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Influence of ultraviolet irradiation of P-Aminobenzoic acid on its subsequent use by acetobacter suboxydans Kitts, Warren Dale 1949

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L £ 3 THE INFLUENCE OF ULTRAVIOLET IRRADIATION OF P-AMINOBENZOIC ACID ON ITS SUBSEQUENT USE BY ACETOBACTER SUBOXYDANS. A T h e s i s s u b m i t t e d i n P a r t i a l F u l f i l l m e n t of t h e Requirements f o r the Degree of MASTER OF SCIENCE IN AGRICULTURE i n t h e Department o f Animal Husbandry. The U n i v e r s i t y of B r i t i s h August, 1 9 4 9 . Columbia INFLUENCE OF ULTRAVIOLET IRRADIATION OF n-AMINOBENZOIC  ACID AND ITS SUBSEQUENT USE BY ACETOBACTER SUBOXYDANS. Warren D . K i t t s ABSTRACT T h i s s t u d y has been based on a w o r k i n g h y p o t h e s i s w h i c h p r o p o s e s t h a t t h e b a c t e r i c i d a l a c t i o n o f u l t r a v i o l e t l i g h t i s a t t r i b u t a b l e t o an a b s o r p t i o n o f ene rgy i n the u l t r a -v i o l e t r e g i o n by one o r more e s s e n t i a l c e l l u l a r m e t a b o l i t e s . I t i s f u r t h e r s u g g e s t e d t h a t t h e a b s o r p t i o n p r o d u c e s a m o l e c u l a r s h i f t i n t h e a b s o r b i n g m e t a b o l i t e ( i n t h i s case p - a m i n o b e n z o i c a c i d ) o f such a n a t u r e t h a t t h e a l t e r e d compound, w h i l e s t r u c t u r a l l y s t i l l q u i t e s i m i l a r t o i t s m e t a b o l i c p r e c u r s o r , i s s u f f i c i e n t l y d i f f e r e n t t o i n t e r f e r e w i t h t h e n o r m a l m e t a b o l i c f u n c t i o n s o f t he c e l l * F a c t e r i -o s t a s i s t h e n f o l l o w s and u l t i m a t e l y d e a t h ensues* The l i t e r a t u r e a s s o c i a t e d w i t h t h e b i o l o g i c a l a c t i o n o f u l t r a v i o l e t l i g h t and t h e s t u d y o f p - a m i n o b e n z o i c a c i d and s u l p h o n a m i d e s as w e l l a s o t h e r r e l a t e d s u b j e c t s g i v e s c i r c u m s t a n t i a l e v i d e n c e t o s u p p o r t t h e above h y p o t h e s i s has been r e v i e w e d and i s p r e s e n t e d by s e c t i o n s i n t h e t h e s i s * The e s t a b l i s h m e n t o f a m i c r o b i o l o g i c a l a s s a y p r o c e d u r e f o r the d e t e r m i n a t i o n o f p - a m i n o b e n z o i c a c i d was done i n o r d e r t o d e t e c t i f u l t r a v i o l e t r a d i a t i o n i s a b l e t o p roduce an a l t e r a t i o n i n t h e p - a m i n o b e n z o i c a c i d m o l e c u l e . A m i c r o b i o l o g i c a l a s s a y p r o c e d u r e f o r t h e d e t e r -m i n a t i o n o f p - a m i n o b e n z o i c a c i d has been recommended, as i t was found n e c e s s a r y t o m o d i f y t h e p u b l i s h e d a s s a y p r o -c e d u r e s . I t was s u g g e s t e d t h a t t h i s recommenda t ion s h o u l d be f o l l o w e d i n f u t u r e work a l o n g the l i n e s o f t h e e x p e r i -m e n t a t i o n s r e p o r t e d h e r e i n . I n t h e s t u d y o f the b i o l o g i c a l a c t i o n o f u l t r a v i o l e t i r r a d i a t i o n i t seems sa fe t o c o n c l u d e t h a t t h e h y p o t h e s i s upon w h i c h the p r e s e n t work i s based i s sound , a t l e a s t , i n p a r t , i n t h a t i t has been d e m o n s t r a t e d t h a t i r r a d i a t i o n o f p - a m i n o b e n z o i c a c i d so a l t e r s the compound t h a t i t c a n no l o n g e r a c t as an " e s s e n t i a l m e t a b o l i t e " f o r A c e t o b a c t e r  s u b o x y d a n s . under the p r e v a i l i n g c o n d i t i o n s * E x p e r i m e n t a t i o n s were c a r r i e d o u t t o show t h e e f f e c t o f u l t r a v i o l e t i r r a d i a t i o n on t h e c e l l s o f A c e t o b a c t e r  s u b o x v d a n s . and t h e subsequent s t u d y t o o b s e r v e i f t he " e s s e n t i a l m e t a b o l i t e " , p - a m i n o b e n z o i c a c i d , was a b l e t o c o u n t e r a c t t h e a c t i o n s o f t h e s e i r r a d i a t i o n s . A s t u d y o f t h e c h e m i c a l a c t i o n o f u l t r a v i o l e t l i g h t on p - a m i n o b e n z o i c a c i d was i n c l u d e d . D e t e r m i n a t i o n o f the a b s o r p t i o n c u r v e s o f n o r m a l and i r r a d i a t e d p - a m i n o b e n z o i c a c i d were c a r r i e d ou t w i t h t h e a i d o f t h e Beckman M o d e l DU S p e c t r o p h o t o m e t e r . I t was shown t h a t t h e a b s o r p t i o n c u r v e o f i r r a d i a t e d p - a m i n o b e n z o i c a c i d d i f f e r e d m a r k e d l y from t h a t o f n o r m a l p - a m i n o b e n z o i c a c i d * • K P r e l i m i n a r y a t t e m p t s t o i s o l a t e t h e i r r a d i a t i o n p r o d u c t o r p r o d u c t s o f p - a m i n o b e n z o i c a c i d were made* V a r i o u s s o l u b i l i t y t e s t s o f t he new compound were c a r r i e d ou t and r e c o r d e d . I t i s i m p o s s i b l e a t p r e s e n t t o s t a t e the i d e n t i t y o f the i r r a d i a t i o n p r o d u c t o f p - a m i n o b e n z o i c a c i d ; however f u t u r e work w h i c h i s p l a n n e d may be s u c c e s s f u l i n i s o l a t i n g t h e compound o r compounds i n a pure s t a t e and w i t h t h i s r e s u l t t he i d e n t i t y o f t h e s e compounds may be e s t a b l i s h e d * The o r i g i n a l h y p o t h e s i s emphas ized t h e emphas ized t h e l i k e l i h o o d t h a t t h e i r r a d i a t i o n p r o d u c t o f p - a m i n o b e n z o i c a c i d w o u l d a c t as. an a n t i - m e t a b o l i t e . The work r e p o r t e d , does n o t s u p p o r t such a c o n t e n t i o n i n e n t i r e t y . I t does sugges t t h a t e i t h e r an a n t i - m e t a b o l i t e i s formed o r more l i k e l y t h a t t h e " e s s e n t i a l m e t a b o l i t e " , p - a m i n o b e n z o i c a c i d , i s d e s t r o y e d by i r r a d i a t i o n as d e t e r m i n a b l e by m i c r o -b i o l o g i c a l a s s a y . i ACKNOWLEDGEMENT The writer wishes to take t h i s opportunity to thank Professor H.M. King, Head of.the Department of Animal Husbandry for providing the f a c i l i t i e s i n order that t h i s work could be accomplished. Sincere thanks are expressed to Dr. A.J. Wood, Associate Professor i n the Department of Animal Husbandry, for suggesting this problem and also for his dir e c t i o n , assistance and c r i t i c i s m during the course of t h i s study. The writer also wishes to express his gratitude to the Defence Research Board of Canada for t h e i r f i n a n c i a l assistance, and to the B r i t i s h Columbia Research Council for the use of certain equipment essential to the conductance of the work. TABLE OF CONTENTS I. Introduction 1 II. Literature Review: A. Action of U l t r a v i o l e t Light on B a c t e r i a l Growth 3 B. A Hypothesis Proposed to Explain the Effects of U l t r a v i o l e t Energy on C e l l u l a r Metabolism 7 C. Enlargement of the Working Hypothesis to Explain the Effects of U l t r a v i o l e t Energy on Cellular. Metabolism 10 D. p-Aminobenzoic Acid; Mode of Action of ' Sulphonamides 14 E. Relation Between Chemical Structure and Bacteriostatic A c t i v i t y of a Compound 19 F. Other Nu t r i t i o n a l Antagonistic Reactions.. 22 G. p-Aminobenzoic Acid and i t s Relat ion to Pteroylglutamic Acid i n Bacterial Nut r i t i o n . 25 H. Review of the Relationship Between p-Aminobenzoic Acid and Sunburn 30 I. Protection from Sunburn by p-Aminobenzoic Acid 39 III. Experimental A. Introduction 40 B. Establishment of the Assay Procedure 41 C. B i o l o g i c a l Aspects in Relation to Assay . Procedure: Growth Requirements for A. suboxydans. . 56 D. Correlation of Turbidity Readings with Actual C e l l Count 64 E. F i n a l Re"commended Assay Procedure 68 F. The Bi o l o g i c a l Action of U l t r a v i o l e t I r r a d i a t i o n 68 G. The Chemical Action of U l t r a v i o l e t Light on p-Aminobenzoic Acid 86 H. Preliminary Tests to Isolate the I r -radiation Product or Products of p-Aminobenzoic Acid 94 IV. Summary 95 Table of Contents continued; V. Appendices: Page A. Appendix I: Review of Photochemical Reaction 98 B. Appendix II: The Use of Microorganisms to Assay Vitamins 103 C. Appendix III: Procedures for Microbiological and Chemical Assay, of p-Amino-benzoic Acid 113 D. Appendix IV: Composition of Media Used i n the Present Work 119 E. Appendix V: Determination of the Number of Bacterial Cells i n the In-oculum . 120 F. Appendix VI: Description and Use of the General E l e c t r i c , Mercury Arc, 15 watt Germicidal Lamp 122 .VI. Bibliography 124 INTRODUCTION . It has been recognized for the past half century that u l t r a v i o l e t l i g h t i s capable of destroying bacteria and molds (Heyroth, 1941). The many studies on the mechanism of the l e t h a l action of u l t r a v i o l e t l i g h t which have been carried out, have f a i l e d to provide an adequate explanation for the b i o l o g i c a l action of these rays. Extensive reviews have been written around this general- subject. • (Blum, 1941; Heyroth, 1941; Salle, 1943.) It i s of interest to note the work of Harris and Hoyt (1919) in r e l a t i o n to the action of u l t r a v i o l e t l i g h t on bacteria. These workers came to the conclusion that.the aromatic amino acids,- tryptophan-e, -t-ryo-si-ne and phenyla-l i n e , present i n the b a c t e r i a l c e l l s , are able to absorb those l i g h t rays that are l e t h a l in action. The absorption of such rays re.sults in the death of the c e l l , or may i n some cases, bring about an i n h i b i t i o n of the c e l l ' s metabolic functions. A concept proposed in recent years attempts to explain the b a c t e r i o s t a t i c action of i n i m i c a l agents on the basis of an interference by these agents with certain "essential metabolites." The now c l a s s i c a l theory of sulphonamide action proposed by Fildes (1940) and by Woods (1940) pro-vides an excellent example of the application of t h i s con-cept. - 2 -The present work i s based on a working hypothesis which proposes that the b a c t e r i c i d a l action of u l t r a v i o l e t l i g h t i s attributable to an absorption of energy i n the u l t r a v i o l e t region by one or more essential c e l l u l a r metab-o l i t e s . It i s further suggested that the absorption pro-duces a molecular s h i f t i n the absorbing metabolite of such a nature that the altered compound while s t r u c t u r a l l y s t i l l quite similar to i t s metabolic precursor i s s u f f i c i e n t -l y d i f f e r e n t to int e r f e r e with the normal metabolic functions of the c e l l . Bacteriostasis then follows and ultimately death may r e s u l t . If such a theory proves tenable then certain points must be established from published work and by new experi-mental proof. The l i t e r a t u r e associated with the bio-l o g i c a l action of u l t r a v i o l e t l i g h t and other related sub-jects has been surveyed and i s given by sections in a review following this Introduction. It i s worthy of mention that u l t r a v i o l e t l i g h t has been u t i l i z e d for many p r a c t i c a l and commercial purposes i n the f i e l d s of Animal Husbandry and Food Technology. This subject has already been re-viewed by others (Steenbock et a l . , 1924; Hess and Weinstock 1924; Moran 1936; Jacobs 1944; Heyroth 1947; Manard 1947; Morrison 1948) and hence i t is f e l t that a r e p e t i t i o n of these reviews need not be included in t h i s report, although i t has been c a r r i e d out as background material. - 3 -L i t e r a t u r e Rev iew A . A c t i o n o f U l t r a v i o l e t L i g h t on B a c t e r i a l Growth T o - d a y i t i s g e n e r a l l y a c c e p t e d t h a t n e a r l y a l l b a c t e r i a may be k i l l e d o r a t t e n u a t e d by u l t r a v i o l e t ene rgy o f c e r t a i n w a v e l e n g t h s bu t t h a t d i f f e r e n t s p e c i e s v a r y c o n -s i d e r a b l y i n t h e i r s u s c e p t a b i l i t y t o d e s t r u c t i o n . I t has been s u g g e s t e d t h a t t h o s e o r g a n i s m s w h i c h o c c u r i n a s s o c i a t i o n w i t h t h e a n i m a l body a r e g e n e r a l l y more e a s i l y k i l l e d t h a n t h o s e whose env i ronmen t i s f l o o d e d w i t h s u n l i g h t ( L u c k i e s h , 1 9 4 6 ) . The l a t t e r may become more adap ted t o exposu re t o s o l a r ene rgy and p a r t i c u l a r l y t o i t s u l t r a v i o l e t component . ( F i g u r e 1 ) . The wave l e n g t h range o f b a c t e r i c i d a l a c t i v i t y i s most c o n v e n i e n t l y i l l u s t r a t e d by what has been t e rmed t h e g e r m i c i d a l c u r v e p r e s e n t e d as F i g u r e 2. I t may be n o t e d t h a t t h e r e g i o n o f b a c t e r i c i d a l a c t i v i t y ex t ends o v e r t h e wave-§ l e n g t h s 2960 and 2100 A w i t h maximum a c t i v i t y o c c u r r i n g between o 2537 and 2662 A . Gates (1929) n o t e d t h a t t he w a v e l e n g t h o f maximum l e t h a l e f f e c t i v e n e s s v a r i e s w i t h t h e s p e c i e s o f b a c t e r i a i r r a d i a t e d . He f o u n d t h a t t he most e f f e c t i v e wave -o l e n g t h f o r t h e d e s t r u c t i o n o f S t a p h , au reus was 2650 A and o f o r E . c o l i . 2537 A . I t i s i n t e r e s t i n g t o m e n t i o n t h e work o f S m i t h b u r n and L a v i n (1937) i n c o n n e c t i o n w i t h the i r r a d i a t i o n o f M y c o b a c t e r i u m t u b e r c u l o s i s . They f o u n d t h a t w i t h s u b l e t h a l o doses o f monochromat ic l i g h t (2537 A) t h i s o r g a n i s m d e c r e a s e d Solar Radiation x-rays y-rays U l t r a -V i o l e t Infra-Red 53000 A .... 3.14 x 10 2 Hertzian Waves^ Radio Waves ) " 1 1 — g 9000 10,000 R Wave Length _ J I : A n t i r a c h i t i c Radiation FIGURE I. Chart Showing Relation of Type and Wave Length of Radiant Energy 0 (Hawk, et a l . 1947) (Figures indicate wave lengths (A ) expressed i n A Footnote: One micron (JJL) - 0.001 m$. One millimicron (mu) - 0.001 u One Angstom unit (A) - 0.1 nyu. i n v i r u l e n c e and f i n a l l y became a v i r u l e n t w i t h o u t b e i n g d e s -t r o y e d . The r e s u l t i n g a v i r u l e n t o r g a n i s m was a b l e t o i n d u c e i m m u n i t y w h i l e t h o s e o rgan i sms o f t h e same s t r a i n t h a t were k i l l e d by t h e same w a v e l e n g t h s were n o t c a p a b l e o f i n d u c i n g a measurab le immuni ty i n v a c c i n a t e d a n i m a l s . U l t r a v i o l e t E n e r g y S 1 0 0 > •H CO H © 80 6 0 40 20 0 F a r M i d d l e ^ rc^rmicidal TCffTtn, 7 \ \ \ * 4* 2000 2400 2800 Angs t rom U n i t s Near 3200 FIGURE 2 : G e r m i c i d a l Curve . ( G e n e r a l E l e c t r i c B u l l e t i n , 1947) . The above r e s u l t s were o b t a i n e d when b a c t e r i a l c e l l s were exposed t o u l t r a v i o l e t i r r a d i a t i o n . There have been many i n v e s t i g a t i o n s t o d e t e r m i n e i f u l t r a v i o l e t i r r a d i a t i o n has an e f f e c t on c u l t u r e m e d i a . B l a n k and A r n o l d (1935) showed o t h a t u l t r a v i o l e t i r r a d i a t i o n ( a t 2537 A) o f aga r o r a g a r -w a t e r g e l s so a l t e r e i c u l t u r e media s u b s e q u e n t l y made f rom them t h a t t h e y w i l l n o t s u p p o r t the g rowth o f B . s u b t i l i s . Baumgar tner (1936) and P r a t t (1936) have shown t h a t i r r a d i a t -i o n o f c a r b o h y d r a t e med ia and c a r b o h y d r a t e s o l u t i o n s b r i n g s about a s h i f t i n pH towards g r e a t e r a c i d i t y . I t was s u g g e s t e d t h a t t h e changes o c c u r r e d as f o l l o w s : p o l y s a c c h a r i d e s ^ d i s a c c h a r i d e s ^ m o n o s a c c h a r i d e s ^ a l c o h o l s , a l d e h y d e s , k e t o n e s and a c i d s . No e x p e r i m e n t a l e v i d e n c e was p r e s e n t e d t o s u p p o r t t h i s c o n t e n t i o n . I f p l a t e s were i r r a d i a t e d and b e f o r e i n o c u l a t i o n t h e pH was r e a d j u s t e d t o the p r o p e r l e v e l f o r t h e p a r t i c u l a r o r g a n i s m t h e n t h e medium w o u l d s u p p o r t n o r m a l b a c t e r i a l g r o w t h . Most t e x t b o o k s o f b a c t e r i o l o g y d e v o t e some space t o the d e s c r i p t i o n o f t h e b a c t e r i c i d a l o r b a c t e r i o s t a t i c a c t i o n o f u l t r a v i o l e t ene rgy ( H e y r o t h , i941j S a l l e , 1943;; T o p l e y and W i l s o n , 1943.X A l t h o u g h t h e g e r m i c i d a l r ange o f 2000 t o 2950 A has been f i r m l y e s t a b l i s h e d f o r a c o n s i d e r a b l e t i m e no c o m p l e t e l y adequa te e x p l a n a t i o n o f the mechanism i n v o l v e d has y e t been o f f e r e d . A number o f w o r k i n g h y p o t h e s e s have been advanced ( H e y r o t h , 1941J S a l l e , 1943). B . A H y p o t h e s i s P r o p o s e d t o E x p l a i n the E f f e c t s o f U l t r a -. v i o l e t E n e r g y on C e l l u l a r M e t a b o l i s m ,  As men t ioned e a r l i e r a new c o n c e p t t o e x p l a i n b a c t e r -i o s t a s i s due t o i n i m i c a l agen t s has been p r o p o s e d . B r i e f l y t h i s r e c e n t c o n c e p t s u g g e s t s t h a t c e r t a i n agen t s w h i c h a r e b a c t e r i c i d a l owe t h e i r a c t i v i t y t o t h e i r a b i l i t y t o b l o c k e s s e n t i a l r e a c t i o n s , F i l d e s (1940) p r o p o s e d t h a t i n h i b i t o r s o f b a c t e r i a l g r o w t h a r e e f f e c t i v e as a r e s u l t o f t h e i r i n t e r -f e r e n c e w i t h an " e s s e n t i a l m e t a b o l i t e " ^ The a p p l i c a t i o n of t he F i l d e s 1 t h e o r y to. t h e g e r m i c i d a l e f f e c t o f u l t r a v i o l e t r a d i a t i o n s u g g e s t s i m m e d i a t e l y t h a t t h e i r r a d i a t i o n o f b a c t e r i a r e s u l t s i n t h e p r o d u c t i o n o f a s u b -s t a n c e o r s u b s t a n c e s w h i c h may i n t e r f e r e w i t h some " e s s e n t i a l m e t a b o l i t e " . I t i s known, i n c e r t a i n c a s e s a t l e a s t , ( M o r t o n , 1942) t h a t a b s o r p t i o n o f r a d i a n t ene rgy by a c h e m i c a l compound w i l l r e s u l t i n the p r o d u c t i o n o f a new compound o f s l i g h t l y a l t e r e d c h e m i c a l c o n f i g u r a t i o n . The a c t i o n o f u l t r a v i o l e t l i g h t on e r g o s t e r o l , f o r example r e s u l t s i n the f o r m a t i o n o f a new compound, namely c a l c i f e r o l ( H e y r o t h , 1941)• £ A c c o r d i n g t o F i l d e s (1940) an " e s s e n t i a l m e t a b o l i t e " i s a s u b s t a n c e w h i c h t a k e s p a r t i n a c h a i n o f s y n t h e s e s n e c e -s s a r y f o r b a c t e r i a l g r o w t h , A " g r o w t h f a c t o r " on t h e o t h e r hand , i s a s u b s t a n c e w h i c h must be s u p p l i e d and may be c o n s i d e r e d an " ' e s s e n t i a l m e t a b o l i t e " w h i c h , t h e c e l l canno t s y n t h e s i z e . - 8 • TACHYSTEROL CALCIFEROL ( D 2 > W i t h t h i s i n f o r m a t i o n a t hand i t does no t seem too i l l o g i c a l t o s u g g e s t t h a t u l t r a v i o l e t i r r a d i a t i o n o f b a c t e r i a may cause a change i n some " e s s e n t i a l m e t a b o l i t e " . T h i s change i s l i k e l y t o be so s l i g h t t h a t t he new compound formed i s s u f f i c -i e n t l y , l i k e i t s n o n - i r r a d i a t e d p r e c u r s o r t o e n t e r i n t o com-b i n a t i o n i n a c e l l u l a r sys tem i n p l a c e o f t h e n o r m a l m e t a b o l i t e . However, a t t h e same t i m e t h e change may be s u f f i c i e n t l y g r e a t t h a t t h e new compound w i l l f a i l t o meet t h e f u n c t i o n s e r v e d by t h i s m e t a b o l i t e . Such a compound s h o u l d p o s s e s s an a b s o r p t i o n maximum c l o s e l y a p p r o a c h i n g t h a t o f t h e most ge rm-- 9 -i c i d a l p o r t i o n o f u l t r a v i o l e t i r r a d i a t i o n . Among t h o s e com-pounds p o s s e s s i n g w e l l e s t a b l i s h e d m e t a b o l i c f u n c t i o n s and a t t he same t i m e p o s s e s s i n g a b s o r p t i o n maxima i n the g e r m i -c i d a l w a v e l e n g t h r ange a r e t h e p a r a d e r i v a t i v e s o f b e n z e n e . P - a m i n o b e n z o i c a c i d (PABA) and t h e amino a c i d s , t r y p t o p h a n e , t y r o s i n e and p h e n y l a l a n i n e , a l l p o s s e s s w e l l marked maxima w i t h i n t h e r ange o f 2960 t o 2100 A . H a r r i s and Hoyt (1919) r e p o r t e d t h a t t h e a r o m a t i c amino a c i d r a d i c a l s a r e among t h e s u b s t a n c e s i n b a c t e r i a t h a t a re a f f e c t e d by the a c t i o n o f u l t r a v i o l e t l i g h t . I n a d d i t i o n t o t h i s i n f o r m a t i o n t h e s e w o r k e r s c o n c l u d e t h a t the u l t r a v i o l e t r a d i a t i o n s p roduced by t h e m e r c u r y a r c o f w a v e l e n g t h s n o t ab so rbed by t y r o s i n e and p h e n y l a l a n i n e a r e r e l a t i v e l y non t o x i c . Kober (1915) has p r o v i d e d i n d i r e c t c o n f i r m a t i o n f o r H a r r i s and H o y t ' s work by showing t h a t t h e a b s o r p t i o n band f o r t y r o s i n e l i e s between 2480 and 2970 1 and t h a t f o r p h e n y l a l a n i n e between 2360 and 2710 8 . The a b s o r p t i o n maximum o f PABA i s a t 2785 X ( F i g u r e 3) w h i l e the a b s o r b i n g band ex tends o v e r t h e r a n g e o f 2350 t o 3100 A . S i n c e t h i s i s t h e c a s e , t h i s a r o m a t i c amine has the a b i l i t y t o abso rb t h o s e w a v e l e n g t h s o f u l t r a v i o l e t e n e r g y t h a t l i e w e l l i n t h e r e g i o n of b a c t e r i c i d a l a c t i v i t y as i l l u s -t r a t e d i n F i g u r e 2 . As f a r as i t i s known t h e r e has been no p r e v i o u s s u g g e s t i o n t h a t t he g e r m i c i d a l e f f e c t o f t h e wave-o l e n g t h r a n g e , 2960 t o 2100 A , i s due t o t h e a l t e r a t i o n o f PABA t o a compound t h a t w i l l p o s s e s s b a c t e r i c i d a l o r b a c t e r -i o s t a t i c p r o p e r t i e s . - 10 -23 24 2 5 26 27 28 29 30 31 32 33 Wave-Leng th (X x 1 0 - 2 ) F i g u r e 3 . U l t r a v i o l e t A b s o r p t i o n Spec t rum o f p -Amino B e n z o i c A c i d ( 0 . 