A COMPARATIVE AND QUANTITATIVE STUDY OF THE VIRULENCE OF SEVERAL CULTURES OF BRUCELLA ABORTUS FOR THE GUINEA PIG AND COW L e s l i e M o r to n H u t c h i n g s A THESIS S u b m itte d t o th e G ra d u a te S c h o o l o f M ic h ig a n S t a t e C o lle g e o f A g r ic u lt u r e and A p p lie d S c ie n c e i n p a r t i a l f u lf i lm e n t o f th e re q u ire m e n ts f o r th e d egree of MASTER OF SCIENCE DEGREE D e p artm en t o f B a c t e r i o l o g y A p r i l , ]Sb2 ProQuest Number: 10008741 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008741 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 A COMPARATIVE AND QUANTITATIVE STUDY OF THE VIRULENCE OF SEVERAL CULTURES OF BRUCELLA ABORTUS FOR THE GUINEA PIG AND COW I. II. In tro d u c tio n . . .....................• . . • • • • . . . 1 ... E x p e r i m e n t a l P r o c e d u r e s a n d R e s u l t s ........................................IfS ources and C h a r a c te r is t ic s o f C u l t u r e s . . • • . . . • • • *i+ M eth o d o f D e t e r m i n i n g Numbers o f L i v e B r u c e l l a . * . . 5 I n V iv o V i r u l e n c e S t u d i e s .................. • ......................... . 1 6 E xposure o f H e if e r s t o B r♦ a b o r tu s III. IV . V. ................ . . . 3 8 S u m m a r y .......................................................... .. A clo iow led gm ent R eferences .................. ................................. 143805 U5 .i-i-6 A COMPARATIVE AND QUANTITATIVE STUDY OF THE VIRULENCE OF SEVERAL CULTURES OF BRUCELLA ABORTUS FOR THE GUINEA PIG AND COW* In th e s tu d y o f an in f e c tio u s d ise a se i t i s alw ays im p e r a ti v e t h a t o n e know t h e minimum n u m b e r o f t h e c a u s a t i v e m i c r o o r g a n i s m s t h a t i s re q u ire d to e s t a b l i s h i n f e c t i o n i n th e s p e c ie s o f a n im a l u n d e r s tu d y . G e n e ra lly i t tiv e is r e c o g n i z e d t h a t im m u n ity t o b a c t e r i a l i n v a s i o n I s a r e l a ­ s t a t e a n d may b e o v e rw h e lm e d b y e x p o s u r e t o l a r g e n u m b e rs o f m i c r o ­ o rg a n ism s . I t i s n o t s u r p r i s i n g t h a t m ore w o rk h a s n o t b e e n done t o m in e t h e minimum i n f e c t i v e a re v ery c o s tly . d e te r­ dose o f B r u c e lla f o r c a t t l e as such s t u d i e s A p a r t f r o m t h e e c o n o m ic c o n s i d e r a t i o n t h e r e a r e a t l e a s t tw o o t h e r e x p l a n a t i o n s f o r t h e l a c k o f s t u d i e s o f t h i s n a t u r e * One i s t h a t c a t t l e f r e q u e n t ly resp o n d i r r e g u l a r l y to e i t h e r n a t u r a l or e x p e rim e n ta l i n ­ f e c t i o n e s p e c i a l l y w hen t h e c r i t e r i a B ru c e lla in th e f e tu s , o f i n f e c t i o n a re th e d e m o n stra tio n of f e t a l m em branes o r c o l o s t r u m m i l k . T h is i r r e g u l a r i t y i n re sp o n se has c o n s e q u e n tly s u g g e ste d t h a t th e s iz e o f th e i n f e c t i v e dose is of l i t t l e , i f any im p o rta n c e , n a tu r a l ro u te s* e s p e c i a l l y w hen t h e a n i m a l s a r e e x p o s e d v i a The s e c o n d e x p l a n a t i o n i s t h a t m o s t w o r k e r s h a v e e x p e c t e d o r h o p e d f o r t h e d e v e l o p m e n t o f a m o re s o l i d im m u n ity i n c a t t l e t h a n h a s t h u s f a r been d e m o n stra te d . C o n s e q u e n t^ a v e ry s e v e re c h a lle n g in g dose has been a d m in is te r e d i n m o st e x p e rim e n ta l a tte m p ts i n im m u n iz a tio n . T h is s tu d y i s p a r t o f t h e c o o p e r a tiv e p r o j e c t betw een t h e B u r e a u o f A n im a l I n d u s t r y , U. S . D e p a r t m e n t o f A g r i c u l t u r e and t h e M ic h ig a n A g r i c u l t u r a l E x p erim en t S ta tio n . - 2- H agan ( l and 2 ) h as s t a t e d t h a t t h e i n f a c t i v i t y o f B r. a b o r t u s f o r g u i n e a p i g s w as e x t r e m e l y g r e a t . t h a n 100 o r g a n i s m s w e r e r e q u i r e d t o I t was c o m p u te d t h a t som ew hat l e s s i n f e c t m ost g u in e a p i g s . O c c a sio n a lly h e e n c o u n te re d g u in e a p ig s t h a t had a g r e a t e r r e s i s t a n c e t o i n f e c t i o n t h a n th e average. The m o s t r e s i s t a n t g u i n e a p i g e n c o u n t e r e d was i n f e c t e d b y a n u m b e r o f o r g a n i s m s c o m p u te d t o b e l e s s t h a n 1 0 , 0 0 0 . H ag an a l s o n o t e d t h a t t h e p r i n c i p a l e f f e c t on t h e c h a r a c t e r o f th e d i s e a s e , caused by v a ry in g th e s i z e o f t h e i n f e c t i n g d o s e , was a c h a n