EFFECTS OF CERT M E ANIONIC, CATIONIC, AND NOE-IONIC AGENTS OE GROWTH OF ESCHERICHIA COLI, SALMONELLA PARATYPHI B, AND STAPHYLOCOCCUS AUREUS by CHARLES GAIEOR A THESIS S u b m itted t o t h e S c h o o l o f Graduate S t u d i e s o f M ichigan State C o l l e g e o f A g r i c u l t u r e and A p p l i e d S c i e n c e in P a r t i a l F u lfillm e n t o f the R equirem ents fo r the Degree o f DOCTOR OF PHILOSOPHY D e p a r t m e n t o f B a c t e r i o l o g y and P u b l i c H e a l t h -1 9 4 9 ACZtTOWLEDGMENT The a u t h o r w i s h e s t o e x p r e s s h i s t o D r . W.L* Mallmann f o r h i s throughout t h i s study. sin ce re ap p reciation e n c o u r a g e m e n t and a d v i c e He i s a lso in d eb ted to the v a r i o u s m a n u f a c t u r e r s who g e n e r o u s l y c o n t r i b u t e d t h e c h e m i c a l compounds u s e d i n t h i s in v estig a tio n . TABLE OF CONTENTS Page I. II. INTRODUCTION . . ......................................... 1 HISTORICAL SURVEY................................................................... 4 I I I . MATERIALS AND EQUIPMENT A. G u l t u r e Medium .............................................................. 9 B. S u r f a c e - A c t i v e A g e n t s ............................................... 10 ............................................... 13 D. E q u i p m e n t .............................................................................. 13 C. B a c t e r i a l C u l t u r e s IV. EXPERIMENTAL PROCEDURE A. P r e l i m i n a r y S t u d i e s 1. C h o i c e o f M e d i u m .................. ........ 14 2. S electio n 14 o f pH R a n g e s . . . . . . . B. M a i n t e n a n c e o f C u l t u r e s 15 C. Growth C u r v e s and R e l a t e d Phenomena . . . 15 V. RESULTS A. C o n t r o l T e s t s .................................................................... 22 B. A n i o n i c Compounds .......................................................... 22 C. C a t i o n i c 22 D. N o n - I o n i c C o m p o u n d s .................................................... Compounds . 41 ............................................... 44 F . S u r f a c e - T e n s i o n and pH C o n t r o l s ..................... 50 E. B a c t e r i o s t a t i c T e s t s VI. ......................... . GENERAL D I S C U S S I O N ........................................................ V II. V III. 58 SUMMARY......................................................................................... 66 LITERATURE C I T E D ............................................................... 68 I. S u rfa ce-a ctiv e INTRODUCTION agents are cu stom arily d e fin e d as s u b s ta n c e s w hich a l t e r t h e e n e r g y r e l a t i o n s h i p s a t i n t e r ­ faces* gents, In t h i s c a t e g o r y are in c lu d e d such a g e n ts a s d e t e r ­ em u lsifiers, w e t t i n g a g e n t s , and s i m i l a r s u b s t a n c e s , some o f w h i c h d e m o n s t r a t e b a c t e r i c i d a l or b a c t e r i o s t a t i c action . Because th e term " s u r f a c e - a c t iv e gen era l one, the p resen t ten dency i s to pounds as w e t t i n g a g e n t s , d e t e r g e n t s , agent" i s such a c l a s s i f y t h e s e com­ etc*, according to the f u n c t i o n s t h e y b e s t perform . S in ce the effectiv en ess of s y n t h e t ic s u r f a c e - a c t iv e a g e n t s a s s o a p s u b s t i t u t e s w a s d i s c o v e r e d d u r i n g World War I , an i n c r e a s i n g amount o f i n d u s t r i a l r e s e a r c h h a s b e e n d e v o t e d to the developm ent of th ese compounds. the l a s t 15 y e a r s a tremendous a p p e a r e d on t h e m a r k e t . d evelop ed , however, As a r e s u l t , w ith in q u a n tity o f such p rod u cts has The r a p i d i t y w i t h w h i c h t h e y w e r e d id n o t perm it the th orou g h , b a s i c search n e c e s s a r y to d e fin e c le a r ly the re­ r e l a t i o n s h i p between t h e s e compounds and t h e i r b i o l o g i c a l a c t i v i t i e s . N ev erth eless, a vast ing them h a s b e e n p u b l i s h e d . amount o f l i t e r a t u r e concern­ Many o f t h e s e p a p e r s h a v e b e e n e x c e l l e n t , c l a r i f y i n g numerous a s p e c t s o f t h e r e l a t i o n s h i p b e t w e e n s u r f a c e - a c t i v e a g e n t s and b a c t e r i o l o g i c a l a c t i v i t y . Some o t h e r s , however, have r e p o r t e d e x p e r im e n ts d e m o n str a tin g b a c t e r i o s t a t i c and g e r m i c i d a l a c t i o n o f s u r f a o e - a c t i v e which c o u ld n o t b e d u p l i c a t e d i n o t h e r l a b o r a t o r i e s , that m atter, in the same l a b o r a t o r y . T h is was d u e, agents or, for i n some 2 o a s e s , t o in a d e q u a t e d e f i n i t i o n o f c o n d i t i o n s under which t h e t e s t s w e r e made, and i n o t h e r s t o t h e i n a p p l i c a b i l i t y o f t h e t e s t em ployed ( f o r e x a m p le, u s e of th e P h e n o lC o e f f i c i e n t Method w i t h q u a t e r n a r y ammonium c o m p o u n d s ) . The m a j o r i t y o f t h e r e s e a r c h on t h e in flu en ce o f s u r f a c e - a c t i v e a g e n t s on b a c t e r i o l o g i c a l a c t i v i t y h a s b e e n d i r e c t e d toward e s t a b l i s h i n g th e c r i t i c a l k i l l i n g concen­ t r a t i o n s f o r t h e s e c o m p o u n d s , d e t e r m i n i n g t h e i r optimum pH range, stu dying t h e ir e f f e c t on b a c t e r i a l m e t a b o l i s m , or i n v e s t i g a t i n g th e r e l a t i o n s h i p between t h e i r c h e m ic a l s t r u c ­ t u r e and t h e i r b a c t e r i o l o g i c a l a c t i v i t y . more r e c e n t s t u d i e s , an a t t e m p t h a s b e e n made t o a s many o f t h e s e o b j e c t i v e s a s p o s s i b l e H owever, In s e v e r a l o f th e th e proced u re h a s been to incorp orate i n one i n v e s t i g a t i o n . ob serve the e f f e c t s s u r f a c e - a c t i v e a g e n t s on b a c t e r i o l o g i c a l a c t i v i t i e s of only over p e r i o d s o f l i m i t e d d u r a t i o n , v a r y i n g from 5 t o 90 m i n u t e s . In s u c h a s h o r t t i m e , k illin g c o n c e n t r a t i o n s or optimum pH r a n g e s f o r t h e compoun ds c o u l d be e s t a b l i s h e d , but the e f f e c t s on b a c t e r i a l a c t i v i t y o f p r o l o n g e d e x p o s u r e t o w e a k e r c o n c e n ­ tration s c o u ld n o t a d e q u a t e l y be s t u d i e d or i n t e r p r e t e d . Moreover, k illin g i n m o st i n v e s t i g a t i o n s , arb itrary k il lin g con cen tration s o f th e se sy n th e tic se lec ted fo r study. of the agen ts' a g e n ts have been T h is has le d to a r e s t r i c t e d effect ap p raisal on b a c t e r i a l g r o w t h . The c h i e f p u r p o s e o f t h i s i n v e s t i g a t i o n i s sent a sy ste m a tic stu dy o f th e e f f e c t s an io n ic, ca tio n ic, and n o n ­ to p r e ­ of c er ta in sy n th etic and n o n - i o n i c a g e n t s on t h e g r o w t h o f 3 c u l t u r e s o f B s o h e r i c h i a c o l l . S a l m o n e l l a - p a r a t y p h i B . and and S t a p h y l o c o c c u s a u r e u s * The s u r f a c e - a c t i v e a g e n t s were t e s t e d i n v a r y i n g c o n c e n t r a t i o n s w i t h a medium o f v a r y i n g pH. M e a s u r e m e n t s o f b a c t e r i a l g r o w t h , pH s h i f t , t e n s i o n w e r e made a t d e f i n i t e ods. and s u r f a c e i n t e r v a l s d u rin g 24-hour p e r i ­ T h r o u g h o u t , t h e i n t e n t h a s b e e n t o e x p l a i n more f u l l y the a c c e p t e d g e n e r a l i z a t i o n s concerning the in flu e n c e th ese a g e n t s e x e r t on b a c t e r i a l a c t i v i t y , s u l t s having p r a c t i c a l a p p l ic a t io n s . r a th e r than t o s e e k r e ­ 4 II. HISTORICAL SURVEY P r io r to 1914, the o n ly s u r f a c e - a c t i v e w e r e s o a p s and s u l f o n a t e d c a s t o r o i l . a g e n t s known S h ortly t h e r e a f t e r , w i t h t h e i n t r o d u c t i o n i n Germany o f a l k y l a t e d n a p h t h a l e n e su lfon ates, an e x t e n s i v e d e v e l o p m e n t o f t h e s e p la ce in the U n ited S t a t e s . P r o d u c t i o n r o s e fro m z e r o p o u n d s i n 1928 t o 1 2 5 , 0 0 0 , 0 0 0 pounds in 1945 ( 6 5 ) . a r e o v e r 500 d i f f e r e n t compounds t o o k At p r e s e n t , t h e r e s y n t h e t ic s u r f a c e - a c t i v e c h em ica l prod­ u c t s c o m m e r c i a l l y a v a i l a b l e on t h e U . S . m a r k e t ( 6 0 ) . Some o f t h e e a r l i e s t s t u d i e s on t h e i n f l u e n c e o f s u r f a c e - a c t i v e a g e n t s on b a c t e r i a w e r e made w i t h s o d i u m o lea te. Lamar ( 4 2 ) dem onstrated in c r e a s e d s u s c e p t i b i l i t y o f p n e u m o c o c e i t o se r u m l y s i s a f t e r t r e a t m e n t o f t h e o r g a n i s m s w it h sodium o l e a t e . Avery ( 5 ) n o t e d t h a t i t s a d d itio n to c u l t u r e m e d i a p r e v e n t e d t h e g r o w t h o f p n e u m o c o c c u s and s t r e p ­ tococcu s, w h ile i n c r e a s i n g t h e growth o f B a c i l l u s in flu en za e. The work o f B a y l i s s and H a l v o r s o n ( 9 ) s u b s t a n t i a t e d t h e s e ­ l e c t i v e b a c t e r i c i d a l a c tio n of the unaaturated so a p . A series o f e x p e r i m e n t s on t h e e f f e c t s ot surface t e n s i o n on b a c t e r i a l g r o w t h w e r e u n d e r t a k e n a b o u t t h e same tim e. Larson e t a l (43) noted t h a t p e l l i e l e - f o r m e r s ceased t o grow a t t h e s u r f a c e when t h e t e n s i o n o f t h e medium w a s r e ­ d u c e d b e l o w 4 5 d y n e s p e r c e n t i m e t e r by s o a p s . Poor growth o f s t r e p t o c o c c i and p n e u m o c o c c i i n m e d ia a t 4 5 d y n e s p e r c e n ­ t i m e t e r was a l s o d escrib ed . F rob ish er (20) suggested a pos­ s i b l e means o f d i f f e r e n t i a t i n g b a c t e r i a on t h e b a s i s o f t h e i r a b i l i t y t o grow a t l o w s u r f a c e t e n s i o n s . M arshall (47) n o te d 5 t h a t i n m edia o f d i f f e r e n t s u r f a c e t e n s i o n , b a c t e r i a appear t o have d i f f e r e n t lism s. However, r a t e s o f g r o w t h and d i f f e r e n t g a s m e t a b o ­ the c o r r e la t io n o f th e s e v a r ia b le s rev ea led no s y s t e m a t i c v a r i a t i o n . regards r e la t io n s h ip He l i k e w i s e p o i n t e d o u t t h a t , b e t w e e n s u r f a c e t e n s i o n and b a c t e r i a l g r o w t h , t h e d a t a can be c o n s i d e r e d a s l i t t l e g estiv e. -After a l l , te n sio n at the "as more t h a n s u g ­ th e r e l a t i o n s h i p between th e s u r f a c e air-m edium i n t e r f a c e i s n o t n e c e s s a r i l y a fu n c tio n of th e s u r fa c e energy r e l a t i o n s h i p s a t th e organism medium i n t e r f a c e i n a n y way . . . " M a rsh a ll's statem ent i s co n sid ered v a l i d to th e p r e se n t day. The r o l e o f pH i n 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 id a l p r o p e r tie s o f s u r fa c e -a c tiv e agen ts has m erited a great deal o f in v e s tig a tio n . E ggerth ( 1 7 ) , in 1926, reported that t h e r e w e r e optimum pH r a n g e s f o r b a c t e r i c i d a l a c t i v i t y f o r a series of soaps. He f o u n d t h e l o w e r members o f t h e s a t u r a t e d s e r i e s more a c t i v e i n an a c i d r a n g e , w h i l e t h e h i g h e r members w e r e more b a c t e r i c i d a l a t i n c r e a s e d pH l e v e l s . in 1957, p o in te d out t h a t , Dunn ( 1 5 ) , a t an a l k a l i n e pH, a l k y l d i m e t h y l b e n z y l ammonium c h l o r i d e w a s a more e f f i c i e n t b actericid e t h a n a t n e u t r a l o r a c i d pH. dem onstrated G ersh en feld (22) g r e a t e r b a c t e r i c i d a l a c t i o n f o r A e r o s o l OT w i t h a d e c r e a s e i n pH. I n a l a t e r p a p e r ( 2 4 ) , h e sh o w e d t h a t T r i t o n K - 1 2 , a c a tio n ic agent, w as m o s t e f f e c t i v e i n an a l k a l i n e range, w h i l e t h e a n i o n i c s T e r g i t o l 4 and 4T w e r e m ore e f f i c i e n t an a c i d r a n g e . these f in d in g s . The work o f B a k e r e t H oogerheid e (3 1 ) a l (6) su b sta n tia tes contrasted th e r ela tiv e in 6 b a c t e r i c i d a l p o w e r o f CTAB a t pH 8 a nd pH 5 t and c o n c l u d e d th at at the a l k a l i n e pH l e v e l t h e q u a t e r n a r y ammonium s a l t c o u l d more e a s i l y w i t h s t a n d d i l u t i o n and s t i l l m a i n t a i n g e r ­ m icid al a c t i v i t y . Sin ce 1935, a number o f p a p e r s h a v e a p p e a r e d i n w h ic h t h e r e l a t i o n s h i p a c tiv itie s is o f su rfa c e-a c tiv e d iscu ssed , K atz agents to b io l o g i c a l ( 3 4 ) found t h a t d i l u t i o n s from 1-10T t o 1-5 0 T o f t h e sodium s a l t o f d i - s e c o n d a r y b u t y l naph­ thalene s u l f o n i c a cid sume i n v o l u t i o n ca u sed M ycobacterium am egm atis t o form s. B a y liss (10) as­ dem onstrated th e a b i l i t y o f s o d i u m o l e a t e and s o d i u m l i n o l e a t e to to x in . sh ow e d t h e l y s i n g e f ­ In 1 9 3 7 , t h e same a u t h o r ( 1 1 ) f e c t o f sodium l a u r y l su lfa te d e to x ify d ip h th eria on b a c t e r i a . G a le a n d T a y l o r (21) l i k e w i s e dem on strated l y s i s w ith su bseq uent r e l e a s e o f amino a c i d s . The d e s t r u c t i o n of p a th o g en ic fu n g i by a l k y l - d i m e t h y l - b e n z y l ammonium c h l o r i d e s power o f c a t i o n i c a g e n t s (19) uses fo r th ese compounds. im portant work, sy n th etic (16), and t h e exten d ed the list of p o ten tia l .Anson ( 3 ) , dem onstrates th e a b i l i t y cy sticid a l in h is very of sy n th e tic deter­ g en ts to d e n a t u r e s u c h p r o t e i n s a s h e m o g l o b i n and e g g a l b u m i n at th eir is o e le c tr ic p o in ts. Kramer ( 3 9 ) treated Strep to­ c o c c u s h e m o l y t i o u s . S a l m o n e l l a t y p h o s a . and B a c i l l u s w ith v a r io u s w e t t in g a g e n t s , b acteria f i lt r a b l e cis and s u c c e e d e d i n r e n d e r i n g t h e s e through s i l i c e o u s f i l t e r s . S t o c k and F r a n ­ (5 9 ) were a b l e t o p r o v e t h a t i n f l u e n z a v i r u s a c t i v a t e d by c e r t a i n su b tilis o f the h igh er f a t a c id s . o f im m un ological r e a c t i o n s was p o i n t e d could be i n ­ The i n h i b i t i o n o u t b y H olm es ( 3 0 ) . 