DOCTORAL DISSERTATION SERIES d e Tensions E r Tri TITLE f Of The Got)OCOCCUS‘ Of her AUTHOR ironmenfs VfM F( UNIVERSITY DEGREE h Z .M 1 icfliqan State College 11 PUBLICATION NO. m 11111111m1 UNIVERSITY MICROFILMS S" A N N ARBOR - MICHIGAN OPTIMAL CAHBO:; DIO XIDE TEXSIOH5 POE PRIMARY ISOLATION OP TER JO TO COCCUS; RESPONSE CP TEC O R C A 'I SM TO OTHER CASEOUS El-iY I r o :DISIT TS OPTIMAL CARBON DIOXIDE TENSIONS FOR PRIMARY ISOLATION OP THE GOHOCOCCUS; HESPOUSE OF THE ORGANISM TO OTHER GASEOUS ENVIRONMENTS A T h e s is S u b m itte d to t h e F a c u l t y o f M ic h ig a n S t a t e C o l l e g e o f A g r i c u l t u r e an d A p p l i e d S c ie n c e i n p a r t i a l f u l f i l l m e n t o f t h e r e q u i r e m e n t s f o r th e d e g r e e o f D o c to r o f P h i l o s o p h y "by W. W. F erg u so n Septem ber, 19^-2 Table of Contents I n t r o d u c t io n ................................................................................................................................1 Review o f the L i t e r a t u r e ................ . . .................... 2 d as A n a ly s e s ............................................................................................................................ 9 P la n o f th e Stud y..................................................................................................................13 P art I Comparison o f th e E f f e c t o f A tm ospheric A ir , Reduced Oxygen T en sion and Carbon D io x id e ................... . . . . . . . . . . .lU M eth o d s..................................................................................................................... 15 R e s u lts o f P a r t 1 ...............................................................................................23 P a r t l a Study o f th e E f f e c t o f M oistu re on "Aerobic" S tr a in s o f Conoco c c i .................................................................................................................2 7 P a r t I I D eterm in a tio n o f th e O ptim al Range o f Carbon D ioxid e S tim u la tio n ................................................................ ............................................29 P a r t I l a E f f e c t o f Carbon D io x id e Beyond th e Optim al Range.................... b l P a r t I I I E f f e c t o f C ases O ther Than CarbonD io x id e on th e Crowth o f G o n o co cci........................................................... ............................................ P a r t I l i a E f f e c t o f N itr o g e n Compared w ith A tm ospheric A ir , Reduced Oxygen T ension and CarbonD io x id e ...................................... . 5 0 D is c u s s io n . ................................ Summary. B ib lio g r a p h y ......................... Acknowledgem ent.............. ........... *53 57 .5 8 &L OPTIMAL CARBON DIOXIDE TENSIONS FOR PRIMARY ISOLATION OF THE GONOCOCCUS; RESPONSE OF THE ORGANISM TO OTHER GASEOUS ENVIRONMENTS S in c e t h e s u c c e s s f u l c u l t i v a t i o n o f t h e g o n o c o c c u s by E. Bumm i n 1885 ( 1 ) a v a s t am ount o f l i t e r a t u r e h a s a p p e a r e d on th e g r o w th r e q u i r e ­ m ents and c u l t i v a t i o n o f t h i s o r g a n is m . Of t h e s e o b s e r v a t i o n s a coin- p a r a t i v e l y s m a ll p a r t h a s d e a l t w i t h th e a t m o s p h e r ic r e q u i r e m e n t s o f g o n o co cci. U n t i l r e c e n t y e a r s r e p o r t s on o p t i m a l g a s e o u s e n v iro n m e n t have b e e n c o n t r a d i c t o r y and b a s e d , i n some i n s t a n c e s , on a s tu d y o f a c o m p a r a t i v e l y few s t r a i n s . The g row ing m ass o f e v id e n c e i n d i c a t e s t h a t g o n o c o c c i a r e b e n e f i t e d d u r i n g p r i m a r y i s o l a t i o n by an a tm o s p h e re e n r i c h e d w i t h c a r b o n d i o x i d e . There h a s grown up among b a c t e r i o l o g i s t s a f e e l i n g , b a s e d on i n c o n c l u s i v e e v i d e n c e , t h a t to a c h i e v e o p tim a l r e s u l t s one m u st s u p p ly a c a r b o n d io x id e t e n s i o n o f a p p r o x i m a te l y t e n p e r c e n t . The m eth od s i n c u r r e n t u s e f o r s u p p l y i n g c a r b o n d i o x i d e e n r ic n m e n t u n d o u b t e d l y g i v e v a r y i n g am ounts o f th e g a s , and a s tu d y ( 2 ) o f g ro w th u n d e r c a r b o n d i o x i d e - t e n s i o n s su p ­ p l i e d by v a jrio u s means i n d i c a t e s t h a t c o m p a ra b le r e s u l t s may b e a c h i e v e d . D u rin g th e c o u r s e o f e x p e r i m e n ts on th e v i a b i l i t y o f g o n o c o c c i u n d e r c o n d i t i o n s o f t r a n s p o r t a t i o n from p a t i e n t to l a b o r a t o r y (3 )» t h e w r i t e r u s e d v a r i o u s means o f s u p p ly i n g c a r b o n d i o x i d e f o r c u l t u r e . The r e s u l t s w ere p r a c t i c a l l y th e ssme by two m etho d s w hich w ere ju d g e d to p r o v id e d i f f e r e n t am ounts o f g a s . A n a ly s e s c o n f irm e d o u r o p i n i o n t h a t th e g a s t e n s i o n s s u p p l i e d w ere c o n s i d e r a b l y d i f f e r e n t . W ith t h i s i n f o r m a t i o n a s an im p e t u s , th e p r e s e n t s tu d y was u n d er­ ta k e n to d e f i n e more e x a c t l y th e c a r b o n d i o x i d e r e q u i r e m e n t s o f gono­ c o c c i d u r i n g p r im a r y i s o l a t i o n an d , a l s o , to exam ine th e e f f e c t o f g a s e o u s e n v ir o n m e n ts o t h e r t h a n c a rb o n d i o x i d e . LITERATURE Wherry and O l i v e r (U) i n 1916 c a l l e d a t t e n t i o n to t h e f a c t t h a t th e g o n o co ccu s i s a p a r t i a l o x y g e n - t e n s i o n o r g a n is m . In p r o d u c i n g a p a r t i a l oxygen t e n s i o n t h e y made u s e o f a method employed hy an e a r l i e r w o rk er i n c u l t i v a t i n g B r u c e l l a a b o r t u s . By means o f r u b b e r t u b i n g t h e y f a s t e n e d t o g e t h e r an a g a r s l a n t t u b e on w hich g o n o r r h e a l m a t e r i a l was i n o c u l a t e d and a n o t h e r s l a n t tu b e c o n t a i n i n g a. t r a n s f e r o f B. s u b t i l i s . The g a s exchange b etw een th e tu b e s to o k p l a c e th r o u g h t h e c o t t o n .p lu g s . Wherry and O l i v e r f e l t t h e s u c c e s s f u l g ro w th o f th e gonococcus was due to th e lo w e r in g o f t h e oxygen t e n s i o n by B a c i l l u s s u b t i l i s . S im ila r in o cu lu m u n d e r a t m o s p h e r i c c o n d i t i o n s f a i l e d to y i e l d g ro w th o f g o n o c o c c i. D uring an i n v e s t i g a t i o n o f th e c u l t u r a l r e q u i r e m e n t s o f t h e gono­ c o c c u s , Chapin ( 9 ) t e s t e d th e i n f l u e n c e o f c a rb o n d i o x i d e . He f e l t t h a t th e c a r b o n d i o x i d e t e n s i o n p r e s e n t i n l i v i n g t i s s u e s , w hich he gave a s f i v e to seven cm. o f m e rc u ry , was th e l o g i c a l t e n s i o n to employ i n grow in g g o n o c o c c i. I n i s o l a t i n g t h e c o c c i from u r e t h r a l e x u d a t e , Chapin fo u n d t h a t a c a r b o n d i o x i d e t e n s i o n o f a p p r o x i m a te ly t e n p e r c e n t c a u s e d a more v i g o r o u s g r o w th o f th e o r g a n is m s th a n norm al a i r . The g a s was s u p p l i e d by t r e a t i n g sodium b i c a r b o n a t e w i t h s u l f u r i c a c i d . s t r e s s e d t h e im p o rta n c e o f a m o is t a tm o s p h e r e . b rie f a r tic le , Chapin He m e n tio n e d , i n h i s t h a t a c a n d l e had b e e n fo u n d to s u p p ly s u f f i c i e n t c a rb o n d i o x i d e f o r good g ro w th . R u e d ig e r ( 6 ) , r e p o r t i n g on t h e s u c c e s s f u l r e c o v e r y o f g o n o co cci from t h r e e c a s e s o f g o n o r r h e a l i n f e c t i o n , announced t h a t a v i g o r o u s g ro w th was o b t a i n e d by e x c l u d i n g th e a i r from c u l t u r e tu b e s by means o f ru b b er s to p o e rs . S chw artz ( 7 ) f o u n d , s i m i l a r l y , t h a t e x c lu d i n g a i r from c u l t u r e tu b e s - 3 - g av e b e t t e r r e s u l t s t h a n i n c u b a t i n g t h e g o n o c o c c i u n d e r a t m o s p h e r i c c o n d itio n s. By h e a t i n g t h e t o p s o f h i s t u b e s i n t h e f la m e b e f o r e s to p ­ p e r i n g , he s e c u r e d a r e d u c t i o n i n a t m o s p h e r i c p r e s s u r e o f a b o u t t e n p er c e n t. He c o n c l u d e d , a l s o , t h a t m o i s t u r e i s e s s e n t i a l f o r g r o w th . R o c k w e ll and McXhann (S ) hav e b e e n q u o te d i n t h e l i t e r a t u r e a s a s s e r t i n g t h a t g r o w th o f g o n o c o c c i i s f a c i l i t a t e d b y t h e p r e s e n c e o f h y d ro g e n g a s . T h is s t a t e m e n t i s l i k e l y to be m i s l e a d i n g u n l e s s i t i s u n d e r s t o o d t h a t t h e p r i m a r y i s o l a t i o n s o f t h e s e w o r k e r s w ere o b t a i n e d i n a t m o s p h e r e s a l t e r e d by g r o w th o f B a c i l l u s s u b t i l i s . In s tu d y in g th e a d a p t a t i o n o f g o n o c o c c i to v a r i o u s g a s e o u s e n v ir o n m e n t th e y f o u n d t h a t c u l t u r e s i s o l a t e d u n d e r p a r t i a l o x y g en t e n s i o n g rew i n p u r e h y d ro g e n b u t n o t i n c a r b o n d i o x i d e and o x y g e n . A f t e r g r o w th f o r some tim e u n d e r a t m o s p h e r e s o f h y d r o g e n t h e o r g a n is m s seemed to become more s e n s i t i v e to t h e i n h i b i t i n g i n f l u e n c e o f c a r b o n d i o x i d e and o x y g en , b u t g r o w th was s t i l l e x c e l l e n t u n d e r p a r t i a l t e n s i o n . They f o u n d t h a t when s t r a i n s i s o l a t e d a t p a r t i a l t e n s i o n w ould n o t grow tin d e r p u r e c a r b o n d i o x i d e o r p u r e o x y g en , t h e y c o u l d b e a d a p t e d to do s o . In 1922 E r i c k s o n and A l b e r t ( 9 ) r e p o r t e d t h e i r e x p e r i e n c e s i n c u l t i v a t i n g g o n o c o c c i. L ik e th e e a r l i e r w o r k e r s t h e y s t r e s s e d t h e i m p o r t a n c e o f m o i s t m e d ia an d m o i s t a tm o s p h e r e . They t e s t e d t h e m ethods d e s c r i b e d by W herry an d O l i v e r and o t h e r s f o r p r o d u c i n g a r e d u c e d oxygen t e n s i o n and f o u n d no a d v a n ta g e o v e r t h e oxygen t e n s i o n o f n o rm a l a i r . They c o n c l u d e d t h a t t h e s a t i s f a c t o r y r e s u l t s o f i n v e s t i g a t o r s u s i n g r e d u c e d oxygen t e n s i o n was due to a n i n c r e a s e d amount o f m o i s t u r e b r o u g h t a b o u t by t h e t e c h n i q u e em ployed to e x c l u d e a i r . I n t h e same y e a r T o r r e y and B u c k e l l ( 1 0 ) p u b l i s h e d a n a c c o u n t o f - u - e x t e n s i v e work on t h e p r i m a r y i s o l a t i o n o f g o n o c o c c i . L ik e E r i c k s o n a n d A l b e r t t h e y f o u n d a r e d u c e d o x y g en t e n s i o n to p r o v e i n no way s u p e r i o r to m o i s t a i r o f n o rm a l p r e s s u r e . E n r ic h m e n t o f t h e a tm o s­ p h e r e w i t h c a r b o n d i o x i d e a c c o r d i n g to C h a p i n 's m ethod l i k e w i s e y i e l d e d no b e t t e r r e s u l t s th a n n o rm a l a i r . The i n t r o d u c t i o n o f th e d i r e c t o x i d a s e r e a c t i o n a s an a i d i n i d e n t i f y i n g th e g o n o c o c c u s i n m ixed c u l t u r e , by McLeod, C o a te s e t a l . ( 1 1 ) i n 193^* f o c u s e d th e a t t e n t i o n o f w o r k e rs on t h e g e n e r a l c u l t u r a l m eth od s o f t h e E n g l i s h i n v e s t i g a t o r s . McLeod and h i s c o l l e a g u e s e n d o r s e d t h e xxse o f an i n c r e a s e d c a r b o n d i o x i d e t e n s i o n o f e i g h t p e r c e n t , i n c u b a t i n g t h e i r c u l t u r e s u n d e r t h e e n r i c h e d c a r b o n d i o x i d e atm os­ p h e r e f o r 2k h o u r s , f o l l o w e d by i n c u b a t i o n i n norm al a i r f o r 2k h o u r s . T h e ir c h o i c e o f a c a r b o n d i o x i d e t e n s i o n o f e i g h t p e r c e n t r e s u l t e d fro m i n c u b a t i n g g o n o r r h e a l m a t e r i a l u n d e r t e n s i o n s o f f o u r , e i g h t and s ix te e n p e r c e n t atm o sp h eres o f carb o n d io x id e . s p e c im e n s , In a sh o rt s e r ie s of t h e r e s u l t s a p p e a r e d to be b e s t i n t h e m id d le am o u n t, to q u o te t h e a u t h o r s . As t h i s a p p r o x im a te d t h e t e n p e r c e n t recommended b y C h ap in i t was a d o p t e d f o r r o u t i n e w ork. A c c o r d in g to t h e i r o b s e r v a ­ t i o n s o l d s t o c k s t r a i n s o f g o n o c o c c i w ere n o t a i d e d i n t h e i r g ro w th by carbon d io x id e . The e f f e c t o f an a tm o s p h e r e o f h y d ro g e n was a l s o t r i e d on p r im a r y c u l t u r e , i n a few 1 . s t a n c e s , b u t no w o r th w h ile a d v a n t a g e s w ere o b s e r v e d . The o b s e r v a t i o n s o f McLeod an d h i s a s s o c i a t e s i n r e g a r d to o p tim a l a tm o so h e ric c o n d itio n s a r e o f i n t e r e s t . "The g e n e r a l i m p r e s s i o n l e f t by t h e s e o b s e r v a t i o n s ( t h o s e sum­ m a r iz e d a b o v e ) i s t h a t t h e r e a r e some s t r a i n s o f g o n o co ccu s w h ich a r e - 5 - fa v o r e d i n t h e i r grow th a t th e o u t s e t by an a tm o sp h ere o f r a i s e d carbon d io x id e c o n t e n t . T h is may h e due to n eed f o r some d e l i c a t e a d ju stm en t o f r e a c t i o n w h ich i s m ost e a s i l y d e v e lo p e d i n a carb on d io x id e atm os­ p h ere o r to th e f a c t t h a t th e g o n o c o c c u s i s a m ic r o -o r g a n ism more h ig h ly a d a p ted than m ost to i t s g a s e o u s en v iro n m en t and one w h ich , t h e r e f o r e , grow s b e s t a t f i r s t i n a c o n c e n t r a t io n o f carb on d io x id e a p p ro x im a tin g t h a t w h ich e x i s t s in th e t i s s u e s . " L eahy a n d C a r p e n t e r ( 1 2 ) i n t r o d u c e d t h e m e th o d s o f McLeod a n d h i s c o - w o r k e r s to t h i s c o u n t r y w i t h some m o d i f i c a t i o n s . They u s e d a n i n ­ g e n i o u s m a n o m e tric m eth o d o f m e a s u r i n g th e c a r b o n d i o x i d e s u p p l i e d by t a n k to s e a l e d c u l t u r e j a r s . Of 6 l s t r a i n s o f g o n o c o c c i i s o l a t e d u n d e r a t e n p e r c e n t c a r b o n d i o x i d e t e n s i o n , o n l y ^2 g re w i n a j a r i n w h ic h t h e g a s e o u s e n v i r o n m e n t h ad n o t b e e n a l t e r e d . In t h i s s e r i e s th e r e l a ­ t i v e h u m i d i t y i n t h e two c u l t u r e j a r s was s u p p o s e d l y t h e sam e. W hether o r n o t th e a u th o r s t e s t e d o th e r c o n c e n tr a tio n s o f carb o n d io x id e b e fo re a d o p t i n g an e n r i c h m e n t o f t e n p e r c e n t i s n o t i n d i c a t e d . Spink and K e e fe r (1 3 )» l i k e C hap in, fou n d t h a t a c a n d le burned in th e c u lt u r e j a r f a c i l i t a t e d prim ary i s o l a t i o n o f g o n o c o c c i. C h r i s t e n s e n an d S c h o e n l e i n ( 2 ) c u l t u r e d 122 c l i n i c s p e c im e n s from go n o rrh eal p a t i e n t s in m u ltip le p la tin g s , s u b je c tin g each s e t o f p l a t e s to t h e f o l l o w i n g c o n d i t i o n s : (1 ) C arbo n d i o x i d e s u p p l i e d b y c a n d l e (2) C arbo n d i o x i d e ( a p p r o x . 