FINAL R E P O R T

STERILITY TESTING A N D ANTIMICROBIAL

ACTIVITY O F C O M M E R C T A L G R A D E GLYCERINE

P r e p a r e d f o r

Mrs. Joyce C . Kern, Manager

G l y c e r i n e P roducer s ' A s s o c i a t i o n -. .

475 Park Avenue South

New York, New York

Warren L i t s k y

~ h r i s t o ~ h e r J . Libbey. and

Eugene J . M a r i a n i , J r .

U n i v e r s i t y of M a s s a c h u s e t t s Amhers t , M a s s a c h u s e t t s C 1002

August 1971

F i n a l Repor t

S u b j e c t :

~ t e r i l i ty t e s t i n g and a n t i m i c r o b i a l a c t i v i t y o f

commercial g r a d e s of g l y c e r i n e .

O b j e c t i v e s :

1 . To d e m o n s t r a t e t h e absence o r p r e s e n c e of m i c r o b i a l

c o n t a m i n a t i o n i n commercial g r a d e s o f g l y c e r i n e ( 9 5 % and

9 9 % ) a t t h e s i t e of p r o d u c t i o n .

2 . To d e t e r m i n e t h e b a c t e r i o s t a t i c and b a c t e r i c i d a l

a c t i v i t y o f g l y c e r i n e a g a i n s t s e l e c t e d b a c t e r i a .

The P r o g r a m

Phase I: U n d e r a g r a n t f r o m t h e G l y c e r i n e P r o d u c e r s '

A s s o c i a t i o n a s t u d y was u n d e r t a k e n t o d e m o n s t r a t e t h e p r e s e n c e

o r a b s e n c e o f m i c r o b i a l c o n t a m i n a t i o n i n 60 s a m p l e s o f

c o m m e r c i a l g r a d e ( 9 5 % a n d 9 9 % ) g l y c e r i n e . T e n s a m p l e s f r o m

e a c h o f s i x p r o d u c e r s , c o l l e c t e d f r o m drums a n d t a n k c a n s ,

w e r e f o r w a r d e d f o r a n a l y s i s b y t h e s p o n s o r .

Phase 11: The a n t i m i c r o b i a l a c t i v i t y o f c o m m e r c i a l

g r a d e g l y c e r i n e was i n v e s t i g a t e d t o d e t e r m i n e t h e c o n c e n -

t r a t i o n s n e c e s s a r y f o r i n h i b i t i o n ( b a c t e r i o s t a t i c ) a n d d e a t h

( b a c t e r i c i d a l ) o f s e l e c t e d b a c t e r i a ( S t a p h y l o c o c c u s a u r e u s ,

Pseudomonas a e r u g i n o s a , E s c h e r i c h i a c o l i , a n d S a l m o n e l l a

t y p h i m u r i u m ) as s p e c i f i e d i n USP s t a n d a r d s .

Phase One - S t e r i l i t y T e s t i n q

B a s i c a l l y t h e s t e r i l i t y t e s t i n g e m p l o y e d i n t h i s s t u d y

c o n s i s t e d o f i n n o c u l a t i n g a1 i q u o t s o f g l y c e r i n e i n t o t w o n o n

s e l e c t i v e n u t r i e n t m e d i a , i .e. , T r y p t i c a s e s o y ( B B L ) a n d

T h i o g l y c o l l a t e b r o t h s ( D i f c o ) . These m e d i a w e r e s e l e c t e d

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

o f m i n i m a l numbers o f a w i d e v a r i e t y o f o r g a n i s m s . T h i o -

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

g r o w t h o f m o s t o f a e r o b i c , f a c u l t a t i v e , a n d a n e r o b i c o rgan i : ; rns ;

w h i l e T r y ~ t i c a s e s o y b r o t h i s an e n r i c h m e n t med ium c a p a b l e

o f s u p p o r 1 . i n g g r o w t h o f .most o r g a n i s m s . common ly f o u n d i n th:!

e n v i ronmer t.

Cont ro l Sys terns :

Two samples fr.om each Producer were randomly chosen and

used i n t h e s u b s t r a t e t o d e t e r m i n e t h e a b i l i t y o f t h e g l y c e r i n e -

media sys tem t o i n i t i a t e and s u p p o r t growth o f a minimal number

o f c e l l s comparable t o t h a t which may be found i n t h e t e s t

p r o d u c t . The t e s t organism employed f o r t h i s p o s i t i v e c o n t r o l

sys tem was a s t r a i n of Pseudomonas f l u a r e s c e n s ( A . T . C . C .