g e i n t h e t i m e r e l a t i o n s . V ery sm a ll doses produced an i n f e c t i o n o f a slo w e r c o u rse th a n l a r g e r d o s e s , b u t th e e v e n t u a l r e s u l t s w e re p r a c t i c a l l y t h e sam e. McEwen ( 6 ) i n a n e x t e n s i v e a n d c a r e f u l l y c o n d u c t e d e x p e r i m e n t has e s tim a te d th e s u ita b le in fe c tiv e d o s e o f B r . a b o r t u s f o r c a t t l e t o be ll+60 x 1 0^ o r g a n i s m s w hen d e p o s i t e d i n t h e c o n j u n c t i v a l s a c . f i v e g ro u p s o f t e n p re g n a n t h e i f e r s b y w ay o f t h e c o n j u n c t i v a . each t o He e x p o s e d d i f f e r e n t d o ses o f B r. a b o r tu s G roup 1 r e c e i v e d 1 ,1 + 6 0 ,0 0 0 ,0 0 0 b a c t e r i a ; g r o u p 2 , ll+, 600 , 000; g r o u p 3» 1 , 1+60 , 000; g r o u p 1+, 11+6,000; a n d g r o u p 5* l,l+6o. I n g r o u p s 1 a n d 2 , n i n e h e i f e r s beca m e i n f e c t e d ; i n g r o u p s 3s h a n d f i v e a n d tw o h e i f e r s , r e s p e c t i v e l y , becam e i n f e c t e d . r e s u l t s t h e minimum i n f e c t i v e dose o f t h i s seven, A cc o rd in g t o t h e s e s t r a i n fo r th e average p re g n an t h e i f e r w as a p p r o x i m a t e l y ll+, 0 0 0 , 0 0 0 o r g a n i s m s . I t h a s l o n g b e e n know n t h a t p a t h o g e n i c b a c t e r i a o f t e n u n d e r g o c h a n g e s i n v i r u l e n c e w hen c u l t i v a t e d on a r t i f i c a l m e d ia f o r l o n g p e r i o d s o f t i m e , an d t h a t i n m o s t c a s e s t h e i r v i r u l e n c e c a n b e e n h a n c e d b y p a s s a g e th ro u g h s u ita b le e x p e rim e n ta l a n im a ls. can be fou n d i n th e l i t e r a t u r e t o H o w e v e r, l i t t l e c o n c re te ev id en ce s u b s ta n tia te th e b e l i e f th a t th e v ir u ­ le n c e of B ru c e lla can be enhanced by p assag e th ro u g h a n im a ls. - 3- McEwen (1+) -mB.de s e r i a l p a s s a g e s i n g u i n e a p i g s o f a s t r a i n o f Br* a b o r t u s o f lo w v i r u l e n c e . s ix th , tw e n tie th , t h i r t i e t h He m e a s u r e d t h e v i r u l e n c e f o r m ic e o f t h e an d f o r t i e t h p a s s a g e a n d f o u n d t h a t s e r i a l p a s sa g e s th ro u g h g u in e a p ig s r e s u l t e d i n an in c re a s e d v i r u l e n c e . same p a s s a g e s w hen i n o c u l a t e d i n t o g u i n e a p i g s a n d p r e g ? i a n t c a t t l e T hese a lso sho w ed a n i n c r e a s e i n v i r u l e n c e . H ag an ( l a n d 2 ) o b s e r v e d t h a t t h e v i r u l e n c e o f B r. a b o r t u s f o r g u in e a p ig s c o u ld be r a i s e d or lo w e re d by a p p r o p r i a t e p a s s a g e th r o u g h th e s e a n im a ls. M e tz g e r and S to k e s ( 5 ) h a v e o b t a i n e d some i n t e r e s t i n g r e s u l t s fro m lo n g c o n tin u e d c u l t i v a t i o n o f B r. a b o r t u s i n t h e Two s t r a i n s o f _Br. a b o r t u s w e r e u s e d ; v iru le n c e . one v i r u l e n t , l i v e c h i c k e m b ry o . t h e o t h e r o f low The o r i g i n a l v i r u l e n t s t r a i n p r o d u c e d l e s i o n s a n d a h i g h a g g lu tin a tio n t i t e r on a r t i f i c i a l m e d i a . i n g u in e a p ig s and r e q u i r e d c a rb o n d io x id e f o r g ro w th At th e t h i r t y - n i n t h p a s s a g e t h i s v i r u l e n t s t r a i n a c q u i r e d t h e a b i l i t y t o g ro w a r t i f i c i a l l y w i t h o u t c a r b o n d i o x i d e . A com­ p a r i s o n o f t h e f o r t y - e i g h t h a n d f i f t i e t h p a s s a g e s sho w ed t h a t t h i s s tra in h a d l o s t some o f i t s v i r u l e n c e a n d a b i l i t y t o s t i m u l a t e a g g l u t i n i n p r o ­ d u c tio n i n th e g u in e a p ig . They s t a t e t h a t t h e v i r u l e n t s t r a i n re m a in e d s m o o th t h r o u g h o u t t h e s e r i a l p a s s a g e s i n c h i c k e m b r y o s . I n v i e w o f t h e i n c o m p l e t e n e s s o f o u r k n o w le d g e p e r t a i n i n g t o t h e m inimum nu m b e r o f B r . a b o r t u s t o p r o d u c e i n f e c t i o n , t h i s s tu d y c o n s t i t u t e s a n e f f o r t t o t h r o w a d d i t i o n a l l i g h t on minimum i n f e c t i n g d o s a g e , c h a n g e s i n v i r u l e n c e u n d e r a r t i f i c i a l c u l t i v a t i o n and by r e p e a t e d g u in e a p ig p a s s ­ age. In a d d itio n a le s s e x t e n s i v e e f f o r t h a s b e e n made t o d e te rm in e th e s u i t a b l e i n f e c t i v e d o s e o f B r . a b o r t u s f o r p r e g n a n t h e i f e r s b y w ay o f t h e c o n ju n c tiv a . EXPERIMENTAL PROCEDURES AND RESULTS S o u rces and C h a r a c t e r i s t i c s of C u ltu re s B. A* I* s t r a i n 1 9 - a e r o b i c B r . a b o r t u s I s o l a t e d f r o m v a c c i n e made b y t h e U. S . B u r e a u o f A n im a l I n d u s t r y . 