i s m

grow n in FD A broth.

culturing m e d iu m

was

T h i s lo t of m e d i u m
further

tests

w ere

l o t of m e d i u m .
dilution w as

sub-

tim e of th e f i r s t t e s t .

r e f r i g e r a t e d to r e t a r d

run after

using

The S pecial FD A

one d a y o ld at the

was

perform ed,

evaporation;

20 a n d 30 d a y s , u s i n g t h i s

sam e

E x a m i n a t i o n of T a b l e X V I I I s h o w s th a t t h e k i l l i n g

1-2000

w hen the

subculturing m ed iu m

o l d , w h i l e i t w a s >1 - 1 0 0 0 < 1 - 1 2 0 0

and

1-1000

was

one day

r e s p e c t i v e l y on the

20- and 3 0 -d ay -o ld m ed iu m .
It i s i n t e r e s t i n g
o b s e rv e d th at the u se

to n o t e

of a f r e s h l y

value in m a in ta in in g the
of t h e t e s t o r g a n i s m . .
tension m ig h t be
ence as

to the

F o t e r a n d G i b b y (2 1)

p rep a re d m ed iu m was

standard re sista n c e

and

of

sm ooth phase

T h e y t h e o r i z e d th a t the p a r t i a l o x y g e n

a factor.

The

e f f e c t of the a g e

the v a lu e s

obtained for

variations

o b s e r v e d in this

There

th at Q uisno,

w r i t e r f a i l e d to f i n d a n y r e f e r ­
of t h e

subculture m ed iu m

phenol coefficients.

is need for fu rth e r

No r e a s o n s

on

f o r the

study can be p r e s e n te d at this

tim e.

study.

I

TABLE
EFFECT
THE

O F AGE O F

XVIII

F D A S U B C U L T U R E M ED IU M ON

C U LTIV A TIO N O F D IS IN F E C T A N T -T R E A T E D
CELLS OF

M. PYOGENES

T e s t c o m p o u n d — D ow icide

VAR. AUREUS

A

T E S T O R G A N ISM G R O W N IN D T M E D IU M

D ate

M edium P r e p a r e d

D ate

of T e s t

K illing D ilution

2-5-51

2-23-51

1-400

2-19-51

2-23-51

1-800

7-30-51

7-31-51

>1-2400

T E S T O R G A N I S M G R O W N IN F D A B R O T H

D ate

M edium P r e p a r e d
7-30-51

D ate

of T e s t

7-31-51
8-20-51
8-30-51

K illing D ilution
1-2000
> 1-1000< 1-1200
1-1000

f

l

60
E.

E f f e c t of C u l t u r e M e d i a o n t h e B a c t e r i a l P o p u l a t i o n a n d
o n t h e R e s i s t a n c e of M . p y o g e n e s v a r . a u r e u s t o P h e n o l

In the
states

standard FDA

th a t the t e s t c u ltu re

"phenol coefficient"
m u s t be t a k e n f r o m

s la n t and t r a n s f e r r e d into b r o th at l e a s t th r e e
before

it is

r e a d y to b e u s e d .

of t h e d a i l y t r a n s f e r s
r u n on the f i r s t ,

from

the

second and th ird

w ere

r u n on the

there

w as a change in the

stock n u trie n t a g a r

a f t e r the

a nutrient agar
successive

to d e t e r m i n e

on the b a c te r i a l population,

w ere

ism

In o r d e r

p r o c e d u r e it

slant.

into b r o t h

coefficient"

second and th ird day tra n s fe r s ,
resistan ce

the e ffe c t

grow th cu rv es

day t r a n s f e r s

"Phenol

to p h e n o l of th e t e s t o r g a n ­

second day tra n sfe r fro m

l i s t e d in T a b le XIX.

from

the d e h y d ra te d f o r m ,

w ere

used; th ree

the

stock culture.

plus one lot m ad e

and one m a d e

one l o t of TS b r o t h w e r e

could be c o m p a r e d .

in the la b o r a to r y

two m a d e

w ith d iffe re n t

w ith A r m o u r peptone,

in c lu d e d in the

e f f e c t of d i f f e r e n t l o t s o f p e p t o n e s
and on the r e s i s t a n c e

study.