#11250) s e l e c t e d because i t i s non f a s t i d i o u s , s u r v i v e s on

minimal n u t r i e n t s and i s r e l a t i v e l y r e s i s t a n t t o chemical

d i s i n f e c t i o n . C u l t u r e s were m a i n t a i n e d by weekly t r a n s f e r s

on T r y p t i c a s e soy a g a r . Twenty-four hour b.roth c u l t u r e s o f

t h i s organism were used a s . innoculum. All o f t h e tubed

media were t e s t e d f o r s t e r i l i t y by i n c u b a t i o n a t 37°C. f o r

24 h r s . prior t o use . T h i s p r e - i n c u b a t i o n minimized t h e f a l s e

p o s i t i v e r e s u l t s from c o n t a m i n a t e d media.

To t e s t t h e p o s i t i v e c o n t r o l sys tem t u b e s c o n t a i n i n g

9.6 ml o f media , t o which g l y c e r i n e was added i n both 1 ml

and 5 ml amounts , were i n n o c u l a t e d w i t h dec imal d i l u t i o n s

o f t h e t e s t o rgan i sm r a n g i n g from ::800 t o 2 . 6 o r g a n i s m s . All

o f t h e g l y c e r i n e - m e d i a t u b e s were : haken t w e n t y - f i v e t i m e s i n

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

c e r i n e . C o n c u r r e n t l y , t u b e s c o n t a ; n i n g 9 . 6 ml o f media w i t h -

o u t g l y c e r i n e were i n n o c u l a t e d a s zbove t o d e m o n s t r a t e t h a t

growth o f t h e t e s t organ'ism a t a p, r t i c u l a r c o n c e n t r a t i o n

cou ld be i n i t i a t e d i n t h e media . I 'nseeded media c o n t r o l s

were a l s o i n c u b a t e d a t each temper, t u r e t o d e m o n s t r a t e

s t e r i l i t y of t h i s component. Al l o f t h e above c o n t r o l s

were i n c u b a t e d i n d u p l i c a t e a t 20°C. and 37°C.. f o r f o u r t e e n

days . D a i l y o b s e r v a t i o n s were made t o d e t e r m i n e p r e s e n c e

o r absence o f growth.

I n a l l c a s e s s t e r i l i t y was obse rved i n t h e unseeded

t u b e s of media. L i k e w i s e , growth o f t h e t e s t o rgan i sms i n

both media i n c u b a t e d a t 37OC. b u t n o t a t 20°C. was obse rved

. w i t h i n 2 4 h r s . Tab le I shows r e s u l t s o b t a i n e d when t h e

seeded g l y c e r i n e was i n c u b a t e d a t 20°C. a n d 37°C. f o r f o u r t e e n

days . In g e n e r a l t h e growth o b s e r v e d i n t u b e s c o n t a i n i n g 1 ml '. -

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

no g l y c e r i n e . In o t h e r words i t was demons t ra ted t h a t a n

innoculum i n t h e range o f 2 . 6 c e l l s s u r v i v e d and f l o u r i s h e d

i n an envi ronment comprised o f one ml of g l y c e r i n e and

9.6 ml of media. The t u b e s c o n t a i n i n g 5 ml o f g l y c e r i n e were

i n h i b i t e d by t h e g l y c e r i n e t o v a r y i n g d e g r e e s dependen t upon

t h e number o f organisms p r e s e n t .

S t e r i l i t y l e s t s :

Havins s a t i s f i e d o u r s e l v e s w i t l ~ an a d e q u a t e sys tem

which i n i t ' a t e d a n d s u p p o r t e d growth o f a minimal number o f

organisms ( t e r i l i t y t e s t s were conducted i n t h e f o l l o w i n g . manner: T r y p t i c a s e soy and T h i o g l y ! : o l l a t e b r o t h s were seeded

w i t h bo th ' m l a n d 5 ml a l i q u o t s from a l l g l y c e r i n e samples

f r o m each ~ l r o d u c e r . I n o t h e r words f o u r t u b e s o f T r y p t i c a s e

soy b r o t h ; .nd f o u r t u b e s of Thiog1yc:ol la te b r o t h were

i n n o c u l a t e d w i t h 1 ml and 5 m l of eilch sample o f g l y c e r i n e .

Two tubes wi th each a l i q u o t were then i ncuba t ed a t 37°C. and

a t 20°C. f o r f o u r t e e n days a n d obse rved d a i l y f o r growth.

One h u n d r e d - s i x t y - t u b e s were used t o t e s t a s e t of t en samples

from each producer and a t o t a l volume of 4 8 ml of each sample

was t e s t e d .