29 5 “ a e r o b i c , B r . a b o r t u s i s o l a t e d f r o m c o w 's m i l k i n M i c h i g a n 5/ 31 / 3 0 . 9 7 9 - a e r o b i c , B r . a b o r t u s i s o l a t e d f r o m human b l o o d i n Iow a 9 / 3/ 3 0 . A* 1099T2 - a e r o b i c , B r . a b o r t u s i s o l a t e d f r o m t h e f i s t u l a o f a h o r s e i n M i n n e s o t a i n 1930* T h i s c u l t u r e g ro w s s l i g h t l y o n t h i o n i n d y e p la te s . 1257 - a e r o b i c , B r . a b o r t u s i s o l a t e d f r o m m i l k o f an i n f e c t e d cow i n M i c h i g a n 1 / 1 3 / 3 9 * 1265 - J 3 r . a b o r t u s i s o l a t e d f r o m l u n g o f a b o v i n e f e t u s i n Illin o is 3 / 9 /b®* T h i s c u l t u r e r e q u i r e s a n i n c r e a s e d CO^ t e n s i o n f o r g ro w th . 1271 - B r . a b o r t u s i s o l a t e d f r o m human b l o o d i n M i c h i g a n 5/ 27/Ll-O. T h i s c u l t u r e r e q u i r e s a n i n c r e a s e d C0S t e n s i o n f o r g r o w t h . 12 80 - B r . a b o r t u s i s o l a t e d f r o m m i l k o f a n i n f e c t e d cow i n M i c h i g a n 10/lj./2+0. T h i s c u l t u r e r e q u i r e s a n i n c r e a s e d C02 t e n s i o n f o r g r o w t h . 1282 - B r . a b o r t u s i s o l a t e d f r o m m i l k o f a n i n f e c t e d cow i n M i c h i g a n 1 2 /1 2 /1 + 0 . T h i s c u l t u r e r e q u i r e s a n i n c r e a s e d C03 t e n s i o n f o r g ro w th . 128i| - B r . a b o r t u s i s o l a t e d f r o m m i l k o f a n i n f e c t e d cow i n M i c h i g a n 'J/2.^/hX* g ro w th . T h i s c u l t u r e r e q u i r e s a n i n c r e a s e d C02 t e n s i o n f o r 1285 - B r . a b o r t u s i s o l a t e d f r o m a n a b o r t e d f e t u s i n M i c h i g a n 2 /1 8 /Ij.l. T h i s c u l t u r e r e q u i r e s a n i n c r e a s e d C02 t e n s i o n f o r g r o w t h . 1287 - B r . a b o r t u s i s o l a t e d f r o m a n a b o r t e d f e t u s 5 /l8 /U l» i n M ic h ig an T h i s c u l t u r e r e q u i r e s a n i n c r e a s e d C0S t e n s i o n f o r g r o w t h . 1677 “ B r . s u i s i s o l a t e d f r o m human b l o o d 5 / 3 / 4 0 . M eth od o f D e t e r m i n i n g l u m b e r s o f L i v e B r u c e l l a C o n s i d e r a b l e p r e l i m i n a r y w o r k w as do n e t o e s t a b l i s h a m e th o d f o r d e t e r m in i n g t h e num ber o f l i v e o rg a n ism s i n a g iv e n s u s p e n s i o n u s e d i n i n o c u l a t i n g e a c h group o f g u in e a p i g s . s u i t a b l e su sp e n d in g f l u i d , I t w as n e c e s s a r y t o f i n d a a m e th o d o f s t a n d a r d i z i n g t h e n u m b e r o f o r g a n i s m s i n t h e s u s p e n s i o n , an d t h e p r o p e r d i l u t i o n s o f t h e s t a n d a r d su sp e n sio n s. The f o l l o w i n g f o u r s u s p e n d i n g f l u i d s w e r e s t u d i e d : N o n - b u f f e r e d p h y s i o l o g i c a l s a l t s o l u t i o n pH 6 . I l B u f f e r e d p h y s i o l o g i c a l s a l t s o l u t i o n pH 6 . 8 1 p e r c e n t T r y p t o s e , 0 . 5 p e r c e n t s a l t pH 6 * 9 0 . 1 p e r c e n t T r y p t o s e , 0 . 5 p e r c e n t s a l t pH 6 . 9 The b u f f e r e d s a l t s o l u t i o n w as made b y d i l u t i n g a s t o c k p h o s p h a t b u f f e r o f pH a b o u t 7 * 0 made a c c o r d i n g t o t h e m e th o d o f C l a r k a n d L ubs ( 7 ) to 1 :6 w ith p h y s i o l o g i c a l s a l t s o l u t i o n . T he c u l t u r e s u s e d w e r e s u s p e n d e d i n t h e s e s o l u t i o n s a n d s t a n d a r d VvO ' i z e d b y m ean s o f t h e C enco p h o t e l o m e t e r u s i n g t h e =$2 g r e e n f i l t e r t o a g alv o n o m e te r re a d in g o f 6 8 . 5 . ^ is MdSfcjfarl&nd n e p h e l o m e t e r . T h is d e n s i t y c o rre s p o n d s t o tu b e 1 o f th e A ll su sp e n sio n s, b r o t h w ere s t a n d a r d i z e d t o t h i s d e n sity . except th e 1 p e r c e n t T ry p to se The 1 p e r c e n t T r y p t o s e b r o t h h a d a d e f i n i t e y e l l o w c o l o r a n d g a v e a g a l v a n o m e t e r r e a d i n g o f 9b 0*1 t h e p h o t e l o m e t e r a s c o m p a r e d w i t h 100 f o r t h e o t h e r s u s p e n d i n g f l u i d s . n e c e s s ita te d a c o rre c tio n of 6 u n its T h is on t h e g a l v a n o m e t e r s c a l e f o r t h i s p a r t i c u l a r su sp e n d in g f l u i d . The s t a n d a r d i z e d s u s p e n s i o n s w e