1 0 p e r c e n t ) s u p p l i e d b y p l a c i n g m o iste n e d o a t s i n th e c lo s e d c o n ta in e r . (3 ) C arbon d i o x i d e s u p p l i e d b y a c t i o n o f s u l f u r i c a c i d on sodium b ic a rb o n a te (H) 8-10 p e r c e n t carbon d io x id e s u p p lie d by tan k (5) M o is t a i r - 6 - The number o f s t r a i n s i s o l a t e d by th e r e s p e c t i v e m ethods w ere: (l) 71 (2 ) They n o te d a l s o 69 (3) 70 (H) 70 Sb (5) t h a t p u re c u l t u r e s o f th e g o n o co ccu s p r e v io u s l y i s o l a t e d do n o t r e q u ir e carb on d io x id e f o r gro w th . D u rin g th e yearB 1935 and 193& some i n t e r e s t i n g work was p u b lis h e d in Q-ermany on th e a tm o sp h e ric r e q u ir e m e n ts o f th e g o n o c o c c u s. Neumann ( lH , 1 3 ) i n v e s t i g a t e d th e a b i l i t y o f p rim ary s t r a i n s o f g o n o c o c c i to grow u n d er a e r o b ic and a n a e r o b ic c o n d i t i o n s , red u ced o x y g e n t e n s io n and in c r e a s e d carb on d io x id e t e n s io n . To o b t a in a n a e r o b ic c o n d it io n s he u se d th e m ethod o f F o r tn e r ( l 6 ) w it h s l i g h t m o d if ic a t io n s . p r o d ig io s u s was th e o rgan ism u se d to consume o x y g e n . B a cteriu m The a n a e r o b ic m ethod was abandoned a f t e r th e o b s e r v a t io n th a t g o n o co ccu s c o l o n i e s in th e F o r tn e r p l a t e a t t a i n e d t h e i r maximum s i z e in 2b h o u r s, w h ereas a s t r i c t a n a e r o b e , in o c u la t e d a lo n g w ith th e g o n o r r h e a l m a t e r ia l, f i r s t becam e v i s i b l e a t a b o u t t h i s tim e . Neumann r e a so n e d th a t th e g o n o c o c c i d e v e lo p e d d u rin g a p e r io d o f red u ced oxygen t e n s io n and c e a s e d grow th when a n a e r o b ic c o n d i t i o n s w ere a t t a i n e d . He term ed th e a n a e r o b ic p l a t e th e "B” p l a t e m ethod. To o b t a in re d u c e d oxygen t e n s i o n , or p a r t i a l vacuum, Neumann in ­ v e r t e d th e m e d iu m -co n ta in in g h a l f o f a P e t r i d is h on a g l a s s p l a t e o f 13 x 13 cm. d im e n s io n s w h ich had b een h e a te d i n a fla m e . d is h was s e a le d i n p l a c e by p l a s t i c i n e . p l a t e m ethod. The P e t r i T his method he c a l l e d th e "C" He d e te r m in e d , by means o f a s e n s i t i v e w ater m anometer, t h a t a p r e s s u r e d e c r e a s e c o n tin u e d i n u n in o c u la te d "0 " p l a t e s d u rin g in c u b a t io n . agar. T h is d e c r e a s e he a t t r ib u t e d to th e r e d u c in g a c t io n o f the Reduced o x y g en t e n s io n in th e "CM p l a t e , t h e r e f o r e , was a t t a in e d by h e a t in g th e g l a s s p l a t e s and by a c t io n o f th e medium. - 7 - F o r th e c a r b o n d i o x i d e e n r i c h e d , o r "D" p l a t e m ethod, Neumann p l a c e d an i n o c u l a t e d h a l f o f a P e t r i d i s h on a n u n h e a t e d g l a s s p l a t e , b u t p u s h e d a p i e c e o f d r y i c e u n d e r t h e d i s h b e f o r e m o ld in g t h e p l a s t i ­ cin e in p la c e . A c c o r d in g to th e a u t h o r , "The c a r b o n d i o x i d e volume n e c e s s a r y f o r g o n o c c u s g ro w th may be a b o u t t e n p e r c e n t . volume i s n o t n e c e s s a r y , how ever. Such a l a r g e Through num erous c o m p a r a tiv e e x p e r i ­ m ents I hav e f o u n d e m p i r i c a l l y , w i t h o u t m e a su re m e n t, t h a t a p i e c e o f d r y i c e th e s i z e o f a p e a s u p p l i e s a l l r e q u i r e m e n t s . . . . w i t h a p ie c e of dry ic e t h e s i z e o f a b e a n t h e r e may d e v e lo p enough to i n h i b i t th e g r o w th o f g o n o c o c c i . " U s in g t h e a e r o b i c , o r "A" p l a t e , carbon d io x id e t h e "C" and "D" p l a t e , Neumann o b ta in e d th e fo llo w in g r e s u l t s in a t o t a l of l 6 l s t r a i n s o f gonococci iso la te d : 113 grew a e r o b i c a l l y ; 30 g re w u n d e r r e d u c e d oxygen t e n s i o n an d c a r b o n d i o x i d e ; li5 grew u n d e r c a r b o n d i o x i d e o n l y . However, a l l s t r a i n s grew u n d e r c a r b o n d i o x i d e . W in k le r ( 1 7 ) u s e d t h e t e c h n i q u e o f Neumann and c o n c lu d e d t h a t t h e r e a r e a e r o b i c g o n o c o c c i w h ich grow o n l y on t h e "A" p l a t e s ; t h a t t h e r e a r e g o n o c o c c i w hich grow o n l y on t h e "C" o r "D" p l a t e s ; th e re a re a lso s tr a i n s w h ich c a n grow on a l l p l a t e s w i t h o u t r e g a r d to oxygen c o n t e n t . The r a t i o i n w h ic h t h e s e t h r e e g r o u p s o f s t r a i n s e x i s t to one a n o t h e r i s a b o u t 1 :1 :1 . The b e s t m ethods o f c u l t i v a t i n g t h e g o n o co ccu s a r e norm al and c a r b o n d i o x i d e e n r i c h e d a tm o s p h e r e s . S in c e t h e work o f McLeod, C o a te s e t a l ( 1 1 ) , t h e r e i s a g re e m e n t among i n v e s t i g a t o r s t h a t c a r b o n d i o x i d e e n r i c h e d a tm o s p h e r e s f a c i l i t a t e g ro w th o f m ost s t r a i n s o f g o n o c o c c i . There i s a g r e e m e n t, a l s o , p e r c e n t a g e o f a p p r o x i m a te ly t e n p r o d u c e s th e b e s t r e s u l t s . th a t a I t i s in ­ t e r e s t i n g to n o t e why t h e d i f f e r e n t w o rk e rs a r r i v e d a t t h a t p e r c e n t a g e . - * - I n th e c a s e o f C h ap in ( 5 ) who p r e c e d e d McLeod, t h e t e n s i o n o f th e carbon d io x id e in l i v i n g t i s s u e s in flu e n c e d h is c h o ic e . A c c o r d in g to him t h e t e n s i o n v a r i e s fro m f i v e to s e v e n cm. o f m e r c u ry . H is f i g u r e s a r e a t v a r i a n c e w i t h t h o s e o f C am pbell ( 1 8 ) who d e t e r m i n e d t h e c a r b o n d io x id e te n s io n in s u b c u ta n e o u s t i s s u e and i n t h e p l e u r a l an d p e r i t o n e a l c a v i t i e s o f man a s e q u i v a l e n t to f o u r to f i v e cm. o f m e r c u r y . u r i n e o f man Cairrpbell f o u n d t h e t e n s i o n s to be t h e same. in t o -p ercen tag e s, to 6 . 6 ; I n n orm al T ransposed th e t e n s i o n s d e t e r m in e d by Cam pbell wotil d be fro m 5*3 t h e t e n s i o n a s s t a t e d b y C h ap in w ould b e fro m 6 .6 to 9*2 p e r c e n t. McLeod and h i s c o l l e a g u e s c i t e t h e work o f C h ap in and t h e s u c c e s s ­ f u l c u l t i v a t i o n o f B r u c e lla a b o r tu s u n d er te n per as re a so n s f o r i n v e s t i g a t i n g th e e f f e c t of t h i s g a s . c e n t carbond io x id e I t has been s ta te d b e f o r e t h a t t h e s e w o r k e r s f i x e d on e i g h t p e r c e n t a s optimum, a f t e r a s h o r t s e r i e s o f co m p ariso n s. L eahy a n d C a r p e n t e r ( 1 2 ) i n v e s t i g a t e d th e work o f McLeod and a d o p t e d t e n p e r c e n t a s th e p e r c e n t a g e o f c h o i c e , p o s s i b l y fro m u n p u b l i s h e d d a t a . Neumann ( lU , 1 5 ) a p p a r e n t l y made no m easurem en t o f t h e c a r b o n d i o x i d e f u r n i s h e d i n h i s ”D” p l a t e s b u t ju d g e d it to b e a b o u t t e n p e r c e n t . I t i s p r o b a b l e t h a t t h e work o f th e f o r e ­ going i n v e s t i g a t o r s i n f l u e n c e d Neumann i n nam ing a t e n p e r c e n t c a rb o n d io x id e te n s io n as th e d e s ir a b l e c o n c e n tr a tio n . I n t h i s c o u n t r y th e m ethods o f c u l t u r e a d v o c a t e d b y C a r p e n t e r a r e w id e ly u s e d . I n h i s s e c t i o n on "The G onococcus” , " D i a g n o s t i c P r o c e d u r e s an d R e a g e n t s , ” l ? U l ( 1 9 ) , C a r p e n t e r a d v i s e s t h e u s e o f a c a r b o n d i o x i d e e n r i c h e d a tm o s p h e r e w h ich may be o b t a i n e d i n t h r e e ways; ( l ) 10 p e r c e n t by t a n k w i t h a p p r o x im a te d e t e r m i n a t i o n o f g a s t e n s i o n by m anometer 4 - 9 - (2 ) By ■burning a c a n d l e i n th e c u l t u r e j a r "before p l a c i n g on th e lid (3 ) 3y u s e o f EaHCO^ and H^SO^ One i n f e r s t h a t e q u a l l y good r e s u l t s may h e o b t a i n e d by a l l o f t h e s e m e th o d s. S in c e , a c c o r d i n g to C h r i s t e n s e n a n d S c h o e n l e i n ( 2 ) , th e ca n d le method p r o v i d e s a d e q u a t e g ro w th s t i m u l a t i o n o f g o n o c o c c i a s w e l l a s e i g h t to t e n n e r c e n t c a r b o n d i o x i d e s u n n l i e d by t a n k , t h e l o g i c a l f i r s t s t e p i n t h i s i n v e s t i g a t i o n was th e d e t e r m i n a t i o n o f t h e c a r b o n d i o x i d e t e n s i o n s u p n l i e d by c a n d l e . GAS ANALYSES The c u l t u r e j a r s u s e d were h eav y b r a s s c y l i n d e r s f i t t e d w i t h i n l e t and o u t l e t n i p p l e s i n t h e l i d s . The l i d s w ere made a i r - t i g h t by means o f s o f t r u b b e r g a s k e t s and wing n u t s . The c y l i n d e r s were o r i g i n a l l y d e s ig n e d f o r m o d i f i e d E i l d e s - M a c l n t o s h a n a e r o b i c j a r s , o s d e s c r i b e d by Cummings ( 2 0 ) , b u t s e r v e d e x c e l l e n t l y f o r g o n o c o c c u s c u l t u r e . By t e s t th e y w ere f o u n d c a p a b l e o f h o l d i n g a p a r t i a l vacuum o f 40 cm. o f m ercury f o r Ug h o u r s , a s m e a s u re d on a U -ty u e op en m an om eter. The t o t a l c a p a ­ c i t y was a p p r o x i m a t e l y s i x l i t e r s . I n p r e p a r i n g th e j a r s f o r t e s t , a " s m o k e le s s " c a n d l e was p l a c e d i n t h e b o tto m o f t h e c o n t a i n e r and l i g h t e d . The c a n d le was a l l o w e d to b u r n f i v e m i n u t e s b e f o r e t h e j a r l i d was f i t t e d i n p l a c e . B u r n in g th e c a n d l e f o r v a r y i n g l e n g t h s o f tim e b e f o r e p u t t i n g on t h e l i d made no marked d i f f e r e n c e i n t h e c a r b o n d i o x i d e c o n t e n t f u r n i s h e d . The j a r s were a llo w e d to s t a n d o v e r n i g h t o r some h o u rs b e f o r e t e s t i n g i n o r d e r f o r th e g a s e s to come to room t e m p e r a t u r e . At 20 to 2U d e g r e e s c e n t i g r a d e i t was n o t e d t h a t a p a r t i a l vacuum e x i s t e d i n th e c a n d l e j a r s . - 10 - T his p a r t i a l vacuum when measured on th e open manometer was found to v a ry betw een fo u r to seven cm. o f m ercury. A m o d ified H enderson-H aldane ap p aratus was u sed in p r e lim in a r y carbon d io x id e and oxygen d e te r m in a tio n s, but c o n s ta n t r e s u l t s were n o t o b ta in e d w ith t h i s d e v ic e . The p o s s ib le so u r c e s o f e r r o r appeared to be due in p a r t to the apparatus and in p a r t to f a u lt y m a n io u la tio n . The samnle o f ten c c . o f gas which the Henderson ap p aratus t e s t s , and th e slo w a b so r p tio n o f both carbon d io x id e and oxygen by n o ta ssiu m h y d ro x id e and a l k a l i n e p y r o g a llo l, r e s p e c t i v e l y , are f a c t o r s w hich ten d to ca u se e r r o r . A fte r r e p e a te d t e s t s w ith d i f f e r e n t j a r s i n which th e volum es v a r ie d s l i g h t l y , the average p e r c e n ta g e s o f th e g a s e s were d eterm in ed a s: Carbon d io x id e Oxygen 1 .7 5 to 1 7 .5 1 .8 5 to 17*23 I t was b rou gh t to our a t t e n t io n s h o r tly a f t e r th e s e p r e lim in a r y t e s t s th a t Hye and Lamb (2 1 ) had made d e te r m in a tio n s o f the carbon d io x id e and oxygen c o n te n t o f s e a le d museum j a r s in w hich a ca n d le had b een burned. These d e te r m in a tio n s were made in th e cou rse o f stu d y on th e e f f e c t o f carbon d io x id e t e n s io n in the prim ary i s o l a t i o n o f s t r e p t o c o c c i , m en in gococci and g o n o co cci from pus and body f l u i d s . ITo m ention was made by the au th ors o f the apparatus u sed in o b ta in in g the f o llo w in g p e r c e n ta g e s o f g a se s: B efo re In cu b a tio n A fte r U8 hours Small ca n d le j a r (1 2 x 20 cm .) p er ce n t carbon d io x id e p er c e n t oxygen 1 .7 ^ 1 7 .8 7 18.00 Large ca n d le j a r (15 * 30 cm .) n er c e n t carbon d io x id e p e r c e n t oxygen 2.9 1 lG . 7o 16.56 1 .8 5 3.08 - l i ­ l t w i l l b e n o t e d t h a t t h e c a r b o n d i o x i d e t e n s i o n d e t e r m i n e d b y Hye a n d Lamb was h i g h e r t h a n t h a t o b t a i n e d by o u r m e a s u re m e n t. The l a r g e c a n d l e j a r i n t h e a b o v e t a b l e h ad a c a p a c i t y o f a b o u t 5300 c c . , w h i l e t h e j a r s u s e d i n t h i s s t u d y v a r i e d fro m 5900 c c . to 6000 c c . B e c a u s e o f t h i s d i s c r e p a n c y i n d e t e r m i n a t i o n s i t woe d e c i d e d to s u b s t i t u t e a n a n p e r a t u s u s i n g a n a s c a r i t e t r a i n f o r t h e KOH a b s o r p t i o n b o t t l e o f t h e H e n d e r s o n - H a ld a n e a n a l y s e r . F ig u re 1 The d ia g r a m g i v e n ab o v e shows t h e a s c a r i t e a p p a r a t u s w i t h i t s v a r i o u s u n i t s s e t up f o r c a r b o n d i o x i d e d e t e r m i n a t i o n . I n o p e r a t i o n , v a l v e B was o p e n e d an d t h e two-way s t o p c o c k * '^ t u r n e d to c o n n e c t t h e 150 c c . sam ple b o t t l e w i t h t h e c a n d l e j a r . The m e r c u ry r e s e r v o i r was lo w e r e d u n t i l t h e m e rc u ry i n t h e sam ple b o t t l e - 12 - r e a c h e d t h e 150 c . c . m ark on. t h e g l a s s shank s e a l e d to th e b o tto m o f t h e sam ple b o t t l e . m eter. A s l i g h t vacuum was t h e n i n d i c a t e d on t h e open mano­ C alcium c h l o r i d e s o l u t i o n * was a l l o w e d to f lo w i n t o t h e c a n d l e j a r u n t i l th e m e rc u ry colum ns o f th e m anometer w ere l e v e l . S to p c o c k C was t h e n t u r n e d to c o n n e c t t h e sam ple b o t t l e w i t h t h e tu b e l e a d i n g to D, a b o t t l e o f c o n c e n t r a t e d s u l f u r i c a c i d . * * o p en ed to th e a i r . A two-way s to p c o c k S was The m e rc u ry r e s e r v o i r was r a i s e d , f o r c i n g t h e g as i n th e sample b o t t l e to b u b b le th r o u g h t h e a c i d i n D. Two 1^0 c c . p o r­ t i o n s o f g a s w ere f o r c e d t h r o u g h D b e f o r e a sam ple was s e n t th r o u g h th e a s c a r i t e t r a i n . T h is was done to c l e a n t h e a c i d b o t t l e and th e t u b i n g o f p r e v i o u s g a s s a m p le s . I n s e n d in g a g a s sanvnle t h r o u g h th e t r a i n th e o p e r a t i o n s j u s t d e s c r i b e d w ere r e p e a t e d , e x c e p t t h a t s to p ­ co ck 2 was t u r n e d to c o n n e c t D w i t h t h e a s c a r i t e b o t t l e s F end G-. The g l a s s v a l v e s i n F an d G w ere o p e n e d and th e g a s th e n f o r c e d th ro u g h th e a s c a r i t e . B o t t l e "F" was w eighed on an a n a l y t i c a l b a l a n c e and th e i n c r e a s e i n w e ig h t due to a b s o r p t i o n o f c a r b o n d i o x i d e was d e te r m in e d by s u b t r a c t i n g f i n a l w e ig h t fro m i n i t i a l w e i g h t . B o t t l e "G" was u s e d to e l i m i n a t e a b s o r p t i o n o f c a r b o n d i o x i d e from a i r i n th e t e s t t r a i n . T h is p r e c a u t i o n was f e l t to b e n e c e s s a r y s i n c e a p a r t i a l vacuum d e v e lo p e d in th e a s c a r i t e b o t t l e s . C a l c u l a t i o n o f t h e p e r c e n t o f c a r b o n d i o x i d e i n a sam ple was made by m u l t i p l y i n g t h e w e ig h t o f c a rb o n d i o x i d e i n gram s by th e c o n v e r s i o n f a c t o r 509. T h is v a l u e i n c u b i c c e n t i m e t e r s was d i v i d e d by 1 5 0 , th e volume o f t h e sa m p le . C o r r e c t i o n f o r b a r o m e t r i c p r e s s u r e and te m p e r a tu r e * HOfi c a lc iu m " c h l o r i d e s o l u t i o n . Carbon d i o x i d e i s a o n r o x i m a te ly l / l 6 a s s o lu b le in t h i s s o lu tio n as in w a te r. ** The s x i l f u r i c a c i d was u s e d to remove m o i s t u r e from th e g a s . - 13 was made an d t h i s v a l u e m u l t i p l i e d b y 1 0 0 . The p e r c e n t a g e s o f c a r b o n d i o x i d e d e t e r m i n e d by t h i s m eth o d w ere f o u n d to v a r y s l i g h t l y , b u t v e r y c l o s e a g re e m e n t was o b t a i n e d i n s a m p le s fro m t h e same j a r . The r a n g e was from 2 . 