The tubes from f o u r samples of each producer which

showed n o growth a f t e r f o u r t e e n days o f i n c u b a t i o n were

reseeded by loop i n t o t h e i r r e s p e c t i v e media and i ncuba t ed

a s above. Th i s was done t o de t e rmine whether t h e n e g a t i v e

growth o b s e r v a t i o n was due t o t h e absence of organisms i n

t h e o r i g i n a l sample o r due t o i n h i b i t i o n e x e r t e d by t 'he. -.. . a l i q u o t of g l y c e r i n e t h a t was t e s t e d . In s h o r t , what t h i s

t e chn ique accompl ishes i s t o d i l u t e t h e c o n c e n t r a t i o n of

g l y c e r i n e i n t h e reseeded t u b e . The l i m i t a t i o n of t h i s t e s t

i s t h a t a n ex t remely low c o n c e n t r a t i o n of microorganisms i n

t h e o r i g i n a l may be missed on loop t r a n s f e r t o t h e second

tube . However, a s suppo r t ed by t h e d a t a i n Phase 11, i t

was demons t ra ted t h a t 1 rnl a l i q u o t s of g l y c e r i n e were n o t

i n h i b i t o r y t o t h e organisms employec'.

Samples d i s p l a y i n g one o r more p o s i t i v e growth t u b e s

a f t e r f o u r t e e n days were r e t e s t e d ucing t h e above d e s c r i b e d

t echn ique i n o r d e r t o conf i rm t h a t t h e g r o w t h o r i g i n a t e d i n

t h e sample of g l y c e r i n e and n o t by chance con t amina t i on . . P o s i t i v e tubes from t h e second t e s t s e r i e s were t r a n s f e r r e d

t o T r y p t i c t s e s ~ y b ro th f o r en r i chmen t and i ncuba t ed a t t he

o r i g i n a l temperature f o r 2 4 h r s . Fcl lowing t h i s they were

s t r e a k e d or T r y p t i c a s e soy a g a r w h i c h was aga in i ncuba t ed f o r

24 h r s . I s o l a t e d c o l o n i e s were examined macro and micro-

s c o p i c a l l y f o r genus i d e n t i f i c a t i o n .

R e s u l t s o f S t e r i 1 i ty T e s t s :

A f t e r c o m p l e t i o n o f t h e e n t i r e s t e r i l i t y p r o c e d u r e i t

was c o n c l u d e d t h a t o n e s a m p l e (8C) o f t h e s i x t y e x h i b i t e d

m i c r o b i a l c o n t a m i n a t i o n . T h i s c o n c l u s i o n i s b a s e d o n t h e

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

w i t h one m l o f t h i s s a m p l e a n d i n c u b a t e d a t 3 7 ° C . showed

g r o w t h a f t e r f o u r d a y s . L i k e w i s e , g r o w t h a f t e ' r s e v e n d a y s

a t 20°C. was o b s e r v e d i n a t u b e o f T r y p t i c a s e s o y b r o t h

i n n o c u l a t e d w i t h f i v e m l o f t h i s s a m p l e . Upon r e - e x a m i n a t i o n

o f t h e o r i g i n a l s a m p l e , o n e t u b e o f T h i o g l y c o l l a t e b r o t h ,

s e e d e d w i t h one m l o f t h i s s a m p l e showed g r o w t h a f t e r 2 4 h r s .

a t 37°C. To f u r t h e r v e r i f y o u r f i n d i n g s t h i s s a m p l e was

e x a m i n e d a g a i n a n d t h i s t i r n e g r o w t h o c c u r r e d a f t e r 2 4 h r s .

a t 37°C. i n 2 t u b e s o f T r y p t i c a s e s o y b r o t h . The c o l o n i a l

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

m i c r o s c o p i c o b s e r v a t i o n s i n d i c a t e d t h a t t h i s c o n t a m i n a n t

was " v i r t u a l l y " a p u r e c u l t u r e o f a g ram p o s i t i v e s p o r e

f o r m i n g r o d o f t h e g e n u s B a c i l l u s .

O c c a s i o n a l l y g r o w t h was a l s o c b s e r v e d i n o t h e r t u b e s

b u t u p o n r s t e s t i n g o f t h e o r i g i n a l s a m p l e no g r o w t h was e v e r Q

o b s e r v e d . N e g a t i v e f i n d i n g s o f t h ~ s e c o n d d e t e r m i n a t i o n

p r o m p t e d us t o c o n c l u d e t h a t t h e o r i g i n a l g r o w t h was d u e t o

an o c c a s i o q a l l a b o r a t o r y c o n t a m i n a t i o n . When o n e c o n s i d e r s

t h a t a t o t a l o f f o r t y - e i g h t t u b e s h e r e u s e d f o r e a c h s a m p l e

t h e c h a n c e o f a c o n t a m i n a n t i s h i g l l y p r o b a b l e . O n l y o n e 01'

t h e 59 r e r n i i n i n g s a m p l e s i n d i c a t e d m o r e t h a n o n e " f a l s e

p o s i t i v e " t u b e .