7 Many r e s e a r c h e r s h a v e f o r m u l a t e d t h e o r i e s (the m a j o r i t y o f w h i c h a r e i n a g r e e m e n t ) c o n c e r n i n g t h e mode o f a c tio n of th e s y n t h e t i c s u r f a c e - a c t i v e a g e n ts on b a c t e r i a and b i o l o g i c a l s y s t e m s . Baker e t a l ( 6 ) favor the t h e o r y t h a t a t t h e optimum pH f o r b a c t e r i c i d a l a c t i o n , t h e r e a l t e r a t i o n o f b a c t e r i a l membrane o r p r o t o p l a s m , t h e b a c t e r i a more s u s c e p t i b l e t o is an ren dering the s u r fa c e -a c tiv e a g en ts. They a l s o c o n t e n d t h a t a t t h e optimum pH t h e f o r m a t i o n o f u n d is s o c ia te d m o le c u le s o f the s y n t h e t i c agent is favored, and t h a t t h e s e u n d i s s o c i a t e d m o l e c u l e s h a v e a g r e a t e r a b i l ­ i t y to e n te r the b a c t e r i a l c e l l H erein they concur w ith th e suggest of the b a c t e r i a l c e l l action : first, su lt su rfa ce-a ctiv e the is d e p e n d e n t on a t w o f o l d a c t i v i t y of the s y n th e tic and grow th a r e d e n a t u r e d . first, In a l a t e r pap er, c e r t a i n p r o t e i n s w h i c h are v i t a l o f m olecular f o r c e s Valko agents, f o r m etabolism (61) su g g e s ts th a t two t y p e s are i n v o lv e d in t h e i n t e r a c t i o n between a g e n t s and p r o t e i n s in tr in sic in c e l l membrane i s d i s o r g a n i z e d a s a r e ­ o f the great su rfa ce and s e c o n d , c e lls. (48) th a t the d is r u p t io n o f the normal fu n ction the d estru ctio n . f in d in g s of O sterhout h i s e x p e r i m e n t s on l a r g e p l a n t Ba k er e t a l ( 8 ) and c a u s e i t s a ffin ity (b a cteria l c e lls ) and s e c o n d , t h e — electro sta tic c o u l o m b i c f o r c e s o f b o t h t h e p r o t e i n m o l e c u l e and t h e a d ­ sorbed i o n s , both o f w h ich c a rr y f r e e in v estig a tio n s o f H otch kiss t h e r e a r e t h r e e m a in s t a g e s su rfa ce-a ctiv e a g en ts. e le ctr ic charges. The ( 3 2 ) h a v e l e d him t o b e l i e v e t h a t in th e b a c t e r i c i d a l a c t i v i t y o f 1) In te r a c tio n o f s u r fa c e -a c tiv e 8 a g e n t s and b a c t e r i a charges* 2) r e s u l t s from t h e a t t r a c t i o n o f o p p o s i t e A l l t h e s o l u b l e n i t r o g e n and p h o s p h o r u s com­ pounds a r e r e l e a s e d from t h e c e ll, p r o v id in g th e hydrophobic group o f the s u r f a c e - a c t i v e a g en t has th e a p p r o p r ia t e a f f i n ­ i t y fo r the b a c t e r i a l s u r f a c e . th o u g h t h e y may s t i l l The c e l l s a r e now d e a d , g i v e e v id e n c e o f a v e r y low m e ta b o lism and a p p e a r u n c h a n g e d m o r p h o l o g i c a l l y . and a g r e a t l y i n c r e a s e d r e l e a s e fo llo w s. a l­ 5) A u to ly sis s e t s in , o f n i t r o g e n and p h o s p h o r o u s 9 III. A. MATERIALS AND EQUIPMENT C u l t u r e Medium The "basic medium was c o m p o se d o f t h e f o l l o w i n g in g red ien ts* : kha p o 7 — 0 .1 peroent e ah p o v - 0 .1 percent NaOl - 0 .1 percent - 0 .1 percent (NHV )A S0V - o . l percent 003 MgSOv - 0 .0 0 5 KOI - 0 .0 0 2 5 p e r c e n t Peptones** 1 .0 percent percent D i s t i l l e d H.JD - 1 0 0 0 m l . ( U n a d j u s t e d pH o f medium w a s 7 . 4 . ) In order to e f f e o t 5 cc. o f 1 IT HG1 p e r l i t e r p rior to a u to c la v in g . com plete s o l u t io n o f b r o t h were o f t h e NaA0 05 , add ed t o t h e b r o t h The medium was a u t o c l a v e d a t 1 5 p o u n d s p r e s s u r e f o r 20 m i n u t e s . T h i s b r o t h was u s e d i n a l l t h e exp erim en ts e x ce p t those s p e c i f i c a l l y noted o th e r w is e . P la in a g a r a t a p p r o x i m a t e l y pH 7 ( 1 . 2 ) w a s u s e d f o r a ll p la tin g purposes. * Its c o m p o sitio n was as f o l l o w s : T r a c e s o f Oa and He w e r e a l s o p r e s e n t . ** O n ly D i f c o P e p t o n e , L ot No. 3 6 3 1 1 3 , w as u s e d i n a l l m edia. 10 Peptone 5 grains NaOl 5 grams B eef E xtract Z grams Agar - 1 5 grams D i s t i l l e d H*0 - 1 0 0 0 m l . B. S u rface-A ctive Agents The s u r f a c e - a c t i v e are l i s t e d a g e n t s employed in t h e t e s t s in T ab le 1 . To f a c i l i t a t e un derstan d in g o f th e s e b r ie f d escrip tio n o f each, agents, a a s s u p p l i e d by th e m a n u f a c t u r e r s , i s h erew ith p r e se n te d . D u p o n c l OS i s tia lly an a m b e r - c o l o r e d , s o l u b l e i n HA0 . I t ■which c o m b i n e s t h e is o ily liq u id , an o i l - s o l u b l e em u lsifyin g p r o p e r tie s on ly par­ e m u lsify in g agent o f the alcoh ol s u l­ f a t e s w ith th e h om ogenizin g p r o p e r t i e s o f t h e f a t t y a l c o h o l s . I g e p o n AP E x t r a i s a fin e, lig h t w h ic h d i s s o l v e s r e a d i l y i n h o t w a t e r . c r e a m - c o l o r e d po w der It is a sy n th etic d e te r g e n t u sed m a in ly f o r the p r o c e s s in g o f w o o l. T ergitol 7 i s a c o lo r le ss, syrupy s o l u t i o n . It is used p r i n c i p a l l y a s a w e t t i n g a g e n t in the p r o c e s s i n g o f w o o l, le a th e r , paper, etc. It is lik e w is e a pow erful p e n e tr a tin g agen t . N a c c o n o l NRSF i s a l i g h t , f l a k y compound. is intended p r im a r ily to be used as a d e te r g e n t, plays a n t i s e p t i c , b a c t e r i o s t a t i c , ties in water A lthough i t it also d is­ and m o t h - p r o o f i n g p r o p e r ­ s o l u t i o n or i n o i l e m u l s i o n s . 11 R e k a l BZ H i g h G o n c e n t r a t i o n i s very rea d ily solu b le a f i n e , w h ite powder, i n warm w a t e r w i t h n e u t r a l r e a c t i o n * . I t i s c h a r a c t e r i z e d by s t r o n g w e t t i n g a c t i o n and p o s s e s s e s d i s p e r s i n g and e m u l s i f y i n g p r o p e r t i e s * R occal i s u s u a l l y s o l d i n a 1 0 -p e r c e n t water s o l u t i o n . It is recommended f o r s a n i t i z a t i o n w ith fo o d -p ro cessin g p la n ts, etc. Phenol c o e f f i c i e n t s o f equipm ent a s s o c i a t e d as a b a c te r ic id e , a t 20 d e g r e e s 0. fu n g icid e, a g a i n s t S* t y p h o s a a n d S . a u r e u s a r e 2 5 0 and 279 r e s p e c t i v e l y . * CfiB i s a fin e, phenol c o e f f i c i e n t a g a in s t S. LPO i s pyridinium is again st typhosa i s ch lo rid e. fu n g icid e, S. aureus i s in hot w a te r . 200 t o 250, Its and 125 to 1 7 5 . a c o lo r le ss, 5 5 0 and a g a i n s t cid e, w h i t e powder s o l u b l e 5 0 -p ercen t w ater s o lu t io n I t s phenol c o e f f ic ie n t S. typh osa i s 165. It is of la u ry l again st S. aureus used as a germ i­ and w e t t i n g a g e n t . Emulphor ON i s a w a x -lik e, non ion ogen ic, w a te r -s o lu b le , o r g a n i c s u b s t a n c e w i t h m ark ed e m u l s i f y i n g , d isp ersin g , and su rfa ce-a ctiv e p r o p e r tie s. T r it o n X-100 i s w ettin g agent, an a m b e r , e m u lsifier, o ily liq u id . detergen t, It is u sed as a and d i s p e r s a n t . * The compound o f R o c c a l u s e d i n m o s t o f t h e t e s t s d e s c r i b e d in t h i s p a p e r , h o w e v e r , was a f i n e , w h i t e powder l a b e l l e d a s fo llo w s: 1 ounce o f powder to 4 g a l l o n s o f w a te r g i v e s a 1 t o 5 0 00 c o n c e n t r a t i o n o f R o c c a l . 12 TABLE 1 SYNTHETIC SURFACE-ACTIVE M T S USED Name of Compound Percentage of Active Ingredient Manufacturer 95 to 100 E .I. DuPont de Nemours and Co., Inc, Wilmington, Del. 51 General Dyestuff Corp. Chicago, 111. 25 Carbide and Carbon Co. D etroit, Mich. Alkyl aryl sodium sulfonate 85 National Aniline Division Allied Chemical and Dye Corp. N.Y. 6, N.Y. Sodium alkyl naphthalene sulfonate 80 General Dyestuff Corp. Chicago, 111. 3 to 3.5 Winthrop Chemicals Co. N.Y. 13, N.Y. 95 to 100 Rhodes Chemical Corp. P lain field , N.J. 26 Hooker Electrochemical Co. Niagra F alls, N.Y, Chemical Designation Anionic Duponol OS (SH-2536)* Igepon AP Extra (10339) (481-15916) Mixture of two p arts of oleyl alcohol and one part cyclic amine lauryl alcohol su lfate Sodium sulfonate of an oleic acid ester of an alip h atic compound 0/7 HjjCOj C^H^SOjNa C ^C H C ^C H C jH ^H fC ,^ Tergitol 7 C4H; SO^Na Nacconol NRSF (B.C. 66129) Nekal BX High Concentration Cationic Roccal . CTAB liquid IPC Alkyl dimethyl benzyl ammonium chloride (alkyl = Cj to C(y) Cetyl trim ethyl ammonium bromide lauryl pyridinium chloride Non-Ionic Emulphor ON (S.O. 297) Polyethylene ether of a long-chain fa tty alcohol 100 General Dyestuff Corp. Chicago, 111. Triton X-100 Alkylated aryl poly-ether alcohol 100 Rohm and Haas Co. Philadelphia, Pa. * All numbers in parentheses signify lo t numbers and/or code numbers. IS 0. B a cteria l 1. C u ltu res S . p a r a t y p h i B # 1 5 4 - f u r n i s h e d b y The M i c h ­ ig a n Department o f H e a lt h L a b o r a t o r y , 2. L a n sin g , M ichigan. H e m o l y t i c S . a u r e u s # 2 - f u r n i s h e d b y The M i c h ­ ig a n Department o f H e a l t h L a b o r a t o r y , L a n s i n g , M ic h ig a n . 3. E. o o li - iso la ted fro m a c o n t a m i n a t e d w e l l - w a te r sam ple s u b m i t t e d to t h e M ic h ig a n S t a t e ment o f B a c t e r i o l o g y ill O o lle g e D epart­ for a n a ly sis. t h r e e organism s were rech eck ed f o r t y p i c a l b i o ­ c h e m i c a l r e a c t i o n s and f o u n d t o b e p u r e , rep resen ta tiv e cu ltu res. D. E q u ip m e n t In a d d i t i o n t o th e r e g u la r la b o r a to r y equipm ent, a Humber 7 0 5 2 0 S i m p l i f i e d O e n co -d u ITouy T e n s i o n o m e t e r , a B e c k ­ man pH M e t e r ( M o d e l G ) , and a L u m i t r o n C o l o r i m e t e r , Number 4 0 0 , m a n u f a c t u r e d b y t h e P h o t o v o l t M o del C orporation , N .Y .C ., were em ployed. A 14 IV. A, EXPERIMENT AL PROCEDURE P relim in ary S tu d ies 1. O h o i c e o f Medium I t was t h o u g h t d e s i r a b l e t o u s e a s i n g l e , medium w h i o h w o u l d p e r m i t g o o d g r o w t h f o r a l l ism s u s e d in t h i s study. Jit f i r s t , e m p l o y a c h e m i c a l l y d e f i n e d m ed iu m . a cid s, dextrose, sim ple th r e e organ­ an a t t e m p t w a s made t o Sodium m a l a t e , casam ino and p e p t o n e w e r e u s e d i n d i v i d u a l l y and i n com binations w ith a b a s ic sa lt so lu tio n . The s i m p l e s t medium w h i c h p e r m i t t e d t h e t h r e e t y p e s o f b a c t e r i a t o grow l u x u r i ­ a n t l y i n a 2 4 - h o u r p e r i o d was t h e o n e d e s c r i b e d i n t h e s e c ­ tio n e n title d " M a t e r i a l s and E q u i p m e n t . ” The c h o i c e o f s a l t s was b a s e d on a m o d i f i c a t i o n o f t h o s e s u g g e s t e d b y den D o o r e n de J o n g ( 1 2 ) 2. S electio n the (58). o f pH R a n g e s On t h e b a s i s o f t h e in g t a b le , and E o s e r data p resen ted t h r e e pH r a n g e s u s e d i n t h i s in th e f o l l o w ­ s tu d y were esta b lish ed . pH R a n g e s 5 .4 +++4 + 44+ 4+4+ +444 + +44 ++4+ • 5.2* E. co li S. p a r a t y p h i B S. aureus * Ranges s e l e c t e d + 17* C D . IS C D 5 .0 8.0* 8.2 8 .6 44 ++ 44 + 4 4 +4 444 44+ + + 44 + 44+ + 4 + + ++ + ++ 4+ 4+ +4 4+ 4 + 4 + 4+4 for th is study. + 44 + +4 + +4+ 15 B. M ain ten ance o f C u ltu r e s The s t o c k b a c t e r i a l agar s l a n t s t e s t tube) (w ith at a p a ra film 5 degrees 0. c u lt u r e s were k ep t on p l a i n cow ering over the l i p o f the S t o c k s u b c u l t u r e s w e r e made every fo u r w eeks. The c u l t u r e s u s e d i n t h e em ployed o n ly a f t e r a minimum o f f o u r d a i l y s u b t r a n s f e r s i n t o the proper b roth a f t e r fou r weeks o f u s e , reco rd ed e x p e r im e n t s were com ing o f f th e s t o c k a g a r s l a n t . test A fter o r g a n i s m s were o n c e a g a i n t a k e n f r o m the m o n th -o ld s t o c k c u l t u r e s . made a t t h r e e pH l e v e l s , S in c e m ost o f t h e t e s t s were the organism s were s u b tr a n s fe r r e d and g r o w n o n l y i n t h e b r o t h medium a d j u s t e d t o t h e p r o p e r pH, i.e ., those organism s t e s t e d f e r r e d i n b r o t h a t pH 5 . 2 , in o cu la tin g 5 cc. it etc. S u b t r a n s f e r s w e r e made b y I t w as f o u n d t h a t , was p o s s i b l e cc. of th is two m i l l i o n o f each o f the c u lt u r e s a f t e r 2 4 h o u r s ’ g r o w t h a t 57 d e g r e e s made w i t h t h e l u m i t r o n as a r e s u lt to a s c e r t a in w ith in t h e nu m ber o f o r g a n i s m s p e r c c . 0. subtrans­ o f t h e b r o t h medium w i t h e x a c t l y 0 . 1 o f broth c u ltu r e . procedure, a t pH 5 . 2 w e r e d a i l y 0. A fu rth er c h e c k was co lo rim eter. G row th C u r v e s and R e l a t e d P heno m ena The g r o w t h - c u r v e t e c h n i c u s e d i n was t h e o r e t i c a l l y d i v i d e d stage, testin g the sta b ility th eir i n t o two s t a g e s . o f the resista n ce th is experim ent In t h e f i r s t organism s was e s t a b l i s h e d th ro u g h t o p h e n o l * by t h e P h e n o l - C o e f f i c i e n t * T h is p r o c e d u r e was f o l l o w e d p r i o r t o m aking an y o f t h e t e s t s undertaken in t h i s stu d y . At t h e same t i m e , t h e o r g a n ­ i s m s u s p e n s i o n w a s s t r e a k e d on a g a r p l a t e s ( f o r i s o l a t e d c o l ­ o n ie s ) t o in s u r e the f a c t t h a t th e organ ism s were m a in ly in th e sm ooth p h a s e . Kethod. A c h e m i c a l - r a n g e t e s t w a s a l s o made a t t h e p a r t i c u ­ l a r pH r a n g e u n d e r c o n s i d e r a t i o n . in g 0 .1 cc. T h i s w a s done b y i n o c u l a t ­ o f a 24-hou r organism s u s p e n s io n i n t o o f d ilu tio n s of the S i n c e many o f t h e a f t e r 24 h o u r s ' (2 l o o p f u l s ) s u r f a c e - a c t iv e agent in su rfa ce-a ctiv e of th is in th e l a t t e r in cu b a tion ch em ical-ran ge t a b l e s . ure to 0 ., su btransfers f o r 24 h o u r s . The r e ­ c o n s t it u t e the data p r e se n te d I n f o r m a t i o n g a i n e d from t h e s e c h e m i c a l - r a n g e t e s t s was u t i l i z e d experim en t, o f broth. i n o c u l a t e d s o l u t i o n w e r e made i n t o F . D . A . b r o t h and i n c u b a t e d a t 37 d e g r e e s 0 . su lts 5 cc. agent s o l u t i o n s were t u r b i d , i n c u b a t i o n a t 37 d e g r e e s o f the a series i n the second sta g e o f the the a c t u a l stu d y o f organism growth a f t e r the v a rio u s sy n th etic agents. The o r g a n i s m s w e r e g r o w n i n 2 5 0 - c c . f l a s k s e t c h e d at a 1 7 5 - c c . m ark. m eniscus a f t e r erlen m eyer The mark w a s made a t t h e a d d in g , by v o l u m e t r i c p i p e t t e s , t h e b r o t h medium a t the s u r f a c e - a c t i v e expos­ 20 d e g r e e s 0 . 