r e d i l u t e d i n t h e i r r e s p e c t i v e d ilu e n ts to 1 : 1 ,0 0 0 ,0 0 0 , 1 :5 * 0 0 0 ,0 0 0 and 1 :5 0 ,0 0 0 ,0 0 0 . D i f f e r e n t a m o u n ts o f t h e f i n a l d i l u t i o n s w ere p la c e d i n s t e r i l e P e t r i d is h e s i n t r i p l i c a t e an d T ry p to s e a g a r p o u re d o v e r th e m . I n c u b a t i o n w as c a r r i e d o u t f o r f i v e d a y s a t 3 7 °C* u n d e r a e r o b i c c o n d i t i o n s a n d i n a n a t m o s p h e r e o f i n c r e a s e d C0S d e p e n d i n g on t h e c u l t u r e u s e d . A f t e r n u m e ro u s t r i a l s C o u n ts w e r e made o f a l l p l a t e s . i t w a s f o u n d t h a t s u s p e n s i o n s made i n 0 , 1 p e r c e n t T r y p to s e b r o t h and d i l u t e d t o 1 :5 * 0 0 0 ,0 0 0 and 1 :5 0 ,0 0 0 ,0 0 0 gave n e a rly re p e a ta b le p la te co u n ts. o r ig in a l su sp e n sio n s, One c c . o f 1 :5 * 0 0 0 ,0 0 0 d i l u t i o n o f th e o n e - h a l f c c . o f 1 : 5 *0 0 0 ,0 0 0 a n d o n e - h a l f c c . o f 1 : 5 0 , 0 0 0 ,0 0 0 g a v e c o n s i s t a n t l y u n i f o r m p l a t e c o u n t s o f a b o u t 2 0 0 , 100 a n d 10 c o l o n i e s r e s p e c t i v e l y . T a b l e 1 shows t h e r e s u l t s o f s u s p e n d i n g c u l t u r e s 1265 a n d 1271 i n b o th b u f f e r e d and n o n - b u f f e r e d p h y s i o l o g i c a l s a l t s o l u t i o n s and s t a n - ^ d a r d i z i n g t h e s e s u s p e n s i o n s t o t u b e 1 Mjfife^hrland n e p h e l o m e t e r b y t h e e y e . I m m e d ia t e d i l u t i o n t o 1 : 1 , 0 0 0 , 0 0 0 a n d p l a t i n g i n v a r i o u s a m o u n ts a r e shown a s w e l l a s d i l u t i o n t o 1 : 1 , 0 0 0 ,0 0 0 a f t e r s t a n d i n g 2l\. h o u r s a t room t e m ­ p e ra tu re . I t c a n b e r e a d i l y s e e n t h a t a d e c r e a s e i n num ber o f o rg a n ism s o c c u r s d u r i n g t h e 2ij. h o u r s i n e i t h e r b u f f e r e d o r n o n - b u f f e r e d s a l t s o l u ­ tio n s. T h i s w o u ld i n d i c a t e t h a t s u c h s o l u t i o n s a r e u n s u i t a b l e f o r s u s ­ p e n d i n g B r u c e l l a w hen l i v e o r g a n i s m s a r e t o b e e s t i m a t e d o r m a i n t a i n e d . The r e s u l t s o f s u s p e n d i n g c u l t u r e s 1257 1271 i n b u f f e r e d s a l t s o l u t i o n a n d s t a n d a r d i z a t i o n o f t h e s u s p e n s i o n t o 6 8 , 5 o n t h e C enco p h o t e l o - - 7- 3 g o *H •H Is •§ rH fcuGl aq o 3 00 -H -P 43 (ri —I aS P-t rH O 3 CO *H -P OJ 3, t3 3 © o p © o 3 P3 3 S3 © o 0J 3 « A o S23 © 1O—In 3 co f3 © C V J o © p o NO ON CO KN KN QN CO tN- NO KN ON KN NO KN INKN 3 o *H -P 3 rH O CO ON fcfl 3 *H P ' 3 rH -P C3 © 3 © *H rH OJ LTN KN VO NO c<0 3 CO © 3 PP © 1 4 CO <—I I— 1 & f~“t -4 00 *• p S Ch M C— was © o 4 NO § © o UN OJ KN © • 3 O 1—I S3 £3 OJ KN -P • 3 O rH S i Ph c o n t a m in a te d OJ £ \j Plate C ultures © P Counts of -p O *H Plate TABLE I 126$ and 1271 CO © CO CO 'CS Pi O 45 3 3 C3 1—1 rH i * * -8 m e te r a r e s e t f o r t h in T ab le 2 . P l a t e c o u n t s w e r e made on v a r i o u s a m o u n ts o f 1 :1 ,0 0 0 ,0 0 0 d i l u t i o n o f o r i g i n a l s u sp e n sio n . The c o u n t s o b t a i n e d a r e r e l a t i v e l y u n if o r m and c o n s i d e r a b l y lo w e r t h a n s i m i l a r c o u n ts i n T a b le I w hen t h e suspended c u l tu r e s w ere s ta n d a rd iz e d by eye t o tu b e 1 o f th e IttCfeJ-hrland n e p h e l o m e t e r . T he r e s u l t s o f s u s p e n d i n g c u l t u r e 1257 i n b o t h b u f f e r e d s a l t a n d 1 p e r c e n t T r y p t o s e s o l u t i o n a r e t a b u l a t e d i n T a b l e 3* These s u sp e n s io n s w ere s t a n d a r d i z e d b y eye t o tu b e 1 M ^ ^ h r la n d and t h e n d i l u t e d t o 000 i n t h e i r r e s p e c t i v e d i l u e n t s . O n e - h a l f a n d 1 c . c . a m o u n ts o f e a c h s u s p e n s i o n w e r e p l a t e d a t i n t e r v a l s o f 1 , 6 , 2lj. a n d J48 h o u r s . strik in g 1 : 5 *0 0 0 , The m ost f e a t u r e o f t h e r e s u l t s w as t h e d i s c r e p a n c y i n p l a t e c o u n t s b e t w e e n t h e tw o s u s p e n s i o n s made b y e y e i n d i f f e r e n t d i l u t i n g f l u i d s , t h u s i n d i c a ­ t i n g t h a t s t a n d a r d i z a t i o n b y e y e w as n o t a d e s i r a b l e m e t h o d . I t was n o t e d a l s o t h a t a s h a r p d e c r e a s e i n p l a t e c o u n t s o c c u r r e d a t t h e 21; a n d lj.8 h o u r in te rv a ls in th e b u ffe re d s a l t s o lu tio n . T a b l e 1+ shows t h e r e s u l t s o f h o l d i n g t h e 1 : 5 , 0 0 0 , 0 0 0 d i l u t