2 5 P©r c e n t t o 2 .3 5* w i t h t h e a v e r a g e 2 .2 9 P©r c e n t . PLAIT OP THE STUDY The c o n c e r n o f w o r k e r s i n t e r e s t e d i n d e m o n s t r a t i n g t h e e f f e c t o f g a s e o u s e n v ir o n m e n t h a s b e e n , c h i e f l y , w i t h th e p ro b le m o f how many s t r a i n s o f g o n o c o c c i i n a s e r i e s o f s p e c im e n s c o u l d b e i s o l a t e d u n d e r carbon d io x id e , a tm o sp h e ric a i r , o r o th e r c o n d itio n s . Prom t h e l i t e r a ­ t u r e i t a p p e a r s t h a t a w ide d i f f e r e n c e e x i s t s b e tw e e n t h e g r o w t h - s t i m u ­ l a t i n g p r o p e r t i e s o f carbon d io x id e and o th e r g aseo u s c o n d itio n s . T h e r e f o r e , no c l o s e e s t i m a t i o n o f t h e p o p u l a t i o n s m a i n t a i n e d b y th e d i f f e r e n t en v iro n m e n ts h as been n e c e ssa ry * Our p u m o s e , a s s t a t e d a t th e b e g i n n i n g o f t h e r a p e r , h a s b e e n to d e f i n e t h e o p t i m a l l i m i t s o f c e r b o n d i o x i d e s t i m u l a t i o n a s w e l l a s to i n v e s t i g a t e th e e f f e c t o f g a s e s o t h e r th an c a rb o n d io x id e . A p la te c o u n t a s a means o f a r r i v i n g a t t h e r e l a t i v e r o r y u l a t i o n s u s t a i n e d by d i f f e r e n t p e r c e n t a g e s o f c a r b o n d i o x i d e seemed a n e c e s s i t y . I t was f e l t t h a t t i i i s same m ethod o f e s t i m a t i o n c o u ld be e x t e n d e d to th e g ro w th o f g o n o c o c c i tin d e r r e d u c e d oxygen t e n s i o n and o t h e r c o n d i t i o n s . The d i f f i c u l t y o f c o u n t i n g an o r g a n is m w h ich c a n n o t b e t r e a t e d by t h e p o u r - p l a t e t e c h n i q u e was r e a l i z e d , a s w e l l a s t h e p o s s i b l e e r ­ r o r s o f th e d i l u t i o n m eth o d . However, o u r e x p e r i e n c e i n t h e c u l t u r e o f t h e g o n o c o c c u s h ad t a u g h t u s t h a t a c o u n t was o o s s i b l e on s u r f a c e p l a t i n g s , an d i t seemed r e a s o n a b l e to su n p o se t h a t any c o n s i d e r a b l e i - lU d i f f e r e n c e i n p o p u la tio n co u ld be d e te c te d . I t was r e a l i z e d t h a t to a r r i v e a t an e s t i m a t i o n o f t h e l i m i t s o f o p tim a l carbon d io x id e s tim u la tio n a f a i r l y la r g e p o p u la tio n o f g o n o c o c c i m u st b e p r e s e n t i n e a c h sp e c im e n . For th a t reason c u ltu re o f a c u t e c a s e s o n l y was p l a n n e d an d c a r r i e d o u t . The s t u d y r e s o l v e d i t s e l f i n t o t h e s e p a r t s : P art I C om parison o f t h e e f f e c t o f a t m o s n h e r i c s i r , oxygen t e n s i o n and c a r b o n d i o x i d e . F a rt la S tu d y o f th e e f f e c t o f m o i s t u r e on " a e r o b i c 11 s t r a i n s o f g o n o co cci. F o rt II D e t e r m i n a t i o n o f t h e o p ti m a l r a n g e o f c a r b o n d i o x i d e stim u la tio n . P a rt Ila E f f e c t o f c a r b o n d i o x i d e beyond t h e o p t i m a l r a n g e . P art III E f f e c t o f h y d ro g e n and c a r b o n m onoxide com pered w i t h a t m o s n h e r i c a i r , r e d u c e d oxygen t e n s i o n and c a r b o n d io x id e . P art I lia E f f e c t o f n i t r o g e n com pared w i t h a t m o s p h e r i c a i r , r e ­ d u c e d oxygen t e n s i o n an d c a r b o n d i o x i d e . PAST I . reduced COKPAF.I SO'" OF THE EFFECT OF AC-10 SPHERIC AIR, REDUCED 0XYGE17 TE17SIOI7 A17D CAH30U DIOXIDE The work o f ITeumann ( lH , 15) and W in k le r ( 1 7 ) d e m o n s t r a te d t h a t r e d u c e d oxygen t e n s i o n o b t a i n e d b y p a r t i a l vacuum c o n t r i b u t e s to t h e g r o w th o f some s t r a i n s o f g o n o c o c c i . The h i g h m o i s t u r e c o n t e n t o f th e s m a ll "C" p l a t e s u s e d by t h e s e w o rk e rs may a c c o u n t , i n p a r t , f o r t h e s u c c e s s o f t h i s method o v e r t h e a e r o b i c c o n d i t i o n . ( I t was n o t e d by Ueuman t h a t i n t h e open i n c u b a t o r g o n o c o c c i g-rew b e t t e r i n sto rm y w e a th e r th an i n c l e a r . ) I t i s n r o b a b l e t n e t a sm a ll amount o f c a r b o n d i o x i d e p r o d u c e d by a t t e n d a n t o r g a n is m s a l s o c o n t r i b u t e d to g r o w th i n t h e "C" p l a t e s . - 15 - B e fo re a t te m p ti n g to d e f in e th e l i m i t s o f o p tim a l c a r b o n -d io x id e s t i m u l a t i o n i n t h i s s tu d y i t was f e l t n e c e s s a r y t o o b s e r v e t h e e f f e c t o f r e d u c e d o x yg en t e n s i o n , s i n c e t h i s c o n d i t i o n n e c e s s a r i l y a c c o m p a n ie s e n r i c h m e n t w i t h c a r b o n d i o x i d e ; a l s o i t was d e s i r e a b l e to l e a r n t h e e f f e c t o f m o istu re , s in c e a h ig h e r m o is tu r e c o n te n t i s p r e s e n t i n th e c l o s e d c o n t a i n e r s t h a n i n t h e op en i n c u b a t o r . I t was j u d g e d t h a t i n c u b a t i o n u n d e r t h e f o l l o w i n g c o n d i t i o n s w ould p e r m i t a c o m p a r is o n o f t h e e f f e c t o f r e d u c e d o x y g en t e n s i o n a n d c a r b o n d io x id e te n s io n : C arbo n d i o x i d e , te n p e r c e n t, s u o n l i e d by t a n k . P a r t i a l vacuum e q u a l to n i n e cm. o f m e r c u ry on an open m an o m eter. ( T h i s r e d u c e d o x y gen t e n s i o n i s e a u i v a l e n t to t h e l o w e r e d t e n s i o n c r e a t e d by i n t r o d u c i n g t e n p e r c e n t c a r b o n d i o x i d e i n t o a c u l t u r e j a r b y th e m a n o m e te ric m ethod o f m e a s u r e m e n t . ) C arbon d i o x i d e , 2 .2 9 ± p e r c e n t , f u r n i s h e d by b u r n i n g a c a n d l e i n an a i r - t i g h t c o n t a i n e r . P a r t i a l vacuum e a u a l t o s e v e n cm. o f m e rc u ry on an o pen m an o m eter. ( I t was o b s e r v e d t h a t a p a r t i a l vacuum v a r y i n g fro m f o u r to s e v e n cm. o f m e r c u ry was c r e a t e d by t h e b u r n i n g o f a c a n d l e In a c lo se d j a r . The mean o f t h e s e f i g u r e s p l u s t h e 1 .5 cm. ( a p p r o x . } o f m ercu ry , r e p r e s e n t i n g 2 p e r c e n t carb o n d io x id e , e q u a l s s e v e n c m .) C arbon d i o x i d e f u r n i s h e d by b u r n i n g a c a n d l e i n a c o n t a i n e r whose n r e s s u r e came to e q u i l i b r i u m w i t h a t m o s p h e r i c a i r . A tm o s p h e r ic a i r . i-STHODS The d e s c r i p t i o n o f m a t e r i a l s , and t e c h n i q u e w h ic h f o l l o w s a p p l i e s . i - f o r th e m ost p a r t , 16 - to t h e e n t i r e s t u d y . Where a method a p p l i e s o n l y to P a r t I , m e n tio n h a s "been made o f t h e f a c t . MEDIA The c h o c o l a t e a g a r o f D ifco was u s e d a s t h e p l a t e medium. The i n g r e d i e n t s f o r t h e a g a r h a s e w ere; P r o t e o s e P e p to n e ^ 3 .........................................2 . 0 p e r c e n t E a c to -d e x tro ...................................... ............. 0 .05 p e r c e n t Sodium c h l o r i d e .................................................. 0 . 5 p e r c e n t D isodium p h o s p h a t e . . ......................................0 . 5 p e r c e n t Two gram s o f B a c to - h e m o g lo h in , a d e h y d r a t e d p r o d u c t from washed h e e f b l o o d c o r p u s c l e s , w ere d i s s o l v e d i n 100 c c . o f d i s t i l l e d w a t e r a t 50° C. The p a r t i a l s o l u t i o n was f i l t e r e d t h r o u g h c o a r s e m o is te n e d c h e e s e c l o t h to remove u n d i s s o l v e d p a r t i c l e s and th e n a u t o c l a v e d f o r 20 m in u te s a t 121° C* The a g a r "base a n d s t e r i l e 5 0 -5 5 ° C. and m ix ed . m a te ly 2© c c . s o l u t i o n o f h em o g lo b in w ere c o o l e d to P l a t e s were p o u r e d w i t h t h i s m i x t u r e , a p o r o x i - to t h e p l a t e . The c h o c o l a t e a g a r p l a t e s w ere a lw a y s n r e p e r e d on th e d ay th e y were to he i n o c u l a t e d . I t was f e l t t h a t t h e m o is t p l a t e s more t h a n comoen- s a t e d f o r o c c a s i o n a l c o n t a m i n a n ts i n t r o d u c e d d u r i n g p r e p a r a t i o n . D i l u t i o n medium; A s te rile two p e r c e n t s o l u t i o n o f p r o t e o s e - o e p t o n e #3 and 0 .5 o e r c e n t sodium c h l o r i d e w ere u s e d i n a l l d i l u t i o n s o f th e o r i ­ g i n a l in o cu lu m s u s p e n s i o n . C o l l e c t i o n medium; The pH o f t h i s s o l u t i o n was 7»3» S t e r i l e a s c i t i c f l u i d , u n d i l u t e d , was a d j u s t e d to a pH o f a p p r o x i m a te ly 6 .2 by b u b b l i n g Carbon d i o x i d e g a s th r o u g h i t . f l u i d was d i s t r i b u t e d i n 2 c c . am ounts i n s t e r i l e 3 x 3/ 8” t u b e s and t h e t u b e s w ere r u b b e r - s t o p p e r e d . The - C o l l e c t i o n Swaba: 17 - T hese w ere t h e u s u a l a b s o r b e n t c o t t o n swabs r o l l e d t i g h t l y on wooden a p p l i c a t o r s t i c k s . The amount o f c o t t o n was a b o u t o n e - f o u r t h t h a t u s e d I n t h e o r d i n a r y t h r o a t swab. I t was p o s s i b l e w i t h a s m a ll swab to e n t e r t h e m ale a n t e r i o r u r e t h r a w i t h o u t b r u s h i n g th e s u r f a c e o f th e g l a n s p e n i s . C o lle c tio n o f sp ecim en s: W ith t h e e x c e p t i o n o f one sp ecim en t h e m a t e r i a l was o b t a i n e d from m ale p a t i e n t s a t t h e V e n e r e a l D i s e a s e C l i n i c m a i n t a i n e d by t h e D e t r o i t D e p a rtm e n t o f H e a l t h , D e t r o i t , M ic h ig a n . I n o r d e r to o b t a i n a m o d erate to l a r g e p o p u l a t i o n o f g o n o c o c c i i n e a c h s p e c im e n , e x u d a te was ta k e n from a c u t e c a s e s o n l y . Male c a s e s o f g o n o r r h e a were s e l e c t e d s i n c e i t i s p o s s i b l e to i s o l a t e n e a r l y p u r e c u l t u r e s o f g o n o c o c c i from t h e e x u d a te i n t h e m a t e r i a l fro m a f e m a le th e v a g i n a l f l o r a f r e q u e n t l y o v e rg ro w s t h e g o n o c o c c i and makes an e n u m e r a tio n o f th e p o p u l a t i o n i m p o s s i b l e . C o l l e c t i o n o f sp e c im e n s was made by t h e a u t h o r i n t h e m ale d i a g ­ n o s tic c lin ic . A f t e r a d i a g n o s i s o f " a c u t e g o n o r r h e a " was p ro n o u n c e d by t h e e x a m in in g p h y s i c i a n , e x u d a t e was o b t a i n e d f ro m t h e p a t i e n t by c o t t o n swab and t h e swab w ashed o f f i n t h e c o l l e c t i o n medium o f a s c i t i c f l u i d . The swab was c a r e f u l l y p r e s s e d to t h e s i d e s o f t h e t u b e , t h e n d i s c a r d e d . The tu b e was l a b e l l e d w i t h t h e p a t i e n t ' s c l i n i c number and s to p p e r e d . From t h e C l i n i c L a b o r a t o r y r e s u l t s o f s l i d e e x a m i n a t i o n on e x u d a te from e a c h p a t i e n t w ere o b t a i n e d . Only sp e c im e n s from p c u t e c a s e s i n w hich a l a b o r a t o r y r e p o r t o f "Oram n e g a t i v e e x t r a - o r i n t r a - c e l l u l a r d i o l o c o c c i fo u n d " w ere r e t a i n e d f o r c u l t u r e . The specim en t u b e s were p a c k e d f o r t r a n s o o r t a t i o n i n a l a r g e therm os b o t t l e c o n t a i n i n g c r a c k e d i c e . S in c e t r a n s p o r t a t i o n from t h e V e n e r e a l D i s e a s e C l i n i c i n D e t r o i t to t h e l a b o r a t o r y i n L a n s in g i n v o l v e d a d r i v e o f two and a h a l f h o u r s . - IS t h e i c e d t h e r m o s " b o t t l e was f o u n d t o b e a n e c e s s i t y i n p r e v e n t i n g o v e r ­ growth o f secondary organism s i n th e c o l l e c t i o n f l u i d . t i c u l a r l y t r u e i n summer w e a t h e r . T h i s was p a r ­ O f t e n a ti m e i n t e r v a l o f s i x h o u r s e l a p s e d from th e talcing o f specimens to i n o c u l a t i o n o f p l a t e s . However, t h e number o f s e c o n d a r y o r g a n i s m s i n a s p e c i m e n was u s u a l l y s m a l l and t h e b a c t e r i a were k e p t i n a n e a r l y s t a t i c c o n d i t i o n . ITo e f f o r t was made to c h e c k o n t h e l o s s i n g o n o c o c c u s p o p u l a t i o n i n specimens t r a n s p o r t e d under t h e s e c o n d i t i o n s . A l o s s o c c u r r e d , un ­ d o u b t e d l y , b u t t h e o r i g i n a l number o f o r g a n i s m s i n p r a c t i c a l l y a l l s n e c i a e n s was s u f f i c i e n t l y g r e a t t o i n s u r e a c o u n t a f t e r c u l t u r e . D i l u t i o n and I n o c u l a t i o n o f Specim ens: Some i n d i c a t i o n o f t h e d i l u t i o n s to b e made on a p a r t i c u l a r s p e c i ­ men w e r e o b t a i n e d f r o m o b s e r v a t i o n o f t h e number o f o r g a n i s m s p r e s e n t i n s m e a r s ex a m i n e d a t t h e c l i n i c . A s p e c i m e n whose smear was h e a v y w i t h g o n o c o c c i was d i l u t e d more t h a n one w h i c h showed a few o r g a n i s m s per fie ld . Specim ens h e a v y w i t h g r a m - p o s i t i v e c o c c i o r b a c i l l i were s eld om c u l t u r e d . O c c a s i o n a l l y o b s e r v a t i o n o f s m e a r s was n o t p o s s i b l e an d o n l y t h e r e s u l t s o f m i c r o s c o p i c e x a m i n a t i o n c o u l d b e o b t a i n e d . I t was f o u n d b y e x p e r i m e n t t h a t t h e f o l l o w i n g r a n g e o f d i l u t i o n s would make p o s s i b l e a. c o u n t on p r a c t i c a l l y a l l soecimens: D i r e c t i n o c u l a t i o n fro m c o l l e c t i o n f l u i d ......................0 . 1 c c . i n o c u lu m 1 -1 0 d i l u t i o n .......................................................................................0.1 c c . in o c u l u m 1 -2 0 d i l u t i o n 0.1 c c . in o c u l u m 1-10 0 d i l u t i o n .................................................................................... 0.1 c c . in o c u l u m 1- 20C d i l u t i o n .................................................................................... 0.1 c c . i n o c u lu m I n m o st c a s e s 0 . 