175 c c . The p r o p e r d i l u t i o n s a g e n t s w e r e made on t h e b a s i s of of o f 175 c c . of broth. As a p r e l i m i n a r y was a d j u s t e d t o tion step, a p p ro x im a te ly the t h e pH o f t h e s t e r i l e broth d e s ir e d range b y t h e a d d i­ o f c o n c e n t r a t e d HOI, 1 F HOI, o r 1 I NaOH, a s t h e might b e . The p a r t i c u l a r s u r f a c e - a c t i v e a g e n t w a s t h e n a d d e d to t h i s broth in th e erlenm eyer f l a s k s , and t h e f l a s k s w e r e h e a t e d f o r 2 0 m i n u t e s i n a n A rnold s t e a m e r . s o l u t i o n was e f f e c t e d 20 d e g r e e s case 0 ., A fter com plete and t h e f l a s k s c o o l e d t o a p p r o x i m a t e l y su ffic ien t s o l u t i o n was rem oved t o b r i n g t h e 17 t o t a l volum e back to t h e 1 7 5 - e c . m a rk . T h is was n e c e s s a r y on ly in con cen tration s o f s u r fa c e -a c tiv e a g e n t g r e a t e r than 1-100. The s o l u t i o n was a g a i n a d j u s t e d t o t h e pH l e v e l w h i c h t h e e x p e r i m e n t was t o b e c o n d u c t e d . F ifteen cc. of t h e s o l u t i o n w e r e u s e d i n t h i s pH a d j u s t m e n t t e s t i n g . f l a s k s w e r e t h e n i n c u b a t e d a t 37 d e g r e e s 0. at The f o r 15 h o u rs t o insure s t e r i l i t y . J u st p r io r to in o c u la tin g the s o lu t io n w ith organism s, 1 cc. t h e o r g a n ism s was p l a t e d , The c o u n t teria l tio n cc. o f a 24-hour s u s p e n s io n of c o n s t it u t e s the ( a f t e r com puting i t p rop ortion ). agent a f t e r a d e q u a t e d i l u t i o n s w e r e m ade. from t h e s e p l a t i n g s count su rfa ce-a ctiv e S in c e 160 c c . on t h e b a s i s "O-hour” b a c ­ of th e ensu ing o f the s u r f a c e - a c t iv e agent so lu ­ r e m a i n e d a t t h e t i m e t h e t e s t w a s t o be i n i t i a t e d , 3 .2 o f a 24-h ou r organism su s p e n s io n w ere in tr o d u c e d i n t o solu tion ( a t 37 d e g r e e s CL). su rfa ce-a ctiv e the T h is p r o p o r tio n o f organism s to a g e n t s o l u t i o n w a s t h e same a s t h a t u s e d i n t h e ch em ical-ran g e t e s t . Im m ediately a f t e r th e a d d i t i o n o f organ­ ism s, and pH m e a s u r e m e n t s w e r e t a k e n . su rfa ce-ten sio n co n stitu ted T h is "0-hour" d a ta f o r b o th m ea su r em en ts. The f l a s k was t h e n w e l l s h a k e n and p l a c e d i n t h e in cu b a to r a t 37 d e g r e e s 0. T hereafter, at 45-m inute i n t e r v a l s fo r the first th e f l a s k was sh aken 10 h o u r s . At t h e s p e c i ­ f i e d p e r i o d s wh en p l a t i n g s w e r e t o b e m a d e , t h e f l a s k was again shaken, 1 c c . d i l u t i o n s m ade.* o f th e s o l u t i o n w ithdraw n, and p r o p e r P l a t e s were poured w i t h p l a i n a g a r , allow ed * To c o m p e n s a t e p a r t i a l l y f o r p o s s i b l e b a c t e r i o s t a t i c d i l u t i o n s up t o 1-1M w e r e m a de. a ctio n , 18 to harden, and t h e n i n c u b a t e d f o r 4 8 h o u r s a t 37 d e g r e e s . C. Gram s t a i n s w e r e made p e r i o d i c a l l y t o insure absence o f contam ination* The m e t h o d f o r d e t e r m i n i n g t h e e f f e c t s i a l growth o f exp osu re to on b a c t e r ­ c a tio n ic agen ts for p erio d s o f s h o r t d u r a t i o n was e s s e n t i a l l y t h e same a s t h a t d e s c r i b e d above* The o n l y d i f f e r e n c e w a s t h a t t h e i n a w ater b a th a t 37 d e g r ee s a t 37 d e g r e e s 0. f l a s k 3 were kep t C. i n s t e a d o f i n an i n c u b a t o r In b o th t y p e s o f t e s t s , the s u r f a c e - a c t i v e a g e n t s o l u t i o n s w ere a t 37 d e g r e e s 0 . p r i o r t o i n o c u l a t i o n of the organism s u s p e n s i o n s . A 12-ec. a l i q u o t was l i k e w i s e w i t h d r a w n a nd u s e d f o r b o t h t h e s u r f a c e - t e n s i o n and pH d e t e r m i n a t i o n s . Surface t e n s i o n was m e a s u r e d b y t h e du Nouy T e n s i o n o m e t e r w i t h s t a n d ­ ard watch- g l a s s e s o f uniform s i z e . The s o l u t i o n s fo r m eas­ u r e m e n t o f s u r f a c e t e n s i o n w e r e k e p t a t 37 d e g r e e s w ater b a th , C. i n a and r e a d i n g s w e r e t a k e n a p p r o x i m a t e l y 1 0 s e c o n d s a f t e r t h e sa m p le was i n t r o d u c e d i n t o pH was d e t e r m i n e d l a s t each tim e i n t e r v a l , of a ll* the w atch g l a s s . The P rio r to d eterm in ation s fo r t h e t e n s i o n o m e t e r w a s r e c a l i b r a t e d and t h e Beckman pH m e t e r was c h e c k e d a g a i n s t a standard b u ffe r so lu tio n . In t h e f o r e g o i n g s e r i e s w a s made t o p r e s e n t t h e b le. The d i f f i c u l t i e s o f experim en ts, an a t t e m p t d a t a i n a s u n i f o r m a m ann er a s p o s s i ­ in v o lv e d in such a p r e s e n t a t io n l i e m ainly w ith th e in h e r e n t d i f f e r e n c e s betw een the organism s t h e m s e l v e s and b e t w e e n t h e compounds u s e d , i . e . , d ifferen ces in p h y sic a l s t a t e and i n p e r c e n t a g e o f a c t i v e in g red ien t. 19 n ev erth eless, wherever p o s s i b l e , o f the s u r f a c e - a c t i v e rela tiv e resista n ce com parable c o n c e n t r a t i o n s a g e n t s were employed t o o f the o rg a n ism s. Such a p r o c e d u r e was m o st s u c c e s s f u l w i t h c e r t a i n o f t h e l e s s a g e n t s and t h e n o n - i o n i c c o m p o u n d s . con cen tration o f th e e f f e c t iv e a n io n ic However, i n d i c a t e t h e g r a d u a l e f f e c t on b a c t e r i a l germ icid al a g e n ts, i t in d ic a te the in order to g r o w t h o f t h e more becam e n e c e s s a r y t o u s e w i d e r a n g e s i n sy n th etic a g e n t s when c o m p a r i n g t h e d i f f e r e n c e s between t h e g r a m - p o s i t i v e and g r a m - n e g a t i v e organism s. Wherever p o s s i b l e , an a t t e m p t w a s made t o i n the t a b l e s the approxim ate d i l u t i o n which j u s t k i l l e d com p letely th e e n t ir e b a c t e r i a l pop ula­ tio n . However, o f sy n th etic in clu d e in th o s e i n s t a n c e s where t h i s b e e n o m i t t e d fro m t h e t a b l e , it is n e x t lo w e r d i l u t i o n from t h a t f i r s t agent d il u t i o n has to be assumed t h a t t h e appearing in th e ta b le c o m p l e t e l y k i l l e d t h e o r g a n ism s i n 24 h o u r s or l e s s . * Since S . p a r a t y p h i B was u s e d p r i m a r i l y f o r p u r p o s e s o f c o m p a r i s o n w i t h E . c o l i . n o a t t e m p t was made t o d e t e r m i n e t h e of c ritic a l the c o n cen tra tio n s o f the s u r f a c e - a c t iv e effects a g e n t s on form er o r g a n ism . It sh ould be noted th a t g r a d a tio n s of d ilu tio n pro­ ceeded b y in c r e m e n ts o f 100 w i t h i n th e hundred r a n g e , by * The o n l y e x c e p t i o n s t o t h i s s t a t e m e n t o c c u r w h e r e compounds are not g e r m ic id a l f o r the b a c t e r i a in q u e s t i o n , or where c o n c e n t r a t io n s h ig h e r than t h o s e reco rd ed are too v i s c o u s to p e r m it com p lete s o l u t i o n o f th e organism s u s p e n s i o n . B e c a u s e o f t h e e x t r e m e v i s c o s i t y o f t h e a n i o n i c c om pounds i n c o n c e n t r a t i o n s s t r o n g e r th a n 1 - 5 0 , th e 1 - 5 0 d i l u t i o n was s e ­ le c t e d as the s t a r t in g p o in t f o r th e s e a g e n ts . A 1-20 con­ c e n t r a t i o n o f n o n - i o n i c compounds w a s s e l e c t e d f o r t h e same reason. 20 in c r e m e n ts o f 1000 w i t h i n t h e thousand r a n g e , ments o f 1 0 ,0 0 0 w i t h i n th e and b y i n c r e ­ ten-thousand range. An a d d i t i o n a l e x p l a n a t i o n i s n e c e s s a r y f o r t h e terp reta tio n lish ed of the resu lts. in - S i n c e th e v a s t m a j o r i t y o f pub­ data co n c er n in g the r e l a t i o n s h i p o f sy n th etic su rface- a c t i v e a g e n t s t o b a c t e r i a a r e b a s e d on t h e a s s u m p t i o n o f a 100-percent a c tiv e in g red ien t o f the s y n th e tic w a s deemed a d v i s a b l e t o m a i n t a i n t h i s However, sin ce i t on t h e b a s i s listin g m ight be d e s i r a b l e to in te rp re t d e n s it ie s o f the liq u id sy n th etic u r e s a r e b a s e d on t h e w e i g h t s o f 1 m l . the resu lts in g red ien t, p r e s e n t e d below to a i d i n s u c h an i n t e r p r e t a t i o n . degrees it type o f r e p o r t i n g . o f a c tu a l percentage o f a c tiv e of the compounds, o f the a agents i s Jill f i g ­ agent a t 20 0. as n e a r ly g red ien t), B a s i c b r o t h medium 0 . 9 9 6 gram D u p o n o l OS 0 . 8 4 3 gram T erg ito l 7 0 . 9 9 0 gram LPO 0 . 9 8 5 gram T r it o n X-100 0 . 9 6 0 gram For e x a m p l e , in as p o s s i b l e , o f T e r g i t o l 7 (25 p e r c e n t a c t i v e the d ilu tio n m u lt ip lie d by the com puting t h e exact con cen tration s t a t e d in the v a r io u s t a b l e s factor 4*024. T his in ­ is f a c t o r was o b t a i n e d by th e f o l l o w i n g com putation: w eigh t of T e r g it o l per cc. w eight o f b a s i c b ro th per c c . 21 Thus t h e a c t u a l e f f e c t i v e con cen tration o f t h i s compound i s a p p r o x im a te ly fo u r t i m e s g r e a t e r than t h a t s t a t e d tab les* The f a c t o r s f o r t h e in the o t h e r c om pounds a r e a s f o l l o w s D u p o n o l OS 1 .2 0 5 I g e p o n .AP 1 .96 N e h a l BX 1 .2 5 N a e c o n o l IffiSF 1.176 OTiffl- 1 .0 2 IPO 3.888 R o c c a l (powder) 3 1 .2 5 Sm u lph o r ON 1 .0 T r it o n X-100 1 .0 3 7 22 V . RESULTS A. C ontrol T e sts In p r e p a r in g t h e s e s t u d i e s , determ ine growth r a t e s , pH s h i f t s , it was n e c e s s a r y t o and s u r f a c e - t e n s i o n m easurem ents in th e a b sen ce o f s y n t h e t i c these d a ta m ight a g e n ts so t h a t s e r v e a s a r e f e r e n c e group f o r w ith data ob tain ed in the en su in g e x p e rim en ts. com parison The r e s u l t s are p r e s e n t e d in T ab le 2 . B. . A n io n i c Compounds The f i r s t series agents t e s te d fo r t h e ir of sy n th etic effects su rfa c e-a c tiv e on b a c t e r i a l g r o w t h w e r e t h e a n i o n i c co mpounds D u p o n o l OS, I g e p o n AP, T e r g i t o l 7 , H a c c o n o l NRSF, and H e k a l BX. The r e s u l t s of these t e s t s appear in T a b le s 3 through 7. When t h e r e s u l t s p r e s e n t e d i n are the c o n tro l ta b le c o m p a re d w i t h t h o s e o b t a i n e d w i t h t h e a n i o n i c a g e n t s , becomes apparent t h a t , in every in sta n c e , use o f th e la tter produced a d im in u tio n o f b a c t e r i a l count du rin g a 24-hour p erio d . sh ift It is also clea r th a t the i n pH i s it lik ew ise cu rta iled . For t h e main p a r t , the accepted g e n e r a liz a tio n t h a t a n i o n i c a g e n t s e x e r t t h e i r maximum b a c t e r i c i d a l e f f e c t i n an a c i d r a n g e (22)(23)(£4}(6) d a ta d e r iv e d from t h e s e a ctio n tests. is s u b s t a n t i a t e d by th e The o n e e x c e p t i o n is o f I g e p o n AP on t h e g r a m - n e g a t i v e o r g a n i s m s . the T h is compound a p p e a r s m o re e f f e c t i v e a g a i n s t E. typh i B in A lthou gh t h e t y p e o f t e s t an a l k a l i n e m e d iu m . c o l i a nd S . p a r a ­ 23 TABLE2 DATASHEETOS CONTROLS pH7 E. coll Phenolic Ranges S paratyphi B Surface* Bact.** Tension Count pH Surface Tension S. aureus Bact. Count Min, E. coli S. para­ S. aureus typhi B PH o o 8 0 0 0 01 O1i H1 01 C-1 (D1 I 1 1 I I 7.0 56.49 ISM4 7.0 56.58 91 7,0 56.58 71 ri ri ri 6.9 56.49 62M 6.9 56.63 361 7,0 56,58 271 4 * t - 4 4 - t 4 6.9 56.11 80M 7.0 56.63 781 7,0 56.49 701 10 7.0 56.02 135M 7.2 56.49 2201 7.1 56,11 1601 24 7.5 55.88 670M 7,7 55,88 6001 7,3 56.02 3301: 5,2 56.02 61 0 tOi 1 ri 6 ■ 10 15 - 0 c>i 1 ri 0 toi 1 pi 4 + - + - 4 rl rl rl + f + - 4 t - - ,t PH Surface Tension Bact, Count pH5.2 In. E. soli S. para- S. aureus fmfT 5.2 56.11 14M 5.2 56,11 5.4 55.88 40M 5.6 56,11 . 301 5,3 56.02 191 rl rl rl 5.6 55.74 98M 6.2 56.02 901 5,3 55,88 331 5 • + 4 4 4 4 4 4 4 10 . . + - 4 4 - 4 4 15 * " 4 - - 4 - 4 4 5.7 55.74 190M 6.4 55,74 1801 5.4 55.88 391 6.6 55.65 630M 6.9 55,65 3851 6.3 55.74 3201 0 0 0 0 0 0 0 o 8 (0 ^ CO toi lt“l 00 toI I01I ri 11 1 1 1 1 1 1 1 9M • ri ri rl ri ri ri 24 pH8 Min. E. coli S. para­ typhi B 0 0 0 0 0 0 0 s* co to to 01 ri i i i rl H rl ri ri ri 6 t + + 4 4 4 10 - 4 4 - 4 4 15 - 4 4 - 4 4 S. aureus 0 0 0 to t> CO 1 1 1 rl rl ri 8.0 56.58 14M 8,0 56.58 91 8.0 56,58 71 7.7 56.58 731 7.7 56,58 501 7.8 56,58 271 7.6 56,49 1091 7,8 56,11 1001 7,8 56.58 701 7.7 56.49 1531 8.0 56.02 1951 7,9 56,49 1181 7.9 56.11 4001 8.2 55,88 3201 8.0 66,36 ' 2901 • + - 4 4 4 • • • * Surface tension is measures in dynes per centimeter. ** Bacterial count is measured in organisms per cc. 4 II signifies millions. 24 T A B IE 3 V J S A SHEET OH D U M JO L OS pH V Phenolic Range Min. E. c o l i S. p ara- S. aureus tv n h i B 0 10 H - 0 0 e- op rl H t + + 4 - 4 1-50 Hre. 0 flhemiaal Range 0 0 0 0 8 8 8 8 HM' rl WWf Jl l Ql O I I | I I I I I I E. c o l i S. p rl rl + + S. p aratyph i B E. c o li 0 0 0 0 0 0 0 e- rl H H H rl H rl 6 + + + 4 + 4 10 - ¥ + - ♦ 4 15 - 4 4 - - + rt H H pH 1-100 S .T .* B.C.** pH 7 .0 2 8 .86 ISM1 S.T. 1-100 1-50 B.C. pH S.T. B.C. pH ISM 6 .8 28.05 15M 6 .8 2 9.04 121! 6 .8 28.86 3.81! 7 6.9 2 9.04 SOOT’1' 6 .8 28.86 2.5M 10 6.9 2 9.04 11K 6 .9 28.86 24 7.0 2 8 .8 6 151 7 .0 28.61 S .T . 9M 7 .0 28.59 7 .0 28.55 4 3 . aureus 7 .0 28.86 B.C. PH S.T. 9M 6 .9 28.56 1-700 B.C. pH S.T. B.C. 7M 7 .1 28.56 7M 6 ,8 28.59 6.31! 6.9 28.56 60T 7 .1 28.56 180T 6 .9 28.59 6.9 28.56 91 7 .0 28.56 17T 9M 7 .0 2 8 .1 5 22M 6.9 28.66 131 7 .0 2 8.66 8ET 7 .2 2 8 .1 5 1711 6.9 28.56 401 7 .0 2 9.04 4.2M 6 .9 2 9.04 14M 8M 7 .0 29,04 1-500 7K H 43M 7 .0 28.86 141! n typn: 3 . aureus - - 4 4 pH 5 .2 P henolic Range Min. E. 5 10 15 S. p aratv r h i B 0011 3. 