PHASE I 1 - EVALUATION O F

.BACTERIOSTATIC - BACTERICIDAL PROPERTIES O F GLYCERINE

Samples o f g l y c e r i n e were t e s t e d f o r b a c t e r i o s t a t i c and

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

Pseudomones a e r u g i n o s a A.T.C.C. #10145 , E s c h e r i c h i a c o l i

A . T . C . C . $11 7 7 5 , S a l m o n e l l a typhirnurium A . T . C . C . # I3311 and

S t a p h y l o c o c c u s a u r e u s A.T.C.C. #12600.

Zone of I n h i b i t i o n T e s t :

I n a p r e l i m i n a r y e x p l o r a t i o n t h e zone o f i n h i b i t i o n t e s t

was used employing a m o d i f i c a t i o n o f t h e c y l i n d e r - p l a t e

method i n which a h o l e , which s e r v e d a s a r e s e r v o i r f o r c a .

2 ml o f . g l y c e r i n e , was c u t i n 2 h e a v i l y s e e d e d T r y p t i c a s e s o y

a g a r p l a t e . S i n c e n o zone o f i n h i b i t i o n was o b s e r v e d a f t e r

24 hrs. a t 20°C. and a t 37°C. t h e r e s u l t s i n d i c a t e d t h a t

g l y c e r i n e was n o t i n h i b i t o r y a g a i n s t t h e above o r g a n i s m s

unde r t h e c o n d i t i o n s o f t h i s t e s t . Moreove r , t h i s chemica l

a c t u a l l y enhanced t h e grow,th o f L c h e r i c h i a c o l i a s demonstrqated

by a h e a v i 3 r growth a t t h e p e r i p h e r y o f t h e r e s e r v o i r .

R e a l i z i n g t h a t t h e p r e s e n c e o r a b s e n c e ( and a l s o t h e d i a m e t e r )

o f t h e zon2 o f i n h i b i t i o n e x h i b i t e c by t h e t e s t o rgan i s ins i!.

d e p e n d e n t J n t h e c o n c e n t r a t i o n of ? h e chemica l compound i n -:he

medium, i t may be c o n c l u d e d t h a t t t e f i n d i n g s o f t h e p r e -

l i m i n a r y s t u d y was t h e r e s u l t o f t l e r a p i d d i f f u s i o n o f t h e

g l y c e r i n e th rough t h e medium d e c r e c s i n g i t s c o n c e n t r a t i o n t o

a n o n - i n h i a i t o r y r a n g e . For t h i s i . ea son a m o d i f i e d t u b e

d i l u t i o n t z c h n i q u e was s u b s e q u e n t l ! employed i n f u r t h e r

e v a l u a t i o n .

M u l t i p l e Tube D i l u t i o n T e s t :

A m u l t i p l e t u b e t e c h n i q u e ( 1 6 t u b e s p e r d i l u t i o n ) was

u s e d i n w h i c h v a r y i n g a l i q u o t s o f g l y c e r i n e a n d m e d i a ,

T r y p t i c a s e s o y b r o t h , w e r e combi ned, These a 1 i q u o t s a l o n g

w i t h t h e i n n o c u l a t i o n p r o d u c e d v o l u m e t o v o l u m e d i l u t i o n s

r a n g i n g f r o m a p p r o x i m a t e l y one p e r c e n t t o one h u n d r e d p e r c e n t

g l y c e r i n e . The u n u s u a l p e r c e n t a g e s o f g l y c e r i n e r e p o r t e d

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

c h e m i c a l ; e.g. , a 1 0 rnl p i p e t t e d e l i v e r e d o n l y 9 . 5 m l o f

g l y c e r i n e a n d t h i s i s r e f l e c t e d i n t h e c a l c u l a t i o n s . The

t u b e s w e r e t h e n s e e d e d w i t h 0'.1 m l o f a 1 :10 d i l u t i o n o f a

24 h r . b r o t h c u l t u r e o f t h e r e s p e c t i v e o r g a n i s m . T h e s e t u b e s

w e r e s h a k e n t o a d e g r e e s u f f i c i e n t t o ' i n s u r e a n e v e n s u s p e n s i o n

o f t h e g l y c e r i ne-medium s y s tern.