0 0 0 0 5 m o l a r ) C . E n l a r g e m e n t o f t he W o r k i n g H y p o t h e s i s t o E x p l a i n t h e E f f e c t s o f U l t r a v i o l e t E n e r g y on C e l l u l a r M e t a b o l i s m . P r e v i o u s l y i t was s u g g e s t e d t h a t t h e u l t r a v i o l e t i r r a d i a t i o n o f an " e s s e n t i a l m e t a b o l i t e " ( e g . PABA) migh t so a l t e r t h a t m e t a b o l i t e t h a t i t c o u l d no l o n g e r s e r v e i t s n o r m a l f u n c t i o n i n c e l l u l a r m e t a b o l i s m . I n h i s r e c e n t r e v i e w on b a c t e r i a l m e t a b o l i s m , Woods (1947) p roposes t h a t the s t u d y o f t he f u n c t i o n a l mechan ics o f an " e s s e n t i a l m e t a b o l i t e " i s no t c o m p l e t e d when i t s c h e m i c a l i d e n t i f i c a t i o n i s e s t a b l i s h e d . He c o n s i d e r s t h a t i t i s e s s e n t i a l t o u n f o l d the p r e c i s e c e l l u l a r p r o c e s s e s i n w h i c h the f a c t o r o r f a c t o r s a r e c o n -c e r n e d and on t h e b a s i s o f t h i s has enumerated f i v e approaches - 1 1 -t o t h e g e n e r a l p r o b l e m . They a r e : ( 1 ) To demons t r a t e t h a t a component o f an I s o l a t e d enzyme sys t em i s i d e n t i c a l w i t h a known g r o w t h f a c t o r o r an 11 e s s e n t i a l m e t a -b o l i t e " . ( 2 ) To s t u d y the m e t a b o l i s m o f o rgan i sms grown on media d e f i c i e n t i n t h e f a c t o r i n q u e s t i o n . T h i s s t u d y t o be f o l l o w e d by a d e t a i l e d i n v e s t i g a t i o n o f any m e t a b o l i c p r o -c e s s w h i c h appears t o have been a f f e c t e d . ( 3 ) To employ s u b s t a n c e s , o f t e n a n a l o g u e s o f the f a c t o r s o r a n t i - m e t a b o l i t e s , w h i c h may s p e c i f i c a l l y i n h i b i t t he u t i l i z a t i o n o f the f a c t o r . (4) To s t u d y the m e t a b o l i s m o f t h e f a c t o r . i t s e l f and i t s l i n k a g e w i t h o t h e r m e t a b o l i c p r o c e s s e s o f t h e c e l l . ( 5 ) To d i s c o v e r , i n g rowth e x p e r i m e n t s , a f a c t o r w h i c h may be r e p l a c e d by a s u b s t a n c e ( X ) o f a d i f f e r e n t c h e m i c a l t y p e . These e x p e r i m e n t s may p o s s i b l y s u g g e s t t h a t the f a c t o r i s i n v o l v e d i n the s y n t h e s i s o f ( X ) o r v i c e - v e r s a . The above f i v e app roaches i n t h e s t u d y o f t he metab-o l i s m o f g rowth f a c t o r s have been used by many w o r k e r s as o u t l i n e d by Woods ( 1 9 4 7 ) . I t has a l r e a d y been r e p o r t e d t h a t t h e m o l e c u l a r change i n PABA on i r r a d i a t i o n r e q u i r e s the p r e s e n c e o f oxygen (Rothman and R u b i n , 1 9 4 2 ) . W i t h t h i s i n f o r m a t i o n i t may be p o s s i b l e t h a t on i r r a d i a t i o n o f PABA a c h a i n o f r e a c t i o n s o c c u r s w i t h the u l t i m a t e p r o d u c t i o n o f p - n i t r o b e n z o i c a c i d as the b a c t e r i o s t a t i c a n t i - m e t a b o l i t e . When p - n i t r o b e n z o i c a c i d i s r e d u c e d , PABA i s the r e s u l t i n g p r o d u c t . M a l a v i y a and Du t t (1936) made some q u a n t i t a t i v e o b s e r v a t i o n s on t h e n a t u r e o f t he p r o d u c t s formed by t h e - 12 -p r o l o n g e d exposure t o t r o p i c a l s u n l i g h t o f a number o f o r g a n i c compounds. They n o t i c e d t h a t o r t h o a m i n o b e n z o i c a c i d gave p h e n a z i n e - 1,5 - d i c a r b o x y l i c a c i d , the meta compound d i d not a l t e r , but t h e p a r a a c i d i n a l k a l i n e s o l u t i o n gave t h e sod ium s a l t o f p , p ^ - a z o b e n z e n e - d i c a r b o x y l i c a c i d . As a g u i d e t o f u t u r e work i t i s h y p o t h e s i z e d t h a t t h e f o l l o w i n g c h a i n o f r e a c t i o n s may o c c u r w i t h t h e p r o d u c t i o n o f p - n i t r o b e n z o i c a c i d o r p , p ^ - a z o b e n z e n e - d i c a r b o x y l i c a c i d as t h e b a c t e r i o s t a t i c a n t i - m e t a b o l i t e . p - a m i n o b e n z o i c a c i d N=0 p - h y d r o x y l a m i n e p - n i t r o s o -b e n z o i c a c i d b e n z o i c a c i d p - n i t r o -b e n z o i c a c i d o r : ^ C O O H - < ^ ^ > - NHHH-^ZJ^ COOH * HgO p , p -hyd razobenzene—dica rb -o x y l i c a c i d COOH -<^ZT^>- N a N - ^ . ^ COOH • H 20 p , p ^ - a z o b e n z e n e - d i c a r b o x y l i c a c i d - 13 -I t must be a d m i t t e d , however , t h a t t he a l t e r a t i o n s p roduced i n the PABA m o l e c u l e by u l t r a v i o l e t ene rgy may be s i m i l a r i n n a t u r e t o t h o s e a r i s i n g i n some o f the amino a c i d s . T ryp tophane and t y r o s i n e , h a v i n g an a b s o r p t i o n maximum b e -v i o l e t l i g h t . They combine w i t h oxygen under i t s i n f l u e n c e ( H a r r i s , 1 9 2 6 ) . The n o n - a r o m a t i c amino a c i d s such as g l y c i n e , a l a n i n e , v a l i n e , l e u c i n e and a s p a r t i c a c i d show no i n c r e a s e d r a t e o f o x i d a t i o n when i r r a d i a t e d o r show i t l e s s r e a d i l y ( H e y r o t h , 1 9 4 1 ) . H e n r i and h i s a s s o c i a t e s (1934) o b s e r v e d t h a t a f t e r i r r a d i a t i o n by a m e r c u r y a r c f o r f i f t e e n h o u r s , a 0.1 N aqueous s o l u t i o n o f g l y c i n e i n c r e a s e d i n pH f rom 4 . 8 t o 7 . 4 5 . They c o n c l u d e d t h a t t h i s change was due t o the f o r m a t i o n of ammonia as a r e s u l t o f h y d r o l y s i s : c h e m i s t r y o f PABA. I t may be t h a t , when t h i s a r o m a t i c amine i s s u b j e c t e d t o u l t r a v i o l e t r a d i a t i o n s , i t i s h y d r o l y z e d w i t h the f o r m a t i o n o f p - h y d r o x y b e n z o i c a c i d and ammonia: tween 2400 and 2900 X ( H e i d t , 1936) a r e s e n s i t i v e t o u l t r a -N H 2 C H 2 C 0 0 H «. H 2 0 • •> H0CH 2 C00H * N H 3 g l y c i n e (•0-amino a c e t i c a c i d ) ( < - h y d r o x y a c e t i c a c i d ) The above i n f o r m a t i o n may a i d i n e x p l a i n i n g the p h o t o -C00H f NH3 The a b i l i t y o f u l t r a v i o l e t r a d i a t i o n s t o p roduce b a c t e r i c i d a l o r b a c t e r i o s t a t i c e f f e c t s has been r e c o g n i z e d by many w o r k e r s as a p h o t o c h e m i c a l p r o c e s s ( H e y r o t h , 1 9 4 1 J - 14 -G l a s s t o n e , 1 9 4 8 ) , and c o n s e q u e n t l y i t i s i m p o r t a n t t o r e v i e w t h e modern v i e w s on p h o t o c h e m i c a l phenomena. A b r i e f r e v i e w o f p h o t o c h e m i s t r y has been i n c l u d e d i n t h i s r e p o r t as A p p e n d i x I , D. p - A m i n o b e n z o i c A c i d : mode o f A c t i o n o f S u l p h o n a m i d e s . I f an a n t i - m e t a b o l i t e i s formed f rom the i r r a d i a t i o n o f PABA t h e mechanism o f i t s a c t i o n may be s i m i l a r t o t h a t p r o p o s e d t o e x p l a i n t h e i n h i b i t o r y a c t i o n o f t h e sulphonamides . As was p r e v i o u s l y m e n t i o n e d , F i l d e s (1940) pu t f o r t h the c o n -c e p t t h a t c h e m o t h e r a p e u t i c agen t s may a c t e i t h e r by c o m b i n i n g w i t h an " e s s e n t i a l m e t a b o l i t e " o r c o m p e t i n g w i t h i t f o r a p l a c e on the s u r f a c e o f t h e enzyme w h i c h a c t i v a t e s i t . H e r e , i t i s n e c e s s a r y t h a t t he m o l e c u l a r s t r u c t u r e o f t h e i n i m i c a l agent s h o u l d r e s e m b l e t h a t o f the m e t a b o l i t e . I n 1940 Woods used t h i s concep t t o e x p l a i n the a n t i b a c t e r i a l a c t i v i t y o f t h e s u l p h o n a m i d e s . I t has s i n c e been used e x t e n s i v e l y t o i l l u s t r a t e the " e s s e n t i a l m e t a b o l i t e " h y p o t h e s i s . He has s u g g e s t e d t h a t t he s u l p h o n a m i d e s , because o f t h e i r s t r u c t u r a l s i m i l a r i t y t o the " e s s e n t i a l m e t a b o l i t e " PABA, f u n c t i o n by b l o c k i n g the enzyme s y s t e m w h i c h u s u a l l y u t i l i z e s t h i s com-pound . Woods was l e d t o t h i s h y p o t h e s i s by t h e f a c t t h a t the s u l p h o n a m i d e s a r e b a c t e r i o s t a t i c r a t h e r t h a n b a c t e r i c i d a l and t h a t . t h e i r a c t i o n can be r e v e r s e d by the a d d i t i o n o f l a r g e amounts o f PABA t o t h e su lphonamide p o i s o n e d c e l l - 15 sys tem.- H i s work i s g i v e n i n g r e a t e r d e t a i l a t t h i s p o i n t . He r e p o r t e d t h a t t h e b a c t e r i o s t a t i c a c t i o n o f s u l p h a n i l a m i d e was r e v e r s e d c o m p e t i t i v e l y by PABA. These two a n t a g o n i s t i c s u b s t a n c e s a r e , as has been m e n t i o n e d b e f o r e , v e r y c l o s e l y r e l a t e d s t r u c t u r a l l y , s i n c e t h e y d i f f e r o n l y i n t h e f a c t t h a t the su lphonamide g r o u p o f t h e f o r m e r i s r e p l a c e d by a c a r b o x y l g roup i n t h e l a t t e r ( T a b l e I I I ) . I t was t h e n s u g g e s t e d t h a t s u l p h a n i l a m i d e i n h i b i t s t h e enzyme r e a c t i o n i n v o l v e d i n t h e s y n t h e s i s of PABA and t h a t i t does so a t t h i s p o i n t by v i r t u e o f i t s c h e m i c a l s i m i l a r i t y t o t h e p r o d u c t of t h e r e a c t i o n . Woods (1940) d e m o n s t r a t e d t h a t t h e i n h i b i t o r y a c t i o n o f s u l p h a n i l a m i d e on the g rowth o f S t r e p t . h a e m o l y t i c u s was a n t a g o n i z e d by PABA. H i s r e s u l t s show t h a t one m o l e c u l e o f t he l a t t e r can n e u t r a l i z e t h e b a c t e r i o s t a t i c a c t i o n o f 500 t o 2 5 , 0 0 0 m o l e c u l e s o f t h e f o r m e r . S u b s t a n c e s r e l a t e d t o PABA were a c t i v e o n l y i n h i g h e r c o n c e n t r a t i o n s ( T a b l e I ) w i t h t h e e x c e p t i o n o f n o v o c a i n e , whose a c t i v i t y a p p r o a c h e s t h a t o f PABA. The r e v e r s a b i l i t y o f t h e a c t i o n of s u l p h a n i l a m i d e by PABA s u g g e s t e d t h a t t h i s a r o m a t i c amine i s i m p o r t a n t i n b a c t e r i a l m e t a b o l i s m and i t was p r e d i c t e d by Woods t h a t i t w o u l d appear a t some t i m e as a b a c t e r i a l g rowth f a c t o r . Rubbo and G i l l e s p i e (1940) i n t h e same y e a r found t h a t t h i s compound was n e c e s s a r y f o r t h e g r o w t h o f C I . a c e t o b u t y l i c u m . A f t e r t h i s d i s c o v e r y , Rubbo e t a l . ( 1941) t e s t e d a s e r i e s o f more t h a n t w e n t y - f i v e o r g a n i c compounds i n o r d e r t o show t h e c o n n e c t i o n between m o l e c u l a r p a t t e r n and g rowth s t i m u l a t i o n . TABLE I . A n t i - s u l p h a n i l a m i d e A c t i v i t y o f S u b s t a n c e s  R e l a t e d t o p - A m i n o b e n z o i c acid~TWoods. 1 9 4 0 ) . C o n c e n t r a t i o n of s u l p h a n i l a m i d e = 3 .03 x 1 0 ~ ^ M . Subs t ance A c t i v e a t M c o n e . p - A m i n o b e n z o i c A c i d 1 .2 - 5 .8 x 1 0 " 8 o - A m i n o b e r i z o i c A c i d m-Aminobenzo ic A c i d 0 . 9 x 10"3 p - N i t r o b e n z o i c A c i d 1.8 x 1 0 - 4 E t h y l - p - a m i n o b e n z o a t e 3 . 6 x 1 0 - 5 5 .8 x 1 0 ~ 8 N o v o c a i n e p - H y d r o x y b e n z o i c a c i d -p - T o l u i c a c i d mm B e n z o i c A c i d mm Benzamide 1.4 x 1 0 " 6 p-Aminobenzamide 2 - ( p - A m i n o b e n z y l a m i n s ) p y r i d i n e 0 . 9 x 1 0 - 3 p - A m i n o p h e n o l + S u l p h a n i l i c a c i d - i n d i c a t e s s u b s t a n c e i n a c t i v e a t 10"3M •+• i n h i b i t s g rowth down t o 3 . 6 x 10-5M + i n h i b i t s g r o w t h a t . 1 0 ~ 3 M The r e s u l t s o f t h e i r e x p e r i m e n t s a r e t a b u l a t e d i n T a b l e I I * These w o r k e r s o b s e r v e d t h a t t he compounds p o s s e s s i n g 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 a r e a r o m a t i c compounds c o n t a i n i n g a d i - s u b s t i t u t e d benzene r i n g i n w h i c h the s u b s t i t u e n t s occupy l r 4 p o s i t i o n s . The o r i e n t a t i o n appears t o be o f f u n d a m e n t a l i m p o r t a n c e , and any d e p a r t u r e s f rom i t r e d u c e s c o n s i d e r a b l y the a c t i v i t y o f t h e compound. The g r e a t e s t a c t i v i t y i s shown when one o f t h e two i s an amino group and t h e o t h e r a c a r b o x y l - 17 -or c a r b o x y m e t h y l g r o u p . The i m p o r t a n c e of th.e c a r b o x y l and the amino s u b -s t i t u e n t s i s b e s t i l l u s t r a t e d by c o m p a r i n g t h e a c t i v e and i n a c t i v e compounds g i v e n i n T a b l e I I . The r e p l a c e m e n t o f t he o f t h e c a r b o x y l g roup i n t h e 1:4 s u b s t i t u t e d r i n g o f PABA by - C H 2 O H , o r by - OH, o r t h e r e p l a c e m e n t of t h e amino g roup by - N ( C H ^ . ) 2 ° r - OH, r e s u l t s i n c o m p l e t e i n a c t i v a t i o n o f the new d e r i v a t i v e . I t has been s u g g e s t e d e a r l i e r i n t h i s r e p o r t t h a t when exposed t o u l t r a v i o l e t r a d i a t i o n s a change o f PABA may o c c u r w i t h the f o r m a t i o n o f a new s u b s t a n c e , namely p - n i t r o -b e n z o i c a c i d or p - h y d r o x y b e n z o i c a c i d . I t i s v e r y i n t e r e s t i n g t o s t u d y T a b l e s I and I I , and n o t i c e t h a t p - n i t r o b e n z o i c a c i d w i l l r e v e r s e t h e i n h i b i t o r y a c t i o n of s u l p h a n i l a m i d e on S t r e p t .  h a e m o l y t i c u s and a l s o has g rowth f a c t o r a c t i v i t y f o r C I . a c e t o b u t v l i c u m . I n a d d i t i o n , ' i t i s n o t e d t h a t p - h y d r o x y -b e n z o i c a c i d a p p e a r s t o be i n c a p a b l e o f r e v e r s i n g the b a c t e r i -o s t a t i c a c t i o n o f s u l p h a n i l a m i d e ( T a b l e I ) and a l s o appea r s t o be i n a c t i v e as a g rowth f a c t o r ( T a b l e I I ) . However , i t cannot be c o n c l u d e d a t t h i s p o i n t t h a t p - h y d r o x y b e n z o i c a c i d i s t h e s o l e i r r a d i a t i o n p r o d u c t o f PABA on t h i s b a s i s because t h e r e may be a r e l a t i o n s h i p o f p - n i t r o b e n z p i c a c i d t o t h e s u b s t a n c e , p , p ^ " - a z o b e n z e n e - d i c a r b o x y l i c a c i d , as d e s c r i b e d by M a l a v i y a and Du t t ( 1 9 3 6 ) . I t i s a l s o p o s s i b l e t h a t t h e i r r a d i a t i o n o f PABA p roduces a s e r i e s o f r e a c t i o n s w i t h t h e f o r m a t i o n o f a number o f s t r u c t u r a l l y s i m i l a r compounds . - 18 -TABLE I I . Growth F a c t o r A c t i v i t y of S u b s t a n c e s S t r u c t u r a l l y R e l a t e d  t o p - A m i n o b e n z o i c A c i d (Rubbo e t a l< 1 9 4 1 K Subs t ance Growth Growth F a c t o r A c t i v i t y * 1 u n i t i n p - A m i n o p h e n y l a c e t i c a c i d 0 .00001 u g . p - A m i n o b e n z o i c a c i d + 0 . 0 0 0 1 u g . p - A m i n o b e n z o i c a c i d (Na e s t e r ) 0 . 0001 Ug. p - A m i n o b e n z o i c a c i d ( e t h y l e s t e r ) * 0 . 0 0 0 1 / l g . p - A m i n o b e n z a l d e h y d e 0 .0001 u g . p - A m i n o b e n z o i c a c i d ( b e n z o y l t 0 . 001 u g . d e r i v a t i v e ) p - A m i n o b e n z o i c a c i d 0 . 0 0 1 •ug. ( d i e t h y l a m i n o e t h y l e s t e r ) p - N i t r o b e n z o i c a c i d 0 .001 / i g . p - N i t r o ' b e n z a l d e h y d e + 0 . 0 1 u g . p - M e t h y l b e n z o i c a c i d + 1.0 u g . o - A m i n o b e n z o i c a c i d 1 0 . 0 >lg. m-Aminobenzo ic a c i d 1 0 . 0 u g . p -Aminobenzamide 1 0 . 0 J i g . p - A m i n o b e n z y l a l c o h o l - — p - H y d r o x y b e n z o i c a c i d - --p - A m i n o p h e n o l - --p - D i m e t h y l a m i n o b e n z a l d e h y d e - mm mm p-Aminobenzene su lphonamide mm — Benzene mm mm mm B e n z o i c a c i d mm mmmm I n o s i t o l - mm mm N i c o t i n i c a c i d - — T h i a m i n - mm mm Tryp tophane - --T y r o s i n e mm — C h o l e s t e r o l 7 mm mm x Growth F a c t o r A c t i v i t y i s t h e s m a l l e s t amount of m a t e r i a l n e c e s s a r y t o s t i m u l a t e v i s i b l e g rowth of C I . a iceto-b u t y l i c u m i n 5 m l . o f b a s a l medium under a n a e r o b i c c o n -d i t i o n s a t 3 7 ° C . f o r 48 h o u r s . - 19 -E . R e l a t i o n Between C h e m i c a l S t r u c t u r e and B a c t e r i o s t a t i c ~ A c t i v i t y o f a Compound > The b a c t e r i o s t a t i c mechanism o f s u l p h a n i l a m i d e p r o -posed by Woods (1940) and F i l d e s (1940) has been w e l l e s t a b -l i s h e d by subsequent w o r k e r s (Rubbo and G i l l e s p i e , 1940 ; Lampen and P e t e r s o n , 1 9 4 1 ; Landy and Wyeno, 1 9 4 1 ) . Some i n v e s t i g a t o r s have c o r r e l a t e d t h e a c t i v i t y o f su lphonamides t o t h e i r a c i d d i s s o c i a t i o n c o n s t a n t ( F o x and R o s e , 1942 ; Schmelkes e t a l , 1 9 4 2 ) , w h i l e o t h e r s seem t o s u g g e s t t h a t the . a c t i v i t y i s r e l a t e d t o the b a s i c d i s s o c i a t i o n c o n s t a n t ( p K b ) . Not o n l y has PABA a s t e r i c s i m i l a r i t y t o s u l p h o n a m i d e s , b u t i t a l s o has a c h a r a c t e r i s t i c a b i l i t y t o form s a l t s s i m i l a r t o t h a t d r u g . B e l l and R o b l i n (1942) made a s t u d y o f t h e p h y s i c o -c h e m i c a l p r o p e r t i e s o f s u l p h a n i l a m i d e . They s u g g e s t e d t h a t t h e b a c t e r i o s t a t i c a c t i v i t y o f s u l p h a n i l a m i d e t y p e compounds may be c o r r e l a t e d w i t h t h e n e g a t i v e c h a r a c t e r of t h e S O 2 group and w i t h t h e a c i d d i s s o c i a t i o n c o n s t a n t . These i n v e s t i g a t o r s p o i n t e d ou t t h a t t h e more n e g a t i v e t h e S O 2 group the more a c t i v e the compound w i l l b e , and a l s o t h e more n e g a t i v e t h e S 0 2 g roup i s , the more i t r e s e m b l e s the C O 2 group i n PABA a t pH %. At pH 7 the c a r b o x y l g roup i n PABA i s over n i n e t y - n i n e p e r c e n t i o n i z e d arid c o n s e q u e n t l y t h e C 0 2 group c a r r i e s a n e g a t i v e c h a r g e . The c a r b o x y l i o n may i n -c r e a s e the b a s i c i t y o f t h e p a r a amino r a d i c a l t h r e e - f o l d o v e r t h e v a l u e o b t a i n e d w i t h u n - i o n i z e d c a r b o x y l i n an a c i d medium. W i t h t h e s e d a t a . B e l l and R o b l i n (1942) p r e d i c t e d t h e r e l a t i v e - 2 0 -a c t i v i t y o f the i o n i c and m o l e c u l a r forms o f t h e s u l p h a n i l -amide t y p e d r u g s . Schmelkes and Wyss (1942) o b s e r v e d t h a t the a n t i - b a c t e r i a l e f f i c i e n c y o f a s u l p h o n a m i d e as measu r -a b l e by i t s a b i l i t y t o overcome PABA was dependent upon t h e pH o f t h e medium. They n o t i c e d t h a t s u l p h o n a m i d e s compete more f a v o r a b l y w i t h PABA a t a h i g h r a t h e r t h a n a t a l o w pH where t h e . a c i d i c d i s s o c i a t i o n i s r e l a t i v e l y s m a l l f o r a weaker a c i d . Kumler and Ha lvers tad t (1941) s u g g e s t e d t h a t t h e a c t i v i t y o f s u l p h a n i l a m i d e compounds may be a s s o c i a t e d w i t h t h e c o n t r i b u t i o n o f t h e r e s o n a t i n g fo rm w i t h a s e p a r a -t i o n o f t he c h a r g e : 0 The o b s e r v a t i o n made by Kumler and D a n i e l s (1943) i n d i c a t e s t h a t any e f f e c t t h a t i n c r e a s e s t h e r e l a t i v e n e g a -t i v e cha rge on t h e S O 2 g roup w i l l i n c r e a s e the c o n t r i b u t i o n o f t h i s f o r m w i t h a s e p a r a t i o n of c h a r g e . The o b s e r v a t i o n t h a t the b a c t e r i o s t a t i c a c t i v i t y i s a s s o c i a t e d w i t h t h e n e g a t i v e c h a r a c t e r o f t h e SO^ g roup i s c o m p a t i b l e w i t h t h e i d e a t h a t t he a c t i v i t y i s a s s o c i a t e d w i t h the r e s o n a t i n g form h a v i n g a s e p a r a t i o n o f c h a r g e . The n e g a t i v i t y o f t h e S O 2 g roup and t h e c o n t r i b u t i o n o f t h e r e s o n a t i n g f o r m w i t h a s e p a r a t i o n o f cha rge a r e c o n c o m i t a n t f a c t o r s r e l a t i n g t o one a n o t h e r . As t h e c o n t r i b u t i o n o f t h e r e s o n a t i n g f o r m w i t h a s e p a r a t i o n o f charge i n c r e a s e s the amino g roup i s a f f e c t e d 0 H N R - 2 1 -t h r e e w a y s . The amino g roup has a t e n d e n c y t o become co?<-planar w i t h the r i n g ; i t t a k e s on a p l u s c h a r g e ; and i t becomes doub le bonded t o c a r b o n , r e s u l t i n g i n a q u i n o i d a l s t r u c t u r e ( K u m l e r and D a n i e l s , 1943). Kumle r and D a n i e l s (1943) b e l i e v e t h a t i t i s t he se p r o p e r t i e s o f t he p a r a amino g roup t h a t i s c h i e f l y r e s p o n s i b l e f o r t h e a c t i v i t y o f t he s u l p h a n i l a m i d e t y p e compounds and n o t t h e n e g a t i v e c h a r a c t e r o f t h e S O 2 g r o u p . The r e a s o n s f o r p r o p o s i n g t h i s concep t were as f o l l o w s . I n the f i r s t p l a c e t h e S O 2 group; i n su lphonamides i s no t an a c t i v e group c h e m i c a l l y ; i t e n t e r s i n t o a l m o s t no r e a c t i o n s , w h i l e on the o t h e r hand, t h e a r o m a t i c amino g roup i s a v e r y r e a c t i v e o n e . The r e a c t i o n s . o f enzyme sys tems are e s s e n t i a l l y c h e m i c a l , and hence t h e c h e m i c a l l y r e a c t i v e components w o u l d more l i k e l y e x e r t t h e i r i n f l u e n c e t h r o u g h t h e p -amino g r o u p r a t h e r t h a n t h e S O 2 g r o u p . I t appears t h a t t h e amino g roup i s t h e f u n c t i o n a l p a r t o f the m o l e c u l e f o r a c t i v i t y and changes i n a c t i v i t y b r o u g h t abou t by p l a c i n g d i f f e r e n t R g roups on t he s u l p h o n -amide group r e s u l t s f rom the e f f e c t t h e R g roup has on t h e amino g roup t h r o u g h r e s o n a n c e and i n d u c t i o n ( K u m l e r and D a n i e l s , 1943). The u l t r a v i o l e t a b s o r p t i o n c u r v e s o f v a r i o u s benzene d e r i v a t i v e s were d e t e r m i n e d i n a c i d i c , b a s i c and w a t e r s o l u t i o n s by Kumler a n d S t r a i g h t (1943). A g e n e r a l r u l e was d e v e l o p e d i n t h a t t h e s p e c t r u m o f any a r o m a t i c amino ( u n s u b s t i t u t e d o r a l k y l d e r i v a t i v e ) compound w i l l r e v e r t i n a c i d ( s t r o n g enough t o c o n v e r t i t t o t h e s a l t ) t o t h e - 2 2 -spec t rum o f the c o r r e s p o n d i n g compound i n wh ich t h e amino g roup i s r e p l a c e d by hydrogen o r an a l k y l g r o u p . S u l p h a n i l -amide and PABA have a h i g h e r e x t i n c t i o n c o e f f i c i e n t i n b a s i c t h a n i n sodium c h l o r i d e o r w a t e r s o l u t i o n . T h i s i n d i c a t e s t h a t t h e main r e s o n a n c e form makes a g r e a t e r c o n t r i b u t i o n i n t he i o n t han i n the u n d i s s o c i a t e d m o l e c u l e . Kumler and H a l v e r s t a d t (1941) showed t h a t s i n c e PABA and su lphonamides have s i m i l a r r e s o n a t i n g s t r u c t u r e s w i t h a s e p a r a t i o n o f c h a r g e t h e t h e r a p e u t i c p o t e n c y may be a s s o c -i a t e d o n l y w i t h forms w h i c h have s u c h a. c h a r g e . F . O the r N u t r i t i o n a l A n t a g o n i s t i c R e a c t i o n s P u r s u i n g the h y p o t h e s i s o f Woods, o t h e r i n v e s t i g a t o r s , i n e f f o r t s t o p roduce b a c t e r i o s t a t i c compounds, a p p l i e d t o o t h e r v i t a m i n s s t r u c t u r a l changes s i m i l a r . t o t h o s e i n v o l v e d i n p a s s i n g f rom PABA t o s u l p h a n i l a m i d e . The c o n c e p t t h a t a d i s e a s e may be n e g a t i v e l y p r o d u c e d by the r e d u c t i o n o r o m i s s i o n o f v i t a m i n s i n t h e d i e t was s t a r t l i n g enough when f i r s t p r e s e n t e d . Y e t even more s p e c t a c u l a r i s t h e f a c t t h a t c e r t a i n compounds s t r u c t u r a l l y r e l a t e d t o e s s e n t i a l v i t a m i n s , p r o t e i n d e r i v a t i v e s and o t h e r i m p o r t a n t m e t a b o l i t e s may a c t u a l l y i n d u c e t h e d e f i c i e n c y s i g n s a s s o c i a t e d w i t h t h e l a c k o f e s s e n t i a l n u t r i l i t e s . Mc I l w a i n (1940) found t h a t p y r i d i n e - 3 - s u l p h o n i c a c i d i n h i b i t e d b a c t e r i a l g r o w t h i n c o m p e t i t i o n w i t h n i c o t i n i c - 23 -a c i d and t h a t <*( - amino s u l p h o n i c a c i d s a c t e d s i m i l a r l y i n c o m p e t i t i o n w i t h ^ - a m i n o a c i d s (Mc I l w a i n , 1 9 4 1 ) . L i k e w i s e S n e l l (1941) and l a t e r Mc I l w a i n (1942) showed t h a t t h i o -p a n i c a c i d i n h i b i t e d b a c t e r i a l g r o w t h i n c o m p e t i t i o n w i t h p a n t o t h e n i c a c i d . Thus f a r t h e w o r k has i n v o l v e d one t y p e o f s t r u c t u r a l change a n d has been l i m i t e d by t h e d e s i r e t o p roduce o n l y i n h i b i t i o n o f m i c r o b i a l g r o w t h . The n e x t advance was made when i t was shown t h a t c e r t a i n compounds r e l a t e d s t r u c t u r a l l y t o v i t a m i n s or o t h e r m e t a b o l i t e s p r o d u c e d t y p i c a l s i g n s o f d e f i c i e n c y d i s e a s e s i n a n i m a l s . Woolley and W h i t e (1943) showed t h a t p y r i t h i a m i n e caused t h e p r o d u c t i o n o f t y p i c a l s i g n s o f t h i a m i n e d e f i c i e n c y i n m i c e . T h i s compound a p p a r e n t l y can p r o d u c e t h e d e f i c i e n c y symptoms i n mice more q u i c k l y and s e v e r e l y t h a n does f e e d i n g t h e mice on a d i e t d e f i c i e n t i n t h e v i t a m i n . From the above i n f o r m a t i o n i t can be c o n c l u d e d t h a t c e r t a i n compounds r e l a t e d s t r u c t u r a l l y t o v a r i o u s b i o l o g i c a l l y i m p o r t a n t s u b s t a n c e s a r e a b l e t o cause s p e c i f i c s i g n s o f i n h i b i t i o n or d e f i c i e n c y d i s e a s e s , and t h a t t h e s e s i g n s a r e r e v e r s e d by t h e m e t a b o l i t e s i n q u e s t i o n when g i v e n i n adequate amoun t s . Many s u c h s u b s t a n c e s have been d i s c o v e r e d or p r e p a r e d , and a r e u s u a l l y r e f e r r e d t o as m e t a b o l i c analogues o r c o m p e t i t i v e i n h i b i t o r s . F o r c o n v e n i e n c e , a few o f t h e s e compounds a r e t a b u l a t e d i n T a b l e I I I . TABLE I I I . N u t r i t i o n a l - Antagonists (Gale. 1947). Growth Fa c t o r Antagonist S u s c e p t i b l e Organism H 2N < COOH p-aminobenzoic a c i d H 2N >" S0 2NH 2 Sulphanilamide CI. a c e t o b u t y l i c u m and many others N i c o t i n i c A c i d P y r i d i n e - 3 - s u l p h o n i c a c i d Pr. v u l g a r i s R.CHNH2.C00H C e r t a i n amino-acids R.CHNH2.S0oH Sulphonic a c i d analogues Staph, aureus HocM^t- c-co-NH-cH-fChlxrtooU Pantothenic A c i d ck OH P a n t o y l - t a u r i n e S. haemolyticus S. l a c t i s L. arabinosus P r o p i o n i b a c t e r i a N - e.H hi A / - a " " Staph, aureus Thiamine. P y r i t h i a m i n e NH 2.CH 2.CH 2.C00H (3 - a l a n i n e NH 2.CH 2,CH(CH 3).C00H methyl, ft-amino-propionic a c i d Yeast " HH-co o i , U r a c i l « OC cHi Hf<- co B a r b i t u r i c A c i d Staph, aureus LcHori\ .