i o n s , made a s d e s c r i b e d i n T a b l e 3* room t e m p e r a t u r e f o r 2ij. h o u r s a n d r e ­ p l a t i n g a t th e end o f t h i s tim e . O b v io u sly t h e b u f f e r e d s a l t s o l u t i o n e x e r t e d a m a r k e d b a c t e r i c i d a l a c t i o n on t h e o r g a n i s m s . t h e n u m b e r o f o r g a n i s m s w as much g r e a t e r i n t h e d i l u t © in th e o r ig in a l su sp e n sio n . The d e c r e a s e i n su sp e n sio n th a n T h e r e was a s l i g h t i n c r e a s e i n t h e nu m b e r o f o rg a n ism s i n th e 1 p e r c e n t T ry p to s e s o l u t i o n . R e s u lts s i m i l a r t o T a b le 3 a p p e a r i n T ab le 5 e x c e p t t h a t c u l t u r e s 293 a n d 9 7 9 w e r e s u s p e n d e d i n p h y s i o l o g i c a l s a l t s o l u t i o n r a t h e r t h a n b u ffe re d p h y sio lo g ic a l s a lt T ab le 6 i s so lu tio n . i n c l u d e d t o show t h e c l o s e a g r e e m e n t o b t a i n a b l e b y s t a n d a r d i z i n g s u sp e n s io n s o f a c u l t u r e i n d i f f e r e n t su sp e n d in g f l u i d s , -9 - TABLE 2 P l a t e C o u n ts o f C u l t u r e s 1257 a nd 1271 C u l t u r e 1257 P la te No. B u ffered S a lt S o lu tio n I m m e d ia te P l a t i n g 1/lj.c .0 lo .e . l/2 c .c . 1 225 36 5 85^ 2 265 k3h 828 3 231 581 790 C u l t u r e 1271 P iste No. B uffered S a lt S o lu tio n I m m e d ia te P l a t i n g l/ i^ c . c . l / 2 c , c . lc . c . 1 265 539 739 2 257 k50 835 3 23U h25 800 -1 0 - TABLE 3 E f f e c t o f B u f f e r e d S a l t S o l u t i o n a n d One P e r C e n t T r y p t o s e S o l u t i o n on C u l t u r e 1257 B u ffered S a lt S o lu tio n 1 c *0 * l /2 c ,c . P la te N o. 1 h r. 1 70 2 3 2b h r . 1+8 h r . 121 73 70 98 98 105 6 h r. 2b h r . 1+8 h r . 213 139 95 168 co n t£ 159 ll l+ 222 190 178 l l i+ 1 h r. 6 h r. L)j 203 89 5k 73 51 One P e r C e n t T r y p t o s e S o l u t i o n P la te No. 1 c *c • l /2 c , c ♦ 2l+ h r . 2I4. h r . I4.8 h r . 1+22 333 396 371 371 397 365 371 Ubi 1+S9 b.19 1 h r. 6 h r. 1+8 h r . 1 1 h r . 1 206 193 21+1+ 206 3 90 2 181+ 200 2 38 219 3 238 209 203 215 P l a t e w as c o n t a m i n a t e d . 6 h r. - 11- TABLE k E f f e c t ; o f B u f f e r e d S a l t S o l u t i o n a n d T r y p t o s e on C u l t u r e 1257 i n Low D i l u t i o n P la te N o. P la te N o. 1 la ! 1 c. C. la * 6a * 2l+a* ij.8a * Too Numerous Too Numerous Too N um erous Too Numerous n 2 t» it n ii 3 tt t! it n g|t D ilu te d s u s p e n s io n s o f T ab le 3 h e ld 2i_|. h o u r s a t room t e m p e r a t u r e b e f o r e r e ­ p la tin g . - 12- TABLE 5 E f f e c t o f P h y s i o l o g i c a l S a l t S o l u t i o n a n d One P e r C e n t T r y p t o s e S o l u t i o n o n C u l t u r e s 295 &n d 9 7 9 C u l t u r e 295 N o n - h u f f e r e d S a l t S o l u t i o n P la te N o. 1 /2 c . c . 2l* h r . 1*8 h r . 1 h r. 6 h r. 1 73 67 c o n t. 2 77 65 3 83 66 1 c .c . 2k h r . 1 h r. 6 h r. 38 129 129 ll*0 81* 78 5k ii*o 127 127 98 70 1*7 13 k ll*2 138 98 1*8 h r . C u l t u r e 295 One P e r C e n t T r y p t o s e S o l u t i o n P la te No. l/2 c .c . 2k h r . 1*8 h r . 1 h r. 6 h r. 1 97 78 61 2 106 95 3 92 77 1 c .c . 2k h r . 1 h r. 6 h r. 85 193 173 183 179 83 103 207 163 151 182 100 90 175 co n t. 153 166 1*8 h r . C u ltu r e 979 N o n -b u ffe re d S a l t S o lu tio n P la te No. 1 /2 c . c . 1 h r. 1 c .c . 6 h i '. 2l* h r . 1*6 179 170 118 83 67 38 166 11*2 c o n t. 81 co n t. 1*7 lltf 11*1 127 92 1 h r. 6 h r. 2l* h r . 1 86 83 62 2 112 77 3 79 90 1*8 h r . 1*8 h r . C u l t u r e 9 7 9 One P e r Cen t T r y p t o s e S o l u t i o n P la te N o. 1 /2 c . c . 1*8 h r . 21* h r . 1 c .c . 21* h r . 1 h r. 6 h r. 93 201* 195 182 212 9l+ 115 206 196 191 208 107 113 192 191* 180 177 1 h r. 6 h r. 1 97 81* c o n t. 2 lol* 87 3 107 9k 1*8 h r . -1 3 - TABLE 6 C o m p a r is o n o f S t a n d a r d i z i n g D i f f e r e n t S u s p e n s i o n s on P h o t e l o m e t e r P la te No. Im m ed iate i P l a t i n g B u f f e r e d Sa! . t S o l u t i o n 1 c .c . 1 /2 c .c * 1 125 21 8 2 112 212 3 13U 237 P la te No. One p e r c e n t T r y p t o s e S o l u t i o n Im m e d ia te P l a t i n g 1 /2 c . c . 1 c .c . 1 110 222 2 112 206 3 138 2J4.O - i . e . b u ffered s a lt 14- s o l u t i o n an d 1 p e r c e n t T r y p t o s e s o l u t i o n * These s u s p e n s i o n s w e r e s t a n d a r d i z e d t o 6 8 * 5 a &d 62*5 r e s p e c t i v e l y on t h e C enco p h o t e l o m e t e r . O n e -h a lf and 1 c .c * am o u n ts o f 1 : 5 5 0 0 0 ,0 0 0 d i l u ­ t i o n o f t h e o r i g i n a l s u s p e n s io n s w ere p l a t e d . The 1 p e r c e n t T r y p t o s e s o l u t i o n p r o v e d t o be s u p e r i o r t o t h e o t h e r s u s p e n d in g f l u i d , b u t i t had t h e d is a d v a n t a g e o f foam in g u p o n s h a k i n g a n d a l s o n e c e s s i t a t e d a c o r r e c t i o n on t h e p h o t e l o m e t e r re a d in g to o b ta in th e d e s ire d t u r b i d i t y re a d in g . I t w as d e c i d e d t o u s e 0 . 