T w o l o t s of D T m e d i u m , m a d e

l o t s of F D A b r o t h ,

l o t s of D ifco p e p t o n e

tests

to d e t e r m i n e if

S e v e n d i f f e r e n t c u l t u r e m e d i a w e r e in c l u d e d i n the
They are

tim es

study.

and

In th is w a y the

on the b a c t e r i a l p o p u latio n

of M . p y o g e n e s v a r . a u r e u s

to p h e n o l

61
The
on G r a p h

b acterial

1.

co u n ts e x p r e s s e d in lo g a r ith m s

E x a m i n a t i o n of t h e

graph

h a s the h ig h e s t b a c t e r i a l count of the
there

is n o t m u c h ch an g e in the

third day tra n s fe r.
24-hour b a c te ria l
m uch difference
DT m ed ia

(A, B ,

C).

They

l o t s of F D A b r o t h m a d e

w ere

second and third

organism

"phenol coefficient"

Table

shows

A culture

Difco p e p to n e

but have

peptone.

a

Low er

second day

are

th a t the

tests, m ade

on the

s h o w n i n T a b l e XX..
resistance

It s h o w e d no l i n e a r

on the

g ro w n in th is m e d iu m

phenol.

(D, E ) ,

of t h e t e s t

relationship

to

co u n t a lth o u g h F D A b r o th (F) had the lo w e s t b a c ­

te ria l population and also ,
ism s

of

seven m ed ia used.

to p h e n o l v a r i e d .

the b a c t e r i a l

lots

co u n t t h a n the two

d a y t h a n on t h e

day b ro th tr a n s f e r s

E x a m i n a t i o n of t h e

T h e re is not

with A r m o u r

o b ta in e d o n the t h i r d

of t h e

tran sfers.

with Difco peptone

counts

R esu lts

s e c o n d to th e

(F) s h o w s the l o w e s t

show a low er

count than FD A b ro th m a d e

f o r a ll of the

the

(G)

and that

p o p u l a t i o n of t h e t h r e e

higher

transfers

seven m edia,

S tandard FD A broth

in the b a c te r i a l

plotted

show s th a t TS b ro th

count fro m

count on all t h r e e

are

(E)

third

day t r a n s f e r ,

the o r g a n ­

s h o w e d the l o w e s t r e s i s t a n c e

to

g r o w n i n a l o t of F D A b r o t h , m a d e w i t h
s h o w e d the

sam e

resistance

to p h e n o l, b u t

62
h ad the th ir d h ig h e s t b a c t e r i a l
TS b r o th ,

w ith the h i g h e s t b a c t e r i a l

resistan ce

to p h e n o l a s

the o n e s

had a m u c h low er b a c te ria l
there

i s no l i n e a r

populations

pyogenes v ar.

aureus

states
FDA

of t h e

on m e th o d s

that any

and a

count,

show ed the

sam e

stu d ie d and the

of v ia b le

resistan ce

to n o t e t h a t o n l y o n e m e d i u m
required

survive

resistan ce
antiseptics

a

dilution fo r

M ost w o rk e rs

difficulty at so m e
resistan ce

is

cells

(D) p r o ­

to p h e n o l.

The

FDA

and disinfectants

(31)

a u r e u s u s e d in the

1 -6 0 d i lu t io n of p h e n o l f o r fiv e
15 m i n u t e s .

tim e.

in th is field have

In o r d e r

d e m a n d e d of the t e s t o r g a n i s m ,

DT m e d i u m

pyogenes v a r. au reu s.

required

e n c o u n te re d this

to d e t e r m i n e if too h i g h

of t h e k i l l i n g d i l u t i o n s o f p h e n o l t h a t w e r e
FD A broth,

that

of M.

I t i s v e r y d i f f i c u l t t o m a i n t a i n a c u l t u r e of t h e
resistan ce.

(B ) w h i c h

T h is w ould in d ic a te

s t r a i n of M . p y o g e n e s v a r .

1-70

g ro w n in

g ro w n in DT m e d iu m

population.

of t e s t i n g

procedure m u st

m inutes

organism s

to p h e n o l .

It i s i n t e r e s t i n g
duced a culture

The

c o n n e c tio n b e tw e e n the n u m b e r

p r e s e n t in the

c ircu lar

count.

a study w as m a d e

obtained w hen sta n d a rd

and TS b ro th w e re u se d fo r

grow ing M.