0 aureus 0 0- O O CD ffl 0 0 0 IB 01 rl <0 C~ 11 H 4 4 4 s . ra ra - 3. aureus ty p h i B 0 CD rl 0 01 1 rl + + * + - 8 H 1 rl 4 4 4 O <0 1 rl • • - 0 0 t- © 1 1 H H 4 4 4 Hrs. 0 0 0 0 0 0 0 0(0 H N (0 ^ 10 1 r If HI HI H IHI IH H S. aureus 1-50 1-50 1-50 B.C. pH S .T . 28.86 131! 8 .0 28.86 7 .7 # 54M 7 .8 7 .7 # 25M 7 .7 pH S.T. 8 .0 4 I 7 10 7 .7 E. o o ll 24 S. Paraty p h l B S. paratyphi B B.C. B.C. PH S.T. 911 8 .0 28.86 # 1 7 .5M 8 .0 # # 41M 8 .0 # SOOT 4 Chemical Range H E. c o l l 0 7 .7 # 30M 2 8 .8 6 — 'SOM 7 .8 7 .9 # 28.86 7 .9 # 28M 7 .9 29.04 SOOT S .T . s i g n i f i e s su rface te n s io n in dynes per cen tim eter. A II s i g n i f i e s m illio n s . * T s i g n i f i e s thousands. # Ho measurement was made. 4001 34M S . aureus ** B.C. s i g n i f i e s b a o te r ia l count per s c . 7M 130T 411 Tubes P o s it iv e * B.C. B.C. 7M 68T 25 T A B IE 4 DATA SHEET OH IOEPOU AP pH 7 P henolic Range - 4 - 4 4 4 + - + + - + + t 1 6 10 15 1 o u) t H S. nara- S. aureus ty o b i B o o o o O o o o o tO t* co E- CD CO (7) rl 1 1 1 1 1 1 1 H H rl H H 1 Min. E. c o li - + t - + + t 1-50 Hrs. -60 ■3 0 -40 o CM 1 i » i rl rl H rl O O o t- id pH 8 3. para- IS. aureaa typn i B 0 oO o O O0 CO 0> H to t- CD 1 Hi Hi rl1 rlI HI rli rli rl 5 1C 16 - 4 + - 4 - 4 4 - 4 4 + 4 t t - + - 4 1 rl + t Hrs. w on o-# iod i l l * rl rl rl rt CD 1 H B. o o ll 8 . naratyph l B 3. paratyphi B S. aureus 1-50 1-60 1-50 S.T. B.C. PH S.T. 8 .0 29.87 B.C. PH S.T . 9M 8 .0 29.99 # B.C. 0 8 .0 29.87 13M 4 8 .0 # 12M 8 .1 # 7.6M 8 .0 7 7.9 # 17H 6 .1 # 16M 7 .9 10 7 .9 # 1 7 .6M 8 .0 # IBM 7.9 # 200T 24 7.9 29.83 31M 8 .0 29.73 36M 7.9 29.62 3401 Chemical Range o E. c o ll pH 6M 2.4M 400T A ll Tubes P o s it iv e S.aureus S .T . s i g n i f i e s su rface ten sio n in dynes per oentiraeter. ** B.C. s i g n i f i e s b a c te r ia l count per co. * M s i g n i f i e s m illio n s . * T s i g n i f i e s thousands. 9 Where no M or T fo llo w s the enum eration, th e fig u r e i s the a o tu a l b a c t e r ia l count p er c c . th a t i s , 10 organisms p er c o . in t h i s c a s e . Ho measurement was made. 7M # 6 .4 P henolic Range B.C. pH 3 81! 4 E. c o l i S. paratyphi B S. aureus 86 TABLE 5 DATA SHEET ON TEBGITOL 7 pH 7 Phenolic Range * 6 10 15 ty p h i B o o o o O O o t- 01 o> CD at rl 1 1 1 rl rl rl rl H rl - - 4 4 - + 4 - - 4 4 - 4 4 4 Chemical Range i& o o o o o H W fi fi H 1 1 1 1 1 H rH rl H rl 4 E. c o l i + + S. parat.yphi B * + 4 S . paratyphi B E. o o li o o o <0 P* CD 1 I I rl rH rl - 4 -'4 - EH O N 1 rl Hrs. 4 4 4 EH o tf) rt Eh o 4 1 H 1-100 1-50 e. o IQ 1 H 4 1 1 4 pH S .T .* B.C.** pH S.T. 1-100 1-50 B.C. B.C. pH pH S.T. S. aureus S.T. 1-20T B.C. 8M 7 .0 27.78 BK pH S.T. 1-30T B.C. pH S.T. B.C. 0 7 .0 27.69 15M* 7 .0 27.79 15M 7 .0 27.69 4 6.9 27.59 35M 6.9 27.79 60M 6 .9 27.55 27M 6 .9 27.55 4511 7 6.9 27.64 5011 6.9 27.59 87M 7 .0 2 7.55 251! 10 7 .0 2 7 .5 0 301! 7 .0 27.69 96M 7 .3 27.69 5M 7 .3 27.55 96M 7.0 36.69 500® 7 .0 3 6 .8 5 . 6M 24 7 .1 27.46 20M 7 .1 27.69 411! 7 .5 27.59 4M 7 .5 27.64 4211 7.0 36.69 7 .0 27.73 49M 7 .0 35.85 6U 7 .0 36.93 6M 7.0 35.64 1.4M 7 .0 36.79 1.2M 7.0 35.64 22F 7 .0 36.79 28 41! 6 .9 36.58 72M \ 1 S.aureus + f t * pH 6 .2 P henolic Range E. c o li 5 10 16 - 4 4 - - 4 - - 4 - 4 4 - - 4 . . . 1- o Ol 1 H 1- 1- typhi B o o o o o CD rl H H 4 4 4 S. aureus 1-50 1-40T pH S.T. 27.45 9M 5.2 37.45 61! 5 .4 # 5.2M 5 .2 # 700T 21 5 .6 # 7M 5.2 # 200T 801 5 .9 # 291! 5 .2 # SOT 6 .6 27.46 521! 5.2 38.02 150 S.T. B.C. pH S.T. B.O. pH S.T. 0 6.2 27.59 13M 5.2 27.59 13M 5.2 4 5 .3 ■ # 701 5.2 #' 37T 7 5 .3 # 91 5.2 # # 27.64 2.7M 4 4 4 B.O. B.C. pH Hrs. 4 - S. paratyphi B 1-60 1-50 Chemical Range o o rt W I I rl rt + + E. o o li S. paraty p h i B Ieh EH 0 0*0 o « lO .rt w 1 I I 1 rl rt 'rt rH + i + * ♦ 4 1 1 3 . aureus ... Eh O W 1 rt EH O <4 1 rl =-( O in i rl 1 !1 4 - - 10 5 .3 # 8 5.3 24 5 .3 27.64 0 5 .4 4 pH 8 P henolic Range Min. E. o o ll 3« p ara- S. aureus typhl B 0 o o 0 o o <0 P- aoH I rtI HI HI HI rt1 H 7M 7.2 36.51 14.611 7 ,3 37.02 101! 4 pH 5 .2 P h en o lic Range Min. E. c o l l o 5 10 16 o 1 rt - o C1 rt 4 - o rt 10 1 H H H rt 4 4 4 + - 4 4 - - 4 aureus o o t- CD 1 1 rt rt * 4 4 E. c o li S. paratyphi S S. aureus 1-50 1-60 1-30T pH S.T. 0 5 .2 31.29 4 5 .4 # 7 5 .4 # 10 5 .4 # 24 5.7 30.97 Hrs ■ + 4 4 Chemloal Range o rti rt i rti E* QOli f 3. para- + typ h l B EH O w i Is § 11 rt rt rt rt Ih I I I rIt H E. o o ll 3. paratyphi B 1-50 1-50 Hrs. pH S.T . B.C. pH S .T . 0 8 .0 31.76 12M 8 .0 3 1 .7 5 4 7 .8 4 76M 7 .8 # 38M 7 7 .7 # 89M 7 .7 # 86. 6M M N 7 « il rtOt rtU)i Irtr liii. rt rt 10 7 .8 # 721! 8 .0 # + t 4 24 7 .9 31.36 1211 8 .2 31/36 3. p ara- 4 ty p h l B 4 4 5 - 10 4 4 3 . aureus 1-10T 1-20T 3 .0 . pH S.T. B.O. pH S.T. B.O. 8M 8 .0 34.47 7M 8 .0 36.52 7M 8.0 # 119T 8 .0 # 1.8M 7 .9 # 2T 8.0 # 1.3M 1141! 7 .9 # 40 7 .9 # 1.1M 1 2 .5M 7 .9 , 34.27 7 .8 3 5 .4 9 420T 16 Chemical Hange H EH o o o o >o Eo o rl B« o o ll 3 .aureus I 0 4 4 4 4 * S .T . s i g n i f i e s surface ten sion in dynes per cen tim eter. ** B.O. s i g n i f i e s b a c te r ia l count per co. * M s i g n i f i e s m illio n s . * T s i g n i f i e s thousands. ® Where no M or T follow s the enum eration, th e f ig u r e 1 b the a ctu a l b a o te r ia l count per c o ., th a t i s , 4 organisms per o e. in t h i s c a se. * Ho measurement was made. 28 T A B IE 7 DATA SHEET OH H EKAI BZ pH 7 P henolic Range 5 10 15 o o <£► o o CO <0 o 8 o <£> O O CD rt rt rt H rt rl H rt H - + + - 4 4 - - 4 - 4 4 t 4 4 4 - - 4 - 4 4 • 4 r t Min.IE. c o li !o. o •o e- rt rt rt rt rt rt rt rt rt 5 :10 15 - 4 + + 4 4 - 4 - 4 • t - 4 - 4 4 + 4 4 - 4 4 4 Fl IO 61 O ri 61 O M E. o o li E. o o li para­ typhi B S . O w O w rt rt rt rt - - . 4 4 o 10 |R iH 1-60 1-10T S.T. B.C. pH S.T . B.C. PH S.T. B.C. 0 B.O 32.06 1311 8 .0 32.06 91! 8.0 41.23 7M 4 7.7 # 1211 7 .9 # 2.7 1 8 .0 # 3.211 7 7 .6 t 19K 7 .8 # 6.41 7.9 # at 10 7 .6 # 91 7 .8 # 5M 24 7 .7 31.20 41! 7 .8 31.20 61 o w ri ri ri ri S. aureus I pH Hrs. Chemical Range o rt 3. paratyphi B 1-50 9M 7.9 # 181! 7.9 40.66 f i 4 i i S.aureus i- - 4 4 4 S.T . s i g n i f i e s surface ten sio n in dynes per cen tim eter. ** B.O. s i g n i f i e s B a cteria l count p er cc. * M s i g n i f i e s m illio n s . •* T s i g n i f i e s thousands, ffi Uhere no M or T f o l lo w the enumeration, the fig u r e i s the actu al B a c te r ia l count per c o .. th a t i s , 300 organisms per oo» in thiB case* # ITo measurement ??as mads. 201 29 u s e d o f f e r s no m eans f o r e x p l a i n i n g t h i s p h e n o m e n o n , in terestin g lin o le ic , to n o te th a t and l i n o l e n i c in flu en za v iru s M iller ( 6) S t o c k and F r a n c i s m ately the in in a c t iv a t in g Baker, H arrison , p o i n t e d o u t t h a t I g e p o n AP (pH 7 ) t e r i a l m etab olism even at d ilu tio n in g red ien t (ap p roxi­ o f I g e p o n AP u s e d in th e experim ent d is c u s s e d in t h i s t h e s i s ) . T h is f a c t s u b s t a n t i a t e d by t h e r e s u l t s o b t a i n e d w i t h E . co li ■paratyph i B a t pH 5 . 2 f o r t h e first and stim u lated bac- a c o n c e n tr a tio n o f 1-100 of a ctiv e is (5 9 ) found o l e i c , a c i d s more e f f e c t i v e a t pH 7 . 6 t h a n a t pH 6 . it 7 hours is and S . o f th e growth p erio d . S u r f a c e t e n s i o n a l o n e a p p e a r s to h a v e l i t t l e n ifica n t in flu en ce on t h e g r o w t h - c u r v e d a t a . T h is i s agreement w ith th e r e s u l t s p r e s e n t e d b y M a rsh a ll ( 4 7 ) b y Mallmann and Darby ( 4 5 ) . that the It has is a measure o f i t s cid e. The d a t a f r o m t h i s tio n . No p o s i t i v e and a g e n t can lo w e r effectiv en ess as a germ i­ s t u d y do n o t s u p p o r t t h i s co rrela tio n in s o m e t i m e s b e e n a ss um ed degree t o w hich a s u r f a c e - a c t i v e surface te n sio n sig ­ conten­ app ears to e x i s t betw een g e r ­ m i c i d a l a c t i v i t y and r e d u c t i o n o f s u r f a c e t e n s i o n f o r t h e a n i o n i c a g e n t s a c t i n g a t s u r f a c e t e n s i o n s b e t w e e n 27 and 52 dynes p er c e n tim e te r . A l t h o u g h N e k a l and N a c c o n o l l o w e r e d the a p p r o x i m a t e l y 31 d y n e s p e r c e n t i m e t e r surface ten sio n to in c o n tr a st to 29 d y n e s p e r c e n t i m e t e r f o r I g e p o n and D u p o n o l , the f i r s t two compoun ds a r e f a r more g e r m i c i d a l t h a n t h e l a t ­ ter In a l l t h e e x p e r i m e n t s , tw o. sh ift in su rfa ce te n sio n t h e r e was a n e g l i g i b l e over the 2 4 -h o u r t e s t . so S i n c e the c h e m ic a l- r a n g e t e s t s e r v e d as th e i n i t i a l b a s is fo r u s in g th e proper d i l u t i o n s in the grow th-curve stu d ies, it is s i g n i f i c a n t t o n o t e t h a t t h e r e was no a b s o l u t e c o r r e l a t i o n between th e r e s u l t s o f t h e two m e t h o d s . I g e p o n AP, i n c o n t r a s t t o t h e chem ical-range t e s t S . a u r e u s a t pH 5 . 2 f a i l e d su rvive a 1-50 co n cen tration to o f the B o t h B. tration . o o l i and i n c o n s i s t e n c i e s when r e ­ a c t i n g w i t h U e k a l BX a t a 1 - 5 0 c o n c e n t r a t i o n . tru e fo r B. resu lt, a 24-hour exposure in an io n ic a g e n t. S . p a r a t y p h i B a t pH 5 . 2 d i s p l a y e d W it h The same was c o l i a t pH 5 . 2 w i t h T e r g i t o l 7 i n a 1 - 5 0 The s u r p r i s i n g f a c t i s t h a t o c c u r r e d a t pH 5 . 2 , t h e a l l these concen­ d iscrep a n cies r a n g e w h e r e t h e s e a n i o n i c compounds w e r e m o st e f f e c t i v e a g a i n s t t h e o r g a n i s m s m e n t i o n e d . the f a c t o r o f e x p o s u r e t i m e was c a r e f u l l y checked, Sin ce a p o ssib le e x p l a n a t i o n f o r t h e s e v a r i a t i o n s may be t h a t i n t h e c h e m i c a l range t e s t , the organism s have a b e t t e r chance o f s u r v i v a l , i n s o f a r a s t h e l i m i t i n g a r e a o f t h e t e s t t u b e may be s u f f i ­ c i e n t l y sm all (in c o m p a r i s o n w i t h an e r l e n m e y e r f l a s k ) a f f o r d p r o t e c t i o n f o r some o f t h e to organism s a g a in s t t h e syn ­ th e tic agents. S in c e t h i s method i s val test, q u i t e p o s s i b l e t h a t one o r tw o s u r v i v i n g b a c ­ it is an " a l l o r non e" s u r v i ­ t e r i a may h a v e b e e n s u b t r a n s f e r r e d s u c c e s s f u l l y , or t h a t one o r two v e r y s m a l l c l u m p s o f o r g a n i s m s w i t h s t o o d t h e b a c t e r i ­ cid a l e f f e c t o f the a n io n ic a g e n ts. Further ev id en ce o f t h e s e v a r i a t i o n s w i l l be p r e s e n te d i n the ca tio n ic d is c u s s io n o f the agents. Of t h e a n i o n i c a g e n t s t e s t e d , T e r g i t o l 7 a t pH 5 . 2 was th e m ost b a c t e r i c i d a l (on t h e b a s i s of e ffe c tiv e concen- 31 tra tio n ) f o r S« c o l l and S . a u r e u s . ■with t h e f i n d i n g s (T e r g ito l 4) o f G ersh en feld (24) it ( a t a 1 -4 0 con­ S. p a r a t y p h i B ). N a c c o n o l NRSF w a s g e r m i c i d a l o n l y f o r S . a u r e u s . D u p o n o l OS and I g e p o n AP f o l l o w , ness, in th a t as g e r m ic id a l a g en ts a g a in st cen tration is th a t of th e s y n t h e t ic F in a lly , order o f e f f e c t i v e ­ S. a u r e u s . Perhaps t h e m ost im portant f a c t w ith a n io n ic agen ts a t pH 5 . 2 , in th ese stu d ies w ith t h e proper con­ c o m p oun ds, T e r g i t o l 7 and H e k a l BZ a r e g e r m i c i d a l f o r t h e g r a m - n e g a t i v e o r g a n i s m s . excep tion compound H e k a l BZ a t pH 5 . 2 a l l t h r e e organism s was g e r m i c i d a l a g a i n s t i n agreement on a s i m i l a r and B a k e r e t a l ( 6 ) ( 7 ) . was g e r m i c i d a l a g a i n s t cen tration T h is i s W ith t h e o f t h e p a p e r h y G e r s h e n f e l d and M i l a n i c k ( 2 4 ) , w hich t h e y i n d i c a t e t h a t and pH 5 can k i l l 5. in A e r o s o l OT and T e r g i t o l 4 a t pH 4 t y p h o s a , t h e common b e l i e f h a s b e e n t h a t a n io n ic a g e n ts are o n ly b a c t e r i c i d a l f o r g r a m - p o s it iv e organism s. B a y liss (11) reported th a t c le a r e d su sp en sio n s o f gram -negative sodium l a u r y l s u l f a t e organism s, but he f a i l e d t o f i n d a p o s i t i v e c o r r e l a t i o n b e t w e e n t h e l y s i n g and l e t h a l action . The i m p o r t a n c e o f pH i s D u p o n o l OS. to At pH 8 , S. aureus su rv iv ed a 24-hour exposure a 1-50 c o n c e n tr a tio n o f t h i s 1-400 sy n th etic agent. At pH 7 , a c o n c e n t r a t i o n was e f f e c t i v e l y g e r m i c i d a l a g a i n s t th e same o r g a n i s m , w h i l e a t pH 5 . 2 , k illed d r a m a tic a lly p r e se n te d by S. aureus i n 24 h o u r s . a 1-4000 d ilu tio n The o n l y p o s i t i v e co m p letely co rrela tio n b e t w e e n t h e t r e n d o f pH and b a c t e r i a l c o u n t o v e r t h e v a r i o u s p e r i o d s o f th e growth cu rve i s th a t once th e b a c t e r i a l count 32 g o e s b e l o w t h e o n e - m i l l i o n mark and s t a y s b e l o w t h a t m a rk , t h e s h i f t i n pH i s a ll eith er n e g lig ib le other in s ta n c e s , l e v e l reached in th e 0. the or n o n - e x i s t e n t . c h a n g e i n pH f a l l s In sh ort o f the co n tro ls. O a t i o n i c Compounds D o m a g k 's ( 1 3 ) p u b l i c a t i o n on t h e l o n g - c h a i n q u a t e r ­ n a r y ammonium s a l t s com p oun ds. stim u la te d a tremendous i n t e r e s t in th ese W orking m a i n l y w i t h z e p h i r o l ( a l k y l d i m e t h y l b e n ­ z y l ammonium c h l o r i d e ) , he r e p o r t e d i t s as w e ll as g erm icid a l a c t i v i t i e s germ icid al a c t i v i t y , f o r o t h e r q u a t e r n a r y ammon­ ium s a l t s w h i c h p o s s e s s e d a l o n g - c h a i n a l i p h a t i c g r o u p (CJjH/7 to )• The m e t h o d s f o r p r e p a r i n g t h e t h r e e t y p e s o f c a t i ­ on ic ag en ts used in the i n v e s t i g a t i o n r e p o r te d in t h i s a r e d e s c r i b e d i n t h e p a p e r s b y Kuhn e t a l ( 4 1 ) m e t h y l b e n z y l ammonium c h l o r i d e ) , (57) (for (for th esis a lk y l d i­ and b y S h e l t o n e t a l (56) OTAB and a l k y l p y r i d i n i u m c h l o r i d e ) . The c o n c e n t r a t i o n s used in t h e s e o f q u a t e r n a r y ammonium compounds experim ents e i t h e r reduced th e c o u n ts o f a l l th ree ty p es o f b a c te r ia or k i l l e d a l s o i n h i b i t e d th e normal s h i f t th e m c o m p l e t e l y . T h ey i n pH o v e r t h e 2 4 - h o u r p e r i o d . The r e s u l t s p r e s e n t e d i n T a b l e s 8 , 9 , and 1 0 a r e i n a g r e e m e n t w i t h t h e g e n e r a l t h e o r y o f maximum g e r m i c i d a l a c t i v i t y o f c a t i o n i c a g e n t s i n an a l k a l i n e r a n g e . o f Kuhn and B i e l i g iza tio n . (40) gave str o n g support to t h i s g e n e r a l­ They p o i n t e d o u t t h a t p r o t e i n s cou ld only be pre­ c i p i t a t e d when t h e y w e r e i n t h e fo rm o f a n i o n s , b asic sid e The work o f the is o e le c t r ic p o in t. i.e ., on_the Th ey l i k e w i s e p r o p o s e d TABLE 8 BATA SEE2T Oil ROC C .E pH 7 Phenol!o Range S. rara- S. aureus tvnhi B o o o o o o o (D Oi ri to C- CD 1 1 1 1 1 1 H H H H H rt - 4 4 - 4 4 - 4 4 - - 4 - - 4 - - 4 Kin. E. o o li 5 10 15 O e 1 H - o id 1 rt 4 4 4 o 0) I H 4 4 4 1-3T S. para- typ h l B 4 +! I r + 4 4| 1 1-5T 1-3T B.O. pH S.T. S. aureus B.C. pH S .T . B.C. pH S.T. 1-60T 1-40T B.C. pH S.T. 81£ 7.0 47.17 B.C. pH S.T . 