1 p e r c e n t T r y p t o s e p l u s 0*5 p e r c e n t s a l t s o l u t i o n t o b o t h t h e c o l o r and u n d e s i r a b l e fo a m in g . I n T ab le 7 a r e s e t f o r t h th e re su lts o f p l a t e c o u n t s made f r o m t h e t h r e e flu id s, i.e., su sp e n d in g and d i l u t i n g 0*1 p e r c e n t T r y p t o s e , b u f f e r e d s a l t , s a lt s o lu tio n . C u l t u r e 1257 The 3 s u s p e n s i o n s w e r e 1 : 5 , 0 0 0 , 0 0 0 i n t h e i r r e s p e c t i v e d i l u e n t s a t i n t e r v a l s o f 1, 6 , 24 and 48 h o u r s . One c . c . a m o u n ts o f t h e 1 : 5 , 0 0 0 , 0 0 0 d i l u t i o n s w e r e p la te d in t r i p l i c a t e . tio n and n o n - b u f f e r e d suspended i n th e 3 s o l u t i o n s and s t a n d a r d i z e d t o 6 8 . 5 o*1 t h e C en co p h o t e l o m e t e r . d ilu te d to e lim in a te I t was n o t e d t h a t t h e 0 . 1 p e r c e n t T r y p t o s e s o l u ­ s u sp e n sio n gave r e l a t i v e l y c o n s ta n t p l a t e c o u n ts o ver th e e n t i r e 48 h o u r p e r i o d w h ile b o t h t h e b u f f e r e d and n o n - b u f f e r e d s a l t s o l u t i o n su sp en sio n s gave d e c r e a s i n g p l a t e c o u n ts . t i o n s made a t i n t e r v a l s and r e p l a t e d o f 1 , 6 , 2 4 a n d 4 8 h o u r s w as h e l d f o r 2 4 h o u r s i n 1 c . c . am o u n ts. and n o n -b u ffe re d s a l t s o lu tio n s th e o rg a n ism s. Each o f t h e 1 : 5 ,0 0 0 ,0 0 0 d i l u ­ H e re a g a i n i t was n o t e d t h a t t h e b u f f e r e d e x e r t e d a m a rk e d b a c t e r i c i d a l a c t i o n on T h e r e w as a s l i g h t m u l t i p l i c a t i o n o f t h e o r g a n i s m s i n th e 0 .1 p e r c e n t T ry p to se s o lu tio n . The r e s u l t s o f s u s p e n d i n g c u l t u r e s 1287 a n d 1 2 8 4 i n 0 . 1 p e r c e n t T r y p t o s e a n d s t a n d a r d i z i n g t h e s u s p e n s i o n s t o 6 8 . 5 on t h e p h o t e l o - -1 5 TABLE 7 C o m p a r is o n o f E f f e c t s o f 0 . 1 P e r C e n t T r y p t o s e S o l u t i o n , B u ffe re d S a l t S o lu tio n and N o n-B uffered S a l t S o l u t i o n o n C u l t u r e 1257 0*3. P e r C e n t T r y p t o s e pH 6 .9 1257 P la te N o. 1 h r. 6 hr. 1 133 2 3 21+ h r . 1+3 h r . la * 6 a* 2l+a* l+8 a* 150 189 184 20 3 308 269 217 121 126 198 174 191 cont • 2kQ 2U l 18 2 218 176 168 21k 251+ 2k3 236 B u f f e r e d S a l t S o l u t i o n pH 6 .8 1257 P la te N o. 2k h r . 1+8 h r . la * 6 a* 2 l+a* 4 8 a* 152 163 81 k 10 16 35 108 153 181 95 10 c o n t. 17 34 132 171 99 co n t. 8 0 7 23 1 h r. 6 h r. 1 l i +6 2 3 1257 i n N o n - B u f f e r e d S a l t S o l u t i o n pH 6 . 4 P la te N o. 2 l+ h r . 1+8 h r . la * 6 a* 2l+a* 48 a* 147 100 52 17 k 0 0 135 112 93 69 hi c o n t. 0 2 117 170 122 80 55 2 0 2 1 h r. 6 h r. 1 13*4- 2 3 1 :5 m i l l i o n d i l u t i o n h e l d 21+ h o u r s a t room t e m p e r a t u r e a n d r e p l a t e d i n 1 c . c . a m o u n ts - m e te r a r e s e t f o r t h i n T ab le 8 . 16T he s u s p e n s i o n s w e r e d i l u t e d t o 1 :5 * 0 0 0 ,0 0 0 and 1 :5 0 ,0 0 0 ,0 0 0 and p l a t e d i n t r i p l i c a t e 1 c .c . o f 1 :5 * 0 0 0 ,0 0 0 d i l u t i o n o f o r i g i n a l s u s p e n s io n , 1 : 5 * 0 0 0 , 0 0 0 an d l / 2 c . c . o f 1 :5 0 ,0 0 0 ,0 0 0 d i l u t i o n . as fo llo w s: l/2 c .c . of T h is p ro c e d u re g a v e p l a t e c o u n t s o f a b o u t 2 0 0 , 100 a n d 10 c o l o n i e s r e s p e c t i v e l y a n d w as u s e d i n t h e g u i n e a p i g i n o c u l a t i o n e x p e r i m e n t s a s s t a n d a r d p r o ­ ced u re. I n V iv o V i r u l e n c e S t u d i e s M ost o f t h e e x p e r i m e n t s c a r r i e d o u t t o d e te rm in e v ir u l e n c e a n d I n c r e a s e d v i r u l e n c e du e t o a n i m a l p a s s a g e h a v e b e e n made b y u s i n g l a r g e num ber'"'of v i a b l e B r u c e l l a o r g a n i s m s . I t was c o n s i d e r e d d e s i r a b l e t o u s e v e r y s m a l l n u m b e rs o f o r g a n i s m s i n t h i s stu d y t o d e f i n i t e l y d e t e r ­ m in e t h e v i r u l e n c e o f a c u l t u r e a s w e l l a s a n y i n c r e a s e i n v i r u l e n c e due to an im al p a s s a g e . By u s i n g s m a l l n u m b e rs o f o r g a n i