63
S p ecial F D A b ro th and DT m e d iu m
culturing.

T a b le XXI show s

of t h e v a l u e s

obtained.

m ost resistan t

culture

three

was not as

cultures

deviation

sh o w s th a t the

r e s i s ta n t as is

s e t up by the F D A

g ro w n in F D A b r o th w as

procedure.

the l e a s t r e s i s t a n t of the

tested.
case

a higher

Special FD A b ro th w as

for

E v e n this

specifications

In e v e ry

dium

the m e a n and s ta n d a r d

sub-

w a s the one g ro w n in TS b r o t h a nd s u b ­

cu ltu red in DT m e d iu m .

The

used for

E x a m i n a t i o n of the ta b le

culture

r e q u i r e d by the

w ere

was u sed .

killing

used for

This is

dilution w as

obtained when

su b cu ltu rin g than w hen DT m e ­

sh o w n n o t only in the v a l u e s

obtained

e a c h in d iv id u a l t e s t c u ltu r e m e d iu m , but a lso f o r the fig u re

o b ta in e d w h e n the k illin g d ilu tio n v a lu e s f o r the t h r e e
m edia w ere

pooled.

culture

64
T A B L E XIX
M E D I A U S E D IN S T U D Y
RESISTA N CE O F

ON B A C T E R I A L P O P U L A T I O N

M. PY O G E N E S VAR. AUREUS

TO P H E N O L

A

Difco

D isinfectant T e s t m ed iu m ,

l o t No.. 3 6 5 8 5 3

B

Difco D i s in f e c ta n t T e s t m e d i u m ,

lo t No. 380974

C

Difco D is in f e c ta n t T e s t m e d i u m , m a d e

D

FD A broth,

m ade

w ith Difco peptone^

lot No.

406935

E

FD A broth, m ad e

w ith Difco p ep to n e,

lot N o .

405934

F

FD A broth, m ad e

w ith A r m o u r

G

BBL

T rypticase

Soy b r o th ,

AND

up i n t h e l a b o r a t o r y

p e p t o n e , l o t No.. 1 0 0 9 7 6

lot No.

3639

%__________________________

5

.____________________________________

OF

NUMBER

OF

VIABLE

ORGANISM S.

l o g . c o u n ts o r
M /C P O C O C C U S
PYOGENES
VAR. A U R E U S A T
O , 6 A N D 2 A H R S . O N I 5T, 2 “° A N D 3*° D A Y
B R O T H TR A N SF E R S FROM S T O C K A GAR S L A N T S .

Is t TRANSFER

2 ho TRANSFER

3 r° t r a n s f e r

TABLE

XX

KILLING D ILU TIO N S OF P H E N O L ON SECOND AND
DAY B R O T H

TRANSFERS

SLANT OF

C ulture m e d ia - - lis te d

Second T r a n s f e r

FR O M N U TRIEN T AGAR

M. PY O G EN ES

in T able

Subculture m e d iu m - - D T

THIRD

VAR. AUREUS

XIX

m edium

T hird

Transfer

A

1-70

1-70

B

> 1 - 7 0 < 1 -80

> l-6 0 < l-7 0

C

1-70

1-70

D

> 1 - 6 0 < 1 -70

1-60

E

> l-6 0 < l-7 0

1-80

F

> 1 - 7 0 < 1 -80

1-80

G

1-60

> l-6 0 < l-7 0

I

67
TABLE
KILLING

T est organism --M .

XXI

DILU TIO NS* O F

pyogenes var.

A.

FD A broth

B.

DT m edium

C.

TS m e d iu m

D.

FD A broth,

DT m e d iu m

S u b c u ltu re d in S p e c ia l FD A

PHENOL

aureus

g r o w n in :

a n d TS b r o t h

B roth

S u b c u ltu r e d in DT M e d iu m

A

1 -8 2 .2 ± 8.55

1-7 4 .0 ± 3.14

B

1 - 7 6 . 5 ± 5. 0

1 - 7 1 .5 ± 5.25

C

1 - 7 8 .0 ± 7.25

1 - 6 7 . 5 ± 7.75

D

1-77.1

± 7.25

1-70.45 i

5.5

* V a lu e s o b ta in e d by c a lc u la t io n of the m e a n of killing d i l u ­
t i o n s of p h e n o l b y ' ' p h e n o l c o e f f i c i e n t ' 1 t e s t s .

68

F.