The s e e d e d g l y c e r i n e - m e d i u m t u b e s , w i t h g l y c e r i n e

c o n c e n t r a t i o n s o f 6 . 7 % t o 31.3% w e r e i n c u b a t e d a t 3 7 O C . f o r

f o u r t e e n d a y s w i t h d a i l y e x a m i ' n a t i o n f o r g r o w t h . Any t u b e s

i n w h i c h g r o w t h c o u l d be d e t e c t e d (no i n h i b i t i o n ) w e r e s o

i n d i c a t e d a n d d i s c a r d e d . A f t e r 48 h r s . a l l t u b e s s h o w i n g

i n h i b i t i o n w e r e r e s e e d e d i n t o T r y p t i c a s e s o y b r o t h a n d r e -

i n c u b a t e d 48 h r s . t o d e t e r m i n e w h e t h e r t h e i n h i b i t i o n was

due t o b a c t e r i o s t a t i c o r b a c t e r i c i d a l a c t i v i t y . T h e s e

r e s u l t s s u m m a r i z e d i n T a b l e I I i n d i c a t e t h a t b a c t e r i o s t a t i c -

b a c t e r i c i d a l d i f f e r e n t i a t i o n c o u l d n i t be d e t e r m i n e d w i t h

t h e c o n c e n t r a t i o n o f g l y c e r i n e e m p l o y e d ' s i n c e a l l t h e r e -

I . seeds w e r e p o s i t i v e .

The above e x p e r i m e n t was r e p e a t e d u s i n g h i g h e r c o n c e n t r d -

t i o n s of 23 .6% t o 9 8 . 9 % g l y c e r i n e . Again t h o s e t u b e s showing

i n h i b i t i o n were r e s e e d e d a f t e r 4 8 h r s . Of t h e f o r t y - e i g h t

h o u r r e s e e d s , t h o s e t u b e s t h a t showed growth demons t ra ted t h a t

b a c t e r i o s t a s i s had o c c u r r e d i n t h e o r i g i n a l t u b e which was

a g a i n r e s e e d e d a f t e r seven days t o de te rmine i f t h e c u l t u r e

was k i l l e d upon p ro longed c o n t a c t . Negat ive r e s e e d s were

recorded a t 4 8 h r s . and o b s e r v e d f o r f o u r t e e n d'ays. A t no

i n s t a n c e d i d a n e g a t i v e r e s e e d become a p o s i t i v e d u r i n g t h i s

p e r i od.

R e s u l t s and D i s c u s s i o n :

As shown i n T a b l e I11 t h e c o n c e n t r a t i o n s of g l y c e r i n e

t h a t can be c o n s i d e r e d b a c t e r i o s t a t i c were t h o s e i n which

f o r t y - e i g h t hour r e s e e d s i n d i c a t e d growth whereas t h o s e

i n d i c a t i n g no growth u p o n r e s e e d i n g were c o n s i d e r e d b a c t e r i -

c i d a l . I t s h o u l d be no ted t h a t t h i s s h i f t from b a c t e r i o s t a t i c

t o b a c t e r i c i d a l e f f e c t o c c u r r e d i n a r ange a s i n d i c a t e d i n

Table 111. A d d i t i o n a l l y t h e d a t a a l s o demons t ra ted t h a t a

b a c t e r i o s t a t i c c o n c e n t r a t i o n o f g l y c e r i n e may become b a c t e r i - c i d a l u p o n p ro longed c o n t a c t w i t h tile organism. For example

37.8 p e r c e n t g l y c e r i n e was found t o be t o t a l l y ( a l l 16 t u b e s )

b a c t e r i o s t a t i c f o r Salmonel l a t y p h i ~ i u r i u m a f t e r f o r t y - e i g h t

h o u r s , w h i l e a f t e r seven days above 2 3 . 6 p e r c e n t was f o u n d

t o be b a c t e r i c i d a l . The d a t a a s t a l ~ u l a t e d i n Tab le I11 a l s o

i n d i c a t e a p o s i t i v e c o r r e l a t i o n between t h e c o n c e n t r a t i o n s o f

g l y c e r i n e r e q u i r e d f o r b a c t e r i o s t a s ' s w i t h t h o s e f o r b a c t e r i - c i d a l a c t i v i t y , e.g. , Pseudomonas ac:ruqinosa was i n h i b i t e d a ~ d

k i l l e d a t t h e l o w e s t c o n c e n t r a t i o n s v ih i l e S t a p h y l o c o c c u s a u r e u s

was i n h i b i t e d a t t h e h i g h e s t c o n c e n t r a t i o n s . T h i s phenomenon

o c c u r r e d b o t h f o r t h e f o r t y - e i g h t h o u r a n d s e v e n d a y r e s e e d s .