8M 7 .0 53.99 B.C. 6M 7 .0 56.07 611 0 7 .0 43.71 13Ma 7 .0 46.02 15M 7 .0 47.31 15M 7 .0 43.66 4 7 .0 4 4 .10 2T* 7 .0 46.13 46T 7 .0 47.92 1011 7 7 .0 43.54 20® 7 .0 45.97 10T 7 .0 4 6.75 100T 7 .0 43.99 141! 6.9 4 7.17 401! 7.0 53.76 480T 7 .0 64.97 1H 10 7 .0 4 3.48 0 7 .0 45.84 2T 6 .9 4 6 .6 6 4.5K 6 .9 43.66 291! 7 .1 4 6.34 104M 7 .0 53.57 450T 7 .0 64.97 1M 24 7 .0 43.48 0 6.9 45.84 5M 7.0 46.66 95M 7 .2 43.66 39M 7 .5 4 6.34 26011 6.9 53.99 21T X X - X - 1-5T 1-4T Hrs. pH S .T .* B.C.** pH S.T. Chemical Kange E-* E-4 EH I O O O & & et & o rl W V) H « tC 1 1 1 1 I I 1 H H H H r! E. c o ll S. paratyphi B S. ooli 1M 6.9 47.17 17M 7 .0 54.35 7 .0 43.99 21! 7 .0 66.04 2.2M 7 .0 66.07 600T I S.aureus 4 + 4 pH 5,2 Phenolic Range S. p ara­ S. aureus typhi B Min'. E. c o li fc1 rl 6 10 16 + - o o o O CD 1 H o o CO 01 1 1 H rl 01 1 H ri 1 H o tO i Ci rl OJ i f t t i - + + rt + 4 4 - 4 4 - - 4 + 4 t 4 - t o 1-4T rl Hrs. + Si H Si « s n e e e rt rt rt rt - - - f E. o o li S. jpara- typh i B - + + pH S.T. 0 is rt rt rt o^ W rt B.O. 5 .3 # 32T pH S.T. B.C. 5.2 46.75 IBM 6 .0 240T # 1-6T 1-6T 1-5T 5 ,2 46.97 15M 4 Chemioal Range £ o o rt « S. aureus 3. paratyphi B E. o o ll O pH S.T . pH B.C. 5 .2 46.71 6 .2 47.92 IBM 5 .3 # S.T. 6 .2 31! # B.C. pH S.T. 6M 461 5.2 # SOOT # 3M # BOOT 5 .0 f 600T # 2001 # 400T # 5T 5 .4 # 4T 6 .3 # 220T 6 .2 # 3T 10 5 .3 # 260 5 .4 # 800 5 .3 # 2T 5 .2 # 18T 5.2 24 5 ,3 45.00 6 .0 46.75 52M 6 .4 46.75 800T B.C. 6 .3 5 .3 5 .4 45.97 112 pH S.T. 6.2 60.02 7 0 B.C. 1-30T 6.2 62.26 6M 8H 5 .2 t ■ 1-BOT 6 .2 61.80 5T 6 .3 5 .3 52.78 661 + S.aureus - 4 4 Phenolic Range Min. E. o o li fc» © 5 10 16 1 rt 1 rt 4 - 4 4 - t . i rt 4 4 4 S. Bara- S. aureus typhl.B o to p- CD 8 S S 1 i 1 i t i rl rt rt rt H H 4 - 4 4 - 4 4 4 4 “ t 4 4 4 Hrs. pH S.T. E. o o li s< rl si W e< E! 3 s e e H H H rt rl - - - - 4 S. para- tJPhi B S.aureus 4 t 4 4 IS IT1 o w Si O m H H S.T. B.C. pH S.T • 0 8 .0 4 6 .98 13M 8 .0 47.91 12M 8 .0 4 7 .4 8 4 8 .1 7 ■8.1 1 B.C. pH 7.8 # 3T 8.0 * 91T # 3T 8 .0 # 16T 7 .8 3T 8 .0 10 8 .1 24 8 .1 46.13 # 12 0 8.0 # S. aureus 1-30T 1-6T 1-6T 1-5T Chemioal Range SI S. paratyphi B E. c o li 8 .0 47.49 10T # B.C. pH 91! S.T. 1-4 0T B.C. PH S.T. 8 .0 63.29 6.5M 8 .0 54.61 151! 8 .0 # 15T 7.9 # 641! 8 .0 # 2T 7.9 # 861! 8 .0 # 116 8 .1 4 7 .1 3 191! 8 .0 51.80 0 7.8 # # t 7 .8 53.29 1-50T B.O. pH S.T. 1-70T B.C. PH S.T. 6M 8 .0 65.49 - 61! 8 .0 6.7.83 # 700T 8 .0 # 0OT 7 .8 # 2001 8 .0 # 1.31! 15T 7.8 # 100T 8 .0 # 400T 7 7 .8 53.97 100 1 4 S.T. s i g n i f i e s surface tension in dynes per cen tim eter. ** B.C. s i g n i f i e s B a c te r ia l count per co. 4 M s i g n i f i e s m illio n s . + T s i g n i f i e s thousands. ® Where no M or T follow s the enumeration, the fig u r e th a t i s , 20 organisms per c c. in t h i s ea se. t Ho measurement was made. la 611 _ 120T 7.8 1 1- 4 4— B.C. the actual b a c te r ia l oount per c o . , 8 .0 56.94 8H 7 61 34 TABEE 9 DATA SHEET ON EPC P henolic Range Min. E. o o li 3 . para­ S« aureus typhi B ^ o o o o o o o o O o c- co m CO O H 10 C- CD 1 1 1 i i i i i i rl r l H r t r t rt r t rt H 6 10 16 - + + + + - + 4 • + 4 - - + + + Chemical &< EH O o& IQ r l 1 | W 1 rl H H Range u1 o £ o oZ 1 o IH tfi 1 1I I H H I H - - i + 4 - + + 4 E. c o l i S. para— typhi B S.aureus 1- 4 4 Pj o o W I H + 4 4 4 E. o o li 1-30T Hrs. pH S.T.* B.C.** pH S.T. 1-30T B.O. pH S.T. S. aureus 1-401 B.C. pH S.T. 1-200T B.C. pH S.T. 1-800T 1-300T B.C. pH B.C. pH S .T . S.T. 0 7 .0 44.98 15M1 7 .0 44.17 8M 6.9 45.56 7M 7 .0 4 4.33 7 7 .0 40.91 400T* 6 .9 45.95 5M 6 .9 43.38 47M 6.9 46.51 66M 7 .0 53.66 2.1M 7.0 64.90 5.5M 7 .0 68.39 481! 10 7 .0 42.87 6 .9 46.07 3M 7 .0 44.83 4711 84 7 .0 43.81 60T 84® 7 .0 4 6 .B7 1511 7.0 46.81 4011 7 .0 44.03 8M 7 .0 46.49 ?H 6M 6 .9 46.87 1611 7 .0 53.04 6M 7 .0 55.76 6M 7 .0 67.36 611 7 .0 62.29 8.111 7 .0 53.56 6.9M 7 .0 68.06 9H 7.2 46.96 110M 7 .0 63.23 800T 7 .1 64.50 5.6M 7.8 56.14 601! 96T 7 .1 53.47 460T 7 .4 66.75 901! 7 .5 45.34 75M 7 .6 47.07 192M 7 .0 52.29 4 pH 6 .8 P henolic Range 3. para­ S. aureus typh i B o o o o o o o 1 i i i i i H H rt o i i o i 5 10 16 + - l 1 1 1 o 4 4 4 + - + E. c o li 4 - 4 4 - 4 4 t 4 - - + + + + + 4 pH S.T. 0 6.2 4 2 .8 3 4 5.2 7 10 Hrs. Chemioal Range Eh O WH Eh rt rt E. c o li - S. para- fyph i B - + o o o °W 210 °^ |. orl oCM oto rt rt rt i H rt rt 4 + 4 | P-i + Eh + S. paratyphi B 1-30T 1 l Min. E. c o li 3. aureus 1-30T 1-2001 1-100T PH S.T. B.C. pH S.T. B.C. 9M 6.2 51.20 6M 5 .2 53.56 6U # 9M 5.2 # IT 5.2 # 7.8M 5 .7 # 9M 5.2 * 6 5 .3 # 5.9H 6 .3 # 78M 5.8 # 0 6.3 # 3.8M 5 .4 51.59 700T B.C. pH S.T. 12K 5 .2 42.27 # 6.7M 5 .2 5.2 # 1.6M 5.2 # BOOT B.C. Eh 1 5 .2 84 41.05 88 4 3 .3 8 6 .8 13611 5 .2 0 50.95 4 i- S. aureus 4 4 pH 6 Phenolic Range S. para­ S. aureua typ h i B o 0 0 ID t(0 0> ri 1< 1 1 i■ iI H rH H rl H 4 4 4 + 4 4 4 - 4 4 4 - - 4 Min. E. c o li o o © ^ cd ci i i i. H rl ri 5 + + 4 10 - + + IB - 4 4 EH o o cj to H H rt rl rl - - - 4 + S. para- typ h l £ - 4 + + E, c o l i S.aureua 8 01 1 rt 4 pH S .T . B.C. PH S.T. B.O. pH S.T. 0 8 .0 4 5.95 14M 8 .0 45.87 9M B.O 63.16 4 8 .0 # BIT 7.8 # 13M B.O # 7 8 .0 # 15T 7 .8 # 13M 8 .0 # Hrs. 1 rt 10 8 .0 t 24 8.0. 45.56 1-300T 1-2001 1-40T 1-401 Eh O o S. aureus S. paratyphi B E. c o l i Chemical Range eh B.O. 4 P-4 o o « 1 H S. paratyphi B 1-40T 700 7 .7 # 47M 8 .0 19 8 .2 4 5.87 13M ' 8 .0 ' B.C. pH S.T. B.C. 6M 8 .0 65.76 6U 200T 8 .0 ‘ 8 .1 . B.O t 160T # IT 8 .0 t 82T 53.56 0 6 .0 66.75 208 s ig n if i e s su rface tension in dynes per cen tim eter. ** B.C. s i g n i f i e s b a c te r ia l count per oo. A + ® # M s i g n i f i e s m illio n s . T s i g n i f i e s thousands. Where no M or T fo llo w s the enumeration, th e fig u r e i s the a ctu a l b a c te r ia l count per c c . , th a t i s , 24 organisms per oc. in t h is c a se. No measurement was made. 4001 6T 4 * f 35 TABLE 1 0 DATA SHEET OH CTAB pH 7 Phenolic Range S» para­ S. aureus typhi B o o o o o o o o o o t- CD 01 CD Oi H tO CD 1 1 1 1 1 1 1 1 1 rH H H + - 4 4 + 4 + 4 1 + 4 4 1 + + - 4 - 4 1 1 5 10 16 1 1 Min. E. o o li - 4 4 - -I 4 + 1-10T B B o cn rl i 1 r rl + B in I H - E. o o li S. para- typhi B + B 11 o* o .o W H 1' I HIH a o o CD 4 - 4 4 4 4 - - 4 1-20 Chemioal Hange 0H C 0U t 0f t ' 0j ' 0i O i0O E0- C0O 1 I I I I II I rtHrtrtrtrtrtrt E. o o ll + + + + + 3. paratyphl B + + + +4 4 4 4 3. aureua + + +4 4 pH Hre, S. paratyphi B 1-20 1-50 S.T.* B.C.*'1' pH S.T. B.C. pH S.T. S. aureus 1-20 1-50 B.O. pH S.T. B.C. 0 7 .0 07.61 12M4 7 .0 07.99 13V 7 .0 37.61 8M 7 .0 37,94 4 6 .7 37.66 45V 6 .7 38.00 75M 6 .8 3 7.56 68M 6 .8 37.94 pH S.T. 8M 7.0 37.64 1-60 pH B.C. S.T. B.C. 6M 7 .0 37.94 6M 6 .9 38.03 6M 70M 6.9 37.64 5.7M 7 6 .8 37.66 82M 6 .8 30.08 105M 7 .1 37.56 100M 7 .1 3 8.03 124M 6 .9 37.89 3.5M 6 .9 38.00 4.1V 10 6 .8 07.56 55M 6 .8 38.08 6.9 07.89 2.2V 6 .9 38.03 2.6M 24 6 .9 07.56 651- 7 .0 38.08 130M 68V 7 .2 37.62 152M 7.2 38.03 180M + 4 4 + + 7 .5 37.52 169M 7 .6 38.03 214M 6 .9 07.89 1 1 .TV 7 .0 3 8.08 23V 4 pH 5 .2 P henolic Range 3. para­ S. aureus typhi B Min. E. co li o o o o > CD Ol co 1 1 1 1 H rl rl rt 5 4 4 4 4 10 - 4 4 15 - 4 4 - E. o o li o o m h 1 1 rt rl o o o (O 013 S. paratyphi B S. aureus 1-20 1-20 1-20 rt 4 Hrs. pH S.T. ■ 4 4 0 5.2 37.75 Chemical Hange 4 5.3 # o o o o o O o o rl N (1 « « <0 t - CO 1 1 1 1 1 1 i 1 H H r t rl r t r t r t r l 4 4 4 4 4 + + 4 7 5.4 10 5.5 4 + 4 4 4 4 rt E. c o li - - rt t 4 4 3. p a ra - 4 typhi £ 4 4 4 4 + + 4 3 .aureus + 4 4 4 t 4 4 + 5.6 24 a o o o o o o o t- CD O) CO o i r t rt rt r t rt rt rt 10 16 4 - + - S. O O ll para­ ty p h i 5.2 37.75 # SBM 5.2 # 175# 6.0 # 107M 6.2 # 2.6V 6.4 # 203V 5.2 # UK 239M 6.1 07.76 90JI 5.2 37.75 86M 5.5 # 10611 # 120M 214V 6.8 07.89 4 t 4 4 + 4 - 4 4 + - o o 10 4 4 o CO rt rt rt • 4 C- - 4 4 4 4 - 4 0 rt 0 N 0 e> rt rt +4 + 0 0 0 10 c - 0 CO ri ri ri H rl rl 4 4 4 4 4 0 10 4 4 t 4 4 4 4 4 4 4 4 t 4 t 4 4 4 E. o o li S. paratyphi B 1-20 1-20 pH S.T. 8.0 37.56 4 7.8 # 7 7.7 10 7.8 24 8 .0 Hrs. 0 . B.C. PH S.T. 16M 8 .0 37.56 110M 8 .0 # 117M 8 .0 # 138H 8 .1 37.61 142M 8.2 4 M s i g n i f i e s m illio n s . ■* I s i g n i f i e s thousands. # No measurement was made. 1-20 B.C. S.T. 8M 8 .0 37.56 # 5111 8 .0 # 20T # 120K 7.9 # 2.1V 239V 7.9 # 6V 161M 8 .1 37.56 # ■ 37.61 3 ,1 . s i g n i f i e s surface ten sion in dynes per oen tim eter. B.C. s i g n i f i e s B a cteria l count per c c . S . aureus PH B S ia u r e u s 6M pH 8 Chemical Hange S i BM 15M B.C. S. p a ra- S. aureue ty p u i 5 S.T. S.T. 37.89 B.O. pH pH P henollo Hange Min. E. ooli B.C. B.C. 6V - 52V 43 TABIE 12 DATA 3HE3T Oil T R IM X-100 P h en olic Range Kin. E. c o l i 5 10 15 S. para­ 3. aureus typh i £ S. c o li o o O o o o oo U> H ® » 8 rl o o 10 t- O m H H H H H t + t 4 + - + + “ - rH 4 H H - - 4 + + H + f “ + + t 1-20 o O o rl H rl 4 4 t O O o a* IO (0 c- C O H rl H rl rl + 4 + + 4 3. p ara- + typ h i B + + + 4 + S.aureus + + 4 4 t + t H E. o o li W W 1-50 1-20 pH S .T .* B.C.** pH S.T. Hrs. B.C. pH S.T. S. aureus 1-50 1-20 B.C. pH S.T. B.C. pH 3.T . 1-50 B.C. pH S.T. B.C. 0 7 .0 32.66 12M* 7 .0 32.75 1411 4 6 .8 32.62 56K 6 .9 32.68 7211 6 .9 32.71 10M 6.9 32.66 1 8 .5M 7 .0 32.62 1 7 0 / 7 .0 32.71 280T 7 6.9 32.62 3111 5.9 32.57 53M 7 .1 32.71 BOM 7 .1 32.66 57M 7 .0 32.57 10 7 .0 32.62 15M 7 .0 32.57 6711 7 .3 32.66 601! 6111 7 .0 32.57 100T 6 .9 38.71 600T 24 7 .0 32.75 9OK 7 .0 32.66 112M Chemical Range o S. paratyphi B o 7 .0 32.66 8K 7 .0 32.75 8M 7 .0 32.62 7 .3 38.66 7 .5 32.85 52M 7 .5 32.66 100M 6 .9 38.75 6M 7 .0 32.71 60T 7 .0 32.71 .6M 6 .8 32.66 6M 60T 1011 ■+ 4 pH 5 .2 P henolic Range S. para­ 3. typh i B Kin. E. c o li 5 10 15 a u re u s o ti -C o CO O m o CO o Oi O o H o e- o ai O o> H H H H H> H rl H - 4 4 4 4 4 - - 4 4 4 4 - - 4 + 4 4 4 4 4 4 f 3. pal para- + h f typnl T .aureua + + + 4 f 1-20 S.T. B.C. B.C. pH S.T. 32.71 5 .2 8M 5 .2 32.71 B.C. 5 .3 # 38M 5 .4 # 2 OK 6.2 # 2.111 7 5.4 # 47M 5 .8 # 51M 5.2 # 1.7M 10 5 .8 # 54M 6 .3 f 9411 5.2 # 3.111 24 5.8 32 .’6 2 58K 6 .7 32.62 1201! 5.1 32.75 6M 4 +4 + f + + + + 4 + 1-20 32.71 o o I I I II I I I H H H H H H H H 1-20 S.T. 4 - S. aureus pH Chemical Range “ S. paratyphi B 12M 5 .2 o o o o o o HMK^O^t-CO E. o o li pH Hrs. 4 4 4 0 - - - + E. c c l i 8M pH 8 P h en olic Range 3* para­ 3* aureue typhi o o o 0 O o o c- CD 01 CO O' rl Min. E. c o l i o o o o S> H H rt H CO 5 10 16 4 - 4 4 4 H H rH rl + t i - + rl - 4 4 - 4 4 - - 4 4 4 4 i H 4 4 4 Hrs. 0 4 Chemioal Range o o rH rl Q o o t£> t" 8 5 4 s rl H H H rl rl 4 4 + t + t S. p ara- 4 typ h i B 4 + i + 4 4 S. aureus 4 4 + t + + rl E. o o li « O o + 4 4 E. OOli S. paratyphi B S. aureus 1-20 1-20 1-20 pH S.T. 8 .0 32.62 7 .8 B.C. 1211 pH S.T. 8 .0 32.62 B.C. pH S.T. 811 8 .0 32.62 611 # 55M 7 .8 # 1411 8 .0 # 4.811 7 .8 # 44M 8 .0 # WOT 311 7 7 .7 # 64M 10 7 .8 # 42M 7 .9 # 341! 8 .0 # 24 7 .9 32.57 31M 8 .0 32.57 2511 7 .9 32.71 4 B.C. 4 * 3 .1 . s i g n i f i e s surface ten sio n in dynes per oen tim eter. ** B.O. s i g n i f i e s h a o teria l count per cc. * M s i g n i f i e s m illio n s . ^ I s i g n i f i e s thousands. * Ho measurement was made. 141! 44 a c u r ta ilm e n t o f the normal s h i f t lesser i n pH, alth ou gh to e x te n t than t h a t o c c u r r in g w ith e i t h e r a much a n i o n i c or ca tio n ic agents. The r o l e itie s S. o f pH e x e r t s o f t h e s e compounds. some i n f l u e n c e On t h e again st S. o th e r hand, a ctiv ­ E m ulp hor OH was m o s t a c t i v e a g a i n s t a u r e u s a t pH 8 and a g a i n s t B. pH 7 . on t h e co li and S. p a r a t y p h i 3 a t T r i t o n X -100 was m ost e f f e c t i v e a u r e u s a t pH 7 , and a g a i n s t B . c o l i and S . p a r a ­ t y p h i B a t pH 8 . A lthou gh th e d a t a p r e s e n t e d i n T a b l e s 1 1 and 12 a r e b y n o m ea n s c o n c l u s i v e , com plete r e v e r s a l it is in terestin g o f th e i n h i b i t o r y pow ers o f t h e two a g e n t s a g a i n s t g r a m - p o s i t i v e and g r a m - n e g a t i v e and pH 8 . F u n ction in g at t h e i r pounds are c a p a b le to note the o p t i m a l pH r a n g e s , b o t h com­ o f red u cin g t h e 3. b e t t e r th a n 97 p e r c e n t for o r g a n i s m s a t pH 7 au reu s p o p u l a t i o n by 4 h o u r s or l e s s . made t o e s t a b l i s h w h e t h e r t h i s a ctiv ity Ho a t t e m p t was is b a cterio sta tic or b a c t e r i c i d a l . I n fo r m a tio n co n c er n in g com m ercial u s e s , p rep aration , e tc ., of n o n -io n ic m ethods o f com pounds can be f o u n d i n t h e p a p e r s by G o l d s m i t h ( 2 6 ) ( 2 7 ) . E« B a c te r io s ta tic T ests D esired d ilu t io n s pared in t e s t cc. w ell tubes in 5 -c c . of su rfa ce-a ctiv e q u a n tities. agent were p r e ­ To e a c h t u b e , o f a 2 4 - h o i i r o r g a n i s m s u s p e n s i o n was a d d e d . s h a k e n and t h e n i n c u b a t e d f o r A fter 24 h o u r s , subtransfers 0 .1 Tubes were 24 h o u r s a t 57 d e g r e e s ( 2 l o o p f u l s ) w e r e made i n t o 0. F.D..