^w'V R i b o f l a v i n th(X T °b An ^O^s^^ Mepacrine S. haemolyticus G. p-Aminobenzoic A c i d and i t s R e l a t i o n t o P t e r o y l g l u t a m i c A c i d i n B a c t e r i a l N u t r i t i o n  The r e c e n t c h e m i c a l d i s c o v e r y t h a t t h e PABA mo i e t y i s an e s s e n t i a l c o n s t i t u e n t of t h e p t e r o y l g l u t a m i c a c i d (PGA) m o l e c u l e p r o v i d e s an i n t e r e s t i n g example o f t h e g e n e r a l approach proposed by Woods ( 1 9 4 7 ) . I t i s suggested t h a t one of t h e f u n c t i o n s o f PABA i n c e l l u l a r m e t a bolism i s t o a c t as a p r e c u r s o r f o r the b i o l o g i c a l s y n t h e s i s of PGA and t h a t PABA may be c o n s i d e r e d an " ' e s s e n t i a l m e t a b o l i t e " o n l y i n the sense t h a t i t i s p a r t of a l a r g e r m o l e c u l e which i s the a c t u a l growth f a c t o r . B e f o r e t h e c h e m i c a l n a t u r e o f PGA was e s t a b l i s h e d t h e r e appeared t o e x i s t a b i o l o g i c a l r e l a t i o n between t h i s f a c t o r and PABA. Mayer (1943) r e p o r t e d t h a t a y e l l o w p i g ~ ment was formed i n c u l t u r e s of Mycobacterium t u b e r c u l o s i s when i t was c u l t u r e d i n media c o n t a i n i n g h i g h c o n c e n t r a t i o n s of PABA. I t was found t h a t t h i s pigment was not i d e n t i c a l t o r i b o f l a v i n and d i d not appear i n the c u l t u r e s when no PABA was p r e s e n t . A y e l l o w pigment was a l s o produced by t h i s organism when i t s growth menstruun c o n t a i n e d c e r t a i n c o n c e n t r a t i o n s o f p r o c a i n e . (A d e r i v a t i v e of PABA). Mayer's l a t e r work (1944) i n d i c a t e d t h a t t h e y e l l o w pigment i s formed by an enzymatic p r o c e s s and i s a p p a r e n t l y a compound d e r i v e d from PABA by o x i d a t i o n . He assumed t h a t the enzyme i s an o x i d a s e and suggested t h a t i t may be a s p e c i f i c enzyme, namely, PABA-oxidase, or i t may be a group of enzymes such as t h o s e i n v o l v e d i n t h e Dopa r e a c t i o n . - 26 -Compounds whose c h e m i c a l s t r u c t u r e s a re s i m i l a r t o PABA, such as a n i l i n e and s u l p h a n i l a m i d e d e r i v a t i v e s when added t o the medium f o r the growth of Mycobacterium t u b e r c u l o s i s d i d not p e r m i t t h e f o r m a t i o n o f t h e y e l l o w pigment (Mayer, 1 9 4 4 ) . In 1944 Mayer dem o n s t r a t e d t h a t s u l p h a n i l a m i d e i n -h i b i t e d the s y n t h e s i s of f o l i c a c i d by b a c t e r i a ; as the c o n c e n t r a t i o n of s u l p h a n i l a m i d e was i n c r e a s e d , t h e s y n t h e s i s of f o l i c a c i d by t h e b a c t e r i a d e c r e a s e d . F u r t h e r m o r e , Lampen and Jones (1946) observed t h a t b a c t e r i a which r e q u i r e : f o l i c j a c i d i n t h e i r media are not s u s c e p t i b l e t o s u l p h o n -amide i n h i b i t i o n . I t may be t r u e t h a t t h e s e b a c t e r i a have no f o l i c a c i d s y n t h e s i z i n g enzyme systems t o be i n h i b i t e d and thus t h e r e w i l l be no b a c t e r i o s t a t i c a c t i o n o f the sulphonamide. In a d d i t i o n t o the above o b s e r v a t i o n s , Lampen and Jones (1946) noted t h a t f o l i c a c i d w o u l d o v e r - r i d e t h e b a c t e r i o s t a t i c e f f e c t s of the sulphonamides as w e l l as would PABA. The antagonism between sulphonamides and PABA i s c o m p e t i t i v e (Ansbacher, 1 9 4 4 ) , w h i l e t h a t between s u l p h o n -amides and f o l i c a c i d i s n o n - c o m p e t i t i v e . I n o t h e r words, the same amount of PGA would overcome a l a r g e dose of t h e drug e q u a l l y as w e l l as a s m a l l e r amount. I t w i l l be i n t e r e s t i n g t o note i f t h e b a c t e r i o s t a t i c e f f e c t ( i f any) of t h e i r r a d i a t i o n p r o d u c t o f PABA w i t h normal PABA i s -c o m p e t i t i v e or n o n - c o m p e t i t i v e . I t does seem l i k e l y t h a t t h e former p r o d u c t w o u l d be competing w i t h PABA f o r t h e enzyme s u r f a c e ^ t h e b a c t e r i a l c e l l . I f t h e i r r a d i a t e d - 27 -product can b l o c k the enzyme system so t h a t normal PABA cannot be u t i l i z e d by the c e l l , t h e n , t h e o r e t i c a l l y the b a c t e r i a l c e l l w i l l be i n h i b i t e d u n t i l a proper c o n c e n t r a t i o n of PABA i s a v a i l a b l e t o o v e r - r i d e the " b l o c k i n g e f f e c t " of the i r r a d i a t i o n p r o d u c t . During t h e p a s t few y e a r s c o n s i d e r a b l e work has been r e p o r t e d on t h e a n t i b a c t e r i a l a c t i v i t y of su b s t a n c e s r e l a t e d t o PABA ( W i l l i a m s , 1944 ; M a r t i n and Rose, 1945 ; Lampen and Jones, 1946a , 1947 ; S a r e t t , 1 9 4 7 ) . Of t h e t h i r t y - t h r e e t e s t e d s u b s tances r e l a t e d to PABA ( M a r t i n and Rose, 1 9 4 5 ) , t h r e e of them were found t o be i n h i b i t o r y , namely, 3-hydroxy- and 3 - c h l o r o - 4 - a m i n o b e n z o i c a c i d , and 3 : 4 - d i a m i n o b e n z o i c a c i d . Lampen and Jones (1947) showed t h a t p - a m i n o b e n z o y l g l u t a m i c a c i d , p t e r o i c a c i d , PGA and p t e r o y l t r i g l u t a m i c a c i d are a l l l e s s a c t i v e on a molar b a s i s t h a n i s PABA. They conclude by p o i n t i n g out t h a t PGA, p u r i n e s and thymine a r e p r o d u c t s of enzyme systems i n which PABA f u n c t i o n s . A diagram was p r e s e n t e d i n t h e i r r e p o r t w h i c h shows the p o s s i b l e r e l a t i o n s of PABA and r e l a t e d compounds i n b a c t e r i a l m e t a b o l i s m ( F i g u r e 4 ) . PGA, i t i s c l a i m e d (Lampen and Jones, 1947) i s s y n t h e s i z e d from PABA and t h i s s y n t h e s i s i s i n h i b i t e d by the a d d i t i o n of sulphonamides. P - a m i n o b e n z o y l - L - g l u t a m i c a c i d (PABG) or p t e r o i c a c i d may be i n t e r m e d i a t e s i n t h i s s y n t h e s i s , but t h e r e i s no e v i d e n c e to support t h i s s t a t e m e n t . However PABG i s u t i l i z e d f o r growth o n l y a f t e r d e g r a d a t i o n of PABA ( R e a c t i o n I ) . R e a c t i o n 2 shows t h a t p t e r o y l - t r i g l u t a m i c a c i d - 2 8 -can be h y d r o l y z e d t o PGA. T h i s h y d r o l y s i s i s e s s e n t i a l b e f o r e the t r i g l u t a m i c a c i d can be u t i l i z e d by some organisms. I t i s suggested i n R e a c t i o n 3 t h a t t h e p r o d u c t o f t h e a c t i o n of PGA i s not thymine ( n or t h e p u r i n e ) as such, but r a t h e r some d e r i v e d or c l o s e l y r e l a t e d s u b s t a n c e . S a r e t t (1947) p o i n t e d out t h e i n t e r - r e l a t i o n s h i p between PABA and PGA as a growth f a c t o r f o r organisms. He r e p o r t e d t h a t t h e PGA was a b l e t o r e p l a c e PABA t o a s m a l l e x t e n t f o r one organism but not f o r a n o t h e r . He a l s o suggested t h a t PABA had o t h e r f u n c t i o n s b e s i d e s i t s use as a p r e c u r s o r o f PGA and t h a t c o n j u g a t e d PABA may have t o f r e e t h e PABA b e f o r e u t i l i z a t i o n by organisms which r e q u i r e t h a t compound i n i t s f r e e form. - 29 -(Amino a c i d s ( M e t h i o n i n e ( L y s i n e Growth Purine:—-»PurineX ^-Precursor PABA (1) I n h i b i t e d by s u l p h -onamides (2 ) • • P t e f o y l g l u t a m i c * pt e r o y l t r i g l u -a c i d tamic a c i d I n h i b i t e d by h i g h sulphon-amide concen-t r a t i o n s P t e r o i c a c i d PABG (added) Thymine-( 3 ) •» ThymineX: V P r e c u r s o r . (3) * Growth F i g u r e 4» P o s s i b l e R e l a t i o n s of p-Aminobenzoic A c i d and R e l a t e d Compounds i n B a c t e r i a l M e t a b o l i s m (Lampen and Jones. 1947*7. - 30 _ H. Review of the R e l a t i o n s h i p Between p-Aminobenzoic A c i d and Sunburn.  The l i t e r a t u r e r e v i e w c o v e r e d t o date has emphasized the r o l e of PABA and i t s s t e r i o c h e m i c a l l y r e l a t e d a n t i -m e t a b o l i t e s i n r e l a t i o n t o b a c t e r i a l growth. That the i r r a d -i a t i o n p r o d u c t s of PABA produce s y s t e m i c e f f e c t s i n a n i m a l s and man i s e v i d e n c e d by the e x t e n s i v e r e p o r t s which have been p u b l i s h e d on the r e l a t i o n s h i p of PABA t o t h e erythema of sunburn. S i n c e the met a b o l i s m of mammalian c e l l s i s p r o b a b l y s i m i l a r t o , i f not i d e n t i c a l w i t h t h a t o f b a c t e r i a , the l i t e r a t u r e d e a l i n g w i t h the b i o c h e m i c a l mechanism of t h e sun -burn r e a c t i o n i s r e v i e w e d below i n the hope t h a t i t may shed l i g h t on t h e wo r k i n g h y p o t h e s i s proposed f o r the guidance of t h i s p r o j e c t . The p h o t o c h e m i s t r y of t h e sunburn r e a c t i o n has been d i s c u s s e d by Blum ( 1 9 4 1 ) . The t h e o r y was advanced t h a t a substance e x i s t s i n the p h o t o s e n s i t i v e l a y e r s o f t h e s k i n h a v i n g an a b s o r p t i o n spectrum i d e n t i c a l w i t h or s i m i l a r t o the sunburn a c t i o n spectrum ( F i g u r e 5 ) . A c c o r d i n g t o t h i s t h e o r y a substa n c e or su b s t a n c e s undergo a p h o t o c h e m i c a l r e a c t i o n by a b s o r b i n g the e f f e c t i v e u l t r a v i o l e t r a y s t h u s g i v i n g r i s e t o a pr o d u c t o r p r o d u c t s w h i c h i s r e s p o n s i b l e f o r t h e b i o l o g i c r e a c t i o n r e f e r r e d t o u s u a l l y as erythema. A p p a r e n t l y sunburn i s caused by a narrow band of u l t r a v i o l e t r a y s i n the r e g i o n of 2900 t o 3100 X. The f i r s t r esponse i n normal s k i n produced by t h i s energy i s termed erythema or r e d d i n g of t h e s k i n . T h i s may p e r s i s t f o r s e v e r a l - 3'1 -days, a f t e r w h i c h i t i s o r d i n a r i l y f o l l o w e d by p i g m e n t a t i o n or t a n n i n g which may appear b e f o r e t h e erythema has c o m p l e t e l y s u b s i d e d (Blum, 1 9 4 1 ) . The pigment w h i c h f o l l o w s exposure t o sunburn r a d i a t i o n s c o n s i s t s of g r a n u l e s of m e l a n i n . 1.0 3400 Wavelength % F i g u r e 5 . A c t i o n Spectrum f o r Erythema P r o d u c t i o n ("Hausser, 1928) Blum (1941) r e v i e w s s e v e r a l s u g g e s t i o n s as t o the f o r m a t i o n of t h i s l a t t e r compound. I n g e n e r a l i t has been r e p o r t e d t h a t an enzyme t y r o s i n a s e c o n v e r t s t y r o s i n e i n t o m e l a n i n . The mechanism by which t h i s t r a n s f o r m a t i o n o c c u r s i s not f u l l y known, however, Raper (1927) suggests t h a t a h y d r o x y l a t i o n of the benzene n u c l e u s p r o b a b l y t a k e s p l a c e f o l l o w e d by r i n g c l o s u r e whereby i n d o l e d e r i v a t i v e s a r e formed: - 32 -NH 2 \ CH 2CH-C00H \ CH 2CHC00H NH CH 2CHC00H A V v OH OH T y r o s i n e 3,4-Dihydroxy-p h e n y l a l a n i n e (Dopa) Dopa-quinone H 0 / \ H o v \ GH, CHCOOH N H 5 ,6 d i h y d r o x y d i -hydro i n d o l -<* -c a r b o x y l i c a c i d ° i / V CHCOOH H 5,6-Quinone ( H e l l a c h r o m e ) COOH 5 , 6 - d i h y d r o indole-.<*> -c a r b o x y l i c a c i d M e l a n i n - 33 -I t i s c l a i m e d by Ansbacher (1941) t h a t PABA m o d i f i e s t h e f o r m a t i o n of m e l a n i n . He s t a t e s t h a t PABA i s one o f a number of t h e more r e c e n t l y d i s c o v e r e d v i t a m i n s which f u n c t i o n s i n the p r o c e s s d e a l i n g w i t h the p i g m e n t a t i o n of t h e s k i n . The othe r v i t a m i n s namely p a n t o t h e n i c a c i d and b i o t i n are a l s o found i n t h i s c a t e g o r y , and i t i s p o s s i b l e t h a t not one of these f a c t o r s , but a l l t h r e e p l a y an a p p r e c i a b l e p a r t i n what appears t o be a v e r y c o m p l i c a t e d problem. M a r c h l e v s k y and Mayer (1929), w h i l e s t u d y i n g the a b s o r p t i o n s p e c t r a o f d i - 3 u b s t i t u t e d benzene d e r i v a t i v e s , showed t h a t t h e p a r a compounds absorb l i g h t t o a much g r e a t e r degree t h a n e i t h e r the o r t h o or meta d e r i v a t i v e s . I t i s not e d i n F i g u r e 6 t h a t t h e a b s o r p t i o n maximum o f PABA i s a t 2785 X, w h i l e t h e o t h e r two d e r i v a t i v e s have much l o w e r maxima, w i t h the maxima s h i f t e d towards t h e v i s i b l e wave -l e n g t h s . T h i s work c o n f i r m s the e a r l i e r r e s u l t s of Rothman (1926) and Behaghel et a l . ( 1 9 2 8 ) , i n which q u a n t i t a t i v e s p e c t r o g r a p h s methods were used. Hausser and Vahle (1922), i n the course o f s t u d i e s on t h e erythema of sunburn, demonstrated t h a t a maximum erythema e f f e c t o c c u r r e d a t a wavelength of 2975 A* w i t h a sharp drop i n e f f e c t i v e n e s s i n the d i r e c t i o n of e i t h e r l o n g e r or s h o r t e r w a v e l e n g t h s . L a t e r , Hausser (1928) r e l a t e d t h i s erythema p r o d u c i n g wavelength t o the a b s o r p t i o n maximum of PABA. Rothman (1926) showed t h a t a PABA d e r i v a t i v e , p r o c a i n e , s e l e c t i v e l y absorbs and f i l t e r s out the r a y s c a u s i n g t h e - 3.4 -p e r s i s t e n t erythema of sunburn. L a t e r work from the same l a b o r a t o r y (Behaghel et a l . , 1928) attempted to a s c e r t a i n the p o r t i o n of the procaine molecule which possesses the s c r e e n -ing v a l u e . These s t u d i e s r e v e a l e d t h a t PABA and i t s derivatives were r e s p o n s i b l e f o r the - screening e f f e c t . I t was shown that the e s s e n t i a l c o n f i g u r a t i o n was para s u b s t i t u t i o n of the benzene r i n g w i t h the amino and c a r b o x y l i c groups. I t was f u r t h e r shown that the s u b s t i t u t i o n of the hydrogens of the amine or the c a r b o x y l groups d i d not a l t e r the sunburn pro-t e c t i n g q u a l i t i e s of PABA. The compounds s t u d i e d are shown i n Table IV and are d i v i d e d i n t o two groups on the b a s i s of t h e i r p r o t e c t i v e v a l u e . As p r e v i o u s l y s t a t e d the a b s o r p t i o n maximum of PABA o i s a t 2785 A.. I t i s known, however, t h a t p r o t e i n s c o n t a i n i n g tryptophane and t y r o s i n e , have a b s o r p t i o n maxima i n the re g i o n of 2700 - 2800 A (Blum, 1941) and a l s o t h a t these amino a c i d s have an a b s o r p t i o n band at 2790 and 2750 X. r e s p e c t i v e l y (Smith, 1929). From t h i s i t i s q u i t e probable t h a t the p r i n c i p a l a b s o r p t i o n of sunburn r a d i a t i o n by the epidermis i s due to p r o t e i n s c o n t a i n i n g the benzenoid amino a c i d s t r u c t u r e i n the c o n f i g u r a t i o n , (Blum, 1941) although other substances, f o r example PABA, may c o n t r i b u t e s i g n i f i c a n t l y . - 35 -12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 L 2200 ; - i i i 'S. » \ \ i \ \ -r 1 1 1 1 1 1 1 1 \ \ \ \ > \ » i i \ _ \ i \ i i i \ » • / •\ I ' \ / ^  1 t ) \ 1 1 1 \ i \ X / \ * s \ ' \ ^ \ \ 2700 3200 Angstrom U n i t s 3700 F i g u r e 6 . U l t r a v i o l e t A b s o r p t i o n S p e c t r a of Amino-b e n z o i c Acid's! ( 1 ) Ortho Meta ( 3 ) Para ( M a r c h l e v s k y and Mayer, 1 9 2 9 ) . - 36 -TABLE IV. P o s i t i v e Compounds Do. N e g a t i v e Compounds Do Not  Absorb U l t - r a v i o l e t I r r a d i a t i o n s i n the Sun- burn Region (Behaghel et a l . 1928). POSITIV 1. A 2. A 3. COOrl p-Amidobenzoesaure A n a e t h e s i n Novocainbase p-Amidobenzoesaure- p - A m i d o b e n z o y l d i -a e t h y l e s t e r a e t h y l - a m i d o a e t h a n o l 4 . 5. 6 . ./v/H-cocH3 C O o f ^ C H f ^ ^ H C t C06H fa) C h l o r h y d r a t der Novocainbase p - D i m e t h y l -aminobenzoesaure NEGATIV p - A c e t y l - a m i n o benzoesaure 7 . 8. 9. COO-M*" A n i l i n D i m e t h y l a n i l i n Benzoesaure-a e t h y l e s t e r - 37 -TABLE IV. ( c o n t i n u e d ) P o s i t i v e Compounds Do, N e g a t i v e Compound Do Not Absorb  U l t r a v i o l e t I r r a d i a t i o n s i n t h e Sunburn Region (Behaghel et a l . 1928). 10. CooH 11. NEGATIV PH CooH 12. A* COOH p - N i t r o b e n z o -esaure p-Oxybenzo-•I i j esaure p-Methoxy-benzoesaure 13. V cooU p - I s o p r o p y l -benzoesaure 14. COOH COOH T e r o p h t a l s a u r e B e n z o l - p - D i -carbonsaure 15. /vlh. S03tf p-Aminobenzol-s u l f o s a u r e 16. COOH 17. CH 2NH 2.C00.C 2H 5 G l y k o k o l l a e t h y -l e s t e r 18, P y r i d i n -carbon saure A m i n o e s s i g s a u r e -A e t h y l e s t e r T y r o s i n p-Oxyphenyl-d-amino-pro-p i o n s a u r e 19. M COOti 20. COOH 21, O-Aminobenzoe-saure M-Aminobenzoe-saure M-Aminobenzoe-saure a e t h y l e s t e r - 38 -TABLE IV ( c o n t i n u e d ) P o s i t i v e Compounds Do. N e g a t i v e Compounds Do Not  Absorb U l t r a v i o l e t I r r a d i a t i o n s i n t h e Sun- burn Region (Behaghel et a l . 1928). NEGATIV 22. cR coo C h l o r h y d r a t des D i -me t h y l a m i d o - b e n z o y l -p e n t a n o l s 23. C h l o r h y d r a t des T e t r a -m e t h y l d i a m i n o - b e n z o y l -p e n t a n o l s - 39 -I. P r o t e c t i o n from Sunburn by p.Aminobenzoic A c i d . During the past few years subsequent workers ( B i r d , 1942; Rothman and Rubin, 1942; Rothman and Henningsen, 1947; Kumler and Dani e l s , 1948) have shown that PABA and i t s d e r i v a t i v e s approach the requirements f o r an i d e a l sunscreen. These aromatic compounds are able to absorb those rays that w i l l produce erythema and remain non-toxic and n o n - i r r i t a t i n g f o r mammalian s k i n . - 40 -EXPERIMENTAL I . I n t r o d u c t i o n As s t a t e d p r e v i o u s l y the p r e s e n t work was d e s i g n e d t o t e s t a new h y p o t h e s i s proposed t o e x p l a i n the p h y s i o -l o g i c a l e f f e c t of u l t r a v i o l e t r a d i a t i o n s on l i v i n g c e l l s . T h i s h y p o t h e s i s proposes t h a t the b i o l y t i c a c t i o n of u l t r a -v i o l e t r a d i a t i o n s can be a t t r i b u t e d t o t h e a l t e r a t i o n s p r o -duced by such r a d i a t i o n s on c e r t a i n " e s s e n t i a l m e t a b o l i t e s " . As a s t a r t i n g p o i n t i t was suggested t h a t t h e " e s s e n t i a l ^ m e t a b o l i t e " i n v o l v e d might w e l l be PABA s i n c e i t s a b s o r p t i o n maximum l i e s i n the range o f maximum b i o l y t i c a c t i o n . The c i r c u m s t a n t i a l e vidence which might s u p p o r t such a hypo-t h e s i s has been r e v i e w e d i n the p r e v i o u s s e c t i o n s of t h i s t h e s i s . A number of means seemed p o s s i b l e t o determine i f u l t r a v i o l e t r a d i a t i o n does, i n f a c t , produce an a l t e r a t i o n i n t h e PABA m o l e c u l e . Such an a l t e r a t i o n s h o u l d be d e t e c t -• a b l e by p h y s i c o c h e m i c a l d e t e r m i n a t i o n s . P h y s i o l o g i c a l l y i t would be of g r e a t e r i n t e r e s t t o demonstrate such a l t e r a t i o n by the use of l i v i n g c e l l s . Both methods have been used i n the p r e s e n t work. A s u r v e y of the l i t e r a t u r e r e v e a l e d t h a t a m i c r o b i o l o g i c a l method f o r the d e t e r m i n a t i o n of PABA would o f f e r a number of advantages over the use of o t h e r c e l l p r e p a r a t i o n s or c h e m i c a l p r o c e d u r e s . -The e x t e n s i v e use i n r e c e n t y e a r s of m i c r o b i o l o g i c a l methods f o r v i t a m i n assay has demonstrated t h a t t h e s e methods p r o v i d e a - 41 -precision and a f a c i l i t y of assay not r e a d i l y obtainable through the use of intact animals. A number of such micro-b i o l o g i c a l procedures are available for PABA determination (Appendix I I ) . Two such methods are recorded here as Appendix I I I . The method of Landy and Dicken (1942) using Acetobacter suboxydans was selected for use i n the present work. II. Establishment of the Assay Procedure; At the outset, attempts were made to repeat the assay procedure of Landy and Dicken (1942) for PABA. From the results of these preliminary assays i t was evident that a number of physical and chemical factors influence the response of <A. suboxydans to PABA. For t h i s reason an attempt was made to study these factors. The experiments carried out and the results obtained are outlined below. Experiment I: Assay Procedure for PABA as outlined by Landy and Dicken (1942).  Eleven assays were ca r r i e d out following the pro-cedure of Landy and Dicken as given in Appendix I I I . The results of a l l these assays are presented i n Table V and a representative curve from one of them i s given i n Figure 7/. TABLE V. The G-rowth Response of A. suboxydans to Varying Increments of PABA Micrograms of PABA per 10 cc culture Assay Numbers 1 2 3 4 5 6 7 8 9 10 11 0 0.01 0.02 0.03 0.04 0.05 0 .06 0.07 0.08 0.09 0.1 0 . 0 0 4 H 0 .032 0 .276 0 . 2 8 4 0 .495 0 .569 0 .509 0 .538 0 .569 0 .569 0 .620 0 .004 0 . 0 3 2 0 .244 0 .149 0 .538 0 .523 0 .495 0 .569 0 .552 0 .585 0 .638 0 .004 0 .027 0.187 0 .244 0 .432 0 .532 0 .509 0 .585 0 .523 0 .538 0 .569 0 .004 0 .027 0 .092 0 .237 0 .444 0 .523 0 . 5 5 2 0 .523 0 .509 0 .585 0 .585 0 .009 0 .009 0 .051 0 .119 0 .161 0 .229 0 .658 0 .569 0 .552 0 .552 0 .638 0.004 0.009 0.041 0.194 0.174 0.194 0.678 0.569 0.638 0.569 0.569 0.004 0 .009 O.065 0 .092 0 .143 0 .155 0 .482 0 .602 0 .678 0 .569 0 .585 0 .009 0.018 0.055 0 .143 0 .143 0 .237 0 .585 0 .638 0 .602 0 .620 0 .602 0.018 0.056 0.222 0 .292 0.201 0 .229 0.319 0.337 0.347 0.409 0.469 0.013 0 .076 0.208 0.319 0.222 0.260 0.301 0.337 0.367 0.420 0.482 0.018 0.066 0.222 0.301 0.201 0 .252 0.284 0.337 0.377 0.444 0.513 x Growth was measured i n the Coleman Junior Spectrophotometer at 600 mu. Figures represent growth of A. suboxydans measured as Optical Density O.D. B Log I s 2-log G (Galvometer reading) I - 43 -R e f e r e n c e t o Ta b l e V and F i g u r e 7 r e v e a l s t h a t t h e response curve of the t e s t organism t o i n c r e a s i n g i n c r e m e n t s of PABA i s somewhat i r r e g u l a r , and o b v i o u s l y u n s u i t a b l e f o r p r e c i s e d e t e r m i n a t i o n s of PABA. The r e s p o n s e c u r v e g i v e n by Landy and Dick e n (1942) i n d i c a t e s a smooth and r e g u l a r r e sponse t o PABA. In a subsequent paper, Landy and S t r e i g h t o f f (1943) p r e s e n t n u m e r i c a l v a l u e s f o r the growth response o b t a i n e d w i t h A. suboxydans i n the presence of v a r i o u s l e v e l s of PABA. When t h e i r d a t a were p l o t t e d i t was i m m e d i a t e l y e v i d e n t t h a t the response curve was i r r e g u l a r , 'It appears t h a t a number of f a c t o r s might be r e s p o n s i b l e f o r t h i s i r r e g u l a r i t y . Experiment 2 t The E f f e c t of S u r f a c e Area of the Medium Upon the Growth of A. suboxydans.  