The r e s u l t s o f o u r s t u d y c o n f i r m i n p a r t t h o s e o f B a r r

a n d T i c e who d e m o n s t r a t e d t h a t S t a p h y l o c o c c u s a u r e u s and

Pseudomonas a e r u g i n o s a w e r e i n h i b i t e d b y 30 a n d 25 p e r c e n t

r e s p e c t i v e l y o f g l y c e r i n e o n a w e i g h t t o w e i g h t b a s i s . 1

T a b l e I V i l l u s t r a t e s t h e s i m i l a r i t y o f t h e f i n d i n g s f r o m

b o t h s t u d i e s .

The d a t a r e p r e s e n t e d i n t h e c u r r e n t s t u d y c l e a r l y i n d i -

c a t e t h a t t h e r e i s g r e a t v a r i a t i o n i n s u s c e p t i b i l i t y a n d / o r -,

r e s i s t a n c e o f t h e t e s t o r g a n i s m s t o g l y c e r i n e . T h i s w o u l d

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

c l i n i c a l s p e c i m e n s may r e q u i r e some r e - e v a l u a t i o n e v e n t h o u g h

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

t h a n t h a t e m p l o y e d i n t h i s i n v e s t i g a t i o n . A f t e r f o r t y - e i g h t

h o u r s o f i n c u b a t i o n S a l m o n e l l a t y p h i m u r i u m a n d E s c h e r i c h i a

c o l i d e m o n s t r a t e d e q u a l s u s c e p t i b i l i t y t o t h e b a c t e r i o s t a t i c

c o n c e n t r a t i o n o f g l y c e r i n e w h i l e the S a l m o n e l l a t y p h i m u r i u m

d i s p l a y e d ~ r e a t e r s u s c e p t i b i l i t y t o t h e b a c t e r i c i d a l c o n -

c e n t r a t i o n . T h e s e o b s e r v a t i o n s togc t h e r w i t h t h e s t i m u l a t o r j *

e f f e c t s o f g l y c e r i n e o n t h e g r o w t h c . f E s c h e r i c h i a c o l i demon-

s t r a t e d b y t h e z o n e o f i n h i b i t i o r f ternst s u g g e s t t h a t c a u t i o n

b e t a k e n w t e n u s i n g g l y c e r i n e as a p r e s e r v a t i v e f o r f e c a l

s a m p l e s f r o m w h i c h t h e i s o l a t i o n o f S a l m o n e l l a i s d e s i r e d .

' ~ a r r , M. a n d T i c e , L., A s t u d y o f f he i n h i b i t o r y c o n c e n t r a - t i o n s o f v a r i o u s s u g a r s and p o l y o l s o n t h e g r o w t h o f m i c r o - o r g a n i s m s . J. P h a r m a c e u t i c a l Assn. , - 46 ( 4 ) : 2 1 9 - 2 2 1 .

C o n c l u s i o n s

1. O f t h e s i x t y s a m p l e s o f g l y c e r i n e t e s t e d o n l y o n e

(8C) was. f o u n d t o b e c o n t a m i n a t e d u n d e r t h e c o n d i t i o n s

o f t h e t e s t e m p l o y e d . T h i s c o n t a m i n a n t was i d e n t i f i e d as

a member o f t h e g e n u s B a c i l l u s . S i n c e t h e i n v e s t i g a t o r s

h a d no c o n t r o l o v e r e i t h e r t h e s t e r i l i z a t i o n o f . t h e c o n t a i n e r s

i n w h i c h t h e s a m p l e s w e r e c o l l e c t e d a n d s h i p p e d o r t h e a c t u a l

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

a s c e r t a i n e d .

2. G l y c e r i n e was f o u n d t o b e b a c t e r i o s t a t i c a n d b a c t e r i c i d a l

a t v a r i o u s c o n c e n t r a t i o n s a g a i n s t Pseudornonas a e r u g i n o s a ,

E s c h e r i c h i a c o l i, S a l m o n e l l a t y p h i m u r i u m a n d S t a p h y l o c o c c u s

a u r e u s .

3. A p o s i t i v e c o r r e l a t i o n was f o u n d b e t w e e n t h e b a c t e r i o -

s t a t i c ' a n d b a c t e r i c i d a l c o n c e n t r a t i o n s o f g l y c e r i n e a g a i n s t

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

4 . The u s e o f g l y c e r i n e a s a p r e s e r v a t i v e o f c l i n i c a l s a m p l e s

w h i c h a r e t o be a n a l y z e d f o r Salmonella a n d o t h e r p a t h o g e n i c

e n t e r i c o r g a n i s m s s h o u l d b e r e - e v a l ~ a t e d i n l i g h t o f o u r

l i m i t e d d a t a .