T ests
stopped the

w ere

m ade

to

see if the n e u tr a liz in g m e d ia u s e d

A culture

w a s u s e d as the te s t o r g a n is m .

the l a r g e s t a m o u n t of d i s i n f e c t a n t - d i l u t i o n th a t w as

u s e d i n a n y of t h e

tests

the n e u tra liz a tio n te s ts .
t h a t ZO m l o f m e d i u m

w as one m l,

am ount was

be u s e d in th e ir

su fficie n t, the t e s t s

20 m l o f m e d i u m
t e n t h m l of t h e

w ere

this

11 s e m i - m i c r o 1' t e s t to

In o r d e r to d e t e r m i n e
w ere

culture

r e c o r d e d a t t h e e n d o f 24 , 4 8 ,
D ata a re

r e p o r t e d o n the

m edium

R esults

72 h o u r s ' a n d s i x d a y s '

S pecial F D A b ro th and DT m e d iu m

way, it w ould be

One-

w a s t h e n a d d e d i m m e d i a t e l y to e a c h of

c o n ta in in g the d i s i n f e c t a n t - m e d i u m m i x t u r e .

specific n eu tra liz in g

if

c o n d u cted by adding

t o e a c h of t h e d i s i n f e c t a n t d i l u t i o n s .

incubation at 37 C.

the

a m o u n t w as u s e d in

K l a r m a n n a n d W r i g h t ( 15 ) r e c o m m e n d e d

stop the a c tio n of the d isin fe c ta n t.

the tu b es

a c t i o n of th e d i s i n ­

of M. p y o g en es v a r . a u r e u s

g r o w n i n DT m e d i u m

Since

this

T ests

germ icidal or bacteriostatic

fec tan t being te s te d .
w hich w as

N eutralization

six-day

were

reading.

used as w ell as

for e ach disinfectant.

In th is

d e m o n s tr a te d w h e th e r the n e u tra liz in g m e d iu m

used had a g re a te r

e ff e c t th a n th e o t h e r two m e d i a on s to p p in g

the g e r m i c i d a l o r b a c te r io s ta tic

a c t i v i t y of t h e d i s i n f e c t a n t .

69
L etheen broth

as f o rm u la te d by Q uisno, G ibby and F o te r

(23) w a s u s e d a s t h e n e u t r a l i z i n g m e d i u m f o r B T C
thio g ly co llate b ro th w as u s e d fo r M etap h en .
0.03 p e r c e n t f e r r i c
was used for

chloride

se en by exam ining

broth and DT m e d iu m

are

as

thus a llo w in g the t e s t o r g a n i s m

DT m e d iu m

C.

as the F D A b r o th p lu s

D ow icide A and D ow icide

C,

to g r o w .

M e ta p h e n i s n e u t r a l i z e d in the
20 m l o f s o d i u m

(8)

T a b l e XXII t h a t b o th F D A

effective

c h lo r id e in n e u t r a l i z i n g

FD A b ro th plus

s u g g e s t e d by F i e t t et al.

D ow icide A and D ow icide

It c a n b e

ferric

as

and sodium

1-500 d ilu tio n w ith the

th io g ly c o lla te b ro th , w hile the F D A b r o th and

a r e n o t a b le to sto p th e b a c t e r i o s t a t i c

a c t i o n of

M e t a p h e n i n a d i l u t i o n of 1 - 2 0 0 0 .
W ith B T C , L e th e e n b r o th is m u c h m o r e
n eutralizing
It w a s

effe c tiv e in

the d i s i n f e c t a n t th a n th e o t h e r two m e d i a u s e d .

effective a t a

1-1000

dilution, while

DT m e d i u m w a s

e f f e c t i v e a t a d i l u t i o n of 1 - 4 0 , 0 0 0 of B T C ,

and Special F D A

b r o t h o n ly a l l o w e d g r o w t h of th e o r g a n i s m

at a 1-80,000 d ilu ­

tio n of B T C .

1

70
T A B L E XXII
RESU LTS OBTAINED

BY N E U TR A LIZA T IO N

TEST

D ow icide A

D ilution

1-200
1-300
1-400

FDA B roth

_

+
+

S u b c u l t u r e d in:
DT M edium

D ilution

1-1000
1-2000
1-4000

FDA B roth

3

_

+
+
+
D ow icide

FDA B roth + FeC l

+
+
C

S u b c u l t u r e d in:
DT M edium

FDA B roth + F e C l3

_

_

_

+
+

+
+

+
+

M etaphen

D ilution

1-500
1-1000
1-2000

FDA B roth

S u b c u l t u r e d in :
DT M edium

_

_

-

-

-

-

T hioglycollate

B roth

+
+
+

BTC

D ilution

1-1000
1-20,000
1-40,000
1-80,000

FDA B roth

_
-

-

+

S u b c u l t u r e d in:
DT m edium
_

+
+

L etheen B roth

+
+
+
+

VI.