A.-suboxydans o b t a i n s i t s energy f o r growth under the a s s a y c o n d i t i o n s from t h e o x i d a t i o n o f g l y c e r o l t o d i -h y d r o x y a c e t o n e : CH 2 0H CH 2 0H I I CHOH de h y d r o g e n a t i o n _ C -0 f 2H* | » \ CH 2 0H CH 2 0H 2 H # 4- J 0 2 - ~ * H 2 0 S i n c e oxygen i s the f i n a l hydrogen a c c e p t o r i t seemed l i k e l y t h a t one of the l i m i t i n g f a c t o r s i n the growth - 44 -of t h i s organism might w e l l be the oxygen l e v e l i n the growth menstruum. S i n c e the assay i s c a r r i e d out i n s t a t i o n a r y f l a s k s the s u r f a c e a r e a of t h e assay medium exposed t o the gas phase i s one of t h e main f a c t o r s d e t e r m i n i n g the oxygen a v a i l a b l e t o the c e l l f o r growth. To determine t h e TABLE V I . The I n f l u e n c e of S u r f a c e Area on the Growth  of A. suboxydans. Amount of Complete Growth Response b a s a l medium added t o S u r f a c e measured as O p t i c a l Density each f l a s k Area (ml.) i n s q . R e p l i c a t e s cm. 1 2 Average 5 1 8 .103 0 . 9 4 0 0 . 9 2 1 0 .930 10 1 6 . 9 8 9 0 .959 0 . 9 4 0 0 . 9 4 9 15 16 .626 0 .796 0 . 8 1 0 0 .803 20 15 .385 0 . 5 5 2 0 .569 0 . 5 6 0 Control (not i n -o c u l a t e d ) 16 .989 0..027 0 .022 0 .025 i n f l u e n c e of t h i s f a c t o r &.n the growth response c u r v e , the t e s t o r g a n i s m was grown i n the b a s a l mediun e n r i c h e d w i t h 0.05 micrograms PABA i n 50 m l . Erylenmeyer f l a s k s w i t h v a r i o u s amounts of medium p r e s e n t i n each f l a s k . By t h i s means the s u r f a c e a r e a of the c u l t u r e s v a r i e d from 15 . 3 8 5 t o 18 .103 s q . cm. The r e s u l t s are g i v e n i n Table V I . From these d a t a i t i s e v i d e n t t h a t t h e s u r f a c e a r e a does a f f e c t the growth r e s p o n s e . The a r e a p r o v i d e d by t e n m l . of - 45 -medium i n a 50 ml. f l a s k p e r m i t s of maximum growth a t t h e 0 . 0 5 microgram l e v e l of PABA. Such r e s u l t s would suggest t h a t the oxygen c o n t e n t of t h e medium i s not l i m i t i n g i n the a s s a y procedure s i n c e 10 m l . of medium are used i n a 50 ml. f l a s k . Experiment 3 • I n f l u e n c e of I n c u b a t i o n Temperature on t h e Growth Response of A. suboxydans. The s t e a d i n e s s of t h e i n c u b a t i o n temperature f o r A. suboxydans may i n f l u e n c e t o a degree the growth response of t h i s t e s t organism, and thus may be the cause of the i r r e g u l a r i t y of t h e r e s u l t i n g a s s a y c u r v e s . From th e l i t e r a t u r e i t i s noted t h a t A. suboxydans r e q u i r e s an i n c u b a t i o n temperature of 30°C (Landy and D i c k e n , 1 9 4 2 ) , u n f o r t u n a t e l y the i n c u b a t o r a v a i l a b l e f o r the p r e s e n t work had a t e m p e r a t u r e . v a r i a t i o n of 28 - 3 5°C. Experiments were conducted t o observe the e f f e c t of i n c u b a t i o n temperature on the growth response o f the t e s t o rganism. I n o c u l a t e d assay media were i n c u b a t e d at 28~35°C, 30°C ( u s i n g a water bath s e t a t t h a t t e m p e r a t u r e ) and-27°C The r e s u l t s of t h i s experiment are p r e s e n t e d i n Table V I I . The g r a p h i c a l r e p r e s e n t a t i o n of t h e s e r e s u l t s ( F i g u r e 8) shows t h a t a v e r y I r r e g u l a r growth response curve t o PABA by sub-oxydans o c c u r r e d when the c u l t u r e s were i n c u b a t e d a t 28 - 3 5°C. The c u l t u r e s grown at the s t e a d y t e m p e r a t u r e of 30°C r e s u l t e d i n a l e s s i r r e g u l a r c u r v e . However t h e r e TABLE V I I . I n f l u e n c e of S t e a d i n e s s of Temperature on the Growth Response of A. suboxydans t o PABA. Cone e n t r a t i o n I n c u b a t o r Temperature Water Bath Temperature 27°C of PABA /10 cc 28 -35°C 30°C B a s a l Medium R e p l i c a t e s Average R e p l i c a t e s Average i n micrograms 1 2 1 2 0 0 . 1 1 4 K 0 .114 0 .114 0 .114 0 .114 0 .114 0 . 0 4 6 0 . 0 1 0.181 0 .168 0 .174 0 .131 0 .161 0 .146 . 0 .114 0 .02 0 .252 0 .237 0 .244 0 .237 0 .227 0 .234 0.149 0 .03 0 .310 0 .387 0 .347 0 .292 0 .310 0 .296 0 .174 0 . 0 4 0 .284 0 .201 0 .244 0 .201 0.208 0.205 0 .187 0 .05 0 .569 0 .495 0 .530 0 .237 0 .260 0 .256 0.194 0 . 0 6 0 .337 0 .367 0 .352 0 .337 0 .328 0 .333 0.208 0 .07 0.3.37 0 .357 0 .347 0 .337 0 .357 0 .347 0.208 0.08 0.387 0 .377 0 . 3 82 0 .377 0 .301 0 .337 0 .222 0 . 0 9 0 .347 0 .347 0 .347 0 .328 0 .292 0 .310 0 . 2 2 2 0 .1 0 .328 0 .284 0 .305 0 .276 0 .328 0 .301 0 .237 K F i g u r e s r e p r e s e n t growth of A. suboxydans measured as O p t i c a l D e n s i t y . - 4 7 -s t i l l e x i s t s a d e f i n i t e drop i n t h e growth response of the t e s t organism a t t h e 0 .04 microgram l e v e l of PABA. As one would have a n t i c i p a t e d the growth of A. suboxydans i n a l l f l a s k s was s u b s t a n t i a l l y d e c r e a s e d on i n c u b a t i o n a t 27°C. I t i s e v i d e n t t h a t a ste a d y i n c u b a t i o n t e m p e r a t u r e i s de-s i r a b l e but i t i s e q u a l l y e v i d e n t t h a t temperature f l u c t u -a t i o n a l o n e i s not s u f f i c i e n t . t o e x p l a i n the v a r i a b i l i t i e s of the response c u r v e . Experiment 4s The E f f e c t of pH of t h e Medium upon t h e Growth of A. suboxydans  U n d e r k o f l e r e t a l . (1943) have s t u d i e d the i n -f l u e n c e of t h e hydrogen i o n c o n c e n t r a t i o n on the growth response of A. suboxydans i n the assay medium. T h e i r r e s u l t s are p r e s e n t e d i n Table V I I I . T h e i r r e s u l t s and those o f o t h e r s (Landy and Di c k e n , 1942j C h e l d e l i n and Benn e t t , 1945; S a r e t t and C h e l d e l i n , 1945) suggest t h a t the optimum pH of the c u l t u r e medium appears t o be a p p r o x i m a t e l y s i x . The media used i n the p r e s e n t work were a d j u s t e d t o t h i s pH. - 48 -TABLE V I I I . E f f e c t of pH of the Medium upon the Growth  of _A. suboxydans"" (Underkof l e r e_b a l . 1943) pH of Medium Growth Measured as O p t i c a l D e n s i t y Be f o r e S t e r i l i z a t i o n A f t e r S t e r i l i z a t i o n 24 hours 48 hours 5 . 3 0 6 .10 6.90 7 . 1 0 7 .75 4 . 5 5 5.-95 6 .75 7.18 7 .78 0 . 8 6 0 1.0'91 1 .041 1.208 0 .139 0 .527 0 .065 0 .033 0 .321 0 . 0 3 1 Experiment 5 : The E f f e c t of Age of I n i t i a l Inoculum of A. suboxydans on the I r r e g u l a r i t y of the Growth Response Assay Curve.  In the a s s a y procedure f o r the d e t e r m i n a t i o n of PABA, Landy and Dicken (1942) used a 24 hour o l d washed mother c u l t u r e of A. suboxydans f o r t h e i n i t i a l i n o c u l u m , w h i l e o t h e r workers ( S a r e t t and C h e l d e l i n , 1945) used a 3 0 - 4 0 hour o l d c u l t u r e . A c c o r d i n g t o Sherman and A l b u s (1923 , 1924) and Hegarty (1939) the p h y s i o l o g i c a l age of c e l l s d e termines t o a g r e a t degree the response of t h o s e c e l l s t o a new e n v i r o n -ment. For t h i s r e a s o n i t was thought t h a t i t would be d e s i r a b l e t o determine whether the age of the i n i t i a l i n -oculum d u r i n g the a s s a y procedure f o r PABA would have any e f f e c t , on t h e growth response c u r v e . - 49 -A s e r i e s of assays were p r e p a r e d i n which the age of the i n i t i a l i n o culum d i f f e r e d . A t w e n t y - f o u r hour and a f o r t y - e i g h t hour mother c u l t u r e of A. suboxydans were used as the i n o c u l a . The age of t h i s parent c u l t u r e has c o n s i d e r a b l e b e a r i n g on t h e adjustment of t h e organisms t o a f r e s h medium as can be seen from the r e s u l t s of t h i s experiment i n Table IX. However from t h e growth response c u r v e s of A. suboxydans as r e p r e s e n t e d i n F i g u r e 9, i t can be observed t h a t the i r -r e g u l a r i t y of t h e s e assay c u r v e s s t i l l e x i s t and t h a t the younger parent i n o c u l u m shows a g r e a t e r response t o the l e v e l of t h e growth f a c t o r , PABA, i n the c u l t u r e medium. In the l i g h t of the above experiments t o determine whether a c e r t a i n f a c t o r o r f a c t o r s would cause the i r -r e g u l a r i t y of the a s s a y growth response curve of A. sub- oxydans , a p o s s i b l e e x p l a n a t i o n can now be brought f o r t h t h a t shows why t h e r e e x i s t s such pronounced i r r e g u l a r i t y i n a l l the a s s a y e x p e r i m e n t s . These e r r a t i c growth response c u r v e s may be due t o the "clumping e f f e c t " of the t e s t organism. P r i o r t o measure-ment of t h e growth of the organism by the use of t h e s p e c t r o -photometer, the c u l t u r e i s d i l u t e d t o double i t s volume w i t h d i s t i l l e d w a t e r . The s o l u t i o n i s t h e n t h o r o u g h l y mixed so t h a t the growth response can be measured by means o f the r e s u l t i n g t u r b i d i t y of the c u l t u r e medium. The t e s t o rganism grows r a p i d l y i n t h e p r e s c r i b e d b a s a l medium f o r m i n g a t e n a c i o u s f i l m upon t h e s u r f a c e of the medium d u r i n g i t s growth p e r i o d . Even a f t e r d i l u t i n g the c u l t u r e and s h a k i n g TABLE IX. \ I n f l u e n c e of Age of Parent Inoculum of A. suboxydans on t h e Growth Response t o v a r y i n g C o n c e n t r a t i o n of PABA. Cone, of PABA per 10 c c . of 24 h r . Parent Inoculum 48 h r . Paren t Inoculum B a s a l Medium i n Micrograms R e p l i c a t e s Average R e p l i c a t e s Average 1 2 1 2 0 0 . 0 2 7 * 0 .032 0 .030 0 .022 0 .027 0 .025 . 0 1 0 .268 0 .284 0 .276 0.181 0 .168 0 .175 .02 0 .319 0 .337 0 .328 0 .276 0 .252 0 .268 .03 0 .398 0 .409 0 .403 0 .337 0 .337 0 .337 :o4 0 .398 0 .284 0 .341 0 .284 0 .284 0 .284 .05 0 .301 0 .292 0 .296 0 .319 0 .328 0 .323 .06 0 .252 0 .244 0 .248 0 .337 0 .337 0 .337 .07 0 .602 0 .552 0 .585 0 .456 0 .495 0 .476 .08 0.658 0 .602 0 .638 0 .409 0 .409 0 .409 .09 0 .602 0 .585 0 .594 0 .357 0 .337 0 .347 .1 0 .569 0 .620 0 .586 0 .367 0 .357 0 .362 x F i g u r e s r e p r e s e n t growth of A. suboxydans measured as O p t i c a l D e n s i t y . - 51 -the r e s u l t i n g m i x t u r e t h o r o u g h l y , i t i s d i f f i c u l t t o a s s u r e u n i f o r m d i s t r i b u t i o n of s i n g l e m i c r o b i a l c e l l s . I n s t e a d , of o b t a i n i n g a s i n g l e c e l l s u s p e n s i o n throughout the c u l t u r e medium, the b a c t e r i a l c e l l s have a tendency t o form groups or clumps which p r e v e n t homogeneous s u s p e n s i o n of the s i n g l e c e l l s . As a r e s u l t o f t h i s c lumping e f f e c t a f a l s e r e p r e s e n t a t i o n of t h e growth r e s p o n s e of t h e t e s t organism i s o b t a i n e d when such i s measured t u r b i d i m e t r i c a l l y . In t u r b i d i m e t r y (Hawk et a l . 1947) the l i g h t t r a n s -m i t t a n c e of a f l u i d i s i n f l u e n c e d not o n l y by t h e amount of l i g h t - a b s o r b i n g m a t e r i a l p r e s e n t i n s o l u t i o n but a l s o by the presence of l i g h t - s c a t t e r i n g or l i g h t - o b s t r u c t i n g m a t e r i a l such as i n s o l u b l e s u b s t a n c e s i n s u s p e n s i o n , ( e x . b a c t e r i a l c e l l s ) . Q u a n t i t a t i v e a n a l y s i s of m i c r o b i a l c e l l s i n s u s p e n s i o n based upon t h i s p r i n c i p l e i s known as t u r b i d i -metry or nephelometry. These two terms a r e s u b s t a n t i a l l y e q u i v a l e n t , a l t h o u g h nephelometry u s u a l l y r e f e r s t o the use of s c a t t e r e d l i g h t ( i e . l i g h t a t r i g h t a n g l e s t o the i n -c i d e n t beam) as a measure of t u r b i d i t y . T u r b i d i m e t r i c measurements can be c a r r i e d out by t h e same procedures and i n s t r u m e n t s used f o r the measurement of substances i n s o l u t i o n . The r e l a t i o n s h i p between th e amount of a substance i n s u s p e n s i o n and the t u r b i d i t y or t r a n s -m i t t a n c e of the f l u i d i s much more e m p i r i c a l t h a n f o r sub-s t a n c e s i n s o l u t i o n depending not o n l y on the amount of m a t e r i a l p r e s e n t but a l s o on the s i z e and shape of the suspended p a r t i c l e s , t h e i r r e l a t i v e o p a c i t y or t r a n s p a r e n c y , - 52 -the r e l a t i o n between p a r t i c l e s i z e and t h e wav e l e n g t h of l i g h t used, and the u n i f o r m i t y w i t h which a g i v e n t u r b i d i t y may be r e p r o d u c e d . I f a beam of l i g h t i s passed t h r o u g h a medium t h a t c o n t a i n s p a r t i c l e s or b a c t e r i a l c e l l s , t he l i g h t w i l l be s c a t t e r e d . In c o l l o i d a l s o l u t i o n s t h i s phenomenon i s known as t h e T y n d a l l e f f e c t . I t i s i p o s s i b l e t o v i s u a l i z e b a c t e r i a l c e l l s i n a homogeneous s u s p e n s i o n , and of t h e same s i z e and shape h a v i n g a r e l a t i v e o p a c i t y or t r a n s p a r e n c y . These c e l l s are a b l e t o absorb some l i g h t and r e f l e c t and t r a n s m i t the rema i n d e r . The r e f l e c t e d l i g h t i s d i r e c t e d on v a r i o u s l i n e s of r e f l e c t i o n . From t h i s i t can be r e a d i l y seen t h a t i f t h e b a c t e r i a l c e l l s a r e not suspended as s i n g l e c e l l s i n the s o l u t i o n , but r a t h e r as clumps, then each clump, b e i n g l a r g e r and of d i f f e r e n t shape as compared t o a s i n g l e bac-t e r i a l c e l l , are more opaque and a l s o w i l l t e n d t o r e f l e c t l e s s of the l i g h t beam. As was mentioned p r e v i o u s l y , A. suboxydans forms a t e n a c i o u s p e l l i c l e on the s u r f a c e of the medium. When th e c o n c e n t r a t i o n * of PABA i n the a s s a y medium i s a t 0.03 t o 0.04 micrograms per 10 c c , t h i s p e l l i c l e f o r m a t i o n appears t o approach a maximum. I t i s worthy of note t h a t t h e drop i n the response curve of the t e s t organism u s u a l l y appears at thes e c o n c e n t r a t i o n s . The growth of t h e organism has not n e c e s s a r i l y d e c r e a s e d a t t h e s e p o i n t s as compared t o the lower c o n c e n t r a t i o n of PABA i n the medium, but t h a t the - 53 -formed p e l l i c l e on the su r f a c e prevents homogeneous t u r b i d i t y when the d i l u t e d c u l t u r e i s shaken. This clumping e f f e c t b r i n g s about a f a l s e r e p r e s e n t a t i o n of the growth response of the t e s t organism when measured by means of t u r b i d i t y . R e c e n t l y a review of the l i t e r a t u r e on the a c t i o n o f Surface A c t i v e Agents was done ( K i t t s , 1949). It was noted that i f b a c t e r i a are not wetted by the medium, t h e y w i l l grow on the s u r f a c e i n the form of a f i l m ; i f b a c t e r i a are wetted, they w i l l not produce a p e l l i c l e but i n s t e a d a uniform clouding o f the medium. In 1946, Dubos and Davis (1946)-found t h a t Tween-80 a f a t t y a c i d ester type compound (a p o l y o x y a l k y l e n e d e r i v a t i v e of s o r b i t a n mono-oleate) caused mycobacteria t o grow d i f f u s e l y throughout the l i q u i d medium ra t h e r than as a su r f a c e p e l l i c l e . That t h i s r e s u l t was due to the wetting p r o p e r t i e s of Tween-80 i s suggested by a number of f a c t s . Growth developing i n the media c o n t a i n i n g non-wetting o l e i c e s t e r s , c o n s i s t e d of l a r g e clumps which were extremely r e s i s t a n t to d i s p e r s i o n . Furthermore, w i t h v e r y small amounts of the w a t e r - s o l u b l e es t e r (0.01 per cent Tween-80) growth was at f i r s t submerged and a f t e r a few days a f i n e p e l l i c l e formed at the s u r f a c e of the medium, probably due to the exhaustion of the su r f a c e a c t i v a t o r by h y d r o l y s i s and u t i l i z a t i o n by the organisms. With f u r t h e r a d d i t i o n of Tween-80 to the medium, causing wetting of the c e l l s , t he m i c r o b i a l p o p u l a t i o n d i s p e r s e d throughout the medium. - 54 -Woods (1949) r e c e n t l y has been u s i n g Tween-80 t o d i s p e r s e the growth of A. suboxydans. He found t h a t by adding t h i s w e t t i n g agent t o t h e c u l t u r e medium a t a f i n a l c o n c e n t r a t i o n of 0.5 per cent t h e f o r m a t i o n of the p e l l i c l e on the s u r f a c e of the medium was p r e v e n t e d . The organism developed t h r o u g h o u t t h e l i q u i d medium. From t h e s e p r e v i o u s o b s e r v a t i o n s by o t h e r w o r k e r s , i t may be p o s s i b l e t o overcome the i r r e g u l a r i t y of t h e growth response c u r v e of A. suboxydans t o i n c r e a s i n g i n c r e m e n t s of PABA by add i n g Tween-80 t o the c u l t u r e medium b e f o r e i n o c u -l a t i o n . Experiment 6r The Use of Tween-80 t o Submerge t h e Growth of A. suboxydans d u r i n g t h e Assay f o r PABA. A s e r i e s of a s s a y s were p r e p a r e d f o r t h e d e t e r m i n -a t i o n of PABA u s i n g the method of Landy and Dicken ( 1 9 4 2 ) , but adding d i f f e r e n t c o n c e n t r a t i o n s of Tween-80. The f i n a l c o n c e n t r a t i o n s of t h i s s u r f a c e a c t i v a t o r i n the growth medium were 0.1, 0.25, and 0.5 per c e n t . The v a r y i n g c o n c e n t r a t i o n s of Tween-80 were used t o determine what l e v e l was s u i t a b l e t o p r e v e n t t h e p e l l i c l e f o r m a t i o n of t h e t e s t o r g a n i s m . F i g u r e "10 r e p r e s e n t s the r e s u l t s t a b u l a t e d i n Table X. I t can be seen t h a t Tween-80 at a l e v e l of 0.5 per cent i n the c u l t u r e medium p r e v e n t s the f o r m a t i o n of a p e l l i c l e by A. suboxydans, and thus the growth response curve of t h i s t e s t organism t o PABA tends t o y i e l d a smooth c u r v e . TABLE X. The I n f l u e n c e of Tween-80 on t h e Growth Response of A. suboxydans t o I n c r e a s i n g Increments of PABA. Cone, of PABA i n micrograms No Tween per 10 c c . 0 .1 per cent Tween- 0.25 per cent Tween- 0 .5 per cent Tween-b a s a l medium 80 80 80 0 0.018H 0 .013 0 .013 0 .013 0 . 0 1 0 .143 0 .140 0 .149 0 .102 0 .02 0 .237 0 .237 0 .252 0 .284 0 .03 0 .306 0 .268 0 .310 0 .319 0 . 0 4 0 .301 0 .314 0 .357 0 .367 0 .05 0 .392 0 .347 0 .398 0 .398 0 .06 0 .420 0 .495 0 . 4 4 4 0 .482 0 .07 0 .523 0 .523 0 .482 0 .469 0.08 0 .516 0 .509 0 .516 0 .482 0 .09 0 .488 0 .523 0 .509 0 .658 0 . 1 0 .482 0 .500 0 .538 0 .670 x F i g u r e s r e p r e s e n t growth of A. suboxydans measured as O p t i c a l D e n s i t y , - 56 -The o t h e r t e s t e d l e v e l s of Tween-80 i n the a s s a y medium, 0.1 and 0.25 per c e n t , were not adequate t o pre v e n t t h i s s u r f a c e f i l m f o r m a t i o n . A d d i t i o n a l s e r i e s of assay d e t e r m i n a t i o n s f o r PABA u s i n g t h e same procedure and i n c o r p o r a t i n g the s u r f a c e a c t i v e agent were done t o check t h e above r e s u l t s . From thes e checks ( T a b l e X I ) the growth response c u r v e s a re drawn t o show t h a t the p r e v i o u s r e s u l t s can be r e p r o d u c e d and f u r t h e r t h a t the assays h a v i n g no Tween-80 c o n t i n u e t o produce an i r r e g u l a r growth response curve ( F i g u r e 1 1 ) . I I I . B i o l o g i c a l A spects i n R e l a t i o n t o Assay P r o c e d u r e : Growth Requirements f o r A. suboxydans.  In a d d i t i o n t o t h e e s t a b l i s h m e n t o f the a s s a y p r o -cedure f o r t h e ' d e t e r m i n a t i o n of PABA i t i s f e l t t h a t a d i s c u s s i o n of the growth r e q u i r e m e n t s of t h i s t e s t organism s h o u l d be i n c l u d e d at t h i s p o i n t . For purposes of c l a r i t y t h e s e b i o c h e m i c a l a s p e c t s w i l l be d i s c u s s e d under s e p a r a t e h e a d i n g s . (a) Carbon Source: The carbon s o u r c e t h a t i s i n c o r p o r a t e d i n t o the c u l t u r e medium f o r A. suboxydans i s g l y c e r o l . T h i s organism s e c u r e s growth energy by t h e o x i d a t i o n of g l y c e r o l t o d i h y d r o x y a c e t o n e : TABLE X I . The I n f l u e n c e of Tween-80 on the Growth Response of A. suboxydans t o I n c r e a s i n g Increments of PABA. Cone, of PABA Added Tween-80 ( . 5 p e r c e n t ) No Added Tween-80 per 10 c c , b a s a l R e p l i c a t e s R e p l i c a t e s medium 1 2 A B 1 A 0 0 , 0 0 4 * 0 .004 0 .013 0 .011 0 .000 0.018 0 . 0 1 0.015 •0.015 0.218 0 .230 0 .004 0.182 0 .02 0 .066 0 .051 0 .367 0 .387 0 .009 0 .347 . 0 .03 0 .119 0.108 0 .469 0 .495 0 .036 0 .398 0 .04 0 .210 0 .206 0 .530 0 .545 0 .056 0 .495 0 .05 0 .328 0 .337 0 .568 0 .576 0 .071 0 .523 0 .06 0 .392 0 .401 0 .595 0 .602 0 .114 0 .530 0 .07 0 .444 0 .452 0 .620 0 .627 0 .237 0 .538 0.08 0.482 0 .495 0.648 0 .640 0 .276 0 .658 0 .09 0 .523 .0 .513 0 .658 0 .655 0 .284 0 .648 0 .1 0 .550 0 .522 O .658 0 .658 0 .284 0 .638 * F i g u r e s r e p r e s e n t growth of A. suboxydans measured i n terms of O p t i c a l D e n s i t y . - 58 -CH 20H CH 20H CHOH * C=0 CH 20H In r e c e n t s t u d i e s i t was n o t e d t h a t g l u c o s e ( o r i t s end-p r o d u c t s a f t e r b e i n g a u t o c l a v e d ) had a marked s t i m u l a t o r y -a c t i o n on k. suboxydans when t h i s c a r b o h y d r a t e source was added i n a d d i t i o n t o g l y c e r o l ( S a r e t t and C h e l d e l i n , 1 9 4 5 ) . However, Woods (1949) c l a i m s t h a t when g l u c o s e i s used as a carbon source the c h a r a c t e r of the growth of t h e organism i s a l t e r e d q u i t e s i g n i f i c a n t l y . of A,, suboxydans. These i n v e s t i g a t o r s n o t e d t h a t of the twenty amino a c i d s s t u d i e d v a l i n e was e s s e n t i a l s i n c e no growth o c c u r r e d i n i t s absence. The o t h e r amino a c i d s were c l a s s e d as p r o b a b l y e s s e n t i a l , s t i m u l a t o r y and n o n - e s s e n t i a l as shown i n Table X I I . The need f o r i s o l e u c i n e may be a b s o l u t e s i n c e t h e s m a l l amount of growth o b t a i n e d i n i t s absence may be due t o a s m a l l amount of i s o l e u c i n e p r e s e n t as an i m p u r i t y i n t h e s y n t h e t i c l e u c i n e of the medium (Hegsted and W a r d e l l , 1 9 4 4 ) . I t i s i n t e r e s t i n g t o note t h a t the growth o b t a i n e d w i t h the twenty amino a c i d s i n c o r p o r a t e d i n the c u l t u r e medium was about e q u a l t o t h a t o b t a i n e d when h y d r o l y z e d c a s e i n was used i n t h e i r p l a c e ( T a b l e X I I I ) . (b) N i t r o g e n Source; Stokes and L a r s e n (1945) succeeded i n d e t e r -m i n i n g f o r t h e f i r s t t i me th e s p e c i f i c n i t r o g e n r e q u i r e m e n t s - 59 -TABLE X I I . E f f e c t of Omission of I n d i v i d u a l Amino A c i d s from  the "20 amino a c i d medium" upon Growth o f A. subxixy-dans (Sto k e s and L a r s en, 1945"!» Es s e n t i a l P r o b a b l y E s s e n t i a l S t i m u l a t o r y Non E s s e n t i a l V a l i n e I s o l e u c i n e -68 H i s t i d i n e - 4 5 L e u c i n e -31 - 9 8 H A l a n i n e - 7 2 C y s t i n e -37 M e t h i o n i n e - 3 2 G l u t a m i c Tryptophane -30 a c i d -41 A s p a r t i c T y r o s i n e -33 a c i d -38 P r o l i n e -39 P h e n y l a l a -n i n e -28 Hydroxy- Threonine -30 p r o l i n e -38 L y s i n e -33 A r g i n i n e - 3 2 S e r i n e -28 N o r l e u c i n e -29 G l y c i n e -33 K P e r c e n t t r a n s m i s s i b l e l i g h t of c u l t u r e s grown w i t h o u t the amino a c i d i n d i c a t e d ; u n i n o c u l a t e d medium s 1 0 0 . C u l t u r e s grown w i t h a l l 20 amino a c i d s gave a r e a d i n g of 3 2 . TABLE X I I I Growth o f A. suboxydans w i t h V a r i o u s Combinations of Amino Ac i d s . (Stoke s and L a r s e n . 