T a b l e I . P o s i t i v e C o n t r o l Sys tem

Average Number o f C e l l s I n n o c u l a t e d P e r Tube

T h i o g l y c o l 1 a t e T r y p t i c a s e s o y

P r o d u c e r A Sample 8A

37°C. 1 m l 5 ml

Sample 4A 37°C. 1 ml

5 ml

P r o d u c e r B Sample 2 B

' . 37°C. 1 ml 5 m l

Sample 10B 37°C. 1 ml

5 ml

P r o d u c e r C 2600 260 26 2.6 2600 260 26 2 .6 Sample 1C

37°C. 1 ml 1 2 2 - 1 1 2 - 5 ml 2 2 - - 2 2 - -

Sample 10C 37°C. 1 ml 1 2 2 - 1 1 1 1

5 m l 2 2 - - 2 ' 2 2 -

Number i n d i c a t e r day on which g rowth was o b s e r v e d .

- i n d i c a t e s no yrowth f o r f o u r t e e n d a y s .

~ a b l ' e I (Con t inued)

Average Number of C e l l s I n n o c u l a t e d Pe r Tube

T h i o g l y c o l l a t e T r y p t i c a s e soy

Producer D 3200 320 32 3.2' 3200 320 32 3.2 Sample 4 0

37°C. 1 ml 1 .1 1 2 1 1 1 2 5 ml - - - - 3 6 - -

Sample 7D 37°C. 1 ml 1 1 2 - 1 1 - -

5 ml - - - - 9 - - -

,P roducer E Sample 10E

37°C. 1 ml 5 ml

Sample 3E 37°C. 1 ml

5 ml

Producer F Sample 7 F

37°C. 1 ml 5 ml

Sample I F 37°C. 1 ml 1 1 1 ' 1 . 1 1 1 1'

5 ml - - - - 3 - - -

Number i n d i c a t e s day o n which growth was o b s e r v e d .

- i n d i c a t e s no g-owth f o r f o u r t e e n d a y s .

I I..

Ill a , + - 0 LI Q) ((1 'I- I- u-

W

--

t --a) rD 0

rn >- Ill . C - a, . - m n t 3 2 'g t

m m m m . . - a - - - -

m m m m

I - - - - . . . . - d b m W N N

- b e d . . . . d - m m m N - - -

- - - -

U1

Kl m - C 0 E

L 2 0 .

r E 1

m C .- t ((1 U .- D C .- > \

Q) C .- Q) U > - a, '3 (0

t; 0

UL a) a

+-l Ln -0 a) a, m a, CL

- t 3

E' a

. .

C 0 --

> t .- .O .- f

L I S : - - m

0 ) t 0

I-

- c 0 .- t .- n .- C - 0 z

- -- rp

E cn .- c (0 0 L 0

t Ill a) I-

*

Table I I I

Bacteriostatic-Bactericidal Concentration ( % I of Glycerine

1 Test Organisc ' 48 hr. reseeds 7 day reseeds I

I. Bacteriostatic--All 38 hr. or 7 day reseeds at that concen-fration of glycerine demonstrated growth after 48 hrs. incubation at 37OC.

2. Bactericidal--No growth in 48 hr. or 7 day reseeds at that concentration of glycerine after 48 hrs. incubation at 37OC. (Observed for 14 days of i ncubatlon).

Staphylococcus aureus

Escherichia coli

Salmonella typhimurium

I Pseudornonas aeruginosa I I 28.7 1 40.5 523.6 - 1 23.6 I

~acter iostat ic'

88.8

37.8

37.8

I Bactericida 1 ' None

58.6

48.1

Bacter i osfat ic

167.8 -

- 437.8 -

23.6

Bacter i ci da I

67.8

37.8

33.6

Raw Data Phase 11:

Evaluation of Bac te r ios ta t i c , Bactericidal

P r o ~ e r t i e s of Glvcerine

C o n t e n t s of A ~ ~ e n d i x :

1. P a r t I

The d e t e r m i n a t i o n o f t h e i n h i b i t o r y c h a r a c t e r i s t i c s o f

g l y c e r i n e a g a i n s t Pseudomonas a e r u g i n o s a A.T.C.C. #10145,

E s c h e r i c h i a c o l i A.T,C.C. #11775, S a l m o n e l l a typhimurium

A.T.C.C. #I3311 and S taphy lococcus a u r e u s A,T.C.C. #12600.

2. P a r t 1 1

The d e t e r m i n a t i o n o f whe the r t h e i n h i b i t i o n demons t ra ted

I n P a r t I i s b a c t e r i o s t a t i c o r b a c t e r i c i d a l , f o r t h e

above organisms.