A study w as m a d e
the r e s i s t a n c e

W hen the
was

a n t to a l l t h e
broth

aureus

c u lt u r e m e d i a on

to d i s i n f e c t a n t s

and

a fte r they had been tr e a te d w ith d isin fe c ta n ts.
''s e m i-m ic ro "

rep resen tativ e-ty p e

m edium

e f f e c t of t h e

s u b c u l t u r e m e d i a on th e c u ltiv a tio n of b a c ­

' ' P h e n o l c o e f f i c i e n t 1' a n d
on four

of t h e

of M . p y o g e n e s v a r .

o f t h e e f f e c t of t h e
te ria l cells

SUMMARY

s h o w n to p r o d u c e

showed a g rea ter

w ere

conducted

disinfectants.

"phenol coefficient"

disinfectants

tests

technic w as used, DT

a culture

that w as m o re

tested w hereas

resistance

the

to p h e n o l .

c u lt u r e in TS
How ever,

the

killing d ilu tio n with the

TS b r o th

standard

the m e a n th an did the k illing d ilu tio n

deviation f r o m

w ith the F D A b r o t h o r the
indicate

that valu es

sisten t as those
In the

DT m e d iu m

showed a g re a te r

cu ltu res.

T h is would

o b ta in e d w ith the TS b r o t h w e r e n o t a s

con­

o b ta in e d w ith th e o t h e r two m e d i a .

c o m p a r i s o n of t h e

that F D A b r o th plus f e r r i c

s u b c u ltu re m e d ia , it w a s found

c h lo r id e w as no m o r e

S p e c ia l F D A b r o t h a lo n e in the
treated

culture

resist­

effective than

c u l t i v a t i o n of t h e d i s i n f e c t a n t -

c e l l s of M . p y o g e n e s v a r . a u r e u s .

T hioglycollate b ro th

72
and L eth een b ro th w ere
and BTC,

respectively,

DT m e d iu m
m edium
tests

was

o b tained u sin g the

not serve

as the
as

be l e s s
the

T his indicates

"phenol

coefficient"

a satisfactory

W ashed

a subculture

"phenol coefficient"

" s e m i - m i c r o '1 technic w ere

c o n s i s t e n t th r o u g h o u t the w hole

reliable

DT m ed iu m .

com pounds.

by the F D A p r o c e d u r e .
as

effective as

than w as S pecial F D A b r o th in

R esults

effective fo r M etaphen

than Special F D A b ro th o r

s h o w n to be m o r e

on a ll fo u r

not as

s h o w n to b e m o r e

cells

study as

those

obtained

th at th is m e th o d is not
technic

and th a t it would

s u b s titu te f o r the F D A p r o c e d u r e .

of M . p y o g e n e s v a r .

aureus

w ere

s h o w n to

r e s is ta n t than those in b ro th c u ltu re s n o rm a lly u se d for

"phenol coefficient"

tests.

This is

d u e to t h e f a c t t h a t t h e

f o r e i g n m a t e r i a l i n t r o d u c e d by the b r o t h i n t e r f e r e d w ith the
a c t i o n of t h e d i s i n f e c t a n t ,

allo w in g the o r g a n i s m

to a p p e a r m o r e

resistant.
The

age of the F D A b r o t h

subculture m ed iu m

appeared

t o h a v e a n i m p o r t a n t e f f e c t u p o n t h e c u l t i v a t i o n of t h e d i s i n f e c t a n t treated

cells

of M . p y o g e n e s v a r . a u r e u s .

M edia that w e re fre sh ly

p r e p a r e d w e r e not a s e ffectiv e in allow ing the o r g a n i s m s
as m ed ia that had been p re p a re d

to g r o w

s e v e r a l d a y s p r i o r to u s e ,

The

1

73
o x y g e n t e n s i o n of th e m e d i u m m a y h a v e - h a d
resu lts

obtained.

som e

e ff e c t on the

VII.