1945) Amino A c i d s i n B a s a l Medium V a l i n e 4 i s o l e u c i n e + a l a n i n e + h i s t i d i n e + c y s t i n e (A.) (A)* + p r o l i n e (A) + h y d r o x y p r o l i n e (A) + s e r i n e (A) + p h e n y l a l a n i n e 20 amino a c i d s H y d r o l y z e d c a s e i n Per cent T r a n s m i s s i b l e L i g h t Experiment A Experiment B. 52x 50 29 34 38 37 32 40 41 37 29 29 17 27 K Per cent t r a n s m i s s i b l e l i g ^ i t of c u l t u r e s . - 60 -Stokes and L a r s e n (1945) were a b l e t o show t h a t a c o m b i n a t i o n of v a l i n e , i s o l e u c i n e , a l a n i n e and h i s t i d i n e r e p r e s e n t e d the s m a l l e s t number of amino a c i d s which can c o n s i s t e n t l y s u p p o r t some growth of A, suboxydans. However by the a d d i t i o n of e i t h e r c y s t i n e or m e t h i o n i n e growth was c o n s i d e r a b l y improved. With the f u r t h e r a d d i t i o n of p r o l i n e , growth a g a i n was i n c r e a s e d t o t h e l e v e l o b t a i n a b l e w i t h the m i x t u r e of the t wenty amino a c i d s or h y d r o l y z e d c a s e i n . However development i s not so r a p i d and somewhat l e s s e x t e n -s i v e than w i t h t h e use of y e a s t e x t r a c t . S a r e t t and C h e l d e l i n (1945) i n c l u d e d N o r i t e A-t r e a t e d peptone and N o r i t e A - t r e a t e d l i v e r c o n c e n t r a t e t o the b a s a l medium f o r A. suboxydans and o b t a i n e d a g r e a t e r growth response of t h i s organism. ( c ) M i n e r a l Source; A l t h o u g h the forms of f o o d r e q u i r e d by m i c r o -organisms show c o n s i d e r a b l e v a r i a t i o n , i t i s b e l i e v e d t h a t t h e y must c o n t a i n t e n known el e m e n t s : (1 ) carbon " (6 ) c a l c i u m (2) hydrogen (7) p o t a s s i u m (3) oxygen (8) magnesium (4 ) i r o n (9 ) s u l p h u r (5 ) n i t r o g e n (10 ) phosphorus The i n o r g a n i c s a l t s are n e c e s s a r y f o r b a c t e r i a l n u t r i t i o n and the exact r e q u i r e m e n t s p r o b a b l y v a r y w i t h each s p e c i e s . The i n o r g a n i c s a l t s added t o t h e b a s a l medium of A. suboxydans are u s u a l l y d e s i g n a t e d i n two groups, namely S o l u t i o n A and S o l u t i o n B. S o l u t i o n A c o n s i s t s of 10 per cent KH 2P0, and 10 per cent K 2HP0^. T h i s s o l u t i o n i s u s u a l l y - 61 -added t o the medium because i t possesses a s t r o n g b u f f e r i n g a c t i o n . S o l u t i o n B c o n s i s t s of 4 per cent MgS0^.7H 20, 0 . 2 per cen t NaCl, 0 .2 per cent FeS0^.7H 20 and 0.2 per cent MnS0^.4H 20. A l l t h e s e s a l t s p r o v i d e t h e n e c e s s a r y elements f o r t h e v i t a l f u n c t i o n s and a c t i v i t i e s of the b a c t e r i a l p r o t o p l a s m . The NaCl i s g e n e r a l l y added t o c u l t u r e media i n o r d e r t o i n c r e a s e t h e i r osmotic p r e s s u r e . A s a t i s f a c t o r y medium i s one t h a t c o n t a i n s riot o n l y t h e n e c e s s a r y f o o d s ubstances i n proper p r o p o r t i o n s but a l s o an osmotic p r e s s u r e a p p r o a c h i n g i s o t o n i c i t y w i t h the p a r t i c u l a r c e l l . D u r i n g t h e p r e l i m i n a r y a s s a y d e t e r m i n a t i o n of PABA u s i n g t h e method of Landy and Dicken ( 1 9 4 2 ) , i t was n o t e d t h a t t h e b a s a l medium f o r t h e t e s t organism a f t e r b e i n g p r e p a r e d appeared o p a l e s c e n t or c l o u d y . T h i s c l o u d i n e s s must be due t o the f o r m a t i o n of a p r e c i p i t a t e by a c h e m i c a l r e a c t i o n of two or more compounds. A s e r i e s o f t e s t s were c a r r i e d out on a l l t h e c o n s t i t u e n t s of t h e b a s a l medium i n order t o f i n d what c o n s t i t u e n t or c o n s t i t u e n t s produced t h i s p r e c i p i t a t i o n . The r e s u l t s of t h i s experiment are p r e s e n t e d i n Table XIV. I t can be seen from t h i s t a b l e t h a t the c l o u d i n e s s of the b a s a l medium a r i s e s on m i x i n g S o l u t i o n s A and B. S i n c e t h e growth response i s measured t u r b i d i m e t r i c a l l y , the b a s a l medium must be as c l e a r as p o s s i b l e , t h e r e f o r e i t was found d e s i r a b l e t o f i l t e r t he medium t h r o u g h a S e i t z f i l t e r i n g pad ( t y p e ST, s i z e L6) t o a v o i d f o r m a t i o n of a p r e c i p i t a t e d u r i n g a u t o c l a v i n g . - 62 -TABLE IV. The P e t e r m i n a t i o n of,, the C a u s a t i v e F a c t o r s t h a t  Produce Opalescence i n t h e B a s a l Medium. C o n s t i t u e n t C h a r a c t e r of C h a r a c t e r of S o l u t i o n B e f o r e S o l u t i o n A f t e r A u t o c l a v i n g A u t o c l a v i n g S o l . A - ( K 2 H P 0 A (KH 2P0, S o l . B-MgS0..7H 2 0 NaCl c l e a r c l e a r F e S 0 4 . 7 H ? 0 c l e a r s l i g h t p a l e MnSO,.4H,0 S o l . A + S o l . B^ y e l l o w ppte w h i t e c l o u d y heavy w h i t e p r e c i p i t a t e ppte Tryptophane s o l n . c l e a r c l e a r C a s e i n H y d r o l y s a t e c l e a r c l e a r N i c o t i n i c A c i d s o l n . c l e a r c l e a r C y s t i n e s o l u t i o n c l e a r c l e a r Ca. P a n t o t h e n a t e c l e a r c l e a r Tryptophane4Sol. A c l e a r c l e a r Tryptophane+Sol. B c l e a r s l i g h t p a l e y e l l o w ppte. T r y p t o p h a n e - S o l . A c l o u d y , w h i t e heavy w h i t e and S o l . B ppte. ppte. C a s e i n Hydrol. + c l e a r s l i g h t w h i t e S o l . A pp t e . C a s e i n Hydrol. + c l e a r s l i g h t p a l e S o l . B y e l l o w p p t e . C a s e i n Hydrol. + heavy w h i t e S o l . A and S o l . B c l o u d y , w h i t e p p t e . p p t e . C y s t i n e S o l . 4 c l o u d y , w h i t e heavy w h i t e S o l . A and S o l . B ppte. ppte. N i c o t i n i c a c i d S o l . c l o u d y , w h i t e heavy w h i t e + S o l . B + S o l . B ppt e . p p t e . (d) V i t a m i n Source; As a r e s u l t of t h e i r s t u d y on the growth r e q u i r e m e n t s of JU suboxydans, U n d e r k o f l e r e t a l . (1942) found t h a t b e s i d e s a s u i t a b l e .carbon s o u r c e , o r g a n i c n i t r o g e n and m i n e r a l s a l t s , t h e medium must c o n t a i n - 63 -p a n t o t h e n i c a c i d , n i c o t i n i c a c i d and PABA, b e f o r e growth would o c c u r . In a d d i t i o n i t was c o n c l u d e d t h a t r i b o f l a v i n was not r e q u i r e d i n t h e medium; i t appeared t h a t A,, suboxy-dans had the a b i l i t y t o s y n t h e s i z e t h i s compound. These workers a l s o t e s t e d S.M.A. C o r p o r a t i o n ' s B i o t i n C o n c e n t r a t e No. 1000 and n o t e d t h a t i t f u r n i s h e d an e s s e n t i a l f a c t o r , not b i o t i n , which was i n d i s p e n s a b l e f o r the growth of the organism. T h i s e s s e n t i a l f a c t o r has been found by o t h e r i n v e s t i g a t o r s t o be n i c o t i n i c a c i d . (e ) P u r i n e Metabolism'; Landy and S t r e i g h t o f f (1943) observed t h a t of a v a r i e t y of compounds examined f o r a b i l i t y t o i n c r e a s e growth of A,, suboxydans a t low c o n c e n t r a t i o n s of PABA i n t h e b a s a l medium, the p u r i n e b a s e s , adenine, quanine, and x a n t h i n e were found t o be e f f e c t i v e . Not o n l y was t h e q u a n t i t y of growth i n c r e a s e d , but a l s o the s e n s i t i v i t y o f A. suboxydans t o PABA was i n c r e a s e d c o n s i d e r a b l y . W h i l e i n the absence of p u r i n e s the t e s t o rganism responds t o 0 . 0 1 m i c r o -grams of PABA, t h e presence of p u r i n e s b r i n g s about r e a d i l y measurable growth w i t h 0 .001 micrograms per c u l t u r e . The above i n v e s t i g a t o r s found t h a t t h e s e p u r i n e s were not e s -s e n t i a l f o r growth but as growth a c c e s s o r i e s . They brought f o r t h t h i s s u g g e s t i o n because they n o t e d t h a t t h e p u r i n e s are w i t h o u t e f f e c t i n the absence of PABA or when PABA was s u p p l i e d i n amounts above 0 .03 micrograms per 10 m l . of c u l t u r e . There i s s t r o n g e v i d e n c e t h a t PABA i s concerned i n - 64 -the s y n t h e s i s of p u r i n e s , as w e l l as f o l i c a c i d , thymine and m e t h i o n i n e (Woods, 1947). S i n c e t h i s i s the case i t may suggest t h e r e a s o n f o r the s t i m u l a t o r y a c t i o n of the t h r e e p u r i n e s d e s c r i b e d above. These compounds or p o s s i b l y one of them, may be s y n t h e s i z e d by A., suboxydans from t h e growth f a c t o r , PABA. Hence when the p u r i n e s are added t o t h e b a s a l medium i n which t h e r e i s a low c o n c e n t r a t i o n of PABA, t h a t would n o r m a l l y be i n s u f f i c i e n t t o s u p p o r t b a c t e r i a l growth, r e a d i l y a c c e s s i b l e p u r i n e s p e r m i t normal c e l l u l a r m e t a b o l i s m of A. suboxydans. The s m a l l amount of PABA i n t h e medium would t h e n be used by t h i s o rganism i n t h e s y n t h e s i s of o t h e r v i t a l f a c t o r s . IV. C o r r e l a t i o n of T u r b i d i t y Readings w i t h A c t u a l C e l l Counts.  From the l a r g e s e r i e s of assays u n d e r t a k e n and t h o s e d i s c u s s e d i n t h i s r e p o r t , i t can be n o t e d t h a t o c c a s i o n a l l y growth of t h e t e s t organism has been o b t a i n e d i n the s y n t h e t i c medium t o which no PABA has been added. The washed c e l l s used as the i n i t i a l i n o c u l u m i n t h e assay procedure-might w e l l c o n t a i n s u f f i c i e n t PABA or a c o n j u g a t e of PABA t o p e r m i t the i n i t i a t i o n of growth i n a medium d e v o i d of added PABA. In the work o f Landy and D i c k e n (1942) and o t h e r workers (Landy and S t r e i g h t o f f , 1943; C h e l d e l i n and B e n n e t t , 1945; S a r e t t and C h e l d e l i n , 1945) no attempt was made t o determine the number of c e l l s p r e s e n t i n the i n o c u l u m f o r the m i c r o -b i o l o g i c a l a s s a y f l a s k s . T h e r e f o r e f o r t h i s r e a s o n i t - 65 -seemed d e s i r a b l e t o determine the number of b a c t e r i a l c e l l s used as i n i t i a l i n o c u l u m . The drop p l a t e t e c h n i q u e of M i l e s and M i s r a (1938) as d e s c r i b e d i n Appendix V was used t o determine the b a c t e r i a l c o u n t . The organism was p l a t e d on g l y c e r o l - y e a s t e x t r a c t agar (Appendix I V ) . Data were ob-t a i n e d from t h r e e s e p a r a t e d e t e r m i n a t i o n s and are p r e s e n t e d i n Table XV. TABLE XV. P l a t e Counts of Number of C e l l s per ml. of Washed Mother C u l t u r e . D e t e r m i n a t i o n Log. of Count/ml C a l c u l a t e d Log. Number of Mother Count of Inoculum C u l t u r e used i n Assay F l a s k s 1 9.20 7.90 2 8.57 7.25 3 8.46 7.15 From t h i s data i t can be e s t i m a t e d t h a t t h e number of c e l l s of A. suboxydans added t o each a s s a y f l a s k i s a p p r o x i m a t e l y 2.5 x 10^. I t would be of i n t e r e s t t o t e s t the i n f l u e n c e of " s i z e " of i n i t i a l i n o c u l u m on the growth response i n a s y n t h e t i c medium c o n t a i n i n g 0.05 micrograms of PABA per 10 ml. A s e r i e s of f l a s k s were i n o c u l a t e d w i t h s e r i a l d i l u t i o n s from a washed mother c u l t u r e o f known c e l l c o n c e n t r a t i o n . Table XVI p r e s e n t s the r e s u l t s of two such d e t e r m i n a t i o n , the data from No. 2 below, are p l o t t e d i n F i g u r e 12. - 66 -TABLE XVI. The I n f l u e n c e o f I n i t i a l Inoculum Count  on t h e Response of A. suboxydans when Grown i n a S y n t h e t i c Medium Containing: 0.05 micrograms of PABA  per 10 m l . of Medium. No. 1 No. 2 C e l l Count per 0.05 c c . of Inoculum T u r b i d i t y of C u l t u r e at 72 h r s . , measured as O p t i c a l D e n s i t y C e l l Count per 0 . 1 c c . of Inoculum T u r b i d i t y of C u l t u r e at 48 h r s . , measured as O p t i c a l D e n s i t y Blank No PABA , 0 .5 x lOr 5 0.25 x 1CK 0 .5 x 1 0 3 1 . 0 x 103 2 . 0 x 1 0 3 4 . 0 x 1 0 3 4 . 0 x 104 8 . 0 x 1 0 ' 0 .003 0 .068 0.180 0 .279 0 .751 0 .770 0 .842 0 .947 Blank No PABA. .0.058 x 1 0 3 x 1 0 3 x 1 0 3 0 .29 0 .58 2 . 9 2 . 9 2 . 9 2 . 9 2 . 9 x 1 0 3 x l o j x. 10? x 1 0 6 x 10? 0.000 0.002 0.009 0.052: 0.108 0.588 1.086 1.188 I t i s e v i d e n t t h a t the growth response i s p r o p o r t i o n a l t o the number of c e l l s i n the i n i t i a l i n o c u l u m . E q u i v a l e n t numbers of c e l l s , as t h a t of No. 2 , above, were i n o c u l a t e d i n t o the b a s a l medium d e v o i d of PABA. In e v e r y f l a s k no measureable amount of growth r e s u l t e d . I n t h i s p a r t i c u l a r case t h e r e i s no e v i d e n c e t o suggest t h a t w i t h heavy i n o c u l a t h e r e i s any c a r r y - o v e r of PABA or PABA d e r i v a t i v e s i n t o t h e b a s a l medium. Co n s e q u e n t l y i f the paren t c u l t u r e i s a d e q u a t e l y washed w i t h s a l i n e b e f o r e b e i n g used as t h e i n i t i a l i n o c u l u m , t h e r e seems t o be l i t t l e or no c a r r y - o v e r of PABA i n t o t h e ass a y f l a s k s . E f Y C E O B 0 05 - 67 -These experiments have i n d i c a t e d , however, t h a t the c e l l c o n c e n t r a t i o n i n the i n i t i a l i n o c u l u m markedly a f f e c t s the subsequent r a t e of growth. I t might be wise t o suggest at t h i s p o i n t f o r f u t u r e work i n t h i s c o n n e c t i o n , t h a t t h e c e l l c o n c e n t r a t i o n of a l l i n o c u l a be s t a n d a r d i z e d t o a known l e v e l . I f t h i s c o u l d be done, u s i n g the same procedure and t e c h n i q u e i n p r e p a r i n g the p a r e n t c u l t u r e , a s t a n d a r d c u r v e f o r t h e response of A. suboxydans t o v a r y i n g i n c r e m e n t s of PABA c o u l d be o b t a i n e d . This' curve c o u l d thus be used as a means of c a l c u l a t i n g l e v e l s of PABA by growth response of t h i s t e s t organism, w i t h o u t p r e p a r i n g a r e f e r e n c e a s s a y each time a d e t e r m i n a t i o n o f t h i s compound i s sought. TABLE X V I I . R e l a t i o n s h i p Between C e l l Count and  C e l l S uspension O p a c i t y . Number of C e l l s O p a c i t y , measured as per c c . O p t i c a l D e n s i t y 0 . 5 0 4 0 . 2 8 4 0 .248 0 .212 0 .161 0 .096 0 .056 I n t h i s c o n n e c t i o n Table X V I I p r e s e n t s the r e l a t i o n -s h i p between c e l l count and c e l l s u s p e n s i o n o p a c i t y u s i n g a 48 hour o l d c u l t u r e o f A. suboxydans. As was a n t i c i p a t e d t h e i n c r e a s e i n o p a c i t y of the s u s p e n s i o n i s p r o p o r t i o n a l t o t h e i n c r e a s e i n numbers of c e l l s ( F i g u r e 1 3 ) . 2 . 9 0 x 10° 1 .45 x 10° 1 .27 x 10° 1 .07 x 10° 0 . 8 4 x 10° 0 .58 x 10* 0 .29 x 1 0 s - 68 -V. F i n a l Recommended Assay P r o c e d u r e : From the r e s u l t s of a l l t h e p r e v i o u s e x p e r i m e n t s , an a s s a y procedure f o r the d e t e r m i n a t i o n of PABA can th u s be recommended. T h i s a s s a y procedure has been adopted f o r many of the experiments t o f o l l o w and i t i s f e l t t h a t such a procedure s h o u l d be used i n f u t u r e work a l o n g the l i n e s of the e x p e r i m e n t a t i o n s r e p o r t e d h e r e i n . The proposed assay procedure o u t l i n e d by Landy and Dicken (1942) s h o u l d be f o l l o w e d v e r y c a r e f u l l y , b e i n g sure a l l g l a s s w a r e i s c l e a n and a l l c h e m i c a l s pure. The bas.al medium d e s c r i b e d by t h e s e workers appears to be s a t i s f a c t o r y f o r the t e s t organism, however i f g r e a t e r response of A. sub-oxydans t o v e r y minute amounts of PABA i s r e q u i r e d , the medium d e s c r i b e d by Landy and S t r e i g h t o f f (1943) or C h e l d e l i n and Bennett (1945) i s recommended. S i n c e the growth response of the t e s t organism i s measured t u r b i d i m e t r i c a l l y , the a d d i t i o n of the S u r f a c e A c t i v e Agent, Tween-80, t o the b a s a l medium b e f o r e s t e r i l i z a t i o n and subsequent i n o c u l a t i o n , i s recommended i n o r d e r t o p r e v e n t the f o r m a t i o n of a p e l l i c l e on the s u r f a c e of the medium. V I . The B i o l o g i c a l A c t i o n of U l t r a v i o l e t I r r a d i a t i o n : The w o r k i n g h y p o t h e s i s p r e s e n t e d p r e v i o u s l y i s based on the s u g g e s t i o n t h a t u l t r a v i o l e t i r r a d i a t i o n of the " e s s e n t i a l m e t a b o l i t e " PABA, may a l t e r t h a t compound t o such an e x t e n t t h a t the new compound formed i s s u f f i c i e n t l y l i k e - 69 -i t s n o n - i r r a d i a t e d precursor that i t may enter i n t o combin-a t i o n i n a c e l l u l a r system i n place of the normal m e t a b o l i t e . This newly formed compound may act l i k e an a n t i - m e t a b o l i t e i n that i t blocks an e s s e n t i a l enzyme system so that the normal metabolic f u n c t i o n concerning the " e s s e n t i a l metabo-l i t e " i s stopped, the f i n a l r e s u l t may be a s t a t e of i n -h i b i t i o n or death of the c e l l i n q u e s t i o n . A s e r i e s of p r e l i m i n a r y experiments were c a r r i e d out on the i n f l u e n c e of u l t r a v i o l e t i r r a d i a t i o n of PABA on i t s subsequent a b i l i t y t o permit the growth of _A. suboxydans i n a s y n t h e t i c medium. Experiment 7: E f f e c t of U l t r a v i o l e t L i g h t on the B i o l o g i c a l Value of PABA. ; • A s o l u t i o n of PABA c o n t a i n i n g 2 micrograms per 10 ml. of d i s t i l l e d water was i r r a d i a t e d under the General E l e c t r i c G e r m i c i d a l Mercury Arc Lamp (see Appendix VI) f o r 30, 45, 60 and 90 minutes. The i r r a d i a t e d PABA s o l u t i o n was then added to the b a s a l medium at a l e v e l of 0.05 micrograms per 10 ml. of medium. A f t e r s t e r i l i z a t i o n and subsequent c o o l i n g the medium was then i n o c u l a t e d with 0.05 ml. of a 48 hour o l d washed mother c u l t u r e of A. suboxydans. The r e s u l t s of these experiments are presented i n Tables XVIII and XIX. Reference t o the above data i n d i c a t e s that the i r -r a d i a t i o n of PABA under the c o n d i t i o n s d e s c r i b e d , r e s u l t s i n a p a r t i a l d e s t r u c t i o n of the PABA i n s o f a r as i t s f u n c t i o n as - 70 -TABLE X V I I I . The E f f e c t of I r r a d i a t i o n of PABA on i t s Sub-sequent Use as an E s s e n t i a l M e t a b o l i t e f o r A. suboxydans. F l a s k No. Normal 30-Minute 45-Minute Replicates No PABA PABA I r r a d i a t e d I r r a d i a t e d Enrichment PABA PABA 1 0.068* 0.961 0.618 0.328 2 0.068 0.959 0 .620 0.367 3 0.068 0.959 0 .620 0.347 4 -- 0.959 0.638 0.367 5 0.959 0,654 0.367 6 — 0.921 0.654 0.301 7 — Q.943 0.654 0.347 8 -- 0.930 0.660 0.076 Average 0.068 0.949 0.639 0.341 TABLE XIX. The E f f e c t of I r r a d i a t i o n of PABA on i t s Sub-sequent Use as an E s s e n t i a l M e t a b o l i t e f o r A. suboxydans. F l a s k No. Normal 60-Minute 90-Minutee Replicates No PABA PABA I r r a d i a t e d I r r a d i a t e d Enrichment PABA PABA 1 0 .029K 0 .783 0 .071 0 .036 2 0 .027 0..783 0 .053 0 .034 3 0 .029 0 .783 0 . 0 5 6 0 .032 4 — 0 .839 0 .052 0 .029 5 - - 0 .839 0 . 0 5 1 0 . 0 4 1 6 — 0.783 0 . 0 7 6 0 . 0 4 3 7 — 0 .839 0 .076 0 .029 8 — 0 .796 0 .076 0 .031 Average 0.028 0.806 0 .064 0 .034 x Figures represent growth of A. suboxydans measured as O p t i c a l Density. - 71 -an " e s s e n t i a l m e t a b o l i t e " i s co n c e r n e d . The e x t e n t of des-t r u c t i o n o b t a i n e d a f t e r 45 minutes of i r r a d i a t i o n i s o n l y p a r t i a l s i n c e s u f f i c i e n t u n a l t e r e d PABA remained t o p e r m i t a p p r e c i a b l e growth of the organism a l t h o u g h t h e growth was not n e a r l y e q u a l t o t h a t o b t a i n e d w i t h n o n - i r r a d i a t e d PABA. A f t e r 90 minute i r r a d i a t i o n of a s o l u t i o n of PABA l i t t l e t o no growth of the t e s t organism o c c u r r e d , which c o u l d be due t o the f a c t t h a t a l l but a s m a l l amount of the PABA was a l t e r e d t o a compound t h a t c o u l d n o t t a k e the p l a c e of normal PABA i n i t s f u n c t i o n s as an " e s s e n t i a l m e t a b o l i t e " . Experiment 8 : T i m e - D e s t r u c t i o n Curve of PABA. The above experiment was r e p e a t e d u s i n g a s e r i e s of i r r a d i a t i o n t i m e s . The d a t a from t h i s experiment are presented i n Tables XX and XXI. The r e s u l t s t a b u l a t e d i n Table XX are p l o t t e d i n F i g u r e 14 and 1 5 . I r r a d i a t i o n of a 2 microgram per 10 m l . aqueous s o l u t i o n of PABA f o r 30 minutes r e s u l t e d i n 50 per ce n t des-t r u c t i o n of the PABA; i r r a d i a t i o n f o r 60 minutes r e s u l t e d i n 86 per cent d e s t r u c t i o n . I n t h e l i g h t of t h e s e r e s u l t s a s i m i l a r experiment was conducted i n which v a r i o u s c o n c e n t r a -t i o n s of PABA were i r r a d i a t e d , by u l t r a v i o l e t l i g h t . Experiment 9 :• T i m e - D e s t r u c t i o n of V a r i o u s C o n c e n t r a t i o n s of PABA by U l t r a v i o l e t I r r a d i a t i o n s  Four aqueous s o l u t i o n s of PABA, 100 , 50, 10 and 2 TABLE XX. Time of I r r a d i a t i o n - D e s t r u c t i o n Data f o r I r r a d i a t e d PABA. R e p l i c a t e Normal Fl a s k No. No PABA PABA Time of I r r a d i a t i o n i n Minutes 15 30 45 60 70 80 90 100 110 1 0 .014K 0 .854 2 0.014 0 .806 3 0.014 0 .798 4 — 0 .824 5 - - 0 .858 0 . 796 0 .494 0 ,324 0 .276 0 .137 0 .076 0 .076 0 .071 0 .077 0 .796 0 .509 0 .315 0 .237 0 .137 0 .094 0.068 0.028 0.030 0 .783 0 .485 O.365 0 .284 0 .117 O.O89 O.O63 0 .032 0 .011 0.819 0.329 0 .346 0.286 0 .121 0.083 0 .071 0.018 0 .000 0 .796 0 .454 0.339 0 ,284 0 .168 O.O83 0 .061 0.004 0 . 