&. b r o t h , f o r 24 h o u r s . days, and b o t h s e t s Eac h 2 4 h o u r s t h e r e a f t e r , s u b t r a n s f e r s from t h e i n t o H .D.A. b r o t h . b asis o f t e s t tu b es were in c u b a te d o r ig in a l set for 2 a d d itio n a l o f t u b e s w e r e made P o s i t i v e g r o w t h was r e c o r d e d on t h e o f v is u a l tu rb id ity . T h is method i s a m od ification o f t h a t s u g g e s t e d b y Ii olm er ( 37 ) • S in ce the group, q u a t e r n a r y ammonium c o m p o u n d s , a r e c o n s i d e r e d more a c t i v e b a c t e r i o s t a t i c than th e a n i o n i c compounds, b a c t e r i o s t a t i c as a agents titres on t h e f o r m e r a g e n t s w e r e d e t e r m i n e d a t t h e t h r e e pH r a n g e s . The r e s u l t s p r e s e n t e d i n T a b l e s I S and 1 4 a r e i n a g r e e m e n t w i t h the accepted g e n e r a liz a tio n s t a t i c pow ers. agents, concerning r e l a t i v e b a c t e r io ­ The a c i d r a n g e o n l y was u s e d f o r t h e s i n c e th e y are g e n e r a l l y m ost a c t i v e a n io n ic again st b a c te r ia a t t h i s pH l e v e l . In t h e b a c t e r i o s t a t i c ism s i n i t i a l l y in trod u ced into th e t i c a l w i t h t h e number i n i t i a l l y range t e s t s tests, t h e number o f o r g a n ­ test t u b e s was a l m o s t i d e n ­ in ocu lated and t h e g r o w ' t h - c u r v e t e s t s . in both the ch em ical- Su ch c o n t r o l o f t h e q u a n t i t y o f o r g a n i s m s o r i g i n a l l y i n t r o d u c e d more c l e a r l y f in e s the con d ition s of these tests de­ and m a k e s p o s s i b l e a m ore e x a c t d u p l i c a t i o n o f r e s u l t s w ith a l l t h r e e m ethods o f t e s t ­ in g. The a g r e e m e n t b e t w e e n t h e tests and t h e 2 4 - h o u r r e a d i n g s o f t h e the b a c te r io s ta tic tests* r e s u l t s o f the chem ical-ran ge ns u b t r a n s f e r " i s v e ry good in s p i t e tub es in o f the fa c t t h a t t h e t e s t s w e r e made 1 t o 6 m o n t h s a p a r t . * I t s h o u l d b e n o t e d t h a t t h e c h e m i c a l - r a n g e t e s t s and t h e 24-hour " s u b tr a n s fe r ” d e te r m in a tio n s in the b a c t e r i o s t a t i c s e r i e s are i d e n t i c a l t e s t s . 46 TABLE 12 BATA FHOl' BAOIEEIOSTATIC TESTS AT pH 5.2 O riginal Hrs. O El Ml H Subtransfer B (jI B 4I B 1I0 R H H H R Hrs. R R CD & I I rl H 24 S. paratyphi S. aureus E. o o li n_„ Duponol OS E loooulation E. o o li S. paratyphi B S. aureus 24 48 72 24 48 72 24 48 72 S. aureus 4 4 4 4 4 4 4 + 4 4 4 4 4 + 4 + 4 4 4 t 4 4 4 1 4 4 4 ± 4 4 4 4 + 4 1 ± ± r| fi 4 4 4 4 4 4 4 4 4 4 + 4 4 4 + 4 4 4 4 + 4 4 + 4 4 4 4 4 4 4 t S. aureus Hrs. E. o o ll S. paratyphi B 3. aureus 24 48 72 24 48 72 24 49 72 Hrs. E. o o li S. paratyphi B S. aureus 24 48 72 24 48 7« 24 48 72 4 4 4 + 4 4 4 4 4 E. o o li Igepon AP S. paratyphi B S. aureus E. c o li 1 1 T e rg ito l 7 S. paratyphi B 1 0 rl rl 4 4 + 4 4 + O 0 w rt 4 4 4 4 4 4 O R O O 0 R R O 8 <41 to <0 H to H 1 1 1 1 rt rl rl rt rt rl rl + t t 4 t + + t f 4 4 f + 4 4 + 4 4 + 4 + f j - - - " - - 4 t 4 4 4 4 R C w R O rt rl rl O O O O . R R Eh S. aureus 1 N aooonol O IfflSP 4 T 4 4 4 i O o o ll S. paratyphi B S. aureus 4 4 4 4 4 4 + 4 4 4 4 4 +1 4 j 41 +1 4| +1 1 I1- E. c o li Hekal BX . . . . . R H rl rt rl - - - - - • . . 4 + t 10 <0 0 R R g CO H CVl rt H H H t t t 0 - 4 + 4 4 4 4 4 4 8 0o oo H N t O ^ W O E - C D a > H W W I r It HI HIr Il r Il HI H Ir lI r Il HIH I A ll Tuhes P o sitiv e All Tubes P o sitiv e Ail Tubes P o sitiv e 0 rl 24 48 72 24 48 72 24 48 72 + S. paratyphi B S. aureus 24 48 72 24 48 72 24 48 72 0 O Pi O O O Cl to to R 0 rl R O Cl g R O <*• R O 10 8 rt rl. rl rl rl rl H rl rl rl rl + + 4 4 + + 4 i + 4 + 4 + 4 4 + 4 + ■1 t 4 4 4 t 4 + 4 + i t 4 4 + 4 1 4 0 H rt 4 4 4 4 4 4 O 0 Cl to H H 4 4 4 4 4 i 4 4 4 4 4 4 O O 10 rl rl 4 4 4 4 t 4 4 4 4 4 * 4 - - - + 4 - - - t 4 - t 4 R R O R O O to S to rl Cl H rt rl rl rl - - t 4 4 R R O O to * H rt 4 4 + 4 — . . . . . . . <0 + . oCl o« JA . . Pi + * Hrs* O <11111,111111 r t r t r t r t r t r l l r l r l r t r t r t r l . . . . . . . . . - - 4 - . 1 - - 4 24 48 72 24 48 72 24 48 72 Hrs. E. - R rt H I I ' Granular 1 R to rl 24 48 72 24 48 72 24 48 72 Hrs. Turbid Turbid R rl rt rt + 4 t 4 t t + 4 + 4 rt R EH O 0 10 U) rt H SOT S. paratyphi B 24 48 72 24 48 72 24 48 72 Eh 0 ^ r-1 4 4 4 4 4 4 4 4 4 24 48 72 24 48 72 24 48 72 Hrs. I I I I 1 I |R R R 0 O O O O 0 'o 0 0 10 to jH W W rt C] ttt rH H rl H H H |rl H r ! E. o o li S. paratyphi B 0 0 0 0 0 0 0 0 0 8 8 0 H N N ^ U J v O t - C O c n HH M t a r l rH r t r l H rl rt rl rt rH r-1 I I II I 0 Cl rl li. o o li 72 24 48 72 24 48 72 0 - - - - - 4 4 4 4 4 - - t . . . 4 + 4 + i 1010 o<0 Rto1 ©R1 l1 H r1 t I rl rl rl 4 4 4 4 * 4 4 4 4 + 4 4 4 i 4 1 4 I 4 * 4 1|- 1........................... t 47 TABLE 14 DATA FROM BACTERIOSTATIC TESTS pH 7 pH 5.2 lauryl Pyridiniuni Chloride Hrs. ooll Original/ S. paratyphi oo li SubtransfgrfS. paratyphi B . aureua Roooal ooll O r i g i n a l / S. paratyphi B .3. aureus ooll Subtransfer/S. paratyphi S. aurens E. ooll Original, paratyphi B Subtranafer/S. paratyphi B ■S. aureus 48 No b a c t e r i o s t a t i c inary t e s t s e f f e c t s were o b serv ed in p r e lim ­ w ith th e n o n -io n ic a g en ts. Under t h e l i m i t a t i o n s im p o s e d b y t h e t u r b i d g r a n u la r appearance in s o l u t i o n as w e ll as by the n atu re b l e t o make a n y a b s o l u t e tent of b a cterio sta tic o f som e o f t h e a n i o n i c o f the t e s t statem ent a ctiv ity and em ployed, it is agents, im p o ssi­ con cern in g th e g e n e ra l ex ­ of these a n i o n i c compounds. The r e s u l t s w i t h N e k a l BX, N a c c o n o l NK8F, a n d I g e p o n AP i n d i ­ c a t e t h a t no s t a t i c n eg a tiv e e f f e c t was a p p a r e n t a g a i n s t organism s. However, the r e s u l t s th e gram- i n Table 5 in d ica te t h a t T e r g i t o l 7 m ight p o s s i b l y have e x e r t e d a b a c t e r i o s t a t i c e f f e c t on B . c o li. The s t r o n g e s t evid en ce o f h a c t e r io s t a s is was d i s p l a y e d b y IP k a l BX a g a i n s t S . a u r e u s . A ll the terio sta sis ca tio n ic a g a in s t S. o c c u r r i n g a t pH 8 . agents T able 14) These c om pounds s i m i l a r l y d isp la y ed b a c t e r io s t a t ic a c tiv it y t h e p r e s e n c e o f serum , to f i v e (51), in h is can b e no c a t i o n i c exp erim en ts w ith observed th a t agent CTAB i n ,rA c o n c e n t r a t i o n o f two T h is o b se r v a tio n i s T ab le 14) by r e s u l t s w ith S. c o li. a g a in s t S. p a r a ty p h i B. tim es th e c r i t i c a l b a c t e r io s t a t ic t o b e b a c t e r i c i d a l . 1' a f f e c t E. c o n c e r n i n g t h e pH r a n g e W ith t h e e x c e p t i o n o f LPO a t pH 5 . 2 , H oogerheid e e x h ib it bac- a u r e u s , th e m o st marked e f f e c t a l w a y s a l t h o u g h no g e n e r a l i z a t i o n made. (see d i l u t i o n was fou n d su b sta n tia ted (see a u r e u s a l o n e wh en e x p o s e d t o three c a t i o n i c a g e n t s a t pH 8 and w i t h N e k a l BZ a t pH 5 . 2 (the o n l y pH r a n g e a t w h i c h N e k a l BX was s u b j e c t e d t o b a c ­ ter io sta tic tests). a ll 49 A com parison o f th e grow th r e s u l t s transfer" tu b e s w ith the (T ables 5 to 10) cut. more c l o s e l y was p o s s i b l e a n io n ic tu b e s t th e The c a t i o n i c in s ta n c e w ith t h e growth cu rv es to rela tio n sh ip p a r a lle led the fro m t h e resu lts in c o l i e x p o s e d t o R o c c a l a nd LPO. m o re c l o s e l y T h is was On t h e a u r e u s r e a c t i n g w i t h R o c c a l and LPC, than tubes th e grow th c u r v es than c a s e f o r E. fro m " s u b t r a n s f e r " c lea r- In e v e r y " orig in a l" from t h e " s u b t r a n s f e r " t u b e s . fo r S. is not agents are lik e w is e v a r ia b le .* OTAB, t h e r e s u l t s In o b ser v e grow th in did r e s u l t s hand, "sub- i n d i c a t e s no s t e a d f a s t c o r r e l a t i o n . t h o s e i n s t a n c e s where i t the " o r ig in a l" 24-hour counts in in the a lso the other resu lts tubes p a r a lle le d grow th-curve r e s u lt s did r e s u l t s Perhaps t h i s from " o r i g i n a l " apparent la c k can p a r t i a l l y be a t t r i b u t e d to the tub es. of p o s itiv e c o rrela tio n l a c k o f a g r e e m e n t among v a r i o u s a u th o r s as to what c o n s t i t u t e s b a cterio sta sis. term " b a c t e r i o s t a s i s " was f i r s t the a c tio n d y e s on b a c t e r i a w a s m ore i n h i b i t o r y o f certa in than g e r m i c i d a l. to in tr o d u c e d to that S i n c e t h a t t i m e , an a t t e m p t h a s b e e n made a r r i v e a t a m o re s p e c i f i c d efin itio n . because, in s tu d y in g the e f f e c t sp e c ific lin e h ib itio n of b a c teria l m u ltip lica tio n of in d ica te The of T h is was n e c e s s a r y ch em icals on b a c t e r i a , no d e m a r c a t i o n c o u l d b e drawn b e t w e e n t h e i n ­ and g e r m i c i d a l a c t i v i t y (49 ) . H offm a n n and Rahn ( 2 9 ) sta te d that b a c te r io s ta s is i s m a n ife s te d by a lo n g la g p e r io d , f o l lo w e d by a norm al * S i n c e S . p a r a t y p h i B w a s u s e d m a i n l y f o r p u r p o s e s o f com­ p a r is o n w ith S . c o l i . t h e form er organism d o e s n ot e n t e r in to t h is p a rticu la r d iscu ssio n . 50 rate of m u ltip lica tio n . death. However, e x te n d e d s t a s i s lead s to R o b e r t s a n d Rahn ( 5 3 ) p o i n t o u t t h a t r e t a r d a t i o n o f b a cteria l tiv ity . g r o w t h may h a v e l i t t l e T h is ob servation S e v a g and R o s s (55). S ly the op p o site d ir e c tio n , had no e f f e c t is o r no e f f e c t on e n z y m e a c ­ in agreem ent w ith (18), a ttack in g sh ow e d t h a t the r e p o r t o f the m a tter in h ib itio n of from resp ira tio n on b a c t e r i a l n u m b e r s . P r e s e n t s t u d i e s on q u a t e r n a r y ammonium com p o un ds em ploy m ethods com parable t o the t h e Shippen m o d i f i c a t i o n P .D .A . P h e n o l - C o e f f i c i e n t M ethod. w ell as in in the of In t h i s m eth od , the method o u t l i n e d by K o lm e r, no v i s i b l e o r i g i n a l tube f o l l o w e d by v i s i b l e growth in as growth a sub- t r a n s f e r tube in d ic a t e s th a t th e ch em ical agent e x e r te d b a cterio sta tic tio n is r a th e r than b a c t e r i c i d a l th at by s u f f ic ie n t ly the b a c t e r io s t a t ic u tiliz es o r ig in a l effect the tub es on ly. I f these th e chem ical a g e n t, .Another m ethod tubes f a i l do show v i s i b l e term b a c t e r i o s t a s i s The a s s u m p ­ to show growth in 3 , 4 , is a p p lied . A ctu ally, d e f i n i t i o n b y H o ffm a n n and Rahn a p p e a r s t o b e m o s t p r a c ­ tica l P. the out can b e m i t i g a t e d . growth i n 1 or 2 d a y s, but or 5 days, d ilu tin g action . at p resen t. S u r f a c e T e n s i o n and pH C o n t r o l s The d i l u t i o n s o f s y n t h e t ic a g en ts s e le c t e d for study rep resen ted the c o n c e n t r a t i o n s w hich f i r s t growth o f E. c o li and S . in v estig a ted in aureus r e s p e c t i v e l y th e grow th-curve s t u d ie s . th is p erm itted a t t h e pH l e v e l s 51 E rlen m eyer f l a s k s tra tio n s of sxtrface-active c o n ta in in g the d e s ir e d ag en t were p rep ared i n m anner a s t h a t d e s c r i b e d u n d e r t h e C u r v e s and R e l a t e d P h e n o m e n a . " betw een the tim e broth pH, in the the f l a s k s and t h e sectio n However, su rfa ce-a ctiv e and t h e tim e t h e the e n title d so lu tio n adju sted to th e d esired "O -hou r" r e a d i n g s w e r e t a k e n . 37 d e g r e e s 0 ., 0. P rior s u r f a c e - t e n s i o n m e a s u r e m e n t s * w i t h t h e du Houy t i o n were t r a n s f e r r e d i n t o grees "Growth a g e n t s were added to th e T en sio n o m eter, a liq u o t p o r tio n s of th e bath at same on ly 1 hour e la p s e d A l l f l a s k s w ere i n c u b a t e d a t 37 d e g r e e s to tak in g concen­ sin ce C, test sy n th etic so lu ­ t u b e s im m ersed i n a w a t e r The m e a s u r e m e n t s w e r e made a t t h a t was t h e t e m p e r a tu r e i s m s w e r e g ro w n i n a l l th e p reviou s t e s t s th e sta n d a r d watch g l a s s . 37 d e ­ a t w h ich th e o r g a n ­ perform ed. i n g s w e r e t a k e n a p p r o x i m a t e l y '10 s e c o n d s a f t e r was p l a c e d i n agent the Read­ so lu tio n The r e m a i n d e r o f t h e a l i q u o t p o r t i o n w a s e m p l o y e d f o r pH d e t e r m i n a t i o n s i n a Beckman pH m e t e r . The r e s u l t s tests o f the s u r fa c e -te n s io n are p r e se n te d in T a b les 15, * The f o r m u l a f o r s u r f a c e - t e n s i o n 16, a nd pH c o n t r o l and 1 7 . c a l i b r a t i o n was a s f o l l o w s : Mg x R X = ------------- 2L w h e r e O' i s Mi s g is r is Ris and L is the the the the the the s u r f a c e t e n s i o n in dynes p er c e n tim e te r , t o t a l w e i g h t i n grams added t o t h e r i n g , a ccelera tio n of g ra v ity , r e a d i n g on t h e s c a l e o f t h e a b o v e w e i g h t s , r e a d i n g on t h e s c a l e o f t h e unknown s o l u t i o n , mean c i r c u m f e r e n c e o f t h e r i n g . 52 TABLE 1 5 MEASUHEMENT S OP SURE AGE TEUSIOIT* M D pH WITH COIJTROL BROTH Su rface T en sion in dynes p er c e n tim e te r pH 7 H rs. PH pH 8 pH 5 . 2 H rs.- pH 7 pH 5 . 2 pH 9 0 5 6 .0 4 54 .4 5 56.27 0 7 .0 5 5 .2 5 8 .0 5 4 5 6 .0 4 54 .4 5 5 6 .2 7 4 7 .1 5 .3 8 .1 7 5 6.04- 5 4 . 4 5 56.2 -7 7 7 .1 5 .3 8 .1 10 5 6 .0 4 54 .4 5 56.27 10 7 .1 5 .3 8 .1 24 5 6 .0 4 5 4 .4 5 56.27 24 7 .1 5 .3 8 .1 30 5 6 .0 4 5 4 .4 5 5 6 .2 7 30 7 .1 5 .3 8 .1 48 5 6 .0 4 54.45 56.27 48 7 .1 5 .3 8 .1 * The s u r f a c e - t e n s i o n r e a d i n g o f s i n g l y d i s t i l l e d w a t e r (pH 5 . 5 ) a t 07 d e g r e e s 0 . w a s 7 0 . 0 4 d y n e s p e r c e n t i m e t e r . Th e p u r p o s e o f t h e tests was t o sta b ility effects su rfa ce-ten sio n o b s e r v e any p o s s i b l e o f the s o l u t i o n s and pH c o n t r o l r e l a t io n s h ip betw een th e of s y n th e tic a g e n t s and t h e i r on b a c t e r i a l g r o w t h a s n o t e d i n t h e g r o w t h - c u r v e exp erim en ts. The r e s u l t s tim e i n T a b le s 16 and 17 i n d i c a t e t h a t req u ired fo r a s o lu tio n reach eq u ilib riu m i s the a g e n t, tra tio n i.e ., o f a su rfa ce-a ctiv e p rop o rtio n a l t o agent to the c o n c e n t r a t i o n o f th e tim e r e q u ir e d i n c r e a s e s as th e decreases. T h is i s the concen­ in agreement w ith t h e f i n d in g s co n cern in g su r fa c e t e n s i o n b y Adam and S h u t e (l). The an io n ic agen ts (in c o n c e n tr a tio n s g r e a t e r than 1 -7 0 0 ) and th e n o n - io n ic a g en ts reached e q u ilib r iu m im m ed iately, whereas 55 fiBIS 16 fergltol 7 Jpol O S 1-50 1-fOO1-50 1-81I1-50 pE7 pH7 pH6.! 