LITER A TU R E

CITED

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1911.- A m e t h o d
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2.

A r m b r u s t e r , E . H ., a n d R id e n o u r , G , M,
1947.
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S a n i t . C h e m . , 23, 11 9.

3.

B a k e r , Z .,
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J. E xptl.
M e d . , 74 , 6 1 1 .

4.

B r e w e r , C.
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V a ria tio n s in phenol coefficient d e ­
t e r m i n a t i o n s of c e r t a i n d i s i n f e c t a n t s .
A m . J. Pub.
H e a l t h , 3_3, 261..

5.
27,

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1944.
554.

6.

D o m a g k , G.
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'7.

F ildes, P .
m ercury.

R e p o r t on d is in f e c ta n ts .

A new
Soap and

The

J . A. O. A . C.,

1935.
E in e neue K la s s e von D e sin fe k tio n D e u t . M e d . W o c h s c h r . . , 61, 8 2 9 1940.
B rit.

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J . E x p t l . P a t h . , 2 1 , 6 7.

8..

F l e t t , L ., a n d H a r i n g , R .; G e u t e r a s , A., a n d S h a p i r o , R.
1945.
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J . B a c t . , 50 , 5 9 1 .

9.

G arrod, L.
1935.
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b a c terio static concentrations.
J . Inf. D i s e a s e s , 5 7 , 247.

10.

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S tu d ie n u b e r die D e s i n f e k t io n w ir k u n g
des Sublim ates.
A r c h , f u r H y g . , 90, 2 3 .

75
11 .

G r u b b , T . C ., a n d E d w a r d s , M .
1946.
A m e t h o d of r e ­
s t o r i n g a n d m a i n t a i n i n g th e p h e n o l r e s i s t a n c e of c e r t a i n
s t r a i n s o f S. a u r e u s .
J . B a c t . , 51, 2 0 5 .

12.

H ein em an n , B;
1943.
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pounds.
J . A m . P h a r m . A s s o c . , S c i. E d ., 32, 298.

13 .

J a m e s , L . H.
1947.
Q uaternaries vs.
a n d S a n i t . C h e m . , N o v . , D e c . , 125.

14.

K l a r m a n n , E . G . , a n d W r i g h t , E . S.
1945.
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Soap and
S a n i t . C h e m . , Jan.-, 113.

15 .

phenolics.

Soap

1946.
A n in q u iry into the g e r m i c i d a l p e r f o r m a n c e
of q u a t e r n a r y a m m o n i u m d is in f e c ta n ts -.
Soap and Sanit.
C h e m . , J a n ., 125.

16.

K lein , M ., a n d K a r d o n , Z. G .
1947.
T h e " r e v e r s a l , 11
n e u t r a l i z a t i o n , a n d s e l e c t i v i t y of g e r m i c i d a l c a t i o n i c
detergents.
J . B a c t . , 54, 2 4 5 .

17.

K l i m e k , J . W., a n d U m b r e i t , L . E .
1948.
O n the g e r m i ­
cid al b e h a v io r of a q u a te r n a r y a m m o n iu m co m p o u n d .
Soap
and San it. C h e m ., Ja n ., 137.

18 .

K ro n ig , B ., and P a u l, T. L .
1897.
Die c h e m i s c h e n
G ru n d lag e n d e r L ib re von d e r G iftw irkung und D e sin fe c tion.
Z e i t s c h r . f u r H y g . , 2 5 , 1.

19 .

M a l l m a n n , W. L . , L e a v i t t , A . H., a n d J o s l y n , D. A .
1948.
E f f e c t o f m e d i u m o n r e s i s t a n c e of S . a u r e u s t o q u a t e r n a r y
am m onium com pounds.
P r o c . Soc. E xptl. B iol, and M ed.,
68 , 4 0 1 .

20.

O strolenk, M.
1950-.
C o m p a r i s o n of in v i tr o g e r m i c i d e
test m ethods.
J . A m . P h a r m . A s s o c ., Sci. E d ., 3 9 , No.
2.

1

76
21.

Q u i s n o , R . , F o t e r , M . J . , a n d G i b b y , I. W .
nique for m ain ta in in g s ta n d a rd r e s is ta n c e
J . B a c t . , 52, 499i

22.

Q u isn o , R., F o t e r , M . J ., a nd R u b e n k o e n ig , H.
1947.
Q u atern ary am m o n iu m g e rm ic id e s.
Soap and Sanit.
C h e m . , J u n e , 145.