000 Average 0.014 0.830 0 .799 0 .476 0.336 0 .273 0 .135 O.O85 0.068 0 .029 0 .022 K F i g u r e s represent growth of A. suboxydans measured as O p t i c a l Density. TABLE XXI. Time of I r r a d i a t i o n - D e s t r u c t i o n Data f o r I r r a d i a t e d PABA. R e p l i c a t e No PABA Normal Time of I r r a d i a t i o n i n Minutes F l a s k No. PABA 15 30 45 60 80 100 120 140 1 2 3 Average O.OOC-x 0.000 0.000 0.000 0.523 0.523 0.523 0.523 0.469 0.301 0.482 0.409 0.155 0.131 0.181 0.155 0.056 0.018 0.051 0.046 0.041 0.009 0.027 0.027 0.004 0.013 0.009 0.009 0.004 0.004 0.004 0.004 0.004 0.000 0.000 0.000 0.004 0.000 0.000 0.000 x ' F i g u r e s r e p r e s e n t gr owth of A. suboxydans measured as O p t i c a l D e n s i t y . - 74 -micrograms per 10 m l . d i s t i l l e d w ater, were i r r a d i a t e d f o r 1, 2, 3 and 4 h o u r s . The i r r a d i a t e d s o l u t i o n s were th e n added t o a b a s a l c u l t u r e medium at the l e v e l of 0.05 m i c r o -grams per 10 ml. of medium. These media were then i n o c u l a t e d w i t h 0.05 ml. of a 24 hour washed mother c u l t u r e of A. suboxy-dans . The c u l t u r e s were s u b s e q u e n t l y i n c u b a t e d at 30°C and the growth response was r e c o r d e d at the end of a 48 and a 72 hour i n c u b a t i o n p e r i o d . The r e s u l t s a r e p r e s e n t e d i n Table X X I I . F i g u r e 16 g i v e s the g r a p h i c a l r e p r e s e n t a t i o n of the r e s u l t s o b t a i n e d . From the two p r e v i o u s e x p e r i m e n t s i t may be p o s s i b l e t o determine t h e r e a c t i o n o r d e r of the d e s t r u c t i o n or a l t e r a t i o n of PABA by u l t r a v i o l e t l i g h t . From the s t a n d p o i n t of t h e q u a n t i t a t i v e c o n s i d e r a t i o n of r e a c t i o n r a t e s , p r o c e s s e s are c l a s s i f i e d by the order of r e a c t i o n , t h a t i s , t h e number of atoms or m o l e c u l e s whose c o n c e n t r a t i o n ( o r p r e s s u r e s ) determine t h e r a t e ( o r k i n e t i c s ) of the r e a c t i o n ( G l a s s t o n e , 1948). Suppose a i s t h e i n i t i a l c o n c e n t r a t i o n of PABA b e f o r e i r r a d i a t i o n , and l e t x be the decrease or change a f t e r the l a p s e o f time t i r r a d i a t i o n , t h e amount r e -maining i n the g i v e n volume w i l l be a-x. I f the p l o t of t a g a i n s t l o g , ( a - x ) s h o u l d be l i n e a r , the r e a c t i o n i s of the f i r s t o r d e r . The v a l u e s of t and a-x were c a l c u l a t e d and t h e r e s u l t i n g graph i s r e p r e s e n t e d as F i g u r e 17. As can be observed from F i g u r e 17, t h e p o i n t s f a l l c l o s e t o a - 75 -TABLE X X I I . Time of I r r a d i a t i o n - D e s t r u c t i o n f o r V a r i o u s  C o n c e n t r a t i o n s o f PABA. Hours Non- Cone, of I n c u - No I r r a d i a t e d of b a t i o n PABA PABA PABA m i c r o -grams per 10 m l . d i s t i l l e d wat er Time of I r r a d i a t i o n Hours 1 2 3 4 48 0.018 0.854 100 0.015 0.824 50 10 2 0.403 0.319 0.051 0.022 0.337 0.051 0.027 0.022 0.284 0.036 0.015 0.018 0.208 0.015 0.018 0.018 72 0.056 0.854 100 0.046 0.921 50 10 2 0.770 0.620 0.367 0.201 0.638 0.215 0.092 0.076 0.569 0.131 0.081 0.066 0.509 0.125 0.076 0.061 F i g u r e s r e p r e s e n t growth o f A. suboxydans. measured as O p t i c a l D e n s i t y . - 76 -s t r a i g h t l i n e . S i n c e t h i s i s the case the a l t e r a t i o n of PABA by u l t r a v i o l e t i r r a d i a t i o n appears t o be a f i r s t o r d e r r e a c t i o n , i e . o n l y one molecule i s i n v o l v e d i n t h i s r e a c t i o n . I t i s obvious t h a t a d d i t i o n a l work on t h i s phase o f t h e problem i s n e c e s s a r y . I t might be proposed t h a t the f o l l o w i n g r e a c t i o n t a k e s p l a c e by the a c t i o n of u l t r a v i o l e t l i g h t on H PABA: N i k fa 0 1 s t R e a c t i o n Order. I JJ U l t r a v i o l e t L i g h t * u cooH C o o H Normal PABA A c t i v a t e d PABA From t h e above two experiments i t seems s a f e t o con-c l u d e t h a t t h e h y p o t h e s i s upon which the p r e s e n t work i s based i s sound i n p a r t a t l e a s t . I t has been demonstrated t h a t i r r a d i a t i o n of PABA so a l t e r s the compound t h a t i t can no l o n g e r a c t as an " e s s e n t i a l m e t a b o l i t e " : f o r A. suboxydans under t h e s e c o n d i t i o n s . Experiment 10: To Test t h e I n f l u e n c e o f U l t r a v i o l e t L i g h t on t h e D i l u e n t of the PABA S o l u t i o n . T h i s experiment was des i g n e d t o determine i f u l t r a -v i o l e t l i g h t has an e f f e c t on the d i l u e n t of the PABA s o l -u t i o n . A washed mother c u l t u r e of A. suboxydans was i n o c u l a t e d i n t o ( l ) 5 c c . b a s a l medium p l u s 5 c c . of PABA s o l u t i o n t o make t h e f i n a l c o n c e n t r a t i o n of PABA 0.05 m i c r o -grams per 10 c c . medium; (2) 5 c c . b a s a l medium p l u s 5 c c . of i r r a d i a t e d PABA s o l u t i o n t o make t h e f i n a l c o n c e n t r a t i o n - 77 -0.05 micrograms per 10 c c . medium; ( 3 ) 5 c c . of b a s a l medium p l u s 5 c c . of i r r a d i a t e d d i s t i l l e d w a t e r , h a v i n g added normal PABA t o the i r r a d i a t e d water a f t e r i r r a d i a t i o n t o make the f i n a l c o n c e n t r a t i o n of PABA 0.05 micrograms per 10 c c . medium; and l a s t l y 5 c c . b a s a l medium p l u s 5 c c . i r r a d i a t e d d i s t i l l e d w a t e r . The media used i n t h i s experiment were t h o s e of Landy and D i c k e n (1942) and Landy and S t r e i g h t o f f (1943), the l a t t e r h a v i n g t h e a d d i t i o n of p u r i n e s i n t h e b a s a l medium (Appendix I V ) . The r e s u l t s o b t a i n e d a r e p r e s e n t e d i n Table X X I I I . From t h e s e r e s u l t s i t i s n o t e d t h a t u l t r a v i o l e t l i g h t a l t e r s PABA t o the e x t e n t t h a t i t cannot be used as an " e s s e n t i a l m e t a b o l i t e " f o r t h e growth of A. suboxydans and does not appear t o a f f e c t t h e d i s t i l l e d w a t e r . Any r e a c t i o n t h a t t a k e s p l a c e when a s o l u t i o n of PABA i s i r r a d i a t e d o c c u r s i n t h e PABA m o l e c u l e and a p p a r e n t l y not In t h e mole c u l e o f the d i l u e n t . I t i s worthy of note t h a t t h e f o r t i f i e d b a s a l medium of Landy and S t r e i g h t o f f (1943) s u p p o r t s l e s s growth of A. suboxydans when i t i s e n r i c h e d w i t h normal PABA as compared t o Landy and Dicken's medium. However w i t h the a d d i t i o n of the p u r i n e s t o t h e b a s a l medium, the l a t t e r s u p p o r t s g r e a t e r growth of the t e s t organism when i r r a d i a t e d PABA or i r r a d i a t e d d i s t i l l e d water i s added t o the medium. P r e v i o u s l y i t has been s t a t e d (Baumgartner, 1936; P r a t t , 1936) t h a t the a c t i o n of u l t r a v i o l e t l i g h t on a s o l -u t i o n a l t e r a its hydrogen i o n c o n c e n t r a t i o n t o such a degree - 78 -TABLE X X I I I . The E f f e c t of U l t r a v i o l e t L i g h t on D i f f e r e n t S o l u t i o n s as Measured by t h e Growth Response of A. suboxydans. Medium R e p l i - Normal I r r a d i a t e d I r r a d i a t e d I r r a d i a t e d Non I r r -c a t e PABA PABA D i s t i l l e d D i s t i l l e d " a d i a t e d No. Water p l u s Water D i s t i l l e d N o n - I r r a d - Water i a t e d PABA Landy and Dicken (1942) B a s a l Medium w i t h no pu r i n e s 1 0.745 0.018 0.770 0.018 0.018 2 0.762 0.011 0.762 0.015 0.013 3 0.721 0.013 0.721 0.013 0.018 4. 0.770 0.004 0.770 0.015 0.018 5 0.745 0.009 0.762 0.018 0.015 Av. 0.742 0.011 0.755 0.016 0.016 Landy and S t r e i g h - ~ t o f f J (1943) B a s a l Medium p l u s p u r i n e s 0.699 0.658 0.569 0.699 0.569 Av. 0.634 0.149 0.161 0.155 0.168 0.125 0.151 0.699 0.638 0.638 0.569 0.699 0.646 0.066 0.063 0.046 0.056 0.036 0.053 0.046 0.061 0.036 0.046 0.056 0.049 - 79 -t h a t the r e s u l t i n g s o l u t i o n has a pH l e v e l which i s u n s u i t a b l e f o r most b a c t e r i a l growth. However t h e y s t a t e t h a t i f the r e s u l t i n g pH i s r e a d j u s t e d t o s u i t the organism i n q u e s t i o n , t h e n the medium w i l l s u p p ort optimum growth. From th e s e o b s e r v a t i o n s a s e r i e s of s o l u t i o n s , , the same t h a t were used i n above e x p e r i m e n t , were i r r a d i a t e d i n o r d e r t o determine whether u l t r a v i o l e t i r r a d i a t i o n would change t h e i r hydrogen i o n c o n c e n t r a t i o n . Experiment 10a To Determine the E f f e c t of U l t r a v i o l e t L i g h t on t h e Hydrogen Ion C o n c e n t r a t i o n of a S o l u t i o n . A s e r i e s of s o l u t i o n s are s u b j e c t e d t o u l t r a v i o l e t i r r a d i a t i o n f o r two h o u r s . The r e s u l t s are p r e s e n t e d below. TABLE XXIV. The E f f e c t of U l t r a v i o l e t I r r a d i a t i o n on t h e Hydrogen Ion C o n c e n t r a t i o n of a S o l u t i o n . S o l u t i o n pH B e f o r e pH A f t e r 2-I r r a d i a t i o n hour I r r a d i a t i o n D i s t i l l e d 5.95 6.4 water PABA S o l - 5.95 6.35 u t i o n (,05 micrograms per 1 cc . ) From t h e s e r e s u l t s i t i s n o t e d t h a t the hydrogen i o n c o n c e n t r a t i o n of a s o l u t i o n i s a l t e r e d by the u l t r a v i o l e t i r r a d i a t i o n , which c o n f i r m s the r e s u l t s o b t a i n e d by Baum-g a r t n e r (1936) and P r a t t (1936). A f t e r the p r e s c r i b e d - 80 -amounts of the s e s o l u t i o n s were added t o t h e b a s a l medium as d e s c r i b e d i n Experiment 10, the r e s u l t i n g pH of the medium was checked and r e a d j u s t e d , i f n e c e s s a r y , t o the l e v e l r e a c -q u i r e d f o r optimum growth of A. suboxydans. The r e s u l t s of Experiment 10 i n d i c a t e t h a t the change of pH of the t e s t e d s o l u t i o n s by u l t r a v i o l e t l i g h t i s not the f a c t o r t h a t reduces t h e growth response of the t e s t organism, but t h e r e e x i s t s an a l t e r a t i o n of the PABA molecule t o such a degree t h a t i t cannot a c t as an " e s s e n t i a l m e t a b o l i t e " i n the n u t r i t i o n of A. suboxydans. Experiment 11: The A b i l i t y of Normal PABA t o O v e r - r i d e the I r r a d i a t e d PABA During t h e Growth of A. suboxydans.  In t h i s experiment A. suboxydans was grown i n media ( l ) c o n t a i n i n g normal PABA, (2) c o n t a i n i n g i r r a d i a t e d PABA, and (3) c o n t a i n i n g i r r a d i a t e d PABA t o which was added normal PABA a t a l e v e l of 0.05 micrograms per 10 m l . of medium a f t e r 48 hours of i n c u b a t i o n . The r e s u l t s are g i v e n i n Table XXV. The growth o b t a i n e d i n the b a s a l medium p l u s normal PABA was e s s e n t i a l l y as e x p e c t e d . A f t e r 60 minutes of i r -r a d i a t i o n , p a r t of the PABA had been a l t e r e d and l e s s growth was o b t a i n e d t h a n i n the normal c u l t u r e . P r o l o n g e d i n c u b a t i o n f o r 96 hours p e r m i t t e d n e a r l y normal growth. A f t e r 90 minutes of i r r a d i a t i o n most o f the PABA had been a l t e r e d as e v i d e n c e d by o n l y s l i g h t growth. Even a f t e r 96 hours of i n c u b a t i o n , growth was. not c o m p l e t e l y normal a l t h o u g h somewhat g r e a t e r - 81 -TABLE XXV. Res pons e o f _A. suboxydans t o I r r a d i a t e d and  N o n - I r r a d i a t e d PABA under V a r i o u s C o n d i t i o n s . Growth i n Media C o n t a i n i n g R e p l i c a t e Growth Normal 60 Minute 90 Minute No No. Measured PABA I r r a d i a t e d I r r a d i a t e d PABA at H rs. PABA PABA 1 48 0.955* 0.770 0.131 0.018 2 48 0.921 0.783 0.155 0.018 3 48 0.940 0.870 0.155 0.018 4 48 0.889 0.854 0.149 0.009 5 48 0.940 0.870 0.155 0.009 6 96 0.979 0.862 0.602 0.018 7 96 1.042 0.862 0.608 0..009 Added to. Media n i l 0.05 micro- 0.05 micro- n i l at 48 Hours grams PABA grams PABA 8 96 1.023 0.921 0.921 0..009 9 96 1.095 0.921 0.886 0.018 10 96 1.071 0.886 0.906 0.018 » F i g u r e s r e p r e s e n t growth of A. suboxydans measured as O p t i c a l D e n s i t y . - 82 -than at the end of 48 hours of i n c u b a t i o n . The a d d i t i o n of normal PABA at 48 hours to r e p l i c a t e c u l t u r e s which f a i l e d t o grow n o r m a l l y i n the b a s a l medium p l u s i r r a d i a t e d PABA, p e r m i t t e d the i n i t i a t i o n and development of the u s u a l number of c e l l s i n the now complete medium. In o t h e r words' t h i s experiment has shown t h a t the a d d i t i o n of PABA t o a c u l t u r e which has f a i l e d t o grow i n the presence of i r r a d i a t e d PABA p e r m i t s normal growth. A subsequent experiment i n which a l o n g e r i r r a d i a t i o n p e r i o d , 100 mi n u t e s , was used, c o n f i r m e d the above r e s u l t s i n e n t i r e t y . In the l i g h t of .the p r e v i o u s experiments i t i s e v i d e n t t h a t PABA i s a l t e r e d by the a c t i o n of u l t r a v i o l e t i r r a d i a t i o n . S i n c e t h i s i s the case a s e r i e s of experiments were conducted t o note the e f f e c t of u l t r a v i o l e t l i g h t on the c e l l s of A_. suboxydans.' The g e r m i c i d a l a c t i v i t y of u l t r a -v i o l e t energy may be due t o the d e s t r u c t i o n of the " e s s e n t i a l m e t a b o l i t e " PABA, or i t s enzyme systems on and w i t h i n the m i c r o b i a l c e l l , because i t has been p r e v i o u s l y shown t h a t the a b s o r p t i o n maximum of PABA l i e s w e l l i n the r e g i o n of g e r m i -c i d a l a c t i v i t y . T h i s f a c t seems t o have been o v e r l o o k e d by e a r l y workers ( H a r r i s and Hoyt, 1919). They s t u d i e d the u l t r a -v i o l e t a c t i o n on t r y p t o p h a n e and t y r o s i n e i n r e l a t i o n t o the d e s t r u c t i o n of b a c t e r i a , and summarily mentioned t h a t aminobenzoic a c i d r e s t o r e d the growth of u l t r a v i o l e t i r -r a d i a t e d m i c r o b i a l c e l l s . T h is statement appeared i n p r i n t t o be v e r y i n s i g n i f i c a n t , however on the c o n t r a r y i t may be a v e r y s i g n i f i c a n t o b s e r v a t i o n . An experiment was conducted. - 83 -t o note i f PABA would r e s t o r e the growth of t h e i r r a d i a t e d c e l l s of A_. suboxydans. Experiment 12: The E f f e c t of U l t r a v i o l e t I r r a d i a t i o n on the Growth of A. suboxydans.  Washed c e l l s of A. suboxydans were i r r a d i a t e d f o r 100 minutes under the G e n e r a l E l e c t r i c G e r m i c i d a l Lamp (Appendix V I ) . These c e l l s were then i n o c u l a t e d at a l e v e l of 0 i 0 5 ml. of washed i r r a d i a t e d mother c u l t u r e i n t o a s e r i e s of media. When the i r r a d i a t e d c e l l s of A. suboxydans were i n o c u l a t e d i n t o t he b a s a l medium d e v o i d of PABA no growth was o b t a i n e d , as was e x p e c t e d . When i n o c u l a t e d i n t o t h e b a s a l medium e n r i c h e d w i t h 0 . 0 5 micrograms of PABA per 10 c c . medium, no growth a f t e r 48 hours of i n c u b a t i o n was a p p a r e n t . F u r t h e r i n c u b a t i o n t o 96 hours r e s u l t e d i n normal growth i n d i c a t i n g t h a t the i r r a d i a t e d c e l l s r e q u i r e d as a p p r e c i a b l e time t o r e t u r n t o t h e i r normal s t a t e i n t h e presence of. PABA. I r r a d i a t e d c e l l s i n o c u l a t e d i n t o the b a s a l medium p l u s 0 . 0 5 micrograms of i r r a d i a t e d PABA per 10 c c . medium f a i l e d t o grow even a f t e r 96 hours of i n c u b a t i o n . When normal PABA was i added a f t e r 96 hours of i n c u b a t i o n , normal growth was ob-t a i n e d a f t e r 48 h o u r s . This would suggest t h a t t h o s e c e l l s had t a k e n up the i r r a d i a t e d p roduct and remained i n a v i a b l e but, r e s t i n g stage f o r the p r e l i m i n a r y 96 hour i n c u b a t i o n p e r i o d . This r e s u l t appears s i g n i f i c a n t when i t i s con-s i d e r e d i n the l i g h t of i r r a d i a t e d c e l l s i n o c u l a t e d i n t o a - 84 -PABA n e g a t i v e medium. These l a t t e r c e l l s not o n l y f a i l e d t o grow d u r i n g the f i r s t 96 hours of i n c u b a t i o n but l i k e w i s e d u r i n g a subsequent 48 hours when 0.05 micrograms of normal PABA per 10 c c . medium was added t o the medium. A 24 hour o l d washed mother c u l t u r e was i r r a d i a t e d f o r a s e r i e s of time i n t e r v a l s . At the end of a time l i m i t of i r r a d i a t i o n , 0.05 m l . of the s u s p e n s i o n of A. suboxydans was i n o c u l a t e d i n t o b a s a l medium c o n t a i n i n g no PABA, i r -r a d i a t e d PABA and normal PABA. The r e s u l t s are p r e s e n t e d below. TABLE XXVI. The E f f e c t of U l t r a v i o l e t L i g h t on the Growth  of k. suboxydans. B a s a l Medium p l u s Time of- I r r a d i a t i o n i n Minutes 0 20 40 60 80 100 120 No PABA I r r a d i a t e d PABA 0.05 micrograms per 10 c c . medium Normal PABA; 0.05 micrograms per 10 cc medium 0 . 0 2 7 * 0 0 0 0 0 0 0.086 0 0 0 0 0 0 0.770 0 0 0 0 0 0 x F i g u r e s r e p r e s e n t growth of A. suboxydans, measured as O p t i c a l D e n s i t y . As i s e v i d e n t by t h e data t a b u l a t e d i n Table XXVI, i r r a d i a t i o n of a s u s p e n s i o n of A. suboxydans f o r 20 minutes r e s u l t s i n complete d e s t r u c t i o n and a l s o t h a t the a d d i t i o n of PABA t o i t s growth medium to a l e v e l of 0.05 micrograms - 85 -per 10 c c . was not s u f f i c i e n t t o r e s t o r e i t s growth. A l l the i n o c u l a t e d media were h e l d over i n the i n c u b a t o r f o r 144 hours, and s t i l l no growth of t h e t e s t organism o c c u r r e d . An experiment s i m i l a r t o the above was c a r r i e d out i n w hich a 24 hour o l d washed c u l t u r e of A. suboxydans was exposed t o u l t r a v i o l e t l i g h t f o r 15, 30, 45, 60 and 90 m i n u t e s . At the end o f each time i n t e r v a l , a p o r t i o n o f the i r r a d i a t e d c e l l s u s p e n s i o n was p l a t e d i n o r d e r t o d e t e r m i n e the number of t h e v i a b l e c e l l s r e m a i n i n g a f t e r each time i n t e r v a l . A f t e r 15 minutes o f i r r a d i a t i o n no growth was e x h i b i t e d on t h e agar p l a t e s , t h u s c o n f i r m i n g t h e p r e v i o u s experiment i n t h a t a f t e r 15-20 minutes of exposure t o u l t r a -v i o l e t l i g h t , A. suboxydans was d e s t r o y e d . T h i s r e s u l t i s of p a r t i c u l a r i n t e r e s t s i n c e the p l a t i n g medium used was g l y -c e r o l y e a s t - e x t r a c t agar (see Appendix I V ) . T h i s medium i s known t o c o n t a i n adequate amounts* of PABA f o r t h e growth of A. suboxydans. A p p a r e n t l y t h e i r r a d i a t e d c e l l s have been damaged beyond t h e p o i n t where the a d d i t i o n of PABA t o t h e i r growth menstruum w i l l p e r m i t normal growth. The above o b s e r v a t i o n s do not c o n f i r m the r e s u l t s o b t a i n e d i n Experiment 12. I t i s f e l t , however, t h a t t h e data of Experiment 12 s h o u l d be p r e s e n t e d i n t h i s r e p o r t f o r purposes o f r e c o r d , because t h e y may be s i g n i f i c a n t t o t h e e x t e n t t h a t f u t u r e work which i s p l a n n e d may c o n f i r m such r e s u l t s . The f a c t t h a t t h e i r r a d i a t e d organism i s i n o c u l a t e d K PABA c o n t e n t of y e a s t - e x t r a c t ( B a c t o ) = 40 micrograms per gram (Landy and D i c k e n , 1942) - 86 -i n t o l i q u i d media may be the f a c t o r t h a t p e r m i t s t h e organism t o r e m a i n i n a r e s t i n g s t a g e u n t i l adequate normal PABA i s a v a i l a b l e t o c o u n t e r a c t the b i o l o g i c a l a c t i o n of u l t r a v i o l e t i r r a d i a t i o n . On the o t h e r hand, when t h e i r r a d i a t e d c e l l s are seeded onto g l y c e r o l y e a s t - e x t r a c t agar, t h e y may have g r e a t d i f f i c u l t y t o o b t a i n the normal PABA and thus cannot overcome the a c t i o n of the u l t r a v i o l e t i r r a d i a t i o n p r e v i o u s l y imposed on them. Rob e r t s and Aldous (1949) s t a t e t h a t t h e a c t i o n of u l t r a v i o l e t i r r a d i a t i o n on m i c r o b i a l c e l l s r e s u l t s i n t h e p r o d u c t i o n o f a p o i s o n w i t h i n the c e l l and t h a t under c e r t a i n c o n d i t i o n s t h i s p o i s o n can be i n a c t i v a t e d or removed thus a l l o w i n g the c e l l t o remain v i a b l e . T h is p o i s o n c o u l d r e a d i l y be t h e i r r a d i a t e d p r o d uct of PABA and t h a t i f t h i s p r o d u c t i s not removed, an i n h i b i t i o n o f c e l l d i v i s i o n i s c r e a t e d and t h e c e l l f i n a l l y d i e s . However i f t h i s p r o d u c t i s r e -p l a c e d by normal PABA, then t h e organism can resume normal d i v i s i o n and g r o w t h . These w o r k e r s found a l s o t h a t r e c o v e r y of b a c t e r i a a f t e r u l t r a v i o l e t i r r a d i a t i o n proceeds i n l i q u i d s but i s stopped or at l e a s t r e t a r d e d on s o l i d media. T h i s may be the e x p l a n a t i o n f o r t h e c o n f l i c t i n g r e s u l t s r e c o r d e d above. V I I . The Chemical A c t i o n o f U l t r a v i o l e t L i g h t on PABA In t h e p r e v i o u s s e c t i o n d e a l i n g w i t h t h e b i o l o g i c a l a s p e c t s o f u l t r a v i o l e t l i g h t on PABA, i t was shown t h a t a - 87 -change o c c u r r e d i n t h i s compound f o l l o w i n g i r r a d i a t i o n as de t e r m i n a b l e by b i o l o g i c a l a s s a y u s i n g A. suboxydans as the t e s t o r g a n i s m . I t i s of i n t e r e s t now t o c o n f i r m t h i s b i o -l o g i c a l f i n d i n g by p h y s i c o c h e m i c a l means i f p o s s i b l e . To ach i e v e t h i s purpose the a b s o r p t i o n c u r v e s o f normal and i r r a d i a t e d PABA were d e t e r m i n e d to note i f t h e a b s o r p t i o n curve o f ( t h e i r r a d i a t e d compound d i f f e r s from t h a t of normal PABA. A l l s p e c t r o p h o t o m e t r i c data r e c o r d e d below were ob-t a i n e d w i t h the a i d o f t h e Beckman Model DU Spe c t r o p h o t o m e t e r . Experiment 13: The D e t e r m i n a t i o n of t h e A b s o r p t i o n Curves of Normal and I r r a d i a t e d PABA.  (a) The D e t e r m i n a t i o n o f t h e A b s o r p t i o n Curve of Normal PABA  An a b s o r p t i o n curve o f normal PABA (0.000036 M b u f f e r e d w i t h KHgPO^ at pH 4.3) was det e r m i n e d , and i s g i v e n as F i g u r e 18(b) from t h e data p r e s e n t e d i n Table X X V I I . Comparing t h i s a b s o r p t i o n c u r v e w i t h t h a t d e t e r m i n e d by Rothman and Henningsen (1947) ( F i g u r e 1 8 ( a ) ) i t i s n o t e d t h a t t h e a b s o r p t i o n maximum of t h e two c u r v e s l i e a t 2785 A. The m o l e c u l a r e x t i n c t i o n c o e f f i c i e n t (S) of the two c u r v e s d i f f e r s , t h e p u b l i s h e d curve showing an £ v a l u e of 15,300, w h i l e the e x p e r i m e n t a l curve shows an J v a l u e of 13,900. The d i s c r e p a n c y between the p r e s e n t f i g u r e of 13,900 and t h a t of 15,300 r e p o r t e d by Rothman and Henningsen cannot be accounted f o r s i n c e t h e s e workers do not g i v e c e r t a i n p e r -- 88 -TABLE XXVII. The O p t i c a l D e n s i t y of a 0.000036 M S o l u t i o n of Normal. PABA when S u b j e c t e d t o V a r i o u s Wave Lengths of L i g h t . Wave Length mp. O p t i c a l D e n s i t y Wave Length mu O p t i c a l D e n s i t y 210 0.322 277 0.499 215 0.331 278 0.499 220 0.321 278.5 0.500 225 0.255 279 0.500 230 0.165 280 0.500 23 5 . 0.105 285 0.490 240 0.094 290 0.458 245 0.131 295 0.393 250 0.188 300 0.315 255 0.265 305 0.232 260 0.345 310 0.147 265 0.416 315 0.081 270 0.465 320 0.035 275 0.490 325 0.009 276 0.495 330 0.000 - 8.9 -t i n e n t i n f o r m a t i o n such as s o l v e n t used and the pH of t h e i r s o l u t i o n s . . S i n c e i t i s known (Kumler and S t r a i g h t , 1943) t h a t t h e pH at w h i c h t h e a b s o r p t i o n curve i s determined i n f l u e n c e s the p o s i t i o n of the curve w i t h r e s p e c t t o w a v e l e n g t h as w e l l as t h e m o l e c u l a r e x t i n c t i o n c o e f f i c i e n t , i t i s f e l t t h a t the d i s c r e p a n c y noted i s not t o o s e r i o u s . I t i s a l s o p o s s i b l e as can be seen from F i g u r e 19 t h a t PABA at pH 4.3 i n aque-ous s o l u t i o n does not obey t h e Lambert-Beer Law. I f t h i s i s t r u e then t h e m o l e c u l a r e x t i n c t i o n c o e f f i c i e n t w i l l t e n d t o be a f u n c t i o n , of c o n c e n t r a t i o n of t h e s o l u t e and hence one would expect v a r i a t i o n s i n t h e r e p o r t e d v a l u e s . (b) The D e t e r m i n a t i o n o f the A b s o r p t i o n Curve of I r r a d i a t e d PABA.  Two s o l u t i o n s of PABA, 0.0036 and 0.000036 M, b u f f e r e d w i t h KH^PO^ a t pH 4.3, were i r r a d i a t e d f o r 4 h o u r s . A f t e r t h i s p e r i o d of i r r a d i a t i o n , t h e f o r m e r s o l u t i o n was d i l u t e d t o make the c o n c e n t r a t i o n of PABA 0.000036 M. The a b s o r p t i o n curves of b o t h s o l u t i o n s were determined and are g i v e n as F i g u r e 18 ( c ) and 18 (d) from t h e d a t a t a b u l a t e d i n Tables X X V I I I and XXIX. The m o l e c u l a r e x t i n c t i o n c o e f f i c i e n t of the more c o n c e n t r a t e d i r r a d i a t e d PABA s o l u t i o n was c a l c u l a t e d t o be 11,000 ( F i g u r e 18 ( c ) ) . From t h i s i L v a l u e i t was c a l c u l a t e d t h a t a p p r o x i m a t e l y 21 per c e n t o f t h e 0.0036 M PABA s o l u t i o n was a l t e r e d by the u l t r a v i o l e t l i g h t . - 90 -TABLE X X V I I I . The O p t i c a l D e n s i t y of a 0.0036 M S o l u t i o n o f I r r a d i a t ed PABA when S u b j e c t e d  t o V a r i o u s Wave Lengths of L i g h t  at _a Concent r a t i o n of  0.000036 M. Wave Length O p t i c a l ' D e n s i t y Wave Length O p t i c a l D e n s i t y mu mu 210 0.277 265 0.335 215 0.288 270 0.371 220 0.268 275 0.395 225 0.214 278 0.396 230 0.139 280 0.396 235 0.094 285 0.385 240 0.086 290 0.360 245 0.112 300 0.254 250 0.157 310 0.120 255 0.215 320 0.033 260 0.278 330 0.004 - 91 -TABLE XXIX. The O p t i c a l D e n s i t y of a 0.000036 M S o l u t i o n o f I r r a d i a t e d PABA when S u b j e c t e d  t o V a r i o u s Wave Lengths of L i g h t . Wave Length O p t i c a l D e n s i t y Wave Length O p t i c a l D e n s i t y mu nyi 210 0.415 279 0.167 220 0.348 280 0.166 225 0.304 285 0.149 230 0.260 290 0.142 235 0.222 . 