1 c c . in o c u lu m d i r e c t and fro m a 1 - 1 0 0 d i l u t i o n were s u f f i c i e n t to o b t a i n a s n a rs e p o p u l a t i o n f o r c o u n ts . The aim n o t 19 - - a l w a y s a c h i e v e d , was t o s e c u r e p l a t e s i n some d i l u t i o n w i t h a c o u n t l e s s t h e n 15 0 . B e c a u s e o f n u c o u s t h r e a d s an d p u s c e l l s p r e s e n t i n t h e e x u d a t e , t h e c o l l e c t i o n f l u i d h a d to b e s h a k e n v i g o r o u s l y to i n s u r e a n e v e n d i s t r i b u t i o n o f o r g a n i s m s an d e x t r a n e o u s m a t t e r . as fo llo w s, s i n c e t h e c o l l e c t i o n f l u i d was 2 c c . o r l e s s : 0.5 cc. ( c . f . ) U.5 c c . of d iluent * 1-10 0.2 cc. ( c . f . ) B.8 c c . o f d i l u e n t » 1-20 0.1 c c . ( c . f . ) -+ 9.9 c c . o f d i l u e n t U p o . (1— 100 d i l u t i o n ) Only D i l u t i o n s w e r e made ■*■ ■ * 1-100 U cc. of d ilu e n t on e s e t o f d i l u t i o n s was made p e r s p e c i m e n . a 1-100 d i l u t i o n , f o r example, » 1—200 T herefore, p l a t e s of to b e i n c u b a t e d u n d e r v a r i o u s a t m o s p h e r i c c o n d i t i o n s w e r e i n o c u l a t e d f r o m t h e same d i l u t i o n t u b e . Inoculation o f a. s e r i e s o f p l a t e s was made w i t h a 1 c c . p i p e t t e g r a d u a t e d i n t e n t h s . I t was fo \m o t h a t s t e r i l e b e n t g l a s s r o d s g a v e a s a t i s f a c t o r y spread of m a te r ia l. entire !To d i f f i c u l t : ' was e x p e r i e n c e d i n c o v e r i n g t h e s u r f a c e o f tlj.e s l i g h t l y m o i s t p l a t e s w i t h 0 .1 c c . i n o c u l u m . PHOV i s i o n POP DIFT'SRT-HT ATMOSPHERIC CONDITIONS, p a r t I P l a t e s to b e i n c u b a t e d a e r o b i c a l l y w e r e p l a c e d i n a l a r g e g l a s s d e s i c c a t o r whose t o n was n u s h e d s l i g h t l y to one s i d e to a l l o w ex ch an g e of a i r . P iv e hundred cc . o f w a te r were p la c e d i n the b o tto n compart­ ment o r d i n a r i l y o c c u p i e d b y t h e d e s i c c a n t . A T a y l o r "H u m id ig u id e " w h ic h h a d b e e n c a l i b r a t e d a g a i n s t wet an d d r y b u l b t h e r m o m e t e r s was used, to t e s t t h e a t r a o s u h e r e i n t h i s c o n t a i n e r . On r e p e a t e d t e s t s t h e r e l a t i v e h u m i d i t y was i n d i c a t e d a s 60 to 10 p e r c e n t . P l a t e s to b e i n c u b a t e d u n d e r p a r t i a l vacuum o r c a r b o n d i o x i d e were - 20 - p la c e d i n th e b r a e s j a r s d e s c rib e d under "gas a n a l y s i s . " One h u n d r e d f i f t y c c to two h u n d r e d c c . o f w a t e r was p l a c e d i n wide—m outh ed b o t t l e s in each of the c o n ta in e r s . I?o m e a s u re m e n t was made o f t h e r e l a t i v e hum idity i n the c lo s e d j a r s ; how ever, f r o m t h e l a r g e amount o f c o n d e n s a t i o n w h i c h was v i s i b l e on t h e i n n e r s i d e s and t o p s o f t h e j a r s follow ing in cu b atio n , it i s p ro b a b le th a t the m oisture co n c e n tra ­ t i o n was c l o s e to s a t u r a t i o n . One j a r was e n r i c h e d w i t h t e n p e r c e n t c a r b o n d i o x i d e by t h e method d e s c r i b e d by Leah;/ and C n r o e n t e r ( 1 2 ) . By means o f r u b b e r t u b i n g a t a n k o f m e d i c a l c a r b o n d i o x i d e , a m e r c u r y open U - t u b e ty p e o f manom eter , t h e c u l t u r e j a r an d t h e vacuum l i n e w e r e c o n n e c t e d to e a c h o t h e r i n t h e o r d e r named. A i r was e v a c u a t e d f r o m t h e j a r u n t i l t h e p r e s s u r e was r e d u c e d by n i n e cm. o f m e r c u r y ( r e m o v a l o f a p p r o x i m a t e l y 12 p e r c e n t of a i r ) . Carbon d i o x i d e was a l l o w e d to f l o w i n t o the j a r u n t i l the manometer r e a d i n g was 1 . 5 cm. b e lo w a t m o s p h e r i c p r e s s u r e . The p a r t i a l vacuum o f 1 .5 cm. a l l o w e d f o r g a s e x p a n s i o n a t i n c u b a t o r t e m p e r a t u r e . Two j a r s were p a r t i a l l y e v a c u a t e d u n t i l t h e p r e s s u r e s on t h e U - t u b e manometer w e r e , r e s p e c t i v e l y , n i n e and s even cm. o f m e r c u r y b e l o w t h a t of atm ospheric a i r . C a n d l e s were p l a c e d i n two j a r s , th e c a n d l e s l i g h t e d an d a l l o w e d to b u r n f i v e m i n u t e s b e f o r e t h e j a r l i d s were cla m p e d i n p l a c e . One j a . r was a i r - t i g h t an d t h e o t h e r h ad a slow l e a k a r o u n d t h e r u b b e r g a s k e t w h ic h p e r m i t t e d a g r a d u a l a d j u s t m e n t o f p r e s s u r e e q u i l i b r i u m w i t h t h e outside a i r . B o t h j a r s were t e s t e d on t h e manometer a f t e r i n c u b a t i o n . A p a r t i a l vacuum o f f o u r to se ve n cm. o f m e r c u r y c o u l d a l w a y s be demon­ s t r a t e d on t h e a i r - t i g h t c o n t a i n e r . In cu b atio n tem peratur e . Thro u g h o u t t h e s t u d y n i l c u l t u r e s were i n c u ­ b a t e d f o r a p p r o x i m a t e l y Us h o u r s a t a t e m p e r a t u r e v a r y i n g from 35 to 36° C . , a c c o r d i n g to t h e t e m p e r a t u r e l i m i t s a d v i s e d b y C a r p e n t e r ( 1 9 ) . f l u c t u a t i o n s i n t e m p e r a t u r e were m arked b y a r e c o r d i n g t h e r m o m e t e r , a nd a. c h e c k was made a t i n t e r v a l s w i t h a m e r c u r y t h e r m o m e t e r . z ::a:.;i ::a t i o :; o r TH3 p l a t e s following i :: ctj3 a t i o :: A n o r o x i m a t e l y hS h o u r s a f t e r i n o c u l a t i o n o f t h e p l a t e s , were o n en ed and t h e n l a t e s ex am ined f o r g o n o c o c c u s c o l o n i e s . u s u a l l y c o n s i s t e d n e a r l y e x c l u s i v e l y o f convex, with u n d u la te d m argins, the j a r s Orowth transparent colonies t y p i c a l of the gonococcus. Colonies o f secondary o r g a n i s m s were r e l a t i v e l y few and t h e s e were u s u a l l y e t a o h y l o c o c c i , / - r e e n - 'o r o d u c i n g s t r e o t o c o c c i o r c o l i f o r n b a c t e r i a . P r o t e u s was encoun­ t e r e d on a few p l a t e s . All plates w ere s p r a y e d by a. D e V i l b i s s " a t o m i z e r " w i t h a 1 p e r c e n t s o l u t i o n of p a r a .- a m in o - d im e th y l- a n ilin e m onohydrochloride (E astm an). E x c e s s dye s o l u t i o n was d r a i n e d f r o m t h e p l a t e s . T h is dye component i n th e p r e s e n c e o f t h e o x i d a s e o f t h e gonococcus produces a p in k colony. and. f i n a l l y b l a c k . On f u r t h e r o x i d a t i o n t h e c o l o r becomes maroon The s p e e d o f t h e c o l o r ch an g e i s n o t c o n s t a n t w i t h d i f f e r e n t s t r a i n s o f g o n o c o c c i b u t t h e s h i f t fro m p i n k to nn.roon to b l a c k u s u a l l y t a k e s p l a c e w i t h i n 12 to 13 m i n u t e s w i t h a l l s t r a i n s . O r g a n i s m s i n a c o l o n y a r e v i a b l e a t t h e p i n k s t a g e b u t n o n - v i a b l e when o x i d a t i o n o f t h e dye i s c o m p l e t e . s u b s e q u e n t Gram s t a i n s . The dye does n o t i n t e r f e r e w i t h The d i r e c t o x i d a s e r e a c t i o n w i t h p n r a - a m i n o - d i u e t h y l - a n i l i n e m o n o h y d r o c h l o r i d e i s n o t p e c u l i a r to t h e gonococcus b u t i s giver, by t h e whole U e i s s e r i a g e n u s , a s w e l l a s Hemophilus - 2 2 - in flu en z ae, c e r ta in gram -positive d ir> lo b acilli, t h e c h o l e r a v i b r i o and membere o f t h e a l k a l i g e n e s g r o u p ( 1 1 , 12, 1 9 ) . Gronau ( 2 2 ) m e n t i o n s c o l o n i e s o f c e r t a i n d i p h t h e r o i d - l i k e o r g a n i s m s which g i v e an o x i d a s e p o sitiv e reaction. g rayish black. The c o l o r o f t h e c o l o n y a f t e r o x i d a t i o n i s a c h a l k y , O r g a n is m s s i m i l a r to t h e s e were f o u n d i n t h i s s t u d y b u t were e a s i l y d i f f e r e n t i a t e d from gon oco cc us, g r o w t h . A gram s t a i n was made o f two o x i d a s e - p o s i t i v e c o l o n i e s on e a c h plate se le c te d f o r counting. The p r e p a r a t i o n s were examined u n d e r th e microscope f o r g ra m -n e g a tiv e , c o ffe e -b e a n -s h a p e d d ip lo c o c c i. A c o u n t was made o f t y p i c a l o x i d a s e - p o s i t i v e c o l o n i e s on p l a t e s which h a d b e e n d e t e r m i n e d by m a c r o s c o p i c e x a m i n a t i o n o f t h e c o l o n i e s an d gram s t a i n t o c o n t a i n g o n o c o c c i . Wherever p o s s i b l e a c o u n t was made o f e v e r y p l a t e i n a d i l u t i o n s e r i e s , p r o v i d e d t h e g r o w th was n o t cr ow d ed. Crowded p l a t e s w i t h a c o u n t above 200 were c o n s i d e r e d " i n ­ n u m e r a b l e ” i f h i g h e r d i l u t i o n s i n t h e same s e r i e s were c o u n t a b l e . It i s p o s s i b l e t h a t o c c a s io n a l c o lo n ie s o f b a c t e r i a o t h e r than gonococci were c o u n t e d b y t h i s a l l - i n c l u s i v e method. However, from t h e v e r y few s t a i n e d s l i d e p r e p a r a t i o n s w hich c o n t a i n e d o t h e r o r g a n i s m s t h a n gramn e g a t i v e d i p l o c o c c i , i t i s p r o b a b l e t h a t e r r o r s o f t h i s k i n d were few. C a r o e n t e r ’ s e x p e r i e n c e would l e a d u s t o b e l i e v e t h a t t h e o c c u r r e n c e o f any o t h e r H e i s s e r i a t h a n th e g o n o c o c c u s i n c u l t u r e s from t h e male u reth ra is rare. A c c o r d i n g t o him ( 1 9 ) . " I t i s s i g n i f i c a n t t h a t d u r i n g t h e n i n e - y e a r n e r i o d t h e a u t h o r h a s made c u l t u r e s o f g o n o c o c c u s , o n l y t h r e e c u l t u r e s o f " e i s s e r i s o t h e r t h a n gonococcus have b een r e c o v e r e d fro m t h e l o w e r b i r t h c a n a l a n d from t n e g e n i t o —u r i n a r y t r a c t o f men." R e c e n t l y C a m e n t e r h a s had to am e n d t h a t s t a t e m e n t somewhat. Carpenter - 23 - an d C h a r l e s ( 2 3 ) h a v e r e p o r t e d i s o l a t i o n , o f m e n i n g o c o c c i fro m t h e g e n i t o ­ u r i n a r y t r a c t o f s e v e n p a t i e n t s w i t h c l i n i c a l symptoms o f g o n o r r h e a . These s t r a i n s a r e t h e f i r s t t o "be r e p o r t e d i n t h e l i t e r a t u r e f r o m such a s o u r c e , an d i t i s f e l t "by t h e a u t h o r s t h a t t h e i r o c c u r r e n c e i s r a r e . Comparison o f C o u n ts (M et hods) The f i g u r e s i n T a b le 1 a r e t a k e n from c o u n t s made on c u l t u r e s one, two, and f o u r ( 7 - 3 1 - U l ) and a r e r e p r e s e n t a t i v e o f t h e r e s u l t s i n P a r t I . - 2h - :?o e f f o r t was made i n t o t a l l i n g t h e c o u n t s to a r r i v e a t t h e number o f o r g a n i s m s grown f r o m 1 .0 c c . o f i n o c u l u m , a s t h e c o n v e n t i o n a l c o u n t i s made f r o m p o u r e d p l a t e s . A c o m p a r i s o n o f n u m b e r s o n l y was s o u g h t i n whatever d i l u t i o n p e r m itte d a com parison. c o u n t was e a s i l y o b t a i n e d f r o m C u l t u r e # 1 . For exam ple, th e c o m p a ra tiv e The number o f g o n o c o c c i o b s e r v e d on t h e v a r i o u s " d i r e c t ” p l a t e s was t a k e n a s r e p r e s e n t a t i v e o f t h e r e a c t i o n o f C u l t u r e #1 to d i f f e r e n t a t m o s p h e r i c c o n d i t i o n s . The nu m b e r s H, 9 8 * 1 5 i 9 0 > 1 8 , and 21 were a s s i g n e d t o t h e r e s p e c t i v e c o n d i ­ tio n s. A c o m p a r a t i v e c o u n t f o r C u l t u r e j*2 was more d i f f i c u l t . The a e r o b i c g r o w t h was z e r o ; g r o w t h on t h e t h r e e c a r b o n d i o x i d e c o n d i t i o n s c o u l d b e c o m p a r e d r e a d i l y ; a. c o m p a r i s o n o f t h e c o u n t s o n t h e p a r t i a l vacuum p l a t e s was e a s i l y made. However, a c o m p a r i s o n o f " p a r t i a l vacuum" c o u n t s a n d t h o s e o n t h e c a r b o n d i o x i d e p l a t e s c o u l d b e made o n l y b y a p p r o x i m a t i n g t h e " i n n u m e r a b l e " c a r b o n d i o x i d e p l a t e f r o m i t s c o u n t on a h i g h e r d i l u t i o n . A c o m p a r a t i v e c o u n t f o r c u l t u r e f o u r was e a s i l y mode s i n c e t h e o r g a n i s m s gr ew u n d e r c a r b o n d i o x i d e o n l y . on t h e 1 -10 d i l u t i o n p l a t e s . C o m parison c o u l d b e o b t a i n e d R e su lts o f P a r t I Out o f some e i g h t y c u l t u r e s w hic h gre w u n d e r one o r more o f t h e c o n d i t i o n s r e c o r d e d i n T a b l e two, 6s were s u i t a b l e f o r c o u n t i n g . Of t h e 68 s t r a i n s c o n s i d e r e d , a l l grew u n d e r t e n p e r c e n t c a r b o n d i o x i d e an d 67 grew u n d e r c a r b o n d i o x i d e s u p p l i e d by c a n d l e i n an a i r - t i g h t container. The one s t r a i n w h ic h g r e w u n d e r t e n p e r c e n t c a r b o n d i o x i d e an d n o t u n d e r " c a n d l e " was fro m a c l i n i c a l c a s e o f g o n o r r h e a . In tracel l u l a r g r a m - n e g a t i v e d i p l o c o c c i were p r e s e n t on a s l i d e t a k e n a t t h e t i n e e x u d a t e was o b t a i n e d from t h e p a t i e n t f o r c u l t u r e . Two o x i d a s e - t j o s i t i v e c o l o n i e s o n l y were p r e s e n t on t h e c u l t u r e p l a t e s . - 26 - On ly 5S 8t r a i n s d e v e l o p e d i n t h e " o p e n 11 c o n t a i n e r i n w h ic h a c a n d l e ha d b e e n b u r n e d . I t was hoped t h a t t h i s c o n d i t i o n i n which c a r b o n d i o x i d e was p r e s e n t , "but no p a r t i a l vacuum, would i n d i c a t e t h e p a r t p l a y e d by t h e f o u r to s e v e n cm. p a r t i a l vacuum p r e s e n t i n an a i r - t i g h t candle j a r . The r e s u l t s were i n c o n c l u s i v e , since i t i s p ro b ab le th a t l o s s o f c a r b o n d i o x i d e th r o i i g h g a s ex ch an g e was r e s p o n s i b l e f o r t h e c o m p a r a t i v e l y moor r e s u l t s o f th e "onen" c o n t a i n e r , r a t h e r t h a n h e i g h t ­ en ed oxygen t e n s i o n . B e t t e r r e s u l t s were o b t a i n e d u n d e r n a r t i a l vacuum t h a n by a e r o b i c incubation. The number o f s t r a i n s i s o l a t e d "under t h e f o r m e r c o n d i t i o n was g r e a t e r by o n l y f o u r th a n t h e number grown a e r o b i c a l l y , b u t t h e d i f f e r e n c e i n p o o u l a t i o n was c o n s i d e r a b l e . On t h e s e c o n d l i n e o f t h e t a b l e a r e r e c o r d e d t h e c o m p a r a t i v e popu­ l a t i o n s o f 3^ s t r a i n s w h i c h grew a e r o b i c a l l y an d u n d e r a l l . o f conditions. i t s reading. the o th e r The c o m p a r i s o n h a s b e e n made i n p e r c e n t a g e s to f a c i l i t a t e The t o t a l p o p u l a t i o n o b t a i n e d u n d e r t e n p e r c e n t c a r b o n d i o x i d e h a s b e e n g i v e n t h e a r b i t r a r y v a l u e o f 1 0 0 , s i n c e g r o w t h was b e s t under th is condition. The p e r c e n t a g e v a l u e s o f t h e o t h e r c o u n t s we re o b t a i n e d , o f c o u r s e , by d i v i d i n g t h e t o t a l c o u n t s o f 3? s t r a i n s grown a e r o b i c a l l y , e t c . , by t h e t o t a l c o u n t o b t a i n e d u n d e r t e n p e r c e n t c a r b o n d i o x i d e and m u l t i p l y i n g by 100 . I t w i l l b e n o t e d t h a t g r ow th s t i m u l a t i o n by th e v a r i o u s c o n d i t i o n s was i n t h e f o l l o w i n g d e s c e n d i n g order: ten p e r c e n t carbon d io x id e , c an d le in a i r - t i g h t c o n ta in e r , c a n d l e i n "open" c o n t a i n e r , p a r t i a l vacuum 7 c m ., p a r t i a l vacuum 9 cm., aerobic condition. first The d i f f e r e n c e b e tw e e n th e g r o w th s u p p o r t e d by the two c o n d i t i o n s an d t h e l a s t f o u r was v e r y g r e a t . - 27 - On l i n e t h r e e a c o m p a r i s o n i s made o f U2 s t r a i n s w h i c h gr ew u n d e r r e d u c e d o xygen t e n s i o n , te n p e r c e n t carbon d io x id e , and carbon dio x id e f u r n i s h e d by c a n d l e i n t h e a i r - t i g h t c o n t a i n e r . A g a in t h e p o p u l a t i o n u n d e r t e n p e r c e n t c a r b o n d i o x i d e was g r e a t e s t , w i t h t h e c a n d l e s u p p l y ­ ing n e a r l y the sane growth s t i m u l a t i o n . Growth u n d e r r e d u c e d oxygen t e n s i o n was l e s s by n e a r l y 70 p e r c e n t t h a n t h a t p r o d u c e d •under c a r b o n dioxide. F i n a l l y a c o m p a r i s o n i s made o f t h e g r o w t h o b t a i n e d t in d e r t e n p e r c e n t c a r b o n d i o x i d e an d t h e c a n d l e i n " c l o s