s m s a n d g r a d i n g t h e s iz e of th e in je c tio n s i t s e e m e d p r o b a b l e t h a t a minimum i n f e c t i n g d o s e c o u l d b e e s ta b L i s h e d a n d e n h a n c e m e n t o f v i r u l e n c e b y a n i m a l p a s s a g e d e te rm in e d . The minimum i n f e c t i n g d o s e w as i n d i c a t e d b y t h e s m a l l e s t n u m b e r o f o rg a n ism s t h a t p ro d u c e d i n f e c t i o n a s d e m o n s tr a te d by t h e a g g l u t i n a t i o n re s p o n s e and c u l t u r a l f i n d i n g s . Any i n c r e a s e i n v i r u l e n c e o f a c u l t u r e du e t o a n i m a l p a s s a g e was m e a s u r e d b y i t s a b i l i t y t o p r o d u c e i n f e c t i o n i n d o s e s s m a l l e r t h a n t h e o r i g i n a l c u l t u r e o r b y p r o d u c t i o n o f m ore e x ­ t e n s i v e g r o s s l e s i o n s a n d a g r e a t e r a g g l u t i n a t i o n r e s p o n s e i n t h e same dosage as th e o r i g i n a l . E ig h t c u l t u r e s o f B r u c e lla w ere s t u d i e d w ith r e s p e c t t o t h e i r v i r u l e n c e f o r g u in e a p i g s and r e s u l t a n t i n c r e a s e i n v i r u l e n c e due t o -1 7 - ta b le 8 R e s u l t s o f S t a n d a r d i z i n g C u l t u r e 1287 a^-d 1281; on C en co P h o t e l o m e t e r U s i n g 0*1 P e r C e n t T r y p t o s e C u l t u r e 1287 P la te N o. 1 c .c . of 1 : 5M l/2 c .c , of 1 : 5M 1 202 11 h 8 2 209 106 10 3 221; 103 15 k 21 2 10J4. 11 l/2 c .c . of 1 : 50M C u l t u r e 1281; 1 c .c . of 1:311 l/2 c .c . of 1 : 5M l / 2 c .c . of 1:50M 1 19k 96 10 2 181 101 9 3 197 83 10 k c o n t. 105 8 P la te No. P l a t e w as c o n t a m i n a t e d w i t h m o l d . - 18- subs e q u e n t g u in e a p ig p a s s a g e . S e v e n p a s s a g e s w e r e made t h r o u g h g u i n e a p i g s w i t h c u l t u r e 1257# s i x w i t h c u l t u r e 1280, f i v e w i t h c u l t u r e 1287, t h r e e w ith c u l t u r e a n d o n e w i t h c u l t u r e s 1285* I 0 9 9 ^ g , a n d 1677* 1281;, C u l t u r e 19 w as u s e d i n e i g h t d i f f e r e n t d o s e s b e f o r e i n f e c t i o n c o u l d be e s t a b l i s h e d . In a l l c a s e s e a c h d o s e o f o r g a n i s m s w as i n j e c t e d s u b c u t a n e o u s l y i n t o t h r e e g u i n e a p i g s o f a p p r o x i m a t e l y t h e same s i z e . The v a r i o u s g r o u p s o f g u in e a p ig s w ere p la c e d in s e p a r a t e cag es i s o l a t e d fro m a l l o th e r groups# A t t h e e n d o f 3 0 d a y s e a c h g r o u p was s a c r i f i c e d a n d a u t o p s i e d . h e a r t b l o o d w as c o l l e c t e d f o r t h e a g g l u t i n a t i o n t e s t , At a u to p sy l e s i o n s w ere n o te d , a n d c u l t u r e s w e r e made o f t h e s p l e e n a n d l i v e r o n T r y p t o s e a g a r p l a t e s c o n ta in in g c r y s t a l v i o l e t i n a 1 :1 ,0 0 0 ,0 0 0 d i l u t i o n . A ll p l a t e s w ere i n ­ c u b a t e d f o r f i v e d a y s a t 37°C# To i d e n t i f y t h e v a r i o u s p a s s a g e s o f t h e c u l t u r e s t h e o r i g i n a l p a s s a g e was g i v e n t h e c u l t u r e n u m b e r, t h e s u b s e q u e n t p a s s a g e s w e r e num­ b e r e d w i t h t h e c u l t u r e num ber f o l l o w e d b y a , b , c , d , e t c . £ la p s i n g b e tw e e n i s o l a t i o n o f a c u l t u r e and i t s The t i m e subsequent r e in j e c t io n i n t o g u i n e a p i g s w as s e v e n day s* T a b l e s 9 t o 12 show t h e r e s u l t o f s e v e n p a s s a g e s o f c u l t u r e 1257 t h r o u g h g u i n e a p i g s . P a s s a g e s o f 1257s.* 1257^# a n d. 1257© ®-re n o t show n s i n c e t h e s e w e r e u s e d on a n o t h e r g r o u p o f g u i n e a p i g s i n r e l a t i v e l y h ig h e r d o ses t h a n w a s th e p r a c t i c e i n t h i s w ork. 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O © 12; pi •H w> 3 O PH All a 9Eh 3 -P by an m OJ rH a g g lu tin a tio n , Pi © © i—i PL, CO © O P-t 43 •H © O Pi © •rH P ai fctO © 6E5 incom plete •H •X3 Pi •H Pi tO O 0i © > •rH in d ic a te s u 3 p rH 3 O tO bD pj © i> •rH P in d ic a te d rH cd 25> •H P •H © O P-4 unless > ►Hi © •H -P •H complete pi ® -2 3 th a n th o s e observed w ith th e o r ig in a l p assag e. ^ T h e r e s u l t s o f f i v e p a s s a g e s o f c u l t u r e 1287 t h r o u g h g u i n e a p i g s a r e sh o w n i n T a b l e s 13 t o 17* I t was n o t e d t h a t t h i s c u l t u r e p ro d u c e d i r r e g u l a r i n f e c t i o n as d e te rm in e d b y c u l t u r a l f i n d i n g s , t i n a t i o n re s p o n s e , and g ro s s l e s i o n s . ag g lu ­ H o w e v e r, c u l t u r e 128 7b p r o d u c e d p o s i t i v e c u l t u r a l f in d in g s I n a l l d o se s, w h ile c u l tu r e s o f 12 8 7 c and 1287d i n d o s e s c o m p a ra b le t o t h o s e h i g h e r d o se s o f 1287, 1287a and 1287 b p r o d u c e d a c o n s i s t e n t l y h i g h a g g l u t i n a t i o n re sp o n se , and e x te n s iv e le s io n s , T h is I n d ic a te d an en­ as w e ll as p o s i t i v e c u l t u r a l f i n d i n g s . hancem ent o f v ir u le n c e f o r t h i s c u ltu re a f te r th re e passages. 