295 0.136 240 0.200 300 0.130 245 0.192 305 0.120 253 0.197 310 0.110 260 0.204 315 0.102 270 0.196 320 0.092 275 0.181 325 0.086 278.5 0.170 330 0.080 - 92 -The a b s o r p t i o n curve of the more d i l u t e d s o l u t i o n of PABA appears t o be ve r y d i f f e r e n t ( F i g u r e 18 (d)) to the previous curves. From t h e work of Doub and Vandenbelt (1947) the a b s o r p t i o n curve of i r r a d i a t e d PABA presented as F i g u r e 18 (d) i s v e r y s i m i l a r t o the a b s o r p t i o n curve of phenol. Since t h i s appears t o be the case i t i s l i k e l y that the amino group of the PABA molecule has been removed by the a c t i o n of the u l t r a v i o l e t l i g h t , or r a t h e r a l l of the normal PABA i n the 0.000036 M s o l u t i o n has been a l t e r e d or changed to another compound by the u l t r a v i o l e t i r r a d i a t i o n . It has been shown e a r l i e r ( F i g u r e 2) t h a t the General E l e c t r i c G e r m i c i d a l Lamp used t o i r r a d i a t e PABA emits 90 per cent of i t s r a d i a t i o n at a wave l e n g t h of 2537 X. From the given a b s o r p t i o n curves o f PABA i t i s i n d i c a t e d t h a t t h i s compound w i l l absorb some l i g h t at t h i s wave l e n g t h , t h e r e f o r e i t i s suggested t h a t i n f u t u r e work i t would be a d v i s a b l e t o use monochromatic l i g h t of 2785 X which i s the abso r p t i o n maximum of' PABA. I f t h i s i s c a r r i e d out, i t would be expected that PABA would be a l t e r e d more r a p i d l y and more s p e c i f i c a l l y than when the General E l e c t r i c G e r m i c i d a l Lamp i s used as the source of u l t r a v i o l e t l i g h t . F i g u r e 19 i n d i c a t e s t h a t t h e r e i s a p o s s i b i l i t y t h a t PABA obeys the Lambert-Beer Law. This shows that at a wave leng t h of 2790 8 t h e r e e x i s t s a p r o p o r t i o n a l i t y between i n -c r e a s i n g s o l u t i o n c o n c e n t r a t i o n of PABA and the o p t i c a l d e n s i t i e s of the s o l u t i o n s (see Table XXX). I f t h i s i s t r u e then an a d d i t i o n a l p h y s i c a l method i s a v a i l a b l e f o r the - 93 -TABLE XXX. The P r o p o r t i o n a l i t y Between I n c r e a s i n g ;  C o n c e n t r a t i o n of PABA and t h e i r O p t i c a l D e n s i t i e s -Measur ed a t 2790 8 C o n c e n t r a t i o n of PABA i n micrograms per c c . O p t i c a l D e n s i t y d i s t i l l e d w a ter 0 0.000 0.5 0.041 1.0 0.075 2.0 0.166 3.0 0.252 4.0 0.366 5.0 0.458 6.0 0.515 7.0 0.662 8.0 0.700 9.0 0.781 10.0 0.875 - 94 -d e t e r m i n a t i o n of PABA. V I I I . P r e l i m i n a r y T e s t s t o I s o l a t e the I r r a d i a t e d P r o d u c t s of PABA. P r e l i m i n a r y attempts were made t o i d e n t i f y the i r -r a d i a t e d p r o duct or p r o d u c t s o f PABA. A s o l u t i o n of normal PABA at a c o n c e n t r a t i o n o f 500 micrograms of PABA per c c . d i s t i l l e d w a t e r was exposed t o u l t r a v i o l e t i r r a d i a t i o n f o r 8 hours. A f t e r t h i s p e r i o d i t was n o t e d t h a t the s o l u t i o n had t u r n e d a dark brown and showed e v i d e n c e of a brown p r e -c i p i t a t e . The d i l u e n t was d i s t i l l e d o f f i n vacuum and t h e r e s u l t i n g r e s i d u e was s u b j e c t e d t o . v a r i o u s s o l u b i l i t y t e s t s . I t was found t h a t t h i s r e s i d u e was s l i g h t l y s o l u b l e i n 95 per c e n t a l c o h o l , a c etone and d i s t i l l e d w ater; and i n s o l u b l e i n e t h e r and carbon t e t r a c h l o r i d e . At p r e s e n t i t i s i m p o s s i b l e t o s t a t e the i d e n t i t y of the i r r a d i a t e d p r o d u c t of PABA, however f u t u r e work which i s planned may be s u c c e s s f u l i n i s o l a t i n g the compound o r com-pounds i n a pure s t a t e and t h e n , w i t h the a i d of c h e m i c a l t e s t s , t h e i d e n t i t y of t h e s e compounds may be e s t a b l i s h e d . - 95 -SUMMARY (1) This work has been based on a w o r k i n g h y p o t h e s i s which proposes t h a t t h e b a c t e r i c i d a l ' a c t i o n of u l t r a -v i o l e t l i g h t i s a t t r i b u t a b l e t o an a b s o r p t i o n of energy i n t h e u l t r a v i o l e t r e g i o n by one or more e s -s e n t i a l c e l l u l a r m e t a b o l i t e s . I t i s f u r t h e r suggested t h a t the a b s o r p t i o n produces a m o l e c u l a r s h i f t i n t h e a b s o r b i n g m e t a b o l i t e ( i n t h i s case p-aminobenzoic a c i d ) of such a n a t u r e t h a t t h e a l t e r e d compound w h i l e s t r u c t u r a l l y s t i l l q u i t e s i m i l a r t o i t s m e t a b o l i c p r e -c u r s o r i s s u f f i c i e n t l y d i f f e r e n t to i n t e r f e r e w i t h t h e normal m e t a b o l i c f u n c t i o n s o f the c e l l . B a c t e r i o s t a s i s then f o l l o w s and u l t i m a t e l y death ensues. (2) The l i t e r a t u r e a s s o c i a t e d w i t h t h e b i o l o g i c a l a c t i o n of u l t r a v i o l e t l i g h t and the st u d y of p-aminobenzoic a c i d and s ulphonamides as w e l l as other r e l a t e d s u b j e c t s whic h g i v e s c i r c u m s t a n c i a l e v i d e n c e to support the above h y p o t h e s i s has been r e v i e w e d and i s p r e s e n t e d by s e c t i o n s i n t h i s t h e s i s . (3) The e s t a b l i s h m e n t of a m i c r o b i o l o g i c a l a s s a y p r o -cedure f o r the d e t e r m i n a t i o n of p-aminobenzoic a c i d was done i n o r d e r to d e t e c t i f u l t r a v i o l e t r a d i a t i o n i s a b l e t o produce an a l t e r a t i o n i n the p-aminobenzoic a c i d m o l e c u l e . (4) A d i s c u s s i o n o f the growth r e q u i r e m e n t s of the t e s t - 96 organism, A c e t o b a c t e r suboxydans, has been i n c l u d e d and i s p r e s e n t e d under separate headings. A m i c r o b i o l o g i c a l assay p r o c e d u r e f o r the d e t e r m i n a t i o n of p-aminobenzoic a c i d has been recommended, as i t was found n e c e s s a r y t o modify the p u b l i s h e d assay procedures,, I t was suggested t h a t t h i s recommendation s h o u l d be f o l l o w e d i n f u t u r e work a l o n g the l i n e s of the e x p e r i -m e n t a t i o n s r e p o r t e d h e r e i n . (6) In the study of the b i o l o g i c a l a c t i o n of u l t r a v i o l e t i r r a d i a t i o n i t seems sa f e t o c o n c l u d e t h a t t h e hypo-t h e s i s upon which the p r e s e n t work i s based i s sound, at l e a s t i n p a r t , i n t h a t i t has been demonstrated t h a t i r r a d i a t i o n of p-aminobenzoic a c i d so a l t e r s the compound t h a t i t can no l o n g e r a c t as an " e s s e n t i a l m e t a b o l i t e " f o r A c e t o b a c t e r suboxydans, j i n d e r the p r e v a i l i n g con-d i t i o n s . E x p e r i m e n t a t i o n s were c a r r i e d out t o show the e f f e c t of u l t r a v i o l e t i r r a d i a t i o n on the c e l l s o f A c e t o b a c t e r * suboxydans, and the subsequent study to observe i f the " e s s e n t i a l m e t a b o l i t e " , p-aminobenzoic a c i d , was a b l e t o c o u n t e r a c t the a c t i o n s of these i r r a d i a t i o n s . (7) A s t u d y o-f- the c h e m i c a l a c t i o n of u l t r a v i o l e t l i g h t on p-aminobenzoic a c i d was i n c l u d e d . D e t e r m i n a t i o n s of the a b s o r p t i o n c u r v e s o f normal and i r r a d i a t e d p-aminobenzoic a c i d were c a r r i e d out w i t h the a i d o f t h e Beckman Model DU S p e c t r o p h o t o m e t e r . I t was shown t h a t the a b s o r p t i o n c u r v e o f i r r a d i a t e d p-aminobenzoic a c i d . - 97 -(7) ( c o n t i n u e d ) : d i f f e r e d markedly f r o m t h a t o f normal p-aminobenzoic a c i d . (8) P r e l i m i n a r y attempts t o i s o l a t e the i r r a d i a t i o n p r o -duct or p r o d u c t s o f p-aminobenzoic a c i d were made. V a r i o u s s o l u b i l i t y t e s t s of t h e new compound were c a r r i e d out and r e c o r d e d . I t i s i m p o s s i b l e a t p r e s e n t t o s t a t e t h e i d e n t i t y of the i r r a d i a t i o n p r o d u c t o f p-aminobenzoic a c i d , however f u t u r e work which i s p l a n n e d may be s u c c e s s -f u l i n i s o l a t i n g t h e compound or compounds i n a pure s t a t e and w i t h t h i s r e s u l t the i d e n t i t y of t h e s e compounds may be e s t a b l i s h e d . (9 ) The o r i g i n a l h y p o t h e s i s emphasized the l i k e l i h o o d t h a t the i r r a d i a t i o n p roduct o f p-aminobenzoic a c i d would a c t as an a n t i - m e t a b o l i t e . The work r e p o r t e d does not support such a c o n t e n t i o n i n e n t i r e t y . I t does suggest t h a t e i t h e r an a n t i - m e t a b o l i t e i s formed or more l i k e l y t h a t the " e s s e n t i a l m e t a b o l i t e " , p-amino-b e n z o i c a c i d , i s d e s t r o y e d by i r r a d i a t i o n as d e t e r m i n a b l e by m i c r o b i o l o g i c a l a s s a y . - 98 -APPENDIX I. Review of Photochemical Reactions (Heyroth, 1941j Glasstone, 1948) The l o s s of r a d i a n t energy of a beam of l i g h t which occurs during i t s passage through a gas or a l i q u i d i s a s c r i b e d t o three processes; (a) r e f l e x i o n at the s u r f a c e s of a system, (b) s c a t t e r i n g of the l i g h t by any dust par-t i c l e s i n the gas or by c o l l o i d a l p a r t i c l e s i n the l i q u i d , and (c) a b s o r p t i o n by the molecules of the gas or l i q u i d . The l a s t of these three i s the only one of importance ph.otochemically • Consequently there should be some r e -l a t i o n s h i p between the l i g h t absorbed and the chemical change o c c u r r i n g i n a photochemical r e a c t i o n . During the 19th century such a connection was g e n e r a l i z e d i n terms known as the Grotthus-Draper law. This law s t a t e s t h a t "only those r a d i a t i o n s which are absorbed by the r e a c t i n g system are e f f e c t i v e i n producing chemical change." Not a l l of the l i g h t absorbed by a system i s e f f e c t i v e chemi-c a l l y because i n some cases the absorbed r a d i a t i o n may be re - e m i t t e d as l i g h t of the same or another frequency, an emission of r a d i a n t energy which occurs s i m u l t a n e o u s l y with the a b s o r p t i o n . This r e - e m i s s i o n of l i g h t has been termed f l u o r e s c e n c e . Lambert i n 1760 showed t h a t a r e l a t i o n s h i p e x i s t e d between the extent of l i g h t a b s o r p t i o n and the depth or _ 99 -t h i c k n e s s of the a b s o r b i n g system. T h i s r e l a t i o n s h i p was put i n t h e f o l l o w i n g form c a l l e d the Lambert's Law: " e q u a l f r a c t i o n s of the i n c i d e n t r a d i a t i o n a r e absorbed by suc -c e s s i v e l a y e r s of e q u a l t h i c k n e s s of the l i g h t - a b s o r b i n g s u b s t a n c e " . ' From t h i s an e q u a t i o n can be formed: where I and I denote the t r a n s m i t t e d and i n c i d e n t l i g h t i n t e n s i t i e s , 1 i n cm. i s the t h i c k n e s s of the a b s o r b i n g l a y e r , and k, the c o n s t a n t which i s c h a r a c t e r i s t i c of t h e l a t t e r . T h i s c o n s t a n t i s r e f e r r e d t o as the a b s o r p t i o n c o e f f i c i e n t of the r e a c t i n g system. I t has r e f e r e n c e t o l i g h t of a p a r t i c u l a r wave l e n g t h , and t h e v a l u e o f "k" v a r i e s w i t h the wave l e n g t h of the absorbed r a d i a t i o n . From the e q u a t i o n g i v e n above the i n t e n s i t y of t h e l i g h t absorbed ( l a b s ) i s e q u a l t o t h e d i f f e r e n c e between the i n t e n s i t i e s of the i n c i d e n t ( l Q ) and the t r a n s m i t t e d ( I ) l i g h t ; t h e r e f o r e : l a b s = I Q - I = 1 - ( I - k l ) = I 0 (1 - e - k l ) Where the a b s o r b i n g s u b s t a n c e i s i n s o l u t i o n , as i n the case of PABA d i s s o l v e d i n a s u i t a b l e s o l v e n t , the r e -l a t i o n s h i p between the i n c i d e n t and t h e t r a n s m i t t e d r a d i a t i o n s i s g i v e n i n the form of Beer's Law. The m a t h e m a t i c a l r e p r e s e n t a t i o n of t h i s law i s as f o l l o w s : I * I- 0 i o - e c l - 100 -where "e" i s the e x t i n c t i o n c o e f f i c i e n t of t h e a b s o r b i n g s o l u t e and " c " the c o n c e n t r a t i o n of the a b s o r b i n g component. A c c o r d i n g t o the Quantum Theory as g i v e n by P l a n c k a n d . E i n s t e i n , a body cannot emit or absorb r a d i a n t energy i n a c o n t i n u o u s manner. The energy can o n l y be t a k e n up or g i v e n out as i n t e g r a l m u l t i p l e s of a d e f i n i t e amount, known as a quantum. I f "E" i s the energy of the quantum f o r a p a r t i c u l a r r a d i a t i o n f r e q u e n c y , Y i n v i b r a t i o n s per second, then the quantum t h e o r y i s E = hV, where "h" i s a u n i v e r s a l c o n s t a n t , known as P l a n k ' s c o n s t a n t , e q u a l t o 6.62 x 10"^ erg s e c . E i n s t e i n (1912, 1913) i n t r o d u c e d an i m p o r t a n t g e n e r a l i z a t i o n i n p h o t o c h e m i s t r y i n t h e form of t h e law of Ph o t o -c h e m i c a l E q u i v a l e n t . T h i s law s t a t e s , t h a t "each m o l e c u l e t a k i n g p a r t i n a c h e m i c a l r a c t i o n , which i s a d i r e c t r e -s u l t of t h e a b s o r p t i o n of l i g h t , t a k e s up one quantum of the r a d i a t i o n c a u s i n g the r e a c t i o n " . T h i s law i m p l i e s t h a t i f -V i s the f r e q u e n c y of the absorbed r a d i a t i o n i n v i b r a t i o n s per second, t h e n the c o r r e s p o n d i n g quantum i s e q u a l t o hV. T h e r e f o r e the energy, "E", absorbed per mole i s t h e n NhY, where "N" i s the Avogrado number e q u a l t o 6.06 x 1023. Hence we have t h e r e p r e s e n t a t i o n as: E = NhV ergs per mole. The f r e q u e n c y "V" of the r a d i a t i o n s i n v i b r a t i o n s per second i s e q u a l t o c/)^ , where " c " i s the v e l o c i t y of l i g h t or 3.0 x 10 1 0 cm. per second and \ i s t h e wave l e n g t h - 101-i n cm. However the l a t t e r i s more f r e q u e n t l y e x p r e s s e d i n ftngstom u n i t s , where 1A = 1 0 ~ 8 cm. and t h u s an e x p r e s s i o n can be w r i t t e n as f o l l o w s : , f = c_ x 1 0 8 From t h i s a l t e r a t i o n t h e above e q u a t i o n can now be w r i t t e n : Q E = Nhc x 10 ergs per mole X The q u a n t i t y E which i s t h e energy absorbed per mole of r e a c t i n g substance i s u s u a l l y r e f e r r e d t o as one e i n s t e i n of r a d i a t i o n of t h e g i v e n wave l e n g t h . The quantum e f f i c i e n c y or quantum y i e l d , which i s d e f i n e d as the number of moles of the l i g h t a b s o r b i n g sub-s t a n c e t h a t r e a c t s f o r each e i n s t e i n of absorbed r a d i a t i o n i s v e r y i m p o r t a n t as f a r as the r e s u l t of a p h o t o c h e m i c a l r e a c t i o n i s concerned. For convenience the quantum e f f i c i e n c y may be r e p r e s e n t e d by t h e e x p r e s s i o n ? Quantum e f f i c i e n c y ( y i e l d ) = Number of moles r e a c t i n g Number of e i n s t e i n s absorbed By the Law of t h e P h o t o c h e m i c a l E q u i v a l e n t g i v e n p r e -v i o u s l y , i t would be expected t h a t i f t h e r e a c t i o n of a system i s one a s s o c i a t e d o n l y w i t h the a c t i o n of l i g h t , t h e n t h e quantum e f f i c i e n c y would be u n i t y . However i n many cases t h i s i s not the a c t u a l r e s u l t . I t was suggested t h a t the f i r s t s t e p of the p h o t o c h e m i c a l r e a c t i o n , i e , the a b s o r p t i o n of l i g h t , s h o u l d be r e f e r r e d t o as t h e p r i m a r y p r o c e s s , and i t i s t h i s p r o c e s s i n which the E i n s t e i n l a w p r o p e r l y p e r t a i n s . In t h i s p r i m a r y p r o c e s s each m o l e c u l e - 102 absorbs one quantum, however, a f t e r such a b s o r p t i o n , a secondary p r o c e s s o c c u r s i n which the m o l e c u l e s of r e -a c t a n t t a k e p a r t . T h i s subsequent secondary p r o c e s s may be q u i t e complex, and w i l l , i n f a c t , cause the quantum y i e l d of the o v e r a l l r e a c t i o n t o v a r y from u n i t y . - 103 -APPENDIX I I . The Use of M i c r o o r g a n i s m s t o As say V i t a m i n s D u r i n g the past few ye a r s v a r i o u s methods have been developed and employed t o a s s a y s u b s t a n c e s f o r t h e i r v i t a m i n c o n t e n t . P h y s i c a l and c h e m i c a l methods a r e u s e f u l , how-ever t h e y are i n c o n t e s t a b l e o n l y i f the r e s u l t s o b t a i n e d from t h e s e methods can be c o r r e l a t e d w i t h b i o l o g i c a l p r o - ' c e d u r e s . As a r e s u l t b i o l o g i c a l methods have been e x t r e m e l y u s e f u l i n the d i s c o v e r y of v i t a m i n s and a l s o as the f i n a l c r i t e r i a f o r q u a l i t a t i v e and q u a n t i t a t i v e v i t a m i n assays as w e l l as b e i n g a means whereby o t h e r methods can be s t a n d a r d i z e d and e v a l u a t e d . B i o l o g i c a l methods u s i n g l a b o r a t o r y a n i m a l s such as the r a t are o f t e n c o s t l y and t i m e consuming, and u s u a l l y cannot be employed when s m a l l amounts of m a t e r i a l a r e a v a i l a b l e t o be a n a l y z e d . The d i s c o v e r y t h a t m i c r o o r g a n i s m s r e q u i r e c e r t a i n v i t a m i n s f o r growth has p r o v i d e d a new, r a p i d and q u a n t i t a t i v e method t o a s s a y v i t a m i n s . For example an organism which r e q u i r e s a g i v e n v i t a m i n i s used, and a l l o t h e r f a c t o r s n e c e s s a r y f o r growth are kept c o n s t a n t i n the medium. A s t a n d a r d curve i s then o b t a i n e d by v a r y i n g the c o n c e n t r a t i o n o f the v i t a m i n i n q u e s t i o n and p l o t t i n g the growth response i n the c u l t u r e medium a g a i n s t the v a r i o u s c o n c e n t r a t i o n s of the v i t a m i n . The amount of a g i v e n v i t a m i n i n an unknown substance can t h e n be - 104 -det e r m i n e d by u s i n g t h i s m a t e r i a l i n p l a c e of the known v i t a m i n . A comparison of t h e r e s u l t s from t h i s s ubstance a g a i n s t the s t a n d a r d a s s a y c u r v e u s i n g t h e pure v i t a m i n i n q u e s t i o n w i l l show the amount of tha(6 f a c t o r i n the un-known. T h i s method of a s s a y i n g v i t a m i n s i s r a p i d l y g a i n i n g f a v o r . At the p r e s e n t time i t s use appears t o be r e s t r i c t e d t o t he B v i t a m i n s , , however as time p r o g r e s s e s , s p e c i a l t e s t s w i l l u n d o u b t e l y be developed f o r a l l the members of the v i t a m i n B-complex and p o s s i b l y o t h e r v i t a m i n s as w e l l . In the work t h a t t h i s r e p o r t i s d e s c r i b i n g , m i c r o -organisms were used t o determine i f any a l t e r a t i o n of PABA r e s u l t s when t h a t s u b s t a n c e i s exposed t o u l t r a v i o l e t r a d i a t i o n s . The l i t e r a t u r e was r e v i e w e d i n o r d e r t o f i n d i f t h e r e e x i s t s a s u i t a b l e m i c r o o r g a n i s m which r e q u i r e s PABA for i t s normal growth. S i n c e t h i s s u r v e y of the l i t e r a t u r e proved e x t r e m e l y u s e f u l , i t i s wise a t t h i s p o i n t t o r e v i e w i t b r i e f l y . The Growth Promoting A c t i v i t i e s of PABA f o r B a c t e r i a ; ( i ) M i c r o o r g a n i s m s t h a t do not r e q u i r e the a d d i t i o n of PABA t o t h e i r growth medium.  Woods (1940) r e p o r t e d t h a t PABA had no s i g n i f i c a n t e f f e c t on the r a t e or mass of growth of S t r e p t o c o c c u s h a e m o l y t i c u s or B a c t e r i u m c o l i under t h e p r e v a i l i n g t e s t c o n d i t i o n s s i n c e i t was not n e c e s s a r y t o add i t t o a medium c o n t a i n i n g o n l y known s u b s t a n c e s . Other i n v e s t i g a t o r s have shown t h a t PABA does not s t i m u l a t e the growth of - 105 -S t r e p t o c o c c u s v i r i d a n s ( M i l l e r , 1941) S t a p h y l o c o c c u s aureus ( S p i n k and Jermata, 1941) or Pneumococcus ( S t r a u s e , et a l . 1941) and a l s o t h a t i t has not been found e s s e n t i a l f o r ot h e r p a t h o g e n i c b a c t e r i a (Fox, 1942). A p p a r e n t l y t h e s e organisms are a b l e t o s y n t h e s i z e t h i s f a c t o r from o t h e r p r e c u r s o r s . I f PABA i s t r u l y an e s s e n t i a l m e t a b o l i t e , the f a c t t h a t i t i s a growth f a c t o r f o r a few organisms s h o u l d be supplemented by ev i d e n c e t h a t i t i s i m p o r t a n t i n the n u t r i -t i o n of b a c t e r i a g e n e r a l l y , t h a t i s t o prove t h a t i t i s s y n t h e s i z e d by organisms a b l e t o grow i n PABA-free c u l t u r e media. Landy et a l (1943), w h i l e s t u d y i n g b a c t e r i a l s y n t h e s i s of PABA, c u l t u r e d a g r e a t v a r i e t y of organisms i n a PABA-f r e e medium. I t was r e v e a l e d t h a t most b a c t e r i a s y n t h e s i z e PABA i n r e a d i l y measurable amounts, and a l s o t h a t i n most i n s t a n c e s the b u l k of PABA produced i s found i n t h e c u l t u r e medium. Few of the s p e c i e s r e t a i n any g r e a t amount of f r e e PABA i n t h e i r c e l l s . The a b i l i t y of b a c t e r i a t o e l a b o r a t e PABA i s not i n t e n d e d t o convey any q u a n t i t a t i v e comparison of v a r i o u s organisms, because i t was observed t h a t s t r a i n s of a g i v e n s p e c i e s c u l t u r e d under a p p a r e n t l y i d e n t i c a l c o n d i t i o n s d i d not n e c e s s a r i l y y i e l d the same amount of PABA. T h i s p o i n t i s shown i n t h e condensed t a b l e g i v e n by Landy and h i s a s s o c i a t e s (1943) ( T a b l e A ) . - io6 -TABLE A. B a c t e r i a l S y n t h e s i s of P_«_ aminobenzoic A c i d (Landy e t a l . 1943) Organism I n c u b a t i o n p-Aminobenzoic A c i d °C Hr. .found (micrograms per cc . c u l t u r e ) In Cells In Medium Tot a l S h i g e l l a d y s e n t e r i a e 37 24 0 0.018 0. 018 E s c h e r i c h i a c o l i 37 24 0.038 0.007 0.045 B r u c e l l a a b o r t u s 37 72 0 0.013 0.013 B a c i l l u s s u b t i l i s 37 24 0 0.019 0.019 B a c i l l u s megatherium 30 24 0 0.007 0.007 C o r y n e b a c t e r i u m diphtheriae 37 24 0.070 0.171 0.241 S t a p h y l o c o c c u s aureus 37 24 0.004 0.063 0.067 S t r e p t o c o c c u s hemolyticus 37 24 0.008 0.004 0.012 L a c t o b a c i l l u s c a s e i 37 72 - 0.016 0.016 L a c t o b a c i l l u s a r a b i n o s u s 30 72 0 0 0 T h i s f i n d i n g l e n d s s u p p o r t t o Wood's t h e s i s t h a t the sulphonamide i n h i b i t i o n i s due t o PABA i n t e r f e r e n c e . I t i s suggested by Landy and h i s c o l l e a g u e s (1943) t h a t the " s e n s i t i -v i t y " of a b a c t e r i u m t o sulphonamides w o u l d depend, a t l e a s t i n p a r t , upon whether i t c o u l d s y n t h e s i z e PABA r e a d i l y or n o t . An organism w i t h l i t t l e s y n t h e t i c a b i l i t y would be more sen-s i t i v e t o sulphonamides t h a n ones w i t h g r e a t e r s y n t h e t i c powers. The f i n d i n g t h a t c e r t a i n b a c t e r i a s y n t h e s i z e PABA (TableA) appears t o make t h i s s u g g e s t i o n a d e f i n i t e p o s s i b i l i t y . In any case i t i s l i k e l y t h a t t h e PABA s y n t h e s i s by a g i v e n organism would p l a y some p a r t i n t h e degree of i t s r e s i s t a n c e t o sulphonamide a c t i o n . - 107 -( i i ) M i c r o o r g a n i s m s t h a t r e q u i r e the a d d i t i o n of PABA t o t h e i r growth medium  I t has been p o i n t e d out (above) t h a t t h e m a j o r i t y of b a c t e r i a can s y n t h e s i z e PABA, w h i l e a few may and do r e q u i r e the presence o f t h i s aromatic amine f o r normal c e l l d e v e l o p -ment and d i v i s i o n . This s i m p l e o r g a n i c compound i s needed f o r growth by A c e t o b a c t e r suboxydans (Lampen, et a l . 