e d " c o n t a i n e r . o f the p r e v i o u s o r d e r w i l l be n o te d , A reversal p r o b a b l y due to t h e c h a n c e o f random s a m p l i n g , g r o w t h was g r e a t e r f o r Gf s t r a i n s u n d e r c a n d l e c o n d i ­ t i o n s t h a n f o r 6S s t r a i n s u n d e r t e n p e r c e n t c a r b o n d i o x i d e . PAHT IA The p o o r g r o w t h o f g o n o c o c c i u n d e r t h e a e r o b i c c o n d i t i o n i n t h e p r e c e d i n g e x p e r i m e n t may be a t t r i b u t e d i n p a r t t o a t m o s p h e r e a n d i n p a r t to th e m o is tu r e c o n c e n t r a t i o n o f t h a t a tm o sp h e re . I n an e f f o r t to d e t e r m i n e t h e e f f e c t o f m o i s t u r e , a s e r i e s o f s p e c i m e n s was c u l t u r e d In d u p lic a te under the follow ing c o n d itio n s: (1) The a e r o b i c c o n d i t i o n o f P a r t I , r e l a t i v e h u m i d i t y 6 0 -70 per cent (2) A tm ospheric a i r i n a c lo s e d c o n t a i n e r , r e l a t i v e hum idity a p p r o x i m a t e l y 100 p e r c e n t (3) Ten p e r c e n t c a r b o n d i o x i d e , s u p p l i e d by t a n k , r e l a t i v e h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t The c a r b o n d i o x i d e a t m o s p h e r e was i n c l u d e d o n l y f o r p u r p o s e s o f com parison. I t was r e c o g n i z e d t h a t t h e c o n d i t i o n s o f ( 2 ) c o u l d n o t be - 28 - c a l l e d t r u e a e r o b i c ) s i s , s i n c e g r o w t h o f g o n o c o c c i a n d any s e c o n d a r y o r g a n i s m s i n t h e s p e c i m e n s would a l t e r t h e c a r b o n d i o x i d e an d oxygen tensions. However, i n a l a r g e c o n t a i n e r t h i s e f f e c t i s p r o b a b l y s m all and o b t a i n s i n an y o f t h e c a r b o n d i o x i d e e n r i c h e d o r r e d u c e d oxygen tension conditions. A t o t a l o f 2U s p e c im e n s were c u l t u r e d , 13 o f which w e r e a e r o b i c . T he 13 s t r a i n s a r e c o n s i d e r e d i n T a ble 3» D u p l i c a t e p l a t i n g s were made o f u n d i l u t e d and d i l u t e d c o l l e c t i o n f l u i d u n d e r e a c h c o n d i t i o n . The in oculum c o n s i s t e d o f 0 .1 c c . o f u n d i l u t e d c o l l e c t i o n f l u i d an d 0.1 c c . of a 1-100 d i l u t i o n . Counts on t h e d u p l i c a t e p l a t e s were made whenever g r o w t h was s u f f i c i e n t l y s p a r s e f o r a c o u n t . - 29 - W it h a l l 13 s t r a i n s o f g o n o c o c c i a g r e a t e r p o p u l a t i o n was o b t a i n e d i n t h e s a t u r a t e d a t m o s p h e r e t h a n i n t h e 60-70 p e r c e n t r e l a t i v e h u m i d i t y . The e f f e c t o f m o i s t u r e a p p a r e n t l y v a r i e d w i t h t h e s t r a i n . F o r example, w i t h c u l t u r e *1 t h e g r o w t h was many t i m e s g r e a t e r u n d e r a s a t u r a t e d atm osphere than u n d e r 60-70 p e r c e n t r e l a t i v e h u m id ity . With c u l t u r e #8 t h e g r o w t h was n o t m a r k e d l y d i f f e r e n t u n d e r t h e two c o n d i t i o n s . Zven t h o u g h t h e 13 s t r a i n s were a e r o b i c , t h e g r o w t h was i n e v e r y i n s t a n c e g r e a t e r u n d e r c a r b o n d i o x i d e t h a n on t h e a e r o b i c - p l a t e s . If f u r t h e r c o n f i r m a t i o n o f t h e b e n e f i c i a l e f f e c t o f c a r b o n d i o x i d e were needed, t h i s p r o t o c o l w ould a p p e a r t o s u p p l y i t . - FAST I I . OPTIMAL LIMITS OF CAKBOh DIOXIDE STIMULATION The r e s u l t s i n P a r t I c o r r e s p o n d to t h o s e o f C h r i s t e n s e n a n d S c h o e n l e i n ( 2 ) i n t h a t t h e number o f s t r a i n s o f g o n o c o c c i i s o l a t e d u n d e r carbon d i o x i d e s u p p lie d by c a n d le i s ab ou t t h e same a s th e number i s o l a ­ te d under ten p e r c e n t carbon d io x id e . In o u r e x p e r i m e n t t h e amount o f grov rth o b t a i n e d by b o t h m e th o d s was p r a c t i c a l l y t h e same. appear, therefore, I t would t h a t one ma.y s u p p l y c a r b o n d i o x i d e i n p e r c e n t a g e s v a r y i n g f r o m a b o u t two to t e n p e r c e n t and e x p e c t g r o w t h to b e t h e same under a l l ten sio n s. I n an e x p e r i m e n t to d e f i n e t h e o p t i m a l l i m i t s o f c a r b o n d i o x i d e stim ulation, two p e r c e n t c a r b o n d i o x i d e sup f i l e d by t a n k was c h o s e n a s t h e s m a l l e s t amount o f g a s to b e t e s t e d and 22 p e r c e n t was s e l e c t e d a s t h e maximum. S n r i c h m e n t by c a r b o n d i o x i d e o f t h e same o r s l i g h t l y l e s s t e n s i o n t h a n t h a t s u p p l i e d by c a n d l e was f e l t d e s i r a b l e , since o th e r g a s e s t h a n c a r b o n d i o x i d e a r e p r o d u c e d by b u r n i n g o f a c a n d l e , p a r t i ­ c u l a r l y carbon monoxide. Twenty—two p e r c e n t c a r b o n d i o x i d e wa6 c n o s e n - 30 - a s t h e maximum t e n s i o n s i m p l y a s a c o n v e n i e n t l i m i t f o r p r e l i m i n a r y t e s t s . The f o l l o w i n g a t m o s p h e r i c c o n d i t i o n s were p r o v i d e d : (1) A t m o s p h e r i c a i r i n open c o n t a i n e r s i m i l a r to t h a t u s e d f o r a e ro b ic growth in P a r t I . R e l a t i v e h u m i d i t y , 60-70 p e r c e n t . (2) Carbon d i o x i d e s u p p l i e d b y c a n d l e i n an a i r - t i g h t c o n t a i n e r . R e l a t i v e h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . (?) Carbon d i o x i d e , two u e r c e n t s u p p l i e d b y t a n k , h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . R elative (*0 Carbon d i o x i d e , s i x o e r c e n t s u p p l i e d by t a n k , h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . R elative (5) Carbon d i o x i d e , 10 p e r c e n t s u p p l i e d by t a n k , h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . (6) Carbon d i o x i d e lU p e r c e n t s u p p l i e d by t a n k , a p p r o x i m a t e l y 100 p e r c e n t . (7) Carbon d i o x i d e , I S p e r c e n t s u p p l i e d by t a n k , hum idity a p p r o x im a te ly 100 p e r c e n t . R elative (8) Carbon d i o x i d e , 22 p e r c e n t s u p p l i e d by t a n k , h u m i d i t y a p p r o x i m a t e l y 10 0 p e r c e n t . R elative R elative R e la t i v e hum idity M ea su re m en t o f t h e c a r b o n d i o x i d e s u p p l i e d by t a n k was b y t h e manom e t r i c method d e s c r i b e d u n d e r " M e t h o d s . " C a l c u l a t i o n o f t h e amount o f a i r to e v a c u a t e f ro m t h e j a r s was made a s f o l l o w s : F o r a f i n a l l h p e r c e n t e n r i c h m e n t o f c a r b o n d i o x i d e , f o r example: 760 mm. x O.lU s 106 mm. o r 1 0 . 6 cm. 1 0 . 6 cm. ♦ - I .5 cm. ( p a r t i a l vacuum to p r o v i d e f o r e x p a n s i o n ) a 1 2 . 1 cm. A i r was e v a c u a t e d u n t i l t h e p r e s s u r e was r e d u c e d by 12 cm. o f mer cu ry t h e n g a s was a l l o w e d to f l o w i n - u n t i l t h e manometer r e a d i n g was 1 . 5 cm. b e lo w a t m o s p h e r i c p r e s s u r e . l a s a n a l y s e s w e r e made on j a r s f i l l e d by t h i s method w i t h amounts o f c a r b o n d i o x i d e v a r y i n g f ro m two to 22 p e r c e n t . Ho c o r r e c t i o n s were made f o r no rmal t e m o e r a t u r e an d p r e s s u r e a t t h e ti m e o f f i l l i n g . The a n a l y s e s d e m o n s t r a t e d t h a t t h e a c t u a l amount o f g a s p r e s e n t v a r i e d a s much a s 0 . 5 to 1 . 0 p e r c e n t fro m t h e c a l c u l a t e d am ou nt. T h i s was n o t f e l t to b e a - serious e rro r, 31 - s i n c e a wide r a n g e o f c a r b o n d i o x i d e t e n s i o n was u n d e r t e s t f u r t h e r m o r e , a d i f f e r e n c e o f f o u r p e r c e n t e x i s t e d between each c o n d i t i o n . The m eth o d s an d t e c h n i q u e o f P a r t I were em p lo y ed . d i l u t i o n s was made f r o m e a c h s p e c i m e n . A single set of E i g h t p l a t e s were i n o c u l a t e d d i r e c t l y f r o m t h e c o l l e c t i o n f l u i d w i t h 0 .1 c c . in o c u l u m , and e i g h t p l a t e s were i n o c u l a t e d fro m e a c h o f t h e d i l u t i o n s . The d u p l i c a t e p l a t e s were m ar ked w i t h s p ecim e n number, d i l u t i o n and a t m o s p h e r i c c o n d i t i o n and p la c e d i n t h e i r ' r e s p e c t i v e j a r s . The p l a t e s a f t e r e x a m i n a t i o n f o r g o n o c o c c i were c o u n t e d a s i n P a r t I . A c o m p a r a t i v e c o u n t o n l y was s o u g h t . The r e s u l t s a r e t a b u l a t e d i n Table U - 32 - A s u r p r i s i n g u n i f o r m i t y w i l l b e n o t e d i n t h e t o t a l c o u n t s an d th e number o f s t r a i n s grown u n d e r t h e f o l l o w i n g c o n d i t i o n s : "Candle” , s ix p e r c e n t c a r b o n d i o x i d e , t e n p e r c e n t c a r b o n d i o x i d e , lH p e r c e n t c a r b o n d i o x i d e , an d I S p e r c e n t c a r b o n d i o x i d e . At t h e e x t r e m e s , u n d e r at m os­ p h e r e s o f two p e r c e n t and 22 p e r c e n t c a r b o n d i o x i d e , less than under the o th e r carbon d io x id e te n s io n s . t h e c o u n t was Growth u n d e r t h e a e r o b i c c o n d i t i o n was so much l e s s t h a n on d u p l i c a t e p l a t e s grown u n d e r c a r b o n d i o x i d e t h a t no a t t e m p t was made to a r r i v e a t a c o m p a r i s o n i n population. The h e a v i e s t g r o w t h was o b t a i n e d on p l a t e s i n c u b a t e d i n " C a n d l e , " and t e n p e r c e n t c a r b o n d i o x i d e a t m o s p h e r e s , p r o b a b l y due to chance in sampling. O nly t h i r t y - o n e s t r a i n s were i s o l a t e d u n d er te n p e r c e n t c a r b o n d i o x i d e , y e t t h e t o t a l g r o w t h was g r e a t e r t h a n i n p l a t e s i n c u b a t e d u n d e r any o t h e r c a r b o n d i o x i d e t e n s i o n s b u t t h a t p r o v i d e d by candle. Whe th er o r n o t an y s i g n i f i c a n c e m ig h t b e a t t a c h e d to t h e l o w e r c o u n t s o b t a i n e d u n d e r two p e r c e n t c a r b o n d i o x i d e and 22 p e r c e n t c a r b o n d i o x i d e c o u l d be d e t e r m i n e d o n l y by f u r t h e r c u l t u r e work. I t was d e c i d e d t h a t a w i d e r r a n g e o f c a r b o n d i o x i d e t e n s i o n s m ig h t a i d i n i n d i c a t i n g a t what t e n s i o n i n h i b i t i o n o c c u r s and s t i m u l a t i o n c e a s e s . For th e second s e r i e s o f P a r t I I , t h e f o l l o w i n g c o n d i t i o n s were provided: (1) A t m o s p h e r i c a i r i n an open c o n t a i n e r s i m i l a r to t h a t u s e d f o r a e r o b ic growth i n P a r t I . R e l a t i v e h u m i d i t y , 60 to 70 p e r c e n t , (2) Carbon d i o x i d e s u p p l i e d by c a n d l e i n an a i r - t i g h t c o n t a i n e r . R e l a t i v e h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . (■=0 Carbon d i o x i d e , two p e r c e n t s u p p l i e d by t a n k . h u m i d i t y a p n r o x i m a t e l y 100 p e r c e n t . R.elative (U) Carbon d i o x i d e , s i x tier c e n t s \ i n p l i e d by t a n k . h u m i d i t y a p p r o x i m a t e l y 100 n e r c e n t . R elative - 33 - (5) Carb on d i o x i d e , 10 p e r c e n t s u p p l i e d b y h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . tank. R elative ( 6) Carbon d i o x i d e , l h p e r c e n t s u p p l i e d by t a n k . h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t R elative (7) Ca rbon d i o x i d e , 18 p e r c e n t s u p p l i e d b y h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . tank. R elative (8) C ar bo n d i o x i d e , 22 p e r c e n t s u p p l i e d by t a n k , h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . R elative (9) C ar bo n d i o x i d e , 26 p e r c e n t s u p p l i e d by t a n k . h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . R elative (10) Carbo n d i o x i d e , 30 P®r c e u t s u p p l i e d by t a n k , h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . R elative I n t h e s e c o n d s e r i e s , 27 s t r a i n s o f g o n o c o c c i were c o u n t a b l e on some o r a l l o f t h e p l a t e s . The r e s u l t s a r e g i v e n i n T a b le ( A e r o b i c g r o w t h , a s u s u a l , was c o n s i d e r a b l y l e s s th a n g r o w t h u n d e r carbon d io x id e. Only lH o f t h e 27 s t r a i n s u n d e r c o n s i d e r a t i o n grew aerobically. As i n th e p r e c e d i n g s e r i e s , f a i r l y u n i f o r m r e s u l t s were o b t a i n e d u n d e r " c a n d l e , " 6 p e r c e n t c a r b o n d i o x i d e , 10 p e r c e n t c a r b o n d i o x i d e , - 35 - lU p e r c e n t c a r b o n d i o x i d e , and 18 p e r c e n t c a r t o n d i o x i d e . The r e l a t i o n ­ ship o f the v a r io u s counts expressed i n p e rc e n ta g e s i s as fo llo w s : Carhon d i o x i d e , 10 p er c e n t ................................100 n er c e n t nCandle "..................................................... 96 Carton d i o x i d e , 6 ner c e n t .................................... 9*+ Carton d i o x i d e , IS p er c e n t ............................... 93 Carton d i o x i d e , lU p er c e n t ................................ 89 Carton d io x id e , 2 p e r c e n t . . . ............................. S? Carton d i o x i d e , 22 p er c e n t .......................... Carton d i o x i d e , 26 n er c e n t ................................ 65 Carton d io x i d e , ?0 per c e n t ................................ 62 79 Maximum g r ow th s t i m u l a t i o n was o t t a i n e d w i t h c a r t o n d i o x i d e t e n ­ s i o n s r a n g i n g fro m 2 . 