26,9620,6528,65 0,82 29.04 29,2229.04 22,04 22,5129,7229.04 28,26 28,5628,66 1-50 !■; .50 I 1-60 1-50 'S.OnHS.9»JTA 1-50 1- 27,52 i j 27,56 56,57 27.2556 55.71 27,56 50.15 23.04 22.62 29.5129,72 44,62 51,52 i 58,0451, 527,56 56,75 60.15 23.04 23.62 29.51 29.72 28,66 28.56 50.15 1-50 I pE5, 29,721)27,52: 527,56 29.72 29,04 29,44 28,5628.56 50.15 22,04 23,62 HacWlIBS! 50,04 51,75 51.7559,55 51,6245,5451,52 51,5240,21 51.5246,6051,52 51.6240,21 27.2555,05 31.7539,01 55.04 51.5245,7751,52 45,17 31,6241,12 27.5256,71 27,56 27.2556,05 35.04 27.52 ’7,2555,05 29,04 55.7127,56 31.75 35,25 51,52 42,92 58,56 31.75 17.2555,05 51,75 58,41 51,75 51.6240,66 Jefgitol 7 1-60 1-700 1-50 7 pE5,2 1-50 1-60 1-50 1-50 pi 7 pE7 pE5,2pE5,2pE8 1-50IMOI ll-SI) iff E.P Bff B.0 r f f 5,2 , 5,2 0 ;1-50 1-401 1.60 1-301 1-50 1-201 1-50 11-501 1-60 1-201 1-59 1-101 ;pE7 pE7 pi 5, pH5i pi 8 pH e pH7■pH 7 pH5, pH5,1pHBpES ■7,0 7,0 5,2 5,2 8,0 0,0 7,0 7,0 5,25 5,2 9,0 8,0 7,0 7,0 5,2 5,25 9,25 8,2 7,05 7,06 6,3 6,2 8,0 8,0 8,0 8,0 8,0 ' 7,0 7,0 5,2 5,25 8,25 8,2 7,06 7,05 5,3 5,2 0,0 8,0 7,0 7,0 5,2 5,25 8,2 8,1 7,05 7,05 5,3 5,2 8,0 j | § 5,25 7,0 7,0 5,2 5,25 8,15 8,1 7,05 7,05 6,3 6,2 6,0 6,0 7,0 7,0 5,2 5,25 8,15 0,1 7,05 7,05 6,3 5,2 8,0 8,|v 7,0 7,015,2 5,25 ,1 8,1 7,06 7,05 6,3 6,2 5,0 8, f f 54 IJB1E 17 SDMCMSSIOf00IR013 IPO Eoocal I 1*41 1*601 1*61 1-i 1*61 1*701 1-301 1*8 pH pH7 pi 7 pH7 1*2001 1*4011*3001 1*201 1*11 1*301 1*9003 1*303 1*11 2pH5,2pH8 pH8 pH7 pH7 pH5,2 pH5,2 pH8 pH8 1*50 1*20 1*20 1*20 pH7 pH5,2pH8 pH7 46,84 64,90 46,89 53,66 47,76 57,19 44,17 56.58 44,01 52,64 47,3355,58 37,9757,6937,84 53,01 37,97 56,35 38,87 38,8 38,87 38,87 32,2 47,49 56,04 45,38 57,17 46,02 51,23 46,51 53,30 37,47 57,7838,20 54,28 37,61 57,13 738,8 38,87 38,87 32,2! 43,52 54,91 46,26 53,62 47,81 56,50 44,74 56.58 43.74 52,90 46,6554,08 37,42 56,8736,69 53,91 37,52 56,35 738,8 38,87 38,87 56,49 42.74 50,73 46,9652,84 37,74 55,3138,29 54,31 37,84 56,29 38,er38,87 38,87 56,96 42,87 54,82 42,38 52,38 47,34 52,38 37,3854,31 37,52 55,20 37,5255,31 38,8738,87 38,87 1 44.61 55,36 46,02 t 44.61 55,91 52,02 31 44.61 64,36 45,66 56,46 43,81 57,16 43,72 51,15 45,47 54,67 37,38 57,65 37,52 54,31 37,5256,87 38,8738,87 38,87 32.21 41 44.61 54,36 45.66 55,69 42,66 56,39 44,49 53,39 45,32 55,83 37,38 56,29 37,52 52,44 37,5255,66 38,87 38,87 38,87 32.21 Eoooal Hrsi 0 M IPO Bmlpiior ffl 1*61 1*31 1*6! pH5,2 pH5,2: 1*30! 1*800! 1*30! 1-i iH7 pH7 pH5,2 1*300 1*201 1*11 1*301 1*1 1*11 1*20 1-50 1*20 1*20 pH8 pH7 pH7' pH5,2 pH5,2 pH8 pH8 pi 7 pH7 pH5,2 pH8 5,2 5.2 7.0 7,0' 5,2 5.2 8,0 7.0 7.0 5.2 5.2 7.0 7,0 5,2 8,0 7.0 4 5.3 8,2 8,1 7.1 7.1 5.15 5.2 8.05 8,1 7.0 7.1 5.2 5.2 8,1 7.0 7,0 6,2 8,0 7.0 7 5.3 5.2 8,2 0.1 7.1 7.1 '5,15 5.2 8.05 8,1 7.0 7.1 5.2 5.2 8,1 7.0 7,0 5,2 8,0 7.0 10 5.3 5.2 8,2 8,1 7.1 7.1 5.15 5.2 8.05 8,0 7.0 7.1 5.2 5.2 7.0 7,0 5,2 8,0 7.0 24 6.3 5.2 8,2 8,1 7.1 7.1 5.15 5.2 8.05 8,0 7.0 7.1 5.2 5.2 7.0 7,0 5,2 8,0 7.0 30, 5.3 5.2 8,2 8,1 7.1 7.1 5.15 5.2 8.05 8,0 7.0 7.1 5.2 5.2 7,0' 7,0 5,2 8,0 46 5,3 5.2 8,1 7,0 7,0 5.15 5.2 7,95 7,95 7.0 7,0 5.2 5.2 7,9. 7,9 7.0 7,0 5,2 8,0 8,0 55 those an ion ic compounds i n c o n c e n t r a t i o n s o f 1 - 1 0 T or l e s s r e a c h e d e q u i l i b r i u m i n from 1 0 h o u r s t o b e y o n d t h e l i m i t of the t e s t p e r i o d . R o c c a l ( T a b l e 1 7 ) s e r v e s a s a go o d e x a m p l e o f t h e r e l a t i o n s h i p b e t w e e n c o n c e n t r a t i o n o f s y n t h e t i c a g e n t and r a p id ity o f reaching e q u ilib r iu m . At a c o n c e n t r a t i o n o f 1 - 4 T , e q u i l i b r i u m was r e a c h e d i n 10 h o u r s ; at 1 -5 T , e q u i l i b ­ rium was a t t a i n e d i n 24 h o u r s ; w h e r e a s a t 1 - 6 T , e q u i l i b r i u m was r e a c h e d i n SO h o u r s . It is i n t e r e s t i n g t h a t at a con­ c e n t r a t i o n o f 1 - 5 0 T e q u i l i b r i u m was a l s o a c h i e v e d i n 30 hours. H ow ever, a t a 1 - 7 0 T d i l u t i o n no e q u i l i b r i u m was a t ­ t a i n e d i n 48 h o u r s . An e x p l a n a t i o n f o r t h i s l a s t - m e n t i o n e d phenomenon m ight p o s s i b l y be f o u n d i n t h e p a p e r b y Lund gren (44). He r e p o r t s t h a t i n h i g h l y d i l u t e chain hydrocarbon s a l t s behave a s s i n g l e so lu tio n s, lon g- io n s , whereas in more c o n c e n t r a t e d s o l u t i o n s t h e s e s a l t s fo rm m i c e l l e s w i t h c o l l o i d a l dim ensions. He a r r i v e d a t t h e s e c o n c l u s i o n s by t a k i n g m e a s u r e m e n t s o f c o n d u c t i v i t y and o s m o t i c c o e f f i c i e n t s . G o n ic k and McBain ( 2 5 ) p o i n t m icelle lik ew ise out c r i t i c a l c o n c e n t r a t i o n s f o r form ation w ith n o n - io n ic a g en ts. r e fe r to c r i t i c a l con cen tration s. Adam and S h u t e ( l ) They i n d i c a t e t h a t a b o v e a c r i t i c a l c o n c e n t r a t i o n o f 0 . 0 0 1 IT f o r a 1 6 - 0 and 0 . 0 1 IT f o r a 12 -C h y d r o c a r b o n c h a i n , i o n i c m i c e l l e s b e ­ g i n t o form. They a l s o add t h a t e q u i l i b r i u m o f s u r f a c e t e n ­ s io n i s reached alm ost im m ed iately at t h e s e c o n c e n t r a t io n s . In o t h e r w o r d s , concentrations it is q u i t e p o s s i b l e t h a t between t h e R o cca l o f 1 - 5 0 T and 1 - 7 0 T , t h e a l k y l d i m e t h y l b e n z y l ammonium c h l o r i d e m o l e c u l e s may b e d i s s o c i a t e d i n t o sim ple 56 ion s, th ereb y d e str o y in g the s t a b i l i t y of the s o l u t i o n . OTAB, a t c o n c e n t r a t i o n s o f 1 - 2 0 T and 1 - 3 0 T , a t ­ t a i n e d e q u i l i b r i u m i n 24 h o u r s , w h e r e a s t h e l o w e r c o n c e n ­ tration s o f OTAB and a l l t h e c o n c e n t r a t i o n s o f IPG t e s t e d did not su cc ee d in reach in g e q u ilib r iu m during th e 48-hour t e s t p eriod . it Under t h e i s n o t p o s s i b l e to o f IPO. H owever, i f conditions o f th ese control t e s t s o f f e r an e x p l a n a t i o n f o r t h e b e h a v i o r Adam and S h u t e are c o r r e c t i n t h e i r co n ten tio n concerning the c r i t i c a l hydrocarbon c h a i n , then i t is c o n c e n t r a t i o n f o r a 12-C q u i t e a p p a r e n t t h a t t h e c o n­ c e n t r a t i o n s o f IPC u s e d i n t h e s e t e s t s were f a r b e l o w t h e 0 . 0 1 N r a n g e , and c o n s e q u e n t l y t h e sta b le con d ition . s o l u t i o n was i n an u n ­ The w r i t e r h a s not s e e n any l i t e r a t u r e on t h e r e l a t i o n s h i p b e t w e e n t h e p y r i d i n e s t r u c t u r e i n a s y n ­ th e tic su rfa ce-a ctiv e a g e n t and t h e s u r f a c e - t e n s i o n s t a b i l i t y . On t h e b a s i s o f t h e s u r f a c e - t e n s i o n and pH c o n t r o l tests, t h e pH o f t h e medium se em e d t o h a v e l i t t l e e f f e c t on t h e r a p i d i t y w i t h w h ic h s u r f a c e - t e n s i o n e q u i l i b r i u m was a t ­ tain ed . With t h e e x c e p t i o n o f H a c c o n o l IJRSP a t pH 8 , a ll t h e s u r f a c e - a c t i v e a g e n t s p r e s e n t e d u n i f o r m s t a b i l i t y i n the pH c o n t r o l t e s t s . Th er e was no s h i f t i n pH t o compare w i t h th e s h i f t s talcing p l a c e in s u r f a c e t e n s i o n . solu tion s ( 1 - 3 0 T ) a t b o t h pH 5 . 2 and pH 8 a c h i e v e d s u r f a c e - t e n s i o n e q u i l i b r i u m w i t h i n 24 h o u r s . w i t h i n t h e pH r a n g e s t e s t e d , on t h e a t t a i n m e n t however, M o r e o v e r , OTAB T his in d ic a t e d t h a t , t h e pH e x e r t e d l i t t l e o f su r fa c e -te n sio n eq u ilib riu m . in flu en ce A dm ittedly, t h e pH r a n g e s t e s t e d were t o o narrow t o j u s t i f y mak­ in g a broad g e n e r a l i z a t i o n . 57 As f o r t h e r e l a t i o n s h i p b e t w e e n s u r f a c e - t e n s i o n s t a b i l i t y and b a c t e r i a l g r o w t h , t h e o n l y g e n e r a l i s a t i o n t h a t can b e made, on t h e b a s i s o f t h e r e s u l t s i n T a b l e s 16 and 1 7 , i s th at a s t a b le s o lu t io n o f a s y n t h e t ic s u r f a c e - a c t iv e agent i s n o t n e c e s s a r y f o r g e r m i c i d a l a c t i v i t y a g a i n s t S. a u r e u s * T h i s s t a t e m e n t i n no way c o n t r a d i c t s t h e f i n d i n g s o f some authors (6 2 )(3 3 ) th a t the u n d is s o c ia te d m o lecu les o f the su rfa ce-a ctiv e agent (con centrated so lu tio n s) m ic id a l than th e io n iz e d m o lec u les ever, a r e more g e r ­ (d ilu te so lu tio n s). How­ i t w ou ld b e d e s i r a b l e t o a s c e r t a i n t h e p o s s i b l e p o t e n c y o f th e d i s s o c i a t e d hydrophobic p o r t i o n o f th e m o l e c u l e . Q uisno and F o t e r ( 5 0 ) ch lorid e) reported th a t Oeepryn ( c e t y l p y r i d i n i u m i n a 1-2 T c o n c e n t r a t i o n was a s e f f e c t i v e c i d e a t pH 3 a s i t was a t pH 8 . l i k e l y be d i s s o c i a t e d a t pH 3 , a germ i­ S i n c e Oeepryn would most it is quite p o s s ib le t h a t , w it h the proper c o n c e n t r a t io n of the s y n t h e t i c a g e n t , th e d issociated c a t i o n m i g h t a l s o be g e r m i c i d a l . 58 V I . GENERAL DISCUSSION A thorough r e v iew o f t h e l i t e r a t u r e p e r t a i n in g to the b a c t e r i c i d a l or b a c t e r i o s t a t i c e f f e c t i v e n e s s of s y n th e tic s u r f a c e - a c t i v e a g en ts i n d ic a t e s th a t the m a jo r ity o f r e se a r c h ­ e r s u s e d a m o d i f i c a t i o n o f t h e P h e n o l - C o e f f i c i e n t Method ( 5 4 ) . in t h e ir s t u d i e s . T h i s t e c h n i c was u s e d m a i n l y a s 'a means o f a sc e r ta in in g the p o te n tia l compounds u n d e r t e s t , q u i c k - k i l l i n g power o f t h e v a r i o u s and t o c on fo rm w i t h p r e v i o u s p r e s e n t a ­ tio n o f data in the l i t e r a t u r e . In 1 9 4 5 , Anderson and M a l l - mann ( 2 ) p o i n t e d o u t t h a t t h e E . D . A . Method ( P h e n o l - C o e f f i ­ c i e n t Method) o f f e r s a f a i r means o f c o m p a r in g p h e n o l i c p r e p ­ a r a t i o n s b u t i s n o t a p p l i c a b l e when o t h e r t y p e s o f g e r m i c i d a l p reparation s are used. tio n , R eddish (52) reaffirm ed t h i s l im i t a ­ and s t r o n g l y warned a g a i n s t u s e o f t h e P h e n o l - C o e f f i c i e n t Method i n t e s t i n g q u a t e r n a r y ammonium compounds. ( 3 2 ) , Klarraann and U r i g h t H otchkiss ( 3 5 ) , a nd D u B o is and D i b b l e e ( 1 4 ) l i k e w i s e p o i n t out d i s c r e p a n c i e s a r i s i n g from u s e o f t h i s method. I t was n o t s u r p r i s i n g t o see a s e r i e s o f papers r e c e n tly p u b lish ed d e a lin g w ith s u g g e s tio n s fo r m odifying th e overworked F .D .A . t e c h n i c . The p u r p o s e o f a l l t h e s e p a p e r s was t o a r r i v e a t a more c o r r e c t e v a l u a t i o n o f t h e g e rm icid a l e f f e c t i v e n e s s of the p r a c tic a l con d ition s. q u a t e r n a r y compounds unde r Reddish s u g g e s t e d th e U s e - D i l u t i o n Method ( 4 6 ) a s t h e m ost p r o m i s i n g a t p r e s e n t . (51) Q u isn o e t a l and Arm bruster and R i d e n o u r ( 4 ) have p r o p o s e d m eth o ds fo r avoiding m isin te r p r e ta tio n s due t o b a c t e r i o s t a s i s . 59 However, in a l l th e se m ethods, th e sh o rt p e r io d o f exposure l i m i t s a c le a r understanding of th e gradual e f f e c t s t h a t a r e e x e r t e d by the s y n t h e t i c a g e n t s on b a c t e r i a . the e x c e p tio n of th e U s e -D ilu tio n t e c h n i c , With a l l the m o d ific a ­ t i o n s o f t h e F.35. A. Method m e r e l y d e s c r i b e t h e a l l - o r - n o n e resu lt, i.e ., com plete h i l l or s u r v i v a l . d e a lin g w ith th e r e la t io n s h ip Even t h o s e p a p e r s o f s u r f a c e - a c t iv e a g e n ts to m e t a b o l i s m o f b a c t e r i a ( 1 8 ) ( 6 ) have l i k e w i s e e m p l o y e d m e t h ­ o d s i n v o l v i n g an e x p o s u r e p e r i o d o f o n l y 1 or 2 h o u r s . An other l i m i t i n g f a c t o r i n t h e e v a l u a t i o n o f t h e s e compounds i s t h e a r b i t r a r y c h o i c e o f c o n c e n t r a t i o n s a t w h i c h to o b s e r v e t h e e f f e c t s on g r o w t h or m e t a b o l i s m o f b a c t e r i a . In many c a s e s , such c h o ic e s r e p r e s e n t the extrem e ranges o f a c t i v i t y o f t h e s y n t h e t i c compound, i . e . , com plete i n e f f e c t i v e n e s s , c o m p l e t e k i l l or le a v in g unreported the p o t e n t i a l a c t i v i t i e s o f a whole s e r i e s o f d i l u t i o n s betw een t h e s e ex­ trem es. One o f t h e main p u r p o s e s o f t h e s t u d y u n d e r t a k e n h e r e was t o f i l l fin e i n s u c h g a p s and a t t h e same t i m e t o de­ a s many o f t h e t e s t c o n d i t i o n s as p o s s i b l e , i.e ., tim e, pH, s u r f a c e t e n s i o n , c o n c e n t r a t i o n o f s y n t h e t i c a g e n t , and i n i t i a l numbers o f o r g a n i s m s . The o ne p o i n t on w h i c h m ost o f t h e i n v e s t i g a t o r s in t h i s f i e l d agree i s t h e i m p o r t a n c e o f pH. I t i s now known t h a t t h e mere r e p o r t i n g o f a k i l l i n g c o n c e n t r a t i o n o f a p articu lar sy n th etic it compound i s n o t s u f f i c i e n t . ?/he re a s i s g e n e r a l l y a c c e p t e d t h a t a n i o n i c a g e n t s a r e m o st e f f e c ­ t i v e a g a i n s t b a c t e r i a i n an a c i d ra ng e and t h e ammonium compounds i n an a l k a l i n e quaternary range, the l a t t e r compounds 60 do n o t a l w a y s f o l l o w s u c h a s i m p l e r u l e . T h e s e compounds a r e d e f i n i t e l y most g e r m i c i d a l a g a i n s t S . a u r e u s i n t h e alk alin e range; however, c e r t a i n o f t h e more c o n c e n t r a t e d s o l u t i o n s o f R o c c a l and OTAB e x e r t n e a r l y a s e f f e c t i v e a b a c t e r i c i d a l and b a c t e r i o s t a t i c a c t i o n on t h e g r a m - n e g a t i v e o r g a n i s m s a t pH 5 . 