23.

Q u i s n o , R . , G i b b y , I. W . , a n d F o t e r , M . J .
1 9 4 6 .. N e u ­
t r a liz in g m e d i u m fo r e v a lu a tin g the g e r m i c i d a l p o ten cy
of th e q u a t e r n a r y a m m o n i u m s a l t s .
A . J . P h a r m . , 118,
320.

24.

25.

26.

27.

1946. A t e c h ­
f o r S. a u r e u s .

R a h n , O.
1947.
A d v i s a b i l i t y of s o m e p r o p o s e d c h a n g e s
f o r t h e e v a l u a t i o n of d i s i n f e c t a n t s .
J. A m . P h a r m .
A s s o c . , S c i . E d . , 36 , 1 3 4 .
. 1945.
I n ju r y and d e a th of b a c t e r i a b y c h e m i c a l
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R e d d is h , G.
1925.- R e s i s t a n c e
J . P u b . H e a l t h , 15, 5 3 4 .
. 1927.

to p h e n o l of S. a u r e u s .
~

E x a m i n a t i o n of d i s i n f e c t a n t s .

Am.

Am.

J. Pub.

H e a l t h , _17, 3 2 0 .
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R e d d i s h , G . , a n d B u r l i n g a m e , E.. M .
1938.
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27, 3 3 1 .

29.

~

R e d d i s h , G . , W o o d , L . , a n d B u r l i n g a m e , V.
1946,
R esist­
a n c e o f S. a u r e u s c u l t u r e d i n s e m i - s y n t h e t i c m e d i a .
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J. P h arm .,

1 1 8 , 4.

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3 4.

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S e p t ., 135.

35 .

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36 .

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Am. J .

Pub.

.
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.5 .

i~ s

CD.LTUPE M ED IA ON T H E R E S IS T A N C E AND

.u

GROWTH OF

M I C R O C O C C U S P Y O G E N E S VA;

AUREUS

By
A nita H a r r i e t

Leavitt

S u b m i t t e d t o t h e S c h o o l o f G r a d u a t e S t u d i e s of M i c h i g a n
S ta te C o lle g e of A g r i c u l t u r e a n d A p p lie d S c ie n c e
i n p a r t i a l f u l f i l l m e n t of th e r e q u i r e m e n t s
f o r t h e d e g r e e of

DOCTOR O F PH ILO SO PH Y

D e p a r t m e n t of B a c t e r i o l o g y a n d P u b l ic H e a lth

Year

1932

Approved^
V

I

1
AN ITA H A R R IE T

LEA V ITT

M icrococcus

ABSTRACT

p y o g e n e s v a r . a u r e u s w as g ro w n in s ta n d a r d

F D A 'br o th ., D i f c o D i s i n f e c t a n t T e s t ( D T ) m e d i u m a n d T r y p t i c a s e
Soy (TS) b r o th .
m icro "

tests

FDA

w ere

m entioned m ed ia .

' 'p h en o i c o e f f ic ie n t” t e s t s a n d

r u n u sin g the o r g a n i s m s g ro w n in the a b o v e A p h e n o lic , a chioro--phenol, a m e r c u r i a l and

a q u a te rn ary am m o n iu m
pounds.
m edium

DT m e d iu m ,
w ere

"sem i--

compound w ere

selected a s te st c o m ­

FDA b ro th and a specific n e u tra liz in g

used for

s u b c u ltu r ih g w h e n the test's w e r e

run.

R e s u lts in d ic a te d th a t the o r g a n i s m s gro w n in DT m e d iu m
w ere m ore

r e s i s t a n t to th e fo u r c o m p o u n d s t e s t e d th an w e r e the

o rg an ism s that w ere
sm oother

gro w n in FD A b ro th o r To broth.

A

c u ltu r e w a s m a in ta in e d in DT m e d iu m and To b r o th

t h a n w a s i n FDA, b r o t h ,

b u t v a r i a t i o n s in the

o c c u r r e d w ith the o r g a n i s m s
R esults

to phenol

g r o w n i n e a c h of t h e t h r e e m e d i a .

o b tain e d w ith the

consistent as those

resistance

' 'se m i - m ic ro ” te s ts w e re

o b tain ed by the

not as

’'phenol c o e ff ic ie n t” m e th o d .