3 to I S p e r c e n t ( v d t n t h e i n c l u s i o n o f t h e " c a n d l e ” condition). While 27 s t r a i n s grew u n d e r 22 p e r c e n t c a r t o n d i o x i d e i t w i l l t e n o t e d t h a t t h e p o o u l a t i o n o t t a i n e d u n d e r t h i s c o n d i t i o n was con­ s i d e r a b l y l e s s t h a n t h a t o f t h e c o n d i t i o n t e l o w i t i n the s e r i e s , 18 n e r cent carto n dioxide. I t would a n n e s r t h a t i n t h i s s e r i e s , a s i n s e r i e s 1 , s t i m u l a t i o n o f g o n o c o c c i d e c r e a s e d somewhat a t o v e th e t e n s i o n o f 18 p e r c e n t c a r t o n d i o x i d e and the d e c l i n e was s u f f i c i e n t l y marked to t e d e t e r m i n e d by n l a t e c o u n t a t a t e n s i o n o f 22 n e r c e n t c a r t o n d i o x i d e . The d e c l i n e i n g r ow th s t i m u l a t i o n , o r i n c r e a s e i n i n h i b i t i o n , was even more marked u n d e r 26 and 3° ? e r c e n t c a r t o n d i o x i d e . Wilson (2 4) has -pointed o u t t h a t to o b t a i n a smal l d i s t r i b u t i o n e r r o r i n t h e n l a t e c o u n t method, a d i l u t i o n g i v i n g t h e n r o p e r number of - 36 - c o l o n i e s p e r p l a t e (lO O -U cO ) must "be a v a i l a b l e i n o r d e r to minim iz e c r o w d i n g o f c o l o n i e s and e r r o r i n s a m p l i n g . W i l s o n ’ s o b s e r v a t i o n , w h i l e a p p l i c a b l e to p o u r e d p l a t e s i n w hich th e m a j o r p o r t i o n o f t h e g r o w t h i s s u b s u r f a c e , i s d i f f i c u l t t o a p p l y to t h e s u r f a c e - p l a t i n g method us ed i n t h i s s t u d y . The s i z e o f th e i n d i v i d u a l gon o co ccu s c o l o n y r a n g e s fro m 0 .5 mm. to 2 .0 mm. and some c r o w d in g o c c u r s w i t h a c o u n t above 1 5 0 . When t h e c o u n t i s above 200, c o n s i d e r a b l e cr o w d in g t a k e s p l a c e and an r m r o x i r a a t i o n o n l y can be made. I n t h e c o m p a r i s o n e x p e r i m e n t s o f P a r t I I , and t h r o u g h o u t t h i s stud.v , d i l u t i o n s h av e b e e n made w i t h t h e i n t e n t i o n o f p r o v i d i n g p l a t e s w i t h c o u n t s o f 150 o r l e s s : f i r s t to p e r m i t e a s y c o u n t i n g , to p e r m i t r e c o g n i t i o n o f t y p i c a l c o l o n i e s . secondly I n a b o u t 70 p e r c e n t o f th e c o u n t s th e number o f c o l o n i e s p e r n l a t e h a s b e e n 150 o r l e s s ; f r e ­ q u e n t l y t h e number h a s b e e n l e e s t h a n 100. W ilson and Kullman ( 25) d e t e r m i n e d t h a t f u r t h e r r e d u c t i o n o f d i s ­ t r i b u t i o n e r r o r may be made by p r o v i d i n g a s u f f i c i e n t number o f d u p l i ­ cate p la te s , t h r e e to f i v e . I t was r e c o g n i z e d by u s t h a t t h e s i n g l e p l a t e c o u n t i s s u b j e c t to e r r o r s , y e t s u f f i c i e n t l y a c c u r a t e to d e t e c t f a i r l y wide d i f f e r e n c e s i n population. I n o r d e r to t e s t t h e v a l i d i t y o f t h e i n d i c a t i o n s o b t a i n e d b y t h e s i n g l e p l a t e c o u n t a s to o p t i m a l r a n g e o f c a r b o n d i o x i d e stimu­ latio n , an e x p e r i m e n t was made i n w h i c h t h e s i g n i f i c a n t atir. oso he ri c c o n d i t i o n s o f P a r t I I were r e p e a t e d and f i v e d u p l i c a t e n l a t e s were in o c u la te d f o r each c o n d itio n . The e x u d a t e s f ro m s i x c a s e s o f a c u t e g o n o r r h e a were c u l t u r e d un der the fo llo w in g c o n d itio n s : - 37 C ar bo n d i o x i d e , 2 p e r c e n t , s u p p l i e d "by t a n k Carbon d io x id e , 10 p e r c e n t,s u p p lie d by tank Carbon d i o x i d e , 18 p e r C ar bon d i o x i d e , c e n t , s u p p l i e d by t a n k 22 p e r c e n t , s u p p l i e d by t a n k E x u d a t e s f ro m s i x o t h e r c a s e s o f a c u t e g o n o r r h e a w e r e c u l t u r e d under: Carbon d i o x i d e , 2 p e r c e n t , s u p p l i e d by t a n k C ar bon d i o x i d e , 10 p e r c e n t , su p-n lied by t a n k Carbon d i o x i d e , 18 p e r c e n t , s u p p l i e d by t a n k Carbon d i o x i d e , 22 p e r c e n t , s u p p l i e d by t a n k Carbo n d i o x i d e , J>0 p e r c e n t , s u p p l i e d by t a n k The r e l a t i v e h u m i d i t y i n e a c h c u l t u r e j a r was c l o s e to s a t u r a t i o n . As i n p r e v i o u s e x p e r i m e n t s one s e t o f d i l u t i o n s was made fro m e a c h s p e c i m e n to e l i m i n a t e e r r o r s which m i g h t a r i s e f r o m u s i n g a s e r i e s o f sim ilar d ilu tio n s, ’f i t h 11 s p e c i m e n s a 1:20 a n d 1:100 d i l u t i o n were made; w i t h one s p e c im e n d i r e c t in o c u l u m fro m t h e c o l l e c t i o n f l u i d and 1 : 2 0 d i l u t i o n were u t i l i z e d . A t o t a l o f 20 p l a t e s was i n o c u l a t e d from e a c h d i l u t i o n t u b e , o r t h e c o l l e c t i o n f l u i d , an d t h e in o c u l u m s p r e a d by g l a s s r o d . I n T a b l e G t h e a v e r a g e o f t h e c o u n t s fro m f i v e p l a t e s i s r e c o r d e d under each co n d itio n . The f i g u r e s g i v e n a r e t h e a v e r a g e o f t h e c o u n t i n one d i l u t i o n o n l y , i n s e v e n i n s t a n c e s t h e 1:100 d i l u t i o n , i n s t a n c e s t h e 1 :20 d i l u t i o n . in fiv e - 38 - In the above ta b le , s e r ie s 3, the to ta l counts and p ercen tag e com­ p a ris o n s 'indicate'-""that s ix c u ltu re s o f gonococci grew uniform ly under two p e r c e n t, ten p e r c e n t, and 18 p er c e n t carbon d io x id e . Growth was con­ s id e ra b ly l e s s under 22 p e r ce n t carbon dioxide than under the o th e r tension - 39 The s i x c u l t u r e s c o n s i d e r e d i n T a b le 6 , s e r i e s U, grew b e s t tinder 18 p e r c e n t c a r b o n d i o x i d e a n d 10 p e r c e n t c a r b o n d i o x i d e . A decided d r o p i n p o p u l a t i o n o c c u r r e d u n d e r two p e r c e n t an d 22 p e r c e n t c a r b o n dioxide. The d r o p i n p o p u l a t i o n was e v e n more n r o n o u n c e d u n d e r 30 P®r cen t carbon d io x id e . These r e s u l t s f o l l o w c l o s e l y t h o s e o b t a i n e d w i t h the s i n g l e - p l a t e c o u n ts. A com parison o f the p e r c e n ta g e r e l a t i o n s h i p s between the t o t a l c o u n t s o b t a i n e d by t h e s i n g l e —p l a t e a n d f i v e - p l a t e c o u n t meth od s b r i n g s o u t c l e a r l y a g r e e m e n t an d d i s c r e p a n c y . The c o m p a r i s o n i s g i v e n i n Table 1 . An ex a m in a tio n o f Table 7 w i l l r e v e a l t h a t , w ith one e x c e p t io n , the c o u n ts o b ta in e d by s i n g l e - p l a t e tech n iq u e were con firm ed by the f i v e p l a t e m ethod. The e x c e p tio n w i l l be fou n d in T able o, s e r i e s 3» under - 1+0 two p e r c e n t c a r b o n d i o x i d e . Growth u n d e r two p e r c e n t c a r b o n d i o x i d e s l i g h t l y e x c e e d e d i n t h i s one s e r i e s g r o w t h u n d e r t e n p e r c e n t and 18 p e r c e n t carbon d io x id e . There a r e s e v e r a l p o s s i b l e e x p l a n a t i o n s f o r t h is exception. 1. I’o r t h e s i x c u l t u r e s i n t h i s s e r i e s , two p e r c e n t c a r b o n d i o x i d e r e p r e s e n t e d t h e ontimum a t m o s p h e r i c c o n d i t i o n . 2. E r r o r s i n sampling r a i s e d the count. 3. The c o n c e n t r a t i o n o f c a r b o n d i o x i d e g a s i n t h e c u l t u r e j a r s was g r e a t e r t h a n two p e r c e n t . Of t h e t h r e e p o s s i b i l i t i e s we a r e i n c l i n e d to f a v o r t h e l a s t . Gas a n a l y s e s e s t a b l i s h e d t h a t c a r b o n d i o x i d e c o n c e n t r a t i o n s v a r i e d a s much a s 0 . 5 to 1 . 0 p e r c e n t when t h e c u l t u r e j a r s were f i l l e d by t a n k . Vfhile t h i s d i f f e r e n c e would n o t be s e r i o u s i n t h e h i g h e r r a n g e s o f s i x a n d 18 p e r c e n t c a r b o n d i o x i d e , an e r r o r o f 0 . 5 to 1 . 0 p e r c e n t m ig h t a f f e c t c o n s id e r a b ly the grow th-prom oting q u a l i t i e s o f a c o n c e n tr a tio n a p p ro a c h in g th e low er l i m i t o f the optim al zone. The e v i d e n c e i n s e r i e s 1 , 2 and. U, P a r t I I i n d i c a t e s t h a t two p e r c e n t c a r b o n d i o x i d e e n r i c h m e n t i s b e l o w t h e optimum, w h i l e t h e d a t a o f P a r t s I and I I i n d i c a t e t h a t th e c o n c e n t r a t i o n o f c a r b o n d i o x i d e f u r ­ n i s h e d by c a n d l e i s w i t h i n t h e zone o f o n t i m n l g r o w t h s t i m u l a t i o n . we a c c e n t , tentatively, If t h e i d e a t h a t g r o w t h s t i m u l a t i o n by a c a n d l e b u r n e d i n an a i r - t i g h t c o n t a i n e r i s due to c a r b o n d i o x i d e g a s a l o n e ( e x c e p t i n g th e f a c t o r of m o i s t u r e ) , i t i s e v id e n t t h a t the low er l i m i t o f t h e o p t i m a l zone o f c a r b o n d i o x i d e s t i m u l a t i o n f o r g o n o c o c c i i s a p p r o x i m a t e l y 2 . 3 Tier c e n t . The e v i d e n c e i n P a r t I I , s e r i e s 1 , 2 and 3* i s t h a t t h e u p p e r l i m i t o f t h e o p t i m a l zone i s some p l a c e b e t w e e n a - 1+1 c o n c e n t r a t io n o f 18 p e r c e n t and 22 p e r c e n t ca rb o n d io x i d e . It is ob v io u B t h a t th e optimum c o n c e n t r a t io n i s n o t l i m i t e d to a d e f i n i t e p e r c e n t a g e o f ca rb o n d i o x id e , b u t may b e o b t a in e d o v e r a f a i r l y w ide zon e o f ca rb o n d io x id e e n r ic h m e n t. PART I l a . EFFECT OF CARBON DIOXIDE BEYOND THE OPTIMAL RANGE V a lle y and R e t t g e r (2 6 ) i n s tu d y in g th e in f l u e n c e o f carb on d io x id e on b a d t e r ia d e m o n stra ted t h a t c e r t a i n b a c t e r i a , such a s L . a c id o p h ilu s , w ere b e n e f i t e d by ca rb o n d io x id e i n p e r c e n t a g e s a s h ig h a s 25 to 30 per c e n t. V ig o r o u s and r e s i s t a n t b a c t e r i a , such a s B a c t e r iu m a e r o g e n e s and S ta p h y lo c o c c u s a u r e u s , grew w e l l i n an atm osp h ere o f 95 to 97 p e r c e n t ca rb o n d io x id e on b o th p l a i n n u t r ie n t a gar and h ig h ly b u f f e r e d a g a r . Cohen and F lem in g ( 2 7 ) d em o n stra ted t h a t p rim ary c u l t i v a t i o n o f th e m en in g o co ccu s was en h an ced by carb on d io x id e en r ic h m e n t. The o p t i ­ mum c o n c e n t r a t io n v a r ie d from te n to J>0 p e r c e n t carb on d i o x id e , depend­ in g upon th e s t r a i n . W ith 50 to 75 P®r c e n t carb on d io x id e th e grow th was s c a n t . I t was f e l t o f i n t e r e s t t o d e t e r m i n e t h e r e a c t i o n o f g o n o c o c c i i n -o rim a r y g r o w t h t o c a r b o n d i o x i d e i n p e r c e n t a g e s ab ove t h e ontimum co ncentration. I n t h i s e x p e r i m e n t t h e c h o c o l a t e a g a r em plo yed i n t h e p r e v i o u s work was u s e d . C u l t u r e was made, a s u s u a l , f r o m g o n o r r h e a l d i s c h a r g e s o f m ale p a t i e n t s . The p e r c e n t a g e s o f c a r b o n d i o x i d e t e s t e d an d o t h e r d a t a a r e s u p p l i e d i n T a b le S. The a v e r a g e s i z e o f t h e c o l o n i e s has b e e n i n s e r t e d b e n e a th the colony count of fo u r s t r a i n s . - H2 - BESULTS OP PiJRT I l a . The t h r e e c u l t u r e s o f s e r i e s 1 , T a b le 8 , grew o v e r th e e n t i r e ra n g e o f c a r b o n d i o x i d e c o n c e n t r a t i o n s , from two p e r c e n t to 5 ° Pe r c e n t . The drop i n p o p u l a t i o n a b o v e a ca rb o n d io x id e c o n c e n t r a t io n o f 22 p e r c e n t w as n o t a b r u p t, a n d , e x c e p t i n th e c a s e o f c u l t u r e #3» th e g ro w th un d er 50 n e r c e n t c a r b o n d i o x i d e was on e—t h ir d to o n e—f o u r t h a s g r e a t a s u nder th e " c a n d le " c o n d i t i o n . The a v e r a g e c o lo n y s i z e a s shown i n c u l t u r e s 1 - U3 and 2, c h a n g e d f r o m a maximum o f 1 , 5 mm. u n d e r two p e r c e n t c a r b o n d i o x i d e t o on a v e r a g e o f 0 .5 nan. u n d e r Ho p e r c e n t c a r b o n d i o x i d e . U n d e r a c o n c e n t r a t i o n o f 50 p e r c e n t c a r b o n d i o x i d e t h e c o l o n i e s were l e s s t h a n 0 . 5 mm. i n d i a m e t e r , b u t d i s t i n c t . The b l a c k e n i n g o f p i n ­ p o i n t c o l o n i e s due to t h e o x i d a s e r e a c t i o n was a s s h a m o s t h a t o f the l a r g e r c o lo n ie s . B e f o r e dy e wa.s o l a c e d on t h e n l a t e s , t r a n s f e r s were made f r o m t h e HO n e r c e n t c a r b o n d i o x i d e p l a t e o f c u l t u r e 1 t o f r e s h c h o c o l a t e a g a r . T r a n s f e r s w e r e a l s o made f r o m t h e Ho n e r c e n t a n d 50 n e r c e n t c a r b o n d io x id e p l a t e s o f c u ltu r e 2. in c u b a te d under "candle.*1 The o r g a n i s m s r e w l u x u r i a n t l y when They w e r e r n o m h o l o g i c a l l y t y n i c a l i n a n p e a r - a n c e and f e r m e n t e d d e x t r o s e o n l y when grown i n s i n g l e s u g a r s . Oram s t a i n s w e r e made o f f i v e c o l o n i e s f ro m t h e 1 : 1 0 0 d i l u t i o n o f e a c h c o n d i t i o n ( " c a n d l e , " 2 p e r c e n t , 10 o e r c e n t , l ’- p e r c e n t c a r b o n d ioxide, e t c . ) of c u ltu re 1. The gram n e g a t i v e d . i o l o c o c c i grown u n d e r t h e "candle** and two p e r c e n t c a r b o n d i o x i d e were t j r n i c a l c o f f e e - b e a n shaned organism s, r e g u la r in shape. O r g a n i s m s f r o m t h e 10 p e r c e n t c a r b o n d i o x i d e p l a t e s w er e f o r t h e m o st n a r t r e g u l a r i n s h o n e , b u t a few b i z a r r e form s were o r e s e n t . quently tria n g u la r , These were i r r e g u l a r i n s h a p e , f r e ­ and r e a d i l y d i f f e r e n t i a t e d f r o m t h e " b a l l o o n " f o r m s o f g o n o c o c c i f r e o u e n t l y e n c o u n t e r e d i n H g - h o u r c u l t u r e s grown a e r o b i ­ c a l l y o r u n d e r lo w c o n c e n t r a t i o n o f c a r b o n d i o x i d e . The b i z a r r e f o r m s v/ere o r e s e r t i n g r e a . t e r num ber s i n s t a i n s made f r o m p l a t e s grown u n d e r high c o n c e n tra tio n s of carbon d io x id e. I n s t a i n s made fro m t h e ? 0 , Ho, a n d 50 n e r c e n t c a r b o n d i o x i d e a b a t e s no r e g u l a r d i n l o c c c c i could, be d i stin.gui shed. - uu - I n S e r i e s 2 , c u l t u r e U f a i l e d to grow ab ove a c o n c e n t r a t i o n o f IS p e r cent carbon d io x id e . C u l t u r e s 5 a n d 6 r e a c t e d d i f f e r e n t l y to t h e maximum c o n c e n t r a t i o n o f c a r b o n d i o x i d e . A sharp drop o c c u r r e d i n t h e g r o w t h o f c u l t u r e 5 above 50 p e r c e n t c a r b o n d i o x i d e , w h i l e c u l t u r e b gr ew w e l l u n d e r a 60 p e r c e n t e n r i c h m e n t . The a v e r a g e c o l o n y s i z e o f c u l t u r e s p ?-nd 6 d e c r e a s e d p r o g r e s s i v e l y u n d e r i n c r e a s ­ ing c o n c e n tra tio n s of carto n dioxide. I n S e r i e s }, carbon d io x id e . t h e maximum c o n c e n t r a t i o n was i n c r e a s e d to JO p e r c e n t hone o f t h e c u l t u r e s o f S e r i e s 3 p r o d u c e d c o l o n i e s u n d e r t h e maximum c o n c e n t r a t i o n o f g a s w hich c o u l d b e seen w i t h t h e unaided eye. I'o g r o w t h c o u l d be s e e n u n d e r t h e low -pow er o b j e c t i v e o f th e m icroscope a g a i n s t the dark background of the p l a t e s . However, when dye was s p r a y e d on t h e p l a t e s m i n u t e b l a c k s p e c k s a p p e a r e d w h i c h sim u lated the d i s t r i b u t i o n p a t t e r n of c o lo n ie s. Stained p re p a r a tio n s w ere o f no a i d i n d e t e r m i n i n g w h e t h e r o r n o t o r g a n i s m s were p r e s e n t , f o r no d i s t i n c t b i z a r r e o r o t h e r f o r m s c o u l d be d i s t i n g u i s h e d . C u l t u r e s 8 a n d 9 , l i k e C u l t u r e 6, a p p a r e n t l y were r e s i s t a n t to t h e e f f e c t s o f 60 p e r c e n t c a r b o n d i o x i d e . C u l t u r e s 10 and 11 were i n h i b i t e d , I t w ou ld a p p e a r a r e a s o n a b l e a s s u m p t i o n f r o m t h e d a t a i n T able 8 t h a t g o n o c o c c i w i l l grow m o d e r a t e l y w e l l u n d e r h i g h c o n c e n t r a t i o n s o f carbon d io x id e . A f e w s t r a i n s i n t h e s h o r t s e r i e s t e s t e d grew u n d e r a c o n c e n t r a t i o n o f oO p e r c e n t c a r b o n d i o x i d e . There i s an i n d i c a t i o n t h a t s t r a i n s o f g o n o c o c c i v a r y i n t h e i r r e s i s t a n c e to c a r b o n d i o x i d e i n high c o n c e n tra tio n s. PART I I I . EFF3CT OF CASHS OTHhd THAH CA3B0:i DI0XID2 Ck THE CHO’.fTH OF COHO COCCI So f a r a s we h av e b e e n a b l e t o d i s c o v e r , t.ne e f f e c t o f g a s e s o t h e r - U5 t h a n c a r b o n d i o x i d e on t h e g r o w t h o f g o n o c o c c i h a s b e e n t r i e d o n l y by R o c k w e l l an d McICann ( 8 ) an d McLeod a n d h i s a s s o c i a t e s ( 1 1 ) . h a s b e e n made b e f o r e to t h e s e e x p e r i m e n t s . Reference I t was m e n t i o n e d t h a t Rockw ell and McKann s t u d i e d t h e a d a p t a t i o n o f g o n o c o c c i to v a r i o u s g a s e o u s e n v i r o n m e n t s a f t e r t h e s t r a i n s were i s o l a t e d u n d e r r e d u c e d oxygen tension. They f o u n d s u c h s t r a i n