Phase I I : B a c t e r i o s t a t i c - B a c t e r i c i d a l Proper t ies

P a r t I

Escher ichia c o l i A.T.C.C. #I1775

Date: i - ? - 3/

I n h i b i t i o n : i i / c$dy~

Cel l count: f- 2; A l c I*

Percent/ No. P o s i t i v e tubes o u t o f 16

G l y ~ e r i n e / ~ ~ ~ I 2 3 4 5 6 7 8 9 10 1 1 I 2 13 14

Reseeds--48 hr .

Date: 6 - / O - 3/

Percent T o t a l Pos. Glycer ine Tubes Tubes

Phase I I : B a c t e r i o s t a t i c -Eac te r i c ida I Propert- ies

P a r t I

Psoudornonas aeruginosa A.T.C.C. #lo145

Date: 6 - / c - 7" I n h i b i t i o n : ,,v' .{L,GJ-

r'

C e l l count: t : ~ ,< / I : -

. Percent / No. P o s i t i v e tubes o u t o f 16

Glycer ine/ d a y l 2 3 4 5 6 7 8 9 10 1 1 12 13 14

Reseeds--48 hr .

Date: 6 . / c - ?/

Percent Tota l Pos. Glycer ine Tubes Tubes

Phase 1 1 : B a c t e r i o s t a t i c - B a c t e r i c i d a l P r o p e r t i e s

P a r t I

Date: G - d J - 2'

I n h i b i t i o n : ,y,JdP~

Cel l coun t : ,s J A , , ~ r

Salmonel la t yph imur ium A.T.C.C. 813311

Percen t No. P o s i t i v e tubes o u t o f 16

G I yce r i ne /day 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14

Reseeds--48 h r .

Date: 6 - / L1 ' .=)/

P r c e n t T o t a l Pos. GI y c e r i ne Tubes Tubes

Phase I I : B a c t e r i o s t a t i c - B a c t e r i c i d a l P r o p e r t i e s

P a r t I

Staphylococcus aureus A.T.C.C. #I2600

Date: (- - ,$'- 7/

I n h i b i t i o n : ,)/ (?.J~J'

Ce l I count : 7 <, A ,-c? 1

Percen t / No. P o s i t i v e tubes o u t o f 16

G l y c e r i n e / day 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14

Reseeds--48 h r .

Date: 6 -/O - 3' Percent T o t a l Pos.

G l yce r i ne Tubes Tubes

a t n 3 s 3 c n O 3 \ \ \

2 k t

cn L * a , L m n

0 3 :3 , g - C X

a, a t \ .o 3 9 c e

5 - - V) 3 m (D 0 + o 3 3 9 3 3 4 4

e ,+ C - - m (0 - 0 - m n t 0 3 - L + O Q C O @ Q Q C , G @ C -

0 c - L Q, U > - -& BP kP = = ' h e as Be 8 a M cn a 3 L n - m o . . a3 Ln a 3 0 . .

O M a 3 ( 3 \ a 3 P CD 0 3 a O t r r \ d U " \ \ O a ) c J ' C m U L a, a

--

9 3 \ \ \

C ' C C

- m m o

a , t n 0 3 k t

m

c n a 0 0 3

a t

- m coo t o 0 3 t-t

In - a , cnn 0 3 a +

- m m a ,

. \ \

O Q

-0 a,

m a, L

>. (D

P

--- u a, a, m a, L

I r N r-

r

u a,

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3

-

c

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4

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o c a ------

. S 3 < \

0 \

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t

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u a, a, m a, L

L IS

- m m a , t o 0 3 I -+

' 0 m a , 0 0 CL 2 t

- cn m a , t o 0 2 I - t

'V) m a , 0 0

00 w

5 - 3 O

C - - lu .- - C -

a,

$ \

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Q 3 t

* - m m a , t n 0 2

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Ln n t o 3

a t

C

\ \

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C - L a, 0 > - cn

t, a, f a, L

BP BQ

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\ \ \ \

Q Q c o u

ae \ O I . \ D a 3 ' n

m c ~ m 1 . 0 C \ l h ( M ~ - .

B?E

n

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7

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c

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W L B P B c e a 3 n a m 1 . ~ 3 a c 0 . V J P C C ' C "

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t

-0 a, a, m a, L

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I. r

73 a, a, cn a, L .

L c

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5 - 3 U

Oc C

- m - 2 c -

0, C - L a, U > - 0)

t C a, U L a, a

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9 . 3 4 9

4

3 3 \ \ ' \ \

G C ) t > C

- m n a t o 0 3 k t

m - a

m a 0 3 a t

- m m a , t o 0 3 k t

m a,

m o 0 3 0-t

- m Q a , t o 0 3 b t

m a,

m 1 ) 0 2 0 - t