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P iri p : • o • 9 rH LTN CO g dno^o PT LfN CO OJ KN rH O t— LfN -W ).- fro m t h e c o l o s t r u m and f e t u s e s o f a l l o f t h e h e i f e r s w h ich c a l v e d o r ab o rte d * The h e i f e r s i n G roup 2 w e r e i n o c u l a t e d w i t h l+,62 7 , 5 0 0 v i a b l e Br* a b o r t u s o r g a n i s m s b y w ay o f t h e c o n j u n c t i v a l s a c . a r e shown i n T a b l e 3 2 . The r e s u l t s The p r e v i o u s l y m e n t i o n e d h e i f e r 883 m u s t h a v e d e v e l o p e d a h i g h d e g r e e o f im m u n ity b y n a t u r a l e x p o s u r e a s i n d i c a t e d by a l a c k o f a g g l u t i n a t i o n r e s p o n s e and a n orm al p a r t u r i ­ tio n . tite r A l l o t h e r h e i f e r s i n G roup 2 d e v e l o p e d a s e ru m a g g l u t i n a t i o n o f 1 :1 0 0 b y th e 9 9 ^ day a f t e r e x p o su re . W i th t h e e x c e p t i o n o f 8 8 3 , a l l h e i f e r s i n G roup 2 a b o r t e d a n d showed B r . a b o r t u s i n th e c o lo s tru m and f e t u s e s . -l^ - SUMMARY A s u i t a b l e s u s p e n d i n g o r d i l u t i n g f l u i d f o r m a k in g p l a t e c o u n t s B r u c e l l a w as p r e p a r e d f r o m 0*1 p e r c e n t T r v p t o s e a n d 0 . 5 P®** c e n t s o d iu m c h l o r i d e i n d i s t i l l e d w a t e r * The pH a f t e r s t e r i l i z a t i o n w as 6*9* G u in e a p i g s w e r e r e a d i l y i n f e c t e d w i t h B r . a b o r t u s a n d B r . s u i s i n v e r y s m a ll n u m b e rs. T en o r g a n i s m s w e r e s u f f i c i e n t t o p r o d u c e i n ­ f e c t i o n i n m ost o f th e g u in e a p ig s . C o n tin u e d p a s s a g e o f B r. a b o r t u s th r o u g h g u in e a p ig s r e s u l t e d in an in c re a s e in v iru le n c e of a l l c u ltu r e s . S t r a i n 19 B . A . I . e x h i b i t e d t h e l o w e s t v i r u l e n c e 'o f a n y c u l t u r e 3 o f B r. a b o rtu s s tu d ie d . ■ ^ ~ S u s c e p t i b l e p r e g n a n t h e i f e r s w ere r e a d i l y i n f e c t e d w i t h 1 |, 6 2 7 j 500 v i a b l e B r . a b o r tu s o rg an ism s d e p o s it e d i n th e c o n j u n c t i v a l s a c . T h e t i m e o f t h e a g g l u t i n a t i o n r e s p o n s e o f t h e two g r o u p s o f p r e g n a n t h e i f e r s e x p o s e d t o tw o d i f f e r e n t d o s e s o f o r g a n i s m s v a r i e d i n ­ v e r s e l y w i t h t h e number o f o rg an ism s u s e d . f ACKNOSilLEDGMEHT T he a u t h o r w i s h e s t o e x p r e s s a p p r e c i a t i o n t o D r . I . F . E u d d l e s o n f o r h i s a s s i s t a n c e i n t h i s w ork. - 14.6 - REFEEEMCES 1. H a g a n , W. A. The s u s c e p t i b i l i t y o f w ith B r. a b o r t u s . 2. H a g a n , W. A. 3. o rig in . 1+. d i s e a s e o f g u i n e a p i g s due J . Exp* M e d ., 5 6 , 697 S c o r g i e , XT. J . to B r. (1 922). I n f e c t i o n o f m ic e w i t h B r . a b o r t u s o f b o v i n e J . P a t h , a n d B a c t . , I46, 1 6 5 - 1 7 1 (d an * 1 9 3 8)* McEwen, A. D. The v i r u l e n c e o f B r . a b o r t u s f o r l a b o r a t o r y a n im a ls and p re g n a n t c a t t l e . 5* in fe c tio n J . E x p . M e d ., J4 6 , J2J ( 1 9 2 2 ) . S tu d ie s on th e a b o rtu s . m ic e a n d r a t s t o M e tz g e r, H. J . V e t . R e c . , 5 2 , 9 7 - 1 0 6 ( I 9 A4JO). a n d F . R. S t o l u s . R e s u lts o b ta in e d b y lo n g c o n t in u e d c u l t i v a t i o n o f B r. a b o r tu s i n l i v i n g c h i c k em b ry o s. Am. J r . 6. o f V e t. R e s e a rc h , V o l. I I , 178-80, (A p ril, McEwen, A. D . , F . W. P r i e s t l e y a n d J . D. P a t t e r s o n . 19 ^1 )• An e s t i m a t e o f a s u i t a b l e i n f e c t i v e dose o f B r. a b o r tu s f o r im m u n isatio n te sts p. 7. on c a t t l e . Jr. o f Comp. P a t h , a n d T h e r . , V o l . 5 2 , 1 1 6 -1 3 1 , ( J u n e , 1939)* C l a r k , W. M ., a n d H . A. L u b s . p h th a la te , H y d ro g e n e l e c t r o d e p o t e n t i a l s o f p h o s p h a te and b o r a te b u f f e r m ix tu r e s . C h e m ., 2 5 , h79 ( 1 9 1 6 ) . J . B io l.