s grew u n d e r p u r e hy d rogen b u t n o t u n d e r c a r b o n d i o x i d e o r oxy g en . S t r a i n s i s o l a t e d u n d e r p a r t i a l oxyg en t e n s i o n w h ic h would n o t grow u n d e r p u r e c a r b o n d i o x i d e o r p u r e oxygen c o u l d be a d a p t e d to do s o . McLeod and h i s c o l l e a g u e s were i n f l u e n c e d to t r y th e e f f e c t o f h y d r o g e n on primary' g r o w th o f g o n o c o c c i from th e r e p o r t s o f Rockw ell and McKann. Mo a d v a n t a g e s were o b s e r v e d from th e u s e o f hydrogen. As f i n a l e x p e r i m e n t s i n t h i s s t u d y , i t was f e l t d e s i r a b l e to t r y t h e e f f e c t o f h y d r o g e n , c a r b o n monoxide, and n i t r o g e n . Hydrogen was s e l e c t e d f o r t e s t b e c a u s e th e d e t a i l s o f i t s u s e by McLeod a r e unknown. Carbon monoxide was i n c l u d e d i n t h e e x p e r i m e n t s s i n c e i t u n d o u b t e d l y i s f o rm e d i n s m a l l q u a n t i t y by t h e b u r n i n g o f a c a n d l e i n a c l o s e d c o n t a i n e r . M i t r o n e n was i n c l u d e d s i n c e i t e x i s t s i n th e l a r g e s t p e r c e n t a g e o f any gas in a ir , reported. and i t s e f f e c t on th e g r o w t h o f g o n o c o c c i has n e v e r b een The e f f e c t o f oxygen was n o t t e s t e d b e c a u s e the c u l t u r a l e x p e r i m e n t s o f many w o r k e r s have d e m o n s t r a t e d t h e m i c r o - e e r o p h i l i c r e ­ q u ire m e n ts o f the gonococcus. I n t h e f i r s t e x p e r i m e n t t h e f o l l o w i n g a t m o s p h e r i c c o n d i t i o n s were provided: ( 1 ) A t m o s p h e r ic a i r i n a c l o s e d c o n t a i n e r . a p p r o x i m a t e l y 100 p e r c e n t . R e la tiv e humidity ( 2 ) Reduced oxygen t e n s i o n o b t a i n e d by a p a r t i a l vacuum e q u a l to n i n e c u . o f m e r c u ry on th e open manometer. R e l a t i v e h u m id ity a p p r o x i m a t e l y 100 pel' c e n t . - 46 ( 3 ) Carbon m onox ide, a p p r o x i m a t e l y 10 p e r c e n t . h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . ( 4 ) Hydrogen, 10 p e r c e n t , s u p p l i e d b y t a n k . a p p r o x i m a t e l y 100 p e r c e n t . R elative R e la tiv e hum idity ( 3 ) Carbon d i o x i d e , 10 p e r c e n t , s u p p l i e d by t a n k . h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . R elative Carb on monoxide was f u r n i s h e d b y t h e a c t i o n o f c o n c e n t r a t e d s u l ­ f u r i c a c i d on sodium f o r m a t e . A s i m p l e g e n e r a t o r was made b y s e c u r i n g t h e s h a n k o f a. s e p a r a t o r y f u n n e l i n t h e n eck o f a s i d e - a r m f l a s k by means o f a o n e - h o l e r u b b e r s t o p p e r . sodium f o r m a t e i n t h e f l a s k . A c i d was p l a c e d i n th e f u n n e l and The s u l f u r i c a c i d was a l l o w e d t o d r i p o n t o t h e sodium f o r m a t e fro m t h e s hank o f t h e f u n n e l a t a c o n s t a n t r a t e . Gas f r o m t h e g e n e r a t o r was p e r m i t t e d t o f l o w t h r o u g h r u b b e r t u b i n g c o n n e c t i o n s f o r t w e n t y m i n u t e s b e f o r e an open U - t u b e manometer and th e c u l t u r e j a r were c o n n e c t e d . The c a r b o n monoxide g a s r e p l a c e d t e n p e r c e n t o f a i r w h ic h h a d b e e n removed by vacuum, pump f r o m t h e c u l t u r e j a r . Ten Tier c e n t c o n c e n t r a t i o n s o f c a r b o n monoxide and h y d r o g e n were s u p p l i e d to c o r r e s p o n d w i t h a p e r c e n t o f c a r b o n d i o x i d e from th e o p t i ­ mum zone o f c a r b o n d i o x i d e s t i m u l a t i o n . I t was r e a l i z e d t h a t t e n p e r c e n t c a r b o n monoxide o r h y d r o g e n g a s m ig ht be c o n s i d e r a b l y above o r b e l o w t h e n o s s i b l e ontimum c o n c e n t r a t i o n s o f t h e s e e a s e s . I n t h i s and t h e f o l l o w i n g e x p e r i m e n t s an i n d i c a t i o n o n l y was s o u g h t o f i n h i b i t i v e o r g r o w t h - o r o r a o t i n g p r o p e r t i e s o f c a r b o n monoxide and h y d r o g e n , a s w e l l as n itro g e n . Two c h o c o l a t e a g a r p l a t e s were i n o c u l a t e d d i r e c t l y from c o l l e c t i o n f l u i d and two fro m a d i l u t i o n t u b e f o r e a c h c o n d i t i o n . - U7 - Thus f o r e a c h s t r a i n and each a t m o s p h e r i c c o n d i t i o n , a t o t a l o f f o u r p l a t e s was i n o c u l a t e d . I n Table 9» t h e r e f o r e , th e s i g n i f i c a n t c o u n t s are g iv en in d u p l i c a t e f o r nine s t r a i n s o f gonococci. RESULTS 0? S3RISS 1 From t h e c o u n t s o f Table 9 th e a t m o s p h e r i c c o n d i t i o n s nay be g r o u p e d t o g e t h e r , o r s i n g l y , a c c o r d i n g to th e growth s t i m u l a t i o n a f f o r d e d . In ascending o rd er they a re : (1 ) A e r o b i c , r e d u c e d oxygen t e n s i o n , hydrogen - p o o r e s t growth (2) Carbon m ono xide................................b e t t e r gro wth than ( l ) (3 ) Carbon d i o x i d e .................................. b e s t gr ow th - Us - I n Group ( l ) conditions l i s t e d , t h e c o u n t s were v e r y much t h e same u n d e r t h e t h r e e e x c e p t i n t h e c a s e o f C u l t u r e 5 w h ic h g r e w u n d e r hydrogen only. Carbo n monoxide d e f i n i t e l y was s u p e r i o r to t h e g a s e o u s c o n d i t i o n s o f g r o u p ( 1 ) an d v e r y n e a r l y on a p a r w i t h c a r b o n d i o x i d e . C u ltu re #1, h o w e v e r , grew l u x u r i a n t l y u n d e r c a r b o n d i o x i d e a n d s p a r s e l y u n d e r c a r b o n m on oxide. C u l t u r e #7 grew o n l y u n d e r c a r b o n d i o x i d e . SEHISS 2 I t a p p e a r e d fro m t h e l i m i t e d d a t a o f T a b l e 9 t h a t c a r b o n monoxide g a s p r o v id e s a s tim u lu s f o r growth o f gonococci g r e a t e r than t h a t of m oist a i r . Such e v i d e n c e c o u l d n o t b e a c c e p t e d , o f c o u r s e , w i t h o u t r u l i n g o u t t h e p o s s i b i l i t y t h a t t r a c e s o f c a r b o n d i o x i d e i n t h e g a s fro m t h e g e n e r a t o r p r o v i d e d g r o w t h s t i m u l a t i o n r a t h e r t h a n c a r b o n mon oxide. s e c o n d s e r i e s o f s p e c im e n s was c u l t u r e d , A t h e r e f o r e , under the condi­ t i o n s o f S e r i e s 1 , w i t h t h e d i f f e r e n c e t h a t c a r b o n monoxide g a s was b u b b l e d t h r o u g h a. column o f UO p e r c e n t p o t a s s i u m h y d r o x i d e s o l u t i o n containing p y ro g a llo l. cu ltured. A t o t a l o f s i x t e e n s t r a i n s o f g o n o c o c c i were D u p l i c a t e p l a t i n g s were made a s i n S e r i e s 1 . The p l a t e c o u n t s o f t h i s s e r i e s a r e p r e s e n t e d i n T able 1 0 . HE SUITS 0? SiSHIES 2 Fne g r o u p i n g o f a t m o s p h e r i c c o n d i t i o n s i n S e r i e s 2, a c c o r d i n g to t h e g r o w t h s t i m u l a t i o n a f f o r d e d by them, had t o be m o d i f i e d from the o rd e r given in S c rie s 1. s h i p was a s f o l l o w s ; Prom t h e d a t a o f Table 10 , th e r e l a t i o n ­ (1) A e r o b i c c o n d i t i o n ....................................p o o r e s t growth (2) Reduced oxygen t e n s i o n , c a r b o n monoxide, and hydrogen .............................................................................b e t t e r growth t h a n (1) (3) Carbon d i o x i d e ........................................... b e s t gr owth Vfith t h e e x c e p t i o n s o f c u l t u r e s 7 and 13. t h e growth o f gonococci u n d e r r e d u c e d oxygen t e n s i o n , ca r b o n monoxide, and hydrogen was g r e a t e r than under the a e ro b ic c o n d itio n . C u l t u r e 2 f a i l e d to grow a e r o b i c a l l y b u t d i d grow u n d e r t h e t h r e e c o n d i t i o n s o f Croup ( 2 ) . ? o u r s t r a i n s ( 1 , S, 9 , and lU) grew u n d e r c a r b o n d i o x i d e b u t d i d n o t grow u n d e r o t h e r a t m o s p h e r e s . With n e a r l y a l l c u l t u r e s , th e - 50 p o p u la t io n u n d er ca rb o n d io x id e w as g r e a t e r th an un d er o th e r c o n d i t i o n s . The s u r p r is i n g g row th s t im u la t io n c a u se d by carb on m onoxide f o r th e d u l t u r e s o f S e r i e s 1 was n o t p r o v id e d by carb on m onoxide b u b b led th ro u g h p o ta s s iu m h y d r o x id e . I t i s h i g h l y p r o b a b le th a t carb on d io x id e w as p r e s e n t i n g a s p ro d u ced by rem oval o f w ater from sodium fo rm a te by s u lf u r ic a c id . J r o m t h i s s h o r t s e r i e s t h e r e se em s l i t t l e evidence t h a t carbon m o n o x i d e a n d h y d r o g e n i n t e n p e r c e n t c o n c e n t r a t i o n s p r o v i d e a n y ad­ v a n ta g e s f o r c u l t u r e o f gonococci t h a t i s not fu rn is h e d by reduced o x y g e n t e n s i o n p r o d u c e d by p a r t i a l vac uu m . be d e t e c t e d from t h e i r u s e . No d e l e t e r i o u s e f f e c t s c o u l d Since b e t t e r grow th developed u n d e r carbon m o n o x id e a n d h y d r o g e n t h a n u n d e r t h e a e r o b i c c o n d i t i o n , i t i s p r o b a b l e t h a t any v a lu e the g a s e s p o s s e s s i n c u l t u r e o f gonococci i s i n t h e i r u s e f o r p r o d u c t io n o f a red u ced oxygen t e n s i o n . PART I l i a . THE EFFECT OP NITROGEN ON GROWTH OF GONOCOCCI The e x p e r i m e n t o n t h e e f f e c t o f n i t r o g e n d i d n o t f o l l o w t h e p l a n of Part III. The g a s e o u s c o n d i t i o n s w e r e somewhat d i f f e r e n t t h a n i n S e r i e s 1 and 2 o f P a r t I I I , s i n c e P a r t I l i a was s u b o r d i n a t e t o a n o t h e r study. The f o l l o w i n g e t r r . o s p h e r i c d o n d i t i o n s were p r o v i d e d : (1) A tm ospheric a i r . R e l a t i v e h u m idity 60-J0 p e r c e n t. (2) R e d u c e d ox y g e n t e n s i o n p r o v i d e d by a p a r t i a l vacuum o f t e n cm. o f m e r c u r y ( o p e n m a n o m e t e r ) . R e la tiv e hum idity a n o r o x irn a te ly 100 p e r c e n t . (3 ) N i t r o g e n 10 p e r c e n t , s u p p l i e d b y t a n k . a p p r o x i m a t e l y 100 p e r c e n t . (b) Carbon d i o x i d e 10 p e r c e n t , s u p p l i e d by t a n k , h u m i d i t y a p p r o x i m a t e l y 100 p e r c e n t . R e la tiv e hum idity relativ e - 51 - (5) Carbon d i o x i d e s u p p l i e d "by c a n d l e . a p p r o x i m a t e l y 100 p e r c e n t . B e la tiv e humidity S i n g l e p l a t e s o n l y were i n o c u l a t e d i n t h i s e x p e r i m e n t f o r ea ch s t r a i n and d i l u t i o n u n d e r t h e r e s p e c t i v e a t m o s p h e r i c c o n d i t i o n s . The r e s u l t s a r e p r e s e n t e d i n Table 11 by t h e same p l a t e c o u n t method u s e d in previous ta b u la tio n s. - 52 RESULTS OP PART I l i a I t i s p o s s i b l e fro m t h e c o u n t s o f T a b le 11 t o group t h e atmos ­ p h e r i c c o n d i t i o n s o f the experim ent i n the fo llo w in g o r d e r ac c o rd in g to t h e g r o w t h s t i m u l a t i o n p r o d u c e d ; (1) A erobic & N i t r o g e n . . . . p o o r e s t g r o w th (2) Reduced oxygen t e n s i o n b e t t e r growth than ( l ) (3) Carbon d i o x i d e 10 p e r c e n t , " c a n d l e " . . . . b e s t gro wth I t w i l l be n o t e d t h a t d e s p i t e t h e d i f f e r e n c e s i n m o i s t u r e c o n t e n t o f t h e a e r o b i c and n i t r o g e n c o n d i t i o n s , r r o w t h u n d e r t h e two was p r a c t i c a l l y t h e same, w i t h th e e x c e p t i o n o f c u l t u r e 5 which grew w e l l u n d e r n i t r o g e n and p o o r l y i n a i r . A l l c u l t u r e s g r e w b e t t e r u n d e r r e d u c e d oxygen t e n s i o n t h a n i n a i r o r in a i r enriched w ith n itro g e n . The b e s t g r o w t h o c c u r r e d u n d e r t e n p e r c e n t c a r b o n d i o x i d e and c a r b o n d i o x i d e s u p p l i e d by c a n d l e . Ho i n d i c a t i o n i s s e e n fro m t h i s e x p e r i m e n t t h a t n i t r o g e n i n t e n p e r c e n t c o n c e n t r a t i o n i s b e n e f i c i a l t o t h e p r i m a r y g r ow th o f g o n o c o c c i . By i n d i r e c t e v i d e n c e t h e r e i s some i n d i c a t i o n t h a t t h i s amount o f g a s a f f o r d s l e s s growth than a i r s a t u r a t e d w ith m o is tu re , since nrowth in an e t m o s n h e r e o n l y p a r t i a l l y m o i s t e n e d a f f o r d e d g r o w t h e q u a l to t h a t of nitrogen. DISCUSSION C h r i s t i a n s e n and S c h o e n l e i n ( 2 ) hav e shown t h a t a c a n d l e burne d i n a c l o s e d c o n t a i n e r p r o v i d e s an e n v ir o n m e n t f o r th e gonococcus q u i t e a s f a v o r a b l e a s t e n p e r c e n t c a r b o n d i o x i d e s u p p l i e d by t a n k . It is b e l i e v e d by some ( 28) t h a t t h e c a n d l e s u p p l i e s a p p r o x i m a t e l y 10 p e r c e n t carbon dioxide; t h e r e f o r e , i t s e f f i c a c y i s t h e same a s a measured amount o f g a s . By g a s a n a l y s i s i t wa.s shown i n t h i s s tu d y t h a t a p p r o x i m a te l y 2 . 3 p e r c e n t c a r b o n d i o x i d e i s formed by th e b u r n i n g o f a c a n d l e i n a closed container of six l i t e r s capacity . T his p e r c e n t a g e i s s l i g h t l y l o w e r t h a n t h a t g i v e n by ITye and Lamb (21) f o r a c o n t a i n e r o f l i k e s i z e . C o n f i r m a t i o n o f t h e o b s e r v a t i o n s o f C h r i s t i a n s e n and S c h o e n l e i n i s g i v e n i n P a r t I o f t h i s t h e s i s where i t was shown by c u l t u r e o f s p e c i ­ mens fro m a c u t e c a s e s o f g o n o r rh e a t h a t t h e " c a n d l e " e nvir onm e nt i s t h e e q u a l o f an a tm o s p h e r e e n r i c h e d w i t h t e n p e r c e n t carbon d i o x i d e . Not o n l y were t h e number o f s t r a i n s o f gonococci p r a c t i c a l l y th e same b u t t h e p o p u l a t i o n s s u s t a i n e d by b o t h atm o s p h e r e s were found by co un t to be n e a r l y t h e same. Two c o n d i t i o n s o f p a r t i a l vacuum p r o v i d i n g r e d u c e d oxygen t e n s i o n s e q u i v a l e n t to t h o s e pr o d u c e d by t e n p e r c e n t c a r b o n d i o x i d e en r ic h m e n t a n d th e " c a n d l e " were found to be i n f e r i o r to ca r b o n d i o x i d e f o r growth stim ulation, h o t o n l y i n P a r t I b u t t h r o u g h o u t th e s t u d y , whe rev er t h e c o n d i t i o n s o f r e d u c e d oxygen t e n s i o n and c a r b o n d i o x i d e were com­ p a r e d , no s t r a i n grew u n d e r re d u c e d oxygen t e n s i o n ( - p a r t i a l vacuum) t h a t d i d n o t grow u n d e r ca r b o n d i o x i d e . On th e o t h e r h a n d , a p u r o x i m a t e l y - 5U 25 p e r c e n t more s t r a i n s were i s o l a t e d u n d e r c a r b o n d i o x i d e t h a n under r e d u c e d oxygen t e n s i o n . I n P a r t I t h e a e r o b i c c o n d i t i o n was f o u n d t o be i n f e r i o r to th e " c a n d l e " and t e n p e r c e n t c a r b o n d i o x i d e c o n d i t i o n s . I t is tru e that t h e r e l a t i v e h u m i d i t y o f t h e a e r o b i c e n v ir o n m e n t was l e s s t h a n t h a t p ro v id e d in the carbon dioxide c u ltu r e j a r s . This i n e o u a l i t y i n m o i s t u r e c o n t e n t was p u r p o s e l y a d j u s t e d , f o r i t was d e s i r e d t h a t t h e a e r o b i c c o n d i t i o n r e n r o d u c e t h e a tm o s p h e r e and h u m i d i t y o f t h e a v e r a g e incubator. Where a c o n d i t i o n o f m o i s t u r e was n r o v i d e d i n th e a e r o b i c method e q u al to t h a t i n a c a r b o n d i o x i d e c u l t u r e j a r , a s shown i n T a b le 5* t h e c a r b o n d i o x i d e method was s u p e r i o r , even though t h e com­ p a r i s o n was made w i t h a e r o b i c s t r a i n s . No s t r a i n grew a e r o b i c a l l y t h a t d i d n o t crow u n d e r c a r b o n d i o x i d e i n any o f t h e e x p e r i m e n t s of the t h e s i s . The e f f e c t o f m o i s t u r e on a e r o b i c s t r a i n s o f t h e go noco cc us i s s t r i k i n g l y i l l u s t r a t e d i n T a b le 3* some i n s t a n c e s growth was 10 o r more t i m e s g r e a t e r u n d e r a s a t u r a t e d a tm o s p h e r e t h a n u n d e r a. r e l a ­ t i v e h u m i d i t y o f 60 to JO p e r c e n t . A c o m p a r is o n o f t h e e f f i c a c y o f a e r o b i c and r e d u c e d oxygen t e n ­ s i o n ( n a r t i a l vacuum) e n v i r o n m e n t s f o r growing t h e gono coccu s can s c a r c e l y b e made f r o m t h e m a t e r i a l i n t h i s t h e s i s , s i n c e th e r e l a t i v e h u m i d i t y was d i f f e r e n t when a l a r g e number o f specim e ns were c u l t u r e d in duplicate. I n T a b l e s 9 and 10 where co m p a r i so n i s made o f a e r o b i c c o n d i t i o n a n a r e d u c e d oxygen t e n s i o n , w i t h th e r e l a t i v e h u m i d i t y t h e same, t h e r e i s a s u g g e s t i o n t h a t r e d u c e d oxygen t e n s i o n w i l l o r o v i d e the b e t t e r growth. 