2 a s t h e y do a t pH 8 . Lauryl pyridinium c h l o r i d e f o l l o w s t h e g e n e r a l r u l e o f b e i n g more a c t i v e i n an a l k a l i n e r a n g e f o r a l l ion ic th r ee organism s. -As f o r t h e n o n ­ compounds, t h e o p t i m a l pH l e v e l s v a r i e d w i t h t h e gram n a tu re o f t h e organism, and w i t h t h e compounds t h e m s e l v e s ( s e e s e c t i o n e n t i t l e d " N o n - I o n i c Compounds1' ) • The i m p o r t a n t p a p e r s by G e r s h e n f e l d e t a l ( 2 2 ) (23) ( 2 4 ) hav e d e f i n i t e l y t i e d maximum b a c t e r i c i d a l e f f i c i e n c y o f syn th etic s u r f a c e - a c t i v e a g e n t s t o t h e optimum pH a t w h ic h th e y are employed. An h i s t o r i c a l r e v i e w o f t h e r e l a t i o n s h i p o f pH t o g e r m i c i d a l a c t i v i t y i s p r e s e n t e d i n t h e l a s t m entioned paper by G e r s h e n f e ld . the e f f e c t As t o t h e e x p l a n a t i o n f o r o f pH on t h e s e g e r m i c i d a l a g e n t s , t h e l i t e r a t u r e o f f e r s many t h e o r i e s . S t e a r n and S t e a r n ( 5 8 ) suggest th at, a t optimum pH l e v e l s f o r g e r m i c i d a l a c t i o n , t h e b a c t e r i a l membrane o r p r o t o p l a s m i s more s u s c e p t i b l e so a l t e r e d a s t o make t h e b a c t e r i a to the a c t i o n o f the c h e m ic a l. H artley (28) dem onstrated the form ation o f m i c e l l e s as a r e s u l t ion c o n c e n tr a tio n . Baker, H a rriso n , and M i l l e r (6) o f hydrogen suggested t h a t pH a c t i v a t i o n ( f o r maximum g e r m i c i d a l e f f e c t ) was i n t h e d i r e c t i o n which fa v o r e d form ation o f u n d i s s o c i a t e d m o l e c u l e s p o s s e s s i n g g r e a t e r a b i l i t y to p e n e t r a t e i n t o cells. Gershen­ f e l d and M i l a n i c k ( 2 4 ) p r e s e n t t h e h y p o t h e s i s t h a t t h e pH 61 e f f e c t may be r e s p o n s i b l e f o r a l t e r a t i o n s o f t h e s o l u b i l i t y o f th e reducing a g e n ts in s o l u t i o n . little is U n fortu n ately, v ery known o f t h e r e l a t i o n s h i p b e t w e e n t h e c hange i n pH o f t h e l i v i n g b a c t e r i a l p r o t o p l a s m and t h e pH c h a n g e s t a k i n g p l a c e i n t h e medium. The i m p o r t a n c e o f t h e i n f l u e n c e o f d i s s o c i a t e d and u n d i s s o c i a t e d m o l e c u l e s o f s u r f a c e - a c t i v e a g e n t s upon b a c t e r i a l g row th and i n h i b i t i o n h a s r e c e n t l y o c c u p i e d t h e a t t e n t i o n o f a l a r g e number o f w o r k e r s i n t h e f i e l d th etic s u r f a c e - a c t i v e compounds. estab lish ed It o f syn­ i s now f a i r l y w e l l t h a t the u n d i s s o c i a t e d m olecule n o t o n ly has greater p en etratin g a b i l i t y ( 4 8 ) , but t h a t i t also accounts fo r the g e r m ic id a l nature of co n cen tra ted s o l u t i o n s . This th e o ry i s f u r t h e r s tr e n g th e n e d by th e f a c t t h a t d i s s o c i a t e d m o le c u le s in d i l u t e s o l u t i o n s e x h i b i t l e s s e f f e c t i v e b a c t e r i c i d a l and b a c t e r i o s t a t i c a c t i o n . men ts on s o l u t i o n s P h y s ic a l- c h e m ic a l measure­ c o n ta in in g varyin g c o n c e n tr a tio n s o f syn­ t h e t i c s u r f a c e - a c t i v e a g e n t s have l e d t o t h e a c c e p t a n c e o f a c r i t i c a l co n cen tration fo r m ic e lla r form ation. I t has l i k e w i s e b e e n e s t a b l i s h e d t h a t above t h i s c r i t i c a l c o n c e n ­ t r a t i o n l i e t h e m ost g e r m i c i d a l l e v e l s pounds. o f the s y n t h e t i c However, t h e r e p o r t by Q u isn o and F o t e r ( 5 0 ) com­ and t h e r e s u l t s o f R o c c a l and Of AB a g a i n s t t h e g r a m - n e g a t i v e o r g a n i s m s , p r e s e n t e d i n f a b l e s 8 and 1 0 , in d ic a te the n e c e s ­ s i t y f o r a more c o m p r e h e n s i v e k n o w l e d g e o f t h e b a c t e r i c i d a l or b a c t e r i o s t a t i c ranges. role o f the d i s s o c i a t e d m o l e c u l e s in acid 62 Many a t t e m p t s h a v e b e e n made t o c o r r e l a t e c h e m i c a l s t r u c t u r e o f s y n t h e t i c compounds t o b a c t e r i c i d a l e f f i c i e n c y . The m o s t s u c c e s s f u l r e s u l t s h a v e b e e n a t t a i n e d w i t h i n an h o m o lo g o u s s e r i e s o f a p a r t i c u l a r s y n t h e t i c a g e n t . The im­ p o r t a n t f a c t o r i n su c h s t u d i e s h a s b e e n t h e l e n g t h o f t h e h y d r o p h o b i c c a rb o n c h a i n . H o o g e r h e i d e ( 3 1 ) and S h e l t o n e t a l ( 5 6 ) , w o r k i n g w i t h q u a t e r n a r y ammonium s a l t s o f the g e n ­ e r a l t y p e P.-fGBj )s -H -Br foun d t h a t a s t h e s t r a i g h t - c h a i n a l k y l gr ou p (R) was i n c r e a s e d from 0< t o a c t iv it y lik ew ise In a s e r i e s 0 /Jt g e r m i c i d a l i n c r e a s e d , w i t h t h e maximum p o t e n c y a t 0 , 6 . o f a l k y l p y r i d i n i u m c h l o r i d e s , t h e same g e r m i ­ c i d a l r e l a t i o n s h i p e x i s t e d w ith t h e in c r e a s e i n the s t r a i g h t c h a i n a l k y l g roup from C g t o 0,* ( 5 7 ) ( 3 6 ) . Sim ilar r e s u l t s w e r e o b t a i n e d from s t u d i e s w i t h d i a l k y l m e t h y l b e n z y l ammon­ ium c h l o r i d e s (41). The work w i t h a n i o n i c a g e n t s l i k e w i s e su b stan tiated the c o r r e l a t i o n betw een c a r b o n -c h a in l e n g t h and b a c t e r i c i d a l efficien cy to structures o f d ifferen t types of su rface- c o r r e la te the (10)(59). However, the attem p ts a c t i v e a g e n t s t o b a c t e r i c i d a l p o w e r s w e re n o t e n t i r e l y s u c ­ cessfu l. H otchkiss (32) TfNo u n i q u e a c t i v e I n d e e d , two a c t i v e su m m a rize s t h e s e e f f o r t s a s f o l l o w s : ch em ical groups have b een r e c o g n i z e d . s u b s t a n c e s may b e a r d i s s i m i l a r g r o u p s , and y e t o t h e r s u b s t a n c e s b e a r i n g t h e s e g r o u p s may be r e l a t i v e l y in a ctiv e, is n e v e r t h e l e s s , w i t h i n a hom ologous s e r i e s , there a t e n d e n c y f o r an o p t i m a l l y b a c t e r i c i d a l s u b s t a n c e t o e x i s t . .." 63 H owever, t h e l i t e r a t u r e i s i n a g r e e m e n t on t h e ■ b a c t e r i c i d a l n a t u r e o f c e r t a i n s y n t h e t i c com pounds. e t a l ( 7 ) and G e r s h e n f e l d T ergitol 4, (24) B a k er r e p o r t t h a t T e r g i t o l 7 and r e s p e c t i v e l y , we re t h e m o st a c t i v e g e r m i c i d e s of a s e r i e s o f an io n s t e s t e d . Domagk ( 1 3 ) and B ak er e t al ( 7 ) p o i n t e d out t h e b a c t e r i c i d a l e f f i c i e n c y o f t h e a l k y l d i m e t h y l - b e n z y l ammonium c h l o r i d e s t r u c t u r e . The r e s u l t s i n t h e g r o w t h - c u r v e t a b l e s s u b s t a n t i a t e t h e f i n d i n g s men­ t io n e d above. Baker e t a l ( 7 ) suggest th at the e f f e c t i v e ­ n e s s o f T e r g i t o l 7 can b e e : z p l a i n e d on t h e b a s i s t h a t is d e r i v e d from a b r a n c h e d - c h a i n , most o f t h e chain, it secon dary a l c o h o l , whereas o t h e r a n i o n i c compounds a r e d e r i v e d from s t r a i g h t - primary a l c o h o l s . They a l s o s u g g e s t t h a t t h e e f f e c t ­ i v e n e s s o f t h e a l k y l d i m e t h y l b e n z y l ammonium c h l o r i d e com­ pou nd s i s b a s e d on t h e i r p o s s e s s i o n o f a b e n z y l group a t t a c h e d t o th e quaternary n i t r o g e n . I t s h o u l d be n o t e d t h a t i n the c o n c e n t r a t i o n s employed i n t h i s s t u d y , N e k a l BZ was t h e o n l y a n i o n i c a g e n t which was b a c t e r i c i d a l organism s. for a l l three typ es o f A p o s s i b l e e x p l a n a t i o n a s t o why t h e b a c t e r i c i d a l a c t i v i t y o f t h i s p a r t i c u l a r compound h a s b e e n g i v e n v e r y little a t t e n t io n in the lit e r a t u r e is th a t o n ly r e c e n t l y has i t be e n m a n u f a c t u r e d i n a v e r y h i g h c o n c e n t r a t i o n . on t h e b a s i s o f p e r c e n t a g e o f a c t i v e was f a r more e f f i c i e n t H owever, in gred ien t, T e rg ito l 7 a g a i n s t E. c o l i and S. a u r e u s t h a n was N e k a l BZ. The a t t e m p t s t o c o r r e l a t e c u r t a i l m e n t o f b a c t e r i a l m etabolism w it h g e r m ic id a l a c t i v i t y have l i k e w i s e f a i l e d produce e x a c t r e l a t i o n s h i p s . C o n t r a r y t o the c o n c l u s i o n s to 64 drawn b y Bak er e t a l ( 6 ) concerning the p o s i t i v e rela tio n ­ s h i p b e t w e e n r e s p i r a t o r y and g l y c o l y t i c i n h i b i t i o n and g e r ­ m icid al a c t i v i t y , a number o f p a p e r s ( 5 5 ) ( 5 3 ) ( 1 8 ) hav e i n d i ­ c a t e d t h e l a c k o f c o r r e l a t i o n b e t w e e n t h e s e phen om en a. were r e p o r t e d b y S l y ( 1 8 ) Bata s h o w i n g a s much a s 50 p e r c e n t i n ­ h i b i t i o n o f r e s p i r a t i o n , w i t h no d i m i n u t i o n o f b a c t e r i a l nu m b e rs . Only at c o m p l e t e c e s s a t i o n o f r e s p i r a t i o n a r e a l l b acteria k ille d . A l t h o u g h t h i s w r i t e r h a s n o t fo u n d any s t e a d f a s t r e l a t i o n s h i p b e t w e e n the s h i f t i n pH and b a c t e r i a l g r o w t h a t s i m i l a r time i n t e r v a l s , as evid en ced in f a b l e s 3 to 10, t h e r e s t i l l e x i s t s u f f i c i e n t exam p les o f d e v i a t i o n s from t h e n o r m a l pH t r e n d t o m e r i t f u r t h e r It i s conceivable t h a t , ly w e ll d efin ed , co n sid era tio n . i n a medium w h ic h i s f a i r ­ a c o n t r o l c u r v e o f pH r e a d i n g s , s i m i l a r t o a c id it y curves in m ic r o b io lo g ic a l a ssa y s, can s e r v e a s a p a r t i a l i n d i c a t i o n o f b a c t e r i a l m etabolism . used in t h i s With t h e medium s t u d y , i t became a p p a r e n t t h a t t h e nor m a l t e n ­ d e n c i e s o f t h e t h r e e b a c t e r i a l t y p e s were to approach neu­ t r a l i t y when i n i t i a l l y grown a t pH 5 . 2 , stab le to rem ain f a i r l y and t h e n r i s e g r a d u a l l y to w a r d a l k a l i n i t y when grown i n t h e medium a t pH 7 , and t o drop to w ar d n e u t r a l i t y o n l y to r i s e a g a i n i n t h e a l k a l i n e d i r e c t i o n when s u b j e c t e d t o t h e medium a t pH 8 . T h ese s h i f t s i n pH m i g h t g a i n i n im­ p o r t a n c e i f t e s t s had b e e n c o n d u c t e d t o d e t e r m i n e t h e e x t e n t o f b a c t e r i a l breakdown o f t h e p e p t o n e under n o r m a l c o n d i t i o n s and a f t e r e x p o s u r e t o s y n t h e t i c su rfa c e-a c tiv e agents. It 65 is quite l i k e l y th a t in th e n ear f u t u r e such co n cu rren t e x p e r i m e n t s w i l l b e made i n o r d e r t o e x p l o r e more t h o r o u g h ­ l y t h e d i f f e r e n c e s b e t w e e n 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 action , as w e ll as the sig n ifica n ce o f su c h r e l a t e d phenom­ e n a a s l o n g l a g p e r i o d s and t o x i c e f f e c t s . 66 VII. SUMMARY A s y s t e m a t i c s t u d y was made o f t h e e f f e c t s o f c e r ­ t a i n a n i o n i c a g e n t s (D u p o n o l 0 S t Ig e p o n AP, T e r g i t o l 7 , N a c c o n o l URSF, U e k a l B Z ) , and n o n - i o n i c a g e n t s c a tio n ic agents (R occal, OTAB, 1PC), (Emulphor OH, T r i t o n Z - 1 0 0 ) on t h e g r o w t h o f E . c o l i , S . p a r a t y p h i B . and S. a u r e u s . An a t t e m p t was made t o e s t a b l i s h t h e r e l a t i o n s h i p s o f pH, s u r f a c e t e n s i o n , b a c t e r i o s t a s i s , and c o n c e n t r a t i o n o f s y n t h e t i c a g e n t , to b a c t e r i a l grow th. G rowth-curve s t u d i e s o v e r p e r io d s o f 0 , 4 , 7, 10, and 24 h o u r s showed t h e f o l l o w i n g : 1. C o n c e n t r a t i o n s o f 1 - 5 0 o f U e k a l 3X and T e r g i t o l 7 c o m p l e t e l y k i l l e d E. c o l i i n 24 h o u r s . H e k a l BZ r e d u c e d t h e c o u n t o f S . p a r a t y p h i B b y more t h a n 99 p e r c e n t i n t h e same p eriod . T his g e r m ic id a l a c t i v i t y o f a n io n ic a g e n ts a g a in s t fa ir ly resistan t in v estig a tio n . th is g r a m -n eg a tiv e organism s m e r i t s a d d i t i o n a l There i s v e r y l i t t l e rela tio n sh ip , 2. litera tu re and t h a t w h ic h d o e s e x i s t i s d e a lin g w ith in co n clu siv e. The q u a t e r n a r y ammonium compounds d i s p l a y e d g e r m i ­ c id a l a c t i v i t i e s a g a in s t a l l t h r e e types o f organism s in accordance w ith th e a cce p ted g e n e r a l i z a t i o n s appearing in the l i t e r a t u r e . T h e se g e n e r a l i z a t i o n s d e a l w i t h optimum b a c te r ic id a l a c t i v i t y at a lk a lin e ranges, greater k i l l i n g power a g a i n s t g r a m - p o s i t i v e o r g a n i s m s , and g e r m i c i d a l e f f i c i e n c y a t low c o n c e n t r a t i o n s . that, in c e r ta in in s t a n c e s , H owever, i t was n o t e d the g e r m ic id a l a c t i v i t i e s o f 67 H o c o a l and GTjiB a g a i n s t t h e g r a m - n e g a t i v e o r g a n i s m s w e r e as marked a t pH 5 . 2 a s a t pH 8 . 3. The o o n c e n t r a t i o n s o f a l l su rfa ce-a ctiv e agents u s e d i n t h e s e s t u d i e s a r r e s t e d growth o f t h e t h r e e b a c t e r ­ i a l c u l t u r e s when compared w i t h nor m a l g r o w t h c o u n t s . 4. D e t e r m i n a t i o n s o f pH made c o n c u r r e n t l y w i t h g r o w t h - c u r v e p l a t i n g s showed a r r e s t e d d e v i a t i o n s from t h e normal t r e n d i n pH s h i f t . T h e s e d e t e r m i n a t i o n s m ig h t s e r v e a s an a d ju n ct to t h e i n d i c a t i o n o f i n t e r f e r e n c e w i t h t h e normal m etab olic a c t i v i t i e s o f th e organism . T est-tube b a c t e r io s t a t ic t e s t s w ith the an ion ic a g e n t s i n d i c a t e d no a b s o l u t e p r o o f o f s t a s i s a g a i n s t t h e gram -negative organism s, a g a i n s t S. aureus. a l t h o u g h b a c t e r i o s t a s i s was n o t e d T u r b ia and p a r t i c u l a t e n a t u r e o f some o f th ese s o lu tio n s prevented exact c o n c lu sio n s . The t h r e e c a t i o n i c a g e n t s e x e r te d s t a t i c e f f e c t s a g a i n s t E. c o l i ana S . a u r e u s , w h e r e a s o n l y LPO a t pH 5 . 2 e x e r t e d b a c t e r i o s t a s i s a g a in s t S. p aratyp h i B. 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