1942) where 0.05 micrograms of PABA per t e n c c . medium s u p p o r t s maximum growth. That t h i s a r o m a t i c amine i s a t r u e growth f a c t o r f o r L a c t o b a c i l l u s a r a b i n o s u s was c o n f i r m e d by sub-sequent i n v e s t i g a t o r s ( i s b e l l , 1942} L e w i s , 1942j Shankman, 1 9 4 3 ) . I s b e l l (1942) found t h a t L a c t o b a c i l l u s a r a b i n o s u s r e q u i r e s 0.001 micrograms of PABA per t e n c c . medium and a l s o t h a t t h i s growth f a c t o r i s 1 0 , 0 0 0 times as a c t i v e as the n e x t most a c t i v e compound. Shankman (1943) observed t h a t t h e r e a r e two s t r a i n s of L a c t o b a c i l l u s a r a b i n o s u s ; one r e q u i r e s PABA w h i l e t h e o t h e r , which i s a mutant, does n o t . Lampen and P e t e r s o n (1943) showed t h a t - PABA i s needed f o r growth by seven s t r a i n s of C l o s t r i d i u m a c e t o -b u t y l i c u m . by C l o s t r i d i u m b u t y l i c u m No. 28, and by C l o s t r i d i u m  f e l s i n e u m . In t h e presence of b i o t i n (1 .5 x 10-4 t o 1.5 x 10""° micrograms per «c. of medium) PABA f u n c t i o n s as a growth f a c t o r f o r t h i s m i c r o o r g a n i s m a t c o n c e n t r a t i o n s as low as 1 x 10"^ micrograms per c c . ( P a r k and Wood, 1 9 4 2 ) , £lo_str_i-dium s a c c h a r o l y t i c u m ( C l a r k and M i t c h e l l , 1944) Coryne-T - 108 -b a c t e r i u m d i p h t h e r i a e (Chattaway, e t a l . , 1942) and Neurospora mutant (Tatum and Be a d l e , 1942) were shown t o r e -q u i r e PABA. I t i s of i n t e r e s t t o note t h a t the e f f e c t i v e n e s s of PABA as a growth f a c t o r f o r t h e Neurospora c r a s s a mutant of Tatum and Beadle (1942) d e c r e a s e s w i t h the i n c r e a s e i n pH 'of t he n u t r i e n t s o l u t i o n , t h u s i n d i c a t i n g a c t i v i t y f o r the m o l e c u l a r and not the i o n i c form of the s i m p l e a r o m a t i c amine. (Wyss et a l . , 1 9 4 4 ) . I t has been demonstrated that-PABA i s a l s o r e q u i r e d by Leuconostoc mesenteroides Pd - 6 0 ( P e n n i n g t o n , 1946) and Rhodopseudomonas p a l u s t r i s . ( H u t n e r , 1946j 1946a) and stimulates growth of C I . b o t u l i n u m ( R o e s s l e r and Brewer, 1 9 4 6 ) . Recent-l y Rainbow (1948) n o t i c e d t h a t two s i n g l e c e l l s t r a i n s of f e r m e n t a t i o n y e a s t s ('Yeast 4 5 ' and 'Yeast 4 7 ' ) r e q u i r e s the presence of PABA i n the medium f o r optimum growth. S i n c e t h e growth i n h i b i t o r y a c t i o n of the m a j o r i t y of sulphonamides i s a n n u l l e d i n a c o m p e t i t i v e manner by PABA, i t i s l i k e l y t h a t the e l u c i d a t i o n of t h e m e t a b o l i c f u n c t i o n of t h i s f a c t o r w i l l i l l u m i n a t e t he b a s i c mode of a c t i o n of t h e s e d r u g s . I t has been suggested (Woods, 1940) t h a t sulphonamides competitively i n h i b i t the enzyme r e a c t i o n i n v o l v e d i n the u t i l i z a t i o n of PABA. S i n c e t h i s s u g g e s t i o n was proposed i n f o r m a t i o n as t o t h e n a t u r e of t h i s u t i l i z a t i o n i s a t hand (Lampen and Jones, 1947) and the i n t e r r e l a t i o n -s h i p s of PABA w i t h p u r i n e b a s e s , m e t h i o n i n e and " f o l i c a c i d " are of p a r t i c u l a r i n t e r e s t . - 1 0 9 -I t may appear t h a t when t h e i r r a d i a t e d p r o d u c t o f PABA i s added t o a c u l t u r e medium, t h i s compound w i l l b r i n g about a growth i n h i b i t o r y a c t i o n t h a t i s s i m i l a r t o t h a t i n h i b i t o r y a c t i o n of sulphonamides. With t h i s i n mind, then i t may p o s s i b l y be the f a c t t h a t t h e newly formed compound w i l l c o m p e t i t i v e l y i n h i b i t an enzyme r e a c t i o n t h a t i s i n -v o l v e d i n the u t i l i z a t i o n of PABA M i c r o b i o l o g i c a l and Chemical Methods f o r t h e D e t e r m i n a t i o n of PABA  ( i ) M i c r o b i o l o g i c a l Methods; A s e m i - q u a n t i t a t i v e m i c r o b i o l o g i c a l a s s a y f o r PABA was d e v i s e d by Rubbo et a l . (1941) u s i n g C l o s t r i d i u m a c e t o -b u t y l i c u m . Other more p r e c i s e methods s i n c e have been deve l o p e d u s i n g the same m i c r o o r g a n i s m f o r t h i s d e t e r m i n a t i o n . (Park and Wood, 1942; Lampen and P e t e r s o n , 1 9 4 4 ) . Landy and Dicken (1942) have employed A c e t o b a c t e r suboxydans f o r t h e t e s t organism i n t h i s d e t e r m i n a t i o n . They measure i n a p h o t o e l e c t r i c c o l o r i m e t e r the t u r b i d i t y r e s u l t i n g from the growth of t h e b a c t e r i a i n r e l a t i o n t o PABA l e v e l i n the -medium. T h i s t e s t i s h i g h l y s p e c i f i c , s i n c e the isomers of PABA and ot h e r r e l a t e d s u b stances were found t o have no b i o l o g i c a l a c t i v i t y . I n 1945 , a m o d i f i c a t i o n i n t h e A c e t o b a c t e r suboxydans assay f o r PABA was p u b l i s h e d ( C h e l d e l i n and Be n n e t t , 1 9 4 5 ) . The medium was changed i n s e v e r a l r e s p e c t s t o p e r m i t g r e a t e r growth response t o added PABA* - 110 -Lewis (1942) d e s c r i b e d a method whereby L a c t o -b a c i l l u s a r a b i n o s u s was used as the t e s t organism i n the m i c r o b i o l o g i c a l a s s a y . M i t c h e l l and h i s a s s o c i a t e s (1943) made use of the Neurospora crasrea mutant of Tatum and Beadle (1942). The l a t t e r method was r e v i s e d by the same workers i n a more r e c e n t p u b l i c a t i o n (Thompson et a l . 1943). Thompson et a l . (1943) found t h a t s t r o n g a c i d or a l k a l i n e h y d r o l y s i s was n e c e s s a r y t o o b t a i n the c o r r e c t PABA c o n t e n t of n a t u r a l s u b s t a n c e s s i n c e i t occurs i n f r e e and bound forms ( B l a n c h a r d , 1941). However as i t i s apparent t h a t PABA o c c u r s i n many n a t u r a l m a t e r i a l s i n an e x t r e m e l y r e -s i s t a n t c o m b i n a t i o n , p a r t of the PABA i s i n a compound r e s i s t a n t t o a c i d h y d r o l y s i s . Recommendations were made t o f r e e PABA from i t s bound form by u s i n g 5N NaOH and sub-j e c t i n g the m a t e r i a l t o 75-80 prounds p r e s s u r e f o r one hour (Lampen and P e t e r s o n , 1944). ( i i ) C h e mical Methods r U s i n g t h i a m i n e (B-^) as a r e a g e n t , a q u a n t i t a t i v e method was developed ( K i r c h and Bergeim, 1943) based upon an i s o a m y l a l c o h o l s o l u b l e , p i n k t o r e d compound o b t a i n e d by r e a c t i n g PABA w i t h d i a z o t i z e d t h i a m i n e . The procedure f o r t h i s method of d e t e r m i n i n g the amount of f r e e PABA i s g i v e n i n Appendix I I I . B r a t t o n and M a r s h a l l ' s sulphonamide d e t e r m i n a t i o n method ( B r a t t o n and M a r s h a l l , 1939) was used f o r a - I l l -q u a n t i t a t i v e d e t e r m i n a t i o n of PABA i n y e a s t ( B l a n c h a r d , 1941) and i n b l o o d ( E c k e r t , 1943). T h i s r e a c t i o n d e s c r i b e d by E k e r t (1943) i s e x t r e m e l y s e n s i t i v e and one microgram of PABA can r e a d i l y be dete r m i n e d i n t e n m l . of f i l t r a t e . The i n v e s t i g a t o r p r e d i c t s t h a t by s u i t a b l e r e d u c t i o n of volumes a s t i l l g r e a t e r s e n s i t i v i t y may be a t t a i n e d , and s i m i l a r l y i t would be p o s s i b l e t o determine 0.1 microgram i n one ml. of f i l t r a t e i f s p e c i a l s m a l l c o l o r i m e t e r tubes are used. Other i n v e s t i g a t o r s ( G a r c i a - B l a n c o and V i n a , 1948;' R i n d i , 1948) have r e c e n t l y p u b l i s h e d methods by which PABA may be determined i n o r g a n i c f l u i d s . From the above r e v i e w of the methods used t o d e t e r -mine PABA and from Table B, i t i s v e r y n o t i c e a b l e t h a t m i c r o o r g a n i s m s can d e t e c t much s m a l l e r amounts of PABA than can the p r e s e n t c h e m i c a l t e s t s . - 112 -TABLE B. M i c r o b i o l o g i c a l Methods f o r the Q u a n t i t a t i v e Assay of PABA M i c r o o r g a n i s m Employed Methods used t o r e c o r d l e v e l s of PABA S m a l l e s t Quan-t i t y D e t e c t -a b l e ( m i c r o -grams per ml. medium) Ref e r e n c e C l o s t r i d i u m ac e t o b u t y l i -cum T u r b i d i t y of c u l t u r e 0.00003 -0.002 Lampen and P e t e r s o n (1944) L. a r a b i n o -sus A c i d P r o -d u c t i o n 0.0002 -0.005 Lewis(l942) A c e t o b a c t e r suboxydans T u r b i d i t y of c u l t u r e 0.001 -0.003 Landy and Dicken(l942) Landy and S t r e i g h t o f f (1943) Neurospora c r a s s a Area of growth 0.0003 -0.001 Thompson et a l . (1943) - 113 -APPENDIX I I I . Procedures f or M i c r o b i o l o g i c a l and Chemical Assays of p_-Aminobenzoic A c i d The methods g i v e n below a r e e s s e n t i a l l y verbatum acc o u n t s from the o r i g i n a l p u b l i c a t i o n s . A. M i c r o b i o l o g i c a l Methods; (a) In r e f e r e n c e t o t h i s work, t h e m i c r o b i o l o g i c a l a ssay f o r t h e d e t e r m i n a t i o n of PABA o u t l i n e d by Landy and Dicken (1942) was chosen because of t h e s p e c i f i c a c t i o n of A c e t o b a c t e r suboxydans t o t h i s a r o m a t i c amine. The p r o c e d u r e g i v e n f o r such an assay i s as f o l l o w s : Organism: The t e s t o r g a n i s m employed i s A. suboxydans. A.T.C. 621, and i s c a r r i e d on y e a s t e x t r a c t - g l y c e r o l - a g a r (0.5 per cent B a c t o - y e a s t e x t r a c t , 5 per cent g l y c e r o l , 1.5 per cent B a c t o - a g a r , pH 6.0). S t o c k c u l t u r e s are t r a n s f e r r e d at monthly i n t e r v a l s and are r e f r i g e r a t e d i n t h e i n t e r i m . Inoculum f o r as s a y i s p r e p a r e d by t r a n s f e r f r o m the s t o c k c u l t u r e t o a f l a s k of b a s a l medium (Table C) t o whi c h i s added 0.05 micrograms p-aminobenzoic a c i d . The i n o c u l u m c u l t u r e i s i n c u b a t e d a t 30°C. f o r 24 hours p r i o r to use. B a s a l Medium; The c o m p o s i t i o n of the b a s a l medium i s g i v e n i n Table C. The f o r m u l a i s t w i c e t h e d e s i r e d con-c e n t r a t i o n . - 114 -Assay P r o c e d u r e : F i v e c c . of b a s a l medium and d i l -u t i o n s o f m a t e r i a l under t e s t made up to 5 c c . w i t h d i s t i l l e d water are p l a c e d i n 50 c c . Erlenmeyer f l a s k s . S i m i l a r l y , a set of r e f e r e n c e f l a s k s i s p r e p a r e d c o n t a i n i n g f rom 0.01. t o 0.1 micrograms of PABA. B l a n k s c o n t a i n i n g no PABA a r e i n -c l u d e d . The f l a s k s a r e plugged w i t h c o t t o n and a u t o c l a v e d at 15 pounds p r e s s u r e f o r 15 m i n u t e s . A f t e r c o o l i n g t h e y are i n o c u l a t e d w i t h a s u s p e n s i o n of k. suboxydans. A 24 hour c u l t u r e grown as d e s c r i b e d above i s c e n t r i f u g e d and washed t w i c e i n 10 c c . of s t e r i l e s a l i n e s o l u t i o n . The washed c e l l s are resuspended i n 15 c c . o f s a l i n e s o l u t i o n . One drop ( a p p r o x i m a t e l y 0.05 c c . ) of t h e r e s u l t i n g s u s p e n s i o n per f l a s k s e r v e s as the i n o c u l u m . The f l a s k s a r e i n c u b a t e d a t 30°C. f o r 48 h o u r s . F o l l o w i n g i n c u b a t i o n , 10 c c . of w a t e r are added t o each f l a s k t o d i l u t e the c u l t u r e s u i t a b l y f o r measurement of t u r b i d i t y and the c o n t e n t s are t h o r o u g h l y mixed by s h a k i n g . The growth response t o in c r e m e n t s of PABA i s d e t e r m i n e d by measurement of t u r b i d i t y w i t h a p h o t o e l e c t r i c c o l o r i m e t e r . A s t a n d a r d curve can be c o n s t r u c t e d by p l o t t i n g the c o l o r i m e t e r r e a d i n g s a g a i n s t the c o n c e n t r a t i o n of PABA. The PABA c o n t e n t of m a t e r i a l under t e s t i s then r e a d from t h e s t a n d a r d c u r v e . (b) Park and Wood (1942) i l l u s t r a t e d a s e m i - q u a n t i t a -t i v e m i c r o b i o l o g i c a l a s s a y f o r PABA. C l o s t r i d i u m a c e t o -b u t y l i c u m , an a n a e r o b i c b a c i l l u s , was used as i t grows l u x u r i a n t l y i n a r e l a t i v e l y s i m p l e s y n t h e t i c medium p r o v i d e d c e r t a i n s p e c i f i c growth f a c t o r s a r e added." The p r o c e d u r e - 115 -TABLE C. B a s a l Medium f o r PABA Assay ( U n d i l u t e d ) . C a s e i n h y d r o l y s a t e 0.6 gm. G l y c e r o l 10.0 it Tryptophane 20.0 mgm. C y s t i n e 15.0 it K 2HP0^ 100.0 M K H 2 P 0 4 100.0 It MgS0 4.7H 20 40.0 It NaCl 2.0 It FeSO, ,7H20 2.0 II. MnS0 4.2H 20 2.0 II C a l c i u m pantothenate 200.0 microgms. N i c o t i n i c A c i d 200.0 it D i s t i l l e d water t o pH a d j u s t e d 6.0 + 1 100.0 cc . o u t l i n e d by t h e s e i n v e s t i g a t o r s i s as f o l l o w s : B a s a l Medium: The b a s a l medium used here was t h a t d e s c r i b e d by Oxf o r d and h i s co-workers (1940) and had the f o l l o w i n g c o m p o s i t i o n : Glucose 20.0 gms. A s p a r a g i n ( o r ( N H 4 ) 2 H P 0 4 ) 1.0 K 2 H P 0 4 0.5 " K H 2 P 0 4 0.5 " MgS0 4.?H 20 0.2 " NaCl 0.01 « FeS0 4.7H 20 0.01 " MnCl 2.4H 20 0.01 » Reduced Fe. 3-5 mgms. D i s t i l l e d w a ter 1000 c c . The medium was tubed i n 30 c c . l o t s i n c h e m i c a l l y c l e a n e d 8 - i n c h t e s t t u b e s and was s t e r i l i z e d by steaming f o r 20 - 116 -minutes j u s t b e f o r e u s e . P r i o r t o s t e r i l i z a t i o n supplements t o t h e b a s a l medium were added. C u l t u r a l Methods; Stock c u l t u r e s of £ 1 . -aceto-b u t y l i c u m were grown i n b a s a l medium e n r i c h e d w i t h peptone. Under t h e s e c o n d i t i o n s the organ-ism grox^s r a p i d l y , p r o d u c i n g l a r g e amounts of gas. Growth was f o u n d t o .be maximum i n 48 hours, at w h i c h t i m e 10 c c . of t h e peptone c u l t u r e was c e n t r i f u g a l i z ed, the s u p e r n a t a n t d i s c a r d e d , and t h e organisms resuspended i n 5 c c . of s t e r i l e b a s a l medium. 0.5 c c . of the s u s p e n s i o n was used as a s t a n d a r d i n o c u l u m . F o l l o w i n g i n o c u l a t i o n , t h e tubes were i n c u b a t e d a n a e r o b i c a l l y . I t was found t h a t the s i m p l e o a t - j a r method ( C a r r o l and H a s t i n g s , 1925) i n which s t a n d a r d a n a e r o b i c j a r s c o n t a i n i n g 3 i n c h e s of moistened oats at t h e bottom, s e a l e d and evac u a t e d by s u c t i o n b e f o r e b e i n g p l a c e d i n the i n c u b a t o r , was s a t i s f a c t o r y f o r the p r o d u c t i o n o f a n a e r o b i o s i s . Supplement s t o B a s a l Medium: C r y s t a l l i n e b i o t i n was d i s s o l v e d i n the b a s a l medium t o make f i n a l c o n c e n t r a t i o n i n th e c u l t u r e tubes from 1.5 x 10"^  micrograms per c c . t o 1.5 x 10~& micrograms per c c . PABA was added t o the b a s a l medium s i m i l a r l y , the f i n a l c o n c e n t r a t i o n s i n t h e c u l t u r e t u b e s 9 6 r a n g i n g from 1 x 10 micrograms per c c . t o 1 x 10" m i c r o -grams per c c . E s t i m a t i o n of B a c t e r i a l Growth: Growth i n the b a s a l medium appears f i r s t between 48 and 72 hours a f t e r i n o c u l a t i o n . When gas p r o d u c t i o n i s noted i n the t u b e s , t h e a n a e r o b i c j a r s - 117 -are r e - e v a c u a t e d by s u c t i o n b e f o r e b e i n g opened. Each tube i s t h o r o u g h l y shaken t o o b t a i n an even s u s p e n s i o n o f o r g a n i s m s . A 5 c c . sample of the c u l t u r e i s t h e n p i p e t t e d i n t o the chamber of a p h o t o e l e c t r i c c o l o r i m e t e r and r e a d i n g s are t a k e n . B. Chemical Methods; The procedure f o r d e t e r m i n i n g t h e amount of f r e e PABA, based-upon an i s o a m y l a l c o h o l s o l u b l e p i n k to r e d compound o b t a i n e d by r e a c t i n g PABA w i t h d i a z o t i z e d t h i a m i n e i s as f o l l o w s ( K i r c h and Bergeim, 1943): To a s u i t a b l e amount o f a s o l u t i o n t o be t e s t e d d i s t i l l e d water i s added t o make a volume o f about 20 c c . 0.3 c c . o f 35 per c e n t a c e t i c a c i d i s added; the r e s u l t i n g pH of t h e m i x t u r e s h o u l d be about 2.9. A 5cc. p o r t i o n of a d i a z o s o l u t i o n w h i c h i s made by m i x i n g e q u a l volumes of 0.2 per cent aqueous s o l u t i o n of t h i a m i n e c h l o r i d e and 2 per cent aqueous s o l u t i o n of sodium n i t r i t e , i s added. Then 4 c c . of a p p r o x i m a t e l y 1 N NaOH s o l u t i o n i s added, b r i n g i n g the pH of the m i x t u r e to about 11.6, f o l l o w e d w i t h 5 c c . of i s o a m y l a l c o h o l . To s h i f t the s o l u b i l i t y of the c o l o r e d compound i n the d i r e c t i o n of t h e amyl a l c o h o l , 0.5 c c . of 35 per cent a c e t i c a c i d i s added, making the f i n a l pH about 5.3. The m i x t u r e i s t h o r o u g h l y shaken, a l c o h o l s e p a r a t e d , and d r i e d over anhydrous sodium s u l f a t e . The s e r i e s of c o l o r e d mix-t u r e s i s t h e n r e a d i n a s p e c t r o p h o t o m e t e r . The s m a l l e s t amount of PABA found t o g i v e a c o l o r under t h e above c o n d i t i o n s was about 10 micrograms. T h i s - 118 -d e t e r m i n a t i o n shows that the m i c r o b i o l o g i c a l a s s a y s a r e more s e n s i t i v e as some mic r o - o r g a n i s m s can d e t e c t as low as 1 x 10~^ micrograms per c c . i n t h e i r growth medium. Methods used to determine t h e amount of PABA i n u r i n e and a l s o t h e d e t e r m i n a t i o n o f c o n j u g a t e d PABA are o u t l i n e d by K i r c h and Bergeim (1943). - 119 -APPENDIX IV. Co m p o s i t i o n of Media Used i n the P r e v i o u s  Experiment s• I . H o l d i n g Medium f o r A. suboxydans. ( U n d e r k o f l e r and Fulmer, 1937) Bacto y e a s t - e x t r a c t G l y c e r o l ( C P . ) Bacto-agar pH a d j u s t e d t o 6.0 + 0.1 S t e r i l i z e d f o r 15 minutes a t 15 pounds p r e s s u r e 0.5 per cent 5.0 per cent 1.5 per Gent I I . C u l t u r e Medium used i n P l a t i n g E x p e r i m e n t s . Bacto y e a s t - e x t r a c t 0.5 per c e n t G l y c e r o l ( C P . ) 5.0 per cent Bacto-agar 1.5 per cent pH a d j u s t e d t o 6.0 1 0.1 S t e r i l i z e d f o r 15 minutes at 15 pounds p r e s s u r e . I I I . F o r t i f i e d B a s a l Medium ( u n d i l u t e d ) f o r PABA Assay (Landy and S t r e i g h t o f f , 1943) C a s e i n h y d r o l y s a t e 0.6 gms. G l y c e r o l 10.0 ti Tryptophane 20.0 mgm. C y s t i n e 15 .0 it Adenine 1.0 ti Quanine 1.0 i i X a n t h i n e 1.0 i i K 2 H P 0 4 KH 2P0, 100.0 it 100.0 I I MgSO, ,7H 20 FeS0?.7H 20 40.0 ti 2.0 it MnSO^,4H20 2.0 it NaCl 2.0 I I C a l c i u m P a n t o t h e n a t e 200.0 "micro N i c o t i n i c A c i d 100.0 it D i s t i l l e d water t o 100.0 ml. pH a d j u s t e d to 6.0 t 0,1 S t e r i l i z e d f o r 15 minutes at 15 pounds p r e s s u r e . - 120 -APPENDIX V. Determinat i o n of the Number of B a c t e r i a l  C e l l s i n the Inoculum. The drop p l a t e t e c h n i q u e of M i l e s and M i s r a (1938) t o e s t i m a t e t h e number of b a c t e r i a l c e l l s p r e s e n t i n a c e r t a i n volume of c e l l s u s p e n s i o n was employed. Methodr Dropping p i p e t t e s were made. They were c a l i b r a t e d by means of w e i g h i n g a c c u r a t e l y a known number of drops of d i s t i l l e d water e j e c t e d f rom them. From t h e average o f f i v e w e i g h i n g s of t h i s t y p e , the a c t u a l number of drops per c u b i c c e n t i m e t e r was c a l c u l a t e d f o r each p i p e t t e . E i g h t f o l d d i l u t i o n s were u s u a l l y made, 1.0 c c . of c e l l s u s p e n s i o n added t o 9.0 c c . o f d i l u e n t , and a f r e s h p i p e t t e used f o r each d i l u t i o n . G l y c e r o l - y e a s t e x t r a c t agar (Appendix IV) p l a t e s were d r i e d f o r 24 hours at room t e m p e r a t u r e . When the agar was s u f f i c i e n t l y d r i e d , f i v e w e l l spaced drops of t h e d i l u t i o n s of A., s uboxy dans p r e v i o u s l y p r e p a r e d were p l a c e d onto the s u r f a c e of t h e medium. A f t e r the a b s o r p t i o n of t h e drops ( u s u a l l y r e q u i r e s 20-30 m i n u t e s ) , the p l a t e s were i n c u b a t e d i n t h e u s u a l manner at 30°C. Counts were made i n the drop a r e a s c o n t a i n i n g t h e l a r g e s t number of c o l o n i e s w i t h o u t s i g n s of c o n f l u e n c e or of - 121 -gross d i m i n u t i o n i n c o l o n y s i ^ e due t o o v e r c r o w d i n g . The number of c o l o n i e s were e s t i m a t e d f r o m t h e mean o f 10 t o 15 c o u n t s . From the mean f i g u r e , the number of m i c r o b i a l c e l l s per c c . of s u s p e n s i o n was c a l c u l a t e d by the f o l l o w i n g f o r m u l a : C x N x D = Number of c e l l s per c c . of s u s p e n s i o n , where C i s the average number of c o l o n i e s counted per i n -i t i a l drop; N, t h e p i p e t t e c a l i b r a t i o n , i e . number of drops per c c . d i s t i l l e d w a t e r ; and D, the d i l u t i o n of the o r i g i n a l s u s p e n s i o n . - 122 -APPENDIX V I . The G e n e r a l E l e c t r i c Mercury Vapor, 15 watt G e r m i c i d a l Lamp. The d e s c r i p t i o n of the lamp used f o r the source of u l t r a v i o l e t energy used i n the p r e v i o u s e x p e r i m e n t s i s as f o l l o w s ( G e n e r a l E l e c t r i c B u l l e t i n , 1947): Designat i o n ^ Nominal L e n g t h , inches 18 Diameter, i n c h e s 1 Bulb T-8 Approximate Lamp Amperes 0.31 Approximate Lamp V o l t s 5 5 C i r c u i t V o l t a g e 110-125 Rated Average L i f e , hours 2500 Base Med. B i p i n U l t r a v i o l e t Output, w a t t s at 2537^ a t 100 hours 2.9 L i s t p r i c e , each #4.50 T h i s G e n e r a l E l e c t r i c Mercury Vapor 15 watt G e r m i c i d a l Lamp, as seen from t h e ' t a b l e g i v e n below r a d i a t e s most o f o i t s energy at t h e 2537A l i n e w h i c h i s v e r y near t h e wave l e n g t h most e f f e c t i v e i n d e s t r o y i n g b a c t e r i a . I r r a d i a t i o n Procedure : A known amount of a s o l u t i o n up t o 20 c c . i s measured i n t o a p e t r i d i s h h a v i n g the d i m e n s i o n s : 9.0 cm. i n diameter and 1.0 cm. deep. The s o l u t i o n i s then i r r a d i a t e d by the p r e v i o u s l y I - 123 -d e s c r i b e d u l t r a v i o l e t source f o r v a r i o u s t i m e i n t e r v a l s . The d i s t a n c e from the lamp to the s u r f a c e of t h e s o l u t i o n i s 14.0 cm. At t h e end o f the i r r a d i a t i o n , t h e s o l u t i o n i s c a r e -f u l l y made up t o i t s o r i g i n a l volume w i t h the d i l u e n t . The apparent l o s s i s due t o t h e e v a p o r a t i o n of the d i l u e n t by the heat of t h e lamp. TABLE D. S p e c t r a l Data: U l t r a v i o l e t L i g h t and L i g h t Output of th e , G e n e r a l E l e c t r i c Mercury Vapor, 15 watt G e r m i c i d a l Lamp  T B u l l . #974117 Source Bulb Below 2800A 2800 -3200A 3 2 0 0 - 3 8 0 0 A G e r m i c i d a l % of % of % of Mercury Watts Lamp Watts Lamp Watts Lamp Lamp Watts Watts Watts 15 watt #9741 2 . 9 19 0 . 0 6 0 . 4 2 0 . 0 0 5 0 . 3 4 Source Bulb G e r m i c i d a l Mercury Lamp 3800-5000A 5000-6000A 6000-7600A • % of % of % of Watts Lamp Watts' Lamp Watts Lamp Watts Watts Watts 15 watt ^9741 0 . 1 7 1 . 1 5 0 . 0 9 0 . 5 7 0 .005 0 . 0 3 - 124 -BIBLIOGRAPHY 1. A n s b a c h e r , S . , S c i e n c e _3_, 164-65, 1941. 2. A n s b a c h e r , S . , V i t a m i n s and Hormones, 2,, 215-54, 1944. 3. B a u m g a r t n e r , J . G . , J . B a c t . , _2_, 75-76, 1936. 4. B e h a g h e l , 0., Rothman, S . , and S c h u l t z e , W . , S t r a h l e n t h e r a p i e 2_, 110-14, 1928. 5. B e l l , P . 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