4 A p l a t e c o u n t was v a lu a b le in a l l th e ex p erim en ts u n d ertak en , b u t th e te c h n iq u e w as, w ith o u t d oub t, a n e c e s s i t y in d eterm in in g the l i m i t s o f o p tim a l carbon d io x id e s t im u la t io n . P roper d i l u t i o n o f the o r i g i n a l specim en and a d eq u ate d i s t r i b u t i o n o f inoculum were req u ired to o b t a in p la te B s u i t a b l e f o r c o u n tin g . B ent g l a s s rod s were the b e s t means found f o r se c u r in g an even spread o f o rg a n ism s. I n P a r t I i t was d e m o n s t r a t e d by c u l t u r e o f 68 s t r a i n s t h a t t h e g o n o c o c c u s grows e q u a l l y w e l l u n d e r a t m o s p h e r e s e n r i c h e d w i t h 2 .3 p e r c e n t and ten p e r c e n t carbon d io x id e . I n P a r t I I , by c u l t u r e o f 60 s t r a i n s o f g o n o c o c c i , e v i d e n c e was p r e s e n t e d to show t h a t g r o w t h o c c u r s e q u a l l y w e l l o v e r an ev en w i d e r zone o f c a r b o n d i o x i d e c o n c e n t r a t i o n s . The l o w e r l i m i t s o f t i l s zone a r e b e t w e e n two and 2.3 p e r c e n t an d th e u p p e r l i m i t s fro m 18 to 2? p e r c e n t . U n til th e a c t u a l n a tu r e o f c a r b o n d io x id e s t im u la t io n o f the g o n o c o c c u s i s known, i t i s f e l t t h a t s e t t i n g d e f i n i t e l i m i t s upon th e o p t i m a l zone must be done w i t h c a u t i o n . b e l i e v e ( 26), I f , ° s V a l l e y and H e t t g e r t h e e f f e c t o f c a r b o n d i o x i d e i s b e n e f i c i a l to m ic ro ­ o r g a n i s m s so l o n g a s t h e oH o f t h e medium r e m a i n s c o n s t a n t , a. medium h i g h l y b u f f e r e d , c o n t a i n i n g n o n - t o x i c b u f f e r s , m ight s u o u o r t l u x u r i ­ a n t g r o w t h o f g o n o c o c c i u n d e r h i g h c o n c e n t r a t i o n s o f ca r b o n d i o x i d e . I t i s n e c e s s a r y to s p e c i f y , t h e r e f o r e , t h a t th e o n t i m a l l i m i t s e s ­ t a b l i s h e d i n t h i s t h e s i s were done w i t h th e s u b s t r a t e known a s "chocolate" agar, D ifco. I t i s f e l t t h a t t h e e s t a b l i s h m e n t o f th e zone o f optimum g r o w th o f t h e g o n o c o c c u s u n d e r c a r b o n d i o x i d e w i l l be o f more t h a n t h e o r e t i c a l - 56 In terest. M ed ic al b a c t e r i o l o g i s t s a c c e p t a l m o s t s l a v i s h l y t h e dic tu m t h a t p r i m a r y c u l t u r e . , prow b e s t u n d e r t e n p e r c e n t o f t h e g a s . Certain w o r k e r s i n th e w r i t e r 1s e x p e r i e n c e have b ee n d i s c o u r a g e d from a t t e m p t ­ i n g c u l t u r a l work i n t h e b e l i e f t h a t methods o f s u p p l y i n g c a r b o n d i o x i d e a.re too e x a c t i n g f o r t h e a v e r a g e b a c t e r i o l o g i s t . While t e n n e r c e n t , o r thereab o u t, i s s t i l l s in ce i t i s i n the th e c o n c e n t r a t i o n o f c h o i c e , m i d d l e o f t h e o p t i m a l zone, measurement need n o t be e x a c t i f g a s i s to be s u n p l i e d by t a n k . F o r r o u t i n e c u l t u r e th e c a n d l e i s to be p r e f e r r e d to ti m e - c o n s u m in g measurement o f g a s by manometer. I t i s n o t s u r p r i s i n g t h a t g o n o c o c c i a r e a b l e to grow w e l l , i f n o t l u x u r i a n t l y , u n d e r 30 and even UO p e r c e n t o f c a r b o n d i o x i d e . Since t h e o p t i m a l zone i s f a i r l y w ide, one would e x p e c t a g r a d u a l i n c r e a s e i n th e i n h i b i t i v e e f f e c t o f t h e g a s above t h i s zone, r a t h e r t h a n an a b r u p t c e s s a t i o n o f g r ow th in p e r c e n t a g e s o f g a s im m ediately above IS to 22 p e r c e n t . The d a t a o f Table 8 on growth u n d e r h i g h c o n c e n t r a t i o n s o f g a s l e n d i n d i r e c t s u p p o r t to P a r t I I . I T e i th e r h y d r o g e n , n i t r o g e n , n o r c a r b o n monoxide i n t e n p e r c e n t c o n c e n t r a t i o n s showed any e v i d e n c e o f s t i m u l a t i n g gr owth o f g o n o c o c c i . As s t a t e d i n P a r t I I I , c a r b o n monoxide was t e s t e d to d i s c o v e r i f t h i s g a s c o n t r i b u t e d to g r o w t h s t i m u l a t i o n i n n c a n d l e j a r . d o e s n o t e n t i r e l y ans wer t h e q u e s t i o n , The exo e r im e n t since the c o n c e n tra tio n of c a r­ bon monoxide i n t h e t e s t s was u n d o u b t e d l y f a r g r e a t e r th a n t h e amount ■nresent i n a c a n d l e j a r . However, s i n c e ca r b o n monoxide i s n o t a n o r ­ mal c o n s t i t u e n t o f a i r and i s n o t e s s e n t i a l to b a c t e r i a l m eta b o lis m , a s i s c a r b o n d i o x i d e , i t i s n r o b a b l e t h a t t h i s i n e r t g a s does n o t con­ t r i b u t e to g r o w t h . SUMMARY By g a s a n a l y s i s i t was e s t a b l i s h e d t h a t a o u r o x i m a t e l y 2 . ^ p er c e n t of carbon d io x id e i s f u r n i s h e d by b u rn in g a candle in a c lo se d co n tain er of six l i t e r s cap acity . T h is p e r c e n t a g e i s s l i g h t l y l e s s t h a n t h r t s t a t e d b y Kye an d Lamb. By p l a t e c o u n t o f s t r a i n s fro m a c u t e male c a s e s o f g o n o r r h e a , i t was f o u n d t h a t e o u a l g r o w t h o f g o n o c o c c i o c c u r s i n a j a r i n which a c a n d l e f u r n i s h e s c a r b o n d i o x i d e and i n a j a r whose a t m o s p h e r e i s en ric h e d w ith ten p e r cent carbon d io x id e. This confirm s the o b s e r v a t i o n s o f C h r i s t i a n s e n and S c h o e n l e i n . An i n c r e a s e i n a t m o s p h e r i c m o i s t u r e c o n t e n t was f o u n d to s t i m u l a t e g r o w t h o f a e r o b i c s t r a i n s o f t h e gonococcu s c o n s i d e r a b l y . In a l l t h e e x p e r i m e n t s i n w hich a c o m p a r i s o n was made o f t h e e f f i c a c y o f a tm o sp h eric a i r , red u ced oxygen t e n s i o n produced by p a r t i a l vacuum, and a i r r e i n f o r c e d v d t h c a r b o n d i o x i d e , t h e l a t t e r c o n d i t i o n was f o u n d to p r o d u c e t h e b e s t g r ow th o f g o n o c o c c i . P r i m a r y s t r a i n s o f t h e g o n o c o c c u s were f o u n d to grow e q u a l l y w e l l on "choccla. t e " arrar i n a zone o f c a r b o n d i o x i d e e n r i c h m e n t w i t h e l o w e r l i m i t b e t w e e n two and 2 .J. p e r c e n t and an u o p e r l i m i t b e t w e e n I d an d 22 p e r c e n t . A m o d e r a t e g r o w t h o f g o n o c o c c i was o b t a i n e d i n p e r c e n t a g e s o f c a r b o n d i o x i d e a s h i g h a s hO p e r c e n t . The morph ology o f th e o r g a n i s m was c o n s i d e r a b l y c h a n g e d by g r o w th u n d e r h i g h c o n c e n t r a ­ tio n s of th is gas. h y d r o g e n , c a r b o n m ono xide, and n i t r o g e n g a s e s i n c o n c e n t r a t i o n s of 10 p e r c e n t i n a i r do n o t c o n t r i b u t e g r o w t h s t i m u l a t i o n to gonococci. BIBLIOGRAPHY 1. Bumm, E. "Der Mikro—O rgani sinus d e r go no r r h o i sc hen Schleiinhaut— Zrkrankungen. 'Gonococcus M e i s s e r ' " , U i e s b . , I S 5. Reviewed by W, B u l l o c h , "A System o f B a c t e r i o l o g y " , V o l . 2 , 21+0, 1929. 2. C h r i s t e n s e n , C. U. and S c h o e n l e i n , H. W. "The C u l t u r e Method f o r t h e D e t e c t i o n and I s o l a t i o n o f t h e Gonococcus , 11 Read b e f o r e t h e Ohio Branch, S o c i e t y o f A m er ican B a c t e r i o l o g i s t s , May, 19h0. A b s t r a c t , J o u r n a l o f B a c t e r i o l o g y , Vol. Uo, l o 2 , l^Uo. 3. F e r g u s o n , U. H. Wherry, W. E . and O l i v e r , " A d a p t a t i o n to Gonococcus and The J o u r n a l o f 5. C hapin, C. W. "Carbon D ioxide i n th e P r i m a r y C u l t i v a t i o n o f the Gonoc oc cu s," The J o u r n a l o f I n f e c t i o u s D i s e a s e s , Vol. 23, 3h2, 191s. 6. R u o d i g e r , E. H. " E x c l u s i o n o f A i r i n th e C u l t i v a t i o n o f th e Gon ococcus," The J o u r n a l o f I n f e c t i o u s D i s e a s e s , V ol. 2U, 37b, 19I S . 7. S c h w a r tz , E. C. "A Mew C u l t u r e Method f o r th e Gonococcus," J o u r n a l o f Urol o g y , V ol. U, 329, 1920. S. R o c k w e l l , G. E. and KcXhann, C. F. "The Growth o f th e Gonococcus i n V a r i o u s Gaseous E n v ir o n ­ m e n t s , " The J o u r n a l o f I n f e c t i o u s D i s e a s e s , V ol. 20, 2^9» 1921. 9. E r i c k s o n , M. J . and A l b e r t , K. " C u l t i v a t i o n o f th e Gonococcus," The J o u r n a l o f I n f e c t i o u s D i s e a s e s , V o l. 70, 2oS, 1922. Unpublished m a t e r i a l . W. W. C e r t a i n T e nsi ons o f Oxygen a s Shown By o t h e r P a r a s i t i c and S a p r o p h y t i c B a c t e r i a , " I n f e c t i o u s D i s e a s e s , Vol. 19, 2SS, 191b. 10. T o r r e y , J . C. and B u c k e l l , G. T. " C u l t u r a l IM-thods f o r th e G onoco cc us," The J o u r n a l o f I n f e c t i o u s D i s e a s e s , Vo l. 31. 129, 1922. 11. McLeod, J . W., C o a t e s , J . C . , Hanpold, F. C . , P r i e s t l e y , D. P . and Wheatley, 3. " C u l t i v a t i o n o f the Gonococcus a s a Method i n th e D i a g n o s i s o f G o n orrh ea w i t h S o e c i e l R e f e r e n c e to t h e Oxydase R e a c t i o n and to A ir R e i n f o r c e d i n i t s Carbon D i o x i d e C o n t e n t , " J o u r n a l o f P a t h o l o g y and B a c t e r i o l o g y , V o l. 39. 2?1 . I ? ? 11* 12. L e a h y , A. D. a n d C a r p e n t e r , C. M. "The D i a g n o s i s o f Gonococcal I n f e c t i o n s lay t h e C u l t u r a l M e th o d , " American J o u r n a l o f S y p h i l i s , G o n o r r h e a an d V e n e r e a l D i s e a s e s . Vol.. 20. W . ~ i S 3 K ~ -------------------------------------------------------- ly . S n i n k , \Jm W. an d K e e f e r , C. S. " S t u d i e s o f Gonococcal I n f e c t i o n ; I A s t u d y o f t h e Mode o f D e s t r u c t i o n o f t h e Gonococcus i n V i t r o , " J o u r n a l o f C l i n i c a l I n v e s t i g a t i o n , V o l. 1 6 . 169, 1937. lH. ITeumnnr, H. " S i n e neue G onoko k k en - K u ltu rm et'n o d e," D e r m a t o l o g i s c h e T,f o c h e n s c h r i f t , Band 1 0 1 , 883, 1 9 39. l c. h e u n m n , H. " D i e K u l t u r d e r Gonokokken; M e th o d ik A-, C-, und DP l e t t e ; B i o l o g i e ; S i n t e i l u n g d e r Gonokokken n a c h ihrern b’a c h s t u m - v e r m o g e n i n n t m o s n h a r i s c h e r X u f t , b e i r e d u z i e r t e r S a u e r s t o f f sonnnung und i n COp — h a l t i g e r A t n o s p h a r e i n A.-, C-, u n d D- Stanrae," A r c h l v f u r D e r m a t o l o g l e und S y p h i l i s , Band 1 7 3 1 ^93» 1936. lo. F o rtn er, J. "Die R e i n z u c h t u n g d e r f u s i f o r m e n B a z i l l e n a u s Z a h n b e l a g , " Z e n t r a l b l a t t f u r 3 a k t e r i o l o g i e , P a r a s i t e n k u n d e und I n f e c t i o n s k r a n k h e i t e n , Band 9^» 1 77» 1929 . 17. n 'in k le r , ? .. " V e r g l e i c h e n d e IT ntersuchung en u b e r d a s T.fa.chstum von Gonokokken a u f den Veumnnnschen A—, C -, und D- F la t t e n und a u f d e r A s z i t e s u e a r o l a t t e , " D a r n a t o l o g i s c h e W o c h e n s c h r i f t , Band 1 0 9 , 8^+9, 1937* 18. C a m o b e l l, J . A. "Gas T e n s i o n s i n t h e T i s s u e s , " R e v i e w s , V o l. I I , 1 , 1931* Physiological 19. C aroenter, 20. Cummings, G. D. "A M o d i f i c a t i o n o f t h e F i l d e s A n a e r o b i c J a r , " Ame r i c a n J o u r n a l o f P u b l i c I d e a l t h . V ol. 22, UlO, 1 9^2. 21. h’y e , 22. G rona u, A. C. M. " D i a g n o s t i c P r o c e d u r e s and R e a g e n t s , " American P u b l i c H e a l t h A s s o c i a t i o n , F i r s t D c i t i o n , 19^-1. r . a n d L a n b , M. S . " I n c r e a s e d Carbon D i o x i d e T e n s io n As An Aid i n t h e P rim ary I s o l a t i o n of C e rta in (M en h itib ic) Pathogenic B a c t e r i a , " J o u r n a l o f t h e Am erican M ed ic al A s s o c i a t i o n , V o l . 1 0 6 , 10?-., 193&. " L a c t e r i o l o g i c D i a g n o s i s i n G o n o r rh e a o f th e M ale ," J o u r n a l o f L a b o r a t o r y an d C l i n i c a l M e d i c i n e , V o l . 2 ^ , 6*’!-3, 19^1 • ?3• C a r p e n t e r , C. M. and C h a r l e s , R. " I s o l a t i o n o f Meningococcus from t h e S e n i t o u r i n e r y T r a c t o f Seven P a t i e n t s , " American J o u r n a l o f P u b l i c H e a l t h , Vol. 32, 6Uo, 19^2. 2H. W ilso n , G-. S. "The P r o p o r t i o n o f V i a b l e B a c t e r i a i n Young Cul­ t u r e s , With S p e c i a l R e f e r e n c e to t h e Technique Employed i n C o u n t i n g , " J o u r n a l o f B a c t e r i o l o g y , Vol. 7, U09, 1922. 29. W i ls o n , P . W. and Kullman, E. 0.* "A S t a t i s t i c a l I n q u i r y i n t o Methods f o r E s t i m a t i n g Uumbers o f R h i z o b i a , " J o u r n a l o f B a c t e r i o l o g y , Vol. 22, 71, 1931 * 26, V a lle y , 27, Cohen, M. 3 . and F lem in g , J . S. "The D i a g n o s i s o f Epidemic M e n i n g i t i s and th e C o n t r o l o f i t s Tre atmen t by Rapid B a c t e r i o l o g i c and S e r o l o g i c M eth ods," 'The J o u r n a l of I n f e c t i o u s D i s e a s e s , V ol. 23, 33T» 191S. 28, S e w e l l , G . , C l a r k e , E . f and Mel son , E. "Use o f t h e C u l t u r e Method i n t h e C l i n i c a l Management o f G o n o r r h e a , " American J o u r n a l o f P u b l i c H e a l t h , V ol. 31, H57. l W . G-, and. R e t t g e r , L . F. "The I n f l u e n c e o f Carbon D ioxide on B a c t e r i a " J o u r n a l o f B a c t e r i o l o g y , Vol. l h , 101, 1927. ACKITOWLLDGLMEHT The a u t h o r w i s h e s to e x p r e s s h i s g r a t e f u l a p p r e c i a t i o n o f the k i n d n e s s and c o o p e r a t i o n shovm him by th e -personnel o f th e D i v i s i o n o f S o c i a l H y g ien e, D e t r o i t De par tm en t o f H e a l t h , where c l i n i c a l m a t e r i a l f o r t h i s s tu d y was o b t a i n e d . To numerous c o l l e a g u e s i n t h e B u r e a u o f L a b o r a t o r i e s , M ic h ig an D epartm ent o f H e a l t h , he w is h e s to e x p r e s s h i s s i n c e r e t h a n k s f o r a d v i c e an d t e c h n i c a l a s s i s t a n c e .